Analytical Profiles of Drug Sub stances Volume 2 Edited by Contributing Editors

Analytical Profiles of

Drug Sub stances Volume 2

Edited by

Klaus Florey The Squibb Institute for Medical Research

New Brunswick, New Jersey

Contributing Editors,

Glenn A. Brewer, Jr. Lester Chafetz Gerald J. Papariello Edward M. Cohen David E. Guttman Frederick Tishler

Stephen M. Olin

Bernard Z. Senkowski

Compiled urider [he auspices of [he Phartnnceutical Analysis ant1 Control Section

Academy of Phnrniac.eiitica1 Sciences

Academic Press NewYork and London 1973

EDITORIAL BOARD

Norman W. Atwater David E. Guttman Glenn A. Brewer, Jr. Eric H. Jensen Lester Chafetz Arthur F. Xichaelis Edward M. Cohen Stephen Id. Olin Jack P. Comer Gerald J. Papariello Klaus Florey Bernard 2. Senkowski Salvatore A. Fusari Frederick Tishler

ACADEMIC PRESS RAPID MANUSCRIPT REPRODUCTION

COPYRIGHT 1973, BY THE AMERICAN PHARMACEUTICAL ASSOCIATION ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

ACADEMIC PRESS, INC. 11 1 Fifth Avenue, New York, New York 1wO3

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NW1 IDD

LIBRARY OF CONGRESS CATALOO CARD NUMBER: 70-187259

PRINTED IN THE UNITED STATES OF AMERICA

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

S. Ahuju, Ciba-Geigy Inc., Ardsley, New York

N . W. A trvater, Searle and Co., Chicago. Illinois

P. P. Begosh, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania

K. W. Blessel, Hoffmann-La Roche Inc., Nutley, New Jersey

G. A . Brewer, Jr . , T h e Squibb Institute for Medical Research, New Brunswick, Now Jersey

L. Clzufbtz, Warner-Lanibert Research Institute, Morris Plains, New Jersey

E: M. C'olzeti, Merck, Sharp and Dohme. Westpoint, Pennsylvania

J. Colirw, Ciba-Geigy lnc., Ardsley, New York

J. P. C o / ~ c r , Eli Lilly and Company, Indianapolis, Indiana

K. 5. Crornhie, Wyeth Laboratories, Philadelphia, Pennsylvania

L. F. Cirlletz, Wyeth Laboratories, Philadelphia, Pennsylvania

R. D. Dalc,y, Ayerst Lahoratories, Rousses Point, New York

R. L'. D U / I ~ . Warner-Lambert Research Institute, Morris Plains, New Jersey

J . EL Fuirhrotlicr, The Squibb Institute for Medical Research, Moreton, Wirral, England

K. Florcy, The Squibb Institute for Medical Research, New Brunswick, New Jersey

S. A . Ft.r.suri. Parke, Davis and Company, Detroit, Michigan

D. E. Girttnian, School of Pharmacy, University of Kcntucky. Lexington, Ke ti t uc k y

vii

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

E. ivashkiv, The Squibb Institute for Medical Research, New Brunswick, New Jersey

E. H . Jensen, The Upjohn Company, Kalamazoo, Michigan

J. Kuzan, American Cyanamid, Bound Brook, New Jersey

A . F. Michaelis, Sandoz Pharmaceuticals, East Hanover, New Jersey

S. M . O h , Ayerst Laboratories, New York, New York

G. J. Papariello, Wyeth Laboratories, Philadelphia, Pennsylvania

T. E. Ricketts, American Cyanamid, Bound Brook, New Jersey

G. D. Roberts, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania

B. C. Rudy, Hoffmann-La Roche Inc., Nutley, New Jersey

R . S. Suritoro, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania

B. Z. Senkowski, Hoffmann-La Roche Inc., Nutley, New Jersey

F. Tishler, Ciba-Geigy Inc., Ardsley, New York

R. J. Warrer?, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania

E. White, V , Smith, Kline and French Laboratories, Philadelphia, Pennsylvania

viii

FOREWORD

The concept for gathering together and publishing pertinent information on the physical and chemical properties of various official and new drug substances had its origin with the members of the Section on Pharmaceutical Analysis and Quality Control of the Academy of Pharmaceutical Sciences. More than two years of consideration preceded the authorization of this ambitious project by the Executive Committee of the Academy in the Spring of 1970. The immediate and virtually spontaneous enlistment of the first group of contributors t o this work attested to its importance and the wisdom of pursuing its publication.

By coincidence, the delegates to the sesquicentennial anniversary meet- ing of the United States Pharmacopeial Convention, Inc., in Washington, D.C. on April 8-1 0, 1970, adopted the following resolution:

Whereas widespread interest has been expressed in the inclusion of additional information about physical and chemical properties of drugs recognized in the United States Pharmacopeia

Be I t Resolved that the Board of Trustees consider publishing in the Pharmacopeia, or in a companion publication, information on such attributes as solubilities, pH and pK values, spectra and spectrophotometric constants, and stability data, pertaining to pharmacopeial drugs.

The U.S.P.C. Board of Trustees unanimously approved the resolution in principle on June 4, 1970 and authorized the Director of Revision t o include in the U.S.P. monographs such physical-chemical information as he deemed proper and also to cooperate with the Academy of Pharmaceutical Sciences to secure the publication of other physical-chemical data.

It was my privilege to be the President of the Academy during the period when Arialyticai Profiles was under consideration. I t is my unusual and unique honor as President of the Academy and Director of U.S.P. Revision to assist in the institution and dedication of this first volume. I trust that it will serve immeasurably in providing the scientific community with an authoritative source of information on the properties of many of our important drug compounds.

Jar1irar.v 19 71 Thomas J. Macek

PREFACE

Although the official compendia define a drug substance as to identity, purity, strength, and quality, they normally do not provide other physical or chemical data, nor do they list methods of synthesis or pathways of physical or biological degradation and metabolism. At present such information is scattered through the scientific literature and the files of pharmaceutical laboratories.

For drug substances important enough to be accorded monographs in the official compendia such supplemental information should also be made readily available. To this end the Pharmaceutical Analysis Section, Academy of Pharmaceutical Sciences, has started a cooperative venture to compile and publish Anulyricul Profiles of Drug Substunces in a series of volumes of which this is the second. It is also planned to revise and update these profiles at suitable intervals.

Our endeavor has been made possible through the encouragement we have received from many sources and through the enthusiasm and cooperative spirit of our contributors. For coining the term Analytical Profile we are indebted to Dr. James L. Johnson of the Upjohn Company.

We hope that this, our contribution to the better understanding of drug characteristics, will prove to be useful. We welcome new collaborators, and we invite comment and counsel to guide the infant to maturity.

Klaus Florey

xi

T

Eiigerie Ivushkiv

1

E U G E N E IVASHKIV

1.

2. 2.1

2.2

2.3 2.4 2.5 3.

4.

5 . 6.

Table of Contents Page

Description 4 1.1 Name:Ampicillin 4 1.2 Formula and Molecular Weight 4 1.3 Isomers 4 1 . 4 Hydrates 4 1.5 Salts 5 1.6 Appearance, Color and Odor 5 Physical Properties 5 Spectra 5 2.11 Infrared Spectra 5 2.12 Nuclear Magnetic 8

Resonance Spectra 2.13 Mass Spectroscopy 12 2.14 Ultraviolet Absorption 16 Crystal Properties 17 2.21 Crystalline Modification of 17

2.22 X-ray Diffraction 18 2.23 Melting Range 18 2.24 Differential Thermal Analysis 18

Ampicillin

2.25 Thermal Gravimetric Analysis 20 Solubility 20 Ionization Constant, pK 20 Optical Rotation 20 Ampicillin Stability 23 3.1 Modes of Penicillin Degradation 23 3.2 Stability of Ampicillin 25

3.3 Stability of Ampicillin Powders 32 3.4 Cupric Ion-Catalyzed Hydrolysis 32 Methods of Manufacture 32 4.1 Microbiological 32 4.2 Chemical 33 Isolation and Purification 37 Methods of Analysis 37 6.1 Identification Tests 37

in Solution

2

AMPICILLIN

Page

6.2 O u a n t i t a t i v e Methods 6 . 2 1 U l t r a v i o l e t Spec t rophoto-

m e t r i c Methods 6.22 F luo romet r i c Determina t ion 6 .23 Po la rograph ic Determina t ion 6.24 Thin Layer Chromatography 6.25 Paper Chromatography 6.26 Iodomet r i c T i t r a t i o n 6.27 Hydroxamic Acid 6 .28 Amperometric T i t r a t i o n 6 .29 M i c r o b i o l o g i c a l Methods

7 . P r o t e i n Binding 8 . Pharmacok i n e t ics 9. References

38 38

39 39 39 40 41 4 1 42 42 4 3 45 47

3

EUGENE IVASHKIV

1. Description

1.1 Name: Ampicillin AmpicillinL,L,jis designated by

Chemical Abstracts as Q- (2-amino-2-phenyl- acetamido)-3,3-dimethyl-oxo-4-thia-l-azabicyclo 17.2.0-7 heptane-2-carboxylic acid. Ampicillin is also known as bb(-)-a-aminophenylacetamidd penicillanic acid, Q ( - ) -a-aminobenzylpenici llin4 and a-aminobenzylpenicillin5.

1.2 Formula and Molecular Weight

C)H 0 l€12 - C-N II I I

C16H1 9N304S 349.41

1.3 Isomers The presence of a symmetric C atom

in the side chain provides optical isomer6. The D-isomer, I)(-)-a-aminobenzylpenicillin, is more active than the &-isomer, L(-)-a-aminobenzyl- penicillin7. has been reported8 3 8 ,

The s nthesis of ampicillin epimer

1.4 Hydrates It has been re orted that ampicillin

can exist in anhydrous sesquihydrate16 and trihydrate Austin, et al. postulate that ampicillin exists in anhydrous or trihydrate forms only. Refer to section 2.21.

4

AMPICILLIN

1 . 5 S a l t s Potass ium and sodium s a l t s of

a m p i c i l l i n 2 0 - 2 9 , human l y s o z me a m p i c i l l i n sal t3! 2 - n i t r o - l , 3 - i n d a n d i o n e sa l t3 ' , and t h e a m p i c i l l i n s a l t o f k a n a m y c 1 r - 1 ~ ~ have been p r e p a r e d .

1 . 6 Appearance, C o l o r and Odor A m p i c i l l i n i s a f r e e - f l o w i n g , w h i t e

c r y s t a l l i n e powder. It h a s an odor c h a r a c t e r i s t i c of p e n i c i l l i n s .

2 . P h y s i c a l P r o p e r t i e s

2 . 1 S p e c t r a

2 . 1 1 I n f r a r e d S p e c t r a S u b s t i t u t e d monocycl ic O-lactams i n

When t h e B-lactam r i n g i s s o l u t i o n show c a r b o n y l a b s o r p t i o n w i t h i n t h e r ange 5 . 6 8 - 5 . 7 8 ~ ~ ~ . f u s e d t o a t h i a z o l i d i n e r i n g , t h e c a r b o n y l a b s o r p t i o n o c c u r s i n t h e r ange 5 .62-5 .651 . Morin and c o - ~ o r k e r s ~ ~ have n o t e d rough c o r r e s - pondence between t h e f r e q u e n c i e s o f t h e f3-lactam c a r b o n y l a b s o r p t i o n and t h e b i o l o g i c a l a c t i v i t i e s of a s e r i e s of p e n i c i l l i n d e r i v a t i v e s .

a n a l o g u e s have been d i s c u s s e d 3 5 . The i n f r a r e d spec t rum of a m p i c i l l i n t r i h y d r a t e was measured on a Perkin-Elmer Model 2 1 double-beam s p e c t r o - p h 0 t o m e t e r 3 ~ . The i n f r a r e d a b s o r p t i o n of p e n i c i l l i n d e r i v a t i v e s h a s been r e c o r d e d and d i s c u s s e d 3 7 . s p e c t r a o f t h e Squ ibb Pr imary Refe rence Subs tances o f a m p i c i l l i n t r i h y d r a t e and anhydrous a m p i c i l l i n r e c o r d e d a s m i n e r a l o i l m u l l and po t - ass ium bromide p e l l e t s w i t h a Perkin-Elmer Model 2 1 s p e c t r o p h o t o m e t e r . I n t e r p r e t a t i o n of t h e spec t rum of a m p i c i l l i n t r i h y d r a t e h a s been re- p ~ r t e d ~ ~ a n d i s g i v e n i n T a b l e 1.

The i n f r a r e d s p e c t r a o f p e n i c i l l i n

F i g u r e 1 and F i g u r e 2 a r e t h e

5

F i g u r e 1. I n f r a r e d Spec t rum o f A m p i c i l l i n T r i h y d r a t e Squ ibb R e f e r e n c e S t a n d a r d .

FREQUENCY ( c M - ~ )

WAVELENGTH IMICRONSJ

F i g u r e 2. I n f r a r e d Spec t rum of Anhydrous A m p i c i l l i n Squ ibb Refe rence S t a n d a r d .

EUGENE IVASHKIV

T a b l e I I n f r a r e d S p e c t r u m o f A m p i c i l l i n T r i h y d r a t e

I R A b s o r p t i o n Band, I n t e r p r e t a t i o n

2 . 9 H20 3 . 1 N' H

5 . 6 5 @ - l a c t a m C = 0 5 . 9 0 Amide C = 0

6 . 7 A r o m a t i c r i n g , Amide 11, NH3+

1 4 . 4 Monosubs ti t u t e d a r o m a t i c r i n g .

weak b a n d s 3.5-4.8 NH3+

+ 6 . 2 and 6 . 3 5 COO-, NH3

I n t h e s o l i d form, a m p i c i l l i n t r i h y d r a t e e x i s t s a s t h e z w i t t e r i o n a n d t h e i n f r a r e d s p e c t r u m shows a b s o r p t i o n s t y p i c a l o f t h i s t y p e of compound38.

2 . 1 2 N u c l e a r M a g n e t i c Resonance S p e c t r a NMR s p e c t r a o f p e n i c i l l i n d e r i v a t i v e s

i n d i f f e r e n t s o l v e n t s were r e c o r d e d on a 100 MHz s p e c t r o ~ n e t e r ~ ~ . R e c e n t l y , s t r u c t u r a l s t u d i e s w i t h l3c n u c l e a r m a g n e t i c r e s o n a n c e w e r e r e p o r t e d 4 0 €or p e n i c i l l i n s . I 3 c Chemica l s h i f t a s s i g n m e n t s were made f o r t h e d i f f e r e n t c a r b o n a toms. NMR s p e c t r a i n D 2 0 s o l u t i o n s o f t h i r t e e n p e n i c i l l i n d e r i v a t i v e s h a v e b e e n r e c o r d e d and i n t e r ~ r e t e d ~ ~ . s p e c t r a of a m p i c i l l i n t r i h y d r a t e i n d e u t e r o d i - m e t h y l s u l f o x i d e (DMSO-d6) and D 2 0 - D C 1 . T e t r a - m e t h y l s i l a n e was u s e d a s a n i n t e r n a l s t a n d a r d . The V a r i a n XL-100-15 NMR s p e c t r o p h o t o m e t e r was u s e d . I n t h e c a s e of D M S O - ~ ~ , B-lactam p r o t o n s c o u l d be e a s i l y d i s t i n g u i s h e d b y t h e i r c o u p l i n g p a t t e r n .

The NMR s p e c t r a of t h e S g u i b b P r i - mary R e f e r e n c e S u b s t a n c e a r e g i v e n i n F i g . 3 a n d F i g . 4 . The a s s i g n m e n t s a r e r e p o r t e d i n T a b l e 11.

Cohen and Puar'' s t u d i e d NMR

8

u)

a I

f n C

.d

a, 4J m

k a $1 c

4

k

E

C

.d

rl

rl

-d

u

-d

$ rCI 0

5 k 4J u a, a

w

x m

a, k 7

0-l .rl Irr

9

c 0

m " e ? , , , , . , , , / , , , , , , , , . , , , , / , , , , , , , . , , , , , I , , , , / , , , , , , , , , , , , 1 , 1 " " ' , " " , , 1 , , , ' , ' , , ' ~ , ' , ' , ' ' ' , ~ ~ ' '

1

Figure 4. NMR Spectrum of Ampicillin Trihydra te in D20-DC1.

T a b l e I1 Pro ton- re sonance L i n e s

DMSO-d6

D 2 0 - D C 1

2 H 3H 5H , 6H 7H 8 H 9 10

1 . 3 9 1 . 3 9 5.39q ( 9 . 0 , 4 . 0 ) 5 .95b 4 .84 7.36m 4.6433 1 . 4 9 5 .39d ( 4 . 0 )

1 . 4 1 4 .47 5 .54 5.26 7 . 5 3 >

6 5 c

e 1 . 4 4 5

EUGENE IVASHKIV

2.13 Mass Spectroscopy The behavior of the penicillins in

the mass spectrometer has been studied by several investigators. The fragmentation of penicillin V has been discussed by Kukolja, et a1 42. Richter and Biemann43examined penicillins G and V methyl esters and made numerous accurate mass measurements at high resolution. The nucleus fragmentation by four different routes is shown in Fig.5.

Bochkarev, et al. 42 studied various penicillin derivatives substituted at the carbonyl group and showed that it was possible to deduce the nature of this substituent from the mass spectrum. The mass spectrum of ampicillin trihydrate was determined by Funke and C ~ h e n ~ ~ and is shown in Figure 6. They prepared a disilyl derivative using N,O-bis (trimethylsilyl) - acid amide dissolved in pyridine. The mass spectrum exhibits the molecular ion, M+, of m/e 493 €or the disilyl derivative. The more intense M+ -15 ion at m/e 478 is due to loss of methyl radical. The m/e 421 ion corresponds to the loss of Si(CH3)2CH2 from the molecule or the mono-silyl derivative while the ion 15 amu lower at m/e 406 has lost a methyl group in addition to the silyl function. The diagnostic fragment ion at m/e 178 €or silyl derivatives of penicillins demonstrates the presence of 6-aminopenicillanic acid ( 6 - A P A ) function. In addition, the ion at m/e 106 also is diagnostic for the non-silanized phenylqlycyl derivative. The intense ion at m/e 232 is diagnostic €or all penicillin derivatives. The mass spectral assignments are summarized in Figure 7.

AMPICILLIN

F i g u r e 5. P e n i c i l l i n F r a g m e n t a t i o n

lt. __+ RCHzCONHCH=C=O

b __+ Me2C==CHCO,Mclt'

Me R C H ~ C O ~ H C H

H? 1 N-'

RCH2CONH c - - c

/

R e p r o d u c e d w i t h p e r m i s s i o n from A d v a n c e s i n Druq R e s e a r c h , Vol. 6, p. 2 0 0 , A c a d e m i c P r e s s , N e w York , 1971.

13

i

EUGENE IVASHKIV

R E L A T I V E INTENSITY

- c - -

~ ~ ~ r n O ) m m l r n

PERCENT TOTAL IONIZATION 39

Figure 6 . Low Resolut ion Mass Spectrum of Ampic i l l in Trihydrate Squibb House Standard.

14

AMPICILLIN

231 + H I

178 I

m/e 178

1

+

t

m/e 106

Fig. 7. Fragmentation of Ampicillin Derivative.

IS

EUGENE IVASHKIV

2 . 1 4 U l t r a v i o l e t A b s o r p t i o n U l t r a v i o l e t s p e c t r a o f a m p i c i l l i n

t r i h y d r a t e , S q u i b b P r i m a r y R e f e r e n c e S u b s t a n c e were r e c o r d e d on a C a r y Model 1 5 s p e c t r o p h o t o - meter46 and E l % v a l u e s c a l c u l a t e d . The r e s u l t s a r e g i v e n i n 4aF€'e 111.

T a b l e I11

U l t r a v i o l e t A b s o r p t i o n of A m p i c i l l i n T r i h y d r a t e

W a v e l e n g t h , nm

P h o s p h a t e b u f f e r p H 5 . 3 2 57 262 268

E 1% 8 . 6 9 7 . 8 1 5 . 6 1 1 c m

P h o s p h a t e b u f f e r pH 7 . 0 257 262 268

1% 7 . 9 4 6 . 6 9 4 . 5 5 1 c m

P h o s p h a t e b u f f e r 9 . 5 2 58 2 68 -

E 1% 7 . 2 8 5 . 0 3 1 c m

With t h e i n c r e a s e o f p H o f b u f f e r s o l u t i o n El% d e c r e a s e s . N o p e a k a t 268 nm i n c m p H 9 . 5 b u f g e r was o b t a i n e d .

t r i h y d r a t e s o l u t i o n s p r o d u c e d maximum a b s o r p t i o n a t 258,

S a t u r a t e d m e t h a n o l i c a m p i c i l l i n

262 a n d 268 nm46.

16

AMPICILLIN

2.2 Crys ta 1 Properties

2.21 Crystalline Modification of Ampicillin Ampicillin crystallizes into two

forms depending on the temperature of the

obtained from aqueous solutions at temperatures above 6OoC; the trihydrate is obtained from aqueous solutions at temperatures below 5OoC. The existence of anhydrous, monohydrate, sesquihydrate and trihydrate fo-ms of ampicillin was It has been shown by x-ray dif f r a ~ t i o n ~ ~ that ampicillin monohydra te existed. of the infrared spectra of various forms of crystalline ampicillin that only the anhydrate and trihydrate exist. They believed the other hydrates were either amorphous or partially dehydrated t r i h ~ d r a t e ~ ~ . Crystals of anhydrous and trihydrate ampicillin were prepared. James and Hall reported crystallographic data for ampicillin trihydrate. They showed that ampicillin exists as a zwitterion with three water molecules extensively involved with hydrogen bonding. distinguished between a crystalline anhydrous form and a monohydrate, which differ in solid- state infrared spectra, density, solubility and th erma 1 stab i 1 i ty . Anhydrous amp i c i 11 in wa s prepared from any form of ampicillin but reason- ably rapid conversion of trihydrate required a temperature 80°-1000C19. Hydrated ampicillin was also converted into an anhydrate by heating a suspension in nitromethane or other nitro- hydrocarbons.

aqueous solution 19 . Anhydrous ampicillin is

Austin -- et.al.19 concluded from a study

Grant and A l b ~ r n ~ ~ have

17

EUGENE IVASHKIV

2.22 X-ray D i f f r a c t i o n S i n g l e - c r y s t a l x - r a y d i f f r a c t i o n

was u s e d t o d e d u c e t h e s t r u c t u r e of b e n z y l p e n i - illi in^^. was a n a l y z e d a n d b o n d l e n g t h s determined5' . X-ray d i f f r a c t i o n a n a l y s i s o f a n h y d r o u s a m p i c i l l i n a n d a m p i c i l l i n m o n o h y d r a t e were p e r f o r m e d by T.Doyne and r e p o r t e d 4 7 . F i g . 8 shows the x - r a y d i f f r a c t i o n o f a m p i c i l l i n and g i v e s t h e d v a l u e s c h a r a c t e r i s t i c o f t h i s c r y s t a l form o f a m p i c i l l i n t r i h y d r a t e 5 1 .

S e v e r a l s a l t s o f b e n z y l p e n i c i l l i n

2 . 2 3 M e l t i n q Ranqe A m p i c i l l i n m o n o h y d r a t e mel t s w i t h

decom o s i t i o n a t 202OC a n d sodium a m p i c i l l i n a t 205°Cf4, s e s q u i h y d r a t e a n d a n h y d r o u s a m p i c i l l i n decompose a t 199-202°C16. The m e l t i n g r a n g e f o r a m p i c i l l i n t r i h y d r a t e w i t h d e c o m p o s i t i o n a t 214.5O - 215.5OC was r e p o r t e d 5 2 and m e l t i n g w i t h d e c o m p o s i t i o n h a s b e e n repor ted a t 202O- 204OC by a n o t h e r i n v e s t i g a t o r5?

2 . 2 4 D i f f e r e n t i a l Thermal A n a l y s i s Jacobson54 r e c o r d e d d i f f e r e n t i a l

therma 1 a n a l y s i s c u r v e s o f a m p i c i l l i n tr i h y d r a te, S q u i b b P r i m a r y R e f e r e n c e S u b s t a n c e , on a DuPont D i f f e r e n t i a l Thermal A n a l y z e r w i t h a t e m p e r a t u r e r i s e o f 15O per m i n u t e . 88O,92O, and a l a r g e e n d o t h e r m a t 100°C were ob- s e r v e d . Some s a m p l e s of a m p i c i l l i n t r i h y d r a t e h a d a s i n g l e l a r g e e n d o t h e r m a t 125OC w h i l e Y i h e r s h a d t h e l a r g e e n d o t h e r m a t a b o u t 105O- llO°C . D i f f e r e n t i a l t h e r m a l a n a l y s i s f o r t h e c o n t r o l o f p h a s e t r a n s f o r m a t i o n o f a m p i c i l l i n i s u s e d 5 5 . I t h a s b e e n f o u n d t h a t thermograms were r e p r o d u c i b l e f o r a g i v e n l o t of a m p i c i l l i n t r i h y d r a t e b u t v a r i e d from lot t o l o t . The p h a s e t r a n s f o r m a t i o n of a m p i c i l l i n t r i h y d r a t e b y t h e d i f f e r e n t i a l t h e r m a l a n a l y s i s u n d e r p r e s s u r e was s t u d i e d 5 6 .

E n d o t h e r m i c steps a t

18

AM

PIC

ILL

IN

w 0

c !4

19

Figure 8. X-Ray Diffraction Pattern of Ampicillin Trihydrate

EUGENE IVASHKIV

2.25 Thermal Gravimetric Analysis A DuPont Thermogravimetric Analyzer,

Model 950, indicated total volatile material to be 13.8% in ampicillin trihydrate Squibb Primary Reference Substance54.

2.3 Solubility The solubilities of anhydrous ampicillin,

ampicillin trihydrate and sodium ampicillin from two manufacturers in various solvents were determined by Marsh and we is^^^. variation in the solubility of sodium ampicillin from two different manufacturers and indicated this could be due to crystalline structure. The results are reported in Table IV.

The theory is 13.39%.

They found a

Anhydrous ampicillin and ampicillin trihydrate were compared for solubility in distill d water at temperatures ranging from 7.50 to 5OoC 58 ,

2.4 Ionization Constant, pK

Rapson and Bird5' reported ionization constants for ampicillin to be: pK1 = 2.53 f 0.004 and pK - 7.24 f 0.02. Jacobson and Russo-Alesi66 calculated pK2 for ampicillin trihydrate to be 7.24. = 2.66 f 0.03 and pK2 = 7.24

Hou and PooleG1 reported pK1 0 . 0 3 .

2.5 Optical Rotation

Ampicillin monohydrateL-d21 + 281°

Ampicillin sesquihydrateL a4 + 283.1°

Sodium Ampicillin/-az20 - - + 209'

Anhydrous AmpicillinL %

Reference

14 D (C = 1 in H20)

(C = 1 in H20) 16

20 - -

( C = 0.2 in H20) 2o 14

( C = 1 in H20) 16

- _ + 287. go

20

Tab le I V S o l u b i l i t y of A m p i c i l l i n (mg/ml)

S o l v e n t Anhydrous

Water Me thano 1 Ethanol I sop ropano l Isoamyl a l c o h o l Cyclohexane Benzene

h) Petroleum E t h e r I s o o c t a n e Carbon T e t r a c h l o r i d e E t h y l Ace ta t e Isoamyl A c e t a t e Acetone Methyl E t h y l Ketone D i e t h y l E t h e r E thy lene C h l o r i d e 1,4-Dioxane Chloroform

i

10.098 2.968 0.390 0.055 0.125 0.048 0.002 0.010 0.0 0.008 0.025 0.030 0.125 0.052 0.022 0.032 0.595 0.095

A m p i c i l l i n

N a S a l t I * Na S a l t 11*

> 20 P 2 0 v 2 0 1. 13 1.902 0.075 0.022 0.025 0.022 0.032 0.035 0.105 0.518 0.178 0.022 0.060 1.375 0.118

> 20 > 20

19.780 6.405 19. 300 0.0 0.0 0.0 0 . 0 0 . 0 0.058 0.048 7 20 7 2 0 0.0 0.032 1.845 0.155

T r ihydra t e

7.558 6.649 2.538

- 0.068 0.032 0.038 0.022 0.025 0.225 0.078 8.952 2.790 0.03 0.068 2.772 0.075

c o n t i n u e d . . .

Table IV. Solubility of Ampicillin (mg/ml), continued

Ampicillin *

Solvent Anhydrous Na Salt I* Na Salt I1 Trihydrate

Carbon Disulfide 0.015

Formamide 20 Ethylene Glycol 18 .415 Propylene Glycol 2 . 2 3 0 Dime thylsul f ox ide

13 0.1N NaOH 20 0.1N H C 1 20

Pyr i dine 2.100

20

h)

0.010 3.256

20 20 20 20 20 20

0 . 0 20 20 20 20 20 20 20

0.022 1 2 . 1 3 1

20

19.128 rn C

rn z rn

<

I x <

4.138 GI

20 20 20

-

R -

* Sodium ampicillin from two different manufacturers.

AMPICILLIN

The optical rotation for ampicillin trihydrate, - /-&26

and ampicillin anhydrous, l -~$*~' + 289O ( C = 1, buffer p H 8.0) has been reported62. D ~ r s c h ~ ~ reported the optical *rjotation for ampicillin trihydrate as L-d 252.7O (C = 1, buffer pH 8.0p. Optical rotation in 2 1 hydrochloric acid, after dissolution of 1% ampicillin and 60 minutes of standing at 25OC is

+ 2 4 5 . 1 0 (C = 1 buffer p H 8.0)

+ 249.5O to

= -85. 5°C64.

3. Ampic i 1 lin Stab i 1 i ty

3.1 Modes of Penicillin Degradation The main course of deterioration

penicillins is hydrolysis. The course of the hydrolysis is shown in Figure 9. reviewed modes of penicillin degradation.

Dunham 65

23

w P

F i g u r e 9. P e n i c i l l i n D e g r a d a t i o n /s\

R-CONHCH-CH C(CHJ,

Pe nic il I i n

I 1 I CO -N - CHCOO H / "/lHIH \

/s\ /s\ HOOC-CH-CH C(CH,),

I RCONHCH-CH C(CH3)z RCONHCHCHO

I I I 1 1 I NH-CHCOOH COOH N N-CKOOH COOH

Penicilloic Ac id Penaldic Ac id

\

\ / C

I R \ Penillic Acid

+ \

S H' 'C(CH3)2 RCONKH2CHO

/s\ I RCONHCH,- C H C (C H3)2

I I NH-CHCOOH H$J- CHCOOH

Penil loaldehyde Penilloic Acid Penlciliamine

Reproduced w i t h p e r m i s s i o n from "Textbook of O r g a n i c M e d i c i n a l and P h a r m a c e u t i c a l Chemis t ry" , e d i t e d b y C . O . Wi lson , 0. G i s v o l and R . F . Doerge, 5 t h Ed. , 1966. L i p p i n c o t t Co., P h i l a d e l p h i a and Toronto .

AMPICILLIN

3.2 S t a b i l i t y o f A m p i c i l l i n i n S o l u t i o n s S a v e l l o a n d Shangraw66 showed t h a t

f r e e z i n g sodium a m p i c i l l i n s o l u t i o n s a t -200 t o -78OC g e n e r a l l y i n c r e a s e d t h e d e g r a d a t i o n o f 1% s o l u t i o n s a n d d e c r e a s e d t h e d e g r a d a t i o n i n 25% s o l u t i o n s . The c o n c e n t r a t i o n of a m p i c i l l i n i n s o l u t i o n a n d t h e t y p e of v e h i c l e c o n t r o l d e g r a - d a t i o n . D e x t r o s e a n d m a n n i t o l a c t a s c a t a l y s t s i n t h e h y d r o l y s i s of a m p i c i l l i n . A m p i c i l l i n a t a 1% c o n c e n t r a t i o n i s i n c o m p a t i b l e w i t h d e x t r o s e Normal s a l i n e i s t h e m o s t s u i t a b l e v e h i c l e f o r i n t r a v e n o u s a d m i n i s t r a t i o n o f a m p i c i l l i n . Ga 1 l e 11 i 6 7 c l a imed t h a t sodium ampi c i 1 l i n i s s t a b l e a t 5OC a n d 2 5 O C i n 1% sodium c h l o r i d e s o l u t i o n s a l s o c o n t a i n i n g 5% d e x t r o s e . J a c o b s -- e t a1.,6' 5% d e x t r o s e , 4.3% d e x t r o s e , 0.18% sodium c h l o r i d e , 0 .9% sodium c h l o r i d e , Hartmann' s s o l u t i o n a n d 1/6 M sodium l a c t a t e . A m p i c i l l i n was l e a s t s t a b l e Tn s o l u t i o n s c o n t a i n i n g l a c t a t e . A m p i c i l l i n was l e a s t s t a b l e i n s o l u t i o n s con- t a i n i n g l a c t a t e i o n , l o s i n g 40% a c t i v i t y a f t e r 4 h o u r s a n d more t h a n 60% a f t e r 24 h o u r s . A m p i c i l l i n i s i n a c t i v a t e d b y s u c r o s e , d e x t r o s e and d e x t r a n s o l u t i o n s a t a l k a l i n e pH6'. s t a b i l i t y of a m p i c i l l i n i n 0.2M a c e t a t e , c i t r a t e , p h o s p h a t e a n d l a c t a t e b u f f e r s at p H 4.570 a n d i n s o l u t i o n s a t pH 6 a n d p H 6.5 was s t u d i e d 7 7 . The d e c o m p o s i t i o n r a t e o f a m p i c i l l i n i n a 1% s o l u - t i o n f o l l o w s f i r s t o r d e r k i n e t i c s and o b e y s t h e A r r h e n i u s e q u a t i o n when compared a t 4OoC 5OoC, a n d 6OoC. T h e s e f i n d i n g s w e r e c o n f i r m e d f2 when t h e d e c o m p o s i t i o n r a t e was s t u d i e d i n t h e p r e s e n c e of H' i o n s . D e g r a d a t i o n o f a m p i c i l l i n i s s e c o n d o r d e r i n t h e p r e s e n c e o f OH- i o n s . The s t a b i l i t y of a m p i c i l l i n i n a q u e o u s s o l u t i o n s a t pH 4 - 9 was d e t e r m i n e d 7 3 . d e g r a d a t i o n o f a m p i c i l l i n i n s o l u t i o n s w e r e i n -

s t u d i e d t h e s t a b i l i t y o f a m p i c i l l i n i n

The

The k i n e t i c s o f

EUGENE IVASHKIV

v e s t i g a t e d a t 35OC and c o n s t a n t i o n i c s t r e n g t h o f 0 . 5 o v e r a pH r a n g e o f 0 .8 t o 1074. The o b s e r v e d r a t e s were f i r s t - o r d e r and s i g n i f i c a n t l y i n f l u - enced by g e n e r a l a c i d and g e n e r a l b a s e c a t a l y s i s . The a p p a r e n t h e a t s o f a c t i v a t i o n f o r a m p i c i l l i n d e g r a d a t i o n i n s o l u t i o n s w e r e 1 6 . 4 , 1 8 . 3 , and 9 . 2 k ca l /mole i n b u f f e r s o f pH 1 . 3 5 , 4 .93 and 9 .78 r e s p e c t i v e l y . The p H - r a t e p r o f i l e i n b u f f e r s o l u t i o n s showed a minimum a t a pH o f 4 .85 . However, a t z e r o b u f f e r c o n c e n t r a t i o n t h e maximum s t a b i l i t y was s h i f t e d t o a pH o f 5 .85 . A m p i c i l l i n was s t a b l e i n s o l u t i o n s a t p H 3 - 9 when s t o r e d f o r 24 h o u r s a t 5OC and 25OC. A m p i c i l l i n was u n s t a b l e i n s o l u t i o n s a t pH 10 when s t o r e d a t above condition^^^. d e m o n s t r a t e d a m e t a s t a b l e i n t e r m e d i a t e i n iso- m e r a t i o n o f p e n i c i l l i n t o p e n i c i l l e n i c a c i d i n aqueous s o l u t i o n s . The d e g r a d a t i o n o f a m p i c i l l i n t r i h y d r a t e i n s o l u t i o n i s g r e a t l y i n f l u e n c e d n o t o n l y b y p H , b u t a l s o b y t h e t y p e s o f b u f f e r s a l t s

p a n e d i o l ( T r i s ) b u f f e r o f pH 7 i s h i g h l y d e l e t e r - i o u s t o t h e s t a b i l i t y o f a m p i c i l l i n , b u t n o t a t pH 5 . 0 . C i t r a t e p r e s e n t s a c o n v e r s e p a t t e r n i n t h a t a m p i c i l l i n i s r e l a t i v e l y s t a b l e a t pH 7 b u t n o t so a t p H 5. Phospha te i s i n t e r m e d i a t e i n a c t i o n b u t t e n d s t o resemble t h e T r i s p a t t e r n . Jacobson and Russo-Ales i7* p r e p a r e d a m p i c i l l i n t r i h y d r a t e s o l u t i o n s a t a c o n c e n t r a t i o n of 5 mg/ml b y d i s s o l v i n g i t i n b u f f e r s o l u t i o n s of pH 5 . 0 o r pH 7 . 0 p r e p a r e d from 0.1g sodium a c e t a t e and a c e t i c a c i d and i n b u f f e r s o f pH 8 . 0 o r pH 8.8 prepared. from 0.1M - 2-amino-2 (hydroxy m e t h y l ) - 1 , 3 -p ropaned io l and a c e t i c a c i d . S o l u t i o n s w e r e k e p t a t room t e m p e r a t u r e and were a n a l y z e d b y i o d o m e t r i c t i t r a t i o n . S t a b i l i t y d a t a i s g i v e n i n Tab le V.

B ~ n d g a a r d ~ ~

77 . 2-Amino-2 (hydroxymethyl ) -1 ,3-pro-

26

Table V Stability of Ampicillin as Function of p H

Percentage Activity Remaining Time, hours pH 5 PH 7 P H 8 p H 8.8

0.5 1 2 3 4 5 24 27 48 96

- 99.4

100.6

92.0

92.0

-

-

-

8 3 . o

- 100.6

100.0

100.3

96. 5 91

-

I

-

96.0 87. 3 73.3 62.0 54.0 44.0

0

- - -

81.9 65.8 43.6

17.4 11.4

28.9

r

EUGENE IVASHKIV

George7’ s t u d i e d t h e s t a b i l i t y of d i l u t e ampic i - l l i n s o l u t i o n s a t pH 5 .8-6 .0 and s t o r e d a t 5OC. R e s u l t s from m i c r o b i o l o g i c a l a s s a y s a r e r e p o r t e d i n T a b l e V I .

T a b l e V I

S t a b i l i t y of A m p i c i l l i n i n D i l u t e Phospha te S o l u t i o n s

A m p i c i l l i n L e f t , % Days

A m p i c i l l i n , mcg/ml 6 8 14

3 1 0 2 - 8 0 6 97 1 0 2 88

1 0 98 99 9 1 1 5 99 99 92 25 1 0 2 - - 40 100 - -

The s t a b i l i t y o f a m p i c i l l i n i n b u f f e r s p r e p a r e d from 0 . 1 M T r i s b u f f e r and a c e t i c a c i d a s a f u n c t i o n o f pH and t e m p e r a t u r e has been s t u d i e d 8 0 . a m p i c i l l i n were made and s t o r e d a t room t e m p e r a t u r e o r 5 C f o r 29 days . S o l u t i o n s w e r e t e s t ed €or t h e r ema in ing a m p i c i l l i n by t h e c o l o r i m e t r i c hydroxylamine method. R e s u l t s a r e r e p o r t e d i n T a b l e V I I .

S o l u t i o n s c o n t a i n i n g 5 mg per m l o f

0

28

Table VII stability of Ampicillin as Function of p H and Temperature

Time in Days 1

2 6 9

15 29

L3 N Time in Days 1 2 6 9

15 29

Percent Activity Remaining at Room Temperature 7 .5 6 . 4 4 . 9 3 .8 2 .9 2 . 3 1 . 7 P H 8 . 4 - - - - - - -

p H 8 . 4 4 3 . 9 23. 1

1 0 0 0

1 6 . 5 5 3 . 1 90 .6 8 9 . 4 7 3 . 1 4 7 . 9 1.8 32.2 8 2 . 1 8 8 . 7 54.2 26 .7

0 4 . 8 6 0 . 1 6 7 . 6 21 .7 3 .5 0 0 45 .2 54 .4 10 .7 1.1 0 0 26 .5 39.5 3 .9 0 0 0 1 1 . 4 1 3 . 9 1 0 Percent Activity Remaininq at 5OC

7 .5 6 . 4 4 . 9 3 . 8 2 .9 2 .3 8 1 . 4 92.7 100 95.2 94 .2 8 5 . 3 71 .2 8 7 . 6 93 .4 95.5 9 2 . 1 8 1 . 6 4 2 . 5 78 .9 94 .6 8 6 . 3 8 2 . 4 6 4 . 7 27 .7 73.6 93.4 91 .9 71 .5 51 .2 1 1 . 9 63 .5 8 5 . 1 8 9 . 9 58 .6 33 .2

0 48 .6 79 .9 8 6 . 3 39.8 13 .6

------

24.7 8 . 0

0 0 0 0

b

? 1 . 7 c

r r 83 .0

67.7 39.8 23.7

9 . 9 1 . 5

z

The stability of ampicillin trihydrate in sodium acetate and Tris buffers as a function of time was studied8l. Data is reported in Table VIII.

Table V I I I S t a b i l i t y of A m p i c i l l i n T r ihydra t e i n S o d i u m A c e t a t e and

T r i s B u f f e r s a s a F u n c t i o n of p H

( A m p i c i l l i n ( 2 m g / m l , H y d r o x y l a m i n e m e t h o d )

0.1M T r i s 0.1g N a O A c 0 . 1 M T r i s

O 0 . 1 M N a O A c 0.1M T r i s 0.1M - N a O A c

u

I n i t i a l p H

4 .9 4.9 5.9 6.0 6.7 6.5

5 24 48 120 216 312 F i n a l H o u r s H o u r s H o u r s H o u r s H o u r s H o u r s p H ---- --

rn 98.1 93.9 92.4 73.4 57.4 40.8 4.8 C

98.3 94.3 94.7 78.9 65.0 49.9 4.9

98.8 90 .1 91.5 66.9 45.6 30.5 5.6 -

$ 102.4 96.7 102 .1 90.8 86.7 77.9 5.8

91.3 65 .6 4 8 . 1 14.3 3.3 0 6.4 99.9 96.9 98.2 90.7 86 .9 75.7 6.3 <

2 rn

<

I E -

AMPICILLIN

Data have been obtained concerning the stability of ampicillin in human stool specimens. Sixty- four to eighty-four percent of the ampicillin was recovered after 3-4 week storage of the stool-buffer blend in dry ice. A recovery cont control consisting of the sample stool-buffer blend dosed with a known amount of ampicillin was prepared f o r each sample. When a correction is made based on the recovered penicillin from the control, full recovery of ampicillin and stability through 3 weeks is obtained from the stool-buffer mixture82. When ampicillin was incubated with the fecal flora of rats and tested for antibiotic activity, only 36.7% oi the ampicillin activity was 1 e F t after 4 1 1 0 ~ ~ ~ 8 3 .

unstable when injected with penicillinase into rabbits. Synergistic action of ampicillin and cloxaci llin has been demons tra ted85. Cloxacillin inhibits the microbia 1 degradation o f ampici llin. The enzymic degradation of ampicillin by extra- cellular and cell-bound penicillinases from an ampicillin resistant strain of Staphylococcus was also inhibited by cloxacillin. The addition of cloxacillin to solutions containing - S.aureus penicillinase greatly reduced @-lactam hydrolysis of ampicillin. B-lactam ring hydroly- hydrolysis with Difco penicillinase is a basis of the spectrophotometric determination of ampicillin75. Aminoalkylcatechols have been shown to catalyze the hydrolysis of ampicillin at relatively rapid rates at neutral pH by a mechanism similar to that postulated for several hydrolytic enzymesa6. ampicillin were measured in the presence of several 3,6-bis(aminoalkyl) catechols where the amino group was varied in both size and basicity. Hou and P ~ o l e ~ ~ r e p o r t e d that ampicillin at a pH equal to the isoelectric point exists as

G r a d r ~ i k ~ ~ showed that ampicillin is

Rates of hydrolysis of

31

EUGENE IVASHKIV

zwitterion and in this form is most stable in aqueous solutions. The stabilization of solutions with acetone has been patented88. Levy8’ showed that ampicillin formed polymers in different molecular sizes in aqueous solutions within 1 day that were separable by filtration on columns of an acrylamide gel.

Kuchinskas and

3.3 Stability of Ampicillin Powders

powders were stable when stored in a closed system at 43% and 81% relative humidity at room temperature for 6 weeks. Ampicillin is also stable at 35OC in the same closed system for nine weeks. Ampicillin showed little change in either moisture content or potency.

Weiss and Palmerg0 showed that ampicillin

3.4 Cupric Ion-Catalyzed Hydrolysis

Cu (11) on the penicillins was to promote their degradation to coordination complexes of Cu (11) and the corresponding penicilloic acids. Complex- ation was assumed to occur between Cu (11) and the intact penicillins, followed by a rate limiting hydrolysis of the complex into the corresponding penicilloic acid - Cu (11) complex, The reaction mechanism and the catalytic siteof complexation of Cu (11) with penicillin have been s tudiedg3.

It has been reported91,92 that the effect of

4. Methods of Manufacture

4.1 Microbiological Ampicillin can be prepared microbiologi-

cally by incubation of microbes, e.g. Pseudomonas species, Kluyvera citrophila, Rhodopseudomonas spheroides, or Micrococcus ureae with 6-aminopenicillanic acid and a-phenylglycineg4, via enzymic acylation of 6-aminopenicillanic acidg5, 96

3 2

AMPICILLIN

or it may be synthesized by the cell-bound pen ic i 11 in a cy la se of Es cher ich ia col i9 7. Factors affecting the synthesis of ampicillin by the cell-bound penicillin acylase of Escherichia - coli have been studied98.

4.2 Chemical Ampicillin has been synthesized by

condensing 6-aminopenicillanic acid (6-APA) with a protected a-phenylglycine. The many groups used to protect the reactive primary amine of a-phenylglycine are summarized in the Table IX. Ampicillin is regenerated by removal of the protective groups. Ampicillin can also be obtained by treating 6-APA with a-bromophenylacetyl bromide131 via azido penicillin and catalytic hydr~genationl~~ 3 133 9 134, the reaction of 6-APA with D ( - 1 -2-phenylglycine chloride135 and tri- me thylsiliconamine and pyr idine136. Ampici 11 in is prepared by treating a-bromobenzylpenicillin with hexamethylenetetramine137>138 and by Schiff base processl39. A flowsheet of a plant for the production of synthetic penicillins containing predetermined side chains attached to 6-APA is givenl40.

33

EUGENE IVASHKIV

Table IX Synthesis of Ampicillin

Protective Group on a-Phenylglycine

2-Nitro-4-methoxyphenylsulfenyl 2,5-0xazolidinedione

- o-Nitrophenylsulfenyl N-Carboxyanhydride E-Toluenesulfenyl N-(l-Methyl-2-phenylcarbamoylvinyl)

N-Formyl 2-Quinol-8-yl-carbonylamino 1-3-0xazine-2,6-dione 6-N-benzoyl-2-tyrosyl a-Benzyloxycarbonylamino 2,4-Pentanedione(Acetylacetone) N-Carbobenzoxy @-Dicarbonyl(BzCH2Ac, Ac2CH2,

N (2-Hydroxy- 1-naphthylme thylene )

6-2-Phenylcyclohex-3-enyl-

anhydride

ACCH2C02E t)

anhydride

carboxamido

Reference

99 100,101,102 103,104 110 107,108,109 105,106

111 112 113 114 115 116 117,118,119 120,121,122

123,124

125

126 Fur fura 1, f urylacro le in, c ro tona ldehyde, anisaldehyde and salicylaldehyde 127

E thoxy f orm i c anhydride 128 Phenylsulfide (PhSH) 129 - o-Acetoacetanisidine 130

34

AMPICILLIN

Production of ampicillin via methyl N( a-carboxy- benzy1)-@-aminocrotonate is illustrated. The process for the preparation of the a-phenylglycine has been patented and can be summarized as follows:

i4fYf22 ted

HCOOH + NaOCH3+ CHCOONa t CH30H

H2 NH2 D- ( - ) - a - sodium ph e ny lg ly c i ne

phenylglycine methylate sodium salt

HCOONa + CH3COCH2COOCHT,

H2

methyl acetoacetate

+ H2°

3s

EUGENE IVASHKIV

The patented process for the synthesis of ampicillin trih~dratell~-~’’ can be outlined:

ClCOOCH2CH3+

mixed anhydride

mixed anhydride + H2N-CHC

protected Ampicillin + H C 1 , N a O H 4

LHCOOH H O=C’

3 H 2 0 Ampicillin trihydrate

+ CH3COCH2COOCH3 + N a C 1

36

AMPICILLIN

5. Isolation and Purification Ampicillin can be purified by acid or

alkaline recrystallizations. It is dissolved in a mineral acid or caustic solution and precipi- tated by the adjustment of pH64. ampicillin is purified by dissolution in methylene dichloride containing triethylamine, filtration and precipitation with p-toluene sulfonic acid 143. Ampicillin is separated from 6-aminopenicillanic acid by the filtration of chloroform or methylene chloride containin triethylamine and evaporation of the solvent 124 . Antigenic impurities are removed from solution by gel filtrati0n~~5-148. A process is described for the isolation of ampicillin via adduct formation with aliphatic amines and precipitation with 6-amino-1,3-naphthalene- disulfonic acid149.

Ampicillin was separated from dicloxacillin and cloxacillin on an ion exchange column, IRA-402 resinl50. Ampicillin was extracted with ion-pairing and adduct-forming agentsl51. Puri- fication of ampicillin via Schiff' s bases, sulfonic acid salts, amine salts and Tergitol salts has been evaluatedl52.

Crude

6. Methods of Analysis

6.1 Identification Tests Ampicillin was identified by

infrared s p e c t r o ~ c o p y l ~ ~ . Chromotropic acid, sulfuric acid, ninhydrin and potassium cupri- tartrate tests2 are used to identify ampicillin. Electrophoresis in an agar gel is used for separation of penicillins. Spots are detected microbiologica 1 1 ~ ~ ~ ~ . described a method for the rapid separation and detection of mixtures of penicillins by low voltage electrophoresis. The penicillins are

Thomas and Broadbr idge155

EUGENE IVASHKIV

id?ntified microbiologically.

bioautography were combined for the identif ica- tion of antibioti~sl~~. antibiotics sensitivity discs were developed157. Several thin layer chromatography methods for identification of penicillin are reportedl58-162. A simple and rapid polyamide chromatography of penicillins is describedl63. (See Section 6.2.4 for additional systems).

tion and identification of different p e n i ~ i l l i n s l ~ ~ - ~ ~ ~ . samples are compared with those of standards. (See section 6.2.5 for additional methods).

Sephadex thin layer chromatography and

Identity tests for

Paper chromatography is used for the separa-

The Rf values of separated

6.2 Quantitative Methods 6.21 Ultraviolet Spectrophotometric

Methods A spectrophotometric method for the

determination of ampicillin involving initial benzoylation of the side chain a-amino group is described. a-Benzamidobenzylpenicillin so formed is treated with mercuric chloride in acid solution and a-benzamidobenzylpenicillenic acid is obtained. This may be assayed spectrophoto- metricallyl69. Ampicillin is degraded in a buffer solution at p H 5.2 at 75OC and the absorbance is measured at 320 nm 170. Smith's method €or the spectrophotometric determination of ampicillin was adapted to the assay of ampicillin in chicken blood, bile and urinel7+ Ampicillin is dissolved in 5 g sodium hydroxide and the absorbance is measured at 279 Ampicillin is determined spectrophotometrically as its copper complexl73. Spectrophotometric and circular dichroism methods for determining the activity of ampicillin are described174. A method.based on,the degradation of.the @-lactam ring with penicillinase and formation of a

38

AMPICILLIN

175 copper chelate is given .

6.22 Fluorometric Determination A method for the fluorometric analy-

Ampicillin forms a strongly fluor- sis of am icillin in biological fluids is describede76. escent yellow product in acid solution at elevated temperatures. A fluorescent thin layer chromatography procedure for determining trace ampicillin involves the reaction of hydrolyzed penicillin with a fluorescent isothiocyanate to form the corresponding fluorescent thioureal77.

6.23 Polarographic Determination Ampicillin can be determined at

p. p. m. levels by differential pulse p~larographyl~~. Ampicillin undergoes reduction at a mercury electrode and the peaks obtained can be used €or both quantitative and qualitative analyses. Peak heights are linear with concentration.

6.24 Thin Layer Chromatography A thin layer chromatographic assay

of ampicillin and cloxacillin in body fluids was developed. Penicillins are separated on silica gel. Spots were obtained using a bioautographic technique177. Mixtures of synthetic penicillins are separated on a silica gel sheet and developed with butano1:ethanol:water (4:1:5) l 8 O and zones of inhibition were obtained with Bacillus subtilis ATCC6633. Mixtures of ampicillin and oxacillin were separated by thin layer chromatography on silica gel and the antibacterial activity was measured against Sarcina luteal8’. chromatographic method for semisynthetic pen ici 11 ins and tetracyclines is descr ibed18 2 . Antibiotics are separated on silica gel plates using water or p H 6.1McIlvaine’s buffer solution

A thin layer

EUGENE IVASHKIV

a s s o l v e n t . B i a g i e t a1 .183 used r e v e r s e d phase t h i n l a y e r chromatography on s i l i c a g e l impreg- n a t e d w i t h s i l i c o n e DC200 t o deve lop Rf v a l u e s f o r v a r i o u s p e n i c i l l i n s . Reversed-phase t h i n l a y e r chromatography was used t o examine t h e e f f e c t o f t h e pH o f a mob i l e phase on t h e p a r t i t i o n i n g o f v a r i o u s p e n i c i l l i n s between a p o l a r mob i l e phase and n o n p o l a r s t a t i o n a r y

g r a p h i c p r o c e d u r e f o r measur ing a m p i c i l l i n i s d e s c r i b e d . S i l i c a g e l p l a t e s impregnated w i t h s i l i c o n e f l u i d a r e used . M c I l v a n e ' s pH 4 . 1 b u f f e r c o n t a i n i n g 1.5% a c e t o n e i s employed f o r development . The s p o t s a r e v i s u a l i z e d w i t h n i n h y d r i n . A c o l o r i s e l u t e d and measured spectrophotometrically~8~.

A q u a n t i t a t i v e t h i n l a y e r chromato-

6 .25 Paper Chromatography A p a p e r ch romatograph ic t e c h n i q u e

f o r d e t e r m i n a t i o n of a m p i c i l l i n i n e p i c i l l i n samples h a s been developed186. grams were deve loped w i t h a w a t e r s a t u r a t e d m i x t u r e o f n -bu tano l and t -amyl a l c o h o l ( 6 : l ) pH 4 . 2 McIlTainel s b u f f e r . P e n i c i l l i n s were d e t e c t e d b i o l o g i ca 11 yI8 6. R o b e r t s and Vah i d i l8 d e s c r i b e d a p a p e r ch romatograph ic p rocedure f o r the d e t e r m i n a t i o n of a m p i c i l l i n . Papergrams a r e deve loped i n n -bu tano l , t -amyl a l c o h o l and w a t e r (7:1:4) f o r 4 8 h o u r s . Bioautograms o f t h e s t r i p s a r e p r e p a r e d u s i n g g . a u r e u s p l a t e s . Pape r chromatography of a m p i c i l l i n samples w a s c a r r i e d o u t on Whatman N o . 1 paper188. The f o l l o w i n g sys t ems w e r e used: b u t a n o l , e t h a n o l , w a t e r ( 4 : 1 : 5 ) , b u t a n o l , a c e t i c a c i d , w a t e r (12 :3 :5 ) , b u t a n o l , p y r i d i n e , w a t e r (l:l:l), methanol , p y r i d i n e , c o n c e n t r a t e d h y d r o c h l o r i c a c i d , w a t e r (160: 20:4: 3 5 ) , ammonia, p r o p a n o l , w a t e r (3 :6 :1 ) . A f t e r descend ing chromatography p a p e r s were s p r a y e d w i t h n i n h y d r i n o r deve loped b i o a u t o g r a p h i c a l l y

Paper chromato-

40

AMPICILLIN

u s i n g S t a p h y l o c o c c u s aureus18*. Two d i m e n t i o n a l p a p e r c h r o m a t o g r a p h y f o r t h e d e t e r m i n a t i o n o f a m p i c i l l i n i n u r i n e was used189. U r i l g O i n o c u l a t e d a g a r p l a t e s i n p a r a l l e l s t r i p e s w i t h 1 6 m i c r o b i a l c u l t u r e s o f g r a m - p o s i t i v e , g r a m - n e g a t i v e a n d a c i d r e s i s t a n t m i c r o o r g a n i s m s and w i t h Candida a l b i c a n s The g r o w t h z o n e s a r e t h e n p a r t i a l l y c o v e r e d w i t h a f i l t e r p a p e r s t r i p c o n t a i n i n g t h e s e p a r a t e d a n t i - b i o t i c s t o be t e s t e d . The z o n e s o f i n h i b i t i o n were m e a s u r e d and c o n c e n t r a t i o n o f a m p i c i l l i n c a l c u l a t e d .

6 . 2 6 I o d o m e t r i c T i t r a t i o n A l i c i n o l 9 1 d e s c r i b e d a n i o d o m e t r i c

method f o r t h e d e t e r m i n a t i o n of p e n i c i l l i n s . O n l y a f t e r a l k a l i n e o r p e n i c i l l i n a s e h y d r o l y s i s t h e p e n i c i l l i n s consume i o d i n e . The d i f f e r e n c e i n c o n s u m p t i o n o f i o d i n e b e f o r e a n d a f t e r h y d r o l y s i s i s p r o p o r t i o n a l t o t h e q u a n t i t y o f t h e a n t i b i o t i c . R u ~ s o - A l e s i ~ ~ ~ u s e d t h e i o d o m e t r i c t i t r a t i o n f o r e s t i m a t i o n o f a m p i c i l l i n i n f o r m u l a t i o n s . Ampi- c i l l i n s o l u t i o n s w e r e h y d r o l y z e d w i t h sodium h y d r o x i d e f o r 30 m i n u t e s . A f t e r a c i d i f i c a t i o n t h e i o d i n e s o l u t i o n was added . A f t e r a n a d d i t i o n a l 30 m i n u t e s , t h e e x c e s s o f i o d i n e was t i t r a t e d w i t h t h i o s u l f a t e s o l u t i o n . A b l a n k c o n s i s t e d o f un- h y d r o l y z e d a m p i c i l l i n . The a m p i c i l l i n s t a n d a r d was t r e a t e d i n e x a c t l y t h e same manner a n d u s e d i n c a l c u l a t i o n s . The h y d r o l y s i s o f a m p i c i l l i n w i t h p e n i c i l l i n a s e i n s t e a d w i t h sodium h y d r o x i d e makes t h e method more s e l e c t i v e .

An a u t o m a t e d s y s t e m f o r t h e iodome- t r i c d e t e r m i n a t i o n o f p e n i c i l l i n s u s i n g a n A u t o A n a l y z e r i s d e s c r i b e d l 9 3 , The a c c u r a c y of t h e method i s from 1 t o 3% and t h e p r e c i s i o n i s 2 t o 3.4%.

6 . 2 7 Hydroxamic A c i d The f o r m a t i o n o f a c o l o r e d c h e l a t e

o f f e r r i c i o n a n d t h e h y d r o x a m i c a c i d o f b e n z y l -

41

EUGENE IVASHKIV

penicillin is the basis of the colorimetric methodl94. into butanollg5. publishedlg6.

The automated methodlg7 for the determination of benzylpenicillin can be used for the determination of ampicillin. Penicillins react with hydroxylamine and ferric ion to produce a color which is measured continuously with an AutoAnalyzer.

The color produced can be extracted Details of the procedure was

6.28 Amperometric Titration Ampicillin is hydrolyzed to

penicillamine. The SH group of penicillamine is titrated amperometrically. The author claims that the method determines only the biologically active penicillins and not the degradation productslg8.

6.29 Microbiological Methods

the laboratory equipment, solutions, culture diffusion and turbidimetric assays used as tests and methods for the assay of ampicillin199. Yakovlev200 determined ampicillin in blood serum by an agar-diffusion technique. Staphylococcus aureus 209P and Bacillus subtilis were used as test organisms. A modified AutoAnalyzer method of Shaw and Duncombe201 is used for the determination of ampicillin202. Escherichia coli 3973 grown on tryptone soya agar, prepared by the method of Dewart203. Srimshaw and Jones204 presented an automated method for the determination of ampicillin. The method produces a linear response for ampicillin over an 80-180 mcg/ml range. An automated turbidimetric system for ampicillin assay is described205.

A complete description is given for

The inoculum was

42

AMPICILLIN

7 ' Ouinn, e t Bindin?06 a l . , f o u n d t h a t a n p i c i l l i n w a s l ess h i g h l y bound t o serum t h a n e i t h e r b e n z y l - p e n i c i l l i n o r phenoxymethylpenicillin. Serum h a d l i t t l e e f f e c t on t h e a c t i v i t y o f a m p i c i l l i n 2 0 7 . When t h e O x f o r d S t a p h y l o c o c c u s a n d a s t r a i n o f E s c h e r i c h i a c o l i w e r e u s e d a s t e s t o r g a n i s m s , t h e m i n i m a l i n h i b i t o r y c o n c e n t r a t i o n s f o r a m p i c i l l i n i n t h e p r e s e n c e o f 40% human serum w e r e o n l y o n e t u b e lower a m p i c i l l i n P h e n o l r e d c o m p a r i s o n p r o t e i n s .

t h a n i n t h e c o n t r o l s . The b i n d i n g of t o p r o t e i n s was i n v e s t i g a t e d 2 0 8 , p r o v e d t o be t h e most s u i t a b l e f o r o f t h e b i n d i n g o f p e n i c i l l i n s t o serum I n v e s t i g a t i o n s w i t h a l b u m i n t r e a t e d

w i t h d i n i t r o f l u o r o b e n z e n e p r o v e d t h a t p r i m a r y r e a c t i v e amino g r o u p s o f t h e a l b u m i n m o l e c u l e s a r e r e s p o n s i b l e €or t h e b i n d i n g of p e n i c i l l i n s . B i n d i n g o f p e n i c i l l i n s , i n c l u d i n g a m p i c i l l i n , w i t h serum p r o t e i n s were s t u d i e d , t o o 209. P e n i c i l l i n s w e r e n o t bound b y human g l o b u l i n b u t a l a r g e amounts , e x c e p t a m p i c i l l i n , was bound w i t h 1% a l b u m i n . L i p i d s o l u b i l i t y a n d serum p r o t e i n b i n d i n g o f v a r i o u s p e n i c i l l i n s w e r e c o r r e l a t e d 2 1 0 . Robinson a n d S u t h e r l a n d 2 1 1 s t u d i e d t h e b i n d i n g of p e n i c i l l i n s a n d o t h e r a n t i b i o t i c s i n serum b y u l t r a f i l t r a t i o n t e c h n i q u e . N e w d a t a w e r e g i v e n on t h e b i n d i n g of p e n i c i l l i n s t o t h e p r o t e i n s i n human s e r u m 2 1 2 . The e x t e n t o f b i n d i n g was d e t e r m i n e d b y d i a l y s i s , p l a t e t e s t s a n d g e l f i l t r a t i o n . A new a p p r o a c h t o s t u d y i n g t h e p r o t e i n b i n d i n g of p e n i c i l l i n s h a s b e e n reported213. a n d c o n t i n u o u s d i a l y s i s m e t h o d s were u s e d t o examine p r o t e i n b i n d i n g o f p e n i c i l l i n s . R e s u l t s showed t h a t p e n i c i l l i n s d i f f e r i n t h e d e g r e e o f b i n d i n g . The r e l a t i o n s h i p b e t w e e n t h e e x t e n t of s e r u m - p r o t e i n b i n d i n g o f p e n i c i l l i n s a n d t h e c o r r e s p o n d i n g e f f l u e n t volume from S e p h a a e x G - 2 5 , a n d t h e r e l a t i o n s h i p b e t w e e n t h e p r o t e i n - b i n d i n g

G e l - f i l t r a t i o n a n d b o t h s t a t i c

43

EUGENE IVASHKIV

e x t e n t and t h e g e l - a f f i n i t y i s desc r ibed214 . P e n i c i l l i n s showing an i n c r e a s e i n serum b i n d i n g a l s o showed an i n c r e a s e i n t h e e f f l u e n t volume. E s t i m a t i o n and u s e of serum l e v e l s i n the e v a l - u a t i o n of t h e new p e n i c i l l i n i s p resen ted215 . T h e d e g r e e o f b i n d i n g was e s t a b l i s h e d b y f i l t r a t i o n under n e g a t i v e p r e s s u r e th rough a d i a l y s i s membrane. The b i n d i n g r a t e s o f p e n i c i l l i n s t o m i x t u r e s o f a lbumin, euglobumin, and pseudo- g l u b u l i n f r a c t i o n were n e a r l y t h e same a s t h o s e o f t h e whole b o v i n e serum216. The b i n d i n g r a t e o f t h e m i x t u r e o f any 2 o r 3 o f t h e serum p r o t e i n f r a c t i o n s i s a lways lower t h a n t h e sum o f t h o s e t o c o r r e s p o n d i n g i n d i v i d u a l f r a c t i o n . Binding r a t e s and t h e modes of b i n d i n g o f p e n i c i l l i n s w i t h v a r i o u s p r o t e i n s i n v i v o and i n v i t r o were i n v e s t i g a t e d b y u l t r a f i l t r a t i o n and d i a l y s i s 2 1 7 . Kunin2I8 r e p o r t e d t h a t o n l y 23% o f a m p i c i l l i n w a s bound t o human serum. Serum p r o t e i n s b i n d a m p i c i l l i n o n l y loose ly219. A m p i c i l l i n was h a r d l y abso rbed on human r e d ce l l s , s l i g h t l y i n a c t i v a t e d w i t h mouse l i v e r homogenates and was combined s l i g h t l y w i t h h o r s e serum p r o t e i n s 2 2 0 - T h e e f f e c t of serum b i n d i n g on t h e d i s t r i b u t i o n of p e n i c i l l i n s i n t h e r a b b i t h a s been s tud ied221 . l 4C- l abe led p e n i c i l l i n s were used and t h e i r d i s t r i b u t i o n i n b r a i n , musc le , l ung and h e a r t was measured and c o r r e l a t e d t o b i n d i n g . The r o l e o f a m p i c i l l i n i n a n t i b i o t i c t h e r a p y and i t s serum b i n d i n g i s g iven222. D i f f e r e n c e s i n serum b i n d i n g among p e n i c i l l i n s e x e r t a p ro found e f f e c t upon the amount of f r e e d rug a v a i l a b l e f o r a n t i m i c r o - b i a l a c t i v i t y 2 2 3 . Kunin224 found t h a t a m p i c i l l i n showed less b i n d i n g t o serum p r o t e i n s t h a n d i d d i c l o x a c i l l i n , c l o x a c i l l i n , o x a c i l l i n , n a f c i l l i n , p e n i c i l l i n s G and V and m e t h i c i l l i n . An a n t i - m i c r o b i a l a c t i v i t y o f p e n i c i l l i n s i s i n c r e a s e d b y c o m p e t i t i v e serum b i n d i n g i n h i b i t o r s 2 2 5 . Immunochemical s t u d i e s on t h e a n t i g e n t i c b i n d i n g s

44

AMPICILLIN

o f a m p i c i l l i n and o t h e r p e n i c i l l i n s w e r e con- d u c t e d 2 2 6 . p r o t e i n was e s t a b l i s h e d b y d i a l y s i s a n d w i t h p c h l o r o m e r c u r i b e n z o a t e t i t r a t i o n o f t h e p e n i c i l l - o y l g r o u p s a n d b y t h e f o r m o l t i t r a t i o n o f p r o t e i n amino g r o u p s 2 2 7 .

The amount o f a m p i c i l l i n bound t o t h e

8 . P h a r m a c o k i n e t i c s

a n d sodium a m p i c i l l i n f o l l o w i n g i n t r a m u s c u l a r i n j e c t i o n w e r e s t u d i e d 228 . Serum l e v e l s o f a m p i c i l l i n a c t i v i t y w e r e d e t e r m i n e d . The r a t e s of a b s o r p t i o n o f t h e d r u g from t h e i n t r a m u s c u l a r i n j e c t i o n s i t e s were c a l c u l a t e d . The a m p i c i l l i n s u s p e n s i o n d a t a showed t h a t a b s o r p t i o n p r o c e s s i s s lower t h a n t h e e l i m i n a t i o n p r o c e s s . A m p i c i l l i n s o d i u m s o l u t i o n s a r e a b s o r b e d more e f f i c i e n t l y t h a n a m p i c i l l i n t r i h y d r a t e . K l e i n e t a l . 229 g a v e t h r e e i n t r a m u s c u l a r i n j e c t i o n s o f a m p i c i l l i n t o e i g h t a p p a r e n t l y h e a l t h y young men. Doses o f 1 . 0 g . , and 0 . 5 g . w e r e g i v e n a t two-day i n t e r v a l s . A b s o r p t i o n a n d e x c r e t i o n o f i n t r a m u s c u l a r d o s e s of a m p i c i l l i n a p p e a r e d t o be somewhat d e l a y e d when compared w i t h p e n i c i l l i n s G a n d V. Peak serum l e v e l s o f a m p i c i l l i n a p p e a r e d l a t e r and s i g n i f i - c a n t a n t i b a c t e r i a l a c t i v i t y was also d e m o n s t r a t e d On t h e a v e r a g e , a b o u t t w i c e a s much a m p i c i l l i n was r e c o v e r e d i n t h e u r i n e a f t e r i n t r a m u s c u l a r a d m i n i s t r a t i o n t h a n when same amounts h a d b e e n g i v e n o r a l l y . f o u n d t h a t p e a k serum c o n c e n t r a t i o n a f t e r a 500 mg i n t r a m u s c u l a r d o s e of a m p i c i l l i n was a t t a i n e d w i t h i n 20-30 m i n u t e s . U r i n a r y e x c r e t i o n amounted t o 40% o f the d o s e w i t h i n t h e f i r s t 8 h o u r s . Bunn, e t a l . f o u n d a l s o t h a t p e a k serum c o n c e n t r a t i o n o f a m p i c i l l i n was a t t a i n e d w i t h i n o n e h o u r a f t e r i n t r a m u s c u l a r i n j e c t i o n of 500 mg, 750 mg o r 1000 mg of a m p i c i l l i n 2 3 1 . P r o b e n e c i d i n c r e a s e s and s u s t a i n s t h e l e v e l s of a m p i c i l l i n i n t h e serum a f t e r i n t r a -

P h a r m a c o k i n e t i c s o f a m p i c i l l i n t r i h y d r a t e

4s

EUGENE IVASHKIV

muscu la r i n j e c t i o n o f t h e a n t i b i o t i c 2 3 2 . e x c r e t i o n o f a m p i c i l l i n i n t o t h e u r i n e i s d e l a y e d , p ro longed and amount reduced . The combina t ion o f i n j e c t e d a m p i c i l l i n and o r a l p r o b e n e c i d h a s been used233,234,235. When a m p i c i l l i n i s g i v e n o r a l l y i n doses of 250 mg, 500 mg, 750 mg, and 1000 mg peak serum c o n c e n t r a t i o n s a r e o b t a i n e d a t a b o u t 2 h o u r s a f t e r dosing236. S i g n i f i c a n t serum c o n c e n t r a t i o n s of a m p i c i l l i n a r e p r e s e n t a t s i x h o u r s of t h e dos ing . Doubling t h e dose d o u b l e s t h e peak serum c o n c e n t r a t i o n . About 30% o f a g i v e n dose i s e x c r e t e d i n t h e u r i n e 6-8 h o u r s . A m p i c i l l i n c o n c e n t r a t i o n i s n o t a f f e c t e d by food237 and a n t i b a c t e r i a l a c t i v i t y of s e r u m samples i s p r o p o r t i o n a l t o dosage .

The

Bunn238 found t h a t 25% o f an o r a l dose of a m p i c i l l i n i s e x c r e t e d i n t h e f i r s t 8 h o u r s , and t h a t t h e b l o o d l e v e l s of a m p i c i l l i n a t 4 h o u r s w e r e t h e same a f t e r doses of 250, 500, or 1000 mg. S t e w a r t e t a l . 239 showed t h a t i n h i b i t o r y l e v e l s of a m p i c i l l i n a r e a t t a i n e d and m a i n t a i n e d between one and one-ha l f and seven h o u r s a f t e r o r a l d o s e s of 100 mg/kg/day. With lower doses i n h i b i t o r y c o n c e n t r a t i o n s a r e m a i n t a i n e d f o r one and one- h a l f t o f i v e h o u r s , About 30-50% of t h e t o t a l dose g i v e n o v e r a p e r i o d of f i v e t o seven days was excreted. Chromatographic b i o a s s a y s of t h e e x c r e t i o n p r o d u c t i n t h e u r i n e s u g g e s t e d t h a t it was l a r g e l y a m p i c i l l i n and n o t a m e t a b o l i t e .

46

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61

CHLORPROTHIXENE

Bruce C. Rudy and Bernard Z. Senkowski

63

6. C. RUDY AND B. Z . SENKOWSKI

I N D E X

Analy t ica l P r o f i l e - Chlorprothixene

1. Descr ipt ion 1.1 Name , Formula, Molecular Weight 1 . 2 Appearance, Color, Odor 1 . 3 Isomeric Forms

Phys i c a l P rope r t i e s 2 . 1 I n f r a r e d Spectrum 2 . 2 Nuclear Magnetic Resonance Spectrum 2 .3 U l t r a v i o l e t Spectrum 2 . 4 Fluorescence Spectrum 2.5 Mass Spectrum 2.6 Opt ica l Rotat ion 2 . 7 Melting Range 2 . 8 D i f f e r e n t i a l Scanning Calorimetry 2 .9 Thermogravimetric Analysis 2 . 1 0 S o l u b i l i t y 2 . 1 1 X-ray Crys t a l P rope r t i e s 2 . 1 2 Dissoc ia t ion Constant

3. Synthes is

4 . S t a b i l i t y Degradation

5. Drug Metabolic Products

6 . Methods of Analysis 6 . 1 Elemental Analysis 6.2 Phase S o l u b i l i t y Analysis 6 .3 Thin Layer Chromatographic Analysis 6 .4 Direct Spectrophotometr ic Analysis 6 . 5 Color imet r ic Analysis 6 .6 Fluorescence Analysis 6 . 7 Non-Aqueous T i t r a t i o n

7 . Acknowledgements

8 . References

64

CHLORPROTHIXENE

1. D e s c r i p t i o n

1 .1 Name, Formula, Molecular Weight C h l o r p r o t h i x e n e i s 2-chloro-N,N-dirnethyl-

thioxanthene-A9 ,Y-p ropy lamine .

CHCH, CH2N (CH&

&fC'

C18H18ClNS Molecu la r Weight: 315.87

1 . 2 Appearance, Co lo r , Odor C h l o r p r o t h i x e n e i s a l i g h t yel low t o yel low

c r y s t a l l i n e powder wi th a l i g h t a m i n e - l i k e o d o r .

1 . 3 I somer i c Forms C h l o r p r o t h i x e n e e x i s t s a s a c i s - o r a t r a n s -

i somer . The c i s - i s o m e r h a s a l s o been r e f e r r e d t o as t h e a - i somer o r t F h i g h m e l t i n g i somer ( 1 , Z ) . The t r a n s - i s o m e r l i k e w i s e has been r e f e r r e d t o a s t h e B-isomer o r low m e l t i n g i somer . T h i s work w i l l d e a l p r i m a r i l y wi th t h e a - i s o m e r .

3 - . Phys i ca l P r o p e r t i e s

2 . 1 I n f r a r e d Spectrum The i n f r a r e d spectrum o f bulk r e f e r e n c e s t a n d a r d

c h l o r p r o t l i ~ x e n e i s shown i n F igu re 1 ( 3 ) . The spectrum o f a 10'" carbon d i s u l f i d e s o l u t i o n o f c l i l o r p r o t h i x e n e (w/v ) was measured v e r s u s carbon d i s u l f i d e i n 0 . 1 mm NaCl l i q u i d c e l l s on a P e r k i n Elmer 621 Spec t ropho tomete r .

The f o l l o w i n g a s s ignmen t s have been g iven t o t h e bands i n F igu re 1 ( 3 ) :

a . C h a r a c t e r i s t i c f o r a r o m a t i c C H : 3063 cm-l b . C h a r a c t e r i s t i c f o r CH2 s t r e t c h i n g : 2939 and 2854

c . C h a r a c t e r i s t i c f o r CHI, s t r e t c h i n g : -7816 and 2767 cm- ' cm-

65

B. C

. RU

DY

AND B

. 2. SE

NK

OW

SK

I

i

-i

0

8 0

0

- 0

0

9

0

e 8 N 0

N

8 8 8

8 Y) *)

* 33N

VU

IHS

NW

l K

66

CH LORPROTH I XE NE

d . C h a r a c t e r i s t i c f o r 4 free H on benzene r i n g :

e . C h a r a c t e r i s t i c f o r 2 f ree H on benzene r i n g : 757 t o 739 cm-l

809 cm-l

2 . 2 Nuc lea r Magnetic Resonance Spectrum (NMR) The NMR spec t rum shown i n F i g u r e 2 was o b t a i n e d

by d i s s o l v i n g 52 .2 mg o f r e f e r e n c e s t a n d a r d c h l o r p r o t h i x e n e i n 0 . 5 m l o f C D C 1 3 c o n t a i n i n g t e t r a m e t h y l s i l a n e as t h e i n t e r n a l reference. The s p e c t r a l a s s ignmen t s are shown i n Tab le I ( 4 ) .

TABLE I

NMR S p e c t r a l Assignments f o r C h l o r p r o t h i x e n e

No. o f Each Chemical Type P r o t o n s P ro ton S h i f t (ppm) M u l t i p l i c i t y

methyl 6 2 .24 S

methy 1 ene 4 2 .35 -2 .80 m (u) v i n y l 1 5 .97 t (u) a r o m a t i c 7 7 .10 -7 .60 m (u)

s = s i n g l e t ; m(u) - unsymmetr ical m u l t i p l e t ; t ( u ) = unsymmetr ical t r i p l e t

2 . 3 U l t r a v i o l e t Spectrum The u l t r a v i o l e t spec t rum o f c h l o r p r o t h i x e n e i n

0.1N HC1 i n t h e r e g i o n 400 t o 210 nm e x h i b i t s t h r e e maxima and t h r e e minima. The maxima are l o c a t e d a t 229 - 230 nm ( E = 3 . 4 x l o 4 ) , 267 - 268 nm ( E = 1 . 3 x l o 4 ) , and 323 - 324 nm (E = 2 . 8 x l o 3 ) . 217 nm, 254 nm, and 307 - 308 nm. The spectrum p r e s e n t e d i n F igu re 3 was o b t a i n e d from a r e f e r e n c e s t a n d a r d s o l u t i o n o f c h l o r p r o t h i x e n e a t a c o n c e n t r a t i o n o f 0 .602 mg p e r 100 m l o f 0.1N H C 1 ( 3 ) .

The minima were obse rved a t

2 . 4 F luo rescence Spectrum F igure 4 shows t h e e x c i t a t i o n and emiss ion s p e c t r a

€or r e f e r e n c e s t a n d a r d c h l o r p r o t h i x e n e from 260 t o 540 nm ( 5 ) . The s p e c t r a were measured on a 0.1N HC1 s o l u t i o n o f c h l o r p r o t h i x e n e (0 .5 mg/ml) u s i n g a Farand M K - 1 s p e c t r o - f l u o r o m e t e r . E x c i t a t i o n a t e i t h e r 310 nm o r 352 nm produced

67

6. C

. RU

DY

AN

D 6

. Z. S

EN

KO

WS

KI

68

CHLORPROTHIXENE

Figure 3

Ultraviolet Spectrum of Chlorprothixene

1.0

.8

.6

NANOMETERS

AlIS

N31N

I

B. C. R

UD

Y A

ND

B. Z

. SE

NK

OW

SK

I

CHLORPROTHIXENE

i den t

2

c a l emission s p e c t r a with a max m u m a t 401 nm.

5 Mass Spectrum The mass spectrum o f ch lorpro th ixene r e fe rence

s t anda rd was obta ined us ing a CEC 21-110 spec t rometer wi th an ion iz ing energy o f 70 eV (Figure 5) ( 6 ) . The low r e s o l u t i o n spectrum showed t h e fol lowing d i agnos t i c peaks:

mass (m/e)

315 313 271 257 255 2 34 2 2 1 189

58

I n t e n s i t y

very weak very weak very weak weak medium - we ak weak s t rong medium-weak very s t rong

The molecular ion a t m/e 315 and t h e fragment ions a t m/e 2 7 1 , 257, and 255 d i s p l a y t h e expected ch lo r ine i so tope peak 2 mass u n i t s h igher . and t h e ion a t m/e 257 a r e both due t o c leavage b e t a t o t h e amine func t ion . l o s s of two hydrogens from m/e 257 poss ib ly lead ing t o a r ing-c losed s t r u c t u r e . The peak a t m/e 2 2 1 i s due t o t h e l o s s of H C 1 from m/e 257. S ince t h e s p e c t r a d id no t show any change with t ime i t i s l i k e l y t h a t m/e 313 i s a fragment ion der ived from m/e 315. Again, a r i n g c losu re type of s t r u c t u r e i s a p o s s i b i l t y ( 6 ) .

The base peak a t m/e 58

The peak a t m/e 255 could be expla ined as

2.6 Opt ica l Rotat ion Chlorprothixene e x h i b i t s no o p t i c a l a c t i v i t y .

2 . 7 Melting Range A sharp mel t ing po in t i s no t observed with

ch lorpro th ixene . ' The mel t ing depends on t h e r a t e of hea t ing . 101.5°c.

The melt ing range r epor t ed i n NF XI11 i s 96.5' t o

71

6. C. RU

DY

AN

D 6

.2. S

EN

KO

WS

KI

72

CH LORPROTH I XENE

2 . 8 D i f f e r e n t i a l Scanning Ca lo r ime t ry (DSC) The DSC curve €or r e f e r e n c e s t a n d a r d c h l o r p r o -

t h i x e n e was o b t a i n e d u s i n g a P e r k i n Elmer DSC - IB C a l o r i m e t e r . With a t e m p e r a t u r e program o f 10°C/min., a m e l t i n g endotherm was obse rved s t a r t i n g a t 92.8OC (shown i n F igu re 6) and a n o t h e r endotherm s t a r t i n g a t 246OC which co r re sponds t o t h e decomposi t ion o f t h e c h l o r p r o t h i x e n e . The Aldf was found t o be 7 . 4 kcal /mole f o r t h e m e l t i n g endotherm ( 7 ) .

2 . 9 Thermogravimetr ic A n a l y s i s (TGA) The TGA performed on r e f e r e n c e s t a n d a r d c h l o r p r o -

t h i x e n e e x h i b i t e d no l o s s o f weight when h e a t e d t o 10SOc a t a h e a t i n g r a t e o f 10°C/min ( 7 ) .

2 . 1 0 S o l u b i l i t y The s o l u b i l i t y d a t a o b t a i n e d a t 2 5 O C f o r

r e f e r e n c e s t a n d a r d c h l o r p r o t h i x e n e i s l i s t e d i n Tab le I1 ( 8 ) .

TABLE I 1

Ch 1 o r p r o t h i x e n e - So l u b i 1 i t y

S o l v e n t So 1 ub i 1 i t y (mg/ml )

3A a l c o h o l benzene ch 1 o r 0 f o rm 95% e t h a n o l e t h y l e t h e r i sop ropano 1 me thano 1 pe t ro l eum e t h e r ( 3 O o - 6 O 0 ) w a t e r

36 .7 30.5

2 8 . 2 7 7 . 6 2 2 . 0 36 .5 1 6 . 9

> S O 0

< 0 . 1

2 .11 C r y s t a l P r o p e r t i e s The x - r a y powder d i f f r a c t i o n p a t t e r n o f r e f e r e n c e

s t a n d a r d c h l o r p r o t h i x e n e i s p r e s e n t e d i n Tab le I11 ( 9 ) . The i n s t r u m e n t a l c o n d i t i o n s a r e g iven below:

Y -a

13 u

Li

d

u)

-w

2

W

x I

-6

g 4 I --u

-

B. C

. RU

DY

AN

D B

. 2. S

EN

KO

WS

KI

74

C H L O R P R O T H I X E N E

Instrumental Condi t ions:

General E l e c t r i c Model XRD-6 Spectrogoniometer Generator : Tube Target : Radia t ion : Opt ics :

Goniometer: Detector :

Recorder: Samples :

50 K V , 12-1/2 MA Copper Cu KCY = 1.542 1 0.1' Detector s l i t M . R . S o l l e r s l i t 3' Beam s l i t 0.0007" N i f i l t e r 4' t ake o f f angle Scan a t 0.2 ' 2 0 per minute Amplif ier ga in - 16 coarse , 8 . 7 f i n e Sealed p ropor t iona l counter tube and DC vo l t age a t p l a t eau Pulse he ight s e l e c t i o n E L - 5 v o l t s

Rate meter T . C . 4 2000 C/S f u l l s c a l e Chart Speed 1 inch p e r 5 minutes Prepared by gr inding a t room temperature

Eu-out

2 . 1 2 Dissoc ia t ion Constant The apparent PKa f o r ch lorpro th ixene has been

determined spec t rophotometr ica l ly to - be 8 . 4 (10). apparent pKa has a l s o been determined from t h e t i t r a t i o n curve i n an iscpropano1:water ( 1 : l ) mixture and found t o be 7.5 (11) . In water , t he t r i a lky lamino type compounds a r e s t ronge r bases , on t h e average, by 0 . 9 pK u n i t s (11,12) . Therefore , t h e es t imated PKa i n water i s 8.4 which is i n good agreement w i t h t h a t found spec t rophotometr ica l ly .

The

75

B. C . RUDY AND B. 2. SENKOWSKI

TABLE I11

X-ray Diffract i o n Pat t e r n o f Ch l o r p r o t h i x e n e

20

8 . 1 8 14 .49 16.39 1 6 . 6 7 19.55 21 .43 22 .71 23.00 24.24 24.55 25.86 26.33 26.82 2 7 . 5 3 28.69 29.69 30 .47 31.37 33.12

33.79 34.40 35 .03 35.92 37.25 37.81 38 .18 39.00 39.65 40 .80 4 1 . 8 0 42.62 4 3 . 2 3 44.36 45 .06 45 .70 49.55 50 .68

-

33.48

d*

10 .81 6 . 1 1 5 . 4 1 5 . 3 2 4 .54 4 .15 3 .92 3 .87 3 .67 3 . 6 3 3 .45 3.38 3 .32 3 .24 3 .11 3 .01 2 . 9 3 2.85 2.70 2 .68 2 .65 2 .61 2 .56 2.50 2 . 4 1 2 .38 2 .36 2.31 2.27 2 . 2 1 2 .16 2 . 1 2 2 .09 2 .04 2 .01 1 .99 1 . 8 4 1 . 8 0

76

I/** i0

100 21 62 45 75 24 8

10 31 29 15 3

28 6

10 3

15 1 8 1 2 3 3 4 2 3 1 1 1 3

17 2 3 3 4 2 2 5

CHLORPROTHIXENE

TABLE I11 (cont.)

X-ray Diffraction Pattern of Chlorprothixene 0 I/**

- 20 d* A I0

52.30 54.10

1.75 1.70

2 2

*d - (interplanar distance) nX 2 Sin 0

**I/ = relative intensity (based on highest intensity of 1.00) I 0

3. Synthesis

shown in Figure 7 . 2-Chlorothioxanthone is reacted with dimethylaminopropyl chloride in the presence of magnesium to form 2-chlorothioxanthenol which is then dehydrated to give chlorprothixene (13).

Chlorprothixene may be prepared by the reaction scheme

3 . Stability Degradation Chlorprothixene has been shown to be stable when heated

for one hour at 100°C in 0.1N HC1, 0.1N NaOH, and water. (14) When it is subjected to ultraviolet radiation or strongly basic conditions, 2 chlorothioxanthene and 2-chlorothioxanthone are formed which can be detected by TLC or paper chromatography. Also the odor of volatile aniines is apparent (15).


can be either N-demethylated or further oxidized to the N - oxide, Figure 8 ( 2 , l S ) . The analytical procedures for the metabolites have been described by de Silva (17).

Chlorprothixene is metabolized to the sulfoxide which

6 . Methods of Analvsis

6.1 Elemental Analysis The results from an elemental analysis of

reference standard chlorprothixene is presented in Table IV (18).

77

Figure 7

Synthesis of Chlorprothixene

CH L

OR

PR

OT

H I X

EN

E

Figure 8

Metabolic

Products of Chlorprothixene

(2)

CH

LO

RP

RO

TH

IXE

NE

CH

I

CH

i I

N

c~

f b‘cH, 0

CH

LO

RP

RO

TH

IXE

NE

-

SU

LF

OX

IDE

-N-O

XID

E

CH

LO

RP

RO

TH

IXE

NE

- S

UL

FO

XID

E

DE

SM

ET

HY

L -

CH

LO

RP

RO

TH

IXE

NE

- S

UL

FO

XID

E

79

B. C , RUDY AND B. Z . SENKOWSKI

TABLE IV

Elemental Analysis of Chlorprothixene

E 1 ement

C H N S c1

% Theory

68.45 5.74 4.43

10.15 11.22

% Found

68.36 5.72 4.45 10.26 11.36

6.2 Phase Solubility Analysis Phase solubility analysis has been carried out

for chlorprothixene using either isopropanol or acetonitrile as the solvent. An example is presented in Figure 9 for the reference standard chlorprothixene along with the conditions under which the analysis was carried out (8).

6.3 Thin Layer Chromatographic Analysis (TLC) The following TLC procedure is useful for

separating chlorothioxanthone and chlorothioxanthene from a-chlorprothixene (19). Using silica gel G plates and benzene:heptane (1:l) a s the solvent system, 0.1 mg of the sample in anhydrous 3A alcohol is spotted on the plate and subjected to ascending chromatography. solvent front has ascended 10 to 15 cm, the plate is a i r dried and sprayed with concentrated sulfuric acid. The plate is then viewed under shortwave ultraviolet radiation. The approximate Rf values are as follows:

After the

a-chlorprothixene 0.0 (orange fluorescence) chlorothioxanthone 0.35 (greenish fluorescence) chlorothioxanthene 0.85 (orange fluorescence)

The TLC procedure found useful for the separation of B-chlorprothixene and 2-chlorothioxanthenol is described below (19). A silica gel C; plate is spotted with 0.1 mg of sample and developed 10 to 15 cm using absolute ethanol: benzene:water ( ? 0 : 2 0 : 1 ) as the solvent system. Thc plate is air dried, sprayed with concentrated sulfuric acid, and viewed under shortwave ultraviolet radiation. The approximate Rf values are as follows:

Figure 9

I

PHASE SOLUBILITY ANALYSIS

SAMPLE 8 CHLORPROTHIXENE SOLVENT: ISOPROBINOL SLOPE: 00% EQUILIBRATION 20 HRS 01 25' C EXTRAPOLATED SOLUBILITY.

28.10 mg/g of SOLVENT

-

25 50 75 100 0

SYSTEM COMPOSITION: mg of SAMPLE per g SOLVENT

n I

X rn z rn

8 . C. RUDY AND 6. 2. SENKOWSKI

a-chlorprothixene 0.65 (orange fluorescence) B-chl orprothixene 0 . 5 0 (orange fluorescence) 2-chlorothioxanthenol 0 .35 (reddish fluorescence)

6 . 4 Direct Spectrophotometric Analysis The chlorprochixene content in tablets may be

determined spectrophotometrically by using the following procedure. The tablets are finely ground, a portion of the powder is weighed, and then dispersed by shaking in a 1% ammonium hydroxide solution. The chlorprothixene is ex- traced with ethyl ether. The chlorprothixene is extracted from the ethereal solution with 0.5N HC1 and diluted to volume with 0.5N HC1. to give a solution containing about 50 ug/ml and the absorbance measured at 324 nm. The concentration of chlorprothixene is calculated using the absorbance obtained from a solution of chlorprothixene reference standard similarly prepared and measured (5,20).

Suitable aliquots are further diluted

The system used with the Technicon Autoanalyzer for measuring the content uniformity of individual chlorprothixene tablets is as follows. Each tablet is crushed and allowed to stand in 5 ml of dimethylformamide for about 20 minutes. This solution is diluted with 0.1N H~SOI, until the concentration is about 50 pg/ml. The absorbance is measured at 324 nm and the amount of chlorprothixene calculated by comparison to a solution of reference standard chlorprothixene similarly prepared and measured.

6 . 5 Colorimetric Analysis Colorimetric analysis may be accomplished by

dissolving the chlorprothixene in concentrated sulfuric acid and diluting an aliquot with 50% sulfuric acid to a final concentration of approximately 0.04 mg/ml. orange color is measured at 490 nm and the concentration obtained by means of a calibration curve (15).

The

6 . 6 Fluorescence Analysis Fluorescence analysis has proven to be a valuable

method for the analysis of chlorprothixene in biological samples, blood has been reported when the blood is extracted and the extract dissolved in cold concentrated sulfuric acid (17).

A sensitivity limit of 0.010 micrograms/ml of

82

CHLORPROTHIXENE

6.7 Non-Aqueous Titration The non-aqueous titration as described in the

NF XI11 is the method of choice for the analysis of b u l k chlorprothixene ( 2 0 ) . The sample is dissolved in chloroform, ethanolic methyl red indicator added, and the titration carried out with HC10, in dioxane. Each ml of 0.1N HClOk is equivalent to 31.59 mg of chlorprothixene.

7. Acknowledgements

Literature Department of Hoffmann-La Roche Inc. and Dr. A. MacDonald for their assistance in the preparation of this analytical profile.

The authors wish to acknowledge the Scientific

83

6. C. RUDY AND 6. 2. SENKOWSKI

8. References

1.

2 . 3 .

4 .

5 .

6 .

7 .

8 .

9 .

1 0 .

11.

1 2 .

13. 14 . 1 5 .

1 6 .

1 7 .

1 8 .

1 9 .

20.

J. D. Dunitz, H. Eser, and P. Strickler, HeZv. Chim. Acta, 4 7 , 1897 (1964) J. Raaflaub,ArzneimitteZ-Forshung, 1 7 , 1393 (1967). M. Hawrylyshyn, Hoffmann-La Roche Inc., Personal Communication. J. H. Johnson, Hoffmann-La Roche Inc., Personal Communication . J. Boatman, Hoffmann-La Roche Inc., Personal Communication. W. Benz, Hoffmann-La Roche Inc., Personal Communication. J. Donahue, Hoffmann-La Roche Inc., Personal Communication. E. MacMullan, Hoffmann-La Roche Inc., Personal Communication. R. Hagel, Hoffmann-La Roche Inc., Personal Communication. E. Lau, Hoffmann-La Roche Inc. , Unpublished Data. V. Toome and G. Raymond, Hoffmann-La Roche Inc., Unpublished Data, B. Gutbezahl and E. Grunwald, J. Amer, Chem. Soc. , 75, 559 (1953) . W . Kimel, Hoffmann-La Roche Inc., Unpublished Data. I. Fischer, Hoffmann-La Roche, Unpublished Data. B. Schmidli, Hoffmann-La Roche Inc., Unpublished Data. C. L. Scheckel, Mod. Probi!. Pharmacopsychiatry, 2 , 1 (1969) . 1.A.F. deSilva and L. D. D'Arconte, J. Forensic Sci., 1 4 , 184 ( 1 9 6 9 ) . F . Schzdl, Hoffmann-La Roche Inc. , Personal Communication. R. Colarusso, Hoffmann-La Roche Inc., Unpublished Data. National Formulary XIII, Submitted August 25, 1971.

-

84

CHLORAL HYDRATE

J o h ~ E. Fairhrotlier

85

JOHN E. FAIRBROTHER

CONTENTS

1. Description 1.1 Name, Formula, Molecular Weight

~

1.2 Appearance, Color, Odor

2.1 Spectra 2. Physical Properties

2.11 Infrared 2.12 Raman 2.13 Ultraviolet 2.14 Nuclear Magnetic Resonance 2.15 Nuclear Quadrupole Resonance 2.16 Mass Spectrum 2.17 Electron Spin Resonance

2.2 Physical Properties of the Solid 2.21 Basic Thermal Constants 2.22 Differential Thermal Analysis

and Differential Scanning Calorimetry

2.23 Loss on Drying and Thermogravi- metric Analysis

2.24 Optical Characteristics 2.25 X-ray Diffraction 2.26 Neutron Diffraction 2.27 Polymorphism

2.31 Water 2.32 Water-Miscible Solvents 2-33 Water-Immiscible Solvents 2-34 Oils of Pharmaceutical Interest

2.3 Solubility

2.4 Physical Properties of Solutions and Melts 2.41 Heats of Solution and Dilution 2.42 Dissociation and pH 2.43 Surface Tension 2.44 Ultrasonic Absorption 2.45 Viscosity 2.46 Cryoscopy 2.47 Refractive Index 2.48 Radiolysis

3. Molecular Complexes

4. Synthesis and Purification

3.1 Types of Known Complexes 3.2 Structure of Complexes

86

CHLORAL HYDRATE

5. Degradation 5.1 Degradation of Solid

5.11 Volatilization 5.12 Chemical Degradation

5.2 Degradation of Solutions 5.21 Action of Light 5.22 Action of Heat 5 . 2 3 Ultrasound 5.24 Autoxidation and Polymerization 5.25 Action of Bases 5.26 Radiolysis 5.27 Microorganisms 5.28 Incompatibilities (solids and

5.29 Interaction with Starch solutions )

6. 5.3 Stabilization

6.1 Identity Tests 6.2 Methods of Analysis

Chemical Properties

6.21 Volumetric/Titrime tric Procedures

6.22 Spectrophotometry 6.23 Ion-Exchange and Column

6.24 Paper and Thin-Layer Chrom-

6.25 Vapor-Phase Chromatography 6.26 Refractometry and other physical

6.27 Polarography 6.28 Determination of Water

Chromatography

at ography

parameters

6.3 Radiolabelled Chloral Hydrate 7. Metabolism 8. R e f e r e n c e s

87

JOHN E. FAIRBROTHER


1.1 Name, F o r m u l a , M o l e c u l a r Weight

C h l o r a l H y d r a t e ( C h l o r a l i s H y d r a s ; C h l o r a t u m Hydra tum) a l s o known as 2 , 2 , 2 - T r i c h l o r - o e t h a n e - l , l - d i o l a n d t r i c h l o r o a c e t a l d e h y d e mono- h y d r a t e .

c1 OH I CH - OH I

I c1

c1 - c -

C 2 H j C 1 0 3 2 Mol.Wt. 165.40

1 . 2 A p p e a r a n c e , Color, Odor , T a s t e

C o l o r l e s s c r y s t a l s w i t h a p u n g e n t , b u t n o t a c r i d , o d o r , Has a p u n g e n t a n d r a t h e r b i t t e r t a s t e . V o l a t i l i z e s s l o w l y on e x p o s u r e t o a i r .


2 . 1 S o e c t r a

2 . 1 1 I n f r a r e d Spectrum

An u n d e r s t a n d i n g o f t h e i n f r a r e d s p e c - t r u m of c h l o r a l h y d r a t e s h o u l d be a p p r o a c h e d f r o m a s t u d y o f t h e s p e c t r u m o f c h l o r a l , wh ich h a s b e e n d e s c r i b e d i n some d e t a i l i n t h e l i t e r a t u r e 1~2,j. C h l o r a l h y d r a t e h a s b e e n shown t o h a v e a g e m - d i o l s t r u c t u r e b y Raman s p e c t r a ? a n d by N . M . R . 5 Compar i son of t h e i n f r a r e d ( m u l l ) s p e c t r a of t h e compound a n d of i t s d e u t e r a t e d a n a l o g 1 7 1 i n d i c a t e ; t h a t t h e two OH g r o u p s a r e n o t e u a l . T h i s i s c o n f i r m e d b y s o l u t i o n s p e c t r a 1 7 , q 8 , 2 3 , 1 6 8 ~ 1 7 1 a n d Ogawa23 s u g g e s t s t h a t c h l o r a l h y d r a t e e x i s t s i n s o l u t i o n i n a n e q u i l i b r i u m s t a t e , wh ich may b e r e p r e s e n t e d as f o l l o w s : -

88

CHLORAL HYDRATE

H kI 0 OH

\ I / C I

J1

/I\ c1 c1 c1

\ Yo H

n L

I /e\ c1 c1 c1

H /O\ H

H I

c i ci c i Near i n f r a r e d s t u d i e s 6 on a s o l u t i o n o f

c h l o r a l h y d r a t e show t h e e x i s t e n c e o f a l a b i l e e q u i l i b r i u m b e t w e e n t h e - g e m - d i o l and a d i m o l e c u l a r 1:l complex o f t h e a l d e h y d e and w a t e r . It i s t h o u g h t t h a t t h e g e m - d i o l s t r u c t u r e r e s u l t s f rom t h e e l e c t r o n i c e f f e c t o f t h e h a l o g e n s t r a n s m i t t e d t h r o u g h t h e m o l e c u l e , r a t h e r t h a n from i n t r a - m o l e c u l a r h y d r o g e n b o n d i n g b e t w e e n t h e h a l o g e n s and t h e h y d r o x y l h y d r o g e n s .

T h e n e a r - i n f r a r e d s p e c t r u m o f c h l o r a l h y d r a t e i n c h l o r o i c r i n ( t r i c h l o r o n i t r o m e t h a n e )

Nu jo17 ,171 a n d i n p o t a s s i u m b r o m i d e 8 , 9 , 1 0 show t h e a b s e n c e o f t h e c a r b o n y l band a t 1 7 6 5 cm- l , b u t s p e c t r a r e c o r d e d i n s o l u t i o n ( c a r b o n t e t r a - c h l o r i d e 7 ~ 1 0 , 1 1 , 1 6 9 b e n z e n e 7 , a n d c h l o r o f o r m l 7 0 , ) show t h e f o r m a t i o n o f t h e c a r b o n y l b a n d , i n d i c - a t i n g some d i s s o c i a t i o n . P i g u e t and J a c o t - G u i l l a r m o d 7 c o n c l u d e d t h a t , i n c a r b o n t e t r a c h l o r - i d e s o l u t i o n , c h l o r a l h y d r a t e d i s s o c i a t e s i n t o c h l o r a l and w a t e r , e v e n t u a l l y f o r m i n g a d i s t i n c t s t a b l e h e m i h y d r a t e . However, E k e b l a d l l f o u n d t h a t s o l u t i o n s c o n t a i n i n g up t o 0.l5g c h l o r a l h y d r a t e p e r 100 ml o f c a r b o n t e t r a c h l o r i d e c o u l d b e p r e - p a r e d w i t h o u t d i s s o c i a t i o n .

has b e e n r e c o r d e d E . I n f r a r e d s p e c t r a r e c o r d e d i n

89

JOHN E. FAIRBROTHER

I n t h e O H r e g i o n , d i s s o c i a t i o n c a u s e s a d e c r e a s e i n t h e O H p e a k e x t i n c t i o n s a n d t h e a p p e a r a n c e o f a new O H p e a k f r o m water a t 3720 cm-l . A s e x p e c t e d , t h e r e i s a s h i f t i n t h e O H s t r e t c h & $ f r e q u e n c y f r o m t h e c r y s t a l l i n e s t a t e

11?1R,5y. I n t h e l a t t e r c a s e , two OH p e a k s ( o f a p p r o x i m a t e l y s imilar i n t e n s i t y ) a p p e a r a t 3580 a n d 3615 cm- . However, f r e s h l y p r e p a r e d s o l - u t i o n s i n t h e c o n c e n t r a t i o n r a n g e t o 10-3u show no s i g n i f i c a n t v a r i a t i o n s i n t h e m o l a r e x - t i n c t i o n s a t t h e two p e a k maxima a n d n o water or c h l o r a l p e a k s a r e o b s e r v e d . The i n f r a r e d s p e c t r a o f t h e S q u i b b House S t a n d a r d Lot 7 6 1 4 7 i n m i n e r o i l and i n K B r a r e p r e s e n t e d i n f i g u r e s 1 a n d 2 .

) o f c h l o r a l h y d r a t e t o d i l u t e s o l u t i o n

$ 3 2 . 1 2 Raman S p e c t r u m

The Raman s p e c t r a o f b o t h c h l o r a l 3 ~ 1 3 , 1 2 2 2 1 , 2 2 2 , 2 2 3 , 2 2 4 and c h l o r a l h y d r a t e l 3 ~ ~ 7 ~ ~ 2 ' 2 : 2 2 3 , 2 2 4 h a v e b e e n r e p o r t e d . F o n t e y n e 2 2 3 f o u n d t h a t t h e i n c r e m e n t a l a d d i t i o n o f water t o c h l o r a l p r o d u c e d a new Raman s p e c t r u m , w h e r e a s t h e l i n e s due t o c h l o r a l d i s a p p e a r e d s l o w l y . The l i n e a t 1760 cm-1, c o r r e s p o n d i n g t o t h e C F 0 d i s p l a c e m e n t , i s n o t e n t i r e l y a b s e n t i n c h l o r a l h y d r a t e , s u g g e s t i n g t h e e x i s t e n c e o f a n e q u i - l i b r i u m .

The most r e c e n t l y r e p o r t e d s p e c t r a a r e t h o s e o f M a t s u s h i m a l 3 .

2 . 1 3 U l t r a v i o l e t S p e c t r u m

The U . V . s p e c t r u m o f c h l o r a l h y d r t e h a s b e e n r e c o r d e d i n a number of s o l v e n t s . 1 ' , 2 2 5 . I n s o l u t i o n i n water or i n d i o x a n c o n t a i n i n g a few p e r c e n t o f water , t h e h y d r a t e i s s o s t a b l e t h a t no c a r b o n y l a b s o r p t i o n i s d e t e c t a b l e . How- e v e r , i f t h e c r y s t a l l i n e h y d r a t e i s d i s s o l v e d i n c y c l o h e x a n e , i t d i s s o c i a t e s s u f f i c i e n t l y t o g i v e a c o n s i d e r a b l e a b s o r p t i o n a t 290mu. 1 4 ( g i v i n g a n E O v a l u e o f 3 8 . 3 ) .

90

CHLORAL HYDRATE

A s p e c t r u m o f t h e U . V . a b s o r p t i o n o f a s o l u t i o n o f c h l o r a l h y d r a t e i n 95% e t h a n o l (6.Og. i n 2 5 m l o f s o l u t i o n ) l 5 shows s t r o n g end a b s o r p t i o n a b o v e 240mu.

2 . 1 4 N u c l e a r Magnet i -c R e s o n a n c e SDe c t r um

T h e N.M.R. s p e c t r u m o b t a i n e d d e p e n d s on t h e p o s i t i o n o f t h e c h l o r a l + . c h l o r a l h y d r a t e e q u i l i b r i u m . C h l o r a l g i v e s a s i m p l e s p e c t r u m (CDCl3) , t h e a l d e h y d e p r o t o n a b s o r b i n g a t 6 9 . 1 . C h l o r a l h y d r a t e , h o w e v e r , c o n s i s t e n t l y g i v e s a p e a k a t 6 9 . 1 , p l u s o t h e r peaks whose p o s i t i o n s a r e more v a r i a b l e , for h y d r o x y l a b s o r p t i o n s a r e g rea t18 i n f l u e n c e d b y t e m p e r a t u r e a n d c o n c e n t r a - t i o n ( s e e f i g . 3 ) .

A b r o a d - l i n e N.M.R. s t u d y 5 has b e e n u s e 3 t o d e f i n e the s t r u c t u r e o f c h l o r a l h y d r a t e a s a g e m - d i o l , d i s t i n c t f rom t h e a l t e r n a t i v e s t r u c t u r e c o n t a i n i n g m o l e c u l a r w a t e r , a s i n gypsum. A t 200Ky c h l o r a l h y d r a t e e x h i b i t s a s l i g h t t r i p l e t s t r u c t u r e d u e t o t h e t h r e e - s p i n C H ( O H ) , g r o u p wh ich is s u b s t a n t i a t e d b y t h e s e c o n d moment o f t h e h y d r o g e n r e s o n a n c e ( 8 . 9 + 0.5 g a u s s * ) . T h e m o l e c u l a r h y d r a t e s t r u c t u r e would g i v e a s e c o n d moment g r e a t e r t h a n 1 5 g a u s s 2 , w h e r e a s w i t h t h e g e m - d i o l s t r u c t u r e , a s e c o n d moment o f a b o u t 3 . 8 = a s 2 would b e e x p e c t e d . The f i g u r e o b t a i n e d - c o n f i r m s t h e g e m - d i o l s t r u c t u r e , b u t i t a l s o f o l l o w s t h a t a a r a e n r o l s o r t i o n o f i n t e r m o l e c u l a r b r o a d e n i n g m u s t be p r e s e n t , wh ich c o u l d a r i s e i f t h e c r y s t a l i n v o l v e d e x t e n s i v e h y d r o g e n b o n d i n g .

2.15 N u c l e a r Q u a d r u p o l e Resonance S p e c t r u m

The N . Q . R . s p e c t r a o f 35Cl i n c h l o r a l h y d r a t e a n d c h l o r a l d e u t e r a t e h a v e b e e n r e o r d e d i n t h e t e m p e r a t u r e z jyge 7’7 t o 323OK. 1 - 7 9 1 g ~ 1 9 9 2 0

( S i m i l a r s p e c t r a o f C1 h a v e a l s o b e e n r e c o r d e d 20). T h e f r e q u e n c i e s o f t h e q u a d r p e l i n e s ob- t a i n e d by i n d e p e n d e n t w o r k e r s l r / , l Y y y ’ a r e i n

91

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Fig. 1. I n f r a r e d spectrum of c h l o r a l h y d r a t e , S q u i b b House S t a n d a r d ( M i n e r a l Oil M u l l ) .

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F i g . 2 . I n f r a r e d s p e c t r u m o f c h l o r a l h y d r a t e , S q u i b b House S t a n d a r d ( P o t a s s i u m Bromide D i s c ) .

L

94

F i g . 3. N.M.R. s p e c t r u m o f c h l o r a l h y d r a t e , Squibb House S t a n d a r d , in d e u t e r o a c e t o n i t r i l e .

CHLORAL HYDRATE

good a g r e e m e n t , a n d show t h a t two OI? t h e l i n e s a r e s e p a r a t e d by o n l y a b o u t 80 K H z whereas t h e t h i r d i s a b o u t 1 . 2 5 M H z l o w e r . T h i s l a r g e r d i f f - e r e n c e s u g g e s t s a d i f f e r e n t C - C 1 b o n d i n g f o r o n e of t h e C l a t o m s .

O t h e r s t u d i e s 304 on t h e t e m p e r a t u r e d e p e n d e n c e o f t h e s p i n - l a t t i c e r e l a x a t i o n t i m e f o r c h l o r a l s u g g e s t t h e c r y s t a l l o g r a p h i c non- e q u i v a l e n c y o f t h e C C l 3 g r o u p s .

2.16 Mass S p e c t r u m

The mass s p e c t r u m o f c h l o r a l h 5 t j r a t e h a s b e e n r e c o r d e d on a n MS-9 i n s t r u m e n t . A s e x p e c t e d , c h l o r a l h y d r a t e u n d e r g o e s t h e r m a l d e - h y d r a t i o n a t t h e s o u r c e t e m p e r a t u r e ( 2 O O 0 C ) , g i v i n g t h e h i g h e s t mass d e t e c t e d as t h e m o l e c u l a r i o n o f c h l o r a l . The f r a g m e n t a t i o n p a t t e r n f r o m t h i s s p e c t r u m , which i s g i v e n i n T a b l e 1, d i f f e r s f r o m t h a t o b t a i n e d by Reyes e t al.25 ( w i t h a H i t a c h i P e r k i n E l m e r R M U - 6 D i n s t r u m e n t ) i n t h e d e t e c t i o n o f a d d i t i o n a l p e a k s a t m / e 118 , 1 1 7 , a n d 110.

TABLE 1 F r a g m e n t a t i o n p a t t e r n p r o d u c e d i n

mass s p e c t r a l e x a m i n a t i o n of c h l o r a l h y d r a t e

m/e

- 1 4 6 118 1 1 7 111 110

83 82 * 47

I o n p r o d u c e d by loss f r o m m o l e c u l a r i o n of c h l o r a l

o f cc13mo+ -

C C 1 2 C H G t c 1 C C 1 2 H t C l + co

C H C 1 2 ' co C C l 3 C H O

C C 1 2 C O t HC 1

c c q C1 t CHO cc1 C H O + C 1 2

* Most a b u n d a n t p e a k

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JOHN E . F A I R B R O T H E R

1 slsl

9sl

8Q

7sl

614

5hl

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F i g . 4 . Mass s p e c t r u m o f c h l o r a l h y d r a t e , S q u i b b House S t a n d a r d .

96

CHLORAL HYDRATE

2 . 1 7 E l e c t r o n S p i n Resonance S p e c t r u m

E l e c t r o n i r r a d i a t i o n i n d u c e s paramag- n e t i c s p e c i e s i n c r y s t a l l i n e c h l o r a l h y d r a t e which c a n b e c h a r a c t e r i z e d b y E.S.R. Two d i f f e r e n t i n t e n s i t y s e q u e n c e s have b e e n ob- s e r v e d t h o u g h t t o c o r r e s p o n d t o t h e CC13C(OH)2 and t h e C C l , C H ( O H ) 2 r a d i c a l s .

S p e c t r u m 2 5 .

2 . 2 P h y s i c a l P r o p e r t i e s o f t h e S o l i d

2 . 2 1 B a s i c T h e r m a l C o n s t a n t s

The f o l l o w i n g h a v e b e e n r e p o r t e d : -

Heat and F r e e E n e r g y o f F o r m a t i o n l l l c r y s t a l l i n e s t a t e )

( f o r t h e _ _ _ _ _ _ _ - _ _ _ _ _ _ _ _ _ - - ---------------

A Hf= - 1 3 2 . 2 ( k g . c a l . p e r m o l e )

S t a t e Temp e r a t u r e S p e c i f i c Heat C r y s t a l l i n e 32oc 0 . 2 1 3 L i q u i d 55 t o 8 a o c 0 . 4 7 0

M e l t i n g - - - - -_ ---- r a n g e -_---------- a n d b o i l i n g - m j n t a r e v a r i a b l e , d e p e n d i n g on d i s s o c i a t i o n a n d , t h u s , on s u c h f a c t o r s as t h e r a t e o f h e a t i n g .

Lle&lipg-ranggs as d i f f e r e n t as 46-47OC and 5 9 - 6 0 0 ~ 113 h a v e b e e n q u o t e d i n t h e l i t e r a t u r e , b u t t h e f i g u r e o f 57OC g i v e n by t h e Nerck I n d e x 1 1 4 a p p e a r s t o be t h e most r e p r e s e n t a t i v e .

__-- - - B o i l i n g p o i n t has b e e n r e c o r d e d b y L a n g e l l l as 9 6 . 3 ( 7 6 4 j d C - a n d by t h e Merck Index114 as 98OC.

JOHN E. FAIRBROTHER

2 . 2 2 D i f f e r e n t i a l Thermal A n a l y s i s and D i f f e r e n t i a l S c a n n i n g C a l o r i m e t r y

House S t a n d a r d o f c h l o r a l h f b r a t e l 2 g a v e a s i n g l e e n d o t h e r m a t 5 9 O C . D.S.C.; ( s e a l e d c o n t a i n e r ) o f a s a m p l e o f B.P. q u a l i t y c h l o r a l h y d r a t e s i m i - l a r l y g a v e i n g l e e n d o t h e r m a t 5 8 O C . S e p a r a t e e x p e r i m e n t s ?” w i t h U . S . P . g r a d e c h l o r a l h y d r a t e g a v e a s i n g l e , w e l l - d e f i n e d m e l t i n g e n d o t h e r m a t 56 t o 5 7 O C a n d a b o i l i n g p o i n t e n d o t h e r m t h a t v a r i e d f rom 1 0 5 t o 1 2 5 O C , d e p e n d i n g on t h e amount e n c a p s u l a t e d . Samples o f c h l o r a l h y d r a t e were h e a t e d t o s p e c i f i e d t e m p e r a t u r e s a b o v e t h e me l t i rg p o i n t , m a i n t a i n e d f o r v a r i o u s p e r i o d s a t t h e t e m p e r a t u r e a n d t h e n a l l o w e d t o c o o l . Re-programming o f t h e c o o l e d s a m p l e s gave u n r e p r o d u c i b l e r e s u l t s t h a t f a i l e d t o show t h e p r e s e n c e o f a d i s c r e t e po l y m o r p h i c f o r m .

D . T . A . ( o p e n c o n t a i n e r ) o f t h e Squ ibb

D i l a t o m e t r i c measu remen t s117 t h a t i n d i - c a t e a p h a s e t r a n s i t i o n o c c u r r i g a t 3 2 O C a r e i n a g r e e m e n t w i t h t h e much l a t e r l l B r e p o r t o f a n e n a n t i o t r o p i c t r a n s i t i o n o c c u r r i n g a t a b o u t 32OC ( o n c o o l i n g ) ,301,302 as o b s e r v e d w i t h t h e p o l a r i p

m i c r o s c o p e . The t r a n s i t i o n i s n o t t h e same i n t h e r e v e r s e d i r e c t i o n ( i . e . on r a i s i n g t h e

t e m p e r a t u r e ) . D . T . A . e x a m i n a t i o n o f c h l o r a l h y d r a t e (room t e m p e r a t u r e t o 7 O o C ) as r e p o r t e d b y O g a ~ a ~ ~ d e t e c t s t h e t r a n s i t i o n i n t o a h igh - t emp- e r a t u r e f o r m a t 5 2 . 6 O C , m e l t i n g o c c u r r i n g b e t w e e n 5 5 . 0 and 6 4 . 5 O C .

7

2 . 2 3 Loss on D r y i n g and Thermo- g r a v i m e t r i c A n a l y s i s

Hea ted i n open c o n t a i n e r s f rom room t e m p e r a t u r e t o i t s m e l t i n g p o i n t , c h l o r a l h y d r a t e d i s s o c i a t e s s l o w l y , w i t h v o l a t i l i z a t i o n o f t h e p r o d u c t s . The v a p o r p r e s s u r e o f c h l o r a l h y d r a t e has b e e n examined b y s e v e r a l a u t h o r s . 1 1 7 , 1 2 2 , 3 0 0 .

CHLORAL HYDRATE

I n t h e t e m p e r a t u r e r a n g e 1 5 . 6 5 t o 3 8 . 5 0 ~ ~ a smooth v a p o r - p r e s s u r e c u r v e was o b t a i n e d , 117 r i s i n g from 6 . 1 2 t o 26.90mm. a - b r o m o n a p h t h a l e n e . A s e p a r a t e s o u r c e 1 2 2 g i v e s t h e v a p o r p r e s s u r e o f c h l o r a l h y d r a t e a t 2 O o C as 12.7mm. Hg.

2 . 2 4 O p t i c a l C h a r a c t e r i s t i c s

C h l o r a l h y d r a t e e x i s t s i n t h e fo rm o f l a r g e m o n o c l i n i c p l a t e s ( h e x a g o n a l t a b u l a r c r y s - t a l s ) 2 3 ~ 1 1 4 , 118- A l e s s s t a b l e h i g h - t e m p e r a t u r e form, o b t a i n e d o n c r y s t a l l i z a t i o n o f t h e m e l t l g ~ 2 3 , or f r o m c h l o r o f o r m s o l u t i o n b y e v a p o r a t i o n 2 3 , e x i s t s i n a n e e d l e - i k e c r y s t a l form. D e n s i t y of c r y s t a l l i n e s o l i d 1 * 3 a t 20°C i s 1 . 9 0 8 .

2 . 2 5 X-Fiay D i f f r a c t i o n

The c r y s t a l and m o l e c u l a r s t r u c t u r e o f c h l o r a l h y d r a t e h a s b e e n i n v e s t i g a t e d b y x - r a y d i f f r a c t i o n 2 3 ~ 1 1 9 ~ 1 2 O ~ 1 2 1 ~ ~ 1 ~ . The c r y s t a l s t r u c - t u r e d i m e n s i o n s o b t a i n e d by a l l t h r e e a u t h o r s 2 3 3 119,121 a r e i n a g r e e m e n t , w i t h i n t h e l i m i t s o f e x p e r i m e n t a l e r r o r . The c r y s t a l h a b i t i s mono- c l i n i c and b e l o n g s t o t h e p21/C s p a c e g r o u p , w i t h f o u r m o l e c u l e s i n a c e l l o f t h e d i m e n s i o n s : -

0 a = 11.50 t 0.03 A

b = 6.04 t 0.02 A 0

c = 9.60 - t 0.03 A

- 0

-

The r e s u l t s o f s t u d i e - 1 e c u l a r s t r u c t u r e a r e l e s s c o n c l u s i v e , 1 9 Y ” Y ” and t h e r e s u l t s o r i g i n a l l y o b t a i n e d b y Kondo and N i t t a 2 2 were found to b e i n e r r o r b y Ogawa23, who re - p e a t e d t h e w o r k and r e p o r t e d bond l e n g t h s a n d bond a n g l e s . From t h e s e r e s u l t s , 3gawa s u g g e s t e d t h a t t h e m o l e c u l e c o n t a i n s two b i f u r c a t e d b o n d s . However, t h e C - C l bond l e n g t h s g i v e n b y Ogawa have s i n c e b e e n d i s p u t e d b y . “ ( i l i a and H a d j o u d i s 2 l on t h e b a s i s o f t h e i r N . Q . R . s t u d i e s , wh ich

99

JOHN E. FAIRBROTHER

sugges t t h a t o n l y one hydrogen-bonded C 1 e x i s t s .

An g g r a y powder d i f f r a c t i o n p a t t e r n h a s b e e n r e c o r d e d f o r a b a t c h o f C h l o r a l H y d r a t e U . S . P . ( s ee F i g . 5 . ) . 0 g a w a 2 3 g i v e s p o w d e r - d i f f r a c - t i o n p a t t e r n s f o r b o t h t h e n o r m a l h e x a g o n a l c r y s - t a l p l a t e s o f c h l o r a l h y d r a t e a n d for t h e h i g h - t e m p e r a t u r e n e e d l e c r y s t a l s .

2 . 2 6 N e u t r o n D i f f r a c t i o n

N e u t r o n d i f f r a c t i o n s t u d i e s c o n d u c t e d by Brown123 h a v e y i e l d e d a d d i t i o n a l d a t a a b o u t bond l e n g t h s and bond a n g l e s i n c h l o r a l h y d r a t e .

2 . 2 7 Polymorphism

S c h i 1 1 9 4 r e j e c t e d t h e p o s s i b i l i t y o f e n a n t i o t r o p y or t h e e x i s t e n c e o f i s o m e r i c f o r m s o f c h l o r a l h y d r a t e . However, h e was a b l e t o p r e p a r e a h e m i h y d r a t e (m.p. 9 5 O C ) r e p r o d u c i n g t h e work of Meyer and Du lk124 . G u i l l a r m o d 7 more r e c e n t l y p r e p a r e d a "hemi- h y d r a t e " t h a t g a v e a w e l l - d e f i n e d i n f r a r e d s p e c - t r u m d i f f e r e n t f r o m t h o s e o f c h l o r a l h y d r a t e or o f a m i x t u r e o f c h l o r a l a n d c h l o r a l h y d r a t e .

P i g u e t a n d J a c o t -

Naveau118, r e j e c t i n g S c h i l l ' s c o n c l u s - i o n s , p r e s e n t e d d a t a s u p p o r t i n g t h e e x i s t e n c e o f a dynamic i s o m e r i s m b e t w e e n two s t e r e o - i s o m e r s o f unknown c o n f i g u r a t i o n .

A l l e n l 7 , i n a n N . Q . R . e x a m i n a t i o n o f c h l o r a l h y d r a t e , n o t e d t h e n e e d t o "age" for 3 months ma te r i a l f r e s h l y r e - c r y s t a l l i z e d f rom a m e l t b e f o r e i t would g i v e a s a t i s f a c t o r y s p e c t r u m . B iedenkapp and W e i s & % n v e s t i g a t e d t h i s phenomenon more t h o r o u g h l y , r e p o r t i n g t h e e x i s t e n c e o f two c r y s t a l m o d i f i c a t i o n s ( s ee s e c t i o n 2 . 1 5 ) . The l e s s s t a b l e m o d i f i c a t i o n ( 1 ) was p r o d u c e d by f r e e z i n g m e l t s or b y h e a t i n g c h l o r a l h y d r a t e t o a t l e a s t 4 8 O C f o r 0 . 5 h o u r . 1 2 5 M o d i f i c a t i o n ( 1 ) changed s l o w l y t o t h e more s t a b l e m o d i f i c a t i o n ( I 1 ) i n a b o u t 2-3 weeks a t room t e m p e r a t u r e , b u t no a c c e l e r a t e d t r a n s i t i o n was f o u n d e v e n a t 32OC.

100

1w

M

80

70

60

so

40

30

90

80

70

bO

50

9 a "n

40

30

0 0 - 28 1

A + - + Y ? - s s : a m $ % q ? . . ? l ? ? * * *

Fig. 5. X-ray powder-diffraction pattern of chloral hydrate, Lot T-102.

JOHN E. FAIRBROTHER

Ogawa23 s i m i l a r l y d e t e c t e d t h e t r a n s i t i o n i n t o a h i g h - t e m p e r a t u r e fo rm a t 52.6OC ( s e e S e c t i o n s 2 . 2 2 , 2 .24 a n d 2 . 2 5 ) . X - r a y p o w d e r - d i f f r a c t i o n p a t t e r n s t a k e n a t d i f f e r e n t t imes a f t e r c r y s t a l l - i z a t i o n o f t h e h i g h - t e m p e r a t u r e form show t h a t t h i s p h a s e c h a n g e s s l o w l y t o t h e r o o m - t e m p e r a t u r e p h a s e .

2 . 3 S o l u b i l i t y

2 . 3 1 S o l u b i l i t y i n Water

The s o l u b i l i t y o f c h l o r a l h y d r a t e i n water a t 2 O o C i s g i v e n by t h e B . P . ( 1 9 6 8 ) as 1 p a r t i n 0 . 3 p a r t s o f w a t e r . However, t h e Merck I n d e x 1 l 4 g i v e s t h e f o l l o w i n g a q u e o u s s o l u b i l i t i e s :

Tetnperat u r e ( O C ) So l u b f l i t y ( m g / m l ) 0 2 400

10 2 0 30 40

3800 6600

10100 14300

2 . 3 2 S o l u b i l i t y i n W a t e r - M i s c i b l e S o l v e n t s

The s o l u b i l i t y f i g u r e s q u o t e d for c h l o r - a l h y d r a t e i n e t h a n o l d i f f e r w i d e l y .

T e m p e r a t u r e

Room Temp. 2 ooc

Volume o f E t h a n o l ( 9 5 % ) r e q u i r e d t o d i s s o l v e lg o f

C h l o r a l H y d r a t e 1.5 m l

R e f e r e n c e

Merck I n d e x 0.2 ml B.P .196899

E q u i l i b r a t i o n o f l 5g o f c h l o r a l h y d r a t e w i t h 1 m l o f e t h a n o l ( 9 5 % ) a t 25OC f o r 2 4 h o u r s r e s u l t e d i n a l i q u i d s y s t e m c o n t a i n i n g a b o u t 2 t o 3 g o f s o l i d c h l o r a l h y d r a t e l 3 7 . A p a r a l l e l s t u d y w i t h m e t h a n o l g a v e s imi l a r r e s u l t s , b u t o n l y a b o u t 1 g o f s o l i d r e m a i n e d l 3 7 . R e s u l t s o b t a i n e d w i t h o t h e r w a t e r - m i s c i b l e s o l v e n t s a r e as f o l l o w s : -

CHLORAL HYDRATE

S o l v e n t T e m p e r a t u r e S o l u o i l i t y R e f e r e m e (mg c h l o r a l h y d r a t e p e r m l s o l v e n t )

G l y c e r o l Room Temp. c a . 1 6 0 0 1 1 4 A c e t o n e - F r e e l y s o l u b l e 1 1 4 Methy l E t h y l Ke tone - F r e e l y s o l u b l e 1 1 4

2 . 3 3 S o l u b i l i t y i n W a t e r - I m m i s c i b l e S o l v e n t s

S o l v e n t

D i e t h y l E t h e r D i e t h y l E t h e r C h l o r o f o r m C h l o r o f o r m ( e t h a n o l - f r e e ) C h l o r o f o r m C h l o r o form ( c o n t g . 2 % e t h a n o l ) C h l o r o f o r m Carbon D i - s u l f i d e Hexane H e p t a n e n-Decane P e t . E t h e r

Carbon T e t r a - c h l o r i d e Benzene

T o l u e n e

Temp e r a t u r e (OC)

Room Temp. 20 2 0

2 5 2 0

2 5 Room Temp.

Room Temp. 25 25 2 5

Room Temp.

Room Temp.

Room Temp.

Room Temp.

S o l u b i l i t y Ref e r e n e

h y d r a t e p e r m l s o l v e n t )

(mg c h l o r a l

c a . 660 1 1 4 > 1 0 0 0 99

155 138

380 137 c a . 330 9 9

5 2 5 137 500 1 1 4

1 1 . 6 1 1 4 4 4 . 4 137 3 9 . 2 1 3 7 33 .0 1 3 7

S p a r i n g l y 1 1 4 s o l u b l e S p a r i n g l y 1 1 4 s o l u b l e S p a r i n g l y 1 1 4 s o l u b l e S p a r i n g l y 1 1 4 s o l u b l e

103

JOHN E. FAIRBROTHER

S o l v e n t ---

2 . 5 4 S o l u b i l i t y i n O i l s of Pharma- c e u t i c a l I n t e r e s t

T e m p e r e t u r e S o l u b i l i t y - - __ R e f e r e n c e - -_ - mg c h l o r a l h y d r a t e p e r m l s o l v e n t )

- - - T o r - - - (

O l i v e O i l R O O K Temp. c a . 710 1 1 4 O l i v e O i l 2 5 8 30 137 C a s t o r O i l 25 10 14 0 137 Corn O i l 2 5 990 137

Tu r p e n t i ri e L i q u i d P z r a f f i r ) 25 1 3 . 3 $37

Room Temp. S p a r i n g l y s o l u b l e 1 1 4

2 . 4 -- Pkiysi c a l P r o p e r t i e s o f S o l u t i o n s and Melts

2 . 4 1 H e a t s o f S o l u t i o n and D i l u t i o n

The h e a t of s o l u t i o n o f c h l o r a l h y d r a t e

- --

i r m e t h a n o l Pas b e e n deternl inc>d mlcrc,cal o r i m e t - ri c a l l y a s a b o u t - 1 . 1 2 K c a l / m o l . l o . S c h i 1 1 9 4 measu red t h e t e n p e r a t u r e r i s e on d i s s o l u t i o n of c h i o r a l arid m e l t e d c h l o r a l h y d r a t e i n water. I n t h e same p a p e r , h e d e s c r i b e s t h e t e m p e r a t c u r e c h a n g e o b s e r v e d on d l s s o l v i n g c h l o r a l h y d r a t e i n water , a n d c o n p a r e s i t w i t h t h o s e p r o d u c e d by c h l o r a l h e m i h y d r a t e ( s e e s e c t i o n 2 . 2 ) and b y c h l o r a l h y d r a t e m e l t s t h a t h a d b e e n r a p i d l y c o o l e d t o rcom t e m p e r a t u r e i m m e d i a t e l y G e f c r e d i s s o l u t i o n .

S c h i 1 1 g 4 s u g g e s t s t h a t i n a m e l t c f c h l o r a l h y d r a t e s e v e r a l h y d r a t e s o f d i f f e r e n t s t r u c t u r e s may e x i s t . By c a l c u l a t i o n from S c h i l l ' s f i g u r e s , a n a p p r o x . h e a t o f s o l u t i o n f o r c h l o r a l h y d r a t e i r water i s -0 .78Kca l / r r , o l . Measu remen t s o f t h e h e a t o f d i l u t i o n o f c h l o r s l h y d r a t e a q u e o u s s o l u t i o n s h a v e b e e n made95. No N e r r s t t e m p e r a t w e c o e f f i c i e n t , i s fourid for t h e s e s o l u t i o n s .

104

CHLORAL HYDRATE

2 . 4 2 D i s s o c i a t i o n a n d pH

The d i s s o c i a t i o n o f c h l o r a l h y d r a t e i n c y c l o h e x a n e h a s a l r e a d y b e e n m e n t i o n e d i n s e c t i o n 2 . 1 3 . D i s s o c i a t i o n o f d i l u t e a q u e o u s s o l u t i o n s y g c h l o r a l h y d r a t e was r e p o r t e d i n t h e same p a p e r

The i o n i z a t i o n c o n s t a n t , pKa, was o b t a i n e d by m e a s u r i n g t h e pH o f b u f f e r e d s o l u t i o n s o f c h l o r a l h y d r a t e g 6 . g a v e pKa 1 0 . 0 4 , which d i f f e r s c o n s i d e r a b l y from t h e v a l u e of 11 o b t a i n e d b y E u l e r and E u l e r 9 7 and i s c l o s e r t o pK 9 . 7 7 , d e r i v e d i E d i r e c t l y f rom k i n e t i c rneasurernents98,

T h e mean o f 25 e x p e r i m e n t s

The d i s s o c i a t i o n o f c h l o r a l h y d r a t e i n v a r i o u s o r g a n i c s o l v e n t s (CCl4, b e n z e n e e t c . ) h a s b e e n s t u d i e d b y i n f r a r e d s p e c t r o s c o p y 7 ( s e e s e c t i o n 2 . 1 1 ) . S t u d i e s o f t h e n e a r i n f r a r e d s p e c t r a 6 h a v e a l s o b e e n u s e d t o c a l c u l a t e e q u i l i b r i u m and r a t e c o n s t a n t s f o r t h e d i s s o c i a t i o n o f s o l u t i o n s of c h l o r a l h y d r a t e .

2 . 4 3 S u r f a c e T e n s i o n

T e i t e l ' b a u m e t a 1 . l o 0 g i v e a c o m p l e t e p i c t u r e o f t h e s u r f a c e t e n s i o n s o b t a i n e d w i t h a q u e o u s s o l u t i o n s o f c h l o r a l ( c o n c . r a n g e 2 t o 1 0 0 % ) w i t h i n t h e t e m p e r a t u r e r a n g e 0 t o 7 5 O C ( a t 5 - O C i n t e r v a l s ) . T h e s e a u t h o r s have a l s o g i v e n a t t e n t i o n t o t h e t e m p e r a t u r e c o e f f i c i e n t , w h i c h , when p l o t t e d a g a i n s t m o l . % o f c h l o r a l , shows a maximum a t 50 m o l . % , c o r r e s p o n d i n g t o t h e form- a t i o n o f c h l o r a l h y d r a t e , and two minima a t a p p r o x . 18 a n d 8 1 m o l . % , r e s p e c t i v e l y . F e r r o n i e t a1 .56 h a v e a l s o r e p o r t e d v a l u e s for t h e sur- f a c e t e n s i o n o f c h l o r a l h y d r a t e s o l u t i o n s t h a t r a n g e f rom 5 9 . 5 f o r 1 m o l a r t o 7 6 . 0 ( d y n e / c m . ) a t i n f i n i t e d i l u t i o n . S u r f a c e t e n s i o n measure- m e n t s made on s o l u t i o n s o f c h l o r a l h y d r a t e i n d i e t h y l e t h e r , a c e t o n e , or b e n z e n e 3 ° i n d i c a t e weak compound f o r m a t i o n i n t h e s e s y s t e m s .

-___--

JOHN E. FAIRBROTHER

The s u r f a c e t e n s i o n o f c h l o r a l h y d r a t e m e l t s 3 1 4 i n t h e t e m p e r a t u r e r a n g e 53 t o 100°C i s e x p r e s s e d ( i n d y n e s / c m . ) b y t h e e q u a t i o n u 4 4 . 6 - 0 . 1 1 0 ( t - 5 3 ° ) . The c o n t a c t w e t t i n g a n g l e o f i t s s o l i d phase i s 1 8 0 4 0 ' - t 1.5O.

2 . 4 4 U l t r a s o n i c A b s o m t i o n

The v a r i a t i o n o f u l t r a s o n i c v e l o c i t y and a b s o r p t i o n w i t h t e m p e r a t u r e has b e e n r e c o r d e d f o r c h l o r a l h y d r a t e me l t s ( f o r 7 MHz e m i s s i o n , v e l o c i t i e s r a n g e f rom 1215 m / s e c . a t 5 2 O C t o 1125 m / s e c . a t 75OC ) l o 1 y 1 O 2 . The u l t r a s o n i c a b s o r p t i o n c o e f f i c i e n t s (a / V 2 ) f o r c h l o r a l hy- d r a t e , when compared w i t h t h o s e o f a m i x t u r e o f c h l o r a l a n d water a t c o r r e s p o n d i n g t e m p e r a t u r e s , show t h a t t h e e f f e c t o f d i s s o c i a t i o n b r o u g h t a b o u t b y i n c r e a s i n g t e m p e r a t u r e i s t o r e d u c e t h e a b s o r p t i o n v a l u e s .

S o l u t i o n s o f c h l o r a l i n non-aqueous s o l v e n t s 2 7 ,3 i show a b s o r p t i o n - c o e f f i c i e n t c u r v e s t h a t p a s s t h r o u g h a maximum i n t h e c a s e s o f assoc ia ted s o l v e n t s s u c h as m e t h a n o l a n d e t h a n o l , c o r r e s p o n d i n g t o t h e f o r m a t i o n o f a m o l e c u l a r compound 308.

2 . 4 5 V i s c o s i t y

The v i s c o s i t i e s o f c h l o r a l h y d r a t e mel ts o b t a i n e d b y u s e o f a n O s t w a l d V i s c o m e t e r h a v e b e e n r e p o r t e d 1 0 1 a s : -

T e m p e r a t u r e ( O C ) Shear V i s c o s i t y ( q / p ) C.S.

52 t o 75

10.10 t o

3 . 4 0

From t h e t e m p e r a t u r e c o e f f i c i e n t o f t h e s e v i s - c o s i t i e s , t h e a c t i v a t i o n e n e r g y of shear v i s - c o s i t y o f c h l o r a l h y d r a t e ( m e l t ) has b e e n c a l c u l - a t e d as 1 0 . 6 K c a l / m o l .

The v i s c o s i t y of s o l u t i o n s o f c h l o r a l

CHLORAL HYDRATE

i n v a r i o u s h y d r o x y l a t e d o r g a n i c s o l v e n t s ( a l i - p h a t i c a l c o h o l s , b e n z y l a l c o h o l , p h e n o l s , e t c . ) , has b e e n p l o t t e d a g a i n s t c h l o r a l c o n c e n t r a t i o n showin maxim wher com ound f o r m a t i o n o c c u r s . 2 8 , 3 3 , 9 4 , 7 5 , 7 % , 7 7 , 7 b 9 , b 3 1

2 . 4 6 Cryoscopy

The f r e e z i n g - p o i n t s i o n c a u s e d

--

l o r a l h y d r a t e i n a q u e o u s ''y'cis a n d a l c o h o l i c 98 ,?b s o l u t i o n s h a s b e e n d e s c r i b e d .

Van Rossem226, s t u d y i n g t h e f r e e z i n g - p o i n t c u r v e s o f c h l o r a l and water f o u n d e v i d e n c e for t h r e e d i f f e r e n t h y d r a t e s : a " s e m i h y d r a t e " (1 mole o f water t o 2 m o l e s o f c h l o r a l ) , a mono- h y d r a t e (1 mole o f e a c h ) , and a h e p t a h y d r a t e ( 7 m o l e s o f water t o 1 mole o f c h l o r a l ) . The h e p t a h y d r a t e h a d a s h a r p m e l t i n g p o i n t a t - 1 . 4 % . E u t e c t i c t e m p e r a t u r e s o f 5 loC w i t h a z o b e n z e n e a n d o f 4 0 0 C w i t h b e n z i l h a v e b e e n r e p o r t e d 3 1 3 .

2 . 4 7 R e f r a c t i v e I n d e x

The r e f r a c t i v e i n d e x o f a c h l o r a l hy - d r a t e m e l t ( a t 6 0 . 0 O C ) r i s e s w i t h t i m e 9 4 , e v e n t u - ally r e a c h i n g a c o n s t a n t v a l u e a t 1 . 4 8 0 7 ( i . e . m o l a r r e f r a c t i o n MR a t 6 0 O c i s 2 9 . 4 7 ) . T h e F e f r z t i v e i n d e x v a l u e s o b t a i n e d w i t h a s e r i e s o f b i n a r y s y s t e m s o f c h l o r a l a n d v a r i o u s s o l v e n t s ( i n c l u d i n g water , e t h a n o l , a n d i n d i - c a t e t h e f o r m a t i o n o f a n a s s o c i a t i o n compound b e t w e e n c h l o r a l and e a c h s o l v e n t .

2 . 4 8 R a d i o l y s i s

Aqueous s o l u t i o n s o f c h l o r a l h y d r a t e , when i r r a d i a t e d w i t h X - r a y s , gamma r a y s , or b e t a r a y s , g i v e r i s e t o h y d r o c h l o r i c a c i d a s t h e end p r o d u c t o f a c h a i n r e a c t i o n 2 5 , I 0 3 t o l l o .

3. M o l e c u l a r Complexes

3 . 1 Types o f Known Complexes -

JOHN E . FAIRBROTHER

Chloral forms a range of molecular complexes o r dducts main1 in a 1:1 alc oho 1 s 22 to 31,$9,92,53 diethy1 benzene, 30, j1 acetone, 3°3 31 meprob tetracycline,38 oxytetrac cline,38 299 and others32 to 36330 E .

ratio) with ether,30,31 amate, 37 acetaminophen,

Similarly, chloral hydrate has been

caffeine, shown to form c mp exes (main lycinamide ,399e0341 betaine,

58,49 diazepam,50,312 ,5-dimethyl dantoin 52,307 phe a~etin,~3,5? phenazon tY,56 to 61, glycine, 1 7 4 , glucosel?6, urea53,%8 and others. 32,42,4 3,4 8,49,51,53,54,62-67 9 69 173,1753219 8

3.2 Structure of Complexes

A number of the molecular complexes of chloral and chloral hydrate have been shown to be hydrogen-bonded association compounds. The techniques employed in the examination of these complexes are summarized in Table 1.

TABLE 1

Techniques used in the characterization of complexes of chloral and chloral hydrate

Technique References

Cryoscopy 26,29,51,55,63,64,66,70,

Differential Thermal Analysis 62 Differential Scanning Calorirne t ry 10,83,84 Heats of Solution 10,49 Refractive Index 74,86 Surface Tension 30,4 3,56 Viscosity 28,33,34,75-81. Infrared Spectroscopy 10,41,82 Raman Spectroscopy 57 X-ray Diffraction 10,85,312 Ultrasound 27,31 Hot-stage Microscopy 86

71,72,73

Chloral hydrate and phenazone form both

108

CHLORAL HYDRATE

(1:l) and (2:l) m o l e c u l a r c o m p l e x e s . The s t r u c - t u r e o f t h e (1:l) complex h a s b e e n s t u d i e d by i n f r a r e d 4 1 and Raman57 s p e c t r o s c o p y , i n a d d i t i o n t o a s u r f a c e - t e n s i o n 5 6 s t u d y t h a t examined b o t h c o m p l e x e s .

The i . r . and Raman d a t a s u g g e s t a s t r o n g l y hydrogen-bonded c o m p l e x , w i t h t h e a s s o c i - a t i o n a t t h e c a r b o n y l oxygen o f t n e p h e n a z o n e a n d n o t a t t h e n i t r o g e n . I n f r a r e d s t u d i e s o f t h e complex i n c a r b o n t e t r a c h l o r i d e s o l u t i o n havi? b e e n u s e d t o c a l c u l a t e a s t a b i l i t y c o n s t a n t f o r t h e complex (l:l), and c r y o s c o p i c m e a s u r e m e n t s on d i l u t e a q u e o u s s o l u t i o n s show t h e m o l e c u l a r compound t o b e c o m p l e t e l y d i s s o c i a t e d i n wa te r41 .

T h e c r y s t a l 2nd m o l e c u l a r s t r u c t u r e o f t h e (1: 1) m o l e c u l a r complex o f c h l o r a l h y d r a t e and bromodiazepam has b e e n d e t e r m i n e d b y x - r a y d i f f r a c t i o n s t u d i e s 3 1 2 .

4 . S y n t h e s i s a n d P u r i f i c a t i o n

C h l o r a l l i yd ra t e wa's f i r s t o b t a i n e d b y L i e b i g ( 1 8 3 2 ) f rom t h e r e a c t i o n o f w a t e r w i t h c h l o r a l , t h e l a t t e r o b t a i n e d v i a t h e c h l o r i n a t i o n o f e t h a n o l . I n d u s t r i a l l y , c h l o r a l i s p r e p a r e d b y p a s s i n g c h l o r i n e i n t o c o o l e d e t h a n o l ( e i t h e r a b - s o l u t e o r 9 5 p e r c e n t ) t o form t h e h e m i a c e t a l o f t r i c h l o r o a c e t a l d e h y d e , f rom which c h l o r a l i s l i b e r a t e d b y t r e a t m e n t w i t h s u l f u r i c a c i d .

It has b e e n f o u n d , e v . r , t h a t t h e herni- a c e t a 1 may b e h y d r o 1 y z e iJ. ' g B ~ 2 k g b y t h e a d d i t i o n o f w a t e r , g i v i n g c h l o r a l h y d r a t e and a l c o h o l .

The o v e r - a l l r e a c t i o n may b e r e p r e s e n t e d b y t h e e q u a t i o n : -

13ther p r o c e s s m o d i f i c a t i o n s i n c f u d e t h e r e p l a c e - ment o f e t h a n o l w i t h e t h a n o l - a c e t i c a c i d m i x - t u r e s i n t h e c h l o r i n a t i o n s t e p 2 T 0 , w i t h r e c y c l i n g o f t h e l e s s v o l a t i l e b y p r o d u c t s , s u c h as c h l o r a l a c e t a t e , c h l o r a l a l c o h o l . a t e , b u t y l c h l o r a l , and

E t O I l + 4Cl2 + l i20 + C l , C C H ( O H ) 2 + 5 H C l

109

JOHN E. F A I R B R O T H E R

t r i c h l o r o a c e t i c a c i d .

Crude c h l o r a l i s s e p a r a t e d by f r a c t i o n a t i o n f rom v o l a t i l e b y p r o d u c t s s u c h as e t h y l c h l o r i d e , e t h y l e t h e r , e t h y l e n e d i c h l o r i d e , and a l c o h o l , and t h e c h l o r a l r ' c u t r r i s t a k e n b e t w e e n 93 and 9 8 O C . T h i s material1g0 c o n t a i n s 2 t o 3% water- i n s o l u b l e m a t e r i a l c o n t a i n i n g e t h y l d i c h l o r o - a c e t a t e , e t h y l t r i c h l o r o a c e t a t e , and t r i c h l o r o - a c e t a l . The t e c h n i c a l c h l o r a l s e p a r a t e d from t h e o i l c o n t a i n s a c e t i c a c i d ( 0 . 2 t o 0 . 8 % ) , d i - c h l o r o a c e t a l d e h y d e ( 2 t o 5%), e t h y l d i c h l o r o - a c e t a t e ( a b o u t 0 . 3 % ) , e t h y l t r i c h l o r o a c e t a t e ( a b o u t 0 . 6 % ) , a n d t r a c e s o f c h l o r a l a l c o h o l a t e 1 g 0 .

The c h l o r a l h y d r a t e formed b y t r e a t i n g t h e c h l o r a l w i t h a n a p p r o p r i a t e q u a n t i t y o f w a t e r may c o n t a i n t r a c e s o f t h e above i m p u r i t i e s a n d a l s o t r i c h l o r o a c e t i c a c i d ( o x i d a t i o n p r o d u c t ) or t r i - c h l o r o e t h a n o l ( r e d u c t i o n p r o d u c t ) . The c r u d e c h l o r a l h y d r a t e may t h e n b e p u r i f i e d f u r t h e r by r e c r y s t a l l i z a t i o n from a n o r g a n i c s o l v e n t s u c h as b e n z e n e .

An a l t e r n a t i v e i n d u s t r i a l r o u t e t o c h l o r a l i s by t c h l o r i n a t i o n o f a c e t a l d e h y d e o r p a r a l - d e h ~ d e ? ~ ~ ~ 271

C H CHO + 3 C 1 2 - C l C C H O + 3HC1

A l t e r n a t i v e l a b o r a t o r y p r e p a r a t i o n s i n c l u d e 3 3

t h e r e a c t i o n o f et l i 1 f o r m a t e w i t h s u l f u r y l c h l o r i d e a t 1 7 O o C 2 y 2 a n d t h e r e a c t i o n o f c h l o r o - a c e t y l e n e w i t h sod ium h y p o c h l o r i t e s o l u t i o n a n d s a t u r a t e d b o r i c a c i d s o l u t i o n 2 7 3 . The p r e a r a t i m o f h i g h - p u r i t y c h l o r a l h a s b e e n a c h i e v e d 2 2 1 by h e a t i n g m e t a c h l o r a l a t 180-2OO0C.

5 . D e g r a d a t i o n

5 . 1 D e g r a d a t i o n o f S o l i d C h l o r a l H y d r a t e

5.11 V o l a t i l i z a t i o n

C h l o r a l h y d r a t e c r y s t a l s , l e f t open t o

CHLORAL HYDRATE

t h e a i r , v o l a t i l i z e s l o w l y as c h l o r a l a n d w a t e r . C o n d e n s a t i o n o f t h e mixed d i s s o c i a t e d v a p o r s g i v e s b a c k c h l o r a l h y d r a t e ? . C h l o r a l h y d r a t e i n s e a l e d c o n t a i n e r s w i l l o n l y v o l a t i l i z e u n t i l e q u i l i b r i u m v a p o r p r e s s u r e has b e e n a t t a i n e d 8 9 .

V o l a t i l i z a t i o n may b e d e t e r m i n e d b y m e a s u r e m e n t o f w e i g h t l o s s 1 7 3 , b y G.L.C or by G.L.C. a s s a y o f t h e u l l a g e gases84 o f t h e c o n t a i n e r .

a s ~ a y * 5 ~

5 . 1 2 C h e m i c a l D e g r a d a t i o n

D e g r a d a t i o n o f c h l o r a l h y d r a t e o c c u r s i n t h e p r e s e n c e o f a n e x c e s s o f f r e e o x y g e n t o f o r m p h o s g e n e , C 0 2 , a n d H C I , b u t o n l y a f t e r a c o n s i d e r a b l e l a g - p h a s e ( 5 2 d a y s ) 2 2 7 , u n l i k e a n - h y d r o u s c h l o r a l , w h i c h d e c o m p o s e s much more r a p i d l y 2 2 ? .

E x p o s u r e o f c h l o r a l h y d r a t e t o s u n l i g h t i n t h e a b s e n c e o f o x y g e n g e n e r a t e s n o s i g n i f i c a n t l e v e l s o f g a s e o u s d e g r a d a t i o n p r o d u c t s , e v e n a f t e r 1 6 0 d a y ~ ~ ~ 7 .

T r a c e s o f water may c a u s e t h e d e g r a d - a t i o n o f c h l o r a l h y d r a t e to d i c h l o r o a c e t a l d e h y d e , t r i c h l o r o a c e t i c a c i d a n d h y d r o c h l o r i c a c i d ; t r a c e of a l k a l i l e a d t o t h e f o r m a t i o n o f c h l o r o f o r m a n d f o r m i c a c i d 2 1 7 91.

5 . 2 D e g r a d a t i o n o f C h l o r a l H y d r a t e i n S o l u t i o n

5 . 2 1 A c t i o n o f L i g h t -

It h a s b e e n p r o p o s e d 2 1 7 , 2 1 ? t h a t ; n e u t r a l a q u e o u s s o l u t i o n s o f c h l o r a l h y d r a t e d e c o m p o s e b y a n o x i d a t i o n - r e d u c t i o n p r o c e s s .

2 C l T C C I I ’ 3 ~ t E20+Ci2CfICHO t IH(‘1 + C l j C C O O H

E x p o s u r e o f s o l u t i o n s t o l i g h t g r e a t l y a c c e l e r ; tes t h i s ~ I ’ O C E S S , as may Le demons t rda ted by m e a s u r e -

JOHN E. FAIRBROTHER

ment of t h e f o r m a t i o n o f f r e e h y d r o c h l o r i c a c i d .

5 . 2 2 A c t i o n o f Heat

On warming, n e u t r e l a n d s l i g h t l y a c i d i c a q u e o u s s o l u t i o n s of chloral h y d r s t e a p p e a r t o deg rEde t o t r i c h l o r o a c e t i c a c i d d i c h l o r o a c e t a l - d e h y d e , a n d h y d r o c h l o r i c a c i d 2 1 f .

5 . 2 3 U l t r a s o u n d ( S o n o c l e a v a g e ) -

E x p o s u r e o f a q u e o u s s o l u t i o n s o f c h l o r a l h y d r a t e t o h i g h - e n e r g y u l t r > a s o u n d ( 1 M H z ; 1.8kV) has b e e n showri220 t o c a u s e a d e g r a d a t i c n o f t h e c h l o r a l h y d r z t e t h a t follows z e r o - o r d e r k i n e t i c s . The main d e g r a d a t i o n p r o d u c t s a r e h y d r o c h l o r i c a c i d and d i c h l o r o a c e t a l d e h y d e .

5 . 2 4 A u t o x i d a t i o n and P o l . y m e r i z a t i o n - -__--

Anhydrous c h l o r a l h a s b e e n shown t o u n d e r g o a u t o x i d a t i o n i n a i r 2 2 7 v i a b o t k of t h e f o l l o w i n g r e a c t i o n s and p o s s i b l y o t h e r s .

C13CCHO t 0 ->Cl3CCOOH

C l 3 C C H O + 02+COC12 t C 0 2 t HCI

The a c t i o n of l i g h t a p p e a r s t o b e n e c e s s a r y for a t l e a s t t h e i r i i t i a l f o r m a t i o n o f t h e p e r o x i d e c a t a l y s t . A n t i - o x i d a n t s , such as h y d r o q u i n o n e , r e s o r c i n o l , and a- and 6 - n a p h t h o l s , d e c r e a s e t h e o x i d a t i o n r a t e , w h e r e a s c o m p l e t e p r o t e c t i o n i s g i v e n 2y a n i l i n e , d i p h e n y l a m i n e , and r e l a t e d compounds r 8 . of a u t o x i d a t i o n , a s o l i d p o l y m e r t h o u g h t t o b e m e t a c h l o r z l i s f o r m e d . C h l o r a l h y d r a t e d o e s n o t a p p e a r t o u n d e r g o a u t o x i d a t i o n un s s it. f i r s t

I n a d d i t i o n t o t h e g a s e o u s p r o d u c t s

d i s s o c i a t e s t o c h l o r a l and water 2 3 9 .

S t r o n g sulfuric a c i d c a u s e s t h e polymer- i y a t i o n of c h l o r a l t o fo rm a- and 8- p a r a c h l o r a l s * 3 l , wkiich h a v e b e e n shown t o b e s t e r e o i s o m e r : o f t h e c y c l i c t r inier of ~ h l o r a . 1 ~ 3 ~ , ~ 3 3 . “lore d i l u t e s u 1 fur i c a c i d p r o d u c e s t ki e amo r 1 i h o 11 s m e t a c h 1 o r a 1

CHLORAL HYORATE

231, w h ' c h may b e a n o n c y c l i c p o l y m e r of c h l o r a l h y d r a t e & , .

The a n i o n i c . p o l y m e r i z a t i o n of c h l o r a l h a s b e e n s t u d i e d a t -78OC, w i t h B u L i and sodl.um r t a p h t h a l e n e 2 3 4 t c l 2 3 6 u s e d a s i n i t i a t o r s .

5.25

C h l o r a l h y d r a t e decomposes i n d i l u t e T h i s a q u e o u s s o l u t i o n s o f sodiuni h y d r o x i d e 9 8 .

f j r s t - o r d e r r e a c t i o n i s c a t a l y s e d by t h e h y d r o x y l i o n s , by t h e c h l o r h l a t e i o n and b y w a t e r . It p r o - c e e d s v i a a n i n t e r m e d i a t e b i v a l ent, c h l o r a l a t e i o n wh ich decomposes f u r t k e r t o g i v e c h l o r o f o r m and t h e f o r m a t e i o n .

F u r t h e r s t u d i e s by P f e i l e t a 1 . 2 3 7 i r i d i - c a t e tflat i n t h e p r e s e n c e o f e x c e s s T a E t h e r e - a c t i o n i s f i r s t o r d e r arid t h e r a t e i r r c r e a s e s a l - m c , s t l i n e a r l y w i t h b a s e c o n c e n t r a t i o n . However, fcir e q u i n o l a r c o n c e n t r * a t i o n s t h e r e a c t i o n becomes secorid o r d e r 2nd w i t k l e x c e s s c h l o r a l h y d r ; t e t h e k l y d r o l y s i s o f t h e C l j C - g r o u p becomes t h e p r e - dorriin;nt r e a c t i o n .

The a d d j t i o n o f s o l v e n t s of low d i e l e c - t r i c c o n s t a n t ( s u c h as d i o x a n ) i n c r e a s e s t h e r a t e o f a c t i v i t y t o w a r d s t h e d e g r a d a t i o n o f c h l o r z l h y d r c t e t o v a r y f o r a r a n g e o f h y d r c l x i d e s 6s f o l l o w s : -

Lj 6 Na < I( 4 C s < Ca < Sr> < Ba 4 T 1

T u c h e l e t a1 .278 ,239 ,24 f i have a l s o e x - amined t h e k i n e t i c s cf t k e d e g r a d a t i o n o f c h l o r a l h y Z r a t e i n s o l u t i o n s c o n t a j n i r . g sodium h y d r c j x i d e , b o r a x , d i s o d i u m h y d r o g e n phospl-,ate and sod ium p h e n o b a r b i t a l . I n a d d i t i o n , these a u t h o r s ex- a r l i ned t h e s t , a b i l i z i n g e f f e c t o f c e r t a i n c o l l o j d s (gum a r c b i c , a g a r - a g a r , a n d g e l a t i n ) or] m i x e d s o l u t i c i n s o f c h l o r a l h y d r a t e and s o d i u n pheno- b a r b i t a l .

JOHN E. FAIRBROTHER

5 . 2 6 R a d i o l y s i s

Andrews and S h o r e l O 7 r e p o r t e d t h a t

- ~ -

a q u e o u s s o l u t i o n s o f c h l o r a l h y d r * a t e were decom- p o s e d on i r r a d i a t i o n w i t h x - r a y s ( 5 0 - 2 0 0 k v . ) t o fo rm h y d r o c h l o r i c a c i d and a smaller amourlt o f a weak1 i o n i s e d a c i d . Many o t h e r a u t h o r ~ ~ 5 , ~ O 3 t o 106,188 t o 1 1 0 , 2 4 1 , 2 4 2 h a v e examined t h e i r r a d i - a t i o n of c h l o r a l h y d r a t e s o l u t i o n s w i t h x - r a y s , gamma-rays or b e t a - r a y s . I n a l l c a s e s it i s SUE- g e s t e d t h a t t h e c h l o r a l h y d r a t e decomposes t o h y d r o c h l o r i c a c i d v i a a c h a i n r e a c t i o n .

5 . 2 7 A c t i o n o f M i c r o o r g a n i s m s --

The degrada t j o h l o r a l h y d r a t e i n s o i 1 s h a s b e e n e x am i n e d 2Q3y’4‘ and t h e r e s u l t s suggest t h a t m i c r o o r g a n i s m s p a r t i c j p a t e i n t h e d e g r a d a t i o n i n c o n j u n c t i o n w i t h c h e m l c a l a n d p h o t o c h e m i c a l p i - o c e s s e s .

5 . 2 8 Incompa t i b i l i t i e s -- ___I-

C h l o r a l h y d r z t e has b e e n shown t o b e c h e m i c a l 1 y i n c o m p a t i b l e w i t h many c o n p o u n d s . It i s p r o b a b l e t h a t it i s i n c o r n p a t i - b l e w i t h a l l s u b - s t a n c e s fo rmi r ig c o m p l e x e s w i t h j.t or w i t h c h l o r a l ( see S e c t i o n 3 . 1 , T a b l e s 1 a n d 2 ) , w i t h most bases ( see S e c t i o n 5 . 2 5 ) and w i t h many compounds b e a r i n g h y d r o x y l g r o u p s ,

The f o l l o w i n g t a b 1 e l i s t s s u b s t z n c e s known t o b e i n c o m p a t i b l e w i t h c h l o r a l h y d r a t e ( o t h e r t h a n known complex f o r m e r s ) .

114

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S u b s t a n c e s Show< ng C h e m i c a l I n c o m p a t i b i l i t y w i t h C h l o r a l x d r a t e

R e f e r e n c e S u b s t a n c e R e f e r e n c e - _-- Sub s t a n c e I__-- -

C a u s t i c A l k a l i e s 2 4 5 , 2 3 7 , 9 8 L a c t o s e 87 A l k a l i n e E a r t h s 245 ,246 G l u c o s e 1 7 6 Alkali C a r b o n a t e 2 4 5 , 2 4 6 P e r o x i d e s 2 2 9 , 2 3 0 S o l u b l e Barb i t u r a t e s 2 4 5 , 2 3 9 , 2 4 0 Met21 Al .ky l s234 ,249 B o r k x D j. s o d i uni Hydrogen P h o s p h a t e

P i p e r i d i n e Q u i n i n e S a l t s L l iu re t i n P h e n o l Mannit 01

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* S u b s t a n c e s c a u s i n g d e g r a d a t i o n o f t h e c h l o r a l h y d r F t e m o i e t y o f a c h l o r a l h y d r a t e complex .

5 . 2 9 I n t e r a c t i o n w i t h S t a r c h

C h l o r a l h y d r a t e may b e r e a c t e d w i t h s t a r c h t o f o r m a p r o d u c t s o l u b i e i n c o l d water25] o r i c l e a r a n t i s e p t i c a d h e s i v e 2 5 0 c o a c e r v a t e 2 5 ? , 2 5 3 .

5 . 3 S t . ab j l i z a t i o n o f C h l o r a l H y d r a t e

C h l o r a l h y d r a t e may b e p r c i t e c t e d from a u t o x i -

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I15

JOHN E. FAIRBROTHER

d a t i o n a n d p o l y m e r i s a t i o n by t h e i n c o r p o r a t i o n o f small q u ; n t i t i e s o a n t i o x i d a n t s s u c h as a n i l i n e or d i p h e n y l a m i n e 2 2 6 . F o l l o w i n g s imi la r l i n e s , c h l o r a l may be s t a b i l i z e d b y t h e a d d i t i o n o f 0 . 2 to 1% forniamide or d j m e t h y l f 0 r r n a m i d e ~ 5 ~ . l a r l y 0 . 1 % t e t r a - a l k y l t h i u r a n l d i s u l f ' i d e or 0.1% t o 0 . 2 % a z o b i s i s o b u t y r o n i t r i l e has b e e n u s e d 2 5 5 . C h l o r a l h a s a l s o b e e n s t a b j l i z e d i n t h e p r e s e n c e o f t r a n s i t i o n metal p o l y m e r i z a t i o n i n i t i a t o r s s u c h as F e , Sb or N i by t h e a d d i t i o n o f (3.05 t o 0.5% E ( o r 6 ) c a p r o l a c t a m 3 0 5 .

6 . Chemica l P r o p e r t i e s

6 . 1 I d e n t i t y Tests --

S i m i -

C h l o r a l h y d r a t e may be i d e n t i f i e d by i t s m e l t i n g p o i n t a n d i t s a b i l i t y t o y i e l d c h l o r o - fo rm wh n r e a c t e d w i t h sod ium h y d r o x i d e s o l u t i o n 1 5 , 9 9 , 1 5 7 .

It may b e i d e n t i f i e d by measurement o f p h y s i c a l p a r a m e t e r s , s u c h a s i n f r a r e d s p e c t r u m 1 2 , l a s e r Ranian s p e c t r u m 2 5 7 , or G . L . C . r e t e n t i o n ti.me 1 9 5 , 1 9 9 .

C h l o r a l h y d r a t e g i v e s r a t h e r u n s a t a s - f a c t o r y c o n v e n t i o n a l a l d e h y d i c d e r i ~ a t i v e s ~ 5 ~ , s u c h as oxime (m.p. 56OC), s e m i c a r b a z o n e (m.p. 90°C ( d ) ) , a n d 2,4-dinit~~ophenylhydrazone (m.p . 1 3 l o C ) , b u t f o r m s many complexes t h a t Y2ave c l e a r l y d e f i n e d m e l t i n g p o i n t s ( see S e c t i o n 3 . 1 ) .

C h l o r a l h y d r a t e g i v e s a s t r > o n g F u j iwara c o l o r r e a c t i o n ( s ee S e c t i o n 6.22), which may b e used as a n i d e n t i t y t e ~ t ~ 9 9 , ~ 5 9 . S i m i l a r l y , i t

e s p o s i t i v e color t e s t s w i t h t h e d i z o - r e a g e n t "', t h e i r , d o l e r e a g e n t f o r a l d e h y d e s 2 % 1 , and t h e

p o s i t i v e t e s t w i t h S c h i f f ' s r e a g e n t a n d some o t h e r a l d e h y d e r e a g e n t z 2 6 3 . d i s t i n g u i s h c h l o r a l h y d r a t e f r o m o t h e r t r i c h l o r o - compounds n o t p o s s e s s i n g a c a r b o n y l g r o u p i n -

o l v e s r e a c t i o n w i t h r e s o r c i n o l i n d i l u t e a l k a l i .

r e a g e n t . However, i t d o e s n o t g i v e a

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8,275.

116

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O t h e r c o l o r t e s t s employ as r e a g e n t s c o d e i n e - s u l f u r i c acid310, t h i o b a r b i t u r i c a c i d 3 I 1 a n d 1 -pl-,eny 1- 3-me t hy 1 - 5 - p y r a zo l o n e 3 l 1 . s p o t t e s t s w i t h s i t i v i t y o f l u g or l e s s h a v e b e e n d e s c r i b e d 268,?&?.

Se Vera 1

6 . 2 Methods o f A n a l y s i s --

6 . 2 1 V o l u m e t r i c / T i t r i m e t r i c P r o c e d u r e s

Crude c h l o r a l , o b t a i n e d b y t h e l i q u i d - p h a s e c h l o r i n a t i o n o f e t h a n o l , h a s b e e n a s s a y e d 1 2 6 b y a p r o c e d u r e i n v c l v i n g t h e v o l u m e t r i c m e a s u r e - ment o f t h e c h l o r o f o r m formed a f t e r h y d r o l y s i s w i t h s t r o n g K O H s o l u t i o n .

C h l o r a l h y d r a t e i s e a s i l y decomposed by a l k a l i i n t c c h l o r o f o r m a n d a l k a l i f o r m a t e . T h i s r e a c t i o n i s used s t o i c h i o m e t i * i c a l l y t o d e t e r r r i n e c h l o r a l h y d r a t e p u r i t y b y m e a s u r i n g t h e amount o f a n e x c e s s o f 1N sod ium h y d r c x i d e consumed in t h e r e a c t i o n ( t i t r a t i o n w i t h 1N s u l f u r i c a c i d t o p h e n 0 l ~ h t h a l ~ i n ) ~ 5 ~ 9 9 ~ ~ ~ 7 ~ ~ ~ 8 ~ ~ 5 ~ t o 166. A l t e r - n a t i v e l y , t h e f o r m a t e g e n e r z - t e d i n t h e r e a c t i o n may be d e t e r m i n e d b y r e a c t i o n w i t h a n e x c e s s o f i o d i n e (G.lN), f ' o l lowed b b a c k t i t r . a t i o n w i t h 0.1N s o d i u m - t n i o s u l f a t e l 2 ~ (or h y d r a z i n e ) l g 5 o r , a l t e r n a t i v e l y , by r e a c t i o n w i t h 0.1N - p o t a s s i u m p e r m a n g a n a t e and p o t a s s i u m i o d i d e f o l l o w e d b y t i t r a t i o n o f t h e l i b e r a t e d L o d i n e i 3 O ~ l 3 ~ . t h e l a t t e r p r o c e d u r e , sod ium c a r b o n a t e i s u s e d i n p l a c e o f sod ium h y d r o x i d e b e c a u s e i t i s less l i k e l y t o h y d r o l y z e t h e c h l o r o f o r m p r o d u c e d .

I n

More v i g o r o u s h y d r o l y s i s of c h l o r a l h y d r a t e w i l l c o n v e r t t h e c h l o r o f o r m ( p r o d u c e d i n i - t i a l l y ) i n t o i o n i c c h l o r i d e , wh ich c a n t h e n b e d e t e r m i n e d by V o l h a r d t t i t r a t i o n . P r o c e d u r e s for t h i s h y d r o l y s i s h a v e b e e n d e s c r i b e d t h a t use ammoniuni p e r s u l f a t e 1 3 2 o r t h e r e f l u x o f c h l o r a l h y d r a t e w i t h a l u m i n i u m powder and a q u e o u s a c e t i c aci .dl 33.

The o x i d a t i o n o f c h l o r a l h y d r a t e (to t r i c h l o r c a c e t ic a c i . d ) 1 6 7 b y a c i d - d i c h r o m a t e l - 3 4 ,

117

JOHN E. F A I R B R O T H E R

f o l l o w e d by b a c k t i t r a t i o n o f t h e e x c e s s d i c h r o - mate b J i t h a s t a b i l i z e d M o h r ' s s a l t s o l u t i o n ( t o p h e n y l a n t h r a n i l i c a c i d ) , has b e e n u s e d as a quan- t i t a t i v e p r o c e d u r e l 3 4 .

T i t r a t i o n o f c h l o r a l h y d r a z o n e w i t h s o ium m e t a v a n a d a t e s o l u t i o n has b e e n d e s c r i b e d 1 3 2 .

O t h e r t i t r i m e t r i c methods i n c l u d e t h e p o t e n t i o m e t r i c l 4 8 t i t r a t i o n o f c h l o r a l h y d r a t e and t h e a c i d i m e t r i c l 4 9 d e t e r m i n a t i o n o f c h l o r a l , f o l l o w i n g i t s o x i d a t i o n w i t h s i l v e r o x i d e .

6 . 2 2 S p e c t r o p h o t o m e t r y

The f o r m a t i o n o f a n i n t e n s e r e d c o l o r b y t h e a c t i o n of p y r i d i n e on c h l o r a l h y d r a t e i n t h e p r e s e n c e o f a l k a l i was f i r s t d e s c r i b e d by F u j i w a r a l 3 9 . The r e a c t i o n i s n o t s p e c i f i c , simi- l a r c o l o r s b e i n g p r o d u c e d by o t h e r o r g a n i c t r i - halogeno-compounds ( c h l o r o f o r m , t r i c h l o r o a c e t i c a c i d e t c . ) .

T h e c o l o r p r o d u c e d i s v e r y s e n s i t i v e t o t h e r e a c t i o n c o n d i t i o n s u s e d , which h a v e t o b e c r i t i c a l l y d e f i n e d f o r t h e co!.or i n t e n s i t y t o be u s e d as a q u a n t i t a t i v e parameter .

The r e a c t i o n h a s found much u s e i n t h e d e t e r m i n a t i o n o f c h l o r a l h y d r a t e i.n b i o o g i c a l t i s s u e s a n d f l u i d s , and a r e c e n t p a p e r l to des - c r i b e s t h e q u a n t i t a t i v e d e t e r m i n a t i o n o f c h l o r a l h y d r a t e i n t h e r a n g e 6 t o 60pg.

T h e c o n d i t i o n s o f r e a c t i o n have b e e n s t u d i e d e x t e n s i v e l y f o r t h e d e t e r m i n a t i o n o f c h l o r o f o r m and c a r b o n t e t r a c h l o r i d e , and t h e p r e s e n c e o f a c e t o n e i n t h e r e a c t i o n m i x t u r e was found t o improve t h e colorl41.

A number of o t h e r m o d i f i c a t i o s o f t h e

F r i e d m a n and Cooper147 found t h a t t h e

F u j iwara p r c c e d u r e have b e e n d e s c r i b e d l C 2 t o 146.

118

CHLORAL HYDRATE

c h l o r a l h y d r a t e r e a c t i o n p r o d u c t a l s o a b s o r b s a t 370mu, w i t h a n i n t e n s i t y t h r e e t o f o u r t imes t h a t o f t h e a b s o r p t i o n a t 540mp. T h i s a s p e c t , and t h e u s e o f t h e r e a c t i o n t o d i f f e r e n t i a t e c h l o r a l hy- d r a t e f r o m t r i c h l o r o e t h a n o l , wh ich g i v e s a p r o d - uc t a b s o r b i 4 4 0 m u , h a v e b e e n examined t h o r o u g h l y 1!8,8h7.

F e i g l l 5 O n o t e d t h a t t h e c o l o r o f t h e F u j i w a r a r e a c t i o n p r o d u c t i s d i s c h a r g e d by a c e t i c a c i d , and t h a t t h e a d d i t i o n o f b e n z i d i n e t h e n c a u s e s t h e f o r m a t i o n o f a v i o l e t c o l o r . H e s u g - ges t s t h a t t h e p r o d u c t s o f t h e F u j i w a r a r e a c t i o n a r e S c h i f f bases o f g l u t a c o n i c a l d e h y d e , fo rmed b y r i n g o p e n i n g o f t h e p y r i d i n i u m a d d u c t s fo rmed b e t w e e n p y r i d i n e a n d t h e p o l y h a l o g e n a t e d com- p o u n d s . H y d r o l y s i s o f t h e o r i g i n a l S c h i f f bases a n d t r e a t m e n t w i t h b e n z i d i n e y i e l d s t h e same der - i v a t i v e i r r e s p e c t i v e o f t h e n a t u r e o f t h e p o l y - h a l o g e n a t e d compound. T h i s m o d i f i c a t i o n o f t h e F u j i w a r a r e a c t i o n h a s b e e n made q u a n t i t a t i v e 1 5 1 and a l t h o u g h i t i s u n s p e c i f i c for c h l o r a l hy- d r a t e i t g i v e s a f i v e t o e i g h t - f o l d i n c r e a s e i n s e n s i t i v i t y o v e r t h a t o f F r i e d m a n a n d Cooper147 a n d i s less s u s c e p t i b l e t o i n t e r f e r e n c e t h a n u s i n g t h e p e a k a t 370mp.

S t e h w e i n a n d KUhmstedt152 d e s c r i b e a more s p e c i f i c c o l o r r e a c t i o n f o r c h l o r a l h y d r a t e wh ich as a q u a n t i t a t i v e p r o c e d u r e h a s a s e n s i t - i v i t y l i m i t o f a b o u t 5 0 p g / m l o f c h l o r a l h y d r a t e . I n t h i s r e a c t i o n c h l o r a l h y d r a t e is q u a n t i t a t i v e u c o n v e r t e d t o c h l o r a l oxime w i t h h y d r o x y l a m i n e h y d r o c h l o r i d e . The c h l o r a l oxime i s t h e n c o n - d e n s e d w i t h 2 , 6 - d i a m i n o p y r i d i n e i n N h y d r o c h l o r i c a c i d t o g i v e a c h e r r y - r e d p y r i - i s a t i n d y e ( A max. 530mp). A r c h e r a n d H a u g a s l 7 7 d e v e l o p e d a c o l o r - i m e t r i c p r o c e d u r e for t h e s p e c i f i c d e t e r m i n a t i o n o f c h l o r a l h y d r a t e ( u p t o 150pg /10ml s a m p l e s o l - u t i o n ) . T h i s p r o c e d u r e u s e s t h e c o l o r ( 6 0 5 m u ) p r o d u c e d by c h l o r a l h y d r a t e o n r e a c t i o n w i t h q u i n - a l d i n e e t h i o d i d e i n a l k a l i n e ( e t h a n o l a m i n e ) i s o - p r o p a n o l .

JOHN E. FAIRBROTHER

The s p e c i f i c i t y o f t h e p r o c e d u r e has b e e n w e l l e x a m i n e d l 7 7 and a w i d e r a n g e o f compounds h a v e b e n s c r e e n e d f o r i n t e r f e r e n c e i n t h e r e a c t i o n 1.g 3 .

Only c h l o r a l / c h l o r a l h y d r a t e hemi- a c e t a l compounds a n d m o l e c u l a r c o m p l e x e s h a v e b e e n f o u n d t o i r l t e r f e r e . The p r o c e d u r e o f f e r s good a c c u r a c y a n d p r e s i d e r a b l e a p p l i c a t i o n as a s t a b i l i t y assay p r o c e d u r e f o r c h l o r s l h y - d r a t e and i t s c o m p l e x e s .

O t h e r col.0 i m e t r i ass y p r o c e d u r e s have b e e n d e ~ c r i b e d ~ ~ 3 to 18e,298.

6 . 2 3 Ion -Exchange and Column Chrona- - - - - - - ~ t o g r a p h y

C h l o r a l h y d r a t e i s n o t a b s o r b e d o n i o n exchange r e s i n s by iLn i o n e x c h a n g e mechanism b u t i t kas b e e n f o u n d t o b e a b s o r b e d o n a c a t i o n - exchange e s j . n (KU-1) b y a p r o c e s s o f m c i l e c u l a r s o r p t i o n 15 9 .

The n o n a b s o r p t i o n o f c h l o r a l h y d r a t e o n a n i o n e x c h a n g e r s has b e e n u s e d t o d e v e l o p a n assay p r o c e d u r e f o r i t i n t h e p r e s e n c e o f a c e t i c , m o n o c h l o r o a c e t i c , a n d t r ' c h l o a c e t i c a c i d s , a n d sodium t r i c h l o r o a c e t a t e 189,28q.

Column c h r o m a t o g r a p h y has a l s o b e e n used t o d e t e r m i n e t h e p u r i t y cf chloral190.

6.24~aper and T h i n - l a y e r Chromatoqraphy

P a p e r c h r o m a t o g r a p h y has b e e n u s e d 2 y 4 t o d e t e r m i n e t r i c h l o r o a c e t i c a c i d i n c h l o r a l h y d r a t e .

T h i n - l a y e r Chromatography has b e e n u s e d t o s e p a r a t e c h l o r a l h y d r a t e f rom a m y l c h l o r i d e a n d d i c h l o r o e t h a n e , f r o m a d m i x t u r e w i t h monoch lo ro - a c e t i c d arid the a c e t a t e e s t e r o f monoch lo ro - e t hano 1. "' a n d f r o m f c 1 r m a l d e h y d e 3 ~ ~ .

120

CHLORAL HYDRATE

Siniple s o l v e n t - s c r e e n i n g s t u d i e s h a v e

I n t h i s s tudy19* ,

b e e n c a r r i e d out on m i c r o c h r o m a t o p l a t e s ( K j e s e l g e l GF254 l ayers 2 5 0 ~ t h i c k ) w j t h t h e r e s u l t s r e - p o r t e d i n T a b l e s 1 and 2ls2, t h e s p o t s o f chloral h y d r a t e were l o c a t e d b y s p r a y i n g w i t h a r e a g e n t ( r e s o r c i n o l , 5 g , p o t a s s i u m h y d r o x i d e , 5g), i n a b s o l u t e e t h a n o l , 50m1, f o l l o w e d by g e n t l e warming.

C h l o r a l h y d r a t e a p p e a r s as a pink s p o t on a w h i t e b a c k g r o u n d ( c f . r e f . 1 0 ) .

TABLE 1 ~-

Approx ima te - R f -. v a l u e s for ch! oral h y d r a t e --

S o l v e n t Sys t em R f V a l u e A p p e a r a n c e o f -- - S p o t --- --

C y c l o h e > a n e Carbon t et rt c h l @ r i d e Be n z e n e D i c h l o r o m e t h a n e C h l o r o f orni Ace tone E t h a n o l (95% ) n-Fropano! D i e t h y l e t h e r E t h y l a c e t a t e bIethyl e t h y l k e t o n e Cioxan n-But 6 no1

9.0 0.0 0.1 0.1 0. I, 0 . 9 0 . 9 0 . 9 1 . 0 1 . 0

1.0 1 .0 1.0

Compact C ompac t Compa c t S1. s p r e a d Conipac t S1. s p r e a d C ompac t S1. sp i i ead Coii~pac t Compact

Compact C omp a c t8 S1. s p r e a d

121


TABLE 2

= r o x i m a t e Rf Values f o r C h l o r a l H y d r a t e i r l Mu1.t i componen t S o l v e n t S y s t e m s

S o l v e n t Sys t em R a t i o R f Va lue A p p e a r a n c e

-. ---

o f S p o t --

B e n z e n e / M e t h a n o l / (9O:lO:l) 0.4 Conpac t Ammonia ( 0 . 8 8 0 ) B e n z e n e / E t h a n o l / ( 4 8 : 1 6 . 5 : 2 . 5 ) 0 . 8 Compact Ammonia ( G . 8 8 0 ) Methanol/A.mmonia (100: 1 . 5 ) 0 . 9 Conpact ( 0 . 8 8 0 )

The v o l a t i l i t y o f c h l o r a l c o n s i d e r a b l y r e d u c e s t h e u s e f u l n p s s o f q u a n t i t z t i v e T . L . C .

6 . 2 5 Vapor -Phase Chromatogr sphy -

E a r l y w o r k e r s 193y194 c o n v e r t e d t h e

( v , P . C . ,GXm c h l o r a l h y d r a t e t o c h l o r o f o r m by r e a c t i o n w i t h a l k a l i a n d t h e n s u b j e c t e d t h e c h l o r o f o r m t o quan- t i t a t i v e G . L . C .

The d i r e c t i r l j e c t i o n o f c h l o r a l h y d r h t e s o l u t i o n s h a s b e e n d e s c r i b e d more r e c e n t l y b y a number o f a u t h o r s , and a sumriary o f t h e i r s y s - tems i s g i v e n i n T a b l e 3 ( s ee a l s o r e f e r e n c e s 200 and 201 ) ,

Methods o f q u a n t i t a t i o n h a v e r a n g e d f rom t h e u s e o f s t a n d a r d p l o t s a n d d l r e c t c o m p a r i s o n w i t h a s t a n d a r d , t o t h e u9e8of i n t e r n a l s t a n d a r d s , s u c h as c h l o r o b u t a n o l l 9 7 ’ and c h l o r o f o r m ] 9 7 .

The s e n s i t i v i t y o b t a i n e d b y u s e o f a f l a m e - i o n i s a t i c n d e t e c t o r i s o n l y s l i g h t l y g r e a t e r t h a n t h a t c b t a i n e d w i t h t h e m o d i f i e d F u j i w a r c p r o c e d u r e d e s c r i b e d by F r i e d m a n and ~ o o p e r l 4 7 .

122

TABLE 3 - _- -

G.L. .C. (V.P.C. ) Determhation c j f Ci.ilo??al Hydrate - ---- ___

C o l m Colunln St at i onmy Columri Support Phase Temperat urt

(OC) _ _ - ~ -

Chron R 20% Apiezon L 100 60/80 (re&,) Firebrick 20Z Cmbowax 20M 130 6O/eO Chromosorb W 10% Apiezon L F'ropamnied 601 80 1.5/miri f r o m - 60 t o 90

w 2 8 h i n from

Chromosort id 2Cs Cmbowax 20Y 100 60/8G Chi-omosorb W 20% Carbowax 20V 125 GO/FO Chromosorb !A: 15% FFAF 10 5 60/80 Glass Feacis 0.6% Apiezon L 75

h)

90 t o 2-60

Retention Detect,or Tjme ( f i n ) System

11-12 Kathero-

7-8 Electron meter

Capt WE' - F.I.D.

c a . 1 F.T.D.

- Electron Capture

ca. 1 Electron Capture

23ml a t a Electron rh t e of Capture 37ml/mir i .

Type of Det emin -

a t i o n

Forensic

Forensic

Metzbol i c

-

Metabolic

Metabolic

Forensic

Metabolic

Reference

195

195 0 I r O n > r

196

I < 0 n >

19; ;;I

197

198

199

TABLE 3 (cont 'd)

Columu, Colunm Stationary Column Retention Detector Type of Reference Support Phase Temper at w e Time System Deteminat icn

(OC 1 (min )

Column Pak T 5% Silicone 40/60 Fluid 550 80 5.5 - 6

Gas-Chrorr! Q 3%JXR - N

P Poropak Q

50 ca. 1

160 21

Chrorxosorb 20% Dow-Corning 60 - 6O/PO Sil icone 200

Kathero- meter

Electron Capt w e

Electrcn Capture

Ka thero- meter

Puri ty 1 2 b I 2

Vapor rn

n

R P

Pressure 8 9

St a b i l i t y 91 n A s say for 2

m

I rn a

Deg-adat ion Products

Study cf 7 Eissociat ion i n Vzpor Phase

CHLORAL HYDRATE

However, t h e u s e o f a n e l e c t r o n - c a p t u r e d e t e c t o r g r e a t l y i n c r e a s e s the s e n s i t i v i t y , but l i n e a r r e s p o n s e o b t a i n e d i s o n l y o v e r a snlall r a n g e . J a i n e t a l . l g 8 r p p o r t e d a w o r k i n g r a n g e o f 0 . 6 t o 2.5 x 10-9 c h l o r a l h y d r a t e ( i . e . van- l i n e a r tibove 1 . 5 x m o l e s ) . Salmon and Heyes89 o Unicarr: N i k 5 d e t e c t o r , s a t u r a t i o n of t h e d e t e c t o r b e i n g o b s e r v e d a t a b o u t moles wcirki ng range b e t w e e n 10-’3 and a b o u t 10-

_ _

a i n e d s i m i l a r r e s u l t s u s i n g a Pye-

givinf2amo2 es.

G . L . C . p r c c e d u r e s hhve a l s o b e e n d e v e l - oped f o r t h e d e t e r m i n a t i o n o f i m u r i t i e s i n c h l o r a l h y d r a t e 1 ~ , 1 5 , 9 1 ~ 1 ~ 7 , 1 9 6 , P 9 7 .

R e a c t i o n of c h l o r a l h y d r a t e h a s b e e n r e p o r t e d w i t h some of t h e no re common r e a g e r i t s u s e d in t h e e r e p a r a t i o n o f d e r i v a t i v e s for C.L.C. DiazomethaneL,02 g i v e s a n u n s t a b l e i n t e r m e d i a t e , C l ~ C C H ( O H ) C H = N ~ , at, < O O C ( i n E t , 2 O ) , wh ich y i e l d s 1 , 1 , 1 , 5 , 5 , 5 - h e x a c h l o r o - 2 , 3 - e p o x y - 4 - h y d r o x ~ p e ~ t a n e O F 1 , 1 , l - t r j c h l o r c - 2 , 3 - e p o x y - p r o p a n e .

C o n d e n s a t i t n o f c h l o r 6 l h y d r z t e 2 0 3 w i t h Me2SiC12 i n p y r i d i n e / c h l o r o f o r m y i e l d s a c o l o r l e s s v i s c o u s o i l .

( C H3 ) 2 -Si- 0 -CE - OH I

c c 1 3 I C:

T1-e r e a c t i o n b e t w e e n c k l l o r o s j l a n e s and c h l o r a l c o m p l e x e s c o n t a i n i n g ni t r c g e n he . g . d i - c h l o r a l - u r e a ) h a s a l s o bi.en E xarnincd2O .

6 . 2 6 R e f r a c t o n e t r y and o t h e r p h y s i c a l __ _____-__ ~ _ _ _ _ _ - _ p a r a m e t e r s

Ne thods h a v e beer & s c r i b e d for t h e qum t i t 2 t i v e de t errninat i o n o f ct-t l o r a l h y d y a t e u s i n g r e f r a c t o m e t r 1 2 0 5 anc-i sur-face t e n s i o n 1 5 5 or in(-asurenient o f s p e c i f i c g r a v i t y;O6,207.

6 . 2 7 P o l a r o g r a p h y -

T h e p o l a r o g r a p h i c r e d u c t i o n o f c h l o r a l .


h y d r a t e was f i r s t r e o t e d f o r s o l u t i o n s i n 0.1N KC1 by Neiman e t a l . so$, who q u o t e d a v a l u e o f EA for c h l o r a l h y d r a t e o f - 0 . 8 ~ . and h i s co-workers r e p o r t e d a s i n g l e r e d u c t i o n wave ( E J a b o u t - 1 . 6 ~ ) f o r c h l o r a l h y d r a t e i n te t ramethylammonium b u f f e r , which t h e y a s c r i b e d t o a d i f f u s i o n - c o n t r o l l e d p r o c e s s invol .v ing r e d u c t i o n o f t h e h y d r a t e d m o l e c u l e t o d i c h l o r o - a c e t a l d e h y d e , which was c o n s i d e r e d t o b e non- r e d u c i b l e .

E l v i n g and Bennett211,212, a f t e r a v e r y t h o r o u g h e x a m i n a t i o n o f t h e p o l a r o g r a p h i c r e - d u c t i o n o f c h l o r a l h y d r a t e i n a s e l e c t i o n o f ammonia, p h o s p h a t e and b o r a t e b u f f e r s , d i s p u t e d F e d e r l i n f s f i n d i n g s . They r e p o r t e d t h a t , i n a l l c a s e s , t h e y had f o u n d d i c h l o r o a c e t a l d e h y d e t o g i v e two k i n e t i c - c o n t r o l l e d waves ( - 1 . 0 ~ and - 1 . 7 ~ ) . They a d v a n c e d t h e t h e o r y t h a t t h e c h l c r a l h y d r a t e wave (EJ a b o u t - 1 . 4 ~ a g a i n s t S.C.F. ) r e s u l t s f rom t h e s y n t h e s i s of d i f f u s i o n and k i n e t i c r e d u c t i o n p r o c e s s e s . T h e i r t h e o r y i s s u p p o r t e d by p o l a r o g r a p h i c and c o u l o m e t r i c da ta s h o w i n g t h e r e d u c t i o n o f c h l o r a l h y d r a t e t o d i c h l o r o a c e t a l d e h y d e h y d r a t e , which t h e n de- h y d r a t e s by a k i n e t i c p r o c e s s .

F e d e r l i n 2 0 9 ~ 2 1 0

The d L c h 1 o r o a c e t a 1 d e h y d e formed r e d u c e s t o c h l o r o a c e t a l d e h y d e , t h e n t o a c e t a . l d e h y d e , a n d , f i n a l l y , t o e t h y l a l c o h o l a n d / o r 2 , 3 - d i h y d r o x y - b u t a n e .

The c o m p l e t e r e d u c t i o n s e q u e n c e t o a c e t a l d e h y d e r e s u l t s i n t h e one wave, b u t t h e r e d u c t i o n of t h e a c e t a l d e h y d e p r o d u c e s a s e c o n d wave ( E J a b o u t - 1 . 7 ~ ) t h a t i s o n l y c l e a r l y r e s o l v e d i n ammonia b u f f e r s . The v a r i a t i o n o f E t w i t h pH and w i t h t h e n a t u r e of t h e s u p p o r t i n g e l e c t r o l y t e i s summarized i n T a b l e s 4 and 5 .

126

CHLORAL HYDRATE

TABLE 4

E f f e c t o f pH on t h e polaro . r a p h i e b e h a v i o r 2 1 1 - --- o f c h l o r a l h y d r a t e --~-- a t 2 5 O C + f r o m E l v i n g a n d ___- B e n n e t t ) _-

B u f f e r pIi Cone . Head - E i n (em.) ( v ) -- _ - -__ -- ~~

( fly )

D j sodiuni p h o s p h a t e - c i t r a t e 7 . 2 0 . 2 5 40 1 . 4 2 1 . 4 Ammonium c h l o r i d e - ammonia 8 . 4 0 . 2 5 40 1 . 3 4 * 1 . 9 Amvo n i u m c h l o r i de - arlmonia 9 . 1 1 . 0 80 1 . 3 6 * 2 . 6 Borate-KC1 9 . 2 1 . 0 80 1 . 4 5 2 . 2 B o r a t e - K C 1 9 . 8 1 . 0 80 1 . 6 7 1.7

* In t h e c a s e o f ammonia b u f f e r s , a s e c o n d wave i s f o u n d a t Ei a b o u t -. 1 . 6 6 ~ .

TABLE 5

E f f e c t o f s u p p o r t i n g e l e c t r n l y t e on t h e p o l a r c - E p l i i c b e h a v i o r o f c h 1 o r ; l h y d r a t e a t 25OC ~- ( f r c m

FF1-der1i-T

S o l v e n t S u p p o r t i n g pH Cone. -E& (mFI - ) ( V )

Water L i C l (0.1M) - 1 1 . 6 3 5 (CH3)4NC1 TO.1M) - - 1 1 . 4 2 0

D i o x a n / W a t e r (C€T3)4 N t 6 . 7 1 1 . 3 4 5 (50:50!

( C H 3 ) 4 N O H 13.0 1 1.520

Use o f t h e p o l a r o g r a p h i c r e d u c t i o n a s a q u a n t i t a t i v e asstiy p ~ . o c e d u r e i s c a p a b l e o f s e l - e c t i v e l y d e t e r m i n i n g c h l o r a l h y d r a t e i n t h p p r e s - e n c e o f dichloro- and c h l o r o a c e t a l d e h y d e 2 1 2 .

E 1 e c t r* o 1 y t e - -- - -~ -

L i + 9 . 6 1 1 .600

A q u a n t i t a t i v e p o l a r o g r a h c a s s a y fcr c h l o r a l h y d r a t e h a s b e e n d e s c r i b e d 3 ] ' t h a t L t i l -

I27

JOHN E. FAIRBROTHER

i z e s t h e r e d u c t i o n o f c h l o r a l oxime a t pH2 i n a H C 1 - K C 1 b u f f e r ( t w o waves a r e o b t a i n e d a t a p p r o x . EJ - 0 . 5 5 and - 1 . 2 O v . ) .

6 . 2 8 D e t e r m i n a t i o n of Water

The water c o n t e n t o f c h l o r a l h y d r a t e has b e e n s u c c e s s f u l l y d e t e r m i n e d b y t h e Karl F i s c h e r t i t r a t i o n p r o c e d u r e 2 1 5 and by r e a c t i o n w i t h c a l c i u m c a r b i d e 2 1 6 .

6 . 3 R a d i o l a b e l l e d C h l o r a l H y d r a t e

T h i n - l a y e r c h r o m a t o g r a p h y o f c h l o r a l h y d r a t e a f t e r n e u t r o n i r r a a t i o n g a v e s e v e r a l r a d i o - a c t i v e c o n p o n e n t s l g i . C h l o r a l h y d r a t e - ( l - 1 4 C ) a n d - ( 2 - 1 4 C ) have b e e n r e a r e d by c h l o r i n a t i o n o f l a b e l l e d e t h a n o 1 2 t 4 , 3 6 6 and used t o m o n i t o r t h e d e g r a d a t i o n o f c h l o r a l h y d r a t e i n s o i 1 2 4 4 .

C h l o r a l h y d r a t e - t ( 3 H - l a b e l l e d c h l o r a l h y d r a t e ) h a s b e e n p r e p a r e d 2 6 7 by t h e Wilzbach t e c h n i q u e , u s i n g 8Ci of 3 H ( 1 2 m r i . ) a n d s u b j e c t i n g t o Tesla e x c i t a t i o n for 1 5 m i n u t e s . The s p e c - i f i c a c t i v i t y o f t h e p r o d u c t was 30 - 110 mCi /g .

7 . M e t a b o l i s m

The h y p n o t i c p r o p e r t i e s of c h l o r a l h were f i r s t d e s c r i b e d by L i e b r e i c h (18 i s r a p i d l y a b s o r b e d from t h e stomach1 r e a c h e s a n e f f e c t i v e b l o d l e v e l w i t h i n 0 . 5 h r . ki man. Mackay and Cooper 282 s u g g e s t t h a t t h e CNS d e p r e s s i o n t h a t follows i n g e s t i o n i s m a i n l y , i f n o t e n t i r e l y , due t o i t s m e t a b o l i t e , t r i c h l o r o - e t h a n o l . Comparison o f t h e e f f i c a c y of c h l o r a l h y d r a t e a n d q i c h l o r o e t h a n o l s u p p o r t s t h i s s u p p o s i t i o n 2 5 ) .

T r i c h l o r o e t h a n o l i s p r o d u c e d 2 7 9 b y a re- a c t i o n c a t a l y z e d by t h e enzyme, a l c o h o l d i h y d r o - genase2869292, which t a k e s p l a c e i n t h e l i v e r , whole b l o o d , and v a r i o u s o t h e r t i s s u e s 2 8 1 . I n man, a m a j o r p r o p o r t i o n o f t h e t r i c h l o r o e t h a n o l

128

CHLORAL HYDRATE

( t r i c h l o r o e t h a n o l g l u c u r o n i d e ) , wh ich h a s b e e n c h a r a c t e r i z e d a s ' 21,21-trichloroethyl-~-~-glu~ o s i d u r o n i c a c i d 2 8 g . 5 9 0 .

A v a r i a b l e p r o p o r t i o n o f c h l o r a l h y d r a t e i s o x i d i z e d i r i t h e l i v e r a n d k i d n e y by a DPN-depen- d e n t enzyme t o g i v e a C N S - i n a c t i v e m e t a b o l i t e , t r i c h l o r o a c e t i c a c i d 2 8 7 . T h i s i s t h e main u r i n a r y m e t a b o l i t e f o u n d i n man, b u t is m i n o r to t r i c h l o r o e t h n o 1 ( f r e e p l u s c o n j u g a t e d ) i n t h e dog and rat185.

may a l s o b e c o n v e r t e d t o t r i c h l o r o a c e t i c a c i d 1 4 6 .

C h l o r a l h y d r a t e i s n o t d e t e c t e d i n t h e b l o o d or u r i n e i n man146 ,197 ,198 , b u t i s f o u n d i n small amount i n t h e b l o o d o f t h e d o g , 1 4 5 r a t , 145 a n d rnouseltO. The r a t e o f m e t a b o l i s m o f c h l o r e l hy- d r a t e i s s o r e p i d t h a t f r e e c h l o r a l h y d r a t e was n o t d e t e c t e d i n human b l o o d i n t h e p e r i o d 5 m i n . t o 6 h r . a f t e r t h e a d m i n i s t r a t i o n o f lg o f c h l o r a l h y d r a t e 2 9 4 ( s e n s i t i v i t y o f a n a l y t i c a l method 0.5~1 c h l o r a l h y d r a t e / m l b l o o d ) , However, i n t h e samc.594 i n d i v i d u a l s , t r i c h l o r o e t h a n o l was s t i l l p r e s e n t i r i t h e b l o o d ( 2 0 t o 65 mg % ) 6 h r . a f t e r d o s e .

A smal l p r o p o r t i o n o f t h e t r i c h l o r o e t h a n o l

The d e t e r m i n a t i o n p r o c e d u r e s f o r t h e l e v e l s o f t h e m e t a b o l j t e s i n b l o o d , u r i n e , and t i s s u e h a v e c e n t e r e d a r o u n d v r i o u s m o d i f i c a t i o n s o f t h e F u j l w a r a r e a c t i o n 1fio . P r o c e d u r e s h a v e b e e n

G a s - l i q u i d c h r o m a t o g r a p h y h a s a l s o b e e n u s e d f o r t h e d e t e r m i n a t , i o n o f c h l o r a l h y d r a t e t r i - c h l o r o e t h a n o l , and u r o c h l o r a l i c a c i d 1 4 6 i n b l o o d , 198 u r i n e , 1 9 7 , 1 9 9 , a n d t i s s u e h o m 0 g e n & t e ~ 9 ~ , ~ 9 5 . T r i c h l o r o a c e t S c a c i d has n o t been d e t e r m i n e d d ; r e c t l y i n t h i s manner , b u t may b e d e t e r m i n e d i r i d i r 2 e c t l y a f t e r it ,s c o n v e r s i o n t,o c h l o r o f ' n r m ~ 9 7 .

JOHN E. FAIRBROTHER

U r o c h l o r a l i c a c i d is f i r s t h y d r o l y z e d t o t r i - c h l o r o e t h a n o l by a c i d h y d r o l y s i s 1 9 7 , 1 9 8 , 2 9 1 or by i n c u b a t i o n w i t h B - g l u c u r o n i d a s e l 9 7 .

The g l y c o n e o b t a i n e d by t h e enzymic h y d r o l - y s i s of u r o c h l o r a l i c a c i d h a s b e e n i d e n t i f i e d 1 9 7 as D - g l u c u r o n i c a c i d by T . L . C . on a l a y e r o f S i l i c a Gel GF254, u s i n g t h e s o l v e n t s y s t e m n- b u t a n o l / a c e t i c a c i d / w a t e r ( 4 : 5 : 1 ) and v i s u a l i z - a t i o n by c h a r r i n g .

130

CHLORAL HYDRATE

8 . Refe rences

1. 2.

3.

4. 5.

6. 7.

8.

9. 10.

11. 12. 13.

14.

15 * 16. 17. 18. 19.

20.

21.

22. 23. 24. 25.

26.

27

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Anon, Squibb Private Cornmication Powell J. W. , Private Communicat ion Allen H . C . , J. Am. Chem. SOC. 74, 6074-6076 (1952) Allen H.C. , 5. Phys. Chern. 57,501-504 (1953) Biedenkapp D. and Weiss A . ,T. Naturforsch., A - 22,

Pietrazak J. , Poznan. Tow. Przyj . Nauk, Pr. Kom. Mat . - Przyr. 5, 143-153 (1969) Milia F.K. and Hadjoudis E.K. , J, Phys. Chern. - 74,

Kondo S. and Nitta I., X-sen 6, 53 (1950) Ogawa F., Bull. Chem. SOC. Jap., s, 610 (1963) Millard B . J . , Private Communication Reyes J . , Reyes S.A., and L h o n M.A. , Rev. Mex. F i s . 17, 263-287 (1968) Udoveso V.V. and Babak S. , USSR 18, 586-593 (1948) B e r d y z A.A. , Vasil 'eva M.G. and Lezhev N.B. , Tr. Fiz . Tekh. I n s t . , Akad. Nauk. Turkm. SSR - 8, 19-48 (1962)

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28.

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4 1 . 42.

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45 * 46. 47.

48.

49.

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Udovenko V.V., Akad. Nauk. SSSR Otdel Tekh. Nauk. I n s t . Mashinoved. Soveshch. Vyazkosti Zhidk. Kolloid. Rastvorov 2, 89-93 (1944) Zhuravlev E.F. and Bychkova M . N . , Uch. Zap. Molotov. Gos. Univ. im. A.M. Gor'k.Mat., Fiz. Khim. - 8, 15-20 (1954) Syuzyaev V . I . , Berdyev A.A. and Kolando N . I . , Izv. Akad. Nauk. Turkm. SSR (1957) No. 6 , 81-84 Syuzyaev V . I . and Berdyev A . A . , Izv. Akad. Nauk. Turkm. SSR (1957) No. 6, 1-15 Zhuravlev E.F. and Sharshkova L.N., J. Gen. Chem. USSR, 39, 1897-1899 (1969) Udove& V.V. and Khomenko R . I., J . Gen. Chem. USSR, - 26, 3639-3642 (1950) Udovenko V.V. and Khomenko R . I . , J . Gen. Chem. USSR, - 27, 357-359 (1957) Patent, Ger. 234, 7 4 1 (June 7, 1910) t o Chem. Fabrik Gedeon Richter, Budapest. Patent, U.S. 1,025,889 (May 7, 1912) t o Sulzberger N Simmons D.L. and Ranz R . J . , Can. J . Pharm. Sci . - 4 , 96 (1969) Patent, U.S. 2 ,974 , 167 (March 7, 1961) t o Stevens C . R . Degerholm E . , Harrison A . , Leiderman T . and Sterner N . , Farm. Fievy. 62 (29), 601-611 (1963) Patent, Belg., 6 3 , 1 2 8 (Sept. 2, 1963) t o Aktiebolag "ferrosan" Ekeblad P . , Acta Pharm. Suec. 2, 239-254 (1965) Patent, Fr. 1,347,235 (Dee. 27; 1963) t o Soci&t& C l h i q u e de R'Yvette. P i r o l i G . and Montariini I . , B o l l . Chfm;Farm. 103 ( 5 ) , 351-357 (1964) Patent, Br i t . 874,246 (Appl. June 26, 1959) t o B r i t i s h Drug Houses Ltd. Anon, Fed. Regist . 31, 12435-12436 (1966) Strumia E., Minerva-ed. 59 ( 8 2 ) , 4348-4351 (1968) The National Formulary XIn, Am. Phamn. Assoc., Washington, (1970). p. 145 Leulier A . , J . Pharm. C h k . - 6, 18-21 (1913) (see C.A. 7 9926) Oliver5 MaEdala V . E . , Gazz. Chim. Ital. - 56, 889-896

Patent, S. African 68-01, 427 (Oct. 30,1968) t o Delmar Chemicals Ltd.

~

(1926 1

132

CHLORAL HYDRATE

51.

52.

53.

54. 55.

56.

57. 58.

59 *

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61.

62.

63.

64.

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66.

67. 68.

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-

(1956)

I33

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74

75.

76.

77.

78.

79.

80.

81.

82. 83. 84. 85. 86.

87.

88. 89.

90. 91. 92. 93. 94 *

95. 96.

97.

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The above references attempt to be comprehensive to the end of Chemical Abstracts, Volume 74.

143

CLIDIN I UM BROMIDE

Brirw C Ri(~l j , tiiicl Bcrnurtl Z. .Sctrko\v.yki

145

B. C. RUDY AND 6. Z . SENKOWSKI

INDEX

A n a l y t i c a l P r o f i l e - Clid in ium Bromide


1.1 N a m e , Formula, Molecular Weight 1 . 2 Appearance , Color , Odor


2 .1 2 .2 2 .3 2.4 2 .5 2 .6 2 .7 2.8 2 .9 2 .10 2 . 1 1 2.12

I n f r a r e d Spectrum Nuclear Magnetic Resonance Spectrum U l t r a v i o l e t Spectrum Fluorescence Spectrum Mass Spectrum O p t i c a l R o t a t i o n Mel t ing Range D i f f e r e n t i a l Scanning Calor imet ry Thermal Gravimet r ic A n a l y s i s S o l u b i l i t y X-ray C r y s t a l P r o p e r t i e s D i s s o c i a t i o n Constant

3. S y n t h e s i s

4 . S t a b i l i t y Degradat ion

5. Drug Metabol ic Products

6 . Methods of Analys is

6 . 1 Elemental Analys is 6 . 2 Phase S o l u b i l i t y A n a l y s i s 6 . 3 Thin Layer Chromatographic A n a l y s i s 6 . 4 Direct Spec t rophotometr ic Analys is 6 .5 C o l o r i m e t r i c Analys is 6 . 6 T i t r i m e t r i c Analys is

7. References

146

CLlDlNlUM BROMIDE


1.1 N a m e , Formula, Molecular Weipht C l id in ium bromide i s 3-hydroxy-1-methylquinu-

c l i d i n i u m bromide b e n z i l a t e .

Molecular Weight: 432.36

1 . 2 Appearance, C o l o r , Odor C l i d i n i u m bromide o c c u r s as a w h i t e o r n e a r l y

w h i t e , a l m o s t o d o r l e s s , c r y s t a l l i n e powder.


2 . 1 I n f r a r e d Spectrum ( I R ) The i n f r a r e d spec t rum of c l i d i n i u m bromide i s p re -

s e n t e d i n F i g u r e 1 (1). Perkin-Elmer 621 Spec t ropho tomete r i n a K B r p e l l e t c o n t a i n - i n g 1 . 7 mg of c l i d i n i u m bromide/300 mg of K B r . l i s t s t h e a s s ignmen t s f o r t h e c h a r a c t e r i s t i c bands i n t h e I R spectrum (1).

The spec t rum w a s measured w i t h a

Tab le I

Tab le I

I n f r a r e d Assignments f o r C l id in ium Bromide

Frequency (cm-') C h a r a c t e r i s t i c of

3226 OH s t r e t c h i n g v i b r a t i o n s 3059 a r o m a t i c CH s t r e t c h i n g

1726 C=O s t r e t c h of t h e ester 1595 and 1489 a r o m a t i c C=C s t r e t c h i n g

1238-1236 C-0-C s t r e t c h of t h e ester

767 and 696

v i b r a t i o n

v i b r a t i o n

l i n k a g e o u t of p l a n e bend ing of 5 ad-

j a c e n t 11's on benzenerings

147

6. C

. RU

DY

AN

D 6

. Z. S

EN

KO

WS

KI

148

CLlDlNlUM BROMIDE

2.2 Nuclear Magnetic Resonance Spectrum (NMR) The spectrum shown i n F i g u r e 2 was o b t a i n e d i n a

J e o l c o 60 MHz N M R by d i s s o l v i n g 54.3 mg of c l i d i n i u m bromide i n 0 . 5 m l o f DMSO-d6 c o n t a i n i n g t e t r a m e t h y l s i l a n e as an i n - t e r n a l r e f e r e n c e ( 2 ) . The s p e c t r a l a s s ignmen t s are g i v e n in Table I1 ( 2 ) .

Tab le I1

NMR Assignments f o r C l id in ium Bromide

CH3 b

No. of P ro tons Derived Chemical S h i f t

P ro tons from I n t e g r a t i o n ( P P d Mu1 t i p l i c i t y

a 5 b 3 C 6 d 1 e 1 f 10

1.4-2.4 Complex M u l t i p l e t 3.05 Sing 1 e t

3.1-4 .3 Complex M u l t i p l e t 5.25 Complex P l u l t i p l e t 6.86 Sharp S i n g l e t 7 . 4 5 S i n g l e t

2 . 3 U l t r a v i o l e t Spectrum (UV) #en t h e W spectrum of c l i d i n i u m bromide was

scanned from 350 t o 210 nm, two s h a r p maxima occur red a t 257 nm (E = 4 . 8 x 102) and 251 nm (E = 4 . 3 x l o 2 ) . f u r t h e r maxima were reached between 240 and 210 nm b u t t h e abso rbance was s t i l l r a p i d l y i n c r e a s i n g a t 210 nm. The spectrum shown i n F i g u r e 3 w a s o b t a i n e d from a s o l u t i o n of 50.735 mg of c l i d i n i u m bromide/100 m l of water (3) .

No

2 . 4 Fluorescence Spectrum No f l u o r e s c e n c e w a s observed f o r c l i d i n i u m bromide

i n water, methanol , 0.1N H C 1 , o r 0.1N NaOH when t h e sample w a s i r r a d i a t e d w i t h polychromatic l i g h t ( 4 ) .

I49

6. C. R

UD

Y A

ND

B. Z

. S

EN

KO

WS

KI

150

CL

lDlN

lUM

BR

OM

IDE

Fig

ure 3

Ultra

vio

let S

pectrum o

f C

lidin

ium

Brom

ide

I a .9

.8

7 E w

0 z

a

m .!

LL 0

(I) m

a 4 1

.C

.

L .I

25

0

30

0

3!

NA

NO

ME

TE

RS

151

B. C . RUDY AND €3. 2. SENKOWSKI

2.5 Mass Spectrum The mass spectrum of c l i d i n i u m bromide was ob-

t a i n e d u s i n g a CEC 21-110 mass s p e c t r o m e t e r w i t h a n i o n i z i r g energy of 70 e V . The o u t p u t from t h e mass s p e c t r o m e t e r was ana lyzed and p r e s e n t e d i n t h e form of a b a r g raph , shown i n F i g u r e 4 , by a Varian 100 MS d e d i c a t e d computer system ( 5 ) . The h i g h e s t mass observed a t m / e 331 i s a the rma l breakdown p roduc t where CH3Br was l o s t from t h e p a r e n t q u a t e r n a r y ammonium compound. The base peak a t m / e 183 i s due t o t h e diphenylhydroxymethyl f ragment . The rest of t h e peaks a r e due t o a m u l t i t u d e of t he rma l breakdown p r o d u c t s r e l a t e d t o t h e p a r e n t c l i d i n i u m bromide molecu le ( 5 ) .

2.6 O p t i c a l R o t a t i o n C l id in ium bromide e x h i b i t s no o p t i c a l a c t i v i t y .

2.7 Mel t ing Range The m e l t i n g r a n g e r e p o r t e d i n t h e NF XI11 f o r

c l i d i n i u m bromide is 240° t o 244O u s i n g t h e c lass I pro- c e d u r e ( 6 ) .

2.8 D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC) The DSC scan €or c l i d i n i u m bromide i s shown i n

F i g u r e 5. The e x t r a p o l a t e d o n s e t of t h e m e l t i n g endotherm o c c u r s a t 242.OoC when t h e t empera tu re program was 10°/min- U t e . The AHf w a s found t o b e 9.2 kca l /mole ( 7 ) .

2 .9 T h e m o g r a v i m e t r i c A n a l y s i s (TGA) The TGA performed on c l i d i n i u m bromide showed no

we igh t l o s s from ambient t o 27OoC. weight l o s s occur red from 270° t o 39OoC e q u i v a l e n t t o abou t 90% of t h e sample ( 7 ) .

A s i n g l e con t inuous

2.10 S o l u b i l i t y The s o l u b i l i t y d a t a f o r c l i d i n i u m bromide ob ta inad

a t 25OC are g i v e n i n Tab le I11 ( 8 ) .

Tab le I11

S c l u b i l i t y of C l id in ium Bromide i n D i f f e r e n t S o l v e n t s

So lven t

3A a l c o h o l benzene ch lo ro fo rm

S o l u b i l i t y (mg/ml)

9 . 3 0.06 1 . 4

152

CL

lDlN

lUM

BR

OM

IDE

153

B. C. RUDY AND B. 2. SENKOWSKI

I

F i g u r e 5

DSC Scan f o r C l i d i n i u m Bromide

I A Hf = 9.2 k cal/mole

260 250 240 230 220 210 i TEMPERATURE OC

154

CLlDlNlUM BROMIDE

95% e t h a n o l 26.4 e t h y l e t h e r <O .05 m e thano 1 102.4 pe t ro l eum e t h e r 30'-60' <0 .05 2-propanol 0.6 water 82 .7

2 . 1 1 X-ray C r y s t a l P r o p e r t i e s The x-ray powder d i f f r a c t i o n p a t t e r n o f c l i d i n i u m

bromide i s p r e s e n t e d i n Tab le I V ( 9 ) . The i n s t r u m e n t a l c o n d i t i o n s are g i v e n below.

I n s t r u m e n t a l Cond i t ions :

Gene ra l E lec t r i c Model XRD-6 Spec t rogon iomete r

Genera to r : 50 KV-12-1/2 MA Tube t a r g e t : Copper R a d i a t i o n : Cu Q = 1.542 2

0.10 D e t e c t o r s l i t M.R. S o l l e r s l i t 3 O Beam s l i t 0.0007 i n c h N i f i l t e r 4O t a k e o f f a n g l e Scan a t 0.2' 20 p e r minu te A m p l i f i e r g a i n - 1 6 c o u r s e ,

8.7 f i n e Sea led p r o p o r t i o n a l c o u n t e r

t u b e and DC v o l t a g e a t p l a t e a u

P u l s e h e i g h t s e l e c t i o n EL-5

Rate meter T . C . 4 2000 c f s f u l l scale

Goniometer : D e t e c t o r :

v o l t s ; EU - o u t

Recorder: C h a r t speed - 1 i n c h p e r 5 m i n u t e s

Samples p repa red by g r i n d i n g a t room t e m p e r a t u r e .

B. C. RUDY A N D B. 2. SENKOWSKI

Table I V

_ _ Powder D i f f r a c t i o n P a t t e r n of Cl idinium Bromide x-ray

20

8 . 6 6 9 .42

1 0 . 5 2 11 * 10 1 2 . 3 6 1 4 . 2 6 1 4 . 7 0 1 5 . 5 5 1 6 . 1 8 1 6 . 8 2 1 7 . 2 0 1 7 . 7 2 1 8 . 2 6 1 8 . 8 0 19 .18 1 9 . 5 6 1 9 . 9 4 2 1 . 1 4 21.64 21.96 22.40 22 .72 23 .13 23 .38 24 .10 24.76 25 .50 26 .35 26 .92 27 .48 2 7 . 6 1 28.25 28.27

-

*

d (8) * 1 0 . 2

9 . 3 9 8 . 4 1 7 . 9 7 7 . 1 6 6 . 2 1 6 .03 5 .70 5 .48 5.27 5 .16 5 . 0 1 4 . 8 6 4 .72 4.63 4 . 5 4 4 .45 4 . 2 0 4 . 1 1 4 .05 3.97 3 . 9 1 3 . 8 5 3 . 8 0 3 .69 3 . 6 0 3.49 3 . 3 8 3 . 3 1 3 . 2 5 3 .23 3.16 3 . 1 1

I/I ** 0-

6 1 3 40 60 4 3 45 1 5

3 7 3 1 4 1 9 1 3 48 1 6

5 32

6 1 4 25 37 44 22 35 7 0 32 35 1 7 25

100 3 3

11 5

28

2 9 . 3 2 29 .60 3 0 . 2 1 30 .82 31.38 32 .28 33 .10 3 3 . 6 5 34.15 3 4 . 9 8 35 .55 3 5 . 8 6 3 6 . 8 8 37 .20 37 .80 38 .30 39 .20 39.46 40 .05 4 0 . 7 5 41 .50 4 2 . 0 3 4 2 . 5 8 4 3 . 2 5 4 3 . 6 8 4 4 . 8 4 45 .48 4 6 . 8 0 47 .60 4 8 . 5 0 48 .90 49 .19 5 0 . 1 9

- d & *

3 . 0 5 3 . 0 2 2 . 9 6 2 . 9 0 2 . 8 5 2.77 2 . 7 1 2 . 6 6 2 .63 2 .57 2 . 5 3 2 . 5 0 2 . 4 4 2 . 4 2 2.38 2 .35 2 .30 2 . 2 8 2 .25 2 . 2 1 2 . 1 8 2 . 1 5 2 . 1 2 2 . 0 9 2 .07 2 .02 1 . 9 9 1 . 9 4 1 . 9 1 1 . 8 8 1 . 8 6 1 . 8 5 1 . 8 2

d - ( i n t e r p l a n a r d i s t a n c e ) nX ** 2 S i n 0

"Io = r e l a t i v e i n t e n s i t y

(based on h i g h e s t i n t e n s i t y of 1.00)

156

=/I ** -0-

1 3 11 11 1 7

6 1 0

6 24 23 11

4 7

11 6 3 6 9 8 6 3 7 2 4 2 8

1 5 4 3 3 11

7 4 3

CLlDlNlUM BROMIDE


shown i n F i g u r e 6. 3 -Qu inuc l id ino l i s r e a c t e d w i t h methyl b e n z i l a t e t o form t h e es te r . The es ter i s then r e a c t e d w i t h methylbromide t o form t h e q u a t e r n a r y s a l t , c l i d i n i u m bromide (10).

C l id in ium bromide i s s y n t h e s i z e d by t h e r e a c t i o n scheme

4. S t a b i l i t y Degrada t ion C l id in ium bromide i s s t a b l e i n aqueous s o l u t i o n between

pH 2 and pH 8. In 6N H C 1 t h e es te r bond g r a d u a l l y hydro- l y z e s a t room t empera tu re and i n a l k a l i n e media r a p i d decompos i t ion o c c u r s (11 ) . I n f o r m u l a t i o n s t h e d r u g h a s been found t o b e s t a b l e f o r a t l e a s t f i v e y e a r s ( 1 2 ) .

5 . Drug Metabo l i c P roduc t s C l id in ium bromide i s me tabo l i zed t o 1-methyl-3-hydroxy-

q u i n u c l i d i n i u m bromide which i s t h e major form of t h e d rug found i n t h e u r i n e o f dog and man (11). Both t h e i n t a c t d rug and t h e above mentioned m e t a b o l i t e are found i n t h e f e c e s of dog and man (11). t h e d rug is n o t me tabo l i zed by N-demethylation (11).

S t u d i e s u s i n g 14C i n d i c a t e t h a t

6. Methods of Ana lys i s

6 . 1 Elemental Ana lys i s The r e s u l t s from t h e e l e m e n t a l a n a l y s i s are

l i s t e d i n Tab le V ( 1 3 ) .

Tab le V

Elemental Ana lys i s o f C l id in ium Bromide

Element % Theory % Found

C 61.12 61.30 H 6 .06 6 . 1 3 N 3.24 3.14

6 . 2 Phase S o l u b i l i t y Ana lys i s Phase s o l u b i l i t y a n a l y s i s may b e c a r r i e d o u t

u s i n g 2-propanol as t h e s o l v e n t . An example i s shown i n F i g u r e 7 which a l s o l i s t s t h e c o n d i t i o n s under which t h e a n a l y s i s w a s c a r r i e d o u t ( 8 ) .

157

B. C. RUDY AND B. Z SENKOWSKI

I

0

I

t I

6 ti

158

F i g u r e 7

23 2.0 1 1 1 1 I l l 1 I I I 1 I

$% PHASE SOLUBILITY ANALYSIS SAMPLE CLlDlNlUM BROMIDE

0 0

gk 1.5- SOLVENT ISOPROPANOL

EOUlLlBRATlON 20 HOURS at 2 5 O C Z EXTRAPOLATED SOLUBJLiTY 077 m g / g of ISOPROPANOL 0

v)

+

SLOPE 003% ..

- '. lo-- -

o n Q I _- - B U - ., .,

z 0 0 0.5 .. z 9 I- 3

-

I I L I I I 1 - 1 - 1 1 1 1 1 1 1 , .

0 r 0 5 C H m n 0 3 - 0,


6 . 3 Thin Layer Chromatographic Ana lys i s (TLC) The f o l l o w i n g TLC p rocedure is u s e f u l f o r

s e p a r a t i n g c l i d i n i u m bromide from 3-hydroxy-1-methylquinu- c l i d i n i u m bromide and 3 - q u i n u c l i d i n y l b e n z i l a t e ( 6 ) . On two s i l i c a g e l G p l a t e s t h a t have been prewashed i n t h e deve lop ing s o l v e n t ( g i v e n below) and a c t i v a t e d 1 5 minutes a t 105OC, apply 2 mg of c l i d i n i u m bromide sample i n methanol . Develop t h e p l a t e s a t l e a s t 1 0 c m i n f r e s h deve lop ing s o l - v e n t made of acetone:methanol:water:concentrated HC1 (14:4:1:1) . c o o l and s p r a y w i t h t h e po ta s s ium i o d o p l a t i n a t e t o d e t e c t any 3 - q u i n u c l i d i n y l b e n z i l a t e (Rf 0 .8) p r e s e n t and s p r a y t h e o t h e r p l a t e w i t h Modif ied Dragendorff r e a g e n t t o d e t e c t t h e c l i d i n i u m bromide (Rf 0 .5) and any 3-hydroxy- 1-methylquinucl idinium bromide (Rf 0 . 4 ) p r e s e n t ( 6 ) .

Remove t h e p l a t e s , d r y a t 105' f o r 1 0 m i n u t e s ,

6.4 Direct Spec t ropho tomet r i c Ana lys i s Direct s p e c t r o p h o t o m e t r i c a n a l y s i s may b e c a r r i e d

o u t i n water u s i n g t h e maximum a t 257 nm. Due t o t h e ex- t remely narrow peak, however, narrow s l i t w i d t h s are re- q u i r e d and g r e a t care must be e x e r c i s e d i n l o c a t i n g t h e e x a c t maximum.

6 . 5 C o l o r i m e t r i c Ana lys i s C l id in ium bromide forms an i o n p a i r complex w i t h

thymol b l u e i n a n aqueous s o l u t i o n b u f f e r e d a t pH 8 .8 . Th i s complex can b e e x t r a c t e d i n t o ch lo ro fo rm and i t s abso rbance r e a d a t t h e maximum a t abou t 410 nm ( 1 4 ) .

6 .6 T i t r i m e t r i c Ana lys i s The p o t e n t i o m e t r i c t i t r a t i o n w i t h p e r c h l o r i c a c i d

i n d ioxane i s t h e method of c h o i c e t o a s s a y c l i d i n i u m bromide ( 6 ) . The sample i s d i s s o l v e d i n g l a c i a l ace t ic a c i d , an e x c e s s of mercu r i c acetate i s added, and t h e t i t r a t i o n w i t h 0.1N HC104 i n d ioxane i s c a r r i e d o u t . e q u i v a l e n c e p o i n t is de te rmined p o t e n t i o m e t r i c a l l y . Each m l of 0.1N HClO4 is e q u i v a l e n t t o 43.24 mg of c l i d i n i u m bromide.

The

160

CLI DI N I UM BROMIDE

7 . References

1.

2 .

3 .

4 .

5.

6 . 7 .

8 .

9 .

10.

11.

1 2 *

1 3 .

1 4 ,

Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal Communication. Johnson, J . H., Hoffmann-La Roche Inc,, Personal Communication. Rubia, L. B., Hoffmann-La Roche Inc., Personal Communication. Boatman, J., Hoffmann-La Roche Inc., Personal Communication. Benz, W., Hoffmann-La Roche Inc., Personal Communication. National Formulary XIII, pp. 172 and 815 (1970) . Moros, S . , Hoffmann-La Roche Inc., Personal Communication. MacMullan, E., Hoffmann-La Roche Inc., Personal Communication. Hagel, R. B., Hoffmann-La Roche Inc., Personal Communication. Sternbach, L. H., Hoffmann-La Roche Inc., US Patent No. 2 ,648 ,667 (1953) . Schwartz, M . A . , Koechlin, B. A . , and Postma, E., Hof fmann-La Roche Inc. , Unpublished Data. Clyburn, T., Hoffmann-La Roche Inc., Personal Communication. Scheidl, F., Hoffmann-La Roche Inc., Personal Communication. Weber, O., Billmeyer, A . , Geller, M., Venturella, V. S., and Senkowski, B. Z., Hoffmann-La Roche Inc., Unpublished Data.

161

DEXAMETHASONE

Edward ill. Coiitw

163

EDWARD M. COHEN

C o n t e n t s


1.1 N a m e , Formula, M o l e c u l a r Weight, R e g u l a t o r y S t a t u s 1 . 2 Appearance , C o l o r , Odor


2 . 1 2 .2 2 . 3 2 . 4 2 .5 2 .6 2 . 7 2 . 8

C r y s t a l P r o p e r t i e s I n f r a r e d S p e c t r a U l t r a v i o l e t Absorbance O p t i c a l R o t a t i o n S o l u b i l i t y Nuc lea r Magne t i c Resonance S p e c t r a Thermal Behav io r Mass S p e c t r a


4 . S t a b i l i t y

5. Methods o f A n a l y s i s

5 . 1 R e f e r e n c e S t a n d a r d s 5 . 2 A-Ring A n a l y s i s 5 .3 B-Ring A n a l y s i s 5 . 4 C-Ring A n a l y s i s 5 .5 D-Ring A n a l y s i s 5 . 6 I n f r a r e d A n a l y s i s 5 . 7 F l u o r e s c e n c e A n a l y s i s 5 . 8 Mass T r a n s p o r t Techniques

6 . Metabol i sm and P h a r m a c o k i n e t i c s

7 . R e f e r e n c e s

DEXAMETHASONE

1. Description

1.1 Name, Formula, Nolecular Weight, Regulatory Status

Dexamethasone (I)* is 9a-fluoro-ll6,17a,21-trihy- droxy-16~-methylpregna-1,4-diene-3,2O-dione. Other names for the compound include 9a-fluoro-16a-methylprednisolone and 16a-methyl-9a-fluoroprednisolone. three other chemical names for dexamethasone. Proprietary names for dexamethasone include Decadron, Dexacortisyl, Oradexon, Hexadrol and Deronil. A number of others are listed in the monograph for dexamethasone in the Merck Index.

The blerck Index lists

0

c22 H29 F05 Mol. Wt.: 392.45

Official monographs for dexamethasone are given in USP XVIII and B.P. 1968.

*Betamethasone is the 166-methyl configurational isomer of dexamethasone.

I6S

EDWARD M. COHEN

1 . 2 Appearance, Co lo r , Odor

White t o ye l lowish -whi t e , o d o r l e s s , c r y s t a l l i n e powder.

2. P h y s i c a l P r o p e r t i e s

2 . 1 C r v s t a l P r o D e r t i e s

The o p t i c a l c r y s t a l l o g r a p h i c p r o p e r t i e s of dexamethasone r e p o r t e d f o r c r y s t a l s o b t a i n e d by c r y s t a l l i z a t i o n o f t h e s t e r o i d from 50% e t h a n o l are as f o l l o w s : 2

System: o r tho rhombic D i s p e r s i o n C r y s t a l Hab i t : Tabu la r R e f r a c t i v e O p t i c S ign : + Axial Angle: 50" O p t i c O r i e n t a t i o n : xxll a Dens i ty :

r > v s t r o n g 1ndexes:a ( a ) = 1.559

B (E ) = 1.579 Y = 1.649

.337 ( c < a < b ) y y l l c Molar R e f r a c t i o n :

z z l l b Experimental = 99.64 C a l c u l a t e d = 98.59

Two c r y s t a l forms of dexamethasone have been observed t o d a t e . The X-ray powder d i f f r a c t i o n d a t a f o r t h e two forms A and B are g i v e n i n Tab le I . 3 coun te red i n peak i n t e n s i t y from sample t o sample, no l i s t i n g of t h e r e l a t i v e i n t e n s i t y of t h e bands i s g iven . The two c r y s t a l forms have e s s e n t i a l l y t h e same s o l u b i l i t y i n water and e s s e n t i a l l y t h e same decomposi t ion t empera tu re . Form B i s t h e more u s u a l l y obse rved form. No s o l v a t e s o r polymorphic m o d i f i c a t i o n s of dexarnethasone were r e p o r t e d by Mesley4 3 f o r dexamethasone c r y s t a l l i z e d from ch lo ro fo rm, ace tone and e t h a n o l u s i n g X-ray d i f f r a c t i o n p a t t e r n s and s o l i d s t a t e I . R . s p e c t r a t o e v a l u a t e samples.

Because of v a r i a t i o n s en-

2 .2 I n f r a r e d Spectrum ( I . R . S . )

Mesley r e p o r t s t h e f o l l o w i n g band a s s ignmen t s f o r t h e s o l i d s t a t e I.R. S . of dexamethasone (mine ra l o i l m u l l ) .

I66

DEXAMETHASONE

TABLE I

X-Ray Powder Diffraction Pattern of Dexamethasone

Sample - Merck Standard 88415-76 Cu-K, Radiation

5 . 7 0 1 5 . 4 8 7 .95 1 1 . 1 0 9 . 9 0 8 . 9 2

12 .45 7 . 1 0 1 3 . 5 0 6 .55 1 4 . 4 5 6 .12 1 4 . 9 5 5 . 9 1 1 6 . 1 0 5 . 4 9 1 7 . 6 5 5 . 0 2 1 9 . 2 5 4 . 6 0 2 0 . 0 0 4 . 4 3 20 .60 4 . 3 0 20 .90 4 . 2 4 21.00 4 . 2 2 25 .00 3.56 22.30 3.98 23.00 3.86 23.65 3.76 24 .00 3.70 24 .70 3 . 6 0 2 5 . 2 0 3 .53 26 .30 3 . 3 8 26 .70 3 . 3 3 27 .20 3 . 2 7 27 .30 3.26 27.70 3 . 2 1 28 .25 3.15 2 9 . 1 0 3.06 29 .65 3 . 0 1 31 .10 2 .87 32 .25 2.77 31 .70 2.82 32.10 2 .78

aMerck Standard 118415-76 bCu-K, Kadiation

Form Ba db - 20"

_c

5 . 9 5 6 . 9 2 7 . 4 5 8 . 5 0 8 . 8 0

1 0 . 5 5 12 .50 1 3 . 6 0 1 4 . 2 0 1 5 . 1 0 1 5 . 6 0 1 6 . 8 0 1 7 . 7 0 1 8 . 1 0 1 8 . 5 0 1 9 . 7 0 20 .40 2 1 . 6 0 22 .20 2 2 . 8 0 23 .50 2 5 . 0 0 26 .10 26.75 2 7 . 9 5 28.50 2 9 . 0 0 3 0 . 1 0 30 .15 3 1 . 7 0 33 .70 37 .40 44 .40

1 4 . 8 0 1 2 . 7 5 1 1 . 8 3 1 0 . 3 8 1 0 . 0 3

8 .36 7 .07 6 . 5 0 6 . 2 3 5 . 9 0 5 .67 5.27 5 . 0 0 4 . 8 9 4.79 4 . 5 0 4 . 3 5 4 . 1 1 4.00 3 . 8 9 3 . 7 8 3 .56 3 . 4 1 3 .33 3 . 1 9 3 .13 3 .07 2.96 2 .96 2 . 8 2 2 . 6 5 2 . 4 0 2 . 0 4

EDWARD M. COHEN

Wavelength ( cm. - ) Vibrational Modes

1043 1135, 1117 1090, 1056 1408, 1297, 1242 952, 930, 893, 852 830, 703

1lB-OH 17a-OH 21-OH lY4-diene-3-one

Clark lists 1655 cm.-l, 1616 cm.”, and 892 ern.-' as dis- tinctive bands for the KBr disc I .R.S. of dexamethasone.6

Figure 1 shows the I.R.S. of Merck Standard #8415-76. Principal bands and their assignments are tabulated below. The spectrum is in substantial agreement with published I.R.S. of dexamethasone.5,6,8

Wavenumber of Assignment (cm.-’) Vibrational Mode

3400-3675 3020, 3050 2 85 0- 3 000 (sever a1 bands ) 1700 1660 1620, 1600 1130-1040 (several bands) 890

OH stretch Olefinic CH stretches Aliphatic CH stretches C20 carbonyl C 3 carbonyl stretch A-ring, C=C stretch Various C-OH stretches A 1 y4-diene-3-ketone

2.3 Ultraviolet Absorbance

Dexamethasone exhibits a single major absorbance band at about 240 nm. in solution attributable to the A-1,4- 3 keto A ring chromophore. The following data have been reported in the literature:

Reference (E) Solvent X Maximum cm.

Methanol 240 nm. 355(13,920) (6) Met hano 1 238 nm. 392 (15 , 400) (8) Methanol 240 nm. 380-410 (9)

168

WAVELENGTH MICRONS 2.5 3 4 5 6 7 8 9 10 12 14 18 22 35 50

I00 100

80 80

8 w 60 60 u z U b-

k 4 0 40 r UJ z U

20 20

1 1 1 0 4000 3500 3000 2500 2000 1700 1400 1100 800 500 200

0

FREQUENCY (CM- ' )

F i g u r e 1. I n f r a r e d S p e c t r u m of Dexamethasone; S p l i t M u l l T e c h n i q u e - F l u o r o l u b e m u l l a b o v e 1 3 3 5 crn.-l M i n e r a l o i l m u l l b e l o w 1335 c m - I (Merck S t d . #8415-76) .

X

EDWARD M. COHEN

Figure 2 shows an u l t r a v i o l e t s c a n of dexamethasone (Merck S td . 118415-76) i n methanol a t a c o n c e n t r a t i o n of 0.00127%. A s i n g l e absorbance maximum a t 240 nm. was o b t a i n e d w i t h a n

of 393 (15,400).1° c m .

In c o n c e n t r a t e d s u l f u r i c a c i d , t h e u l t r a v i o l e t abso rbance spectrum of dexamethasone undergoes a bathochromic s h i f t c h a r a c t e r i s t i c of a number of s t e r o i d s . S p e c t r a l d a t a re- p o r t e d f o r dexamethasone i n c l u d e :

1% Max. cm. Reference

262 nmSa -444 11 305 nm.a - 308 11

422-455 6 263 nm. b

aTwo h o u r s i n c o n c e n t r a t e d H2S04 bNo c o n d i t i o n s s p e c i f i e d

The s p e c t r a l change no ted f o r dexamethasone i n c o n c e n t r a t e d s u l f u r i c a c i d i s p robab ly due t o t h e i n i t i a l p r o t o n a t i o n of t h e A-1,4-3 k e t o fo l lowed by a coupled chemical r e a c t i o n s i n c e t h e s p e c t r a l changes a r e a p p a r e n t l y i r r e v e r s i b l e . l 2

2.4 ODtical R o t a t i o n

The f o l l o w i n g s p e c i f i c r o t a t i o n s have been r e p o r t e d : 25"

IUID = +75 t o 80" (C = 1 i n dioxane) ' [u]22-240C = 86" (C = 1 i n d ioxane )8 [ a I h 5 " = +77.5" ( C = 1 i n d ioxane ) l

D

= +72 t o 80" ( C = 1 i n d i ~ x a n e ) ~

2.5 S o l u b i l i t y

The f o l l o w i n g s o l u b i l i t y d a t a a r e g i v e n i n t h e l i t e r a t u r e :

0 rn

200 250 300 350 WAVELENGTH, n m

Figure 2. Ultraviolet Absorption Spectrum of Dexamethasone in Methanol at a Concentration of 0.00127% - 2.max. 240 nm. and E1% = 393

(Merck Std. #8415-76). lcm.

EDWARD M. COHEN

So l v e n t

D i s t i l l e d Water D i s t i l l e d Water D i s t i l l e d Water I s o p r o p y l

Myris t a t e L i g h t Mine ra l

O i l E thano l Chloroform Acetone E t h e r

E t h y l Acetate P y r i d i n e

Tempe ra t u r e

37°C. 25°C. 25 " C.

37°C.

37°C. -- -- -- --

25°C. --

S o l u b i l i t y

11 .6 mg/100 m l 8 .4 mg/100 m l

10.0 mg/100 m l

23.3 mg/100 m l

0 .01 mgI100 m l 1 i n 42 1 i n 165 s o l u b l e

v e r y s l i g h t l y s o l u b l e

4 . 1 mg/g - 10%

Refe rence

1 4 15 1

1 4

14 9 9 9

1 3 16

7

2 .6 Nuclear Magnet ic Resonance Spectrum (NMRS)

The 60 MHz NMRS of a 10% s o l u t i o n of dexamethasone i n d e u t e r a t e d p y r i d i n e (d5) i s shown i n F i g u r e 3. Tabu la t ed below are t h e p r o t o n a s s ignmen t s f o r t h e observed chemical s h i f t s and coup l ing c o n s t a n t s . 7

Chemical S h i f t ppm'

7 . 5 5 / d o u b l e t 6 .6? / c rude d o u b l e t 6 .5 4 / q u a r t e t 6 . 4 3 / ~ i n g l e t 6 .35 /un reso lved

m u l t i p l e t 5 . g 4 / b r o a d s i n g l e t 4.9?/AB q u a r t e t 4 .73 /c rude d o u b l e t 1 . 7 4 / s i n g l e t 1.4 / s i n g l e t 1.1 / d o u b l e t 0.88-3.80 complex

ove r l apped s i g n a l s

R e l a t i v e // of P r o t o n s Assignments

-1.1 C W E f 1 1 - O H

3.9

1.1

3.1 C ( 2 1) -Hz

(19) -H3 '61 8i:" 3

19 .8 1 -c J

A l l remain- i n g p r o t o n s

172

500

100

2 5

I

I

F i g u r e 3. NMR S p e c t r u m of Dexamethasone i n D e u t e r a t e d P y r i d i n e ; O p e r a t i n g C o n d i t i o n s S i g n a l : 60 MHz Sample: Merck S t d . #8415-76, 5 1 . 5 mg. / .5 cc. of CgDgN, Sweep Time: 250 sec. S p e c t r u m A m p l i t u d e : 1 . 2 5 x 10. I n t e r n a l S t a n d a r d : T e t r a m e t h y l s i l o n e

-I I P cn 0 z m

EDWARD M. COHEN

'TMS used as i n t e r n a l r e f e r e n c e 2Coupling c o n s t a n t 3Based on 29 p r o t o n s f o r sum of t o t a l s p e c t r a l i n t e g r a l 4Assignment confirmed v i a D 2 0 exchange

2.7 Thermal Behavior

M e l t i n g P o i n t - The m e l t i n g p o i n t of dexamethasone i s r e p o r t e d t o depend i n p a r t on t h e ra te o f h e a t i n g and t h e deg ree of powder f i n e n e s s . " v a l u e s i n c l u d e :

Some of t h e r e p o r t e d

Mel t ing P o i n t -250" -255"

263" - sample 256-258"

Refe rence 1 3

9 r e c r y s t a l l i z e d from e t h e r 1 7

8 262-264" - c r y s t a l s from e t h e r 1 2 6 8-2 72 " 1

D i f f e r e n t i a l Scanning Ca lo r ime t ry - The thermogram o b t a i n e d on a P e r k i n E l m e r DSC 1-B , 20" lminu te f o r dexamethasone (Merck S t d . #8415-76) e x h i b i t s t h e f o l l o w i n g the rma l e v e n t s ( F i g u r e 4 ) :

1. Small exotherm a t -254-258" 2. Complex m e l t i n g endotherm - 258" - o n s e t temp.

- 267" - peak temp.

2 .8 Mass S p e c t r a (MS)

The low r e s o l u t i o n MS of dexamethasone h a s been de- Impor t an t f e a t u r e s of t h e s c r i b e d i n t h e l i t e r a t u r e . l e ~ l 9

spectrum i n c l u d e :

1. Absence of a m o l e c u l a r i o n peak. 2 . Major peak a t m / e 343 ( M . W . - 49) a t t r i b u t e d

t o l o s s of water from t h e D-ring fo l lowed by c l e a v a g e of t h e C 1 8 - C 2 1 bond.

m / e 122 accompanied by a major peak a t m / e 121 a r i s i n g from c l e a v a g e of cg-c7 and Cg- C l 0 bonds and t r a n s f e r o f two (m/e = 122)

3 . The most i n t e n s e peak of t h e MS o c c u r s a t

174

+- 4 'A

4 0 - 5 U

w 1

X W

0 l ~ 1 l l I 1 I I I 1 I 1 1 1 1 1 1 1 ,

280 250 200 150 100 1 in "C

Figure 4. D . S . C . Thermogram of Dexamethasone (Merck S t d . #8415-76); Perkin Elmer - D . S . C . - 1 B ; 20°/min.; sealed p a n with N 2 sweep.

P

EDWARD M, COHEN

o r one hydrogen ( m / e = 1 2 1 ) t o t h e charged fragment .

The a u t h o r s 1 8 * l9 c l a i m t h a t betamethasone, t h e 166-methyl iosmer of dexamethasone, can be u n e q u i v o c a l l y d i s t i n g u i s h e d from dexamethasone by t h e v i r t u a l absence of t h e m / e peak a t 343.

Formation of t h e m / e 343 d i a g n o s t i c f ragment was a t t r i b u t e d t o a f a c i l e t r a n s - e l i m i n a t i o n of water from dexamethasone fo l lowed by c l e a v a g e o f t h e a l l y l i c Czo-Cz l bond. q u i t e c o n c e i v a b l e t h a t i n i t i a l e l i m i n a t i o n of water o c c u r r e d under the rma l stress r a t h e r t h a n under e l e c t r o n impact a l o n e i n t h e r e p o r t e d d a t a . l 8 > l 9

I t is

Attempts t o c o r r o b o r a t e t h e r e p o r t e d l i t e r a t u r e f i n d i n g s w i t h r e s p e c t t o t h e p re sence of m / e 343 as a major peak f o r dexamethasone were n o t s u c c e s s f u l i n Merck l a b o r a t o r - i e s . 2 0 sone o b t a i n e d on an LKB-5000 mass s p e c t r o m e t e r a t 70 e V i o n i z a t i o n ene rgy , S i g n i f i c a n t d i f f e r e n c e s between t h e s p e c t r a of dexamethasone and betamethasone w e r e found i n t h e r e l a t i v e peak h e i g h t a t m / e 315 and 3 4 3 , as l i s t e d below ( i n p e r c e n t o f t h e base peaks a t m / e 1 2 2 ) .

F i g u r e 5 shows t h e low r e s o l u t i o n MS of dexametha-

176

l-

-I W tx

a

c . I I

m /e

Figure 5. Mass S p e c t r u m of Dexamethasone (Merck S t d . # 8415-76) LKB-5000 - 7 0 e V i o n i z a t i o n e n e r g y .

EDWARD M. COHEN

-0 0

- GH3

rnfe 392 rnfe 374 m / e 372 m / e 362 m f e 354 m / e 343 rnfe 342 m/e 333 m / e 315 m / e 312

-

Dexamethasone Betamethasone

-1% - 1% - 1% - 1% - 1%

5% 19%

6% 1 2 %

9%

- 1% M+ - 1% M-H20 - 1% M-HF - 1% M-CH20 -1% M-(H20 + HF) 15% see below 13% 362 - HF

3% M- (CO-CH 20H) 3% see below

10% 372-(CO-CH20H + H )

A r e a s o n a b l e i n t e r p r e t a t i o n of t h e s e two f r agmen t s i s b a s e d on t h e w e l l known a c i d c a t a l y z e d water e l i m i n a t i o n from 17- a-hydroxy s t e r o i d s w i t h concomitant m i g r a t i o n of t h e 18- methyl group from C - 1 3 t o C-17. I n t h e c a s e of dexamethasone t h e c i s - c o n f i g u r a t i o n of t h e C-17 k e t o l and C-16-methyl groups i n t h e r e a r r a n g e d i o n rnfe 374 f a v o r s t h e d i r e c t breakdown t o rnfe 315 w h i l e t h e t r a n s - c o n f i g u r a t i o n (be t a - methasone) a l l o w s € o r a h i g h e r p e r c e n t a g e of t h e i n t e r m e d i - a t e i o n m / e 343.

CH20H

co I

d- H

M @ 1 L m / e 374

3 3

178

DEXAMETHASONE

3 . Synthesis

Dexamethasone can be synthesized starting from 16a- methyl hydrocortisone acetate, an available intermediate of bile acid origin. 21 (Figure 6). While the original synthetic scheme required a selenium oxide l-dehydrogenation2*, subsequent work established that 1-dehydrogenation could be accomplished for any number of appropriate intermediates by using microorganisms of the class Schizomycetes. An entirely different synthetic scheme for dexamethasone starting from diosgenin has also been reported.2"

The synthesis of both tritium labeled and carbon-14 labeled dexamethasone has been given in the literature. The following labeled material has been prepared:

1. 'H at positions 1, 2 and 4 . 2. General tritium labeling. 3. 4 . 'H in the l6B-hydrogen.

14C in the 16-i-methyl carbon.

This type of labeled material provides a very sensitive handle for use as a tracer in subsequent chemical and biological studies.

4 . Stabilitv

Chemical properties of the reactive mioeties of dexamethasone - A consideration of the stability information reported for dexamethasone is best viewed in connection with the molecular changes reported for dexamethasone as well as structurally related adrenocorticosteroids.

A-Ring - can undergo variety of

The A' 9 43-keto grouping of prednisone acetate photocatalyzed transformations leading to a compounds whose composition depend on the con-

ditions of the experiments. 26 products are shown below. Further irradiation of Compound VIII can lead to a photoinduced diene-phenol rearrangement. I t is reasonable to assume that dexamethasone can undergo similar photochemistry with the course of reaction duely influenced by the presence of the 92-fluoro group.

Two such characteristic

179

EDWARD M . COHEN

F i g u r e 6 .

CH2 OC- CH3

c = o I ;

KC2H302

1-21 -ACETATE

NaOCH3/GH30H S e 0 2 1 0

180

DE XAME THASONE

V I I V l l l

(A max. -233 nm. in ETOH) (Lumiprednisone, X max. -218 nm. and 265 nm.)

Sodium bisulfite adds reversibly to the A - 1 position of dexamethasone-21-phosphate sodium forming a 1-sulfonate as shown below. 27

so’,

HSOS

1-21 PHOSPHATE S O D I U M IX

From the data obtained, it is assumed that the active nucleophile is the dianionic sulfite ion. This reaction is typical of conjugate sulfite addition to a,@-unsaturated ketones. It should be inferred that other active nucleo- philic agents may behave in a similar manner.

B-Ring - The 9a-fluoro aliphatic substituent would be expected to be quite stable to hydrolytic displacement. Indeed, there are no reported instances of liberation of fluoride from the 9cu-f luoro adrecocorticosteroids in-vivo. *

181

EDWARD M. COHEN

C-Ring - The 11-6-hydroxyl group of s e v e r a l c r y s t a l l i n e a d r e n o c o r t i c o s t e r o i d es ters unde rgoes a i r o x i d a t i o n , c a t a - l y z e d by b o t h t e m p e r a t u r e and l i g h t , t o form t h e c o r r e s - ponding 11-one compounds. 2 9 i n g t h i s t r a n s f o r m a t i o n are t h e f o r m a t i o n of a non- s t o i c h i o m e t r i c s o l v a t e which undergoes f a c i l e d e s o l v a t i o n w i t h o u t change i n t h e X-ray d i s c e r n i b l e c r y s t a l l i n e l a t t i c e s t r u c t u r e . It was a l s o shown t h a t 11-p-hydroxy s t e r o i d s c a n b e a i r o x i d i z e d i n s o l u t i o n under somewhat r i g o r o u s condi - t i o n s . I n g e n e r a l , t h e r e a c t i o n r e q u i r e s m o l e c u l a r oxygen and produces water. It i s a c c e l e r a t e d by h e a t , g r e a t l y a c c e l e r a t e d by f r e e - r a d i c a l i n i t i a t o r s o r by U.V. l i g h t , and i s quenched by f r e e - r a d i c a l i n h i b i t o r s . These a u t h o r s d i d n o t r e p o r t on any f r e e C - 2 1 a l c o h o l s .

S t r u c t u r a l r e q u i r e m e n t s f a v o r -

D-Ring - The C-17 d ihydroxy a c e t a t e s i d e c h a i n , a cha r - a c t e r i s t i c s t r u c t u r a l f e a t u r e of a l l a d r e n o c o r t i c o s t e r o i d s , i s q u i t e s u s c e p t i b l e t o b o t h a e r o b i c and a n a e r o b i c t r a n s - f o r m a t i o n s . The a n a e r o b i c changes of t h e s i d e c h a i n are c o n s i d e r e d by Wendler as f o l l o w s :

C H 2 0 H CHOH

c.0 C - O H I II

HC.0 I

CHOH 0

X X I X l l l

Io2 COOH

OH X V I

COOH I

CHOH

xv X I V

182

DE XAMETHASONE

Conversion of X to the C-17 ketone (XIII) is considered as a reverse aldol reaction. Conversion of the keto-aldehyde (XIV) to the hydroxy acid (XV) is considered a benzillic acid rearrangement.

The predominant reaction, under base catalyzed aerobic conditions, appears to be oxidative cleavage of the side chain to the corresponding etianic acid (XVI). Formation of C-17 ketone is only a minor reaction product.31 alternative mode of oxidative change at the C-21 position is the formation of a glyoxal (XVII) side chain which was found to be metal catalyzed.32 expected to undergo coupled chemical reactions consistent with the reactivity of that functionality.

An

This glyoxal might be

H C = O I c = o

OH X V I I

The D-homo rearrangement reported for 17n-hydroxy-20-keto steroids is adequately described by Wendler. 33 products of this rearrangement are shown below.

Possible

c = o HO ,CH3

- M A J O R MAJOR

x x X V l l l X I X

While not specifically described in the literature, the D-homo rearrangement may be a possible occurrence for dexamethasone under certain reaction conditions by analogy with other steroids such as triamcinolone. 34

183

EDWARD M. COHEN

Other possible reactions involving the C-17 side chain include acylation of the C-21 hydroxyl group with available acylating agents. Prednisolone was reported to transesteri- fy with aspirin to form prednisolone acetate in a pharmaceutical formulation matrix. 3 5 Finally, reactive reagents such as formaldehyde can attack the C-17 side chain to form the bis-methylene dioxy derivatives (BMD) . 3 6 Formaldehyde is a potential impurity and/or reaction product present in such commonly used pharmaceutical adjuvants as polyethylene glycols.

Stability of Dexamethasone - The following reported stability information is entirely consistent with the potential reactivity discussed above. Dexamethasone solid is reported to be stable in air13 but should be protected from light.’ C-17 a-ketol side chain within 6-8 minutes in the presence of a base catalyst.17 dexamethasone in a variety of marketed pharmaceutical dosage forms.2 methasone in four different tablet formulations. l 7

Solutions of dexamethasone lose about 50% of the

Excellent stability is shown by

Wahba has compared the thermal stability of dexa-


5.1 Reference Standards

Both the U.S.P. and B.P. supply standard samples of dexamethasone. A highly purified sample of dexamethasone was prepared by equilibrating dexamethasone with ethyl acetate. l6 described above, had a phase splubility analysis slope of 0.1% ? 0.1 in ethyl acetate.

Merck Standard #8415-76 , which was prepared as

5 . 2 A-Ring Analysis (without derivatization)

Ultraviolet Absorption - Direct measurement of the U.V. absorbance at the X maximum - 2 4 0 nm. is an official physical measurement in both the U.S.P. and B.P. In addition, U.V. absorption has been used as an analytical measurement for dexamethasone after previous separation techniques such as liquid partition and/or chromatography have been employed to isolate dexamethasone from a pharmaceutical matrix. 9 39 Non-specif ic interference in the U . V .

184

OE XAMETHASONE

measurements may sometimes be e l i m i n a t e d by a d i f f e r e n t i a l t e c h n i q u e i n which a s t r o n g r e d u c i n g a g e n t , s u c h as boro- h y d r i d e , c o n v e r t s t h e A1y4-3 k e t o chromophore t o a s a t u r - a t e d sys t em devo id of abso rbance a t a b o u t 240 nm.40 D e t e r m i n a t i o n of t h e U . V . abso rbance of an u n t r e a t e d a l i - quo t v e r s u s a r educed a l i q u o t y i e l d s i n f o r m a t i o n on t h e s t e r o i d U . V . a b s o r b a n c e i n t h e p r e s e n c e of e x t r a n e o u s ab- s o r b a n c e . The f e a s i b i l i t y o f t h i s approach was r e c e n t l y demons t r a t ed f o r b e t a m e t h a ~ o n e . ~ ~ I t s h o u l d be n o t e d t h a t A4-3 k e t o s t e r o i d s e x h i b i t t h e same U . V . abso rbance b e h a v i o r a s t h e A1y4-3 k e t o s t e r o i d s and d i r e c t measurements c a n n o t d i s t i n g u i s h be tween them. The j u d i c i o u s u s e of b o r o h y d r i d e r e d u c t i o n may e n a b l e one t o measure L4-3 k e t o i m p u r i t i e s i n t h e p r e s e n c e of A1'4-3 k e t o s t e r o i d s such as dexamethasone .

N . M . R . Measurements - An N . M . R . a s s a y method f o r b o t h b u l k d rug and f o r m u l a t i o n s b a s e d on measu r ing t h e i n t e n s i t y o f t h e r o t o n s i g n a l from t h e C - 1 p r o t o n a t a b o u t 2 . 1 Tau f o r A1y9-3 k e t o s t e r o i d s i n c l u d i n g dexamethasone w a s re- c e n t l y d e s c r i b e d . 42 The t e c h n i q u e a p p e a r s t o b e c a p a b l e o f measu r ing b o t h 0-1-3 k e t o and A194-3 k e t o compounds s e p a r a t e l y o r t o g e t h e r i n t h e same sample . The r e l a t i v e l y h igh l e v e l s of d rug r e q u i r e d f o r a n a l y s i s (80-100 mg.1 sample ) w i l l limit a p p l i c a t i o n of t h e p r o c e d u r e as des- c r i b e d w i t h o u t a p p r o p r i a t e m o d i f i c a t i o n s t o i n c r e a s e t h e a s s a y s e n s i t i v i t y by a t l e a s t two o r d e r s of magn i tude .

Po la rography - The p o l a r o g r a p h i c d e t e r m i n a t i o n of dexamethasone i n t a b l e t dosage Lorms i n 80% e t h a n o l , pH 4 . 2 Flc I lva ine b u f f e r h a s been The r e d u c t i o n s t e p e x h i b i t e d by Alr4-3 k e t o s t e r o i d u s u a l l y o c c u r s a t more a n o d i c p o t e n t i a l s , and i s e a s i l y d i s c e r n i b l e from t h e r e - d u c t i o n s t e p e x h i b i t e d by t h e c o r r e s p o n d i n g C4-3 k e t o s t e r o i d s . 4 L t

A-King A n a l y s i s ( w i t h d e r i v a t i z a t i o n )

Hydrazone Format ion - I s o n i c o t i n i c a c i d h y d r a z i d e

A number of s u b s t a n c e s t h a t i n t e r f e r e w i t h (INH) r e a c t s w i t h t h e A-ring c a r b o n y l t o p roduce a y e l l o w hydrazone . 1 3 t h e I N H a s s a y are d i s c u s s e d i n R e f e r e n c e 39. Ta i sho h a s recommended t h e u s e of INH-2HC1 r a t h e r t h a n I N H

R e c e n t l y ,

IS5

EDWARD M. COHEN

i t s e l f as a r e a g e n t f o r t h e d e t e r m i n a t i o n of dexamethasone. 4 5

I t i s a n t i c i p a t e d t h a t o t h e r ca rbony l r e a g e n t s w i l l l i k e - w i s e p rov ide a b a s i s f o r a n a l y t i c a l methodology w i t h dexamethasone. F o r i s t and Johnson d i s c u s s a number of t h e s e . 4 6

The i n t e g r i t y of t h e A-ring of s t e r o i d s i s g e n e r a l l y con- s i d e r e d t o be r e l i a b l y determined by e i t h e r U.V. o r I N H a s s a y . 3 9 , 4 7 I n view of t h e r e p o r t e d p roduc t s of A-r ng p h o t o l y s i s of p redn i sone having c1,B u n s a t u r a t e d ke tone s t r u c t u r e s ( s e e S e c t i o n 4 ) , which may i n t e r f e r e i n t h e measurement of t h e u n a l t e r e d A-ring s t e r o i d , c a u t i o n i n u s i n g t h e s e measurements shou ld be e x e r c i s e d . It i s i n t e r - e s t i n g t o n o t e t h a t d a t a p r e s e n t e d i n Reference 47 s u g g e s t t h a t bo th d i r e c t U . V . a n d / o r I N H a s s a y gave h i g h e r r e s u l t s f o r photolyzed s o l u t i o n s of p redn i sone (A1y4-3 k e t o s t e r o i d ) t h a n d i d a q u a n t i t a t i v e pape r chromatographic a s s a y of t h e same s o l u t i o n s .

5 . 3 B-Ring Ana lys i s

There a r e no l i t e r a t u r e r e p o r t s t h a t d e a l s p e c i f i - c a l l y w i t h a s c e r t a i n i n g t h e i n t e g r i t y of t h e 9a - f luo ro s u b s t i t u e n t i n dexamethasone.

5 . 4 C-Ring Ana lys i s

While t h e r e are no s p e c i f i c r e p o r t s conce rn ing dexamethasone, a number of p rocedures based on t h e react i - v i t y of t h e oxygen f u n c t i o n a t C - 1 1 a r e d i s c u s s e d by F o r i s t and Johnson f o r r e l a t e d s t e r o i d s . 4 6

5 . 5 D-Ring Ana lys i s

The a - k e t o l group (CH20H-CO) p o s s e s s e s r educ ing p r o p e r t i e s and i t s r e a c t i v i t y towards b l u e t e t r a z o l i u m (B.T.) i s u t i l i z e d e x t e n s i v e l y as a n a s s a y p rocedure f o r dexamethasone.39 a s s a y f o r dexamethasone. However, t h e U.S.P. u t i l i z e s a p r i o r t h i n l a y e r chromatographic s e p a r a t i o n which i n c r e a s e s t h e s e l e c t i v i t y and s p e c i f i c i t y o f t h e f i n a l B.T. a s s a y measurement f o r i n t a c t dexamethasone. The p resence of

Both t h e U.S.P. and B.P . employ t h e B.T.

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DEXAMETHASONE

e a s i l y o x i d i z e d e x c i p i e n t s can c a u s e i n t e r f e r e n c e i n t h e B . T . a s s a y p r o c e d u r e . A t a b u l a t i o n o f a number o f s u c h i n t e r f e r e n c e s i s g i v e n i n R e f e r e n c e 39. Both p o s i t i v e and n e g a t i v e i n t e r f e r e n c e s are p o s s i b l e . The fo rmer a r e due t o a g e n t s which are o x i d i z e d unde r t h e c o n d i t i o n s of t h e B.T. r e a c t i o n . The l a t t e r are a g e n t s which can a l t e r t h e pH o f t h e a l k a l i n e B.T. r e a c t i o n m i x t u r e t h e r e b y d e c r e a s i n g t h e e x t e n t o f c o l o r f o r m a t i o n . E f f e c t i v e e l i m i n a t i o n o f i n t e r - f e r e n c e s can o f t e n b e a c h i e v e d by s e l e c t i v e e x t r a c t i o n and ch romatograph ic p r o c e d u r e s . An au tomated B.T. a s s a y h a s been d e s c r i b e d f o r dexamethasone t a b l e t s . 4 8

The 17 ,21-d ihydroxy-20-ke to g roup of dexamethasone reacts w i t h phenylhydrazine-sulfuric a c i d ( P o r t e r - S i l b e r ) r e a g e n t t o form a y e l l o w chromogen s u i t a b l e f o r a s s a y p u r p o s e s . 3 9 A s i n t h e c a s e o f t h e B.T. a s s a y , a number of s p e c i f i c c o l o r i n t e r f e r e n c e s , which can ar ise from t h e r e a c t i o n o f t h e r e a g e n t w i t h e x c i p i e n t s , may b e e l i m i n a t e d by s e l e c t i v e e x t r a c t i o n and ch romatograph ic p r o c e d u r e s .

A number of o t h e r a s s a y a p p r o a c h e s based on t h e r e a c t i v i t y o f t h e i n t a c t C - 1 7 s i d e c h a i n d e s c r i b e d f o r r e l a t e d ster- o i d s are presumably a p p l i c a b l e t o dexamethasone; These i n c l u d e : r e a g e n t was found t o b e a p p r o x i m a t e l y t w i c e as s e n s i t i v e as B . T . a s s a y f o r p r e d n i s o n e b u t i s n o t q u i t e as s e l e c t i v e as B . T . r e a g e n t f o r i n t a c t p r e d n i s o n e i n t h e p r e s e n c e o f de- g r a d a t i o n p r o d u c t s ; b ) formaldehyde e s t i m a t i o n f o l l o w i n g p e r i o d a t e o r b i s m u t h a t e o x i d a t i o n . 46

a ) 2,4-dinitrophenylhydrazine r e a g e n t 49 - t h i s

The u s e o f any o f t h e a s s a y p r o c e d u r e s d e s c r i b e d above as an a b s o l u t e i n d e x o f dexamethasone i n t e g r i t y i n a pharma- c e u t i c a l m a t r i x s h o u l d n o t b e assumed w i t h o u t i ndependen t v e r i f i c a t i o n u t i l i z i n g h i g h r e s o l u t i o n mass t r a n s p o r t p ro - c e d u r e s s u c h as t h i n l a y e r chromatography, b e c a u s e o f t h e complex n a t u r e of t h e chemica l changes p o s s i b l e a t t h e C - 1 7 s i d e c h a i n . For example , a TLC s e p a r a t i o n of a sample m i x t u r e f o l l o w e d by a d e m o n s t r a t i o n of t h e absence o f c o l o r r e s p o n s e t o a p a r t i c u l a r r e a g e n t of a l l f o r e i g n s p o t s , s a v e f o r i n t a c t dexamethasone , would l e n d a s s u r a n c e as t o t h e s p e c i f i c i t y o f t h a t r e a g e n t .

187

E D W A R D M. COHEN

5 . 6 I n f r a r e d A n a l y s i s

B e l l ~ m o n t e ~ ~ d e s c r i b e d a s o l i d s t a t e I R a s s a y p roce - d u r e (KBr m u l l ) f o r measu r ing dexamethasone i n t h e p r e s e n c e of t r i a m c i n o l o n e u t i l i z i n g as a n a l y t i c a l wave leng ths 915 cm.-' and 1140 c m . - l .

5 .7 F l u o r e s c e n c e A n a l y s i s

Un l ike A4-3 k e t o s t e r o i d s , t h e A1,4-3 k e t o compounds

R e c e n t l y , a f l u o r e s c e n t a s s a y f o r c o r t i c o s t e r o i d s do n o t e x h i b i t s i g n i f i c a n t s u l f u r i c a c i d induced f l u o r e s - cence . 5 1 w a s d e s c r i b e d based on making a f l u o r e s c e n t D-ring d e r i v a - t i v e . 66

5 . 8 Mass T r a n s p o r t Techn iques

Assay p r o c e d u r e s d e s c r i b e d below depend on t h e s e l e c t i v e m i g r a t i o n o f e i t h e r t h e i n t a c t m o l e c u l e o r a re- c o g n i z a b l e d e r i v a t i v e o f t h e i n t a c t molecu le be tween p h a s e s fo l lowed by a s p e c i f i c f u n c t i o n a l group o r n o n - s p e c i f i c q u a n t i t a t i v e measurement f o r t h e molecu le i n t h e a p p r o p r i a t e phase .

Phase S o l u b i l i t y A n a l y s i s - See S e c t i o n 5 .1 f o r d i s - c u s s i o n of a phase s o l u b i l i t y s y s t e m f o r dexamethasone .

L iqu id -L iqu id E x t r a c t i o n - S e p a r a t i o n of i n t a c t a d r e n o c o r t i c o s t e r o i d s from a c i d i c decompos i t ion p r o d u c t s h a s been accompl i shed by p a r t i t i o n i n g a sample be tween a n e u t r a l o r s l i g h t l y a l k a l i n e aqueous phase and ch lo ro fo rm. 3 1 y 39 v e r y u s e f u l d i s c u s s i o n of t h e i n t e r p r e t a t i o n o f d i f f e r e n c e s n o t e d f o r s t e r o i d c o n t e n t of s amples o b t a i n e d by B . T . , P o r t e r - S i l b e r , I N H and U . V . a s s a y i s g i v e n i n Refe rence 39.

A

Column Chromatography - The u s e of h i g h p r e s s u r e l i q u i d chromatography as a c o n v e n i e n t s t a b i l i t y i n d i c a t i n g a s s a y p rocedure f o r t h e a n a l y s i s o f c o r t i c o s t e r o i d c reams and o i n t m e n t s w a s r e c e n t l y d e m o n s t r a t e d . 5 2 show a sample chromatogram o b t a i n e d f o r dexamethasone u s i n g a column of 6, R l - o x y d i p r o p i o n i t r i l e on Zipax and 1% e t h a n o l i n hexane as a mob i l e p h a s e a t 500 p . s . i . ( e l u t i o n t i m e -11 m i n u t e s ) . A g e n e r a l i z e d sys t em f o r t h e p r e d i c t i o n o f

The a u t h o r s

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of elution curves for corticosteroids based on partition coefficients for a hexane-chloroform-dioxane-water (90-10- 40-5) solvent system on diatomaceous earth was also des- ~ r i b e d . ~ ~ The authors give data for betamethasone. A column partition system has been described by Graham which can effectively trap the corticosteroid in an acetonitrile layer on a diatomaceous earth column whi1.e interferences are removed by washing with heptane. 54 costeroids are then removed from the column with chloroform. The method may be modified readily for the removal of acidic, basic, and/or water soluble interferences. Data are given for both formulated and unformulated dexamethasone.

Acetonitrile and corti-

Paper Chromatography - Johnson and Fowler give Rf data for dexamethasone and related steroids utilizing the following system: 55

Stationary Phase: Whatman No. 1 impregnated with 40% V / V formamide in methanol.

Mobile Phase: Saturated solution of formamide in

Mode of Operation: Descending development for 35 cm

Detection: Diphenyl styryl phenyl tetrazolium t

chloroform.

in a saturated tank.

heat = violet spots for steroids.

Compound - R f

Betamethasone .16 Cortisone .62 Dexamethasone .21

Prednisolone .15 Prednisone .55

Hydrocortisone .26

Thin Layer Chromatography (TLC) - U.S.P. XVIII utilizes TLC as both a qualitative and quantitative assay procedure for dexamethasone. l 3 N . F . XI11 employs TLC as an identity test for dexamethasone in official dosage forms. ’‘ Wahba has demonstrated the utility of quantitative TLC as a stability indicating approach to the estimation of dexamethasone in aged samples of experimental tablet formulations.” There is little doubt that TLC offers the analyst

EDWARD M. COHEN

an e x c e l l e n t h i g h l y s e l e c t i v e method, u s u a l l y wi thou t re- q u i r i n g any d e r i v a t i z a t i o n , f o r a s s e s s i n g t h e i n t e g r i t y of dexamethasone i n common pha rmaceu t i ca l m a t r i c e s . Thin l a y e r chromatography can o f t e n f u n c t i o n as a pr imary o r r e f e r r a l a s s a y p rocedure t o c o r r o b o r a t e t h e v a l i d i t y of a non-chromatographic a s s a y p rocedure . Some of t h e r e p o r t e d TLC d a t a f o r dexamethasone, i n a d d i t i o n t o t h e i n f o r m a t i o n i n t h e U.S.P. and N.F., are g iven i n Table 11. Other TLC systems f o r dexamethasone a r e d i s c u s s e d i n References 1 7 , 59 and 60. Of s p e c i a l i n t e r e s t i s a r e p o r t which d e s c r i b e s t h e s e p a r a t i o n of dexamethasone from betamethasone.61

Gas Chromatography - S t e r o i d s p o s s e s s i n g t h e C-17 dihydroxyacetone s i d e c h a i n u s u a l l y undergo molecu la r a l t e r a t i o n a f t e r a p p l i c a t i o n t o GLC columns t o y i e l d as a major p roduc t t h e co r re spond ing 1 7 - k e t o s t e r o i d . 6 2 Approaches t o a t t a i n q u a n t i t a t i v e conve r s ion t o a s u i t a b l e n o n - l a b i l e d e r i v a t i v e of s t e r o i d s r e l a t e d t o dexamethasone have been r e c e n t l y d e s c r i b e d by B a i l l i e and co-workers. 6 3 One of t h e most s t a b l e d e r i v a t i v e s s u i t a b l e f o r gas chromatography i s t h e 20-0-methyloxime-17,2l-trimethylsilyl ether of t h e a d r e n o c o r t i c o s t e r o i d .

6 . Metabolism and Pharmacokinet ics

Normal human s u b j e c t s g i v e n 1.6 mcg. of 1,2,4-3H dexamethasone o r a l l y e x c r e t e d about 15% of t h e t o t a l r ad io - a c t i v i t y i n t h e u r i n e w i t h i n f o u r hour s a f t e r admin i s t r a - t i o n . 6 4 Approximately 50% of t h e e x c r e t e d r a d i o a c t i v i t y was i n con juga ted form, presumably a g lucuron ide and 50% of t h e e x c r e t e d r a d i o a c t i v i t y was i n non-conjugated form. The u r i n a r y l e v e l s of t o t a l and con juga ted dexarnethasone i n p a t i e n t s on c h r o n i c d ipheny lhydan to in the rapy were s i g - n i f i c a n t l y i n c r e a s e d compared t o the l e v e l s shown by normal human s u b j e c t s .

I n a n o t h e r s t u d y , t h e mean r ecove ry of u r i n a r y r a d i o a c t i - v i t y a f t e r f o u r hour s and 24 hour s was 16% and 64%, re- s p e c t i v e l y € o r doses of 0.5 t o 1 .5 mg. of l a b e l e d dexamethasone a d m i n i s t e r e d i n t r a v e n o u s l y as a s o l u t i o n i n s a l i n e t o human s u b j e c t s . 6 0 The major pathway of dexamethasone metabolism, f o l l o w i n g I . V . a d m i n i s t r a t i o n , a p p e a r s t o i n v o l v e fo rma t ion of p o l a r unconjugated d e r i v a t i v e s . A

190

TABLE I1

Silica Gel G

Solvent: Rf:b 1.00' .;2d . 51d 1.40d .EOd . i5d . t 5d .:2d .50d .67 Reference: 57 58 58 58 58 58 58 58 58 58

a

J d A C D I a

A = Methylene Ch1oride:dioxane:water (100:50:50) - lower layer; B = Chloroform-ethanol (9:l); C = Chloroform-90% methanol (9:l); D = Cyclohexane-ethyl acetate (1:l); E = Chloroform-acetone (9 : 1) ; F = Chloroform-acetone ( 4 : l ) ; G = Cyclohexane-chloroform-acetic acid (7:2:1); H = Methylene chloride-acetone ( 4 : 1) ; I = Chloroform-acetic acid (9:l); J = Kethylene chloride-acetic acid (9: 1).

bDexamethasone separated from other related steroids. Reference 58 also includes TLC data for dexamethasone on aluminum oxide and magnesium silicate stationary phases as well as useful information on detection system.

Relative to hydrocortisone = 1.00. C

dRelative to cortisone = 1.00.

EDWARD M. COHEN

t% of 252 minutes was no ted f o r t h e e l i m i n a t i o n of i n t a c t dexamethasone from plasma.

I t i s a n t i c i p a t e d t h a t development of a p p r o p r i a t e s e n s i t i v e a s s a y s f o r dexamethasone w i l l e n a b l e s t u d i e s i n human sub- j e c t s t o be c a r r i e d ou t u s i n g conven t iona l pha rmaceu t i ca l dosage forms, made w i t h “co ld” dexamethasone, t o y i e l d a d d i t i o n a l i n f o r m a t i o n on t h e o r a l b i o a v a i l a b i l i t y of dexamethasone. Cur ren t a n a l y t i c a l methodology f o r dexamethasone i s n o t s u f f i c i e n t l y s e n s i t i v e t o a s say a t t h e nanogram p e r m l . l e v e l , which r e p r e s e n t s expec ted plasma concen t r a - t i o n s fo l lowing o r a l a d m i n i s t r a t i o n of t h e r a p e u t i c doses o f dexamethasone. Techniques such as r a d i o i m m u n ~ a s s a y ~ a r e l i k e l y t o p rov ide t h e a s s a y s e n s i t i v i t y and s e l e c t i v i t y re- q u i r e d f o r t h e s e s t u d i e s i n t h e p re sence of t h e normal s t e r o i d background i n b i o l o g i c a l f l u i d s .

A procedure f o r t h e d e t e c t i o n and i d e n t i f i c a t i o n of dexamethasone i n h o r s e u r i n e h a s been d e s c r i b e d and h a s a sen- s i t i v i t y l i m i t of 2 mcg. p e r m l . o f u r i n e t aken f o r a n a l y s i s . 5’

192

DEXAMETHASONE


1. "Merck Index" , E i g h t h Ed. , Merck and Co. , I n c . , Rahway, N . J . , 1968 , p . 334.

2 . B i l e s , J . A . , J. Pharm. K., 50, 464 (1.961).

3. McCauley, J., Merck Sha rp and Dohme R e s e a r c h Labora- t o r i e s , Rahway, N.J., P e r s o n a l Communicat ion.

4 . Mesley , R . J., S p e c t r o c h i m . G., 22, 889 ( 1 9 6 6 ) .

5. Mesley , R . J . , and J o h n s o n , C . A . , J. Pharm. Pharma- __ c o l . , 17, 329 ( 1 9 6 5 ) .

6 . C l a r k e , E. G. C . , " I s o l a t i o n and I d e n t i f i c a t i o n s of Drugs", P h a r m a c e u t i c a l P r e s s , London, p . 287 , 737 (1969) .

7. Doug las , A . W. and S i n g l e t o n , B . , Merck S h a r p and Dohme Resea rch L a b o r a t o r i e s , Rahway, N . J . , P e r s o n a l Com- m u n i c a t i o n .

8 . Nouder t , H. and Ropke, H . , " A t l a s o f S t e r o i d S p e c t r a " , S p r i n g e r - V e r l a g , New York, 6 2 1 ( 1 9 6 5 ) .

9 . B r i t i s h Pha rmacope ia , P h a r m a c e u t i c a l P r e s s , London, p . 295 ( 1 9 6 8 ) .

10. McCauley, J., Merck Sha rp and Dohme R e s e a r c h Labora- t o r i e s , Rahway, N.J., P e r s o n a l Communicat ion.

11. Downing, G . V . , Merck Sha rp and Dohme R e s e a r c h Lab- o r a t o r i e s , Rahway, N . J . , P e r s o n a l Communicat ion.

1 2 , Sadge , W., Riegelman, S . and .Johnson, L . F . , S t e r o i d s , 17, 595 ( 1 9 7 1 ) .

13. U .S .P . X V I I I , Mack P u b l i s h i n g Co. , E a s t o n , P a . , 1 7 4 (1970) .

14 . Dempski, K. E., P o r t n o f f , J . 13. and Wase, A . I d . , ~- J Pharm. S c i . , E, 579 (1969) . _c__

EDWARD M. COHEN

15. Kabasaka l i an , P . , B r i t t , E . , and Yudis , M. D . , 2. -- Pharm. S c i . , 55, 642 (1966).

16. Smith, G . , Merck Sharp and Dohme Research L a b o r a t o r i e s Rahway, N . J . , P e r s o n a l Communication.

17. Wahba, S . K . , Amin, S. W . and R a f a e l , N., J. Pharm. - S c i . , 57, 1231 (1968).

18. Lodge, B. A. and T o f t , P . , J. Pharm. S&., 2, 1045 (1970).

19. Lodge, B. A. and T o f t , P . , J. Pharm. Pharmacol. , 23, 196 (1970).

20. Albers-Schonberg, G . , t ierck Sharp and Dohme Research L a b o r a t o r i e s , Rahway, N . J . , P e r s o n a l Communication.

2 1 . F i e s e r , L. F. and F i e s e r , M. , " S t e r o i d s " , Reinhold P u b l i s h i n g Co., New York, p. 694-695 (1959).

22. Ar th , G . E . , e t a l . , 2. Am. E. =., 80, 3161 (1958).

23. Ar th , G . E . , U.S. P a t e n t #3,375,261 (1968) .

24. O l i v e t o , E. P . , e t a l . , 2. &. Chem. SOC., 80, 4431 (1958).

25. Mertel, H. E . , Ge rbe r , A. N. and Meriwether , H . T., J. Labeled Compounds, V I , 250 (1970).

26. Barton, D. H. R. and T a y l o r , W. C . , J. Chem. SOC. , 2500 (1968).

27. Smith, G. B . , e t a l . , J. Pharm. s., 61, 708 (1972) .

28. Bush, I . E. and Mahesh, V . B . , Biochem. J., 69, 9 (1958) .

29. Brenner , G . , e t a l . , Angew, Chem. (Engl. E d . ) , 8, 975 (1969).

194

DEXAMETHASONE

3 0 . W e n d l e r , N. L . , " M o l e c u l a r R e a r r a n g e m e n t s " , e d i t e d b y P. Mayo, I n t e r s c i e n c e , New Y o r k , P a r t ' h o , 1 0 6 8 ( 1 9 6 7 ) .

31. Gut tman, D . E. a n d Meister, P. O . , J. Am. Pharm. - - - A s s o c . s. Ed. , 4 7 , 773 ( 1 9 5 8 ) .

32. C o n b e r e , J . P . , U . S . P a t e n t 8 3 , 7 7 3 , 0 7 7 ( 1 9 5 6 ) .

33. Wendler , 9. e., p . 1119.

34. S m i t h , L . L . , e t a l . , J. A". e. z., 82, 4676 (1960).

35. Meister, P. D . , -- e t a l . , 2. E. Pharm. A S S O C . , s. Ed. 4 7 , 576 ( 1 9 5 8 ) . -

36. F i e s e r , L . F. a n d F i e s e r , M . , &. c&., p . 6 9 7 .

37 . U n p u b l i s h e d Data, Merck S h a r p a n d Dohme R e s e a r c h L a b o r a t o r i e s , West P o i n t , P a .

38. Z a b r i s k i e , J . , P e r s o n a l Communica t ion , Merck S h a r p a n d Dohme, West P o i n t , P a .

39 . Graham, R. E . , W i l l i a m s , P . A . , and K e n n e r , C. T . , - - - J . Pharm. S c i . , 59, 1 1 5 2 ( 1 9 7 0 ) .

40 . C h a f e t z , L . , J. Pharrn. S c i . , 60, 335 ( 1 9 7 1 ) . --

41. C h a f e t z , L . , T s i l i f o n i s , D . C . a n d R i e d l , J . M., 2 Pharm. S c i . , 61, 1 4 8 ( 1 9 7 2 ) . _ _ _ -

42. Avdonovich , H. W . , Hanbury , P . a n d Lodge, B. A . , 2. Pharm. S c i . , 2, 1 1 6 4 ( 1 9 7 0 ) . --

43. Imr6n6, B . a n d M i h a l y n e , M . , G y o g y s z . , 14, 97 ( 1 9 7 0 ) .

44 . K a b a s a k a l i a n , P. a n d M c G l o t t e n , J . , J. 5. e. SOf., - 78, 5032 ( 1 9 5 6 ) .

4 5 . T a i s h o , K . T . , e t a l . , Chem. Pharm. B u l l . , 20, 5 8 9 ( 1 9 7 2 ) .

105

EDWARD M. COHEN

46. F o r i s t , A. A. and Johnson, J. L . i n "Pha rmaceu t i ca l Ana lys i s " , ed . H iguch i , T. and Brochmann-Hanssen, E r , I n t e r - s c i e n c e , New York, 1961, pp. 71-85.

47. Hamlin, W . E . , et &., J . &. Pharm. ASSOC. , g. Ed., - 49, 253 (1960) .

48. Brower, J . F . , J. Ass. O f f i c . Anal. Chem., 52, 842 (1969).

49.

50. (1962

51. - 5 , 26

52.

Woodson, A . L . , J. Pharm. S&., 60, 1539 (1971).

Bellomonte, G . , m. &. Super. S a n i t a , 25, 581 ; Chem. A b s t . , 58, 12370h, 1962. _L-

Noujaim, A . A. and J e f f e r y , D. A . , Can. 2. Pharm. g. (1970).

M o l l i c a , J . A. and S t r u s z , R. F . , 2. Pharm. g., 5, 444 (1972).

53. Weber, D. J . , e t a l . , J. Pharm. s., 61, 689 (1972) .

5 4 . Graham, R. E . , e t a l . , 2. P h a r m . S c i . , 2, 1473 (1970) --

55. Johnson, C . A . and Fowler, S . , J . Pharm. Pharmacol . , - 16 , Supp. 17T (1964).

56. N.F. X I I I , American Pharm. A S S O C . , Washington, 1970, pp. 208-209.

57. H o l l , A . , J . Pharm. Pharmacol. , 16, Supp. 9T (1964) .

58. K i r s c h n e r , J. G . , "Thin-Layer Chromatography", I n t e r - s c i e n c e , New York, 1967, pp. 596-599.

59. Moss, M. S . and Rylance, H. J . , J. Pharm. Pharmacol . , 18, 13 (1966).

60. Haque, N . , e t a l . , 2. C l i n . Endocr . , 34, 44 (1972) .

61. Scavino, C . and Chiaramonti , D . , J. Pharm. B e l g . , 20, 204 (1965).

DEXAMETHASONE

62. VandenHeuvel , W. J. A . i n "Theory and A p p l i c a t i o n s o f Gas Chromatography in I n d u s t r y a n d M e d i c i n e " , e d . b y Kroman, H . S . and B e n d e r , S . R . , G r e e n e a n d S t r a t t o n , N e w York, 1 9 6 8 , p p . 120-134.

6 3 . B a i l l i e , T. A., e t a l . , A n a l . Chem., 44, 30 ( 1 9 7 2 ) . _ _ _ _ I

6 4 . J u b i z , W . , e t a l . , New E x . - J. x., x, 11 (1970). 65. I l i d g l e y , A . K. and N i s w e n d e r , G . D . , Acts E n d o c r i n o l .

S u p p l . , 1 4 7 . 320 ( 1 9 7 0 ) .

6 6 . Chayen, R., e t al., G. Biocheni . , 39, 5 3 3 ( 1 9 7 1 ) .

DIOCTYL SODIUM SULFOSUCCINATE

Sut Ahuja and Jerold Cohen

Reviewed by J . Kazan and T. E. Ricketts

199

S. AHUJA AND J. COHEN

CONTENTS

1.

2 .

3. 4 . 5 . 6.

7. 8.

1.

Description 1.1 Name, Formula, Molecular Weight, Elemental

1.2 Appearance, Color, Odor Physical Properties 2.1 Infrared Spectrophotometry 2.2 Nuclear Magnetic Resonance 2.3 Mass Spectrometry 2 . 4 Differential Thermal Analysis 2.5 Thermogravimetric Analysis 2.6 Differential Scanning Calorimetry 2.7 Solubility 2.8 Solubilization 2.9 2.10 Crystal Properties Synthesis Stability Met abu 1 ism Methods of Analysis 6 . 1 Titrimetric Analysis 6.2 Colorimetric Analysis 6 . 3 Infrared Analysis 6 . 4 Turbidimetric Analysis 6 . 5 Determination of Micellar Weight 6.6 Thin-Layer Chromatography References Acknowledgements

Composition

Effect On Surface Tension of Liquids

DESCRIPTION

1.1 Name, Formula, Molecular Weight, Elemental Composition Chemically, dioctyl sodium sulfosuccinate is sul-

fosuccinic acid bis(2-ethylhexyl) ester S-sodium salt or sodium 1, 4-bis(2-ethylhexyl) sulfosuccinate o r bis(2-ethylhexyl) sodium sulfosuccinate. It is also known as sodium dioctyl sulfosuccinate; DSSj Aerosol OT; Alphasol OT; Co- lace; Complemix; Coprol; Dioctylal; Dioctyl-Medo Forte; Diotilan; Diovac; Disonate; Doxinate; Doxol; Dulsivac; Molatoc; Molofac; Nevax; Norval; Regutol; Softili; Solusol; Sulfimel DOS; Vatsol OT; Velmol and Waxsol (1). It has a molecular weight of 444.57 (C20H3707SNa).

200


0

// CH2-C. C2H5

I O-CH2-CH(CH2)3CH3

\

I O-CH2 -CH (CH2) 3CH3 N a03S -CH-/ I

% C2H5 0

The t h e o r e t i c a l e l e m e n t a l compos i t ion of d i o c t y l s u l f o s u c - c i n a t e i s a s fo l lows :

Carbon 54.03% Hydrogen 8.39% Oxygen 25.19% S u l f u r 7.21% Sodium 5 . 1 7 %

1 .2 Appearance, Color , Odor D i o c t y l sodium s u l f o s u c c i n a t e i s g r a y i s h t o w h i t e ,

wax-l ike, p l a s t i c s o l i d , hav ing a c h a r a c t e r i s t i c odor sug- g e s t i v e of o c t y l a l c o h o l .

2 . PHYSICAL PROPERTIES

2 . 1 I n f r a r e d Spec t ropho tomet ry The i n f r a r e d spectrum ( 2 ) o f a 5% ch lo ro fo rm s o l u -

t i o n o f d i o c t y l sodium s u l f o s u c c i n a t e r eco rded w i t h a Pe r - kin-Elmer I n f r a r e d Spec t ropho tomete r , Model 225 i s shown i n F i g u r e 1. Band a s s ignmen t s f o r t h e spectrum i n 5% c h l o - roform s o l u t i o n a r e l i s t e d i n Tab le I .

2 . 2 Nuc lea r Magnet ic Resonance The NMR spectrum ( 3 ) of d i o c t y l sodium s u l f o s u c -

c i n a t e ( F i g u r e 2 ) was r eco rded i n DMSO-db on a v a r i a n A60 ~ O M H Z NMR s p e c t r o m e t e r . S t r u c t u r a l a s s ignmen t s f o r t h e NMR s i g n a l s a r e t a b u l a t e d i n T a b l e 11.

2 . 3 Mass Spec t romet ry R e l i a b l e mass s p e c t r a l d a t a ( 4 ) cou ld n o t be ob-

t a i n e d f o r d i o c t y l sodium s u l f o s u c c i n a t e a s t h e spectrum changes w i t h t empera tu re , i n d i c a t i n g the rma l d e g r a d a t i o n .

20 1

S. A

HU

JA A

ND

J. CO

HE

N

rl h

w

0

C

0

.PI u 3

rl

0

v)

H

W

202

FIGURE I. I n f r a r e d Spectrum of a 5% chloroform s o l u t i o n of Dioc ty l Sodium Su l fosucc ina te

. .. .. . ~~ ..

FIGURE 2. Nuclear Magnetic Resonance Spectrum in DMSO-d6 of Dioctyl Sodium Sulfosuccinate


TABLE I

INFRARED BAND ASSIGNMENTS FOR DIOCTYL SODIUM SULFOSUCCINATE

I N 5% CHLOROFORM SOLUTION

W av enumb e r ( cm- A s s ignmen t

2960, 2935, 2865 1728 1465 1391, 1381 1245 1200 1048

a l i p h a t i c C H 2 ; CH3 C = O a l i p h a t i c C H 2 ; CH3

C H 3 asymmetric C-0-C ( e s t e r ) asymmetric s o 3 8 symmetric C-0-C ( p o s i t i o n ) co r re sponds t o C-O-CH2 a n d / o r symmetric S O ~ B

2 . 4 D i f f e r e n t i a l Thermal A n a l y s i s The thermal a n a l y s i s (5) of d i o c t y l sodium s u l f o -

s u c c i n a t e was conducted on a Du Pont Model 900 the rma l an- a l y z e r i n a n i t r o g e n atmosphere. A t h e a t i n g r a t e s of 1 0 ° C / minute and 3"C/minute, t h e thermogram ( F i g u r e 3) shows no endotherm, b u t an i r r e g u l a r c u r v e a f t e r 120°C. No t r a n s i - t i o n was ohserved excep t foaming of t h e sample.

2 .5 Thermogravimetr ic A n a l y s i s A sample o f d i o c t y l sodium s u l f o s u c c i n a t e ( F i g -

u r e 4) loses weight (5) c o n t i n u o u s l y above 35°C: 3.4% between 35°C and 152"C, approx ima te ly 0.5% between 152°C and 220°C. Rapid we igh t l o s s occur s above 220"C, which amounts t o approx ima te ly 5.8% loss up t o 248°C. A r e s i d u e o f approx ima te ly 15% i s l e f t a t 360°C.

2 . 6 D i f f e r e n t i a l Scanning Ca lo r ime t ry A thermogram run a t lO"C/minute shows two broad

endotherms (5) between 119°C and 194°C ( F i g u r e 5 ) .

2 . 7 S o l u b i l i t y D i o c t y l sodium s u l f o s u c c i n a t e i s s o l u b l e i n t h e

f o l l o w i n g s o l v e n t s : carbon t e t r a c h l o r i d e , pe t ro l eum e t h e r , naphtha, xylene, d i b u t y l p h t h a l a t e , l i q u i d pe t ro l a tum,

204


F I G U R E 3. Differential Thermal Analysis in nitrogen atmosphere of Dioctyl Sodium Sulfosuccinate

Smn - w h

t - m

* " O 50 (00 110 Z!O 2SO 300 '350 400 4W WO'

' ' 1 *C lcomcmo ton CllnaMtL U W l L ?nEI*ocouKcs~

F I G U R E 4 . Thermogravimetric Analysis of Dioctyl Sodium Sulfosuccinate

205


1. *C (CORRECTED FOR CHROMEL ALUMEL THERYOCOUPLESI

FIGURE 5. Differential Scanning Calorimetry thermogram of Dioctyl Sodium Sulfosuccinate

206


TABLE I1

NMR SPECTRAL ASSIGNMENTS FOR DIOCTYL SODIUM SULFOSUCCINATE

Number of (ppm) M u l t i p l i c i t y P ro tons A s s ignmen t

H -

00 c€3 - c-’o 2 . 8 - 3 . 1 undetermined 2 I

S-CH-C:’ 0

3.35 s i n g l e t 1.8 E20

3 .6 -4 .1 undetermined 5 S -CH -C- 0- CFl2 - lo

g l y c e r o l , p i n e o i l , o l e i c a c i d , ace tone , ke rosene , a l c o h o l , benzene, d i a c e t o n e a l c o h o l , methanol, e t h a n o l , i sop ropano l , sec. b u t a n o l , methyl a c e t a t e , e t h y l a c e t a t e , amyl a l c o h o l , f u r f u r y l a l c o h o l , t e t r ahydro f u r f u r y l a l c oho 1, PO l y e thy 1 ene g l y c o l , c o r n o i l , and v e g e t a b l e o i l s (1 ) . Table 111 p r e - s e n t s t h e s o l u b i l i t y of d i o c t y l sodium s u l f o s u c c i n a t e i n s e l e c t e d s o l v e n t s . The s o l u b i l i t y f i g u r e s shown do n o t i n d i c a t e t h e l i m i t s of s o l u b i l i t y , s i n c e d i o c t y l sodium s u l f o s u c c i n a t e appea r s t o be ve ry h i g h l y s o l u b l e i n most o r g a n i c s o l v e n t s ( 6 ) .

207

TABLE I11

SOLUBILITY OF DIOCTYL SODIUM SULFOSUCCINATE (DS S ) I N SOME ORGANIC SOLVENTS$:

S p e c i f i c G r a v i t y V i s c o s i t y g/ml a t 25°C DSS i n S o l u t i o n O f

D i s s o l v i n g R e s u l t i n g P e r c e n t S o l u t i o n Method of S o l u t i o n L iqu id S o l u t i o n g/100 m l by Weight ( P o i s e s )

!n D I

> > z 0

0

Disso lved a t Carbon T e t r a c h l o r i d e 1.55 1 . 3 1 73 .8 56 .4 1 . 6 5

Heated t h e n S o l v e n t Naphtha 0.864 0 . 7 0 1 7 0 . 5 69 .8 0 . 6 5 D i b u t y l P h t h a l a t e 1 .03 1 .07 70 .7 6 6 . 1 8 .84 P a r a f f i n O i l

Room Temp. Petroleum E t h e r 0. 688 0.950 70.1 75 .0 0.65 5

0 . 8 8 1 1 .00 69.5 69 .5 1 2 . 9 f-

s

1 3

oc c Cooled

rn 2

7’: R e p r i n t e d by t h e c o u r t e s y of American Cyanamid Co


D i o c t y l sodium s u l f o s u c c i n a t e d i s s o l v e s s l o w l y i n c o l d w a t e r , b u t i t s s o l u b i l i t y i n w a t e r i n c r e a s e s w i t h an i n - c r e a s e i n t e m p e r a t u r e , as i n d i c a t e d i n T a b l e I V .

T a b l e V p r e s e n t s t h e c o n c e n t r a t i o n o f e l e c t r o l y t e s o l u - t i o n i n which 1% of d i o c t y l sodium s u l f o s u c c i n a t e i s s o l u - b l e a t a t e m p e r a t u r e o f 25°C.

TABLE I V

SOLUBILITY OF DSS I N WATER9c

T e m p e r a t u r e "C

25 30 40 50 60 70

Grarns/100 r n l o f Water

1 . 5 1 . 8 2 . 3 3 . 0 4.0 5 . 5

R e p r i n t e d by t h e c o u r t e s y o f Amer ican Cyanamid Co.

~~ ~- TABLE V

SOLUBILITY OF 1% DSS I N ELECTROLYTE SOLUTIONS;':

C o n c e n t r a t i o n o f E l e c t r o l y t e s , %

NaCl NH4CL (NH4)2 HP04 NaN03 N a 2 SO4 Ap pe a r anc e o f S o l u t i o n

0 . 5 0 . 5 2 . 0 ;'< :,: 0 . 5 1.0 C l e a r 3.0 2.0 3.0 1 .0 3 . 0 T u r b i d

;': R e p r i n t e d by t h e c o u r t e s y of Amer ican Cyanamid Co. S 1 i g h t 1 y t u r b i d _LJ_ ,. I~


2.8 S o l u b i l i z a t i o n T a b l e V I p r e s e n t s t h e amount of s o l u b i l i z i n g a g e n t

( s o l v e n t ) n e c e s s a r y t o produce 10% and 25% c l e a r s o l u t i o n s (w/w) i n t h e s o l v e n t and wa te r a t room t empera tu re . The v a l u e s r e p r e s e n t t h e amount of DSS and s o l v e n t t o be added.

TABLE V I

SOLVENTS CAPABLE OF SOLUBILIZING AQUEOUS SOLUTIONS OF DSS”

S o l v e n t Required S o l v e n t Required For 10% S o l u t i o n , Fo r 25% S o l u t i o n ,

S o l v e n t % By Weight % By Weight

Acetone 17 .5

( c l e a r t h i c k g e l ) E t h y l a l c o h o l 15.0 2 - But ano 1 11.5 Bu t y 1 12.0 Bu ty l c e l l u s o l v e ~ ~ ” 1 2 . 0 Diace tone a l c o h o l 18.0 D i e t h y l e n e g l y c o l 15 .0 E thy l l a c t a t e 10.0 F u r f u r y l a l c o h o l 7 . 4

Amy1 a l c o h o l 5 . 9

c a r b i t o 1 $cgc

I s o p r o p y l a l c o h o l 1 7 . 5 Methanol 1 2 . 5 Methyl a c e t a t e 1 5 . 0 P i n e o i l Not s a t i s f a c t o r y Synosol”?; s o l v e n t 17.5 T e t r a h y d r o f u r f u r y l a l c o h o l 9 . 9

12 .0

15.0 1 5 . 0 1 5 . 0 12.0

-

- 15.0 13.0 15.0 5 .0

15.0

Repr in t ed by t h e c o u r t e s y o f American Cyanamid Co. Trademark, Ca rb ide and Carbon Chemicals Company J- 4-

I . I.

D i o c t y l sodium s u l f o s u c c i n a t e c a n a c t a s a solu- b i l i z i n g agen t and can a l s o be a c t e d upon by o t h e r s o l u b i - l i z i n g a g e n t s ( 7 ) . D i o c t y l sodium s u l f o s u c c i n a t e can b r i n g i n t o s o l u t i o n , i n water, m a t e r i a l s which are normally i n -

210


s o l u b l e o r o n l y s p a r i n g l y s o l u b l e ( e . g . o r g a n i c a c i d s , d y e s , p r o t e i n s , g e r m i c i d e s , gums, e t c . ) . Good s o l u b i l i z a - t i o n c a n b e a c h i e v e d b y e n s u r i n g t h a t DSS i s u n i f o r m l y d i s - t r i b u t e d t h r o u g h o u t t h e b u l k o f p a r t i c l e s t o b e d i s s o l v e d , The p r o p o r t i o n o f DSS u s e d s h o u l d p r e f e r a b l y b e be tween 0.1-1.0% of t h e m a t e r i a l t o be d i s s o l v e d ( 6 ) . The e x a c t mechanism o f s o l u b i l i z a t i o n i s n o t known. However, i t a p p a r e n t l y i n v o l v e s m i c e l l a r a c t i v i t y ( 8 - 1 1 ) .

2 . 9 E f f e c t On S u r f a c e T e n s i o n o f L i q u i d s DSS l o w e r s t h e s u r f a c e t e n s i o n o f l i q u i d s e . a . -

w a t e r and v a r i o u s e l e c t r o l y t e s o l u t i o n s . The e f f e c t o f DSS on s u r f a c e t e n s i o n may b e measured by a v a r i e t y o f me thods ; however , t h e P e n d a n t Drop Method ( 1 2 ) seems t o b e most s a t i s f a c t o r y f o r m e a s u r i n g c h a n g e s i n s u r f a c e t e n s i o n . The e f f e c t o f c o n c e n t r a t i o n o f DSS on s u r f a c e t e n s i o n a s d e t e r m i n e d by P e n d a n t Drop Method i s shown i n T a b l e V I I .

TABLE VII

SURFACE TENSION OF DIOCTYL SODIUM SULFOSUCCINATE

TEMPERATURE, 25°C. SURFACE AGE, 5 SECONDS" E Y PENDANT DROP METHOD

S u r f a c e T e n s i o n ( d y n e s / c m . ) Concen t r a t i o n 0 . 2 5 % 0 . 5 % 1.0% 2 . 0 %

DSS % S o l i d s Wate r NaCL N a C l Na2S04 Na2SOq

0 0 . 0 0 1 0 . 0 2 0 . 1 0 . 2 5 0 . 5 1.0 2 . 0

7 2 . 0 6 2 . 8 3 8 . 9 2 8 . 7 2 8 . 5 2 7 . 5 2 6 . 0

C 1 oudy

- - 5 2 . 4 4 0 . 1 2 6 . 3 2 5 . 3 2 4 . 9 2 4 . 8 2 4 . 3 2 5 . 2 2 5 . 3 2 5 . 5

Cloudy Cloudy Cloudy Cloudy

7 2 . 5 7 2 . 8 4 2 . 0 41 .5 2 5 . 9 2 6 . 0 2 4 . 6 2 5 . 2 2 5 . 6 2 5 . 4 2 5 . 2 2 5 . 2

Cloudy Cloudy Cloudy C loudy

;'< R e p r i n t e d by t h e c o u r t e s y of Amer ican Cyanamid Co

21 1

S . AHUJA A N D J. COHEN

2.10 C r y s t a l P r o p e r t i e s X-ray d i f f r a c t i o n a n a l y s i s (5) of d i o c t y l sodium

s u l f o s u c c i n a t e was c o n d u c t e d on a P h i l i p s N o r e l c o D i f f r a c t - ome te r U n i t ( s c i n t i l l a t i o n c o u n t e r ) , u s i n g t h e f o l l o w i n g c o n d i t i o n s :

R a d i a t i o n : Cu Kol, 35 KV. 15 ma F i l t e r : N i c k e l S c a n n i n g R a t e : 1 / 2 " (20 ) / m i n u t e C h a r t Speed: 1 / 2 i n c h / m i n u t e D i v e r g e n c e S l i t : 1" R e c e i v i n g S l i t : 0 . 0 0 6 i n c h e s S c a t t e r S l i t : lo Time C o n s t a n t : 2 s e c o n d s Coun t s , F u l l S c a l e : 1000 c p s

The r e s u l t s of X-ray d i f f r a c t i o n a n a l y s i s a r e i n T a b l e V I I I ( F i g u r e 6 ) .

TABLE V I I I

X- RAY DIFFRACT ION ANALYSIS

Bragg A n g l e s ( d e g r e e 2 Q) I n t e n s i t y

4 . 2 9 100

Re l a t i v e

3 . SYNTHESIS

S e v e r a l p a t e n t s h a v e b e e n i s s u e d c o v e r i n g t h e p r e p a r a t i o n o f DSS (13, 1 4 ) . The s y n t h e s i s of d i o c t y l sod - ium s u l f o s u c c i n a t e i s g e n e r a l l y accompl i shed by t h e t r e a t - ment of m a l e i c a c i d a n h y d r i d e w i t h 2 - e t h y l h e x a n o l t o p r o - d u c e d i o c t y l m a l e a t e which i s t h e n r e a c t e d w i t h sodium b i - s u l f i t e u n d e r c o n d i t i o n s c o n d u c i v e t o s a t u r a t i o n of t h e o l e f i n i c bond, w i t h s i m u l t a n e o u s r e a r r a n g e m e n t o f t h e b i - s u l f i t e t o t h e s u l f o n a t e s t r u c t u r e (15 ) .

212

DIO

CT

YL

SO

DIU

M S

UL

FO

SU

CC

INA

TE

213

F I G U R E 6. X-Ray Diffraction Analysis of Dioctyl Sodium Sulfosuccinate

S A H U J A A N D J COHEN

CH-CO CHCOOC8H 1 7 'o 2 -e thy lhexano l . I ( e s t e r i f i c a t i o n r

CH-CO Male i c a c i d anhydr ide

NaHS03 ( A d d i t i o n s & '

h

Rearrangement )

CHCOOC8H 17

D i o c t y l ma l e a t e

CH-COOC8H 17 I S03Na

D i o c t y l sodium s u 1 f o succ i n a t e

Calcium, po ta s s ium, magnesium, aluminum hydrox ide , and ammonium s a l t s of d i o c t y l s u l f o s u c c i n a t e have been prepared (16-20) b u t have n o t found a p p l i c a t i o n s comparable t o DSS.

4 . STABILITY D i o c t y l sodium s u l f o s u c c i n a t e i s a f a i r l y s t a b l e

compound; a f t e r 12 weeks of s t o r a g e a t 60°C, no decomposi- t i o n was obse rved by TLC ( 2 1 ) . S i n c e d i o c t y l s u l f o s u c c i n - a t e i s a n es te r , i t can be hydro lyzed i n t h e p r e s e n c e of e i t h e r s t r o n g l y a c i d i c o r s t r o n g l y a l k a l i n e media. Exten- s i v e i n f o r m a t i o n on t h e s t a b i l i t y of DSS a s a w e t t i n g a g e n t i s a v a i l a b l e ( 6 ) . F i g u r e 7 shows t h e s t a b i l i t y of DSS a t v a r i o u s pH v a l u e s . The s t a b i l i t y was measured by Draves T e s t which measures t h e w e t t i n g power i . e . r a t e of s p r e a d i n g and i m b i b i t i o n .

of o t h e r m a t e r i a l s such a s : DSS may be used t o improve t h e p h y s i c a l s t a b i l i t y

a ) a s t a b i l i z e r f o r cocoa b u t t e r i n

b ) improving dye u p t a k e and the rma l s t a b i l i t y

c ) s t a b i l i z a t i o n and c o a g u l a t i o n of

d ) improving t h e s t a b i l i t y of po ly ( v i n y l

beve rages ( 2 2 )

of p o l y a c r y l o n i t r i l e y a r n (23)

Ag B r s o l s ( 2 4 )

a c e t a t e ) emul s ions t o f r e e z i n g and thawing(25)

214


Stable

pH of Bath at 86'F.

1 2 3 4 5 I I I I iL3

Figure 7 . Stability of Solutions of Dioctyl Sodium Sulfosuccinate (0.025% in solution at various pH values)"

"Reprinted by the courtesy of American Cyanamid Co.

215


5. METABOLISM

I n human be ings , d i o c t y l sodium s u l f o s u c c i n a t e i s no t absorbed o r a l t e r e d i n t h e i n t e s t i n a l t r a c t ( 6 ) . Ex- per iments w i t h mice appea r t o confirm t h i s . Furthermore, DSS does n o t a i d i n t h e a b s o r p t i o n of o t h e r m a t e r i a l s i n s o l u t i o n such a s i n s u l i n , h i s t amine , diamino-diphenyl s u l - fone ( 2 6 ) . However, i t enhances t h e p e n e t r a t i o n o f s u l f a - ni lamide, s u l f a t h i a z o l e , s u l f a d i a z i n e and s u l f a p y r i d i n e i n t o t h e cornea, s c l e r a and aqueous humor o f human b e i n g s ( 2 7 ) .

6. METHODS OF ANALYSIS

6 . 1 T i t r i m e t r i c A n a l y s i s D i o c t y l sodium s u l f o s u c c i n a t e can b e assayed by

t i t r a t i o n a g a i n s t s t a n d a r d tebrabutylammonium i o d i d e w i t h bromphenol b l u e a s an i n d i c a t o r , i n t h e p re sence of c h l o - roform ( 2 8 ) .

DSS can a l s o be determined by t i t r a t i o n a g a i n s t ce ty l a lkon ium c h l o r i d e s o l u t i o n wi th bromophenol b l u e as an i n d i c a t o r , i n t h e p re sence of chloroform ( 2 9 ) .

6 .2 C o l o r i m e t r i c Ana lys i s DSS forms a complex w i t h methyl g r e e n which i s

s o l u b l e i n benzene and can be determined spectrophotomet- r i c a l l y a t 615 nm ( 6 ) .

on fo rma t ion of t h e d i o c t y l s u l f o s u c c i n a t e s a l t of methy- l e n e b lue , e x t r a c t i o n of t h e s a l t i n t o chloroform, and s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n of t h e e x t r a c t e d s a l t a t 650 nm ( 3 0 ) .

De te rmina t ion of DSS by r e a c t i o n w i t h b a s i c fuch- s i n t o form a chloroform s o l u b l e complex, which i s measured on a c o l o r i m e t e r h a s been r e p o r t e d (31 ) .

Another method of d e t e r m i n a t i o n of DSS i s based

6 .3 I n f r a r e d A n a l y s i s An I R method of a n a l y s i s f o r DSS h a s been pro-

posed (32 ) . T h i s method i n v o l v e s p r e c i p i t a t i o n of DSS a s i t s barium s a l t w i th barium c h l o r i d e , fol lowed by a n a l y s i s of i t s I R band a t 9 . 5 v.

6 . 4 T u r b i d i m e t r i c Ana lys i s A t u r b i d i m e t r i c method of a n a l y s i s based on a d d i -

t i o n of sodium c h l o r i d e s o l u t i o n t o a DSS s o l u t i o n w i t h

216

DlOCTY L SODIUM SU LFOSUCCI NATE

measurement o f s u b s e q u e n t t u r b i d i t y a t 320 nm h a s b e e n re- p o r t e d ( 3 3 ) .

6 . 5 D e t e r m i n a t i o n o f M i c e l l a r Weight D e t e r m i n a t i o n o f m i c e l l a r w e i g h t s f o r DSS i n anhy-

d r o u s and h y d r o u s h y d r o c a r b o n s o l u t i o n s h a s b e e n made by means o f l i g h t s c a t t e r i n g ( 3 4 ) .

6 . 6 T h i n - l a y e r Chromatography DSS may b e ch romatographed on a S i l i c a G e l G p l a t e

w i t h e t h y l ace ta te /ammonium h y d r o x i d e / e t h a n o l ( 5 0 / 2 0 / 2 0 ) s o l v e n t s y s t e m . D e t e c t i o n i n v o l v e s s p r a y i n g w i t h 50% s u l - f u r i c a c i d f o l l o w e d by h e a t i n g t h e p l a t e a t 120°C f o r 1 h o u r ( 3 5 ) .

7 . REFERENCES

1.

2 .

3.

4.

5 .

6 .

7 .

8 .

9 .

10.

11.

"Merck Index" , 8 t h Ed. , Merck & Co., I n c . , Rahway,

E. G r e g o r i a n , Ciba-Geigy C o r p o r a t i o n , Ards l e y ,

R. G r u l i c h , Ciba-Geigy C o r p o r a t i o n , A r d s l e y , N e w

J . K a r l i n e r , Ciba-Geigy C o r p o r a t i o n , A r d s l e y , New

A . Page , Ciba-Geigy C o r p o r a t i o n , A r d s l e y , N e w York,

DSS-NF, D i o c t y l Sodium S u l f o s u c c i n a t e NF, Amer ican

J . H . Van Ness, U.S. P a t . 3 ,151 ,986 (Monsanto Co . )

A . K i t a h a r a , T . Kobayshi , and T. T o r c h i b a n a ,

A . M . Schwar t z , J . W . P e r r y , and J . Berch, S u r f a c e

New J e r s e y , 1968, p . 382-383.

N e w York, p e r s o n a l communica t ion , 1970.

York, p e r s o n a l communica t ion , 1970.

York, p e r s o n a l communica t ion , 1970.

p e r s o n a l communica t ion , 1970.

Cyanomid Company, P e a r l R i v e r , New York, 1967

1964.

J . Phys . Chem., 66, 363, 1962.

Act ive Agen t s and D e t e r g e n t s , l i s t e d , I n t e r s c i e n c e P u b l i s h e r s , I n c . , N e w York, N e w York, 1958.

T . I s emura , C o l l o i d a l S u r f a c t a n t s . Some P h y s i c s - c h e m i c a l P r o p e r t i e s , 1st Ed., Academic P r e s s , N e w York, N e w York, 1963.

A . G . Ward, C o l l o i d s , T h e i r P r o p e r t i e s and A p p l i c a - t i o n s , 1s t Ed. , I n t e r s c i e n c e P u b l i s h e r s , I n c . , N e w York, New York, 1946.

K. Sh inoda , T . Nakagawa, B. Tamamushi, and


12.

13.

14.

15.

16.

1 7 .

18.

19.

20.

2 1 .

2 2 . 23.

24.

25.

26.

2 7 .

28.

29.

30.

31. 32.

33.

C. R . Ca ry l , and W. P. Er ic , Ind . Eng. Chem., 2,

A . 0. Jaege r , U . S . P a t . 2,176,423 t o American

A . 0. J a e g e r , U . S . P a t . 2,028,091 t o American

"Remington's Pharm Sc i " , 14 th Ed., Mack P u b l i s h i n g

L . J . Klo tz , U. S. P a t . 3,035,973 (Lloyd Bros. , I n c )

E . A V i t a l i s , U . S . P a t . 2,441,341 (American

E . F. Wil l iams, and N . T . Woodberry, U . S . P a t .

P.M. L i sh , H . D . Bryan, and N . H . Mercer, U . S . P a t .

N . H . Mercer, and H.D Bryan, U . S . P a t . 3,155,577

S . Ahuja, Ciba-Geigy Corp . , Ards l ey , New York,

E . B . H o t e l l i n g , F r . P a t . 1,543,204, 1968. Lonza, Ltd. (by Cami l l e Nordmann, P a u l Ha lb ig , and

Roland Dagon), Swiss P a t . 396, 303, 1966. S. M . Levi and T . K . Stepanova, K o l l o i d n Zh. 27

K u n s t h a r s f a b r i e k Syn these N . V V e r f k r o n i c k 29, 108, 1956.

Summary of T o x i c i t y S t u d i e s , 1963, American Cyanamid Co., through r e f e r e n c e 6.

J. G . Bellow, and M. Gutmann, Arch. Oph tha l . 30, 352, 1943.

"Na t iona l Formulary", 1 2 t h Ed., Mack P u b l i s h i n g Co. Easton, Penna. p. 138-139, 1965.

"The Un i t ed S t a t e s Pharmacopeia", 18 th Ed., Mack P u b l i s h i n g Co., Easton, Penna. , p . 206, 1970.

J . D . F a i r i n g and F. R . S h o r t , Anal. Chem., 28, 1 1 8 2 7 , 1956.

G . R. Wall in , Anal. Chem., 2 2 , 616, 1950. S. Ahuja, D. S p i e g e l and S . W i l d s t e i n , Ciba-Geigy

44, 1939.

Cyanamid Co., 1939.

Cyanamid Co., 1936.

Company, Easton, Pennsy lvan ia , p. 805, 1970.

1962.

Cyanamid Co.) , 1948.

3,002,995 (American Cyanamid Co . ) , 1961.

3,155,576 (Mead Johnson & Co.), 1964.

(Mead Johnson & Co. ) , 1964.

unpub 1 i s h e d i n f o r m a t i o n , 19 70.

( l ) , 57-63, 1965.

Corpora t ion , Ardsley, N e w York, unpub l i shed i n f o r - mat ion, 1970.

B o l l . Chim. F a r . , 106, 307, 1967. G. L i n a r i ( 1 s t . Farm. B io l . S t r o d e r , F l o r e n c e ) ,


34. S. G. F rank , and Z o g r a f i , J . Pharm. S c i . , 58, 993,

35. S. Ahuja, Ciba-Geigy Corpora t ion , Ardsley, N e w York 1969.

unpub l i shed in fo rma t ion , 1970.

8 . ACKNOWLEDGEMENTS

The a u t h o r s would l i k e t o thank M r . C. B . Johnson, D r . J . Kazan and M r . T. E . R i c k e t t s of American Cyanamid Co. f o r t h e i r c o o p e r a t i o n i n r ev iewing t h i s manusc r ip t and Mrs. M. Kahn f o r s e c r e t a r i a l h e l p .

FLUOROURACIL

Brircp C. Rudy uiid Bcrnurd Z. Seiikowski

2 2 I


I N D E X

Analy t ica l P r o f i l e - Fluorourac i l

1. Descr ipt ion 1.1 Name, Formula, Molecular Weight 1 . 2 Appearance, Color, Odor

2 . Physical P rope r t i e s 2 . 1 I n f r a r e d Spectrum 2 . 2 Nuclear Magnetic Resonance Spectrum

2 . 2 1 Proton Spectrum 2 . 2 2 19F Spectrum

2 .3 U l t r a v i o l e t Spectrum 2 . 4 Fluorescence Spectrum 2.5 Mass Spectrum 2.6 Op t i ca l Rotat ion 2 . 7 Melting Range 2.8 D i f f e r e n t i a l Scanning Calorimetry 2.9 Thermogravimetric Analysis 2.10 S o l u b i l i t y 2 . 1 1 X-ray Crys t a l P rope r t i e s 2 . 1 2 Dissoc ia t ion Constant

3. Synthes is



6 . Methods of Analysis 6 .1 Elemental Analysis 6.2 Phase S o l u b i l i t y Analysis 6 . 3 Thin Layer Chromatographic Analysis 6 . 4 Gas Liquid Chromatographic Analysis 6 .5 Direc t Spectrophotometr ic Analysis 6 .6 F luor ine Analysis

6 .61 Organica l ly Bound Fluorine Analysis 6.62 Free FLuori.de Analysis

6 . 7 Volumetric Analysis

7 . References

222

FLUOROURACI L

1. Descr ipt ion

1.1 Name, Formula, Molecular Weight F luorourac i l i s 5 - f l u o r o u r a c i l .

Molecular Weight: 130.08 C4H3FN202

1 . 2 Appearance, Color, Odor F luorourac i l i s a white t o p r a c t i c a l l y whi te ,

p r a c t i c a l l y odor l e s s , c r y s t a l l i n e powder.

2 . Physical P roue r t i e s

2 . 1 In f r a red Spectrum (IR) The I R spectrum of re ference s tandard f l u o r o -

u r a c i l * i s shown i n Figure 1 (1 ) . The spectrum of a mineral o i l suspension of f l u o r o u r a c i l between cesium iodide d i s c s was measured on a Perkin Elmer 621 Spec t ro- photometer. The assignments given i n Table I t o t he bands i n Figure 1 (1) a r e i n good agreement wi th t h e assignments made by Brownlie ( 2 ) .

TABLE I

IR Spec t r a l Assignments f o r F luorourac i l

C h a r a c t e r i s t i c o f Frequency (cm-l)

NH s t r e t c h 3124 C=O s t r e t c h 1716 and 1657 CH i n p lane deformation 1245 CH o u t o f p lane deformation 813

*The r e fe rence s tandard f l u o r o u r a c i l r e f e r r e d t o i n t h i s work i s No. 354029.

323

6. C

. RU

DY

AN

D 6

. 2. S

EN

KO

WS

KI

-8 0

-2

c5

T 6 t,

-$! -

-E z

w

-E @

0

-8

224

FLUOROURACI L

2 . 2 Nuclear Magnetic Resonance Spectrum (NMR)

2 . 2 1 Proton Spectrum The NMR spectrum shown i n F igu re 2 was

o b t a i n e d by d i s s o l v i n g 5 6 . 3 mg o f r e f e r e n c e s t a n d a r d f l u o r o u r a c i l i n 0 . 5 m l o f d i m e t h y l s u l f o x i d e - dg c o n t a i n i n g t e t r a m e t h y l s i l a n e a s t h e i n t e r n a l r e f e r e n c e ( 3 ) , The s p e c t r a l ass ignments a r e shown i n Tab le I1 ( 3 ) .

TABLE I1

NMR S p e c t r a l Assignments f o r F l u o r o u r a c i l

No. o f Each Chemical S h i f t Type Proton Proton (PPm) M u l t i p l i c i t y

C-H 1 7 .80 d , ( J H - F = 6 . 5 Hz)

N - H 2 11 .40 s (b)

d= d o u b l e t ; S (b ) = broad s i n g l e t ; J = s p l i t t i n g c o n s t a n t i n Hz

2 .22 I 9 F Spectrum The 1YF spectrum shown i n F igu re 2 was

o b t a i n e d wi th a J e o l c o C-60 H L i n s t rumen t w i t h I 9 F module c r y s t a l modif ied t o a f requency o f 56 .446 MHz. Seventy mg o f r e f e r e n c e s t a n d a r d f l u o r o u r a c i l were d i s s o l v e d i n 0 . 5 m l o f dimethylacetamide c o n t a i n i n g CC13F a s t h e i n - t e r n a l r e f e r e n c e ( 3 ) . The spectrum c o n s i s t s o f a d o u b l e t a t -183 ppm which has a s p l i t t i n g c o n s t a n t ( J F - ) o f 6 . 5 Hz. The c h o i c e of CC13F as t h e i n t e r n a l r e y e r e n c e a long wi th t h e assignment o f -183 ppm i s i n accordance w i t h Bovey ( 4 ) .

2 . 3 U l t r a v i o l e t Spectrum ( U V ) The UV spectrum o f f l u o r o u r a c i l i n a c e t a t e

b u f f e r (pH 4 . 7 ) i n - t h e r eg ion 350 t o 220 nm e x h i b i t s one maximum a t 266 nm (E = 7 . 0 7 x l o 4 ) and one minimum a t 232 nm. The spectrum p r e s e n t e d i n F igu re 3 was o b t a i n e d us ing a s o l u t i o n o f 1 . 0 0 4 mg f l u o r o u r a c i l p e r 100 m l o f pH 4 . 7 a c e t a t e b u f f e r ( 5 ) .

225

F i g u r e 2

m 0

-180 -181 -162 -183 -w -185

, , JL,, , , , 0 -I

pgm

l ~ . i ' ~ " ~ l ' ' " ' " ' " ' " ' " ' " ' " ' " ' " ' " ' " ' " ' I ' ' I I I " I ' ' I ' I " 1 " I " I " I '

n C 0 <

m Tu

FLUOROURACI L

Figure 3

Ultraviolet Spectrum of F l u o r o u r a c i l

NANOMETERS

227


2 . 4 Fluorescence Spectrum Figure 4 shows t h e e x c i t a t i o n and emission

s p e c t r a f o r re ference s tandard f l u o r o u r a c i l from 250 t o 600 nm ( 6 ) . The s p e c t r a , measured i n a methanol s o l u t i o n o f f l u o r o u r a c i l (1.0 mg/ml) us ing a Farrand M K - 1 Spec t ro- f luorometer , showed one e x c i t a t i o n peak a t 315 nm and one emission peak a t 391 nm.

2.5 Mass Spectrum The mass spectrum of f l u o r o u r a c i l shown i n

Figure 5 was obta ineh using a CEC-21-110 mass spec t ro - meter with an ion iz ing energy of 70 eV ( 7 ) .

The spectrum shows a s t rong molecular ion peak a t m/e 130. The peak a t m/e 97 i s due t o t h e loss of HNCO from t h e parent and t h e fragment a t m/e 60 i s formed by a hydrogen rearrangement g iv ing h a l f t h e r i n g with t h e f l u o r i n e s u b s t i t u e n t (C2H3FN) (7 ) .

2.6 Opt ica l Rotat ion F luorourac i l e x h i b i t s no o p t i c a l a c t i v i t y

2 . 7 Melting Range The mel t ing range f o r f l u o r o u r a c i l depends on

the r a t e o f hea t ing . (8) i s used, t h e melt ing po in t l i e s between 280" and 284°C.

When t h e USP XVIII Class I procedure

2 .8 D i f f e r e n t i a l Scanning Calor imetry (DSC) The DSC curve shown i n Figure 6 f o r re ference

s tandard f l u o r o u r a c i l was obta ined ;sing a Perkin E l m e r DSC - 1 B Calor imeter . With a temperature program of 1O0C/min., a mel t ing endotherm was observed s t a r t i n g a t 288.2OC w i t h a AHf = 6 . 7 Kcal/mole. s t a r t i n g a t 298.5OC corresponds t o t h e decomposition o f t he f l u o r o u r a c i l ( 9 ) .

The exotherm

2 .9 Thermogravimetric Analysis (TGA) The TGA performed on re ference s tandard

f l u o r o u r a c i l exh ib i t ed no l o s s o f weight when heated from room temperature t o 1 0 5 O C a t a hea t ing r a t e of lO"C/min. (9 )

228

FL

UO

RO

UR

AC

IL

a(

d

kk

3

c,

M

U

.rl

a, u

.a

(I)

a, U

c a

U

m a

k

0

3

4

LL

AlIS

N31N

I

22Y

6. C. R

UD

Y A

ND

6. 2. S

EN

KO

WS

KI

230

FLUOROURACI L

Figure 6

D. S . C . Curve €or F 1 uorouraci 1

I I I 1 1 I I 1 AHf = 6.7 kcal/mole

I I 250 270 290 310

TEMPERATURE "C

23 1

0 . C. RUDY AND 0 . 2. SENKOWSKI

2.10 S o l u b i l i t y The s o l u b i l i t y d a t a obta ined a t 25°C f o r r e f e r -

ence s tandard f l u o r o u r a c i l i s l i s t e d i n Table 111 (10) .

TABLE 111

Fluorourac i l S o l u b i l i t y

Solvent S o l u b i l i t y (mg/ml)

3A a lcohol benzene chloroform 95% e thanol e thy l e t h e r i sopropyl a lcohol met hano 1 petroleum e t h e r (30" water

3.44 <. 1 c . 1 5.54 < o . 1 2.15 9 .37

.60°) < 0 . 1 12.20

2 . 1 1 Crys t a l P rope r t i e s The x-ray powder d i f f r a c t i o n p a t t e r n o f r e fe rence

s tandard f l u o r o u r a c i l i s presented i n Table I V (11) . The ins t rumenta l condi t ions a r e given below:


General E l e c t r i c Model XRD-6 Spectrogoniometer

Generator : 50 KV 12 .5 MA Tube Targe t : Copper 0

Radiat ion : Cu Ka = 1.542 A Opt ics : 0.1" Detector s l i t

M.R. S o l l e r s l i t 3" Beam s l i t 0.0007" N i f i l t e r 4" t ake o f f angle Scan a t 0.2" 20 p e r minute Amplif ier gain - 16 coarse , 8 . 7 f i n e , Sea led p ropor t iona l counter tube and DC vo l tage a t p l a t e a u , Pulse he ight se- l e c t i o n EL-5 vol t s ;Eu - out Rate meter T . C . 4 , 2000 C/S f u l l s c a l e

Goniometer: Detec tor :

232

FLUOROURACI L

Recorder: Samples :

Char t speed 1" p e r 5 minutes P repa red by g r i n d i n g a t room t emp e r a t u r e

TABLE IV

X-ray D i f f r a c t i o n P a t t e r n o f F l u o r o u r a c i l

2 0

12.84 13.32 15 .78 16 .10 17.72 18.90 19.62 20.38 21.72 22.30 22.72 23.60 24.64 25.28 26.70 27.84 28.46 30.98 31.80 32.72 33.88 34.84 36.20 37.01 37.54 38.72 39.14 42 .88 44.08 45.54 46.52 47.26 50.06 50.78

- 0

dA*

6 .89 6 . 6 5 5.62 5 .50 5 .01 4.70 4.52 4 . 3 6 4 . 0 9 3.99 3 . 9 1 3 . 7 7 3.61 3.52 3 .34 3.20 3.14 2.89 2 .81 2 .74 2 .65 2 . 5 8 2 . 4 8 2 . 4 3 2.40 2 . 3 3 2.30 2 . 1 1 2 .05 1 .99 1 .95 1 .92 1 . 8 2 1 . 8 0

233

I/Io**

2 1 4 4 2 4 2 5

10 10 4 5 5 9 4

28 100

17 8 4

<1 2 2 2

<1 <1 <1 <1 <1 <1 <1 1 1 1


53.56 1 .71 <1 54.28 1 .69 2

nX 2 S i n 0

o f 1 * 00)

*d = ( i n t e r p l a n a r d i s t a n c e )

**I/I, = r e l a t i v e i n t e n s i t y (based on h i g h e s t i n t e n s i t y

2 . 1 2 D i s s o c i a t i o n Constant The pKa's f o r f l u o r o u r a c i l have been determined

s p e c t r o p h o t o m e t r i c a l l y t o be 8 . 0 + 0.1 and 13.0 + 0 . 1 ( 1 2 ) . When t h e pKa's f o r u r a c i l were defkrmined i n a sTmi la r manner, t h e y were found t o be 9 . 4 + 0 . 1 and >13.5. These l a t t e r v a l u e s a r e i n good ag reemen t w i t h t h e pKa's r e p o r t e d by Shugar and Fox f o r u r a c i l (13).

3 . S y n t h e s i s F l u o r o u r a c i l may be p r e p a r e d by t h e r e a c t i o n scheme

shown i n F igu re 7 . E thy l f l u o r o a c e t a t e i s condensed i n a C l a i s e n condensa t ion w i t h methyl fo rma te . The e t h y l sodium f luormalonaldehyde formed i s condensed wi th 2 - e t h y l - 2 - t h iopseudourea hydrobromide t o g i v e 2 -e thy lmercap to -5 - f l u o r - 4 (3H) -pyrimidinone which y i e l d s f l u o r o u r a c i l on h y d r o l y s i s ( 1 4 ) .

4 . S t a b i l i t y Degradat ion F l u o r o u r a c i l i s s t a b l e i n s o l u t i o n s which a r e no t

s t r o n g l y b a s i c (pH less t h a n 9 ) . When s u b j e c t e d t o s t r o n g l y b a s i c c o n d i t i o n s , t h e f l u o r o u r a c i l i s hydrolyzed t o u r e a , f l u o r i d e , and an aldehyde. This h y d r o l y s i s i s enhanced by i n c r e a s e d pH and t e m p e r a t u r e . formed on h y d r o l y s i s r e a c t s f u r t h e r g i v i n g ammonia and C 0 2 (15) .

5 . Me tabo l i c P roduc t s The m e t a b o l i c pathway o f f l u o r o u r a c i l i s p r e s e n t e d

s c h e m a t i c a l l y i n F igu re 8. In man, t h e major biochemical e f fec t o f f l u o r o u r a c i l i s t h e i n h i b i t i o n o f DNA s y n t h e s i s , s i n c e c o n c e n t r a t i o n s which i n h i b i t DNA s y n t h e s i s may s t i l l pe rmi t RNA s y n t h e s i s . F l u o r o u r a c i l i s conve r t ed t o f l u o r o u r i d i n e and t h e n t o t h e mono-,di- , and t r i p h o s p h a t e s o f f l u o r o u r i d i n e . T h i s i s t h e n i n c o r p o r a t e d i n t o t h e f r a u d u l a n t FNA. F l u o r o u r i d i n e monophosphate i s a l s o re - duced t o f l u o r o - 2 ' -deoxyur id ine monophosphate. There i s no f u r t h e r metabol ism t o t h e d i - and t r i p h o s p h a t e n u c l e o t i d e s

Some o f t h e u r e a

234

FLUOROURACIL

F i g u r e 7

S Y N T H E S I S O F P L U O P O U P A C I L

P O I

0 0

p C H Z - E - O - C 2 H 5 + H E - o - c H ~ C H N r 30H > N ~ O - C H - C - E - O - C ~ H ~ f l u o r o r c a t a t e f o r r r t e f l u o r m a l o a r l d e h y d r

e t h y l methy l e t h y l - r o d i o -

(1) (11) (111)

NH H - N NI I 1 1 + H 5 C 2 - S - C ’ *HBr CH30H+

‘NH

R 2 - e t h y l - 2 - 2 - e t h y l m c r c r p t o -

t h i o p r e u d o u r e r 5 - f l u o r o - 4 ( 3 H ) - hydrobromide p y r l m i d i n o a r

( 1 V ) ( V )

i h y d r o l y z e , H - N ’ ‘c-P

o - C \ N / C - H I

I l l

H

f l u o r o u r r c i l ( V I )

235

Figure 8

MTAlOLIC PATHWATS O? ?LUOlOUlACIL (16)

a~?LuOlo-~-ALANIIIL + U l L A + C02

T ~-?LUOlO-~-UlKIDOIlOIIONlC ACID t ( I - ? L U O I O - 6 - C U A I I I D O I l O I l O N I C ACID

f DIUTDlO?LUOlOUlACIL

t 5-?LUOlOUlIDI~E T l I I H O S I H A T l

T 5-?LUOlOUlACIL 5-?LIIOlOUlIDIIIK 5-ILUOIOUlIDIIIL M O l l O I Y O S I H A T ~

I

H!!!

t IITIIIDTLIC ACID + S-?LUOlO-Z'-DKOITUlIDINK -

IOHOIHOSIHAIK

DKOITUlIDlLIC ACID

T UIIDTLIC ACID

Fo 0 n C 0 < D 2 0

5-FLUOlOUlIDIHE 5-FLUO10- 2'-ULOXIUlIDIIIX

FLUOROURACI L

o f f l u o r o - 2 - d e o x y u r i d i n e . ‘Therefore t h e f o r m a t i o n o f t h e f l u o r o - 2 ‘ - d e o x y u r i d i n e monophosphate i s c o n s i d e r e d t o be t h e b a s i s f o r t h e a n t i n e o p l a s t i c a c t i o n o f f l u o r o u r a c i l s ince i t i n h i b i t s DNA s y n t h e s i s by b l o c k i n g t h e enzyme t h y m i d y l a t e s y n t h e t a s e . I t i s t h i s enzyme t h a t c a t a l y z e s t h e m e t h y l a t i o n o f d e o x y u r i d y l i c a c i d t o t h y m i d y l i c a c i d ( 1 6 ) .

F l u o r o u r a c i l i s c a t a b o l i z e d i n an ana logous manner t o u r a c i 1, forming t h e f o l lowing d e g r a d a t i v e p r o d u c t s : d i h y d r o f 1 u o r o u r a c i 1 , u - f luoro - 6-u re i d o p r o p i o n i c a c i d , a - f l u o r o - a -guan idoprop io r i i c a c i d , iy - f 1 uoro-d -a1 ani i ie , u r e a , and CO2 ( 1 6 ) .

6 . Methods of A n a l y s i s

6 . 1

re f e r en c e ( 1 7 ) *

6 . 2

E l ement a1 A n a l y s i s The r e s u l t s from an e l e m e n t a l a n a l y s i s o f s t a n d a r d f l u o r o u r a c i l i s p r e s e n t e d i n ‘I‘ahle V

’I’AULE v E 1 ement a1 Aria 1 ys i s o f 1-1 u o r o u r a c i 1

k 1 ement % Theory ”” Found

C 3 b . 93 36 .94 11 2 . 3 2 2 . 5 0 F 1 3 . 6 1 1 4 . 5 7

Phase So 1 ub i 1 i t y . h a 1 y s i 5

Phase s o l u b i l i t v a n a l y s i s has been c a r r i e d o u t f o r f l u o r o u r a c i l u s i n g methanol a s t h e s o l v e n t . An example i s p r e s e n t e d i n F i g u r e 9 f o r a t ) , p i c a l l o t o f f l u o r o u r a c i l a l o n g w i t h t h e c o n d i t i o n s unde r which t h e a n a l y s i s was c a r r i e d o u t ( 1 0 ) .

6 . 3 T h i n Layer Chromatographic Analysis (T’LC) The ‘I’LC o f f l u o r o u r a c i l a n d o t h e r f l u o r o -

p y r i m i d i n e s h a s been s t u d i e d a t l e n g t h by Mawrylyshyn, Senkowski, and W o l l i s h ( 1 8 ) . ‘The ltf v a l u e s p r e s e n t e d i n ‘I’ablc V I were o b t a i n e d i n v a r i o u s d e v e l o p i n g s o l v e n t s when 10 pgm o f f l u o r o u r a c i l was s p o t t e d on n s i l i c a g e l GI:

231

B. C

. RU

DY

AN

D 6

.2 S

EN

KO

WS

KI

238

FLUOROURACI L

plate. After development for at least 10 cm the plates were air dried and viewed under short wave ultraviolet radiation.

TABLE VI

Rf Values for Fluorouracil in Various Developing Solvents (18)

Sol vent System

ethyl acetate:acetone :water (70 : 4 0 : 10)

ethyl acetate:methanol:conc. ammonium hydroxide (75 : 25 : 1)

K Value -f-

0.7

0.4

acetone 0.8

ether 0.12

ethyl acetate 0.4

met h an0 1

2-propanol

0.8

0.7

ethyl acetate :methanol (80 : 2 0 ) 0.8

ethyl acetate:methanol (75 :25) 0.7

ethyl acetate :water (100 : 1) 0.2

ethyl acetate:water (100:3) 0.4

ethyl acetate:mcthanol :glacial acetic acid (75:25:1)

chloroform : glaci a1 acetic acid (65 : 35)

0.9

0.45

6.4 Gas Liquid Chromatographic Analysis (GLC) GLC has been carried out for fluorouracil using

the instrumental conditions listed below. A single peak is observed for reference standard fluorouracil after about two minutes (19).

239


Instrumental Conditions:

Column : 6 f t . copper tub ing Column Packing: Column Temperature: 25OoC C a r r i e r Gas: Nitrogen Flow Rate: 40 cc/minute Detec tor : Hydrogen Flame Ion iza t ion

5% SE-30 on Chromosorb G

6 .5 Direct Spectrophotometr ic Analysis Di rec t spectrophotometr ic ana lys i s i s c a r r i e d

out on f l u o r o u r a c i l i n j e c t a b l e s o l u t i o n s . A volume of t h e f l u o r o u r a c i l i n j e c t i o n i s p i p e t t e d i n t o a volumetr ic f l a s k and d i l u t e d with pH 4 . 7 a c e t a t e b u f f e r t o o b t a i n a f i n a l concent ra t ion of about 10 pgm/ml. The absorbance of t h i s s o l u t i o n i s measured a t 266 nm along with t h e absorbance of a s i m i l a r concent ra t ion of r e fe rence s tandard f l u o r o u r a c i l . From t h i s d a t a the concent ra t ion o f f l u o r o u r a c i l i n t h e i n j e c t a b l e s o l u t i o n i s ca l cu la t ed ( 2 0 ) .

6 . 6 F luor ine Analysis

6 .61 Organica l ly Bound Fluorine Analysis There a r e seve ra l methods a v a i l a b l e t o

determine t h e amount o f carbon-bonde; f l u o r i n e . t he a a r l i e r methods employed t h e Schoniger Combustion technique followed by thorium n i t r a t e o r cerous c h l o r i d e t i t r a t i o n us ing sodium a l i z a r i n s u l f o n a t e o r murexide a s t h e i n d i c a t o r ( 2 1 ) .

One o f

With t h e advent of good s p e c i f i c ion e l ec t rodes , methods were developed t o l i b e r a t e t h e bound f l u o r i n e and d i r e c t l y measure the f l u o r i d e concent ra t ion . The reagent sodium-biphenyl followed by oxida t ion with hydrogen peroxide i s used t o l i b e r a t e t h e o rgan ica l ly bound f l u o r i n e i n f l u o r o u r a c i l . A f l u o r i d e s p e c i f i c ion e l e c - t rode i s used, i n conjunct ion with a h igh - ion ic - s t r eng th - bu f fe r s o l u t i o n , f o r d i r e c t measurement of t h e l i b e r a t e d f luo r ide ( 2 2 ) .

The l a s t method t o be presented f o r t h e ana lys i s o f t h e carbon-bonded f l u o r i n e i n f l u o r o u r a c i l i s 19F Nuclear Magnetic Resonance spectrometry (3 ) . f l u o r o u r a c i l re fe rence s tandard and an i n t e r n a l s t anda rd ,

A

240

FLUOROURACI L

r e a g e n t g r a d e o r t h o f l u o r o b e n z o i a c i d , i s d i s s o l v e d i n N,N-dimethylacetamide and t h e "F spectrum o b t a i n e d . t h i s d a t a an i n t e r n a l s t a n d a r d f l u o r i n e c o n v e r s i o n f a c t o r i s o b t a i n e d as f o l l o w s :

From

A, x C, x 14.62 x P v I

= CF where A x Cb f A

A = h e i g h t o f r e f e r e n c e s t a n d a r d f l u o r o u r a c i l s i g n a l

C = mg/ml o f r e f e r e n c e s t a n d a r d f l u o r o u r a c i l

C = mg/ml o f o r t h o f l u o r o b e n z o i c a c i d

= h e i g h t o f o r t h o f l u o r o b e n z o i c a c i d s i g n a l b

f

f

b 14.62 = t h e o r e t i c a l f l u o r i n e c o n t e n t i n f l u o r o u r a c i l

P = a s s a y o f f l u o r o u r a c i l r e f e r e n c e s t a n d a r d 100 =1

CF = i n t e r n a l s t a n d a r d f l u o r i n e conve r s ion f a c t o r

Now a 1 9 F spectrum o f each f l u o r o u r a c i l sample i s o b t a i n e d and t h e % f l u o r i n e c a l c u l a t e d u s i n g t h e f o l l o w i n g fo rmula .

where: As = h e i g h t o f f l u o r o u r a c i l sample s i g n a l

C = mg/ml o f f l u o r o u r a c i l sample S

6.62 Free F l u o r i d e A n a l y s i s The d e t e r m i n a t i o n o f any f r e e f l u o r i d e

p r e s e n t i n f l u o r o u r a c i l bulk samples o r i n f l u o r o u r a c i l ampuls are c a r r i e d o u t by d i r e c t measurement u s i n g a f l u o r i d e s p e c i f i c i o n e l e c t r o d e . The measurements a r e made i n a t r i s (hydroxymethy1)amino-methane b u f f e r s o l u t i o n (pH = 7 . 5 ) . The e l e c t r o d e r e sponse was found t o be l i n e a r throughout t h e working r ange o f 0 .08 t o 0 . 2 5 mg F( - ) /100 m l s o l u t i o n (23 ) .

6 . 7 Volumetr ic Ana lys i s F l u o r o u r a c i l may be a s s a y e d by d i s s o l v i n g an ac-

c u r a t e l y weighed sample i n dimethylformamide and t i t r a t i n g i t w i t h 0.1N tetra-n-butyl-ammonium hydrox ide i n benzene t o a b l u e end p o i n t u s i n g thymol b l u e a s t h e i n d i c a t o r .

B. C. RUDY AND 6.2. SENKOWSKI

Each milliliter of 0.1N tetra-n-butylammonium hydroxide is equivalent to 13.01 mg of fluorouracil (24).

242

FLUOROURACIL

7 . References

1.

2 . 3.

4 .

5.

6 .

7 .

8.

9 .

10.

11.

1 2 .

13.

14 . 15 .

16.

17 .

18.

19.

20. 2 1 .

22.

M. Itawrylyshyn, Hoffmann-La Roche I n c . , Persona l Communication. I . A . Brownlie, J . Chem. Soc . , 1950, 3062 (1950) . J . H . Johnson, Hoffmann-La Roche Inc . , Pe r sona l Communication. F . A. Bovey, Nuc lea r Magnetic Resonance Spec- t r o s c o p y , Academic Press, N e w York C i t y , pp. 211- 214. H. K . S o k o l o f f , Hoffmann-La Roche I n c . , P e r s o n a l Commun i c a t i on. J . Boatman, Hoffmann-La Roche I n c . , Pe r sona l Communication. W . Benz, Hoffmann-La Roche I n c . , Pe r sona l Communi- c a t i o n . Pharrnacopeia of the United S t a t e s XVIII, 935, (1970) . J . Donahue, Hoffmann-La Roche I n c . , Pe r sona l Communication. E . MacMullan, Hoffmann-La Roche I n c . , Pe r sona l Communication. R . B . Hage l , I-loffmann-La Roche I n c . , P e r s o n a l Communication. A . Motchane, Hoffmann-La Roche I n c . , Unpublished Data. I . Shugar and J . J . Fox, Biophysica e t Biochemica

-

Acta, 9 , 199 and 369 (1952). M. Cole, Hoffmann-La Roche I n c . , Unpublished A . Motchane, and H. J enny , Hoffmann-La Roche Unpub 1 i s hed Data. E. Miller , J . Surgical Oncology, - 3 ( 3 ) , 309 and r e f e r e n c e s w i t h i n . A . Steyermark, Hoffmann-La Roche I n c . , Unpub Data.

Data . I n c . ,

1971)

i s h e d

M . fjawrylyshyn, B . Z . Senkowski, and E . G . W o l l i s h , Microchem. J . , 8, 15 (1964) . F . Mahn, Hof fmazn -La Roche I n c . , Persona l Communication. Pharmacopeia of t he United S t a t e s XVIII, 269 (1970). A . Steyermark, " Q u a n t i t a t i v e Organic Mic roana lys i s " 2nd Ed . , Academic, N e w York, N . Y . , 1961, p p . 326- 332. B . C . J o n e s , J . E . Heveran, and B . Z . Senkowski,

243

6 . C. RUDY AND 13. 2. SENKOWSKI

J, Pharm. Sci., 60 , 1036 (1971).

J. Pharm. S c i . , 58, 607 (1969).

(1970) .

2 3 . B . C . Jones, J . E. Heveran, and B. Z . Senkowski,

24. Pharmacopeia of z e United States XVIII, 268

244

FLUPHENAZINE ENANTHATE

245

KLAUS FLOREY

CONTENTS

1. DESCRIPTION 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor

2.1 Infra Red Spectra 2.2 Nuclear Magnetic Resonance Spectra 2.3 Ultraviolet Spectrum 2.4 Mass Spectra 2.5 pKa, pH 2.6 Boiling Point 2.7 Differential Thermal Analysis 2.8 Solubility 2.9 Refractive Index

2. PHYSICAL PROPERTIES

3. SYNTHESIS

5. DRUG METABOLISM 6. METHOD OF ANALYSIS

4. STABILITY - DEGRADATION

6.1 Elemental Analysis 6.2 Non-aqueous Titration 6.3 Spectrophotometric Analysis 6.4 Spectrofluorometric Analysis 6.5 Colorimetric Analysis 6.6 Chromatoqraphic Analysis

6.61 Paper 6.62 Thin Layer 6.63 Gas-Liquid

7. IDENTIFICATION AND DETERMINATION

8 . References IN BODY FLUIDS AND TISSUE

246



1.1 Name, Formula Molecular Weight

F luphenaz ine e n a n t h a t e i s 4 - L T - D - ( t r i - f l uo romethy l ) p h e n o t h i a z i n - l 0 - y g p r o p y g -1 p i p e r a z i n e e t h a n o l h e p t a n o a t e (es te r ) : a l s o f l u p h e n a z i n e h e p t a n o a t e , SQ 16,144: d i h y d r o c h l o r i d e s a l t : SQ 10,479

CH2-CHz-CH2- I

Mol. W t . 549.70 2 gH 38 3N30 2

1 . 2 Appearance, Odor, Color

F luphenaz ine e n a n t h a t e i s a p a l e ye l low t o ye l low orange , c l e a r t o s l i g h t l y t u r b i d , v i s c o u s l i q u i d w i t h a c h a r a c t e r i s t i c odor .


2 . 1 I n f r a Red S p e c t r a

The i n f r a r e d s p e c t r a o f a s e m i - s o l i d smear and of a chloroform s o l u t i o n a r e p r e s e n t e d i n f i g u r e s 1 and 2 .

2 . 2 Nuclear Magnetic Resonance S p e c t r a

The NMR Spectrum i s p r e s e n t e d i n f i g u r e 3. The fo l lowing ass ignments can be made f o r chemical s h i f t s of t h e spectrum of t h e f r e e b a s e . 2

247

i N P 00

50004

~ 2

4

Figure 1 I n f r a r e d S p e c t r a of F luphenaz ine e n a n t h a t e , Squibb House S tanda rd b a t c h #10 s e m i s o l i d smear. Ins t rument : Perkin-Elmer 621. Curve #29377

x r D C rn n r 0 I] rn <

FREQUENCY (L'I- 1 )

0000 50004000 3000 2500 2000 1800 I600 1400 1200 1100 1000 950 900 850 800 750 700 M I I ' ' i h.

i

I O 0 I

1 0 0 ~ 1 1 I I

I

I

I 2 3 4 5 6 7 8 9 10 i i 12 13 14 I5 WAVELENGTH IMlCRONSl

F i g u r e 2 I n f r a Red Spec t rum o f F l u p h e n a z i n e e n a n t h a t e Squ ibb House S t a n d a r d b a t c h #10 i n c h l o r o f o r m s o l u t i o n . I n s t r u m e n t : Perkin-Elmer 6 2 1 . Curve # 2 9 3 7 7 A

n r C D I rn z > z m rn z D z -I I D

N

d

w ul 0

I I , , , . . I , . . , . . . . I . I . . ,

7 0 4 0 ? , 2 0 I 0

F i g u r e 3: NMR Spec t rum of f l u p h e n a z i n e e n a n k h a t e , S q u i b b b a t c h #10 i n CDC13. Spec t rum # 10450. I n s t r u m e n t : V a r i a n A - 6 0 .

> 7c c T n r C -0 I rn z > N z rn

F i g u r e 4: Low R e s o l u t i o n Mass Spectrum of F l u p h e n a z i n e e n a n t h a t e . Squ ibb House S t a n d a r d b a t c h 10 . 1nstrument:MS-9.

KLAUS FLOREY

Proton Chemical S h i f t s ( )

6 . 0 5 8 . 0 7 7 . 5 5 5.84 8.10 8 . 7 0 9.10 3 . 0

t r i p l e t mu 1 t i p l e t a b s o r p t i o n envelope t r i p l e t t r i p l e t mu1 t i p l e t s mu 1 t i p l e t a romat i c

2 . 3 U l t r a v i o l e t Spectrum

Squibb House S tanda rd b a t c h #7 , curve #22272 i n e t h a n o l 3

Wavelength (nm) a b s o r p t i o n (EF&,) 240 ( s h o u l d e r ) 2 2 1 2 6 1 625 312 6 8

252

2 . 4 Mass Spec t rum

The l o w r e s o l u t i o n mass spec t rum is p r e s e s e n t e d i n f i g u r e 4 . The spec t rum i n d i c a t e s t h e f o l l o w i n g f r a g m e n t a t i o n p a t t e r n , c o n s i s t e n t w i t h t h a t o b t a i n e d f o r f l u p h e n a z i n e :

KLAUSFLOREY

The s i d e c h a i n f ragment and i t s f u r t h e r f r a g m e n t a t i o n p r o d u c t s were a l s o found:

- 1H

-CH2 T2 CH - ~ ~ ~ H 1 ~ c J o H

m / e 1 7 1

Another prominent f ragment i s t h e f o l l o w i n g :

2 . 5 pKa, pH.

The pKa, v a l u e was de t e rmined b y t i t r a t i o n ; t h e pKa2 v a l u e was e s t i m a t e d from t h e pH of t h e f i r s t e q u i v a l e n t p o i n t b e c a u s e f l u - phenaz ine e n a n t h a t e p r e c i p a t e d a t pH v a l u e s s l i g h t l y above t h i s p o i n t . 8

PKa 9 ~ K a 2 F luphenaz ine e n a n t h a t e 3 .50 ; 3.29 8 . 2 ; 7.7 Fluphenaz ine 3.90; 3.60 8.1; 7.8

A s e x p e c t e d t h e v a l u e s a r e v e r y s imi l a r t o t h o s e o f f l u p h e n a z i n e (see A n a l y t i c a l P r o f i l e ) , s i n c e t h e a d d i t i o n o f an es ter two c a r b o n s removed from t h e t e r t i a r y amine n i t r o g e n s h o u l d have a minimal e f f e c t8.

2 .6 B o i l i n g P o i n t

F luphenaz ine e n a n t h a t e base i s a v i s c o u s l i q u i d and a t t e m p t s t o d i s t i l l it and t o d e t e r m i n e a b o i l i n g p o i n t by c o n v e n t i o n a l

254


6 methods h a v e been u n s u c c e s s f u l . 2.7 D i f f e r e n t i a l Thermal A n a l y s i s

F luphenaz ine e n a n t h a t e samples s u b j e c t e d t o DTA a t a t m o s p h e r i c p r e s s u r e u n d e r N2 and i n vacuo (25mm) showed n e i t h e r exo- t h e r m i c o r endo the rmic peaks u p t o a t e m p e r a t u r e o f 350°C7.

2 . 8 S o l u b i l i t y S 0 l u b i 1 i t y gm/m 1

S o l v e n t s o l v e n t a t 250C8

Water i n s o l u b l e E t h a n o l 41 ( V . S . )

Chloroform <1 ( V . S . ) E t h e r 2

2 . 9 R e f r a c t i v e Index

The R e f r a c t i v e Index o f Squibb House S t a n d a r d b a t c h #7 i s de te rmined a s : 1 .5495 (23OC)

3 . S y n t h e s i s

F luphenaz ine e n a n t h a t e (I , F i g u r e 5 ) h a s been p r e p a r e d b y r e a c t i n g f l u h e n a z i n e (11) w i t h h e p t a n o y l c h l o r i d e . 6,lO

255

KL

AU

S F

LO

RE

Y

x ?

a, c,

a, c .d

N

W

m

h

x" u I

x 9

L

H 4

7

h

N

iz V u

I M

I

u I i?

ci 7 N x

m

util I

W

3

N

i? ? p- u- Cf

0

3:

a, L4

256


4. S t a b i l i t y - D e g r a d a t i o n .

F l u p h e n a z i n e e n a n t h a t e , l i k e i t s p a r e n t compound f l u p h e n a z i n e h y d r o c h l o r i d e i s l i g h t s e n s i t i v e . E x p o s u r e of a 10% s o l u t i o n o f f l u - p h e n a z i n e e n a n t h a t e i n i s o - b u t y l m e t h y l k e t o n e f o r o n e month r e s u l t e d i n 5% decompo- s i t i o n . The mechanism o f l i g h t c a t a l y z e d d e c o m p o s i t i o n i s p r o b a b l y s i m i l a r t o t h a t o f f l u p h e n a z i n e h y d r o c h l o r i d e ( s e e A n a l y t i c a l P r o f i l e ) . F l u p h e n a z i n e e n a n t h a t e h y d r o l y z e s t o f l u p h e n a z i n e i n a l k a l i n e medium.

5. Drug M e t a b o l i c P r o d u c t

The d r u g m e t a b o l i s m and t i s s u e d i s t r i b u t i o n o f f l u p h e n a z i n e e n a n t h a t e , l a b e l e d w i t h C1* i n t h e e t h a n o l p o r t i o n o f t h e s i d e c h a i n was s t u d i e d . I n a d d i t i o n t o unchanged f l u p h e n a - z i n e e n a n t h a t e t w o m e t a b o l i t e s f l u p h e n a z i n e (11) a n d f l u p h e n a z i n e s u l f o x i d e (111) were i d e n t i f i e d i n t h e excre ta a n d a l l the t i s s u e s s t u d i e d . However i n t h e b r a i n o n l y f l u p h e n a - z i n e was found. The h y d r o l y s i s i n t h e b r a i n o f f l u p h e n a z i n e e n a n t h a t e t o f l u p h e n a z i n e was d e m o n s t r a t e d . 1 2

I n a l a t e r s t u d y the B - g l u c u r o n i d e of 7 - h y d r o x y f l u p h e n a z i n e ( I V ) was i s o l a t e d from dog b i l e a f t e r a d m i n i s t r a t i o n o f l 4 C l a b e l e d f l u p h e n a z i n e e n a n t h a t e .

6 . Method o f A n a l y s i s

6 . 1 E l e m e n t a l A n a l y s i s C a l c .

C 6 3 . 3 7 H 6 . 98 N 7 . 6 4

9 Found 63 .09

7 . 1 0 7 . 9 9

KLAUS FLOREY

6 . 2 Nonaqueous T i t r a t i o n

F luphenaz ine e n a n t h a t e can be t i t r a t e d w i t h 0 . 1 N HCl04 i n g l a c i a l a c e t i c a c i d . I n d i c a t o r : c r y s t a l v i o l e t . l4 e q u i v a l e n t : c a l c . 285: Found: 271 (Squibb House S t a n d a r d ) ,

Neu t r a 1 i z a t i o n

6 . 3 S p e c t r o p h o t o m e t r i c A n a l y s i s

The U.V. abso rbance a t 261 nm (see S e c t i o n 2 .3) can be u s e d f o r q u a n t i t a t i v e a n a l y s i s (200 s e c t i o n 6 . 7 1 ) .

6 .4 S p e c t r o f l u o r o m e t r i c A n a l y s i s

Although no d a t a a r e a v a i l a b l e , s p e c t r o - f l u o r o m e t r i c a n a l y s i s of f l u p h e n a z i n e e n a n t h a t e s h o u l d be p o s s i b l e , s i m i l a r t o t h a t o f t h e p a r e n t compound (see A n a l y t i c a l P r o f i l e on F luphenaz ine H y d r o c h l o r i d e ) .

6 .5 C o l o r i m e t r i c A n a l y s i s

11 The f o l l o w i n g c o l o r t e s t s were a p p l i e d :

Reagent : c o l o r :

50% S u l f u r i c a c i d red N i t r o p r u s s i d e f a i n t g r e y

Hydroxamic a c i d ye l low-orange Bromo-cresol g r e e n b l u e Phenol r e d r e d Phosphomolybdic a c i d g r e e n grey-

G i b b s g r e y

g r e e n ye l low

258


6 . 6 Chromatographic A n a l y s i s

6 .61 Paper chromatographic A n a l y s i s

The fo l lowing systems have been r e p o r t e d .

Systems: F luphenaz ine F luphenaz ine Fluphena- e n a n t h a t e s u l f o x i d e z i n e

System 112 0.96 0 .55 0 .10

System 3l* 0 .13 0 .68 --

System 112: ma tman pape r

System 2 1 2 0 .00 0 .35 0.62

benzene - ace t ic a c i d - w a t e r (2:2:1)

matman pape r #3 MM System 2 1 2 : 1 N sodium formate ( a scend ing)

System 316: Paper Whatman#l ( e t h a n o l washed)

e t h e r ) S t a t i o n a r y phase: C a s t o r o i l (2% i n

Developing s o l v e n t : Methanol-water

(85-15) Development

t ime : 16 hour s

T h i s l a s t system can b e used f o r q u a n t i - t a t i o n by l o c a t i n g t h e s p o t s under U.V. l i g h t , e l u t i o n w i t h 95% e t h a n o l and measuring U . V . absorbance a t 261 nm a g a i n s t a s t a n d a r d , l6 fo l lowing t h e g e n e r a l procedure . 1 7

25Y

KLAUS FLOREY

6 . 6 2 Thin l a y e r chromatography:

See Table I

6 . 6 3 Gas - l iqu id chromatography

Fluphenaz ine e n a n t h a t e can be chromatographed i s o t h e r m a l l y a t 25OoC on 8/100 mesh g l a s s beads coa ted wi th 0.5% of SE-30. With a r e t e n t i o n t i m e of 6-7 minutes t h e minimum d e t e c t a b l e q u a n t i t y i s 0 . 5 ~ gm.

Reproducib le q u a n t i t a t i o n was n o t ach ieved - non - l i n e a r e r r a t i c r e sponse i n d i c a t e d a b s o r p t i o n of t h e e n a n t h a t e w i t h p o s s i b l e a t t e n d a n t d e g r a d a t i o n . 18

I d e n t i f i c a t i o n and Determina t ion i n Body F l u i d s and T i s s u e s .

Determina t ion by s e c t r o f l u o r o m e t r y i n r a t b r a i n and plasma. '' Thin l a y e r chromatography ( s e e S e c t i o n 5 ) 1 2 ~ 13.

260

Table I

Thin Layer Chromatography

The following systems have been reported.

Rf values

Fluphenaz ine Fluphenazine Fluphenazine 7-Hydroxy- n System en an th a te d ih ydr och 1 or ide sulfoxide f luphenazine U

I .J Benzene- ammonia - 1 2 9 1 3 0.84 0. 26 n - dioxane (60 : 5 : 35)

C h lorof orm-95% ethanol-ammonia (10:85:2. 5)13 0.87 0.75

Chloroform-abs. e thano l-ammonia (80:10:1)19 0.82 0.47

Acetone-cyclohexane- 0.75 0.40 ammonia(50:40:1) 11

0.06 0.05

-- 0. 66

-- 0.15

rn

11 Silica gel GF thin layer plates were used. Detection and spray reagents. U . V . light 50% sulfuric acid.

KLAUS FLOREY

REFERENCES

1.

2.

3. 4. 5.

6. 7. 8. 9.

10.

11. 12.

13.

14. 15.

16. 17.

18.

B. Keeler, Squibb Institute, Private Communication. M. S. Puar and G. M. Jennings, Private Communication. G. A. Brewer, Private Communication. A. I. Cohen, Private Communications. J. N. T. Gilbert and B. J. Millard, Org. Mass Spectrom. 2,17 (1969). H. L. Yale, Pri-ate communication. H. Jacobson, Private Communication. H. Jacobson, Private Communication. H. L. Yale, A. I. Cohen and F. Sowinski, J. Med. Chem. 6,347 (1963). H. L. Yale and-R. C. Merrill, U. S. Patent - 3,194,733 (1965). J. E. Fairbrother, Private Communication. A. G. Ebert and S. M. Hess, J. Pharmacol. and Exp. Therap. 148, p.412 (1965). J. Dreyfuss, J. J. Ross, Jr., and E. C. Schreiber, J. Pharm. Sci. - 60,829(1971). J. F. Alicino, Private Communication. M. A. Mahju and R. P. Maickel, Biochem. Pharmacol. 2,2701 (1969). H. R. Roberts, Private Communication. H. R. Roberts and K. Florey, J. Pharm. Sci. - 51, 794 (1962). A. 0. Niedermayer, Private Communications.

262

FLUPHENAZINE HYDROCHLORIDE

Klaus Florey

263

KLAUS FLOREY

CONTENTS

1.

2 .

3. 4 . 5 . 6 .

7 . 8.

9. 10.

DES C R I PTI ON 1.1 N a m e , F o r m u l a , Molecular Weight I. 2 A p p e a r a n c e , Co lo r , O d o r

2 . 1 I n f r a r e d Spectra 2 . 2 N u c l e a r Magnetic R e s o n a n c e Spectra 2 . 3 U l t r a v i o l e t Spectra 2 . 4 Mass Spectra 2 . 5 pka, p H 2 . 6 Melting R a n g e 2 . 7 D i f f e r e n t i a l T h e r m a l A n a l y s i s 2 . 8 T h e r m o g r a v i m e t r i c A n a l y s i s 2 . 9 S o l u b i l i t y 2 . 1 0 c rys ta l Propert ies

PHYSICAL P R O P E R T I E S

SYNTHESIS S T A B I L I T Y - DEGRADATION DRUG METABOLIC PRODUCTS METHODS OF ANALYSIS

6 . 1 E l e m e n t a l A n a l y s i s 6 . 2 N o n a q u e o u s T i t r a t i o n 6 . 3 Polarographic A n a l y s i s 6 . 4 S p e c t r o f l u o r o m e t r i c A n a l y s i s 6 . 5 C o l o r i m e t r i c A n a l y s i s 6 . 6 E l e c t r o p h o r e s i s 6 . 7 C h r o m a t o g r a p h i c A n a l y s i s

6 . 7 1 Paper 6 , 7 2 Thin-Layer 6 . 7 3 C o l u m n 6 . 7 4 Gas - L i q u i d

COUNTERCURRENT SEPARATION I D E N T I F I C A T I O N AND DETERMINATION I N BODY F L U I D S AND T I S S U E S MISCELLANEOUS REFERENCES

2 64


1. DESCRIPTION 1.1 Name Formula, Molecular Weight

Fluphenazine Hydrochloride is 4 - { 3 - [ 2 - krifluoromethy1)-phenothiazine-10-yll propyl) piperazine ethanol dihydrochloride: also 4-(3-[lo -2(-trifluoromethy1)-phenothiazinyl I propy1)-1- piperazine-ethanol dihydrochloride; 1- (2-hydroxy ethyl)-4- [2-trifluoromethyl-lO-phenothiaz~nyl) propyl ] piperazine dihydrochloride; 10-(3'- [4"- (B-hydroxy ethyl)-1"-piperazinyl] propyl-2-trifluoromethyl-phenothiazine dihydrochloride: S94; SQ 4918.

1.2 Appearance, Color, Odor White to off-white, odorless crystalline

powder.

2. PHYSICAL PROPERTIES 2.1 Infrared Spectra

The infrared spectra of Squibb House Standard #48101-001, in mineral oil mull and KBr

1 from MeOH, are presented in figures 1 and 2. The infrared spectrum of the free base, and a discuss ion of spectra-structure correlation of phenothiazines have been presented. 9 The spectra published by Sammul, et al.4 are in es- sential agreement with those presented here.

265

FREQUENCY (CM-1)

2

Fig. 1 Infrared Spectrum of fluphenazine hydrochloride in mineral oil mull. Instrument: Perkin-Elmer 621

x r t.

n r 0 D rn <

5

3 4 5 6 7 8 I0 II 12 13 is is WAVELENGTH (MICRONS1

Fig. 2 Infrared Spectrum of fluphenazine hydrochloride in KBr from methanol. Instrument: Perkin-Elmer 621

n r

5 I rn z B

2 rn

I < o n 0 c, I r 0 n 0 rn

N

KLAUS FLOREY

2 . 2 Nuclear Maqnetic Resonance S p e c t r a The NMR spectra o f f luphenaz ine base and

h y d r o c h l o r i d e are p r e s e n t e d i n f i g u r e s 3 and 4 .5 The spec t rum of t h e f r e e base i s e s s e n t i a l l y i d e n - t i c a l w i t h t h a t pub l i shed p r e v i o u s l y . 3 The f o l - lowing ass ignments can be made for chemica l s h i f t of t h e spec t rum of t h e f r e e base. 6

a b

d e f

C

2 . 3

corded:

6 .05 t r i p l e t 8 . 0 9 m u l t i p l e t 7 . 5 7 a b s o r p t i o n envelope 6 . 4 1 t r i p l e t 6 .93 s i n g l e t

2 .9-3 .0 a romat i c

U l t r a v i o l e t S p e c t r a The fo l lowing U . V . d a t a have been re-

1. i n methanol’ ( Ins t rumen t Cary 15 )

Amax 261 mp E i E m 646

A m a x 310 mp, E1% 69 1 cm

268

Fiq. 3 NMR Spectrum of fluphenazine base in deuterochloroform. Inscrument: Varian A-60

-*+

-I L r I , . . . . . . l . . . . , . , . , ~ . , . , , , , , , ~

I . . I . I 7a A4 3 0 .(I

1 1 1 , , 1 , , , , , , , , , 1 , , , , , , , , , ~ I I . . I . . . I

I I I . . . I . . . . ' . . I I

0 M i,, 3 0 1 0 I 0 a*

ii r B C v)

n r 0 n rn <

Fig. 4 NMR Spectrum of fluphenazine hydrochloride in deuterochloroform. Instrument: Varian A-60


2 . 3 U l t r a v i o l e t S p e c t r a Cont d . 2 . i n 95% e t h a n o l 3

Amax 264 Log E 4 . 5 9 Amax 316 Log E 3.69 Amin 239 Log E 4.28 Amin 300 Log E 3 . 5 1

3. i n 50% a l c o h o l ( Ins t rumen t Beckman

1 c m

DU) ElPPm

4 .

Xmax 259 0.052 Xmax 305 0 .006 Xmin 224 0 .013 Xmin 280 0 . 0 0 2 i n methanol8 Xmax 259 Log E 4 . 5 3

2 .4 Mass S p e c t r a The low- reso lu t ion m a s s spec t rum o f f l u -

The h i g h - r e s o l u t i o n spectrum shows t h e phenaz ine base is r e p r e s e n t e d i n f i g u r e 5 . 9

fo l lowing f r agmen ta t ion p a t t e r n of s i d e c h a i n and nucleus’, which is i n agreement w i t h t h e r e s u l t s o f an independent i n v e s t i g a t i o n . 69

?zyz 307-1H

2 8 0 I157 t

H2 *CH -CH2

I

350

.+)

27 1

Fig. 5 Low-resolution Mass Spectrum of fluphenazine. Instrument: MS-9


Another prominent f ragment is t h e fo l low- i n g :

248

The s i g n i f i c a n t m a s s numbers shown above

Mass U n i t s D i f f . can be t a b u l a t e d a s f o l l o w s :

C H N 0 S F Unsat O/E* Found- C a l c u l a t e d 437 2 2 26 3 1 1 3 10 0 0 . 3 419 2 2 24 3 - 1 3 10 0 - 0 . 7 406 2 1 23 3 - 1 3 10.5 E - 1 . 6 363 19 18 2 - 1 3 10.5 E -0.3 307 16 1 2 1 - 1 3 10 0 -1.1 306 16 11 1 - 1 3 1 0 . 5 E - 0 . 3 280 14 9 1 - 1 3 9.5 E - 0 . 1 266 1 3 7 1 - 1 3 9.5 E 0.5 248 14 9 1 - - 3 9.5 E -0.1 1 7 1 9 19 2 1 - - 1.5 E -0.1 157 8 17 2 1 - - 1.5 E - 0 . 7 143 7 15 2 1 - - 1.5 E -0.1

"0 = odd e l e c t r o n ion : E = even e l e c t r o n i o n

2 . 5 p K a ; p H The appa ren t pKa of f luphenaz ine d ihydro-

c h l o r i d e i n aqueous a l c o h o l i c s o l u t i o n has been de termined as 8.05 f o r t h e second b a s i c group. For t h e f i r s t basic group, a v a l u e o f 3.90 w a s ob- t a i n e d . lo Independent ly , v a l u e s o f 7 . 8 (estima- t e d ) and 3 .60 , r e s p e c t i v e l y , w e r e o b t a i n e d .

The p H of an aqueous s o l u t i o n o f t h e d i - hydroch lo r ide i s 2 . 4 ; t h a t o f monohydrochlor ide is

71

273

KLAUS FLOREY

11 5 .5 .

2 . 6 Mel t inq Ranqe The fo l lowing m e l t i n g p o i n t s o f t h e hydro-

c h l o r i d e have been r e p o r t e d :

238.5-239.5O (Rec rys t . from abs , e t h a n o l USP Methodp 2 31-2 33' 224-226OI3, l4 224.5-226O (Rec rys t . from methanol -e ther ) 15 2 3 5-2 3 701 7 l7 231-232OI8

(Recrysc. from abs. e t h a n o l ) l2

2 . 7 D i f f e r e n t i a l Thermal Analys is D i f f e r e n t i a l Thermal Ana lys i s of Squibb

1 House S tandard #48101-001 showed an endotherm a t 237OC. ( Ins t rumen t : DuPont)

2 . 8 Thermoqravimetric Ana lys i s Thermogravimetric a n a l y s i s o f Squibb

House S tanda rd #48101-001 gave no weight loss up t o 15Ooc ( Ins t rumen t : DuPont)

2 9 S o l u b i l i t y F luphenaz ine hydroch lo r ide is hygroscopic

and ex t remely s o l u b l e i n water (70% w/v a t 25OC pH 2 . 4 ) .

S o l u b i l i t y i n e t h a n o l : 15% w/v a t 25OC. L e s s t h a n 2% (w/v) i n e t h e r , ace tone ,

I n s o l u b l e i n ch loroform and e t h e r . 67 benzene, l i g r o i n . 11

2.10 crystal P r o p e r t i e s For f o r e n s i c i d e n t i f i c a t i o n , a p i c t u r e of

f luphenaz ine d i h y d r o c h l o r i d e c r y s t a l s from 95% e t h a n o l has been pub l i shed w i t h t h o s e of o t h e r

An x-ray powder d i f f r a c t i o n p a t t e r n of pheno th iaz ines . 19'

f l uphenaz ine d i h y d r o c h l o r i d e is p r e s e n t e d i n

274


t ab l e l .20 S l i g h t l y d i f f e r e n t d-values ( 1 8 . 2 ; 9.07;

8 . 7 1 ; 6 .07 ; 5 .03 ; 4 .85 ; 4 .32; 4.02; 3. 7 5 ) 7 and (17 .20; 11.50; 8 .40 ; 5 .90 ; 5 .50; 5 .40 ; 4 .70 ; 4.50; 4 .20 ; 4 .10; 4 .00 ; 3 .90; 3.75; 3.60; 3.35; 3.20; 3 .10; 2.85; 2 .80; 2.65; 2 .55; 2.45; 2 .40; 2 . 2 2 ; 2 .15) 77 w e r e a l s o r e p o r t e d .

3 . SYNTHESIS S y n t h e t i c pathways t o f luphenaz ine ( I ) are

shown i n f i g u r e 6. The s y n t h e s i s u s u a l l y invo lves t h e a d d i t i o n of t h e p r o p y l p i p e r a z i n e e t h a n o l s i d e c h a i n t o t h e 3-trifluoromethyl-phenothiazine nu- c l e u s (11). The fo l lowing r o u t e s t o ( I ) havebeen r e p o r t e d :

(1) Direct a d d i t i o n of 4 (3-ch loropro y l ) -1-pip- e r a z i n e e t h a n o l t o (11) t o y i e l d ( I ) .

( 2 ) Formation of i n t e r m e d i a t e 10 (3-chloro- propy1)-3 t r i f l u o r o m e t h y l pheno th iaz ine (111) un- d e r a v a r i e t y of c o n d i t i o n s , and condensa t ion of (111) w i t h p i p e r a z i n e e t h a n o l t o ( I ) . l2 J 16, l7

(3)Condensa t ion of 3(2- t r i f luoromethyl -10- p h e n o t h i a z i n y l ) p r o p y l p i p e r a z i n e ( I V ) w i t h e t h y l - ene oxide15, w i t h @-bromo-ethyl acetate v i a (VI) 1 3 9 14, o r w i t h 2-bromo e t h a n o l . method has a l s o been used t o p r e p a r e ( I ) , l a b e l e d w i t h 14C i n t h e two e t h a n o l ca rbons .

( 4 ) Reduction of 4 (f3- (10-phenoth iaz inyl ) t h i o - p r o p i o n y l ) p i p e r a z i n e e t h a n o l w i t h l i t h i u m a l u m i - num hydr ide t o ( I ) .

2 P

Th i s l a s t

6

2 2

4 . STABILITY - DEGRADATION F luphenaz ine hydroch lo r ide is hygroscopic .

When moi s tu re is exc luded , t h e c r y s t a l l i n e m a t e - r i a l is s table f o r a t least 6 months a t tempera- t u r e s as h igh as 5Ooc, b o t h under a i r and n i t r o - gen. The compound is p h o t o s e n s i t i v e , s i n c e t h e s u r f a c e of c r y s t a l s exposed t o incandescen t l i g h t f o r pe r iods o f more t h a n 4 weeks d i s c o l o r e d t o

215

KLAUS FLOREY

2 . 1 0 C r y s t a l P r o p e r t i e s (Continued)

TABLE I X-ray Powder D i f f r a c t i o n p a t - t e r n of

Squibb House Standa rd #48101-001

d (?i) * Rel. I n t e n s i t y * * Curve N O . 1 7 . 6 5 2 0 . 0 5 0 . 2 6 357

9 . 8 0 8 . 9 0 8 . 7 0 6 . 0 2 5 . 5 6 5 . 4 6 5 . 3 6 5 . 1 5 4 . 8 3 _f 0 . 0 2 4 . 7 5 4 . 4 9 4 . 3 1 4 . 2 3 4 . 1 2 4 . 0 0 3 . 9 0 3 . 7 2 3 . 6 9 3 . 5 7 3 . 4 0 3 . 2 7 3 . 2 3 3 . 1 1 3 . 0 6 3 . 0 0 2 . 9 3 2 . 8 7 2 . 7 2 2 . 6 7 2 . 5 4 2 . 4 2 2 . 3 4 2 . 2 7 2 . 0 2

0 . 1 2 0 . 5 8 ( 5 ) m 0 . 4 2 0: 36 0 . 2 4 0 . 1 4 0 . 3 1 0 . 4 7 0 . 5 9 ( 4 )

0 . 4 8 0 . 2 2 0 . 1 2 0 . 6 3 ( 3 ) 1 . 0 0 (1) u-x5 0 . 4 7 0 . 3 2 0 . 2 1 0 . 1 6 0 . 2 4 0 . 4 2 0 . 1 9

0.71 ( 2 )

0 . 2 2 0 . 1 2 0 . 1 5 0 . 1 6 0 . 1 6 0 . 1 4 0 . 1 5 0 . 1 5 0 . 1 4 0 . 1 5

Q d = ( i n t e r p l a n a r d i s t a n c e ) AB s i n A - 1 . 5 3 9 2 Radia t ion : ka and ka2 copper

Ins t rument : Phillips 1 ** Based on h i g h e s t i n t e n s i t y of 1 . 0 0

276

I < O m 0 D I r 0 a o rn

F i g . 6 S y n t h e t i c Pathways.

KLAUS FLOREY

l i g h t t a n . I n aqueous s o l u t i o n , f l uphenaz ine hy- d r o c h l o r i d e degrades when exposed t o l i g h t . Solu- t i o n s a t 0.01% i n e t h a n o l , water, 0.1g H C 1 and 0.5N H2SO4 w e r e k e p t i n t h e d a r k o r exposed t o f l u o r e s c e n t c e i l i n g l i g h t a t room t e m p e r a t u r e (see s e c t i o n 2 . 3 ) . A f t e r 1 7 days i n t h e d a r k , a l l so- l u t i o n s r e t a i n e d t h e i r U . V . absorbance i n t h e 255-265 m p r e g i o n . showed a s l i g h t d e c l i n e i n absorbance . I n t h e l i gh t - exposed samples , o n l y t h e e t h a n o l i c s o l u - t i o n remained unchanged, w h i l e i n t h e remain ing s o l u t i o n s d i s c o l o r a t i o n and t h e appearance of m u l t i p l e bands a t 240 and 255 mw w e r e no ted .64 Exposing a 0.05% f luphenaz ine h y d r o c h l o r i d e i n 0.1g H C 1 s o l u t i o n i n pyrex g l a s s t o s u n l i g h t caused a 40% drop o f absorbance i n 2 h o u r s . 2 3 s t u d y of t h e pH dependency o f t h e l i g h t i n s t a b i l - i t y showed a s imi l a r d e c l i n e i n absorbancy from p H 0 . 3 t o 9, whereas above pH 11 t h e d e g r a d a t i o n w a s r e t a r d e d . 2 4 Again, it w a s found t h a t so l - v e n t s such as methanol , e t h a n o l , dimethylformam- i d e , e t h y l e n e g l y c o l , and e t h e r p r e v e n t o r r e t a r d d e g r a d a t i o n .

The l i g h t - c a t a l y z e d d e g r a d a t i o n i s n o t pre- ven ted by e x c l u s i o n o f oxygen25 and proceeds th rough format ion of t h e f r e e r a d i c a l ( c f 2 6 ) . The r a d i c a l can a l s o be formed by an o x i d i z i n g a g e n t , such as cer ic ammonium s u l f a t e . 26 I n pheno th iaz ines g e n e r a l l y , t h e semiquinone r a d i c a l undergoes d i s p r o p o r t i o n a t i o n . The p roduc t s o f o x i d a t i o n are t h e s u l f o x i d e o r t h e 3 (or 7 ) -hy- d r o x y - d e r i v a t i v e s . However, format ion of t h e l a t t e r is s e v e r e l y i n h i b i t e d i n 1 0 - s u b s t i t u t e d p h e n o t h i a z i n e s . 26 The second-order decay r a t e c o n s t a n t of t h e semiquinone r a d i c a l of f luphena- z i n e w a s found t o be 1100 (l/mole/min) , as mea- s u r e d by e l e c t r o n s p i n resonance . 26 The s u l f - o x i d e c o n t e n t of s e v e r a l commercial b a t c h e s of f luphenaz ine w a s de te rmined to be around 0.1% b y

Only t h e 0 . 5 g H2S04 s o l u t i o n

A

218

FLUPHENAZINE HY DROCH LORI DE

a s p e c t r o f l u o r o m e t r i c method ( e x c i t a t i o n wave- l e n g t h 350 m@; f l u o r e s c e n c e wavelength 400 mp; see a l s o s e c t i o n 6 . 4 ) .27 a l s o be measured by a t h i n - l a y e r method (see a l s o s e c t i o n 6.72). 28 So f a r , t h e r e i s no ev idence f o r t h e d e g r a d a t i v e format ion of 7 - h y d r o x y f l u p h e n a z i ~ which has been i d e n t i f i e d as a m e t a b o l i c p roduc t (see s e c t i o n 5 ) . 5 . DRUG METABOLISM

yzed e n z y m a t i c a l l y t o f luphenaz ine ( I ) i n v i t r o by homogenates o f s m a l l i n t e s t i n a l mucosa, l i v e r , and b r a i n from e i t h e r t h e r a t o r t h e monkey.29 (See a l s o s e c t i o n 7 ) . When f l u p h e n a z i n e w a s i n - cuba ted w i t h a "microsomal and s o l u b l e " f r a c t i o n o f ra t l i v e r s , h y d r o x y l a t i o n on t h e p h e n o t h i a z i n e r i n g was obse rved .30 The s u l f o x i d e w a s n o t de- t e c t e d i n t h e e x t r a c t , and it seems u n l i k e l y t h a t b i o l o g i c a l s u l f o x i d a t i o n p recedes h y d r o x y l a t i o n i n t h e s e sys tems.30 The b i o l o g i c a l d i s p o s i t i o n and me tabo l i c f a t e of fluphenazine-14C i n t h e dog and rhesus monkey w a s s t u d i e d . 7 6

When dogs w e r e t r e a t e d w i t h f luphenazine-14C, 7-hydroxyfluphenazine ( V I I , F i g u r e 6 ) w a s i from t h e u r i n e and i d e n t i f i e d by s y n t h e s i s . human b lood and u r i n e s m a l l amounts o f f luphena- z i n e and i t s s u l f o x i d e , b o t h i n f r e e and conjuga-

and e x c r e t i o n w e r e s t u d i e d i n t h e rabbi t . 6. METHODS OF ANALYSIS

The s u l f o x i d e c o n t e n t can

Ace ty l f luphenaz ine ( V I , F i g u r e 6 ) w a s hydro l -

Wa:f t e d form w e r e i d e n t i f i e d . 7 9 T i s s u e d i s t r i b u t i o n

73

6.1 Elementa l Ana lys i s

Element % C a l c . Ref . 11 12 15 % Found

C 51.76 51.72 51.73 51.68 H 5.53 5.62 5.86 5.42 F 1 1 . 1 7 - - - N 8.23 8.04 8.00 - 0 3.13 - - - S 6.28 6.05 - - c1 1 3 . 8 9 13.66 - -

KLAUS F L O R E Y

6 . 2 Nonaqueous T i t r a t i o n Nonaqueous t i t r a t i o n i n g l a c i a l a c e t i c

a c i d w i t h 0.01g p e r c h l o r i c a c i d and Qu ina ld ine Red or c r y s t a l v i o l e t i n d i c a t o r gave a n e u t r a l - i z a t i o n e q u i v a l e n t t o 259 (calc. 2 5 5 . 2 2 ) . 32

6 . 3 P o l a r o g r a p h i c Ana lys i s The half-wave p o t e n t i a l i n a l c o h o l i c s u l -

f u r i c a c i d s o l u t i o n w a s found t o be 0 .714 ( v o l t v s . normal calomel e l e c t r o d e ) , comparable t o t h a t o f o t h e r p h e n o t h i a z i n e s . 8

6 . 4 S p e c t r o f l u o r o m e t r i c Analys is F luphenaz ine h y d r o c h l o r i d e , l i k e o t h e r

pheno th iaz ine d r u g s , e x h i b i t s f l u o r e s c e n c e . Th i s phenomenon w a s f i r s t exp lo red i n 0 . 2 g s u l f u r i c a c i d . 33 range o f 450 t o 475 mp, s h i f t i n g t o s l i g h t l y l o w - er wavelength upon o x i d a t i o n w i t h potass ium permanganate. A s l i t t l e as 0 . 0 1 t o 0 . 0 5 1-14 of t h e p u r e s u b s t a n c e per m l . of 0 . 2 g s u l f u r i c a c i d cou ld be d e t e c t e d . D u e t o i n t e r f e r e n c e by o t h e r s u b s t a n c e s , t h e s e n s i t i v i t y i n b i o l o g i c a l f l u i d s was lower (about 0 .8 pg/ml f l u i d ) . This method i s s u p e r i o r t o d e t e r m i n a t i o n b y U . V . , where about 4 p.g/ml a r e r e q u i r e d .

o b t a i n e d , w i t h o u t or w i t h o x i d a t i o n w i t h 30% hydrogen peroxide34:

The f l u o r e s c e n c e peak w a s found i n t h e

I n 50% ace t ic a c i d , t h e fo l lowing d a t a w e r e

A c t i v a t i o n maximum,m~ F luorescence maximum,my Unoxidized Oxidized Unoxidized Ox i d i zed

Ref 34 330 345 460 380 Ref 35 - 3 50 - 410

I n t h e unoxid ized form, a s h a r p increase i n f l u o r e s c e n c e i n t e n s i t y occur red above p H 7 ; i n t h e o x i d i z e d form, i n t e n s i t y w a s about e q u a l from

280


p H 2 t o p H 1 2 and f e l l p r e c i p i t o u s l y a t p H 1 2 . I n t h e o x i d i z e d form, as l i t t l e as 0 . 5 wg/ml c o u l d be de termined q u a n t i t a t i v e l y . F luphenaz ine w a s r e l a t i v e l y less f l u o r e s c e n t t h a n some o t h e r p h e n o - t h i a z i n e s .

I n c o n c e n t r a t e d s u l f u r i c a c i d , t h e a c t i v a t i o n maximum w a s found a t 325 m p and t h e f l u o r e s c e n c e maximum a t 500 mp.

t h e Aminco-Bowman S p e c t r o f l u o r o m e t e r .

36 The in s t rumen t used i n a l l i n v e s t i g a t i o n s w a s

6 . 5 C o l o r i m e t r i c Ana lys i s - Color T e s t s f o r

The g e n e r a l method of Ryan37 t o q u a n t i - t a t e unoxid ized p h e n o t h i a z i n e d e r i v a t i v e s by c o l o r complex fo rma t ion w i t h pa l l ad ium c h l o r i d e ( 5 0 0 - 550 mw m a x i m a ) has a l s o been adopted f o r f luphen- a z i n e h y d r o c h l o r i d e (see N.F .XII1) and can b e u s e d f o r s t a b i l i t y measurements.

h y d r o c h l o r i d e reacts w i t h ceric s u l f a t e , f i r s t b y forming a red-colored semiquinone f r e e r a d i c a l (420 ml), fo l lowed by fo rma t ion o f t h e c o l o r l e s s s u l f o x i d e d e r i v a t i v e . Th i s r e a c t i o n has been made t h e basis o f a pho tomet r i c t i t r a t i o n .

p o r t e d . (For a d d i t i o n a l t e s t s , see a l s o s e c t i o n s 6 . 7 1 and 6 . 7 2 ) .

I d e n t i f i c a t i o n

Like o t h e r p h e n o t h i a z i n e s , f l uphenaz ine

38

The fo l lowing c o l o r t e s t s have been re-

FOR COLOR TESTS SEE TABLE I1

6 . 6 E lec t rophores is E l e c t r o p h o r e s i s w a s c a r r i e d o u t on f l u -

phenaz ine h y d r o c h l o r i d e and s e v e r a l o t h e r pheno- t h i a z i n e t r a n q u i l i z e r s i n v a r i o u s b u f f e r s pro- posed by Werrum. 41 Whatman 3MM paper 30 c m i n wid th , and a p o t e n t i a l d i f f e r e n c e of 500 t o 800 v w e r e used . De tec t ion of s p o t s w a s c a r r i e d o u t w i t h a 40% s u l f u r i c a c i d

A Gordon-Misco a p p a r a t u s ,

z x 1

TABLE I1 Reaqent 0.001N Mercur ic n i t r a t e i n h y d r o c h l o r i c acid 0 .5% aq . ammonium molybdate 0 . 5 % aq. ammonium vanada te 3 7% formaldehyde i n conc . s u l f u r i c a c i d (1:20) 0 . 5 % aq. s e l e n i o u s a c i d 1% as. sodium t u n g s t a t e 0 .5% t i t a n i u m d i o x i d e i n c o n c . s u l f u r i c a c i d Fuming n i t r i c a c i d , t h e n e t h a n o l i c po ta s s ium hydroxide ( 3 % ) 10% aq . s u c r o s e Conc. s u l f u r i c acid p-dimethylaminobenzaldehyde 10% i n g l a c i a l

1% c o b a l t acetate: 10% i sop ropy lamine i n a c e t o n e 10% aq. ch loramine T 1% aq. pa l l ad ium c h l o r i d e a c i d i f i e d w i t h h y d r o c h l o r i c a c i d N i t r i c a c i d Conc. s u l f u r i c a c i d , 6% uranium n i t r a t e i n 95% e t h a n o l ( 3 : 1 7 ) 0.5% ammonium vanada te i n conc . s u l f u r i c a c i d 4% aq. s i l v e r n i t r a t e s o l u t i o n a c i d i f i e d w i t h 0 . 1 N s u l f u r i c a c i d 2% Zmmonium f e r r o u s s u l f a t e i n conc . s u l f u r i c a c i d

32 N acet ic acid

R e f Color

b l u e - p i n k - v i o l e t 40 grey-yel low 40 1 ight red 40 grey-green 40 p u r p l e 40 o range 40

- p i n k - v i o l e t 39

yel low-green 40 grey-ye l low 40 l i g h t brown 40 5

5 l i g h t brown 40 l i g h t b l u e 18 l i g h t y e l l o w 18 <

x

orange 18 orange-brown 18

l i g h t o range 18 y e l l o w brown 18

c r e a m wh it e 18

- 7 5


spray (orange color) or fluorescence under W light. 42

migration (cm) after 40 to 60 minutes was: For fluphenazine hydrochloride, anodic

pH : 3.3 4.7 6.0 7.2 8.0 9.3 Migration: 6.8 3.7 4.3 4.5 2.7 1.2

6.7 Chromatoqraphic Analysis 6.71 Paper Chromato- ra hic Analysis

The following chromatographic systems have been reported:

So lvent Systems :

1% Sodium formate 1% Sodium formate 90/n-Propanol 10 1% Sodium formate 90/1N Ammonia 10 1g Sodium formate 97/Formic acid 3 1% Sodium acetate 1% Sodium acetate 90/n-Propanol 10 10% Sodium chloride 92/n-Propanol 8 Formamide + Ammonium formate/Benzene Formamide + Ammonium formate/Cyclo-

Petroleum (b. p. 195-220°) /Ethanol- hexane-benzene (9: 1)

Ammonia-water (60: 2: 38)

Rf Ref - - 0.23 42 0. 32 42 0.19 42 0.42 42 0.17 42 0.48 42 0.45 42 0.71 43

0.22 43

0.59 43

Ref Detect ion systems : color -

Dragendorff-reagent orange 43

1% gold (111) chloride red 43

in ethano1:chloroform (95:5)red 43

40% H2S04 spray orange 42,43 Fluorescence under W light bluish yellow 42,43

Cerium sulfate in 2g sulfuric acid red 43

1% dimethylaminobenzaldehyde

A quantitative paper chromatographic method62 can be used to measure the purity of

' 8 3

K L A U S FLOREY

fluphenazine, following published procedures63. The paper is impregnated with castor oil ( U S P ) in ether. Developing solvent is methanol-water 85:15. Rf values of fluphenazine base approx. 0.79, fluphenazine hydrochloride 0.83. The zones are eluted from the chromatogram with 95% ethanol and the absorbances of the eluates are determined at 261 mp in a spectrophotometer.

6.72 3 The following thin-layer chromato-

graphic systems have been reported:

FOR ABOVE SEE TABLE I11

6.73 Column (Ion-exchanqe) Chromato- graphic Analysis~ Ion-exchange analysis of fluphen-

azine hydrochloride and other phenothiazine tranquilizers on a column of Dowex sulfuric acid resin 50W-X8 was possible, but not too practical for analysis of tablets. 56

6.74 Gas-Liquid Chromatoqraphic Analysis Fluphenazine was not eluted after

90 minutes at 270°, using a 210 SE-30 silicone polymer on a 80-100 mesh Gas-chrom S diatomaceous earth column57; with a 3% SE-30 on 80-100 mesh Gas-chrom Q at 25Ooc, it eluted in 4. 9 minutes. 28 Fluphenazine was also well separated from other tranquilizers on a 120 mesh silanized Gas-chrom P column coated with 1% Hi-Eff-8B (cyclohexanedimethanol succinate) at a temperature of 22Ooc and a retention time of 5 minutes. 58 fication of fluphenazine and other phenothiazine tranquilizers by gas chromatography of their pyrolysis products has also been reported. 59

The identi-

284

TABLE I11

Ads orbent Silica Gel

11

I

1 1

I 1

I 1

I '

1'

11

I 1 N m vI Cellulose

Silica Gel 11

11

11

I 1

11

11

S o 1 vent S vs t em tert. Butyl alcohol 90/1g Ammonia 10 n-Propanol 88/1g Ammonia 1 2 Ether, sat. with water 70% Methanol 85% n-Propanol n-Butanol sat. with 1s Ammonia Benzene-Dioxane-aq. Ammonia (60 :35 :5) Ethanol-Acetic acid-water ( 5 0 : 30: 20) Methanol-butanol (60 : 4 0 ) Cyclohexane-Diethylamine (9: 1) 5% aq. Ammonium sulfate sat. with

Cyclohexane-methanol (50 :50 and 25 :75 ) Chloroform-acetone (50 :50 ) Chloroform-methanol (50 : 50) chloroform-methanol (90: 10) Chloroform-acetone-methanol (50 :25 :25) Chloroform-acetone-methanol (25 :50 :25 ) Chloroform-acetone-methanol-ammonia

Acetone Acetone Acetone-ammonia (100: 1)

Isobutanol

(50 : 25: 25: 1)

R f 0 . 3 7 0 . 5 6 0.09 0 . 6 8 0 . 2 7 0 . 5 7 0 . 3 4 0 . 5 8 0 . 6 8 0 . 0 5

0 . 0 3 0 . 3 6 0 .03 0 . 5 9 0 . 4 4 0 . 4 2 0, 32

0 . 5 4 0 . 0 6 0.16 0 . 3 3

Re f 42

11

11

11

I t

11 n r C -o 1 rn z

11 B N z 53 rn 1

52 11

0 n 0

54 4 4 c

1 r

44 0

51,44,50 45 44 4 4

rn

4 4 44 45 44 ,51

TABLE I11 Cont'd.

Adsorbent Silica Gel

I1

I 1

I 1

I 1

I 1

11

1 1

I 1

13 rn m I 1

II

'1 * 11 * '1 * 11 ** ' 1 **

Silica Ge1,Plain

Chromagram,Plain

solvent System Acetone-ammonia (95: 5 ) Acetone-methanol (50 : 50) Acetone-methanol-ammonia Methanol Methanol-ammonia ( 98 : 2 ) Methanol-ammonia (98: 2 ) Chloroform-methanol-ammonia (80: 20 : 1) Chloroform-ethanol-ammonia (80: 20 : 1) Acetone-benzene-ammonia (30:70:5) Butanol-pyridine-water (1: 2: 1) Cyclohexane-benzene-diethylamine

Cyclohexane-benzene-diethylamine

Methanol Acetone Benzene-ethanol-ammonia (95: 15: 5) Methanol 95% ethanol Chloroform + cyclohexane-diethylamine

Chloroform + cyclohexane-diethylamine

(50 : 50 : 1)

(75:15:10)

(75: 15: 20)

(50:40: 1 0 )

Rf Ref - - 0.54 44 0 . 3 8 0 . 5 6 0 . 3 4 0 .54 0 . 6 7 45 0 .68 30 0 . 7 6 23 -- 46

0.52 47

0 . 0 6 48

0 . 1 8 45 0 .60 48 0 . 2 5 0 .30 45 0 .15 48 0 .06

0 . 2 4 49 0 .48

TABLE I11 Cont d

Rf Ref - - A d s or p t i o n S o l v e n t System Chromagram

+ f l u o r i n d i c a t o r ch lo ro fo rm + cyclohexane-diethylamine 0 . 4 0 49 S i l i c a G e l Chloroform-hexane (1: 1) 0 . 4 8 50

I t 15% aq.ammonium ace ta t e -me thano l (20:80) 0 . 7 5 28***

* p r e p a r e d w i t h 0 . 1 M KOH ** prepa red w i t h 0.1M NaHS03 *** I n t h i s system, f l u p h e n a z i n e - s u l f o x i d e had an Rf o f 0 . 5 9

2 m I

0

0 0

The fo l lowing d e t e c t i o n sys tems have been used: < Colo r a

Reac t ion Ref I System F luorescence under U.V. lamp - 42,44 ,51 ,30 ,28 ,50 6

z 40% s u l f u r i c a c i d s p r a y o range 42,46,47 0 rn 50% a q . s u l f u r i c a c i d i n e t h a n o l ( 4 : l ) orange-red47,52

P o t a s s i u m i o d o p l a t i n a t e v i o l e t 52,54,44,51,49,28 5% f e r r i c c h l o r i d e , 20% p e r c h l o r i c a c i d , 50% ( f l e s h o r

n i t r i c a c i d (5:45:50) (FPN;Forrest r e a g e n t ) (pa le p i n k 44 ,45 ,51 ,53 ,54 ,48 I o d i n e vapors brown 54 ,44 ,48 1% potass ium permanganate 51 Dragendorff r e a g e n t brown 51,48

Detect ion systems used Cont' d.

System 5% p-dimethylaminobenzaldehyde i n 1 8 s

s u l f u r i c ac id Furf u r a l reagent 1% c e r i u m s u l f a t e i n 2 g s u l f u r i c a c i d 1% gold (111) - ch lo r ide Fol in-Ciocal teau reagent 1% ammonium vanadate i n 10 m l . conc.

5% cinnamic aldehyde and 5% HC1 i n e thanol p-benzoquinone i n d ich loroe thane

s u l f u r i c a c i d

c o l o r R e a c t i o n pink o r orange cameo red red cameo

f l e s h cameo

-

Ref - 45,47,51 51,45 47 47 45

45,48 45 55 n

r 0 n rn <

F LUPH E N A2 I N E H Y D R OCH LOR I DE

7 . c o u n t e r c u r r e n t S e p a r a t i o n

phenaz ine ( V I , f i g u r e 6 ) by c o u n t e r c u r r e n t d i s t r i - b u t i o n (see a l s o s e c t i o n 5 ) a c r o s s 25 t u b e s i n a s o l v e n t system o f 0.1M a c e t a t e b u f f e r , pH 3 .82 , and 1 .5% isoamyl a l c o h o l i n h e p t a n e . 2 9

Fluphenaz ine w a s s e p a r a t e d from a c e t y l f l u -

8 . I d e n t i f i c a t i o n and Determina t ion i n Body F l u i d s and T i s s u e s Many o f t h e r e f e r e n c e s c i t e d i n p rev ious sec-

t i o n s concern methods d e v i s e d t o i d e n t i f y and de termine f luphenaz ine h y d r o c h l o r i d e and o t h e r pheno th iaz ines f o r pharmacologica l , t o x i c o l o g i c a l , and f o r e n s i c purposes ( c f . 6 0 ) . These methods can be c l a s s i f i e d as fo l lows :

Color r e a c t i o n s S e p a r a t i o n schemes S p e c t r o f luorometry E l e c t r o p h o r e s i s Paper chromatography Th in - l aye r chromatog-

Gas - 1 i q u i d chromat og-

So 1 u b i 1 i t y X-ray d i f f r a c t i o n bands M i c r o c r y s t a l l i n e iden-

raphy

raphy

t i f i c a t i o n

43 ,45 ,55 ,39 ,18 ,40 ,74 ,75 61,43,45,51,74,79 33,34,35,36,68 42 43 ,43

(28,42-49, 51-54, 65,74, (78 ,79

28,57,58 65 7

19,72

9 . Misce l laneous The a b s o r p t i o n o f f l u p h e n a z i n e d ihydro-

c h l o r i d e and o t h e r p h e n o t h i a z i n e d e r i v a t i v e s by s o l i d a d s o r b a n t s , such a s k a o l i n , t a l c and cha r - coal, h a s been s t u d i e d by Sorby e t a l . i0 v o l u m e t r i c d e t e r m i n a t i o n o f f luphenaz ine by s t o i - ch iometr i c combinat i o n w i t h a n i o n i c su r face -ac - t i v e a g e n t s , such as sodium l a u r y l s u l f a t e and sodium d i o c t y l s u l f o s u c c i n a t e , has been

The

28Y

KLAUS FLOREY

described. 66 ity of fluphenazine and other tranquilizers h a s been studied. 70 scribed. 71

The surface tension-lowering activ-

Ion-pair extraction has been de-

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(2) W. E Thompson, R. J.Warren, J. B. Eisdorfer and J. E. Zarembo, J. Pharm. Sci.3, 1819 (1965) .

(3) R. J. Warren, J. B. Eisdorfer,W. E, Thompson and J. E.Zarembo, J. Pharm. Sci .55, 144 (1966) .

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(14) G. E. Ullyot, U. S. Patent 3,058,979 (1962) , C.A. 58, 68419 (1963).

2Y0

F LUPHENAZINE HYDROCHLORIDE

( 1 5 ) E . I . Anderson, G . B. B e l l i n z o n a , P . N . C r a i g , G . E . J a f f e , K . P . Janeway, C .Kaiser , B . M . Lester , E . J . N i k a w i t z , A.M. P a v l o f f , H . E .Re i f f and C . L . Z i r k l e , Arzne imi t te l -Forschung -9 1 2 937 ( 1 9 6 2 ) .

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p e r s o n a l communication.

( 1 6 ) S c h e r i c o L t d . , B r i t i s h P a t e n t 833,474 (1960)

(17) H . L . Y a l e , F . A . Sowinski and J . B e r n s t e i n , U . S .

(18)D.K.Yung and M.Pernarowski, J .Pha rm.Sc i . 52,

( 19 ) P . R a j eswaran and P . L . Kirk , B u l l . Narcot ics ,

(20) J .Q .Ochs and N . H . Coy, Squibb I n s t i t u t e ,

(21)Michio Nakanish i , J apan . P a t e n t 18,512 (1966) , C . A . 66, 3 7 , 9 3 5 ~ ( 1 9 6 7 ) .

( 2 2 ) M . Izumi and M.Nakanishi, Japan . P a t e n t 4537

(23) J . E . F a i r b r o t h e r , Squibb I n s t i t . u t e , p e r s o n a l

(24) H . Kadin, Squibb I n s t i t u t e , p e r s o n a l communi-

(25) H . Kadin, Squibb I n s t i t u t e , p e r s o n a l communi-

( 2 6 ) T . ~ . T o z e r and L . D.Tuck, J . Pharm. S c i . 54, 1169

( 2 7 ) J . E . F a i r b r o t h e r , Squibb I n s t i t u t e , p e r s o n a l

( 2 8 ) Z . Kofoed, C . Fab ie rk iewicz and G . H . W . L u c a s , J .

( 2 9 ) C . I . Smith and J . C . Burke, Toxic01 . Appl .

(30)A.E.Robinson, J .Pharm.Pharmac. l8, 19 ( 1 9 6 6 ) . (31 ) J . Dreyfuss and A . I . Cohen, J . Pharm. S c i . -9 60

(32 ) J . A l i c i n o , Squihb I n s t i t u t e , p e r s o n a l

( 1 9 6 2 ) , C . A . 58, 10,209h ( 1 9 6 3 ) .

communication.

c a t i o n .

c a t i o n .

( 1 9 6 4 ) .

communication.

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Pharmacol. 2, 553 ( 1 9 6 0 ) .

826 ( 1 9 7 1 ) .

communication.

79 I

KLAUS FLOREY

(33)T.J.Mellinger and C.E.Keeler, Anal.Chem. 35,

(34)J.B.Ragland and V.J.Kinross-Wright, Anal.

(35)S.L.Tompsett, Acta Pharmacol.Toxico1. 2 6 , 298

(36)E.A.Martin, Can. J. Chem. 44, 1783 (1966). (37) J.A.Ryan, J. Pharm. Sci. 48, 240 (1959) . (38)S.P.Agarwal and M.I.Blake, J.Pharm.Sci. 48,

(39)F.M.Forrest, J.S.Forrest and A.S.Mason, Amer.

(40)P.Rajeswaran and P. L.Kirk, Bull. on Narcotics

(41) L. N. Werrum, H. T. Gordon and W. J. Thornburg, J.

(42) T. J.Mellinger and C. E. Keeler, J. Pharm. Sci.51,

(43)K.Macek and J.Vecerkova, Pharmazie 20 , 605

(44)A.NoirfaliseY J.Chromatogr. l9, 68 (1965). (45) I.Zingales, J. Chromatogr. 3 l , 405 (1967). (46)T.Fuwa, T.Kido and H.Tanaka, Yakuzaigaku 25,

(47)Y.Kiroshita, H.Koike, K. Iwashima and

554 (1963).

Chem. 36, 1356 (1964).

(1968).

1011 (1969).

J.Psychiat. 116, 549 (1959).

13, No. 3 , 1 (1961), C.A. 56, 11705b (1962).

Chromatogr. 2, 125 (1960).

1169 (1962).

(1965).

138 (1965), C. A. 64, 6405f (1966) .

S.Oriyama, Nagayu Shiritzu 12, 33 (1964), C.A. 64, 19324h (1966).

(48)W.W.Fike, Anal. Chem. 38, 1697 (1966). (49)P.Schweda, Anal.Chem. 2, 1019 (1967) , C.A.

(50)F. Dondzila, Squibb Institute, personal

(51)A.Noirfalise, J.Chromatogr. 20, 61 (1965). (52)J.Cochin and J.W.Daly, J.Pharmaco1.Exp.Ther.

(53) J.Sunshine, Amer. J.Clin. Pathol. - 40, 576

(54)A.Noirfalise and M. H.Grosjean, J.Chromatogr.

67, 50918j (1967).

communication,

- 139, 154 (1963).

(1963).

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292


(68

(69

(70

(55) J. Jeunier, Ann. Pharm.Fr. 26, - 25 (1968) , C.A. - 68, 117170a (1968).

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(57)M.W.Anders and G . J.Mannering, J.Chromatogr. 7, 258 (1962).

(58)N.C.Cain and P.L.Kirk, Microchem. J. l2, 256 (1967).

(59)C. R. Fontan, N.C. Cain and P. L.Kirk, Mikrochim. 1chnoanal.Acta 1964, 326.

(GO)J.Sunshine, Handbook of Analytical Toxicology. The Chemical Rubber Co. (1969).

(Gl)S.L.Tompsett, Acta Pharmacol.Toxico1. 2% 303 (1968).

(62)H.R. Roberts, Squibb Institute, personal communication.

(63)H.R.Roberts and K.Florey, J.Pharm.Sci. 5 l , 794 (1962).

(64) R. B. Stasiak and N.H. Coy, Squibb Institute, personal communication.

(65)J.L.Kiger and J.G.Kiger, Ann.Pharm.Franc. 23, 489 (1965), C.A. 64, 6407e (1966).

(66) L. A. Araya Ro jas, An. Fac. Quim. Farm. Univ. Chile 15, 101(1963), C.A. 62, 12976~ (1965).

(67)LJacobson, Squibb Institute, personal communication. M.A.Mahju and R.P.Maicke1, Biochem.Pharmaco1. - 18, 2701 (1969). J. N. T.Gilbert and B. J.Millard, 0rg.Mass. Spectrom. 2, 17 (1969). P.M.Seeman and H.S.Bialy, Biochem.Pharmaco1. - 12, 1181 (1963).

(71) B.A. Persson, Acta Pharm. Suec. 2, 335 (1968) . (72)C.N.Andres, J.Ass .Offic.Anal.Chem. - 51, 1020

(73)H.Weissenstein, E. Feller and G.Schelle,

(74)M.Pinkasova, Cesk.Farm. l7, 181 (1968).

-

(1968) and ibid. 53, 834 (1970).

Arzneimittel-Forschung l8, 685 (1968) .

K L A U S FLOREY

(75)B.Gothelf and A.G.Karczmar, 1nt.J.Neuro- pharmacol. 2, 95 (1963).

(76) J. Dreyfuss, J. J.Ross, Jr. and E.C. Schreiber, J.Pharm.Sci. 60, 821 (1971).

(77) A. deLeenheer and A. Heyndrickx, J. Ass. O f f ic. Anal.Chem. 54, 625 and 633 (1971).

(78) C . Korczak-Fabierkiewicz, G. C imbura, J. Chromatogr. 53, 413 (1970).

(79)A.Viala, J.P.Cano and A.Philippe, Ann.Pharm. Franc. 27, 511 (1969).

294

ISOCARBOXAZID

Bruce C. Rudy and Bernard Z. Senkowski

295

B. C. RUDY A N D B. 2. SENKOWSKI

INDEX

A n a l y t i c a l P r o f i l e - I s o c a r b o x a z i d

1. D e s c r i p t i o n 1.1 Name, Formula, Molecular Weight 1 . 2 Appearance, C o l o r , Odor

2 . P h y s i c a l P r o p e r t i e s 2 . 1 I n f r a r e d Spectrum 2 . 2 Nuclear Magnetic Resonance Spectrum 2 . 3 U l t r a v i o l e t Spectrum 2 . 4 F luo rescence Spectrum 2 . 5 Mass Spectrum 2.6 O p t i c a l R o t a t i o n 2 . 7 M e l t i n g Range 2 . 8 D i f f e r e n t i a l Scanning Ca lo r ime t ry 2 . 9 Thermogravimetr ic A n a l y s i s 2.10 S o l u b i l i t y 2 . 1 1 X-ray C r y s t a l P r o p e r t i e s 2 . 1 2 D i s s o c i a t i o n Cons tan t


4 . S t a b i l i t y Degradat ion

5. Drug Metabo l i c P roduc t s

6 . Methods o f A n a l y s i s 6 . 1 Elemental A n a l y s i s 6 .2 Phase S o l u b i l i t y A n a l y s i s 6 . 3 Th in Layer Chromatographic A n a l y s i s 6 . 4 C o l o r i m e t r i c Ana lys i s

6 . 4 1 Ammonium Molybdate React ion 6 .42 T e t r a z o l i u m S a l t React ion

6 . 5 P o t e n t i o m e t r i c Sodium N i t r i z e T i t r a t i o n

7 . References

296

ISOCARBOXAZI D


1.1 Name, Formula, Molecular Weight I s o c a r b o x a z i d i s 5-methyl-3-isoxazolecarboxylic

a c i d 2 -benzy lhydraz ide .

0 -NH-CH

Molecular Weight: 231.26 C12H1 3N302

1 . 2 Appearance, C o l o r , Odor

I t has a s l i g h t , c h a r a c t e r i s t i c o d o r . I s o c a r b o x a z i d o c c u r s as a w h i t e c r y t a l l i n e powder.


2 . 1 I n f r a r e d Spectrum (IR) The IR spec t rum o f bu lk r e f e r e n c e s t a n d a r d i s o -

ca rboxaz id was o b t a i n e d from a p e l l e t made by d i s p e r s i n g 1 .1 mg o f i s o c a r b o x a z i d i n 300 mg o f K B r ( 1 ) . T h i s spec t rum, shown i n F igu re 1, was measured on a P e r k i n Elmer 621 Spec t ropho tomete r . A l i s t o f t h e a s s ignmen t s made f o r some o f t h e c h a r a c t e r i s t i c bands i s g iven i n Tab le I (1) .

TABLE I

C h a r a c t e r i s t i c Bands i n t h e IR Spectrum o f I s o c a r b o x a z i d

Wavelength (cm-l) C h a r a c t e r i s t i c o f

3269 and 3212 2000 t o 1700

1669 1593 and 1453 750 and 704

NH s t r e t c h i n g v i b r a t i o n s Overtone bands o f s i n g l y

C=O s t r e t c h C=C s t r e t c h o f a r o m a t i c s Deformations o f 5 a d j a c e n t

hydrogens on benzene r i n g

s u b s t i t u t e d benzene

297

6. C

. R

UD

Y A

ND

6. 2

. SE

NK

OW

SK

I

cd u :

a

a, k

cd k

ccc c H

lo-

;--

0-

a--

a-

+--

(D--

m--

e--

-

33

NV

lllWS

NV

U %

298

ISOCARBOXAZI D

2 . 2 Nuclear Magnetic Resonance Spectrum (NMR) The NMR spec t rum shown i n F i g u r e 2 was o b t a i n e d

by d i s s o l v i n g 5 7 . 3 mg o f reference s t a n d a r d i s o c a r b o x a z i d i n 0 . 5 m l of C D C 1 3 c o n t a i n i n g t e t r a m e t h y l s i l a n e as t h e i n t e r n a l r e f e r e n c e . The spectral assignments a r e shown i n Tab le I1 ( 2 ) .

TABLE I1

NMK S p e c t r a l Assignments f o r I s o c a r b o x a z i d

Type P ro ton

- CI-I -3

-CH2 - -

-MI-

=CH- -

-

-C& -CONH- -

No. o f Each Chemical S h i f t P ro ton (PP)

3 2.45

3 4 .07

1 4 . 8 5

1 6 . 4 7

5 7.39

1 9 .75

Mult i p 1 i c i t y

S

s s ( b ) S

S

s (b)

S= s h a r p s i n g l e t ; S ( b ) = broad s i n g l e t

2 . 3 U l t r a v i o l e t Spectrum (UV) The UV spec t rum of i s o c a r b o x a z i d i n 2-propanol i n

the r e g i o n 350 t o 210 nm e x h i b i t s n o maximamor minima bu t a s h o u l d e r o c c u r s a t 228-229 rim (~=6.6 x l o 3 ) . The spectrum p r e s e n t e d i n F igu re 3 was o b t a i n e d from a r e f e r e n c e s t a n d a r d s o l u t i o n o f i s o c a r b o x a z i d a t a concen t r a - t i o n o f 1 .014 mg p e r 100 m l o f ?-propano1 ( 3 ) .

2 . 4 F luo rescence Spectrum S o l u t i o n s o f i s o c a r b o x a z i d i n methanol , 0.1N H C 1 ,

and 0.1N NaOli d i s p l a y e d no f l u o r e s c e n c e when t h e e x c i t a t i o n energy used was w h i t e l i g h t ( 4 ) .

2 . 5 Mass Spectrum The mass spectrum o f r e f e r e n c e s t a n d a r d i s o c a r b o x -

a z i d shown i n F i g u r e 4 was o b t a i n e d u s i n g a CEC 21-110 s p e c t r o m e t e r w i t h an i o n i z i n g ene rgy o f 70 eV ( 5 ) . Tab le I11 l i s t s t h e d i a g n o s t i c peaks a p p e a r i n g i n t h e low r e s o l u t i on spec t rum.

299

a .r( N

B. C. RUDY AND B. Z. SENKOWSKI

300

ISOCARBOXAZI D

I .o

.8

.6 w 0 z 3 a

m a

0 v)

.4

.2

0

F i g u r e 3

U l t r a v i o l e t Spectrum o f I s o c a r b o x a z i d

1 I I I I I

200 I , 250 -,L 300

2uu L 3 W JUU

WAVELENGTH, nm,

30 1

6. C. R

UDY AND B

. 2. SEN

KO

WSK

I

302

ISOCARBOXAZI D

TABLE 111

Mass (m/e) I n t e n s i t y Fragment I o n s

231 216 171 154 147 127 110 106 91

Medium Very weak Very weak Very weak Weak S t r o n g Medium Me d i um Very s t r o n g

2 . 6 O p t i c a l R o t a t i o n I s o c a r b o x a z i d e x h i b i t s no o p t i c a l a c t i v i t y .

2 . 7 Mel t ing Range T h e m e l t i n g r ange i s dependent on t h e h e a t i n g

r a t e . Using t h e Class I m e l t i n g p r o c e d u r e o u t l i n e d i n NF X I I I , i s o c a r b o x a z i d melts between 105" and 108°C ( 6 ) .

2 . 8 D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC) A P e r k i n Elmer DSC-1B C a l o r i m e t e r was used t o

o b t a i n t h e DSC c u r v e € o r r e f e r e n c e s t a n d a r d i s o c a r b o x a z i d shown i n F i g u r e 5 . a m e l t i n g endotherm was obse rved s t a r t i n g a t 103.7"C and a broad exotherm due t o decompos i t ion s t a r t i n g abou t 150°C. The A l l f c a l c u l a t e d from t h e m e l t i n g endotherm was 7 . 4 k c a l / mole ( 7 ) .

With a t e m p e r a t u r e program o f lO"C/min.,

2 . 9 The rmograv ime t r i c A n a l y s i s (TGA) The T G A per formed on r e f e r e n c e s t a n d a r d i s o c a r -

boxazid e x h i b i t e d no l o s s o f weight when h e a t e d t o 120°C a t a r a t e o f 1O"C/min. ( 7 ) .

2 .10 S o l u b i l i t y The s o l u b i l i t v d a t a o b t a i n e d a t 25°C f o r r e f -

e r e n c e s t a n d a r d i s o c a r b o x a z i d i s l i s t e d i n 'Table I V ( 8 ) .

303

-0

N

rd X

0

,.c k e

0

0

m

H

rci

0

a, >

k

3

U

U

‘a 0

.ri

B. C. R

UD

Y A

ND

B. 2

. SE

NK

OW

SK

I

0

v c

(w

I u

0

0

9

0

w,

0

Q)

0

0

0

c! 0

d

-

0

co 0

03

0

0

N

Y W

I): 3

I): W

a

5

w

I- G

304

ISOCARBOXAZI D

Solven t

TABLE I V

I soca rboxaz id - S o l u b i l i t y

3 A a l c o h o l benzene ch 1 o r o form 95% e t h a n o l e t h y l e t h e r methanol pe t ro l eum e t h e r (30"-60") 2 -propano1 wa te r


39.6 4 4 . 8

348.6 46.8 16 .5 9 5 . 1

0 . 2 1 6 . 5

0 . 8

2 . 1 1 X-ray C r y s t a l P r o p e r t i e s The x - ray powder d i f f r a c t i o n p a t t e r n o f re f -

e r e n c e s t a n d a r d i s o c a r b o x a z i d i s p r e s e n t e d i n Tab le V ( 9 ) . The i n s t r u m e n t a l c o n d i t i o n s are g iven below:

I n s t r u m e n t a l Cond i t ions :

General E l e c t r i c Model XRD-6 Spec t rogon iomete r

G e n e r a t o r : 50 K V , 1 2 - 1 / 2 MA Tube t a r g e t : Copper 0

Kad ia t ion : Cu Ka = 1.542 A O p t i c s : 0 .1 ' D e t e c t o r s l i t

M . R . S o l l e r s l i t 3" Beam s l i t 0.0007" N i f i l t e r 4" t a k e o f f a n g l e

Goniometer: Scan a t 0 .2" 20 p e r minute D e t e c t o r : A m p l i f i e r g a i n - 16 c o a r s e ,

8 . 7 f i n e S e a l e d p r o p o r t i o n a l c o u n t e r t u b e and DC v o l t a g e a t p l a t e a u , P u l s e h e i g h t s e l e c t i o n E L - 5 v o l t s ; Eu - o u t , Rate meter T . C . 4 , 2000 C/S f u l l scale Char t speed 1"/5 minutes Pre a r e d by g r i n d i n g a t room t em#e r a t u r e

Recorde r : Samples :

305


TABLE V

X-ray Di f f r ac t ion Pa t t e rn o f Isocarboxazid

20

9.100 12 .440 13 .460 14 .860 15 .240 17 .640 18 .440 19 .040 2 0 , 7 0 0 21.260 21.720 23.240 23.480 23.920 24.960 25 .780 27 .300 28.660 29.060 2 9 , 6 8 0 29 .780 30.160 30.280 32.480 34 .520 34 .800 35.780 36 ,540 37.400 37 .940 38.120 38 .760 39 ,340 40 .920 41.420 41.860 4 2 , 3 2 0 43 .080

-

0

d*A

9.7177 7.1151 6 .5781 5.9614 5 .8136 5 .0276 4 .8113 4.6610 4.2908 4.1790 4 .0916 3 .8273 3 .7887 3 ,7200 3 .5673 3 .4557 3 .2666 3 .1146 3.0726 3 .0099 3 .0000 2 .9630 2 .9516 2.7565 2 .5981 2 .5779 2 ,5095 2 .4590 2.4044 2.3714 2.3606 2 .3231 2.2902 2 .2053 2 .1799 2 ,1580 2.1356 2 ,0996

306

I/Io**

41 5 9

1 3 9 5 4

16 44

9 7

15 18 28

100 9 3 5 6 1 2 2 3 4 7 7 2 4 1 1 1

<1 1 3 2 3 2 1

ISOCAR BOXAZI D

45.480 45.880 46.480 46.860 47.980 49.140 50.140 51.680

1.9943 1 .9778 1.9537 1 .9387 1.8960 1.8539 1 .8193 1.7686

nX 2 S i n 0 *d = ( i n t e r p l a n a r d i s t a n c e )

* * I / I o = r e l a t i v e i n t e n s i t y (based on h i g h e s t i n t e n s i t y o f 1 .00 )

2 . 1 2 D i s s o c i a t i o n Cons tan t The a p p a r e n t pKa f o r i s o c a r b o x a z i d has been

de te rmined s p e c t r o p h o t o m e t r i c a l l y t o be 1 0 . 4 ( 1 0 ) .


shown i n F igu re 6 . Methyl 5-methyl-3-isoxazolecarboxylate i s r e a c t e d w i t h benzy lhydraz ine t o form i s o c a r b o x a z i d ( 1 1 ) .

I s o c a r b o x a z i d may be p r e p a r e d by t h e r e a c t i o n scheme

4 . S t a b i l i t y Degradat ion I s o c a r b o x a z i d i s s t a b l e when p r e s e n t i n c r y s t a l l i n e

form. NaOfl, h y d r o l y s i s o c c u r s . After one hour o f h e a t i n g , 7% of t h e i s o c a r b o x a z i d was hydro lyzed i n t h e 0.1N H C l s o l u - t i o n , 1 2 % i n t h e water s o l u t i o n , and 100% i n t h e 0.1N NaOH s o l u t i o n (12 ) .

When h e a t e d a t 100°C i n O . 1 N H C 1 , water, and 0.1N

5 . Drug Metabo l i c P roduc t s

man i s t h e c l e a v a g e and o x i d a t i o n o f t h e benzyl moiety t o benzo ic a c i d fo l lowed by a c o n j u n c t i o n w i t h g l y c i n e t o g i v e h i p p u r i c a c i d ( 1 3 ) . A second m e t a b o l i c mechanism i s t h e h y d r o l y t i c c l e a v a g e o f i s o c a r b o x a z i d forming benzy l hydra - z i n e . Th i s l a t t e r pathway i s more predominant i n g u i n e a p i g s t h a n i n man ( 1 4 ) . F igu re 7 o u t l i n e s t h e s e m e t a b o l i c pathways.

The predominant pathway o f i s o c a r b o x a z i d metabol ism i n

b . Methods o f A n a l y s i s

6 . 1 Elemental A n a l y s i s

307

B. C

. R

UD

Y A

ND

B. 2

. SE

NK

OW

SK

I

'01

Y I 0

.-

& N

I

I B

z I

f yz\o

'-

2

0 =u

a

.r-

N

z .I

0

-c k cd

30

0

am

m

.d

v) a, c

c, f

x

Cn

I )Y

0 0

I

T 6 01

I

z I I

z

I

L

+ m

I

V

?

308

ISO

CA

RB

OX

AZ

ID

I

0

0

0 c\l

-

3

0

kc

,

30

M

0

rd n

c1

0

z

I

0 I

z

q==j \

/

W

I- 2 a

n

-

X 0 Q ‘hl

I

0 I

n

a

-

0

I hl -#-

e N

W

z N

>- I

J

>

N

m z

W

r

a

m

a

I

\ I”

0=0--0-0

0 0

300


The r e s u l t s from an e l e m e n t a l a n a l y s i s o f re fe r - ence s t a n d a r d i s o c a r b o x a z i d are p r e s e n t e d i n Tab le IV ( 1 5 ) .

TABLE VI

Elemental Ana lys i s o f I soca rboxaz id

Element

C H N

% Theory % Found

62 .33 62.22 5 . 6 7 5 . 6 6

18.17 18.20

6 . 2 Phase S o l u b i l i t y Ana lys i s Phase s o l u b i l i t y a n a l y s i s h a s been c a r r i e d o u t

f o r i s o c a r b o x a z i d u s i n g 2-propanol a s t h e s o l v e n t . An example i s p r e s e n t e d i n F igu re 8 f o r r e f e r e n c e s t a n d a r d i s o c a r b o x a z i d a l o n g wi th t h e c o n d i t i o n s under which t h e a n a l y s i s was performed ( 8 ) .

6 . 3 Thin Layer Chromatographic Ana lys i s (TLC) The f o l l o w i n g TLC p rocedure i s u s e f u l f o r

s e p a r a t i n g methyl 5-methyl-3-isoxazolecarboxylate and 1- benzyl-3-methyl-5-aminopyrazole from i s o c a r b o x a z i d (6) . Using s i l i c a g e l GF p l a t e s and e t h y l a c e t a t e : n-heptane (60:40) a s t h e s o l v e n t system, 1 . 0 mg o f t h e sample i n methanol i s s p o t t e d on t h e p l a t e and s u b j e c t e d t o a scend ing chromatography. After t h e s o l v e n t f r o n t has ascended a t l e a s t 1 2 cm, t h e p l a t e i s a i r d r i e d and examined under shortwave u l t r a v i o l e t l i g h t . I f any methyl 5-methyl-3- i s o x a z o l e c a r b o x y l a t e i s p r e s e n t i t w i l l appea r a s a dark s p o t a t about Rf 0 .85 . f r e s h l y p r e p a r e d s o l u t i o n o f 10% FeC13:5% K3Fe(CN)6 ( 1 : l ) . I f any l-benzyl-3-methyl-5-aminopyrazole i s p r e s e n t i t w i l l appea r a s a b l u e s p o t a t about Rf 0 .25. The i s o c a r b o x a z i d produces a b l u e s p o t a t about Rf 0 . 6 .

Next s p r a y t h e p l a t e w i t h a

6 . 4 C o l o r i m e t r i c Ana lys i s

6 . 4 1 Ammonium Molybdate React ion The i s o c a r b o x a z i d c o n t e n t i n t a b l e t s may be

determined by t h e f o l l o w i n g p rocedure ( 6 ) . The t a b l e t s a r e f i n e l y ground, a p o r t i o n e q u i v a l e n t t o about 10 mg o f i s o c a r b o x a z i d i s weighed i n t o a 50 m l v o l u m e t r i c f l a s k and

310

Figure 8

w c e

t / 1

i PHASE SOLUBILITY ANALYSIS

SAMPLE: ISOCARBOXAZID

SOLVENT: 2 - PROPANOL

su)pE: 0.0 %I

EQUILIBRATION: 20 HRS. ot 25O C

EXTRAPOLATED SOLUBILITY: 20.5 W/g af 2- PROPANOL -

-

0 I I I 0 25 50 75 I00

- ffl 0 0 > D m 0


8. C. RUDY AND 6. Z. SENKOWSKI

d i l u t e d t o volume w i t h a c e t o n e . After thorough mixing, t h e s o l u t i o n i s c l a r i f i e d by c e n t r i f u g i n g and 5.0 m l i s p i p e t t e d i n t o a s t o p p e r e d f l a s k . To t h e f l a s k are a l s o added 1 . 0 m l o f water, 50.0 ml o f a c e t o n e , and 1 . 0 m l o f ammonium molybdate r e a g e n t ( 1 gm o f ammonium molybdate d i s s o l v e d i n 100 m l o f d i l u t e HC1). The f l a s k i s s t o p p e r e d and t h e s o l u t i o n mixed and t h e n a l lowed t o s t a n d 30 minu tes w i t h o c c a s i o n a l s w i r l i n g . The absorbance o f t h i s s o l u t i o n i s de te rmined a t 420 nm a g a i n s t a b l a n k . Reference s t a n d a r d i s o c a r b o x a z i d i s p r e p a r e d a t approx ima te ly t h e same c o n c e n t r a t i o n and i n a similar manner as above. The mg o f i s o c a r b o x a z i d p e r t a b l e t i s c a l c u l a t e d by t h e fo l lowing fo rmula :

(A sample) (mg s t a n d a r d ) (Av. t a b l e t w t . i n mg) (A s t a n d a r d ) (mg (5)

where 5 i s t h e d i l u t i o n f a c t o r .

6 .42 T e t r a z o l i u m S a l t Reac t ion The i s o c a r b o x a z i d c o n t e n t i n blood may be

determined i n t h e r ange o f 0.5 pg/ml o f plasma by t h e fo l lowing p r o c e d u r e . Add 1.5 m l o f pH 7 . 3 phospha te b u f f e r t o 1 m l o f b lood and l e t s t a n d f o r 10 minu tes . Add 4 ml of b u t y l a c e t a t e : b u t a n o l ( 9 : l ) and 0 . 7 5 gm o f Na2S04: MgO powder (30: 1 ) . Shake f o r 30 minutes and t h e n c e n t r i f u g e u n t i l t h e phases s e p a r a t e . i n t o a t e s t t u b e , add 0 .1 ml o f t h e t e t r a z o l i u m r e a g e n t (0 .125% 3,3'-dianisole-bis-4,4'-(diphenyl) - t e t r a z o l i u m c h l o r i d e i n methanol) and 0.1 m l o f O.UL N KOH. Place i n a b a t h o f b o i l i n g w a t e r e x a c t l y one minute t h e n c o o l r a p i d l y by immersing i n c o l d w a t e r . The i s o c a r b o x a z i d h a s reduced t h e t e t r a z o l i u m s a l t t o a b l u e formazan. Read t h e absorbance o f t h e s o l u t i o n a t 520 nm w i t h i n 30 minu tes . A t t h e same t ime run t h e above p rocedure on a 1 ml blood b lank and a ' l m l b lood sample c o n t a i n i n g 5 ugm o f ref- e r e n c e s t a n d a r d i s o c a r b o x a z i d . C o r r e c t f o r t h e blank and use t h e r e f e r e n c e s t a n d a r d t o c a l c u l a t e an e x t i n c t i o n c o e f f i c i e n t which i s used t o c a l c u l a t e t h e unknowns (16,17),

P i p e t 3 m l o f t h e o r g a n i c phase

6 . 5 P o t e n t i o m e t r i c Sodium N i t r i t e T i t r a t i o n The p o t e n t i o m e t r i c sodium n i t r i t e t i t r a t i o n as

d e s c r i b e d i n t h e NF XI11 i s t h e method o f c h o i c e f o r t h e a n a l y s i s o f t h e i s o c a r b o x a z i d fiiie chemical ( 6 ) . A sample

312

ISOCARBOXAZI D

of about 700 mg is accurately weighed and dissolved in 20 m l of glacial acetic acid. Following dissolution 20 ml of I1C1 arid 40 nil o f water is added and the solution cooled to room temperature. NaN02 solution using a calomel-platinum electrode system. Each ml of 0.1N NaN02 is equivalent to 23 .13 of isocarboxaz id.

Titrate potentiometrically with 0.1N

313


7 . References

1.

2 .

3.

4 .

S .

6 . 7 .

8 .

9.

10 * 11.

1 2 .

13.

1 4 .

15.

1 6 .

1 7 .

M. Hawrylyshyn, Hoffmann-La Roche Inc., Personal Communication. J. H. Johnson, Hoffmann-La Roche Inc., Personal Communication. L. B. Bandong, Hoffmann-La Roche Inc., Personal Communication. J. Boatman, Hoffmann-La Roche Inc., Personal Communication. W. Benz, Hoffmann-La Roche Inc., Personal communication. National Formulary XIII, 378-379 (1970) . S . Moros, Hoffmann-La Roche Inc., Personal Communication. E. MacMullan, Hoffmann-La Roche Inc., Personal Communication. R. Hagel, Hoffmann-La Roche Inc., Personal Communication. E. Lau, Hoffmann-La Roche Inc., Unpublished Data. T. Gardner and E. Wenis, Hoffmann-La Roche Inc., Unpublished Data. R. Colarusso, Hoffmann-La Roche Inc., Unpublished Data. B. Koechlin, M. Schwartz and W. Oberhznsli, J . PharmacoZ. ExptZ. Therap. 138, 11 (1962) . M. Schwartz, Proc. Soc. Exp. G l . Med. 107, 613 (1961) . F . Scheidl, Hoffmann-La Roche, Personal Communica- tion, M. Roth and J. Rieder, Biochem. Pham. - 1 2 , 445 (1963). B . Koechlin, L. D'Arconte, F. Rubio and R. Engleberg, Hoffmann-La Roche Inc., Roche Product ManuaZ (1971) .

-

314

ISOPROPAMIDE

Rulph S. Suntoro, Rickurcl J. Wurrcn, Geruld D. Roberts. Edward White, V , utid Peter P. Begosh

315

RALPH S. SANTORO e t a /

CONTENTS

1. Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor, Taste

2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Melting Range 2.6 Differential Thermal Analysis 2.7 Thermogravimetric Analysis 2.8 Solubility 2.9 Polarography

2. Physical Properties

3. Synthesis I+. Stability - Degradation 5. Drug Metabolic Products 6. Methods of Analysis

6.1 Isopropamide Chemical 6.11 Elemental Analysis 6.12 Titration of Iodide Functional Group 6.13 Non-Aqueous Titration 6.1 I+ S pe ct r ophot omet r i c Anal y 8 is 6.15 Chromatographic Analysis

6.21 Spectrophotometric Analysis 6.22 Spectrophotometric Analysis in Presence of

6.23 Colorimetric Analysis

6.2 Dosage Forms

Interferences

7. Notes and References

316

ISOPROPAMI DE

1. Description

1.1 Name, Formula, Molecular Weight Isopropamide is (3-Carbamoyl-3,3-diphenylpropyl)-

diisopropylmethy1)amonium iodide. Darbid; Priamid; Tyrimide; 2,2-diphenyl-4-diisopropyl- aminobutyrimide methiodide.

It is also known as

C23H331N20 Mol. wt.:48O.443

1.2 Appearance, Color, Odor, Taste A white to off-white (very pale yellow) crys-

~~

talline or amorphous powder, odorless, with an extremely bitter taste.


2.1 Infrared Spectrum Figure 1 is the infrared spectrum of isopropamide

(SK+F standard SJB-4Q6-226A ) taken in a mineral oil dispersion from 4000 - 625 cm -' on a Perkin-Elmer Model 457. R. Warren assigns the following bands (cm to isopropamide:

3530 and 3475; free NH 3300; bonded NH2 1665; amide C=O 1585 and 14%; aromatic C=C and NH2 770-710; aromatic CH out-of-plane deformation mono-substituted phenyls

317

w c m

T O 8 0 P O 10 I2 I 4 I 6 18 10 1 5 1 0 3 5 4 0 MICRONS

3 0 . O s o 6 0 0 0 2 5

WAVENUMBER I C M ~ l l

Figure 1. Isnpropamide - SK+F standard SJB-4406-22&, mineral oil, dispersion, Instrument: Perkin-Elmer 457.

ISOPROPAMIDE

2.2 Nuclear Magnetic Resonance Spectrum I a i n e d i n a

deutero chloroform' solution of SK+F standard SJB-4406- 226-A which contained about 100mg/ml and tetramethyl- s i lane as internal reference on a JEOL ModelC 60 H.The following a S S i g n ~ ~ ~ ~ t € i (Hz) were made by R. Warren:

@ @

8 + @ @ 63

0 9 f J @ By3 NH2 - C - C - CH2 CH2 N D H ( C H J ) J ~ -1 -

I

82; doublet, protons a t @

177; protons a t @ @ @ 244.5 ; multiplet, protons a t @

340 and 387; protons a t

447; protons a t @ @

319

W F 3 C

60 M C : ~ ~ O 480 420 360 300 240 180 120 60 0 CPS I 1 I I 1 I I I I

I I I I I I I I I 9 8 7 6 5 4 3 2 1 0

Figure 2. NMR Spectrum Isopropamide, SK+F standard SJB-4406-226A in

PPm

deuterochloroform tetramethylsilane internal reference, Instrument: JEOL C 60 H .

ISOPROPAMI DE

2.3 Ultraviolet Spectra The ultraviolet absorption spectrum of iso-

propamide (SK+F standard SJB-4406-226A ) in water is shown in Figure 3. 258.5 nm (a = 1.053; (= 505.9) are bands characteristic for the mono-substituted phenyl moiety (1). Figure 4 is the ultraviolet spectrum in water under conditions suitable for observing the iodide maximum absorbance at 222.5 nm (a = 37.0;E = 17,757) (2, 3 , 4 ). Beginning at 220 nm the automatic slit begins to rapidly open. Neither maximum wavelengths nor absorptivities different from those determined in water solution were observed in C.1 N hydrochloric acid and 0.1 N sodium hydroxide solutions.

Maxima at 265 nm (a = 0.745; € = 357.9) and

- -

Figure 5 is the ultraviolet absorption spectrum of isopropamide in methylene chloride. The maximum at 245 nm (a = 33.7; f = 16,204) is due to the iodide. The automatic slit begins to rapidly open below 227.5 nm.

Figure 6 is the ultraviolet absorption curve of isopropamide in water after the solution was passed through an anionic exchange resin (5) in which the iodide was converted to the chloride; this allows the mono- substituted phenyl spectrum to be more readily observed. Maxima appear at 265 nm (a = 0.721 ; f = 346.4), 258.5 nm (a = 0.9k;C = 451.8), and 252.5 nm (a = 0.856; Neither maximum shifts nor absorptivity differences were observed in the above spectrum when the solution pH was rendered either acidic or basic.

= 411.5)

321

RALPH S. SANTORO e t a / .

Fig. 3

322

ISOPAOPAMIDE

Fig . 4

323

RALPH S. SANTORO e r a /

WB-44C6-226A

Concen tmtmn: 0.0338 n d m l

blethylcne Chloride

cell I’ath: 1 Cm

Fig . 5

324

ISOPROPAMIDE

F i g . 6

325

RALPH S. SANTORO era/.

2.4 Mass Spectrum The mass spectrum of SK+F 4740-5 (SK+F standard

SJB-’t’t06-226-A) was obtained by direct insertion of the solid into an Hitachi Perkin-Elmer RMU-6E low resolution mass spectrometer. The results are presented in tabular form in Table I and as a bar graph in Figure 7. and G. Roberts provided the following observations and ass i gnment s :

E. White

The compound thermally decomposes in the instrument, and consequently no molecular ion is observed for the quaternary salt. The spectrum consists of the sum of the spectra of six thermal products which are listed in Table 11. All of the structurally significant peaks are discussed below.

Compounds I, 11, and 111, tertiary amines show a molecular ion as noted in Table 11. Each of the compounds cleaves p to the amino nitrogen by losing a methyl group resulting in peaks at m/e 100, rnfe 295 and mfe 323 respectively. Compounds I1 and 111 also exhibit cleavage p to the amino nitrogen with strong peaks at rnfe 86 and mle 114 respectively. Following the loss of the methyl groups from the molecular ions of I1 and I11 there is loss of NH, CHO resulting in peaks at mf e 250 and m/e 278. Following the loss of the methyl group from I there is loss of propene resulting in a peak at rnfe 58 as transition is supported by a metastable at rnfe 33.6

Compounds IV, V, and VI are alkyl iodides and as noted in Table 2 a molecular ion is observed for IV and V. Although no molecular ion is observed for VI, its presence is indicated by fragment ions. mle 237 represent the loss of an iodine atom and hydrogen iodide. The ion at m/e 237 further decomposes t o m/e 236 by loss of its hydrogen atom, this transition being supported by a metastable at m/e 235.0. origins of mle 193 and m/e 194 are the loss of NH2CH0 from mle 238 and the loss of NHCO from m/e 237. mle 178 and m/e 179 are possibly formed by loss of a methyl group from mle 193 and mle 194.

The peaks at m/e 238 and

The probable

The ions at

Other structurally significant peaks which could have originated from more than one of the six compounds

326

ISOPROPAMIDE

MIS INTCN. 15.8 0.s 16.9 19.0 P9.0 8i.a P7.R 8R.R R9.0 3m.m 31.0 3e.m

34.a 33.c

36.1 37.R 3 5 . 0 39.63 4e.e 41.0 a8.m 43.R 04.8 45.R 46.8 5A.B $1.8 S2.R s3.0 54.R 55.0 56.0 S7.0 58.8 59.0 68.0 68.0 63.8 64.69 6S.8 67.8 68 .a 69.R 70.8 71.9 7Q.8 73.8 z4.9 7S.8 76.8 71.9

a.p R . 5 R.S e.9

B3.5

3. s 11.7 8.9

s.n

m.4 @.a I .9 R.9 9.n 3.4

7. A 49.7 94.5 5R.R P9.b 15.Q

R . 4 1 . 1 (1.-

I .Y

V . 6 I .4 7 . I

PI .4 0.9

1 Q f l . R 4 . 3 0.5 1.6 3.7 8.7 3.4 0.5 1.1 (1.9 6.3 5.9

16.8 1.5

~ m . 4

0.9 e. I

18.8 4.8

m.9 8.8

MASS INTEN. 79.8 1.9 8m.R “ 1 .G 42.6 83.8 u4.0 R5.R 46.R 97.G R 8 . R

9 0 . A R9.m

91.n 3P.n 91.n 94.m y5.m q6.a 97.0 9q.m 99.0

1nn.a I C I 1 . A

1 ~ 3 . m 1ma .R l0S.Q 1w.a IF7.R I09.R 11e.n Il1.R

113.0 114.8 I IS.#

ll7.R

I149.W

I 1P.m

I i6.m

I i 8 . m I 19.m 180.0 l 2 l . R 105.e l26.R

lPR.8 l29.Q 138.8 131.8 132.0 133.0

i27.m

A . 5

4.7 3.7 5.6 9.7

77.5 5.3 P.!? 9.B 1 .R

m.9

14.9 t .n 0.4 I .5 I .4 P. I R.4 1.7

89.9

3.? 13.9 4 .1 I .5 Q.6 0.4

e.4

R.5 1.4

31.5 (14.8 P5. I 17.3 3.7

8.5 I. 3 8.4 1.5

18.9 18.1

1.8

0.4 I .4 0.4

9.a

7.n

m.4

a.4

8.0

I .m

MASS INTCW. 137.0 0.4 I 3 R . R 139.0 14R.R I 4 I . R 14P.8 I 4 3 . P I4h.R 149.R 15R.R 1 5 1 . R 14P.P 1 5 i . m I -4.R I 5 5 . P 14n.0 1 5 9 . ~ 1nm.R 16i .a

in?.# i610.m I ns.a i 6 n . R I 6’1.13

17a.e 171.m

1LP.R

164-R

1’)P.R 174.0 f7S.P I 7n.e 1w.a

179.0

i 6 i . a

1113.0

l t 9 . R

I R 0 . 8

1RP.R

IS5.8 lR7.0 I B R . 0 189.0 190.0 191 .0 192.8 193.8 194.8 195.8 196.0

m.a 4. I 1.3 1.9

12.5 a.3 a.4 R.5 1.1 P.2 4 , s 9.3 6.4 R.7 R.5 R.5 2.3 R.6

4.7 5.5

49.7 13.2 7.6 I .4

86.3 (1.9

8.5 (1.4 P- 6 4.1 33.8 31 -9 11.7 2.3 3. I 0.8 8 . 3 0.4 0.6

4.4 7.5

2 R - 6 81.6 33.3

8.4

8.0

(1.3

3.0

4.2

203. 8 Q84.8 Q8S.0 286.8

209.0 209.0 Ql0.0 QlL a 0

Ql2.0 R 1 R . A PI9.G

ea7.0

BP(1.a eei .0 Qe2*0 $23.8 815.8 R34.8 835.9 936.8 Q37.8 238.8 P39.0

850.0 851.8 250.8

R64.0 265.0 261.8 868.8 876.8 e77.0 278.8 879.8 280.8 292.8 895.8 e96.0 297.0 305.8 309.0 310.0 311.0 323.0 384.8 358.8 339.8

848.0

1353.9

0.9 0.4 8.8 0.9 1.6

2. 1 6.8 fl.8 0.4 1 . 1 0.6 8.3 8.9 0.3 8.4 0.4 3.Q

86.0 35.1 36.4 6.5 a. 7 6.8

8.7 9.3 1 r 4

8.3 3.3 0.8 1.2 8.3 4.8 1.7 8.4 8.8

1.2 O.P 0.3 0.4 0.8 0.0 8.8 0.4 0.8 8.3

I .a

e.3

5.0

321

20

I i " ' I " ' I " ' I " ' I " ' I I I I

50 90 130 170 210 250 290 330 370 410 450 490

fn 9 z -I

0 ::

Figure 7. Low resolution mass spectrum of isopropamide, SK+F standard SJB-4406-226A.

ISOPROPAMIDE

are m l e 43 [CH(CH,);?, m / e 127 (I4), m/e 128 ( H I ? ) and m/e

,

TABLE I1

T h e r m a l Products of I s o p r o p a m i d e

--- -

I CH, NLWCH~ )272 M + 115

y 3

M + 310 9 ?

I1 NH2 C C CH2 CH2 N CH(CH3 )2 I 8

+ IV CHj I M 142

VI

0

+ M not observed

329

RALPH S. SANTORO e t a /

2.5 Melting Range -- Isopropamide (SK+F standard SJB-4h6-226-A)

melted between 189.0 - 191 .pC. without decomposition under USP conditions for class I substances (6).

2.6 Differential Thermal Analysis The differential thermal analysis of isopropamide

(SK+F standard SJB-4406-226A ) beginning at 43O C and- with a heating rate of 20°C/min. resulted in a single major me1 t ing endotherm at 200' C .

Other chemical lots tested under the same conditions showed minor endothermic transitions below the major endotherm.

tentatively attributed to polymorphic forms known to exist with this compound (7).

These minor transitions have been

2.7 Thermogravimetric Analysis A thermogravimetric analyeis performed on iso-

propamide (SK+F standard SJB-4406-2264) showed a 0.5' f 0

loss in weight complete at about 105'C. The measurement was performed under nitrogen sweep at a heating rate of 10'Clrnin. sample began melting 8t approximately 18pc.

Additional weight was rapidly lost as the

330

ISOPROPAMIDE

2.8 Solubility -- The following solubilities were obtained at room

temperature:

Solvent

ether benzene cycl ohexane pH 7.5 buffer water 0.1 N hydrochloric acil 0.1 % sodium hydroxide acetzne 9 9 1 0 ethanol methylene chloride chloroform methanol

Solution -- col or 1 es s col or1 ess colorless color 1 es s color 1 es s

conc. mg / ml 0.07 0.09 0.10 20.7 21.2

faint yel-Jw 23.6 col or1 es s 23.6 bright yellow 24.9 faint yellow 109 .O

deep brownish yellow >500 deep brownish yellow > 500

brownish yellow 330.4

In addition, the following solubilities at room temperature have been reported ( 8 ):

Solvent Conc. mdml

Ethyl acetate 0.1 1,1, dichloroethane 1 *3 isopropanol 3.2 Methyl ethylketone 4.0 1 g hydrochloric acid 29.0

331

RALPH S. SANTORO et a/.

2.9 P olar ogr aphy The polarogram was obtained on isopropamide (SK+F

standard standard

SJB-kh06-28A ) in 0.1 potassium nitrate with a calomel IdropDing mercury electrode system at a

-0.25k V. The E 112 for iodide determined under similar concentration of 5 x 10 -’& molar ( 9 ) .

conditions was [email protected] V.

The E 112 was

7. Synthesis

following steps (Figure 8): The synthetic pathway to isopropamide includes the

2-Diisopropylaminoethyl chloride (I) is condensed with diphenylacetonitrile (11) in the presence of an alkaline condensing agent and the product 4-diisopropylamino-2,2- diphenvlbutyronitrile (111) purified by distillation.

By reaction with acid, the 4-diisopropyLamino-2,2- diphenylbutyronitrile (111) is partially hydrolyzed to 4- diisopropylamino-2,2-diphenylbutyramide (IV) which is Durified by recrystallization.

Finally (3-carbamoyl-3,3-diphenylpropyl) -diisopropyl- methylammonium iodide (V) is prepared by reacting 4- diiso~ronylamino-2,2-diphenylbutyramide (IV) in solution with methyl iodide. The product is collected by filtration, washed, dried and assayed.

IsoproDamide is covered by U.S. Patent Number 2823233. The original synthesis is described by Janssen et al. ( lo).

l~ Stability - Degradation - - _---- _I_--_I

The dry chemical stored in glass bottles at room temnerature is stable for at least ? years. In aqueous eolution (1 - 5 rngtrnl) stored at room temperature the drug is stable for at least 6 months 01). Because the drug is stable, degradation products have not been observed to occur either under normal or exaggerated storage conditions.

5. g ~ u g Metabolic Products No isoDropamfde metabolic products have been reported

thus far.

332

Figur . : .!

SYNTHETIC PATHWAY TO ISOPROPAMIDE -

NC -kH + C 1 C H 2 C H P i alkaline > condensing \ /CH, agent

HC,

1



6.1 Isopropamide Chemical

6.11 Elemental Analysis

Element O l a Theory Range Obtained

57.50 57.80 - 57.92 6.90 7.00 - 7.06

C H N 5.83 5.69 - 5.78

6.12 Titration of Iodide Functional Group -. An accurately weighed sample (About 1 g.) is

dissolved in a 5:5: 1 mixture of water:methyl alcohol: glacial acetic acid and titrated with 0.1 and Eosin Y as indicator until the precipitate becomes rose red. to 0.01269 of iodide or 0.04804 g. of isopropamide.

silver nitrate

Each milliliter of 0.1 N silver nitrate is equal

6.13 Non Aqueous Titration -_. . _-.-- ~ ~ ---- -- An accurately weighed sample (about 0.75 g.)

is dissolved in glacial acetic acid, mercuric acetate and methyl violet indicator added, and titrated with 0.1 N Derchloric acid in acetic acid to a blue endpoint. milliliter of 0.1 N perchloric acid is equivalent to 0.0'+804 R. of isopropamide.

Ezch

6.14 - _ _ Spectrophotometric ~ Analysis _- The ultraviolet absorption spectrum

obtained either by direct dilution or preliminary treatment with resin (see section 2.3) may be used for determining purity.

6.15 Chromatographic Analysis The following thin layer method may be used

for the qualitative purity evaluation of isopropamide:

Equilibrate a mixture of 60 ml. each of isoamyl and tert-amyl alcohols with 20 ml. of 88'1, formic acid dissolved in 100 ml. of water. layer and use the alcohol layer as the chromatographic solvent. dissolved in methanol two cm. from the edge of an 'Avicelf

Discard the aqueous

Spot 25 and 50 micrograms of isopropamide

334

ISOPROPAMIDE

or cellulose plate (12)~ place the prepared plate in a suitable chromatographic chamber lined with filter paper saturated with the developing solvent, and allow to equilibrate for 45 minutes. to a line drawn across the plate 10 cm. from the origin, remove the plate, and air dry in a fume hood until solvent vapore are no longer detectable. gram may be visibilized under ultraviolet light (254 and 365 nm), visible light, and iodoplatinate reagent (13). The quaternary moiety has an approximate Rf of 0.8, while the iodide appears at approximately 0.2.

Allow the solvent to rise

The developed chromato-

6.2 Dosage Forms Isopropamide is found in dosage forms both alone

and in combination with primary and tertiary amines. methods include ultraviolet analysis after extraction of interfering amines and treatment with an anionic exchange resin, and colorimetric analysis by ion-paring with acid dyes and extraction of the complex into an organic solvent.

Assay

6.21 S pect rophot met r i c Ana 1 y s is The ultraviolet assay of tablets which con-

tain only isopropamide as the active ingredient is the most direct method employed (14). Twenty tablets ( 5 mg/ tablet) are placed in a 250 ml. volumetric flask, dis- integrated in about 150 ml. of water, and diluted to volume with water. The solution is filtered through Whatman No. 1 filter paper (15),W.O ml. of the filtrate percolated through an anion exchange column (16), and the column rinsed thoroughly with water. A l l eluates are collected in a 100 ml. volumetric flask and diluted to volume with water. of this solution is compared with that of a known standard of isopropamide under the same spectrophotometric

The ultraviolet absorption spectrum

condit i

contain ac compl

ns and the assay value per tablet calculated.

6.22 spectrophotometric Analysis in Presence of Interferences The ultraviolet assay of dosage forms which

nther amines removable by solvent extraction is shed by treating the eluate collected in 6.21 with

1

ammonium hydroxide until basic and extracting with ether to remwe the Interfering amines (17).

335


6.23 Color imet r ic Analysis The s e l e c t i v e de te rmina t ion of isopropamide . .

by ion-pair ingwith methyl orange dye has been descr ibed (18). ex t r ac t ed i n t o chloroform. Primary, secondary, and t e r t i a r y amines t e s t e d through t h e procedure d id not cqmplex or i n t e r f e r e wi th t h e subsequent spectrophotomet r ic determinat ion a t 520 nm.

The quaternary amine i s ion-paired a t pH 10.2 and

336

ISOPROPAMI DE

7. Notes and References

1. 2.

3. 4.

5 .

6. 7. 8.

9.

10.

11.

12.

13.

14. 15 -

16. 17. 18.

U . S . P . XVIII, p. 825. Brode, W.R., Chemical Spectroscopy, 2nd Ed., John Wiley and Sons Inc., New York, (1947). Brode, W.R., J. Am. Chem. SOC., 48, 1877, (1926). Bolz, D.F., Colorimetric Determination of Non- metals, Volume V I I I , Interscience Publishers Inc., 1947, p. 218. Amberlite IRA !+GO, chloride form, Mallinkrodt Chemical Works.

I. B. Eisdorfer, Personal Communication. Paul Janssen, Private Communication, Eupharma-Ned- Chem Pharmaceutical Laboratories. Kolthoff, I.M., and Lingane, J . J . , Polarography, Second Edition, Interscience Publishers, New York and London, 1952, p. 579. Janssen et al., Arch. Intern Pharmacodyn., 103, 1955, 82. Private Communication, Eupharma-Nedchem Pharmaceutical Laboratories. 'Avicel'-microcrystalline cellulose - American Viscose. the compound and is to be avoided. One gram of chloroplatinic acid, 10 ml. of 1; hydrochloric acid, and 5 g. of potassium iodide diluted to 250 ml. with waterasa stock solution. Refrigerate. equal volumes of stock solution and water made 112" l o with 88" lo formic acid. M. Kushner, Personal Communication. If the solution is not clear at this point it will be necessary to add 5 ml. of l@ /o aluminum chloride and 2 ml. of concentrated ammonium hydroxide to the l5G ml. of water before diluting to volume. Amberlite IRA LOO, chloride form. P. DeLuca and W. F. Witmer, Personal Communication. R. Santoro, J. Am. Pharm. ASSOC., S c i . Ed., 49, 1960, 666.

U . S . P . XVIII, p. 935.

Silica gel may cause decomposition of

Prepare a working solution with

337


Acknowledgements

The authors wish to thank Joan Rudin for her search of the literature and Patricia Brittingham for her invaluable secretarial help.

338

LEVALLORPHAN TARTRATE

Bruce C. Rudy and Berriurd Z. Senkowski

339

B. C RUDY AND B 2 . SENKOWSKI

I N D E X

A n a l y t i c a l P r o f i l e - L e v a l l o r p h a n T a r t r a t e

1 . D e s c r i p t i o n 1 . 1 N a m e , Formula , M o l e c u l a r Weight 1 . 2 Appearance , C o l o r , Odor

2. P h y s i c a l P r o p e r t i e s 2 . 1 I n f r a r e d Spec t rum 2 . 2 N u c l e a r Magne t i c Resonance Spec t rum 2 . 3 U l t r a v i o l e t Spec t rum 2.4 F l u o r e s c e n c e Spec t rum 2 . 5 Mass Spec t rum 2 . 6 O p t i c a l R o t a t i o n 2 .7 M e l t i n g Range 2 . 8 D i f f e r e n t i a l Scann ing C d l o r i m e t r y 2 . 9 Thermal G r a v i m e t r i c A n a l y s i s 2.10 S o l u b i l i t y 2 . 1 1 X-ray C r y s t a l P r o p e r t i e s 2.12 D i s s o c i a t i o n C o n s t a n t


4 . S t a b i l i t y D e g r a d a t i o n

5 . Drug M e t a b o l i c P r o d u c t s

6. Methods o f A n a l y s i s 6 . 1 E l e m e n t a l A n a l y s i s 6 . 2 P h a s e S o l u b i l i t y A n a l y s i s 6 . 3 Th in Layer Chromatograph ic A n a l y s i s 6 . 4 Gas L i q u i d Chroma t o g r a p h i c A n a l y s i s 6 . 5 Direct S p e c t r o p h o t o m e t r i c A n a l y s i s 6 . 6 C o l o r i m e t r i c A n a l y s i s 6 . 7 N e p h e l o m e t r i c A n a l y s i s 6 . 8 T i t r i m e t r i c A n a l y s i s


340



1.1 Name, Formula, Molecular Weight Leva l lo rphan t a r t ra te is (-)-17-allylmorphinan-3-

I I7 CH*CH=CH,

01 t a r t r a t e (1:l).

HO q y H 2 C 4 H 4 0 6

6 C H N O . C 4 H 6 O 6 Molecular Weight: 433.51 1 9 25

1 . 2 Appearance, Color , Odor Leva l lo rphan t a r t r a t e o c c u r s as a w h i t e o r p rac -

t i c a l l y w h i t e , o d o r l e s s , c r y s t a l l i n e powder.


2 . 1 I n f r a r e d Spectrum ( I R ) The I R spectrum of l e v a l l o r p h a n t a r t r a t e is

g r e a t l y compl i ca t ed by t h e p r e s e n c e o f t h e t a r t a r i c a c i d . To o b t a i n t h e I R spectrum c h a r a c t e r i s t i c of t h e a c t i v e s p e c i e s , t h e f r e e l e v a l l o r p h a n b a s e was e x t r a c t e d from a b a s i c s o l u t i o n , d r i e d , and measured as a K B r d i s p e r s i o n ( 1 . 7 mg l e v a l l o r p h a n / 3 0 0 mg KBr) on a P e r k i n E l m e r 621 Spec t ropho tomete r . T h i s spectrum is shown i n F i g u r e 1 and t h e a s s ignmen t s f o r t h e c h a r a c t e r i s t i c bands are g i v e n i n Tab le I (1).

Tab le I

I n f r a r e d Assignments f o r Leva l lo rphan

Frequency (cm-1) C h a r a c t e r i s t i c of

3580* OH s t r e t c h 3076 Aromatic CH s t r e t c h 2924-2919 and 2851 Asymmetric and symmetric

1644 C=C s t r e t c h of v i n y l group s t r e t c h of CH2

* D e t e c t a b l e o n l y i n s o l u t i o n spec t rum, n o t i n K B r p e l l e t .

341

F i g u r e 1

Infrared Spectrum of Levallorphan

WAVELENGTH MICRONS

2.5 3 4 5 6 7 8 9 10 12 15 1822 3550 I

10 I I I 1 I I 1 I l l 1 I l l

I I I I I I I I I 1 4000 3500 3000 2500 2000 1700 1400 1100 800 500 200

FREQUENCY (CM-')

m

0 n C [7 -< b 2 0

m N

v) m 2 x

2 m 5

LEVALLORPHAN T A R T R A T E

1607

14 54 992 and 910

Aromatic s k e l e t a l v i b r a - t i o n s

CH de fo rma t ions 2 CH o u t o f p l a n e bend ing of v i n y l group

2.2 Nuclear Magnet ic Resonance Spectrum (NMR) The s p e c t r a of l e v a l l o r p h a n t a r t r a t e (A) and

l e v a l l o r p h a n b a s e (B) are shown i n F i g u r e 2. These s p e c t r a were o b t a i n e d by d i s s o l v i n g abou t 63 mg of t h e a p p r o p r i a t e sample i n 0.5 m l o f DMSO-d6 c o n t a i n i n g t e t r a m e t h y l s i l a n e as an i n t e r n a l s t a n d a r d (2). The s p e c t r a l a s s ignmen t s f o r t h e l e v a l l o r p h a n b a s e are g iven i n Tab le 11. Aside from t h e a d d i t i o n a l peaks from t h e p r o t o n s on t h e t a r t r a t e i t s e l f , * t h e major d i f f e r e n c e between t h e b a s e and t h e t a r t r a t e s a l t o c c u r s i n t h e methylene r e g i o n . The b road , n o n - d e s c r i p t bands a re a t t r i b u t e d t o t h e change i n t h e n i t r o g e n quadro- p o l e moment caused by t h e p o s i t i v e c h a r g e p r e s e n t on t h e n i t r o g e n .

Tab le I1

NMR Assignments f o r Leva l lo rphan Base

No. o f Chemical S h i f t Type o f P r o t o n s P r o t o n s ( PPm) M u l t i p l i c i t y

methylene p r o t o n s on ca rbons 5,6,7, 8, and 15 10 1.00-1.95

methylene p r o t o n s on carbon 1 0 and methyne p r o t o n on carbon 14 3 2.10-2.65

methylene p r o t o n s on carbon 1 6 and methyne p r o t o n on carbon 9 3 2.75-3.15

*The t a r t r a t e h a s 2 C-H p r o t o n s which show a s h a r p s i n g l e t a t 4.10 ppm and 2 OH p l u s 2 COOH p r o t o n s which a l o n g w i t h t h e OH p r o t o n from t h e l e v a l l o r p h a n molecu le g i v e a broad s i n g l e t a t 8 . 4 9 ppm.

343


F i g u r e 2

NMR S p e c t r a of Leva l lo rphan and Leva l lo rphan Tartrate

344


methylene p r o t o n s a d j a c e n t t o t h e N i n a l l y l c h a i n 2 3 .10 Doublet

(J=7Hz)

methylene p r o t o n s a t end o f a l l y l group 2 4.95-5.25 Mu1 t i p l e t

methyne p r o t o n i n a l l y l group 1 5.50-6.10 Mu1 t i p l e t

a r o m a t i c p r o t o n s a t ca rbons 2 and 4 2 6 .40-6.61 Mu1 t i p l e t

a r o m a t i c p r o t o n a t carbon 1 1 6.90 Doublet

(J=8Hz)

OH p r o t o n 9 .oo Broad 1 s i n g l e t -

2 . 3 U l t r a v i o l e t Spectrum ( U V ) The UV s p e c t r a of l e v a l l o r p h a n t a r t r a t e i n water

(A) and i n 1 N N a O H (B) are shown i n F i g u r e 3 ( 3 ) . The maxima and minima as w e l l as t h e molar e x t i n c t i o n coe f - f i c i e n t s a t t h e imax are l i s t e d i n Tab le 111. The v a l u e s o b t a i n e d a g r e e w e l l w i t h t h e ones r e p o r t e d by Farmilo and Genest (4) and Mu16 ( 5 ) .

T a b l e I11

UV Absorpt ion S p e c t r a l Data f o r Leva l lo rphan Tar t ra te

Wavelength o f Molar Wavelength of S o l v e n t Maximum (nm) A b s o r p t i v i t y Minimum (nm)

water 279 2.04 103

216 7.25 103 1N H C 1 2 7 8 2 .08 103

216 7 . 4 2 103 1N N a O H 299 3.18 103

239 9.18 1 0 3

245

244

268

6 . C. R U D Y A N D 8 . Z . SENKOWSKI

Figure 3

Ultraviolet Spectrum of Levallorphan Tartrate

10

8

6 W u z a at Lz 0 rn at a

4

2

0

4 : Levol lorphon T a r t r o l e In Wot i ( I : 20,000 I

8 : Levallorphan Tarfrore ~n I N N

( I : 50.0001

210 250 300 3 5 0

NANOMETERS

346


2 .4 F l u o r e s c e n c e S p e c t x The e x c i t a t i o n and emiss ion s p e c t r a f o r l e v a l -

l o r p h a n t a r t r a t e ( 1 mg/ml of methanol) i s shown i n F i g u r e 4 ( 6 ) . Two maxima appea r i n t h e e x c i t a t i o n spectrum a t 306 nm. When 0 . 1 N H C 1 is used as t h e s o l v e n t t h e maxima occur a t t h e same wavelengths b u t a 5 f o l d i n c r e a s e i n s e n s i t i v i t y i s o b t a i n e d .

2 .5 Mass Spectrum The mass spectrum o f l e v a l l o r p h a n ta r t ra te w a s

o b t a i n e d u s i n g a CEC 21-110 m a s s s p e c t r o m e t e r w i t h a n ion- i z i n g ene rgy of 7 0 eV. The o u t p u t from t h e mass s p e c t r o - meter w a s ana lyzed and p r e s e n t e d i n t h e form of a b a r graph, shown i n F i g u r e 5, by a Var i an 100 MS d e d i c a t e d computer sys t em ( 7 ) . The mass spectrum i s a s u p e r i m p o s i t i o n of t h e s p e c t r a o f t h e l e v a l l o r p h a n b a s e and t a r ta r ic a c i d . The molecu la r i o n of l e v a l l o r p h a n a t m / e 2 8 3 i s t h e s t r o n g e s t peak. The peak a t m / e 256 (M-27) cor re sponds t o t h e loss of CH=CH2. The o t h e r peaks a t m / e 2 6 8 , 2 4 2 , 2 4 0 , and 226 are due t o t h e l o s s of o t h e r hydrocarbon m o i e t i e s . S i n c e t h e p o l y c y c l i c s a t u r a t e d r i n g system a l l o w s a m u l t i t u d e of f r a g m e n t a t i o n pathways, an assignment of t h e s e i o n s t o c e r - t a i n p a r t s of t h e molecu la r s t r u c t u r e cou ld a t b e s t t o v e r y t e n t a t i v e ( 7 ) .

2 . 6 O p t i c a l R o t a t i o n The s p e c i f i c r o t a t i o n of l e v a l l o r p h a n t a r t r a t e i n

water a t 25OC i s p l o t t e d v e r s u s wavelength i n F i g u r e 6 (3 , 8 ) . The s p e c i f i c r o t a t i o n observed a t 589 nm w a s -39.0 ' . The c h a r a c t e r i s t i c p a r t s of t h e c u r v e a re a peak a t 290 nm ( - 1 1 6 5 O ) , a t rough a t 267 nm (+240°) and a peak a t 2 2 0 nm ( - 5 8 5 4 ' ) . Zero i n t e r c e p t s o c c u r a t 270 and 264 nm (8 ) .

The s p e c i f i c r o t a t i o n r ange r e p o r t e d i n t h e USP X V I I I f o r l e v a l l o r p h a n t a r t r a t e i s from - 3 7 . 0 t o -39.2' a t 589 nn- ( 9 ) .

2.7 M e l t i n g Range The m e l t i n g r a n g e r e p o r t e d i n t h e USP X V I I I f o r

l e v a l l o r p h a n t a r t r a t e i s 174' t o 177O u s i n g t h e c l a s s Ia p rocedure ( 9 ) .

2 . 8 D i f f e r e n t i a l Scanning Ca lo r ime t ry (DSC) The DSC c u r v e shown i n F i g u r e 7 f o r l e v a l l o r p h a n

t a r t r a t e w a s o b t a i n e d u s i n g a P e r k i n E l m e r DSC-1B C a l o r i - meter. With a t empera tu re program o f 1O0C/min, a m e l t i n g

347

6. C. RUDY AND B. 2. SENKOWSKI

Figure 4

Fluorescence Spectrum of Levallorphan T a r t r a t e

1 Exci lol ion /

2 00 250 300 350 400 450

NANOMETERS

348

LE

VA

LL

OR

PH

AN

TA

RT

RA

TE

I. In

349

6. C . RUDY AND 6 . 2. SENKOWSKI

F i g u r e 6

P l o t of S p e c i f i c R o t a t i o n vs . Wavelength of Leva l lo rphan Tar t ra te

350

L E V A L L O R P H A N T A R T R A T E

Figure 7

DSC Curve of Levallorphan Tartrate

1 A H f = 10 4 kcol/mole

I 140 150 160 170 180 190

TEMPERATURE OC

35 1

6. C . RUDY AND 6. Z. SENKOWSKI

endotherm was observed s t a r t i n g a t 174.8' w i t h a AHf = 10.4 kca l fmole (10) .

2.9 Thermogravimetr ic A n a l y s i s (TGA) The TGA s c a n run on l e v a l l o r p h a n t a r t r a t e showed

no weight l o s s from 30' t o 217OC. l o s s o c c u r r e d from 2 1 7 O t o 47OoC which accounted f o r es- s e n t i a l l y a l l o f t h e sample (10 ) .

One con t inuous we igh t

2.10 S o l u b i l i t y The s o l u b i l i t y d a t a o b t a i n e d f o r l e v a l l o r p h a n

t a r t r a t e a t 25OC are l i s t e d i n Tab le I V (11).

Tab le I V

S o l u b i l i t y of Leva l lo rphan Ta r t r a t e i n D i f f e r e n t S o l v e n t s

S o l v e n t S o l u b i l i t y (mgfml)

3 A a l c o h o l benzene ch 1 o r o f o r m 95% e t h a n o l e t h y l e t h e r methanol pe t ro l eum e t h e r 2-propanol w a t e r

(30'-60')

7 . 8 0 . 2 0 . 3

26.5 0 . 2

79.5 ~ 0 . 0 3

1 . 5 60.0

2 . 1 1 X-ray C r y s t a l P r o p e r t i e s The x-ray powder d i f f r a c t i o n p a t t e r n of l e v a l -

l o rphan t a r t r a t e i s p r e s e n t e d i n Tab le V ( 1 2 ) . These v a l u e s a re ve ry s i m i l a r t o t h o s e g i v e n by Farmilo and Genest ( 4 ) . The i n s t r u m e n t a l c o n d i t i o n s are g iven below.

Ins t rumen t C o n d i t i o n s :

Gene ra l E l e c t r i c Model XRD-6 Spec t rogon iomete r

Genera to r : 50KV, 12-112 MA Tube t a r g e t : Copper R a d i a t i o n : Cu K,= 1 .542 El

352


O p t i c s : 0.1' D e t e c t o r s l i t M.R. S o l l e r s l i t 3' B e a m s l i t 0.0007 i n c h N i f i l t e r 4O t a k e o f f a n g l e Scan a t 0.2O 28 p e r minu te Ampl i f i e r g a i n - 16 c o u r s e ,

8 . 7 f i n e S e a l e d p r o p o r t i o n a l c o u n t e r



Rate meter T.C. 4 2000 CIS f u l l scale

Goniometer: D e t e c t o r :


Recorder : Char t Speed - 1 i n c h p e r 5 minu tes

Samples p repa red by g r i n d i n g a t room t empera tu re .

T a b l e V

X-ray Powder D i f f r a c t i o n P a t t e r n of

20 -

9.18 11.90 1 2 . 1 8 1 3 . 0 6 14 .12 1 4 . 2 8 1 5 . 3 2 1 5 . 8 4 17.22 1 8 . 5 0 1 9 . 0 0 19 .16 1 9 . 7 0 1 9 . 9 0 20.18 2 0 . 7 8 2 1 * 00 2 1 . 4 8

9 . 6 3 3 0 7 .44 33 7 .27 15 6 . 7 8 7 8 6.27 1 0 6 . 2 0 32 5 . 7 8 100 5.59 32 5 .15 6 4 .80 2 1 4.67 1 2 4 . 6 3 34 4 . 5 1 1 0 4 . 4 6 34 4 . 4 0 32 4.27 22 4 .23 49 4 . 1 4 14

29 .18 29.66 30.10 30 .24 30 .38 30 .92 31 .08 31 .74 31.86 34 .14 3 4 . 3 0 35 .02 35.16 35 .32 35 .70 35 .86 36 .22 3 6 . 7 2

353

Leva l lo rphan Tar t ra te

d (1) * I! I,**

3.06 23 3 .01 10 2.97 1 2.96 2 2 . 9 4 2 2.89 10 2 . 8 8 11 2 .82 14 2 .81 17 2 . 6 3 1 2 . 6 1 1 2.56 11 2 . 5 5 1 3 2.54 1 2 2 . 5 1 5 2.50 5 2 .48 2 2 . 4 5 5


21.76 23.12 23.88 24.06 24.62 24.86 25.58 26.00 26.16 26.42 26.78 27.22 27.58 27.98 28.74 28.84

* d -

**- I

4.08 3.85 3.73 3.70 3.62 3.58 3.48 3.43 3.41 3.37 3.33 3.28 3.24 3.19 3.11 3.10

39 37.38 2.41 6 38.08 2.36 11 38.40 2.34 11 38.80 2.32 32 39.40 2.29 85 39.96 2.26 5 40.06 2.25 18 40.24 2.24 18 40.52 2.23 34 43.92 2.21 1 41.74 2.16 9 42.04 2.15 8 43.62 2.07 4 43.82 2.07

11 44.08 2.05 16 45.14 2.01

1 4 6

33 1 4 6 7 4 7 1 6 3 5 13 6

nh 2 S i n 0

( i n t e r p l a n a r d i s t a n c e )

I/I, = r e l a t i v e i n t e n s i t y (based on h i g h e s t i n t e n s i t y of 1.00)

2.12 D i s s o c i a t i o n Cons tan t The pKa's at room tempera tu re f o r l e v a l l o r p h a n

t a r t r a t e have been de te rmined by a pH t i t r a t i o n w i t h sodium hydrox ide t o b e 4.5 and 6.9 (13). The f i r s t pKa observed € o r l e v a l l o r p h a n t a r t ra te r e s u l t s from t h e i o n i z a t i o n of t h e c a r b o x y l i c p r o t o n from t h e t a r t a r i c a c i d . The second pKa i s due t o t h e i o n i z a t i o n o f t h e p r o t o n from t h e ammonium moiety i n t h e l e v a l l o r p h a n ta r t ra te .

3 . S y n t h e s i s Leva l lo rphan t a r t r a t e may b e p repa red by t h e r e a c t i o n

scheme shown i n F igu re 8. (+)-l-(p-hydroxybenzyl)-l,2,3,4, 5,6,7,8-octahydroisoquinoline is r e a c t e d w i t h a l l y l b r o m i d e t o g i v e (-)-l-(p-hydroxybenzyl)-2-allyl-l,2,3,4,5,6,7,8- o c t a h y d r o i s o q u i n o l i n e which i s c y c l i z e d i n phosphor i c a c i d t o (-)-3-hydroxy-17-allyl-morphinan. Tartar ic a c i d i s t h e n added t o make l e v a l l o r p h a n t a r t r a t e (14).

4. S t a b i l i t y Degrada t ion Leva l lo rphan t a r t r a t e s u b s t a n c e i s s t a b l e i n t h e

p re sence o f l i g h t and oxygen from t h e a i r . I n aqueous

354


F i g u r e 8

SYNTHESIS OF LEVALLUWHIW TARTRATE

355

6. C . RUDY AND 8. Z . SENKOWSKI

s o l u t i o n l e v a l l o r p h a n ta r t ra te w a s shown t o b e s t a b l e when hea ted t o 45OC f o r 12 weeks. Only when t h e s o l u t i o n w a s s a t u r a t e d w i t h oxygen and s u b j e c t e d t o u l t r a v i o l e t r a d i a - t i o n d i d s l o w decomposi t ion occur . The l e v a l l o r p h a n tar- t r a t e shows no e f f e c t s from s t e r i l i z a t i o n a t 120°C f o r 20 minutes (15 ) .

5. Drug Metabo l i c P roduc t s

h a s been s t u d i e d by Mannering and Schanker (16 ) . Two meta- b o l i c p r o d u c t s were i s o l a t e d from u r i n e , f e c e s , and t h e l i v e r of mice. One of t h e m e t a b o l i t e s w a s p o s i t i v e l y iden - t i f i e d as t h e N-dealkylated p r o d u c t , 3-hydroxymorphinan. The o t h e r m e t a b o l i t e w a s n o t i d e n t i f i e d u n t i l r e c e n t l y when i t was shown t o be (-)-17-allyl-3,6-dehydroxymorphinan by x-ray c r y s t a l l o g r a p h y ( 1 7 ) . The 3-hydroxymorphinan meta- b o l i t e w a s a l s o found i n t h e u r i n e of gu inea p i g s , r a b b i t s , and dogs ( 1 6 ) .

The m e t a b o l i c f a t e of l e v a l l o r p h a n ta r t ra te i n an ima l s

6 . Methods of Ana lys i s

6 . 1 Elemental Ana lys i s The r e s u l t s from t h e e l e m e n t a l a n a l y s i s of l e v a l -

l o r p h a n ta r t ra te a r e l i s t e d i n Table V I (18).

Table V I

Elemental Ana lys i s of Leval lorphan T a r t r a t e


C 63.73 63.77 H 7 .21 7.26 N 3.23 3.22

6.2 Phase S o l u b i l i t y Ana lys i s Phase s o l u b i l i t y a n a l y s i s i s c a r r i e d o u t u s i n g

a b s o l u t e e t h a n o l a s t h e s o l v e n t . A t y p i c a l example is s h m i n F igu re 9 which a l s o l i s t s t h e c o n d i t i o n s under which t h e a n a l y s i s was c a r r i e d o u t (11).

6.3 Thin Layer Chromatographic Ana lys i s (TLC) S e v e r a l TLC systems have been pub l i shed which can

b e used t o s e p a r a t e l e v a l l o r p h a n b a s e from i ts m e t a b o l i t e s

356

F i g u r e 9

PHASE SOLCBILITY ANALYSIS 1 SAMPLE: LEVALLORPHAN TARTRATE

SOLVENT: ABSOLUTE ETHANOL

SLOPE: 0 .15% 1 ly ~~ EQL'ILIBRAT;: 20 HRS. at 25'C ~~ 1 EXTRAPOLATED SOLUBILITY:

0 .. 7 .18 mg/g of ETHANOL i i 2

n

0 100

bYSTEH C O W O S I T I O N : mg of SAHPLE per g SOLVENT

r rn < F c


and other similar compounds (5,191. In each case about 25 ug of sample was spotted on a Silica gel G plate (250 microns thick) and subjected to ascending chromatography. After development of at least 10 em, the plate is air-dried f o r 10 minutes, dried in an oven at 100°C f o r 15 minutes, and sprayed with iodoplatinate reagent made by adding 10 ml of a 10% solution of platinum chloride to 250 ml of 4 % potassium iodide and diluting to 500 ml with water. The solvent systems as well as the approximate Rf of the levallorphan spot are listed in Table VII (5).

Table V I I

TLC Data for Levallorphan

Solvent System

benzene:dioxane:ethanol:ammonium hydroxide (10: 8: 1: 1)

n-butano1:conc. hydrochloric acid, saturated with water (9:l)

ethanol: glacial acetic acid:water (6 : 3: 1)

ethano1:dioxane:pyridine:water (10: 5: 4 : 1)

n-butano1:water:glacial acetic acid ( 4 : 2 : 1 )

t-amyl alcoho1:water:n-butyl ether (80:13: 7)

methano1:n-butano1:water:benzene ( 1 2 : 3: 3 : 2 )

Rf of Levallorphan

.98

. 73

.70

. 65

.64

.44

. 4 1

6 . 4 Gas Liquid Chromatographic Analysis (GLC) A GLC method for the separation and quantitation

of levallorphan base in the presence of its metabolites and similar structured compounds has been reported by Mulk (5, 19). The pertinent experimental conditions as well as the retention time for levallorphan are given in Table VIII (5).

358


T a b l e V I I I

GLC Method f o r L e v a l l o r p h a n as t h e F r e e Base

Co 1 umn : Suppor t : S t a t i o n a r y Phase : D e t e c t o r : Tempera ture -

I n j e c t i o n P o r t : Column: D e t e c t o r :

Flow Rate: Q u a n t i t i e s I n j e c t e d :

R e t e n t i o n T i m e :

6 f t , 3 mm i d , c o i l e d g l a s s Gas Chrom S (80-100 mesh)

Radium - 226 a rgon i o n i z a t i o n

260' 215O 215' 4 7 c c l m i n u t e of a r g o n 2-25 pg of l e v a l l o r p h a n b a s e

5 .72 m i n u t e s

2% SE-30

i n methanol

6 . 5 Direct S p e c t r o p h o t o m e t r i c A n a l y s i s D i r e c t s p e c t r o p h o t o m e t r i c a n a l y s i s of l e v a l l o r p h

t a r t r a t e h a s n o t been found t o b e a p p l i c a b l e i f any meta- b o l i t e s o r s imi l a r s t r u c t u r e d compounds are p r e s e n t . How- e v e r , i f no i n t e r f e r i n g s p e c i e s a r e p r e s e n t , t h e r e p o r t e d maximum a t 279 nm i n water and i n 1 N H C 1 o r a t 299 nm i n 1 N N a O H may b e used f o r q u a n t i t a t i v e measurements .

6 . 6 C o l o r i m e t r i c A n a l y s i s

6 . 6 1 I o n P a i r E x t r a c t i o n o f Bromocreso l Green Complex A c o l o r i m e t r i c method which t a k e s a d v a n t a g e

o f t h e q u a t e r n a r y amine moie ty i n l e v a l l o r p h a n t a r t r a t e i s t h e e x t r a c t i o n o f t h e i o n p a i r formed w i t h b romocreso l g r e e n . The l e v a l l o r p h a n t a r t r a t e i n i n j e c t d b l e s i s a s s a y e d b y t h i s method ( 9 ) . The complex i s formed i n an aqueous 5 . 3 p h o s p h a t e b u f f e r and e x t r a c t e d i n t o ch lo ro fo rm. The abso rbance i s r e a d a t t h e 420 nm maximum a l o n g w i t h t h e a b s o r b a n c e o f a r e f e r e n c e s t a n d a r d l e v a l l o r p h a n t a r t r a t e p r e p a r e d i n t h e same way. These abso rbance v a l u e s a r e used t o c a l c u l a t e t h e c o n c e n t r a t i o n o f l e v a l l o r p h a n t a r t r a t c p e r ml p r e s e n t i n t h e i n j e c t a b l e .

6.62 F o l i n - C i o c a l t e u C o l o r i m e t r i c Analysis A c o l o r i m e t r i c method which t a k e s d d v m t a g e

of t h e phenol moie ty i n l e v a l l o r p h a n t a r t r a t e u t i l i z e s t l i t

35Y

6 . C. RUDY AND B. 2. SENKOWSKI

Fol in -C ioca l t eu r e a g e n t ( 2 0 ) . This r e a g e n t i s p repa red by by mixing 100 gm of sodium t u n g s t a t e , 25 gm of sodium molybdate, 700 m l of water, 50 m l of 85% phosphor i c a c i d , and 100 m l of c o n c e n t r a t e d h y d r o c h l o r i c a c i d . A f t e r re- f l u x i n g f o r 10 hour s , 150 gm of l i t h i u m s u l f a t e , 50 m l o f wa te r , and a few d rops of l i q u i d bromine are added and t h e r e s u l t i n g s o l u t i o n b o i l e d a few minutes t o d r i v e o f f e x c e s s bromine. This s o l u t i o n i s cooled, d i l u t e d t o 1000 m l and f i l t e r e d (20 ) . When t h i s r e a g e n t i s added t o l e v a l l o r p h a n ta r t ra te i n a s l i g h t l y b a s i c s o l u t i o n (sodium ca rbona te ) a b l u e c o l o r deve lops which i s measured a t t h e maximum a t about 700 nm.

6 .7 Nephelometric Analysis A nephe lomet r i c method w a s developed f o r t h e

a n a l y s i s of l e v a l l o r p h a n t a r t r a t e i n t h e p re sence of levorphanol t a r t r a t e . The method t a k e s advantage of t h e k i n e t i c s o f b romina t ion of t h e a l l y 1 f u n c t i o n on l e v a l l o r - phan which i n a c i d i c aqueous medium i s f a s t e r t han t h e brominat ion of t h e l evorphano l . The brominat ion product i s i n s o l u b l e and hence l e n d s i t s e l f t o a t u r b i d i m e t r i c d e t e r - mina t ion ( 2 1 ) . The p rocedure i n v o l v e s adding 1 mg of leval- lo rphan ta r t ra te (a long w i t h up t o 10 mg of l e v o r p h a n o l t a r t r a t e ) t o 5 m l of 2.5N H C 1 and 15 m l o f water i n a g l a s s s toppe red 25-ml g radua ted c y l i n d e r . 3.0 m l of 0.1N bromide-bromate s o l u t i o n , shake and m a i n t a i n a t 25OC f o r e x a c t l y 45 minutes . absorbance a t 640 nm. A p l o t of absorbance v s concen t r a - t i o n i s l i n e a r between 0 . 5 and 1 .5 mg of l e v a l l o r p h a n ta r t ra te ( 2 1 ) .

Bring t o 25OC and add

Shake and measure t h e

6 .8 T i t r ime t r i c Ana lys i s The p o t e n t i o m e t r i c , non aqueous t i t r a t i o n i s t h e

method of c h o i c e € o r a n a l y z i n g l e v a l l o r p h a n t a r t r a t e f i n e chemical ( 9 ) . About 250 mg of l e v a l l o r p h a n t a r t r a t e i s d i s s o l v e d i n 100 m l of g l a c i a l ace t ic a c i d and t i t r a t e d w i t h 0.02” p e r c h l o r i c a c i d i n dioxane. Each m l of 0.02N p e r c h l o r i c a c i d is e q u i v a l e n t t o 8 .67 mg of l e v a l l o r p h a n t a r t r a t e .

360


7 . Re fe rences

1.

2 .

3 .

4 .

5 . 6.

7 .

8.

9 . 10.

11.

1 2 .

1 3 .

1 4 .

1 5 .

16 .

1 7 .

18 .

19 . 20.

21.

Hawrylyshyn, M . , Hoffmann-La Roche I n c . , P e r s o n a l Communication. Johnson, J . , Hoffmann-La Roche I n c . , P e r s o n a l Com- mun ica t ion . Bandong, L. , Hoffmann-La Roche I n c . , P e r s o n a l Com- mun ica t ion . Fa rmi lo , C . G . and Genes t , K . , Progress in Chemical Tox ico logy , Vol . 1, Academic Press , N e w York, N.Y., 1963, pp. 199-295. Mule, S . J . , AnaZ. Chem., 36, 1907 (1964) . Boatman, J. , Hoffmann-La Roche I n c . , P e r s o n a l Com- mun ica t ion . Benz, W . , Hoffmann-La Roche I n c . , P e r s o n a l Communi- c a t i o n . Toome, V. , Hoffmann-La Roche I n c . , P e r s o n a l Com- mun ica t ion . United S t a t e s Pharmacopeia XVIII, 360-362 (1970) . Moros, S . , Hoffmann-La Roche I n c . , P e r s o n a l Com- mun ica t ion . MacMullan, E . , Hoffmann-La Roche I n c . , P e r s o n a l Communication. Hagel , R . , Hoffmann-La Roche I n c . , P e r s o n a l Com- mun ica t ion . Lau, E . and Yao, C . , Hoffmann-La Roche I n c . , Unpub 1 i s he d Data. H e l l e r b a c h , J. , Hoffmann-La Roche I n c . , Unpublished Data. H a f l i g e r , 0. , Hoffmann-La Roche Inc . , Unpublished Data. Mannering, G . J. and Schanker , L . S . , J . Pharmacol. E x p t l . Therap., - 1 2 4 , 296 (1958) . Mohacsi, E . , Bloun t , J .F . and Mannering, G . J . , 1 3 t h N a t i o n a l Med ic ina l Chemistry Symposium i n Iowa, June 18-22, 1972. S c h e i d l , F . , Hoffmann-La Roche I n c . , P e r s o n a l Com- mun ica t ion . Mule', S . J . , J . Chromatog. S c i . , 10, 275 (1972) . F o l i n , 0. and C i o c a l t e u , V . , J . BioZ. &em., 73, 629 (1927) . Schmall , M. , P i f e r , C.W. and Wol l i sh , E . G . , Hoffmann-La Roche Inc . , Unpublished Data.

361

METHYPRY LON

An Aiialytical Profile

363


INDEX

A n a l y t i c a l P r o f i l e - Methyprylon

1. D e s c r i p t i o n 1.1 N a m e , Formula, Molecu la r Weight 1 . 2 Appearance, C o l o r , Odor

2 . P h y s i c a l P r o p e r t i e s 2 . 1 I n f r a r e d Spec t rum 2 . 2 Nuc lea r Magnet ic Resonance Spec t rum 2 . 3 U l t r a v i o l e t Spec t rum 2 . 4 F l u o r e s c e n c e Spec t rum 2 .5 Mass Spect rum 2 . 6 O p t i c a l R o t a t i o n 2.7 M e l t i n g Range 2 . 8 D i f f e r e n t i a l Scanning C a l o r i m e t r y 2 .9 Thermal G r a v i m e t r i c A n a l y s i s 2 .10 S o l u b i l i t y 2 .11 X-ray C r y s t a l P r o p e r t i e s 2 . 1 2 D i s s o c i a t i o n Cons tan t


4 . S t a b i l i t y Degrada t ion

5 . Drug M e t a b o l i c P r o d u c t s

6 . Methods o f Ana lys i s 6 . 1 E lemen ta l A n a l y s i s 6 . 2 Phase S o l u b i l i t y A n a l y s i s 6 . 3 Thin Layer Chromatographic A n a l y s i s 6 . 4 Gas L iqu id Chromatographic Ana lys i s 6 . 5 Di rec t S p e c t r o p h o t o m e t r i c A n a l y s i s 6 .6 C o l o r i m e t r i c Ana lys i s 6.7 T i t r i m e t r i c A n a l y s i s

7 . Re fe rences

364

METHYPRYLON


1.1 N a m e , Formula , M o l e c u l a r Weight Methypry lon i s 3,3-diethyl-5-methyl-2,4-

p i p e r i d i n e d i o n e .

0

H

M o l e c u l a r Weight: 1 8 3 . 2 5 '1 O H 1 7 No 2

1 . 2 Appearance , C o l o r , Odor Methypry lon o c c u r s as a w h i t e t o n e a r l y w h i t e

c r y s t a l l i n e powder. I t h a s a s l i g h t c h a r a c t e r i s t i c o d o r .


2 . 1 I n f r a r e d Spec t rum ( IR) The I R s p e c t r u m o f m e t h y p r y l o n i s p r e s e n t e d i n

F i g u r e 1 (1). The s p e c t r u m was measured w i t h a P e r k i n - E l m e r 6 2 1 S p e c t r o p h o t o m e t e r i n a K B r p e l l e t c o n t a i n i n g 0 . 5 mg of me thypry lon /300 mg of K B r . The a s s i g n m e n t s f o r t h e c h a r a c t e r i s t i c bands i n t h e I R s p e c t r u m a r e l i s t e d be low ( 1 ) :

3345 cm-1 NH s t r e t c h i n g v i b r a t i o n s 2966, 2933, and

2877 c m - 1 a l i p h a t i c CH s t r e t c h i n g v i b r a t i o n s

1693 and 1656 cm-l C=O s t r e t c h i n g v i b r a t i o n s

2 . 2 N u c l e a r Magne t i c Resonance Spec t rum (NMR) The s p e c t r u m shown i n F i g u r e 2 w a s o b t a i n e d on a

J e o l 60 MHz NMR by d i s s o l v i n g 5 9 . 0 mg o f me thypry lon i n 0 . 5 m l o f CDC13 c o n t a i n i n g t e t r a m e t h y l s i l a n e as a n i n t e r n a l r e f e r e n c e ( 2 ) . The s p e c t r a l a s s i g n m e n t s a re g i v e n i n T a b l e I ( 2 ) . In o r d e r t o make t h e s e a s s i g n m e n t s and e s t a b l i s h t h e c h e m i c a l s h i f t s , e x t e n s i v e s p i n - s p i n d e c o u p l i n g e x p e r i - ments were c a r r i e d o u t . By i r r a d i a t i n g t h e sample a t t h e

3 65

F i g u r e 1

I n f r a r e d Spectrum of Methyprylon

WAVELENGTH (MICRONS)

7 8 9 10 12 15 20 3550 2.5 3 4 5 6

I I 1 I I ' I I I I I 4000 3500 3000 2500 2000 1700 1400 1100 800 500 200

I I I I I I I 1 I I I 1 1 1 I I J 100

80

60

40

20

0 4000 3500 3000 2500 2000 1700 1400 1100 800 500 200

m r, II C 0

m ?J

FREQUENCY (CM-'

ME

TH

YP

RY

LO

N

3 67


f r e q u e n c i e s 4 8 , 65, 1 1 4 , and 206 Hz, t h e spectrum w a s s i m p l i f i e d and a s s ignmen t s cou ld then b e made ( 2 ) .

Table I

NMR Assignments f o r Methyprylon

S t r u c t u r e P ro tons S h i f t (ppm) Mu1 t i p 1 i c i t y Chemical

a 0 . 8 T r i p l e t

(JH a e -H

Doublet

= 7 .5 Hz)

b 1 . 1 4 I H f

= 7 .0 Hz) %-Hd

(J

C Ql .90 Complex M u l t i p l e t ( J ~ e - ~ a = 7.5 Hz)

d ~ 2 . 5 0 Complex M u l t i p l e t = 6.8 Hz)

= 4 .0 Hz) ( JHd-Hb

(JHd-He

e 3 .41 Nonet ( J ~ e - ~ f = 7.0 Hz)

(JHe-Hd = 6.0 Hz)

(J = Unknown) He-Hb

f 8.00 Broad S i n g l e t

2.3 U l t r a v i o l e t Spectrum (UV) When t h e UV spectrum o f methyprylon i n 2-propanol

w a s scanned between 400and 220 nm, one maximum and one minimum were obse rved . The maximum is l o c a t e d a t 294 nm (E = 3 3 ) and t h e minimum a t 353 nm. The spectrum shown i n F i g u r e 3 w a s o b t a i n e d from a s o l u t i o n of 300.1 mg of methyprylon/lOO m l of 2-propanol ( 3 ) .

368

METHYPRY LON

F i g u r e 3

U l t r a v i o l e t Spec t rum o f Methypry lon

.9 -

.8 -

.7 -

.6 - W 0 z U

0 v)

E .5-

m a

4-

.3 -

NANOMETERS

3 69


2.4

pry lon (1

Fluorescence Spectrum The e x c i t a t i o n and emission s p e c t r a f o r methy- mg/ml methanol) are shown i n F i g u r e 4 (4) The

e x c i t a t i o n spectrum e x h i b i t s a broad maximumat about 302nm and a s h o u l d e r about 239 nm. one maximum about 396 nm.

The emission spectrum h a s

2 .5 Mass Spectrum The mass spectrum of methyprylon w a s ob ta ined

u s i n g a CEC 21-110 mass spec t rometer w i t h a n i o n i z i n g energy o f 70 eY. The o u t p u t from t h e mass spec t rometer was analyzed and presented i n t h e form of a b a r graph , shown i n F igure 5 , by a Varian 100 MS d e d i c a t e d computer system which each peak t o t h e most i n t e n s e peak i n t h e spectrum (5) .

i o n . The b a s e peak o c c u r s a t m / e 155 and is due t o t h e l o s s of CO and/or C2H4 from t h e p a r e n t . peaks are compat ib le w i t h t h e known f ragmenta t ion p a t t e r n of methyprylon (5) .

c a l c u l a t e s re la t ive peak i n t e n s i t i e s by comparing

The weak peak a t m / e 183 i s due t o t h e molecular

The remaining

2.6 O p t i c a l R o t a t i o n Methyprylon c o n t a i n s a n a s s y m e t r i c c e n t e r a t t h e

5-carbon i n t h e r i n g . However, t h e racemic mixture i s ob- t a i n e d from t h e s y n t h e s i s and no o p t i c a l a c t i v i t y i s e x h i b i t e d .

2 . 7 Mel t ing Range The m e l t i n g range r e p o r t e d i n t h e N.F. XI11 f o r

methyprylon is 7 4 O t o 77.5O u s i n g t h e class I procedure (6).

2 .8 D i f f e r e n t i a l Scanning Calor imetry (DSC)

A m e l t i n g endotherm was observed a t 76.l0C when t h e The DSC s c a n f o r methyprylon i s shown i n F i g u r e

The AHf w a s found t o 6. t e m p e r a t u r e program w a s 10°/minute. b e 4 .I kcal jmole ( 7 ) .

2.9 Thermogravimetric Analysis (TGA) The TGA performed on methyprylon e x h i b i t e d no l o s s

of weight from ambient t o 115OC. One cont inuous weight l o s s was observed from 115' t o 26OoC which accounted f o r e s s e n t i a l l y a l l of t h e sample ( 7 ) .

370

w -4 c

F i g u r e 4

F l u o r e s c e n c e S p e c t r a of M e t h y p r y l o n

Excitotion

I

200 250 300 350 400

NANOMETERS

I I I

450 500 550

z rn -I I < -0 W

B. C. R

UD

Y A

ND

B. 2

. S

EN

KO

WS

KI r’

372

METHYPRYLON

I I I

AH, = 4.1 kca l /mole

373


2.10 S o l u b i l i t y The s o l u b i l i t y d a t a f o r methyprylon obta ined a t

25OC a r e presented i n Table I1 (8) .

Table I1

S o l u b i l i t y of Methyprylon i n D i f f e r e n t S o l v e n t s


3A a l c o h o l benzene chloroform 95% e t h a n o l e t h y l e t h e r methanol petroleum e t h e r (30'-60') 2-propanol water

2 >5.0 x l o 2 r5 .0 x 1 0 >5.0 x l o 2 >5.0 x l o 2 2 . 7 x 10' >5.0 x l o 2 1 . 7 x 10 >5.0 x l o 2 7.6 x 1 0

2 .11 X-ray C r y s t a l P r o p e r t i e s The x-ray powder d i f f r a c t i o n p a t t e r n of methy-

pry lon is presented i n Table I11 ( 9 ) . The i n s t r u m e n t a l c o n d i t i o n s are g iven below.

Ins t rument Condi t ions :

General Electr ic Model XRD-6 Spectrogoniometer

Genera tor : 50 KV-12-112 MA Tube t a r g e t : Copper Radia t ion : Cu & = 1.542 8

O . l o Detec tor s l i t M.R. S o l l e r s l i t 3O Beam s l i t 0.0007 i n c h N i f i l t e r 4 O t a k e o f f a n g l e Scan a t 0.2' 29 p e r minute Goniometer:

D e t e c t o r : Ampl i f ie r g a i n - 16 c o u r s e , 8 .7 f i n e

Sea led p r o p o r t i o n a l c o u n t e r tube and DC v o l t a g e a t p l a t e a u

3 74

METHYPRYLON

Pulse height selection EL - 5

Rate meter T.C.4 2000 CIS full scale Chart speed - 1 inch per 5

volts; EU - out

Recorder: minutes

Samples prepared by grinding at room temperature.

X-ray

2e -

10.00 1 3 . 7 8 1 4 . 6 4 15 .62 1 6 . 2 9 1 8 . 9 0 1 9 . 5 4 20 .24 20.72 21.96 22.00 2 4 . 0 0 2 4 . 5 5 2 5 . 3 2 2 6 . 1 4 2 6 . 8 8 27 .36

2 8 . 6 8 28.78 28 .90 2 9 . 5 1 3 0 . 4 2 3 1 . 6 0 33 .14 3 4 . 3 2

27.86

Table I11

Powder Diffraction Pattern of Methyprylon

d (2) * 8 . 8 5 6 . 4 3 6 .05 5 .67 5.44 4 . 7 0 4 . 5 4 4 . 3 9 4 . 2 9 4 . 0 5 4 . 0 4 3 . 7 1 3 .63 3 .52 3 . 4 1 3 .32 3.26 3 .20 3 . 1 1 3 . 1 0 3.09 3 . 0 3 2 .94 2 . 8 3 2 . 7 0 2 . 6 1

III ** 0 -

1 3 100

53 7

50 7 3

28 6

11 11 1 2

4 29 17 23

2 1 4

3 3 3

12 1 8

4 6 4

28 -

3 5 . 0 2 35.36 35 .77 3 6 . 2 2 38 .18 3 8 . 8 8 3 9 . 1 0 40 .10 40 .49 40 .82 4 1 . 9 8 4 2 . 3 2 4 2 . 7 2 4 3 . 0 2 4 3 . 1 2 4 4 . 0 6 4 4 . 5 2 44 .66 44 .80 4 5 . 1 8 4 6 . 7 0 4 8 . 3 4 4 8 . 5 8 50 .00 5 0 . 3 0

III ** - d(&* -0-

2.56 8 2.54 3 2 .51 4 2 . 4 8 1 2.36 3 2.32 1 2 . 3 1 1 2 .25 8 2 . 2 3 6 2 . 2 1 4 2 . 1 5 2 2 . 1 4 2 2.12 2 2 . 1 0 1 2 . 1 0 2 2 .06 2 2 . 0 4 2 2 . 0 3 2 2 .02 2 2 . 0 0 2 1 . 9 5 1 1 . 8 8 2 1.87 1 1 . 8 2 1 1 . 8 1 2

nh *d - (interplanar distance)

**IlI0 = relative intensity 2 Sin 8

(based on highest intensity of 1.00)

315

B. C. RUDY AND 5 . 2. SENKOWSKI

2.12 Dissociation Constant The pKa for methyprylon has been determined

spectrophotometrically to be approximately 12.0 (10).

3 . Synthesis Methyprylon is prepared by the reaction scheme shown in

Figure 7. 3,3-Diethyl-1,2,3,4-tetrahydro-2,4-pyridinedione, Presidon, i s reacted with formaldehyde yielding 3,3-diethyl- 5-hydroxymethyl-1,2,3,4-tetrahydro-2,4-pyridinedione (HMP) which is then hydrogenated using a Raney nickel catalyst to methyprylon (11).

4. Stability Degradation When methyprylon i s refluxed i n acidic, neutral, or

basic aqueous solutions i n the presence of oxygen, it break down into several carboxylic acids; e.g. formic acid, acetic acid, propionic acid, and diethyl acetic acid. Traces of unidentified amines and ketones are a l s o observed (12). When pure methyprylon is stored in well-closed, light resistant containers, the substance is quite stable.


humans is shown in Figure 8 (13-18). Methyprylon as the intact drug as well as its 5,6-dehydrogenated metabolite are found in blood (13,14), I n addition to the 5,6-dehydrogenated metabolite, several other metabolites due to oxidation are found in urine. These include the 6-oxy- metabolite from the direct oxidation of methyprylon and the oxidation of the methyl group to the alcohol and then to the acid i n the 5,6-dehydrogenated metabolite (16,18).

The major metabolic pathways of methyprylon i n dogs and


6.1 Elemental Analysis The results from the elemental analysis are

listed i n Table IV (19).

Table IV

Elemental Analysis of Methyprylon Element Theory % Found

C 65.54 65.40 H 9.35 9.46 N 7.64 7.62

316

F i g u r e 7

S y n t h e s i s o f M e t h y p r y l o n

0

CH20H II 0 2 6 + H-C-H N - 2:o

0 I I

H H

0 4

HMP PR E SlDON FORMALDEHYDE

I H2 Raney Nickel I

c2H5&$ C2H5 H

O N H I

H

METHYPRYLON

F i g u r e 8

ti

M e t a b o l i c P r o d u c t s of Methyprylon

m 0 7J C 0 <

3.3- diethyl-5-methyl-1.2.3,4- tetrohydro- 2.4- pyridinedione

I ti

3.3- di ethyl -5- hydroxyrnethyl- 1,2.3,4 - t etrohydro- 2,4- pyridinedione

OXIDATION

A

3.3-diethyl-5-methyl-2,4.6-piperidinc- trione

($$$J-COOH 0

I ti

5.5-diethyl- 3.4,5.6-tetmhydro- 4.6-dioxonicotinic acid

METHYPRYLON

6.2 Phase S o l u b i l i t y A n a l y s i s Phase s o l u b i l i t y a n a l y s i s may b e c a r r i e d o u t

u s i n g n-heptane as t h e s o l v e n t . A t y p i c a l example f o r methyprylon is shown i n F i g u r e 9 which a l s o lists t h e con- d i t i o n s under which t h e a n a l y s i s w a s c a r r i e d o u t (8) .

6 .3 Thin Layer Chromatographic Ana lys i s (TLC) A TLC system used t o i d e n t i f y methyprylon from

o t h e r h y p n o t i c s w a s p u b l i s h e d by Frahm e t a l . ( 2 0 ) . T h i s sys t em u t i l i z e d a K i e s e l g e l C p l a t e and a deve lop ing s o l - v e n t of 2-propanol:chloroform:25% ammonium hydrox ide ( 9 : 9 : 2 ) . The Rf v a l u e s were v e r y s e n s i t i v e t o t h e amount of s o l v e n t s a t u r a t i o n i n t h e t a n k and t h e r e f o r e a r e f e r e n c e s t a n d a r d w a s always run a long w i t h t h e sample f o r i d e n t i - f i c a t i o n pu rposes .

i t s m e t a b o l i t e s and o t h e r s imi l a r compounds employs e t h y l acetate as t h e deve lop ing s o l v e n t . The methyprylon sample, approx ima te ly 100 mcg, i s s p o t t e d on a s i l i c a g e l G p l a t e and s u b j e c t e d t o a scend ing chromatography u n t i l t h e s o l v e n t f r o n t h a s moved a t l e a s t 15 cm. The p l a t e i s a i r d r i e d and sp rayed w i t h f r e s h l y p repa red po ta s s ium f e r r i c y a n i d e (1.6 gm K3Fe(CN)6 i n 100 m l 2N KOH) and examined immediately under long-wavelength u l t r a v i o l e t r a d i a t i o n . Methyprylon h a s an Rf of 0 .40 i n t h i s sys t em (21).

A TLC sys t em used t o s e p a r a t e methyprylon from

6.4 Gas-Liquid Chromatographic Ana lys i s (GLC) The f o l l o w i n g GLC method i s u s e f u l f o r t h e

s e p a r a t i o n and q u a n t i t a t i o n of methyprylon i n t h e p r e s e n c e o f i t s m e t a b o l i t e s and similar s t r u c t u r e d compounds. The p e r t i n e n t expe r imen ta l c o n d i t i o n s as w e l l as t h e r e t e n t i o n t i m e f o r methyprylon are g iven i n Tab le V ( 2 2 ) .

Tab le V

GLC Method f o r Methyprylon

Column : 6 f t , 1 /8 i n O . D . , s t a i n l e s s

Suppor t : D i a t o p o r t S (60-80 mesh) S t a t i o n a r y Phase: 8% Cra ig P o l y e s t e r S u c c i n a t e

De tec to r : Hydrogen Flame I o n i z a t i o n

s t e e l t u b i n g

(BDS)

379

F i g u r e 9

W I I > -I 0 cn

?25- W I- 3 J 0

-

- " n - - - - - " I\ - c ..- 0

D

.. 15- PHASE SOLUBILITY ANALYSIS -

SAMPLE : METHYPRYLON

SOLVENT : HEPTANE - SLOPE : 0.39%

EQUILIBRATION : 72 HOURS at 25 OC

EXTRAPOLATED SOLUBILITY : 19.65 m g 4 of HEPTANE

-

I I 0 25 50 75

w oc c

100

F 0 I] C 0 < b z 0

Fo ?J

METHYPRY LON

Temperature O C - I n j e c t i o n P o r t : 210 Column : 180 D e t e c t o r : 2 50

Flow Rate: 40 c c l m i n u t e o f N i t rogen Q u a n t i t i e s I n j e c t e d : 10-100 pg of methyprylon i n

R e t e n t i o n T i m e s : 11 minu tes 95% e t h a n o l

6 .5 Direct S p e c t r o p h o t o m e t r i c A n a l y s i s D i r e c t s p e c t r o p h o t o m e t r i c a n a l y s i s i s r a r e l y used

b e c a u s e o f t h e v e r y l o w molar a b s o r p t i v i t y o f methyprylon. However, i n t h e absence of any i n t e r f e r i n g s p e c i e s , t h e maximum a t 294 nm ( i n 2-propanol) cou ld b e used f o r d i r e c t s p e c t r o p h o t o m e t r i c a n a l y s i s .

6 .6 C o l o r i m e t r i c A n a l y s i s Methyprylon undergoes e n o l i z a t i o n i n t h e p r e s e n c e

o f a l k a l i t h u s p e r m i t t i n g t h e u s e of t h e F o l i n C i o c a l t e u r e a g e n t t o form t h e b l u e c o l o r e d complex ( 2 3 ) . T h i s method i s u t i l i z e d f o r t h e d e t e r m i n a t i o n o f methyprylon p r e s e n t i n blood o r plasma. A f t e r a p p r o p r i a t e e x t r a c t i o n o r o t h e r s e p a r a t i o n , t h e F o l i n C i o c a l t e u r e a g e n t i s added t o a b a s i c s o l u t i o n of t h e methyprylon and t h e abso rbance of t h e b l u e complex i s measured a t t h e maximum abou t 700 nm ( 1 4 ) . The s e n s i t i v i t y l i m i t is abou t 5 iJg/ml of b lood o r plasma.

6 . 7 T i t r i m e t r i c Ana lys i s The p o t e n t i o m e t r i c t i t r a t i o n of a b a s i c , aqueous

s o l u t i o n of methyprylon w i t h 0.1N po tas s ium f e r r i c y a n i d e i s t h e method of c h o i c e f o r t h e a n a l y s i s of methyprylon i n t h e b u l k form as w e l l as i n c a p s u l e s and t a b l e t s ( 2 4 ) . The end p o i n t i s de te rmined p o t e n t i o m e t r i c a l l y by t h e u s e o f a calomel-plat inum e l e c t r o d e system. Each m l of 0.1N p o t a s - sium f e r r i c y a n i d e is e q u i v a l e n t t o 9 .162 mg of methyprylon.

38 I


7. References

1.

2.

3 .

4.

5.

6.

7 .

8.

9.

10. 11.

12. 13.

14.

15.

16. 1 7 .

18. 19 *

20.

21.

22. 23.

24.

Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal Communication. Johnson, J. H., Hoffmann-La Roche Inc., Personal Communication. Rubia, L. B., Hoffmann-La Roche Inc., Personal Communication. Boatman, J., Hoffmann-La Roche Inc., Personal Communication. Benz, W., Hoffmann-La Roche Inc., Personal Communication. National Formulary XIII, First Supplement , p . 1020 (1970). Moros, S . , Hoffmann-La Roche Inc., Personal Communication. MacMullan, E., Hoffmann-La Roche Inc. , Personal Communication. Hagel, R. B., Hoffmann-La Roche Inc., Personal Communication. Lau, E., Hoffmann-La Roche Inc., Unpublished Data. Pecherer, B., Hoffmann-La Roche Inc., Unpublished Data. Frick, H., Hoffmann-La Roche Inc., Unpublished Data Pellmont, B., Studer, A . , and Jurgens, R., Schweiz. Med. Wschr. , 85, 350 (1955). Randall, L. O., Iliev, V., and Brandman, O., Arch. In t . Pharmacodynamie , 106, 388 (1956). Bernhard, K., Just, M., Lutz, A . H., and Vuilleumier, J. P. , HeZv,. Chim. A c t a , 40, 436 (1957). Pribilla, O., Archiv. f;*r Toxikologie , 1 8 , 1 (1959). Boesche, J. and Schmidt, G . , Arzneim. FGsch. , 16, 548 (1966). Boesche, J., Arzneim. Forsch. , 2, 123 (1969). Scheidl, F., Hoffmann-La Roche Inc., Personal Communication. Frahm, M., Gottesleben, A. , and Soehring, K. , Arzneim. Forsch. , 11, 1008 (1961). Beratti, N., Hoffmann-La Roche Inc., Unpublished Data. Mahn, F., Hoffmann-La Roche Inc., Unpublished Data. Folin, O., and Ciocalteu, V., J . B io l . Chem., 73, 627 (1929). National Formulary XIII, pp. 458-459 (1970).

382

PH ENELZINE SU LF ATE

Reviewed b y L. Chafetz

383

ROBERT E. OALY

CON TEN T S

A n a l y t i c a l P r o f i l e - P h e n e l z i n e S u l f a t e

1.

2.

3. 4 . 5. 6.

7 ,

D e s c r i p t i o n 1.1 N a m e , F o r m u l a , M o l e c u l a r W e i g h t 1 . 2 A p p e a r a n c e , C o l o r , Odor P hy s i ca 1 P r o p e r t i es 2 . 1 I n f r a r e d S p e c t r u m 2.2 N u c l e a r M a q n e t i c Resonance S p e c t r u m 2 . 3 Mass S p e c t r u m 2 . 4 U l t r a v i o l e t S p e c t r u m 2 . 5 D i f f e r e n t i a l T h e r m a l A n a l y s i s 2 . 6 T h e r m o g r a v i m e t r i c A n a l y s i s 2 .7 M e l t i n g Range 2 . 8 S o l u b i l i t y 2 . 9 X-Ray D i f f r a c t i o n Powder A n a l y s i s S y n t h e s i s S t a b i l i t y Metabolism Methods o f A n a l y s i s 6 . 1 S p e c t r o p h o t o m e t r i c A n a l y s i s 6 . 2 P o l a r o g r a p h y 6 . 3 T i t r i m e t r y 6 . 4 Chromatography

6 . 4 1 Gas Chromatography 6 . 4 2 T h i n L a y e r Chromatography

6 . 5 S p o t T e s t s R e f e r e n c e s

384

PHENELZINE SULFATE

1. D e s c r i D t i o n

1.1 N a m e , Formula , M o l e c u l a r Weight P h e n e l z i n e S u l f a t e i s B - p h e n y l e t h y l -

h y d r a z i n e d i h y d r o g e n s u l f a t e . I t i s a l s o known as p h e n e t h y l h y d r a z i n e s u l f a t e .

1 . 2 A p p e a r a n c e , Color , Odor

powder h a v i n g a c h a r a c t e r i s t i c o d o r . W h i t e t o y e l l o w i s h w h i t e c r y s t a l l i n e


2 . 1 I n f r a r e d S p e c t r u m The i n f r a r e d s p e c t r u m ( F i g u r e 1) of

p h e n e l z i n e s u l f a t e was d e t e r m i n e d as a K B r p e l l e t i n a P e r k i n - E l m e r model 6 2 1 s p e c t r o p h o t o m e t e r . The s p e c t r u m o b t a i n e d i s i d e n t i c a l t o t h a t o f p h e n e l z i n e s u l f a t e which a p p e a r s as S a d t l e r i n f r a r e d s p e c t r u m #24825. The broad band a t a b o u t 2500 c m . - ’ may b e a t t r i b u t e d t o t h e h y d r a z i n e s a l t . The b a n d s a t 758 c m . - ’ a n d 705 c m . - ’ may b e a t t r i b u t e d t o t h e m o n o s u b s t i t u t e d b e n z e n e m o i e t y .

2 . 2 N u c l e a r M a g n e t i c Resonance S p e c t r u m The NMR spec t r i im o f p h e n e l z i n e s u l f a t e

w a s d e t e r m i n e d i n a V a r i a n A - 6 0 i n s t r u m e n t employing DMSO as t h e s c l v e n t ( F i g u r e 2 ) . The s p e c t r u m d i s p l a y e d s i n g l e t s a t 6 7 . 7 5 ( h y d r o g e n bonded t o n i t r o g e n ) and a t 6 7.2 (a romat ic ) . O t h e r a b s o r p t i o n w a s c e n t e r e d a r o u n d 6 3 .0 ( m e t h y l e n e bonded t o m e t h y l e n e and N H ) and 6 2 . 5 ( m e t h y l e n e bonded t o m e t h y l e n e and p h e n y l ) . D e u t e r i u m e x c h a n g e ( F i g u r e 3 ) w a s p e r f o r m e d on t h e s a m p l e . The a b s o r p t i o n band a t 6 7 . 7 5 d i s a p p e a r e d v e r i f y i n g t h e a s s i g n m e n t o f t h i s band t o hydrogen bonded t o n i t r o g e n .

2.0 2.5 3.0 4.0 5 .0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 10.0 11.0 12.0 13.0 14.0 15.0 100 I 1 I I - - I . - . . . .

I 1 1 I 0 I

5000 4000 3000 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 640 I 1

W A V E NUMBERS I N CY -'

F i g u r e 1 - I n f r a r e d Spectrum o f Phene lz ine S u l f a t e .

500 400 300 200 z 00 0 cpr

rn C r n b --I rn

I I I I 1 I 1 I 1 I I I I I I I

4.0 3.0 2.0 1.0 0 PPm 8.0 7.0 6.0 5.0

F i g u r e 2 - NMR S p e c t r a of P h e n e l z i n e S u l f a t e

500 x 400 300 200 100

1 1 I 1 I I I I I I I I 1 I I 1 I

8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0 PPm

F i g u r e 3 - NMR S p e c t r a of P h e n e l z i n e S u l f a t e - Deuter ium Exchange

PHENELZINE SULFATE

2 . 3 Mass S p e c t r u m A m a s s s p e c t r u m of p h e n e l z i n e s u l f a t e

c o u l d n o t b e o b t a i n e d d e s p i t e s e v e r a l a t t e m p t s . An A E I 902 w a s employed w i t h a n i o n i z i n g v o l t a g e of 7 0 ev and a t a t e m p e r a t u r e of 1 0 0 ° C . On r u n - n i n g t h e m a t e r i a l d i r e c t l y i n t h e p r o b e , a s p e c - t r u m which showed l a r g e amounts of s u l f u r d i o x i d e r e s u l t e d . A t t e m p t s a t r u n n i n g t h e f r e e b a s e p r o v e d f r u i t l e s s a l s o . The s a m p l e w a s i n t r o d u c e d i n t o t h e mass s p e c t r o m e t e r t h r o u g h t h e h e a t e d i n - l e t b u t t h e r m a l d e c o m p o s i t i o n o c c u r r e d and no s p e c t r u m w a s o b t a i n e d .

2 . 4 U l t r a v i o l e t S p e c t r u m R a j e s w a r a n and K i r k (I) r e p o r t e d h max

( E 1 8 6 2 ) of 258 nm. and X min o f 228 nm. i n 50% e t h a n o l .

P h e n e l z i n e s u l f a t e ( 0 . 0 5 4 1 % i n 5 0 % e t h a n o l ) when s c a n n e d i n a C a r y 1 4 s p e c t r o p h o - tometer be tween 350 and 2 2 0 nm. ( F i g u r e 4 ) , e x h i b i t e d t h r e e p e a k s a t 252, 258 and 263 nm. w i t h A max a t 258 nm. (a = 0 . 7 9 8 : F 1 8 6 9 ) .

2 . 5 D i f f e r e n t i a l T h e r m a l A n a l y s i s A d i f f e r e n t i a l thermoqram h a s b e e n

o b t a i n e d i n a Dupont model 9 0 0 DTA I n s t r u m e n t e m p l o y i n g a DSC c e l l . Two e n d o t h e r m s , one a t 134°C. ( c r y s t a l l i n e c h a n g e ) and t h e s e c o n d a t 1 7 2 ° C . ( m e l t i n g ) were o b s e r v e d ( F i g u r e 5 ) . H e a t i n g r a t e was 10' C . / m i n u t e . The d e t e r m i n a - t i o n w a s c a r r i e d out i n a n i t r o g e n a t m o s p h e r e . A l t e r a t i o n i n t h e r a t e of h e a t i n g would t e n d t o s h i f t t h e e n d o t h e r m s .

2 . 6 T h e r m o g r a v i m e t r i c A n a l y s i s A t h e r m o g r a v i m e t r i c a n a l y s i s w a s p e r -

formed ( F i g u r e 6 ) i n a Dupont model 950 TGA I n s t r u m e n t . The measurement was p e r f o r m e d u n d e r n i t r o g e n sweep a t a h e a t i n g r a t e o f 1 0 " C . / m i n u t e . N o w e i g h t loss w a s n o t e d u n t i l a b o u t 165" C . ( i n i t i a t i o n o f m e l t i n g ) . A f t e r t h i s p o i n t , w e i g h t w a s r a p i d l y lost. T h i s i n f o r m a t i o n t o g e t h e r w i t h t h e DTA i n d i c a t e s t h a t t h e

389

ROBERT E. DALY

0. a

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0.2

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210 320 W A V E L E N G T H

F i g u r e 4 - W Spect rum o f P h e n e l z i n e S u l f a t e

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392

PHENELZINE SULFATE

e n d o t h e r m a t 134O C . w a s d u e t o a n o n d e s t r u c t i v e a l t e r a t i o n i n t h e m o l e c u l e ( c r y s t a l c h a n g e ) .

2 . 7 M e l t i n g Range The m e l t i n g r a n g e h a s been r e p o r t e d ( 2 )

t o be 1 6 4 t o 168" C .

2 . 8 S o l u b i l i t y P h e n e l z i n e s u l f a t e i s s o l u b l e i n wa te r

and p r a c t i c a l l y i n s o l u b l e i n e t h a n o l , c h l o r o f o r m and e t h e r .

2 . 9 X-Ray D i f f r a c t i o n Powder A n a l y s i s Rajeswaran and K i r k (1) h a v e r e p o r t e d

x - r a y d i f f r a c t i o n da t a €or p h e n e l z i n e s u l f a t e . The powdered s a m p l e which h a d b e e n a p p r o p r i a t e l y mounted on a r o t a r y s p e c i m e n h o l d e r , w a s s u b - j e c t e d t o c o p p e r k - a l p h a r a d i a t i o n i n a N o r e l c o G e i g e r C o u n t e r X-Ray S p e c t r o m e t e r . The d i f f r a c - t i o n p a t t e r n s w e r e o b t a i n e d over a n a r c of 4 5 " . The i n s t r u m e n t w a s c a l i b r a t e d o v e r t h i s r a n g e , u s i n g powdered q u a r t z c r y s t a l s , a n d t h e " d " v a l u e s computed. V a l u e s are g i v e n i n T a b l e I .

T a b l e I - Ild" D i s t a n c e s U s i n g

Copper K-Alpha R a d i a t i o n

D i s t a n c e s - 1 8 . 9 , 9 . 8 5 , 6 . 5 1 , 5 . 4 2 , 4 .89 , 3 . 9 0 , 3 . 2 4 , 2 .79 & 2 . 4 4


B i e l ( 3 ) h a s s y n t h e s i z e d p h e n e l z i n e by r e a c t - i n g h y d r a z i n e h y d r a t e and p h e n e t h y l b r o m i d e u n d e r r e f l u x €or 7 h o u r s . A y i e l d of 77% w a s o b t a i n e d . S a c h a and M a l a s n i c k i ( 4 ) c o n d e n s e d p h e n e t h y l c h l o r i d e w i t h h y d r a z i n e u n d e r r e f l u x i n a n h y d r o u s e t h a n o l i n a n i t r o g e n a t m o s p h e r e €or 1 2 h o u r s . The s o l u t i o n of t h e f r e e base which w a s o b t a i n e d a f t e r removal o f h y d r a z i n e H C 1 b y f i l t r a t i o n was t r e a t e d w i t h s u l f u r i c a c i d i n a n h y d r o u s e t h a n o l

393

ROBERT E. DALY

(l.l/l W/W) . A y i e l d of 2 1 % of P-phenyle thyl - h y d r a z i n e d ihydrogen s u l f a t e w a s o b t a i n e d .

B i e l , e t . aZ. ( 5 ) has r e p o r t e d t h a t a r a l k y l a - t i o n o f h y d r a z i n e w i t h p r imary a r a l k y l h a l i d e s a f f o r d s mono- ( a r a l k y l ) -hydraz ines i n y i e l d s of 6 0 t o 75% p r o v i d e d a t h r e e t o f i v e fo ld molar excess of h y d r a z i n e h y d r a t e i s employed. P r o d u c t i o n o f d i p h e n y l e t h y l h y d r a z i n e h a s been r e p o r t e d ( 6 ) as a by-product i n t h e r e a c t i o n .

0- :;:::;::;2:\> Ref lux 7 h o u r s

anhyd. E t O H

O H P - C H Z - N H - N H ~

H2S04 : E t O H 12R'/;d. N P a t m . 1 (l*l:l)

C H 2 - C H 2 C 1 + S u l f a t e N H ~ - N H P * H ~ O

4. S t a b i l i t y

P h e n e l z i n e base i s e a s i l y ox id ized . Decompo- s i t i o n o c c u r s when b a s i f i e d s o l u t i o n s are ex- t r a c t e d , p r o b a b l y due t o a u t o o x i d a t i o n of t h e b a s e .

S c h l i t t , e t . aZ. (7) have r e p o r t e d t h e a u t o o x i - d a t i o n of p h e n e l z i n e s u l f a t e i n pH 5 . 9 phospha te b u f f e r . A u t o o x i d a t i o n i n pH 6 . 5 , 0 . 1 M sodium c h l o r a t e a t 37' C. has a l so been r e p o r t e d ( 8 ) . Decomposi t ion p r o d u c t s were n o t c h a r a c t e r i z e d .

5. Metabol ism

Drabner and Schwerd ( 9 ) i d e n t i f i e d unchanged

394

PHENELZINE SULFATE

p h e n e l z i n e and p h e n y l a c e t y l g l u t a m i n e i n t h e u r i n e o f humans who had r e c e i v e d p h e n e l z i n e s u l f a t e .

C l i n e s c h m i d t , e t . a l . ( 1 0 , 11) s t u d i e d t h e in v i t r o b i o t r a n s f o r m a t i o n as w e l l as t h e in v i v o metabo l i sm o f C"-phenelzine i n ra ts . i c a c i d w a s i d e n t i f i e d as t h e ma jo r m e t a b o l i t e .

e f f e c t o f p h e n e l z i n e on t h e e x c r e t i o n of 6- phene thy lamine .

Phenylace t -

F i s c h e r , e t . al. ( 1 2 ) have d e s c r i b e d t h e

6. Methods of A n a l y s i s

6 . 1 S p e c t r o p h o t o m e t r i c A n a l y s i s

6 . 1 1 F e i g l (13 ) h a s r e p o r t e d t h e reduc- t i o n of l i t h i u m and sodium molybdophosphotung- s t a t e s o l u t i o n by h y d r a z i n e d e r i v a t i v e s . T h i s r e a c t i o n h a s been a d a p t e d t o t h e d e t e r m i n a t i o n of p h e n e l z i n e s u l f a t e r a w ma te r i a l o r t a b l e t s by Bose and V i j a y v a r g i y a (14). Reduc t ion i s c a r r i e d o u t i n a l k a l i n e s o l u t i o n w i t h t h e p r o d u c t i o n o f a deep b l u e c o l o r which is measured a t 650 nm.

6.12 A color imetr ic method b a s e d on t h e f o r m a t i o n o f t h e hydrazone w i t h v a n i l l i n h a s been d e v i s e d ( 1 5 ) . The r e a c t i o n i s c a r r i e d o u t i n 0 .5 N m e t h a n o l i c h y d r o c h l o r i c a c i d . Absorbance o f t h e y e l l o w s o l u t i o n i s r e a d a t 4 0 0 nm. S i n c e p h e n e l z i n e d e g r a d e s by o x i d a t i v e d e s t r u c t i o n of t h e h y d r a z i n e f u n c t i o n , t h i s method i s s t a b i l i t y i n d i c a t i n g .

6 . 2 P o l a r o g r a p h y The h a l f wave p o t e n t i a l v e r s u s

s t a n d a r d calomel e l e c t r o d e ) of t h e a c e t o n e d e r i v a t i v e o f p h e n e l z i n e w a s r e p o r t e d ( 7 ) as - 1 . 3 3 V i n pH 5 . 9 p h o s p h a t e b u f f e r ( 0 . 1 M KH~POI, : N a 2 H P O 4 * 2 H 2 O ) employing a 0 . 0 1 % g e l a t i n s o l u t i o n as a maximum s u p p r e s s o r . The d i f f u s i o n c u r r e n t c o n s t a n t ( I d ) w a s a p p r o x i m a t e l y 2 .5 . Four e lec- t r o n s w e r e t r a n s f e r r e d f o r t h e r e d u c t i o n of t h e hydrazone d e r i v a t i v e . Waveheight was p r o p o r t i o n - a l t o c o n c e n t r a t i o n i n t h e r a n g e of 2 . 5 x lo-' t o 2 . 5 x M i n pH 5 .9 p h o s p h a t e b u f f e r .

3Y.5

ROBERT E . DALY

6.3 T i t r i m e t r y Procedures based on t h e r e a c t i o n of

phene lz ine s u l f a t e wi th i o d i n e i n b i c a r b o n a t e s o l u t i o n ( 2 ) and i n sodium hydroxide ( 1 6 ) and w i t h bromate and bromide i n a c i d s o l u t i o n ( 1 6 ) w i t h subsequent t i t r a t i o n of excess r e a g e n t ( i o d i n e o r bromide conver ted t o i o d i n e ) i n a c i d s o l u t i o n w i t h t h i o s u l f a t e have been r e p o r t e d .

B a s i c a l l y , t h e r e a c t i o n invo lved may be i l l u s t r a t e d a s fo l lows:

Radecka and Nigam ( 1 7 ) have r e p o r t e d t h e d i r e c t t i t r a t i o n of phene lz ine w i t h N-bromosuccinimide i n a c i d i c s o l u t i o n employing methyl r e d a s t h e i n d i c a t o r . However, t h e i r r e s u l t s show a 5% pos- i t i v e b i a s . T h i s b i a s i s a t t r i b u t e d t o a l l y l i c b romina t i on by N -b r omos uccin imide .

6 . 4 Chromatography

6 . 4 1 Gas Chromatography C a r d i n i , e t . a l . (18, 1 9 ) and

McMartin and S t r ee t ( 2 0 ) have r e p o r t e d gas chrom- a t o g r a p h i c methods which a r e r epu ted t o be quan- t i t a t i v e . However, no q u a n t i t a t i v e d a t a a r e g iven . Cond i t ions are r e p o r t e d i n Tab le 11.

6 . 4 2 Thin Layer Chromatography Q u a l i t a t i v e methods f o r t h e ident i -

f i c a t i o n of p sycho t rop ic a g e n t s have been reported ( 2 1 , 2 2 , 23, 2 4 , 2 5 ) . Rf v a l u e s a r e summarized i n Table 111.

6 . 5 Spo t T e s t s A v a r i e t y of s p o t t e s t s have been re-

p o r t e d ( 2 6 ) . These are summarized i n Table v.

396

T A B L E I1 - P a r a m e t e r s f o r G a s C h r o m a t o g r a p h y

C o l u m n C a r r i e r F l o w D e t e c t o r I n j e c t o r R e f . Column Temp. G a s R a t e D e t e c t o r T e m p . T e m p .

1 8 g l a s s column p r o g r a m 1 . 8 M x 2 m m . ; 7 0 - 2 5 0 ' 2 % GE-SE 30 on a t 1 0 . 4 O / A e r o p a k 3 0 , 80- m i n u t e 1 0 0 m e s h

1 9 S t a i n l e s s s t e e l 1 4 0 ' C . c o l u m n 1 . 2 M. x 2 mm.; 15% c a r b o w a x 20 M o n C h r o m o s o r b W a l k a l i n i z e d w i t h 5% KOH

w

c 4

N2 50 m l . / FID 220 ' C . 250 ' C . m i n .

Q I rn z H e 40 m l . / F I D 250 ' C . rn

m i n . P z rn m C r n > 2

20 S t a i n l e s s s t e e l 1 4 0 ' C . N2 30 ml./ F I D c o l u m n 6 ' x 1/8" ; m i n . 2 % SE 3 0 + 0 . 1 % t r i s t e a r i n on s i l a n i z e d ac id w a s h e d C h r o m o s orb W

ROBERT E. DALY

TABLE I11 - Suppor t , S o l v e n t System and Rf

Values f o r TLC of Phene lz ine S u l f a t e

S o l v e n t Reference Suppor t System Rf ( X 1 0 0 )

2 1 s i l i c a g e l G chloroform: m e t hano 1 ( 8 : 2 )

2 1 s i l i c a g e l G chloroform: ace tone : d i e t h y lamine ( 5 : 4: 1)

2 1

2 2

2 2

2 2

2 2

s i l i c a g e l G cyclohexane: chloroform: d i e t h y l a m i ne ( 4 : 5 : 1)

s i l i c a g e l G hexane:benzene: impregnated d i e thy lamine w i t h 0 . 1 M (75:15: 10) N aOH

s i l i c a g e l G methanol impregnated w i t h 0 . 1 M N a O H

s i l i c a g e l G ace tone imp re gn a t ed w i t h 0 . 1 M NaOH

s i l i c a g e l G methanol impregnated w i t h 0 . 1 M NaHSO

100

88

6 2

5 1

72

75

41

398

PHENELZINE SULFATE

TABLE I11

R e f e ren c e

22

23

23

24

2 4

24

2 4

f c o n t . 1 S o l v e n t

Suppor t System Rf ( x 100)

s i l i c a g e l G 9 5 % e t h a n o l impregnated w i t h 0 . 1 M NaHSO ,+

s i l i c a g e l G ch loroform: a c t i v a t e d a t m e t hano 1 110" C. f o r (1:l) 30 minutes

s i l i c a g e l G ch loroform: a c t i v a t e d as methanol : above 2 0 % ammonium

hydroxide ( 2 : 1:l)

s i l i c a g e l G methanol :12 N a c t i v a t e d a t ammon i um 100' C . f o r hydroxide 1 hour ( 1 0 0 : 1 . 5 )

s i l i c a g e l G cyclohexane: impregnated d i e t h y l a m i n e : w i t h 0 . 1 N benzene NaOH and ( 7 5 : 2 0 : 15 ) a c t i v a t e d a s above

s i l i c a g e l G a c e t o n e impregnated and a c t i v a t e d as above

s i l i c a g e l G ch loroform: impregnated methanol and a c t i v a t e d ( 9 0 : 10) as above

25

37

56

7 4

4 4

65

75

399

ROBERT E. DALY

TABLE I11 ( c o n t . )

Re fe rence S u p p o r t Sys tem Rf ( X 100) S o l v e n t

2 4 s i l i c a g e l G benzene: 50 e t h a n o l : 1 2 N ammonium h y d r o x i d e (95: 15: 5)

25 s i l i c a g e l G 95% e t h a n o l imp re gna t e d w i t h 0 . 1 N KHSO 4

2 5 s i l i c a g e l G cyc lohexane : impregna ted benzene : w i t h 0.1N d i e t h y lamine N aOH ( 7 5 : 15: 10)

25 s i l i c a g e l G me thano l imp r e g n a t e d w i t h 0 . 1 N N a O H

25 s i l i c a g e l G a c e t o n e impregna ted w i t h 0 . 1 N N a O H

25 s i l i c a g e l G me thano l imp r e g n a t ed w i t h 0 . 1 N KHSO I,

25 s i l i c a g e l G m e t h y l impregna ted acetate w i t h 0 . 1 N N a O H

49

52

72

75

62

53

400

PHENE LZI NE SULFATE


R e f e r e n c e S u p p o r t System Rf (x 100) S o l v e n t

2 5 chromedi a n - b u t a n o l : 51 (Whatman #41) 5% c i t r i c impregna ted ac id w i t h 5% sodium d i h y d r og e n c i t r a t e ( s o m e K2HP04 added t o p r e v e n t t a i l i n g )

ROBERT E. DALY

TABLE I V - Reagents and R e a c t i o n s For TLC P r o c e d u r e s

Refe rence

21

21

22

23

23

23

24

24

24

Reagent R e a c t i o n

F o l i n - C i o c a l t e a u ' s b l u e v i o l e t Reagent w i t h h e a t i n g

5% phosphomolybdic a c i d b l u e i n e t h a n o l f o l l o w e d by e x p o s u r e o f t h e p l a t e t o ammonia vapor

1% i o d i n e i n me thano l brown

50 m l . o f 0 . 2 % r o s e o r a n g e n i n h y d r i n i n me thano l + 1 0 m l . of g l a c i a l a ce t i c a c i d + 2 m l . of 2,4,6-trimethylpiperidine

1 0 % F e r r i c c h l o r i d e ; 1% f l u o r e s c e i n : ammonia

S a t u r a t e d ammonium molybdate ; s a t u r a t e d o x a l i c a c i d

Fo l i n - C i o c a l t e a u ' s Reagent

Mandelin s Reagent

C i nn ama ldehy de R e a g e n t 5 m l . c innamaldehyde + 9 5 m l . e t h a n o l + 5 m l . conc . H C 1

f l u o r e s c e n t b l u e

celeste

b l u i s h a t RT, b l u e when h e a t e d t o 1 0 0 ' c. p i n k a t R T , f l e s h a t 100 ' C. , f l u o r e s c e n t a f t e r h e a t i n g y e l l o w a t RT

402

PHENELZINE SULFATE

TABLE IV ( c o n t . )

Re fe rence Reagent R e a c t i o n

2 4 0 .25 g . p-d imethyl - lemon chromo aminobenzaldehyde + a f t e r h e a t i n g 5 g . of 85% p h o s p h o r i c t o 1 0 0 ' C . a c i d + 2 0 m l . o f w a t e r f o r 1 0 min.

and t h e n exposed t o ammonia v a p o r

2 4 Fur f u r a l r e a g e n t s o l ' n A . ) 1 m l . f u r f u r a l i n 99 m l . o f a c e t o n e s o l ' n B . ) 4 m l . of conc . H 2 S 0 4 i n 96 m l . o f a c e t o n e

brown a t 1 0 0 ' C.

25 P l a t e examined unde r sho r twave W l i g h t and t h e n s p r a y e d w i t h t h e f o l l o w i n g sequence o f r e a g e n t s :

i o d i n e brown Dragendorf f ' s o r a n g e sodium n i t r i t e deep brown i odop l a t i n a t e v a r i o u s l y c o l o r e d u n t i l a c o l o r d e v e l o p s

403

ROBERT E. DALY

TABLE V - S p o t Tests f o r P h e n e l z i n e S u l f a t e

Reagent Response 0 . 5 mg.

Froehde ' s Reagent b l u e * It . b l u e

Mandelin ' s Reagent

Marquis ' Reagent

Mecke's Reagent

g r e e n b l u e * brown

r e d orange* g r e y

R e i c k a r d ' s Reagent I t . b l u e

F l u e c k i g e r ' s Reagent brown

V i t a l i ' s Reagent a . 1 b l u e b.) o r a n g e

Was i ck y ' s R e agen t red o r a n g e

2 0 1-14.

b l u e

g r e e n

f a i n t o range

pink

It. b l u e

- --

--- ---

- --

* = a f t e r warming

404

PH E N E LZ I NE SULFATE

TABLE V I - Composi t ion o f S p o t T e s t Reagents

Reagent Composi t ion

Froehde I s Reagent 0 . 5 % aqueous ammonium m o 1 yb d a t e

Mande l i n I s Reagent 0 . 5 % aqueous ammonium v a n a d a t e

Marquis Reagent 1 p a r t 3 7 % formaldehyde i n 2 0 p a r t s c o n c e n t r a t e d s u l f u r i c a c i d , f r e s h l y p r e p a r e d b e f o r e u s e

Mecke ' s Reagent 0 . 5 % aqueous s e l e n i o u s a c i d (H2SeO 3 1

Reickard I s Reagent 1% aqueous sodium t u n g s t a t e

F l u e c k i g e r ' s Reagent 0 . 5 % s o l u t i o n o f t i t a n i u m d i o x i d e ( T i 0 2 1 i n concen- t r a t e d s u l f u r i c a c i d

V i t a l i s Reagent a . ) fuming n i t r i c a c i d b . ) 3 % p o t a s s i u m h y d r o x i d e

i n e t h a n o l

Wasicky s Reagent 1 0 % s o l u t i o n o f p-d imethyl - aminobenzaldehyde i n g l a c i a l a c e t i c a c i d

305

ROBERT E. DALY

1.

2. 3. 4 .

5 .

6 .

7.

8 .

9 .

10.

11. 1 2 .

13.

1 4 .

1 5 . 1 6 .

1 7 .

1 8 .

1 9 .

20 .

7. References

406

P . R a j e s w a r a n and P . L. K i r k , B u l l . N a r c o t i c s , U . N . , D e p t . S o : L 4 i l ~ f f ~ i r s 1 4 , No. I, 1 9 ( 1 9 6 2 ) . U.S .P . X V I I I . J . H . B i e l , US 3 , 0 0 0 , 9 0 3 , S e p t . 1 9 , 1 9 6 1 . A. S a c h a a n d L . M a l a s n i c k i , P o l 4 6 , 2 3 7 , O c t . 3 0 , 1 9 6 2 , C . A . 60, 4 5 4 f ( 1 9 6 3 ) . J . H . B i e l , A. E . D r u k k e r , T . H . M i t c h e l l , E . P . S p r e n g e l e r , P . A . N u h f e r , A. C . Conway and A. Hor i ta , J . A m . Chem. S o c . 8 2 , 2 8 0 5 ( 1 9 5 9 ) . E . Votocek a n d 0. L e m i n g e r , ColI. C z e c h . Chem. Commun. 4 , 2 7 1 ( 1 9 3 2 ) , C. A . 2 6 , 5294 ( 1 9 3 2 ) . L . S c h l i t t , M. Rink a n d M. von S t a c k e l b e r g , J , E Z e c t r o a n a l . Chem. 2 3 , 1 0 ( 1 9 6 7 ) . L . E . E b e r s o n and K . P e r s s o n , , J . Med. Pharm. Chem. 5, 738 ( 1 9 6 2 ) . J . D r a b n e r and W . Schwerd , F r e s e n i u s ' Z. A n a l . Chem. 2 4 3 , 9 2 ( 1 9 6 8 ) . C. A . 70, 558941 ( 1 9 6 9 ) . B . Van C l i n e s c h m i d t and A. Horita, B i o c h e m . P h a r m a c o l . 1 8 , 1 0 1 1 ( 1 9 6 9 ) . I d e m . , i b i d . , 1 0 2 1 ( 1 9 6 9 ) . E . F i s c h e r , B . Hel ler , and A. H . Miro, A r z n e i m i t t e l - F o r s c h . 18, 1 4 8 6 ( 1 9 6 8 ) . F . F e i g l , " S p o t T e s t i n O r g a n i c A n a l y s i s " , E l s e v i e r Company, London, 1 9 5 6 , p . 1 2 8 . B . C. Bose and R. V i j a y v a r g i y a , I n d i a n J . Pharm. 2 8 , 3 2 8 ( 1 9 6 6 ) . R. E . D a l y , u n p u b l i s h e d r e s u l t s M. M a r z a d r o a n d A. D e C a r o l i s , R e n d . I s t . S u n i t a 2 6 , 629 ( 1 9 6 3 ) . C . Radecka and I . C. Nigam, Can. J . P h m m S c i . 2 , 1 7 ( 1 9 6 6 ) . C . C a r d i n i , V . Q u e r c i a and A. C a l o , :J . Chromatog . 37, 1 9 0 ( 1 9 6 8 ) . U. Q u e r c i a , C . C a r d i n i and A . Calo, i i ( ' l . Chim. Farm. 1 0 7 , 3 8 3 ( 1 9 6 8 ) . C. McMartin and H . V . S t r e e t , J . Chromatog. 2 2 , 274 ( 1 9 6 6 ) .

PHENELZINE SULFATE

7. R e f e r e n c e s ( c o n t . )

21. E. Schmid, E. Hoppe, C . M e y t h a l e r and L.

22. W . W. F i k e , A n a l . Chem. 38, 1 6 9 7 ( 1 9 6 6 ) . 23. A. A l e s s a n d r o , F. Mari and S . S e t t e c a s e ,

Farmaco, Ed. Prat. 22, 437 ( 1 9 6 7 ) . 24. I. Z i n g a l e s , J . C h r o m a t o g . 3 1 , 405 (1967) . 25. I. S u n s h i n e , W. W . F i k e a n d H . Landesman,

26. P. R a j e s w a r a n and P . L. K i r k , B u l l .

Z i c h a , A r z n e i m i t t e Z - F o r s c h , 1 3 , 9 6 9 (1963).

J . F o r e n s i c S c i . 1 1 , 428 ( 1 9 6 6 ) .

N a r c o t i c s , U . N . , D e p t . S o c i a l A f f a i r s 1 3 , No. 3, 15 ( 1 9 6 1 ) .

407

PRIMIDONE

Raymond D. Daley

409

RAYMOND D. DALEY

CONTENTS

1. Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor, Taste

2. Physical Properties 2.1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectra 2.3 Ultraviolet Spectra 2.4 Mass Spectra 2.5 Crystal Properties 2.6 Melting Points 2.7 Solubility

3 . Synthesis

4. Stability-Degradation


b. Methods of Analysis 6. I Elemental Analysis 9.2 Chromatography

6.21 Gas Chromatography 6.22 Thin Layer Chronatography 6.23 Paper Chromatography

6.31 Ultraviolet Absorption 6.32 Ultraviolet Determination as Phenobarbital 6.33 Colorimetric Methods

6.3 Absorptiometric Methods

6.4 Polarography 6.5 Titration 6.6 Other Analytical Tests

7. References

PRIMIDONE

1. Description

1.1 Name, Formula, Molecular Weight

Primidone i s 5-ethyldihydro-5-phenyl-k,6(lH, 5 H ) - pyrimidinedione.

H

\ / N / ‘‘b H

Mol. W t . 218.26 ‘1.2~ 4N2 ‘2

1.2 Appearance, Color, Odor, Taste

White, odorless powder, w i t h a s l i g h t l y b i t t e r t a s t e .

2. Physical Propert ies

2.1 Infrared Spectra

Infrared spectra 3f two c r y s t a l forms of pr im- idone (here designated as Form I and Farm 11) a r e shown i n Figures 1 and 2. o i l mulls between potassium bromide plates . A Beckman Model 111-12 instrument was used. Similar spec t r a a r e obtained with potassium bromide dispers ions (1,2), b u t t he pressing operation usually causes d i s t o r t i o n s of the absorption bands.

The samples were prepared as mineral

Some of the absorption bands may be assigned as follows: region, amide C=O; 1600, &$lo, 760, 700, and 520 cm-’, aromatic ring. The absorption i n t he 2900 cm’ region and pa r t of the absorption at &60 and 1380 cm’ i s due t o .ninera.l o i l .

3200 cm’ region, bonded N-!I s t r e t c h ; 1700 cm”

A

N L

Fig. 1. Infrared spectrum of primidone, Ayerst Laboratories, Inc., house reference standard P24636B, Form I, mineral oil mul l between KBr plates .

a >

0 z 0

2

P 0 P r m <

P, w

Fig. 2. Infrared spectrum of primidone, Form 11, mineral oil mull between KBr plates.

RAYMOND D. DALEY

2.2 Nuclear Magnetic Resonance Spectra

Figure 3 shows the proton magnetic resonance spectrum of primidone. d6-dimethylsulfoxide solution, using a Varian HA-100D instrument, with a tetramethylsilane reference. assignment of t he spectrum i s shown i n Table I (3 ) .

This spectrum was obtained on a

The

The resonances at 3.96 and 4.12 ppm a r e due t o the fragment NH-CH,TNH i n which the methylene protons have d i f f e r e n t chemca l s h i f t s and thus show geminal coupling, and i n which one of t he methylene protons (probably t h e equator ia l ) is coupled t o both the NH protons and i s thus a doublet of t r i p l e t s . can be described as AB$ (3 ) .

The system

TABLE I

Proton Magnetic Resonance Spectrum Peak Assignments (3)

Relative - PPM I n t e n s i t y Mul t ip l i c i ty

0. 84 3 t r i p l e t 1.99 2 quartet

2.50 - qu in te t

3.35 - s i n g l e t 3.96 1 doublet

4.12 1 doublet of

7 . 3 0 5 mult i p l e t €!. 64 2 doublet

t r i p l e t s

2.3 Ultraviolet Spectra

AssiRnmen t

methyl of e thy l group methylene of e thy l group r e s idua l d5-DMSO i n solvent water i n solvent one of t h e r ing methylene protons the other r ing methylene proton phenyl group NH protons

Figure 4 shows the u l t r a v i o l e t absorption spectrum of primidone (Ayerst Laboratories, Inc., house standard P a 6 3 6 B). (86.6 mg/100 m l ) and scanned VS. methanol.

The sample was dissolved i n methanol Scans of

414

81

I

'1 I , 1

I

, . . . . l . . . . I . . . . I . . . . I . . . . l . . . . I . . . . I . . . . , . . . . I . . . . , . . . .

60 PPMWOMHz 50 4 0 2 0 10

Fig. 3. Nuclear magnetic resonance spectrum of primidone in dg-dimethylsulfoldde, tetramethylsilane reference, courtesy of Dr. G. R. Bedford (3).

0 9

rn < r

Fig. 4. Ultraviolet spectrum of primidone, Ayerst Laboratories, Inc., house reference standard

fold dilutions, in methanol, 1 cm cells. P24636B, 86.6 mg/lOO ml and 10- and 100-

PRIMIDONE

10-and 100-fold d i l u t i o n s of t h i s so lu t ion a r e a l s o shown, A Cary Model U, spectrophotometer was used. M&rna at 264, 258, ar,d 252 nanometers have a b s o r p t i v i t i e s of 167, 216, and 183 l i t e r s per mole cm, respectively, i n reasonable agreement with previously published da ta (1,4). There are no o the r maxima above 210 nanometers.

The absorption bands i n the 260 nanometer region a r e caused by the phenyl group, while t he absorption a t sho r t e r wavelengths i s due t o a combination of the phenyl and a,rni.de group absorpticns.

2.4 Mass Spectra

Figure 5 shows the low resolut ion mass spectrum This data w a s obtained on a Perkin-Elmer/ of primidone.

Hitachi FMU-6E mass spectrometer (3).

The spectrum matches t h a t measured and i n t e r -

Loss of ethylene y i e l d s m/e preted by Locock and Coutts ( 5 ) . C a H N202 i s a t m/e 21P. 190 %l@l$202), and loss of an e t h y l r ad ica l y i e l d s m/e 1W (C1#~N202). m/e 161 (CgHr$02). ion y i e l d s m/e 4 6 (C1oHlOO), the most abundant fragment. Loss of CO from m/e I46 y ie lds m/e 118 (C H ~ o ) , which

The mol.ecular i on

L,oss of CH2NH from m/e 190 y i e l d s Loss of C2HkN20 from t h e molecular

l o ses H and H2 successively t o form m/e 1 9 7 and m/e 115 (5).

A t high resolut ion, m/e 161 i s reported t o be a doublet of compositions C5H7NC2 and C1@11NO, with r e l a t i v e abundances 5 :1 respectively; the l a t t e r , l e s s abundant, ion can be derived from the molecular ion by loss of ~ $ 3 ~ 0 (6).

2.5 Crystal Fropert ies

X-ray powder d i f f r a c t i o n pa t t e rns (7,r) and infrared spectra ( see Section 2.1) i nd ica t e t h a t pricddone may c r y s t a l l i z e i n at Least two d i f f e ren t forms. c r y s t a l l i z e s when a solut ion of primidone i n 95 percent ethanol i s evapcratec! a t room temperature. The second (here designated Form 11) usually forms i f primidone

One of these (here designated Form I ) usually

417

BASE 146

1 2 0 - W LL I 2 M' I 8

P

I , I 1 , I 1

rn <

Fig. 5. Mass s p e c t r m of primidone, da t a courtesy of Dr. G. 2. Bedford ( 3 ) .

PRIMIDONE

c r y s t a l l i z e s from hot water o r from the melt.

The powder d i f f r a c t i o n pa t t e rns of t he two forms are given i n Table 11. These pa t t e rns were obtained with a Norelco diffractorneter, using n i cke l - f i l t e r ed copper KQ' radiation. The pat tern of Form I is the same as t h a t reported by Roy e t a1 (€?>, while t h e pat tern of Form I1 i s s imi l a r t o t h a t reported by Penprase and Biles (7).

TABLE; 11

X-ray Powder Diffract ion Pa t t e rns

Form I d

10.96 7.37 6.57 5.9e 5.46 4.96 4. 83 4.44 4.06 3.68 3.73 3.66 3.56

3.21 3.16 3.12 2.9s 2.82 2.72 2.63 2.61 2.55 2.35 2.33

-

3.42

2.26 2.25 2.19

17 100 46 70 5 1 li.: 50 26 1

58 20 18 1 5 15 20 10 11 22 1 5 10

5 5 7 4 4 6 5 8

Form I1 ,

1/10 - d

13 77 6.88 6.16 5.75 5. l b 4.59 4.18 4.16 3.95 3.93 3.72 3.65 3.44 3.27 3-13 3.06 2.9e

2.66 2.50

2.75

u, 100 20 35

6 55 28 46

6 5 6 4 6 5

12 4 4 4 6 4

419

RAYMOND 0. DALEY

2.6 Melting Points

The following melting points have been reported:

260-281OC (15) 2e10c (11, 12, 13) 2111-282OC (9, 10) 282OC (16) 2G9OC (7)

The value 289°C was reported by the same inves t iga to r s who reported the Form I1 d i f f r a c t i o n pa t t e rn (see Section 2.5) (7 ) .

2 . 7 S o l u b i l i t y

The s o l u b i l i t y of primidone at room temperature i s as f O l l 0 w S :

Solvent

Methanol Ethanol (95%) Acetone Water Chloroform Ether Benzene Petroleum, e the r

Appror;imate Solubi l i ty . mg/ ml

5. 6.2 2.0 0. 5 0.1 0.1

< 0.1 < 0.1

3 . Synthesis

Primidone can be prepared by:

( a ) t h ioba rb i tu r i c acid w i t h Raney nickel (9,12) o r z inc and acid (9,21) ; (b) condensation of phenylethyl- malondiarnide w i t h formamide (lo), o r formic o r oxa l i c acid (I..!+) ; ( c ) reduction of 2-ethoxy-5-phenyl-5- ethylhexahydro-~,6-pyri11idinedione with hydrogen o r formic acid o r formamide o r zinc and acid (11,22); (d) hydrogenatZon of 2-methoxy-5-ethyltet rahydro- pyrimidi.ne-l+,6-dione (13) ; (e) e l e c t r o l y s i s of

Reductive desulfur i zat.ion of 5-ethyl-5-phenyl-

420

PRIMIDONE

phenobarbital (12) or of the 2-imino or 2-cyanimino derivatives of 5-ethyl-5-phenylhexahydropyrimidine- 4,6-dione (15) ; ( f ) reduction of 2-cyanimino-5- ethyl-S-phenylhemhydropyrimidine-4,6-dione w i t h zinc and acid (15).

To help the reader visualize the processes above, (a), (b), and (c) may be depicted as follows:

0 H

U

S Ni q( 0 H

HC -NH2 - \

0 H

421

RAYMOND D. DALEY

4. Stabi l i ty-Degradat ion

Primidone i s q u i t e s t a b l e , as might be pred ic ted from t h e absence of r eac t ive func t iona l groups, low s o l u b i l i t y , and high melting point. No r e p o r t s of degradat ion under ord inary condi t ions were found, although i t i s metabolized (Sec t ion 5) , and w i l l react under d r a s t i c chemical condi t ions (Sect ion 6).


The repor ted metabolic products of primidone i n var ious spec ie s are as follaws:

Hetabol i te

Phenylethylmalondiamide

Ph enobar-bi tal

p-Ilydroxyph enobarbi ta l

p-Hydrowph enoba rb i t a l glucurcni de

Species

man r abb i t

rat

man

rat r a b b i t mouse

r abb i t

dog

dog man

r abb i t

S t r u c t u r a l formulas f o r these rnetabol.ites are shown i n Figure 6.

o. Nethods of Analysis

6.1 Elemental k a l y s i s

PRIMIDONE

Figure 6

Metabolism of P rimidone

RAYMOND D. DALEY

Theoretical ($1 Found (%)( 12 )

Carbon 66. ab Hydrogen 6.47 Nitrogen l2.83 Ovgen UC. 66

66.1 6.5 12.8 ----

6.2 Chromatography

6.21 Gas Chromatography

Many invest igators have used gas chromatography t o analyze f o r primidone. work i s concerned almost e n t i r e l y with analyses f o r primidone i n plasma cr urine, many of the methods could be modified e a s i l y for analyses of pharmaceutical dosage forms.

Although the publishe

A number of t he systems used f o r gas chromatography are l i s t e d i n Table 111; add i t iona l information i s given below.

Bogan and Smith ( 2 9 ) determined primidone and phenobarbital i n blood and urine by gas chromatography. The samples were adjusted t o pH 3 and extracted with chloroform. The drugs were f u r t h e r solvent par t i t ioned, and the f i n a l solut ion i n chloroform w a s evaporated t o a small volume before i n j e c t i n g an a l iquo t i n t o the gas chromatograph.

Pippenger and Gil len (32) determined primidone, phenobarbital, phenylethylmalondiami.de, and a number of o the r anticonvulsants i n plasma, urine, and cerebrospinal f luid. chloroform after buffering t o pH 6.8. The chloroform ex t r ac t was evaporated t o dryness and t h e residue dissolved i n a small amount of absolute e thanol f o r inject ion. Recoveries of known amounts of drugs added t o the body f l u i d s were 90 t o 100 percent.

They extracted the sample w i t h

424

PRIM1 DONE

S t r e e t (62) i d e n t i f i e d primidone and o ther drugs by in jec t ing a mixture of t h e drug and N,O-bis- ( t r imethyls i ly l ) -acetamide i n t o t h e gas chromatograph. The s i l y l a t i n g reagent was drawn i n t o a 10 micro l i te r syringe, then the sample solut ion w a s drawn i n t o the same syringe, and t h e mixture was in jec ted d i r e c t l y i n t o t h e gas chromatograph. Trimethylsilyl derivative8 were formed i n the column. He used 4 micro l i te rs of 10% N,O- bis-( trimethylsily1)-acetamide i n acetone f o r 0.8 micro- l i t e rs of primidone solut ion containing 0.8 pg of t h e drug.

Clarke ( 3 9 ) a lso lists a gas chromatographic system i n which t h e re ten t ion t i m e f o r primidone i s 0.65 r e l a t i v e t o codeine.

Evenson, Jones, and Darcey (33) extracted ac id i f ied serum with chloroform, evaporated the ex t rac t , took up the residue i n a chloroform solut ion of dehydroisoandrosterone ace ta te i n t e r n a l standard, and analyzed t h e solut ion f o r both primidone and diphenylhydantoin by gas chromatography. The average recovery of primidone from serum was 98 percent. chloroform was used t o avoid extraneous peaks.

High grade

F k t e t a1 (34) and Kupferberg (35) found t h a t primidone could be converted t o a methylated der ivat ive i n t h e gas chromatograph by in jec t ing a solut ion containing primidone and trimethylanilinium hydroxide. primidone, phenobarbital, and diphenylhydantoin i n plasma samples. The drugs were extracted with ethylene dichlor ide from samples buffered t o pH 7.2, f u r t h e r solvent par t i t ioned and mixed w i t h cholestane i n t e r n a l standard, and f i n a l l y mixed w i t h trimethylanilinium hydroxide so lu t ion and in jec ted i n t o t h e gas chromatograph. Recovery of primidone from plasma o r water was only 30 percent, probably because primidone was l o s t i n the multiple solvent exchanges (35).

Kupferberg used the react ion t o determine

V a n Meter, Buckmaster, and Shelly (41) used primidone as an internal standard i n t h e gas chromatographic determination of phenobarbital and

425

RAYMOND D. DALEY

diphenylhydantoin i n plasma, and suggested t h a t pheno- b a r b i t a l could be used as an i n t e r n a l standard i n assaying primidone. as an i n t e r n a l standard for primidone assays. invest igators have a lso used primidone as an i n t e r n a l standard i n gas chromatography (43).

Van Meter (42) recommended using mephenytoin Other

Gardner-Thorpe e t a1 (36) determined primidone and a number of other a n t i e p i l e p t i c drugs i n plasma by gas chromatography. f r o m ac id i f ied plasma samples with chloroform and injected without preparing derivatives. gators observed 30 percent recovery of primidone from plasma by t h e i r technique.

rhe drugs were extracted

These invest i -

Proelss and Lohmann (37) developed a rapid gas chromatographic screening procedure for 40 drugs, including primidone, i n serum. The drugs were separated i n t o acidic , neutral , and basic f rac t ions by solvent extraction. gas chromatography.

The three f rac t ions were then analyzed by

Kupferberg (38) reported tha t primidone

Primidone was formed a t r imethyls i ly l der ivat ive with N,O-bis- ( t r imethyls i ly l ) -acetamide i n pyridine. extracted from plasma buffered t o pH 7.2, using chloroform. After several solvent par t i t ion ing s teps , the primidone wa8 t rea ted with the reagent for 20 minutes. The s i l y l a t e d der ivat ive was s t a b l e for severa l hours. Recovery of primidone a f t e r t h e solvent par t i t ion ing s teps was incomplete.

Toseland, Grove, and Berry (40) developed a routine procedure for gas chromatographic analyses of primidone and several other anticonvulsant drugs i n blood. fur ther solvent par t i t ioned before chromatography. doing double extract ions, they were ab le t o recover 58 t o 67 percent of the primidone; they report not being ab le to reproduce the good recovery claimed by some other investigators. They preferred not t o prepare der ivat ives , f inding b e t t e r reproducibi l i ty i n day-to-day routine work w i t h t h e underivatized drugs.

The drugs were extracted i n t o chloroform and By

42 6

PRIMIDONE

TABLE I11

Gas Chromatographic Systems Used f o r Primidone Analvsis

Note: A l l systems l i s t e d used flame i o n i z a t i o n de tec to r s .

Column

6 f t . long x 118 in. O.D. stainless s t ee l tubing 5% SE-30 on Aeropak 30 (100-120 mesh)

6 f t . long x 4 mm I.D. g l a s s tubing, 1% HIEFF-8-BP on Gas Chrom Q, 100-120 mesh

125 crn long x 4 mm I.D. g l a s s tubing, 3.8% W-98 on Diatoport S, 80-100 mesh

6 f t . long x 4 mm I.D. g l a s s tubing, 3% OV-17 on Chromosorb W (HD)

3 f t . long x 4 mm I.D., 1% CDMS ( a ) on Diatomite CQ

6 f t . long x 4 mm I.D. g l a s s tubing, 3% OV-17 on 80-100 mesh Chromosorb W (HP)

Column Temp O C

180'

2 50°

215'

Pro- grammed f rom 160° t o 275' at 1o0/min

2 50'

Pro- grammed from 160' t o 250' a t 1o0/min

RAYMOND D. DALEY

Column

5 ft. long x 4 m I.D. g lass tubing, 2.5% SE-30 on 80-100 mesh Chromosorb W AWHMDS

2 f t . long x 2 mm I.D. g lass tubing, 2% SP-1000 on Gas ChromW

5 ft. long x 1/4 in. I.D. glass tubing, 2% SP-lo00 on Varaport

6 ft. long x 4 mm g l a s s tubing, 1% OV-17 on HP Chrornosorb G (80-100 mesh)

185 cm long x 3 mm I.D. , 3% OV-1 on Chromosorb W (HP) (80-100 mesh)

180 cm long x 4 mm I.D. g l a s s tubing, 3.5% XE-60 on Gas krom Q (100-120 mesh)

Carrier Gas

N 5 8

ml/min

He 50

ml/min

A 90

d/min

--

3 ml/min

He 80

ml/rnin

Column T e m ~ OC

225'

2 50'

2 50"

Pro- grammed from 190' t o 230'

220°

240°

( a ) CDMS is c y c l o h e m e dimethanol succinate.

428

PR IMI DONE

Gallagher and bumel (44) determined primidone by gas chromatography a f t e r preparing a methylated de r iva t ive w i t h N,O-bis-( t r imethyls i ly1)- tr if luoroacetamide and dimethylformamide ( 5 : l ) at room temperature.

6.22 Thin Layer Chromatography

Huisman (45) extracted primidone and o the r anticonvulsant drugs from serum with chloroform/methyl a c e t a t e (60:40) after adding hydrochloric acid and sodium chloride. The ex t r ac t was evaporated and the residue n i t r a t e d t o form the nitrophenyl de r iva t ive of the drug. The n i t r a t e d material was chromatographed on f luorescent s i l i c a g e l t h i n l aye r chromatographic p l a t e s with isobutyl alcohol/chloroform/25% ammonia (72:48:L2) ; t h e developed p l a t e was dr ied 10 minutes a t 100°C and developed a second time. t i on ; t he n i t r a t e d primidone R was 0.76 t o 0.80, The spots were scraped off , the nierophenyl de r iva t ive reduced with zinc, t h e r e su l t i ng amino group de r iva t ive diazotized with n i t rous acid and coupled with a-naphthyl- ethylenediamine, and the determination f inished by measuring the absorbance at 550 m. (The color imetr ic procedure was adapted from t h a t of D i l l e t a1 (50) f o r diphenylhydantoin. )

The spots were located under u l t r a v i o l e t illumina-

Fujimoto, Mason, and Murphy (24) extracted

Chromatography was on s i l i c a g e l impregnated primidone from rabb i t ur ine with chloroform a f t e r adding a c e t i c acid. glass f i b e r sheet , using 2,2,4-trimethylpentae/glacial a c e t i c ac id (5:l). t he sheet a f t e r spraying with concentrated s u l f u r i c acid. The Rf of primidone was 0.7. were estimated from the s i z e and i n t e n s i t y of t he spots. The inves t iga to r s reported 100 percent recovery of t he drug from urine, and an accuracy of about 1 & primidone i n 3 t o 10 pg spots.

The spots were detected by heating

The amounts of t he drug

Gardner-Thorpe, Parsonage, and Toothi l l (46) examined t h i n l aye r chromatographic systems f o r primidone and 20 o the r drugs. from plasma with chloroform after acidifying with hydrochloric acid.

The drugs were extracted

The chloroform ex t r ac t w a s d r i ed and

RAYMOND D. DALEY

evaporated. on s i l i c a g e l plates. ten detect ion systems were tested. detect ion system f o r primidone among those t e s t e d was exposure t o iodine vapor, followed by spraying with a one percent aqueous so lu t ion of mercurous n i t r a t e ; t h i s detected 5 pg of primidone. was not tested.) solvent systems t e s t e d were as follows:

The residue was dissolved and chromatographed Nine d i f f e r e n t solvent systems and

The most s e n s i t i v e

(Sulfur ic acid charring The Rf values f o r primidone i n the

1.

2.

3.

4. 5. 6. 7.

8. 9.

Solvent

Methanol/O.P8 ammonia (38.5:1.5) (60 min equ i l ib ra t ion ) Di-n-butyl ether/l-butanol/acetic acid (80:40: 10) Benzene/dioxane/O. 88 ammonia (60:35: 5 ) Ethanol/acetic acid/water (50:30:20) Methanol/l-butanol (60:40) Chloroform/acetone (9O:lO) Chloroform/acetone (90:lO) 60 min equi l ibrat ion, f i l t e r paper l i n i n g tank Benzene/acetic ac id (9O:lO) Benzene/dioxane/O. 88 ammonia (75:20: 5)

Pippenger, Scott , and Gil len described (47) a rapid semi-quantitative method f o r determining primidone and seve ra l other drugs i n plasma and urine. The drugs were extracted from a c i d i f i e d samples with chloroform. dissolved i n a small amount of ethanol. spotted on th ree d i f f e r e n t s i l i c a g e l p l a t e s , along with standards. The p l a t e s were developed i n th ree d i f f e r e n t solvent systems and examined under short wavelength u l t r a v i o l e t illumination. The lower limit of detect ion f o r primidone was 2 CLg. The solvents and Rf values f o r primidone were as follows:

The ex t r ac t was evaporated and the residue Aliquots were

Solvent

Chloroform/acetone (9: l ) Carbon tetrachloride/acetone ( 7 : 3 ) Benzene/acetone (4: 1)

19 . 26 . 11 430

PRIMIDONE

Yamamoto, Suzuki, and Okuo report (48) a s e n s i t i v i t y of one Crg primidone by chromatographing on s i l i c a g e l with chloroform/acetone (9:l). was 0.06 and de tec t ion was with iodine vapor and one percent mercurous ni t ra te .

The Rf value

Garrettson and Dayton (49) described a t h i n l aye r chromatographic method i n which the spots were scraped off the p l a t e , t h e drug extracted with 20 percent a lcohol i n e ther , t h e ex t r ac t evaporated, and t h e drug determined ca lo r ime t r i ca l ly by the method of D i l l e t a1 (50). (See method of Huisman above, i n t h i s Section.) Their method is about 10 times as s e n s i t i v e as t h a t of Huisman.

6.23 Paper Chromatography

Rusiecki, Henneberg, and Ostrowska (25)

The drugs were extracted with reported a paper chromatographic separat ion of primidor:e and phenobarbital. chloroform from a c i d i f i e d blood, urine, o r homogenized organs. buffer and evaporated t o dryness. The residue was dissolved i n methanol and chromatographed on Whatman No. 1 paper impregnated with formamide. With ascending chromatography using t h e solvent system isopropyl alcohol/chloroform/25$ ammonia (45:45:10), t h e Rf f o r primidone was 0.97-0.98, w h i l e t h a t f o r phenobarbital was 0.67. and eosin i n 95 percent ethanol. amount of primidone was 50 pg.

The e x t r a c t s were washed with pH 7.4 phosphate

Detection was by spraying w i t h mercuric chloride The minimum detectable

6.3 Absorptiometric Methods

6.31 Ultraviolet Absorption

The u l t r a v i o l e t absorption i n t h e 250 run region i s t h e bas i s f o r a number of methods f o r deter- mn ing primidone (1,4,17,18,39). The IJSP XVIII method (17) u s e s a 254 t o 261 nm baseline i n measuring the 257 nm peak absorbance; t h i s w i l l reduce in t e r f e rence from some u l t r a v i o l e t absorbing materials, but o the r s w i l l have an absorption pa t t e rn s imi l a r t o t h a t of primidone. ‘ \ .e method is the re fo re not spec i f i c enough fo r marly

RAYMOND D. DALEY

purposes. Furthermore, t h e abso rp t iv i ty is not high, and t h i s increases the probabi l i ty of interference i f more than small amounts of u l t r a v i o l e t absorbing materials a r e present.

Wysokowski and Rewerski (51) reported determination of primidone i n blood by measuring t h e absorbance of e x t r a c t s at 225 nm, but they a l s o suggested t h a t metabolites might interfere .

6.32 Ultraviolet Determination as Phenobarbital

Bush and Helman (16) determined primidone i n urine and blood as phenobarbital, a f t e r oxidation of the former w i t h dichromate. Primidone and any pheno- b a r b i t a l i n the samples were extracted with ether. The e the r e x t r a c t s were extracted with 0.1 ,M ethanolamine t o remove phenobarbital, and then evaporated t o dryness. The residue was t r e a t e d with 0.042 ,M potassium dichromate i n 9.5 ,M s u l f u r i c acid t o oxidize primidone t o pheno- barbi ta l . The phenobarbital formed was extracted i n t o ether, and the e the r washed with pH 6.2 buffer, 0.1 hydrochloric acid, and water. Finally, the phenobarbital formed was extracted i n t o 0.1 g ethanolamine and determined from its u l t r a v i o l e t absorption at pH 9.3, 7.3, and 5.3. s eve ra l solvent systems were a l s o determined.

P a r t i t i o n coe f f i c i en t s f o r primidone i n

6.33 Colorimetric Methods

Early assays f o r primidone i n blood and t i s s u e were done color imetr ical ly (52). The drug was extracted from the sample at pH 5.5 i n t o a mixture of methyl a c e t a t e and chloroform. The ex t r ac t was washed with pH 11.8 buffer and evaporated. t r ea t ed by a method similar t o t h a t of D i l l et a1 (SO) f o r diphenylhydantoin. The metabolite phenylethylmalondiamide i n t e r f e r e s , but can be removed a f t e r conversion with n i t rous acid t o a-phenyl-n-butyric acid, which i s alkali-soluble (52).

The residue was

Primidone can be hydrolyzed i n 60 t o 80 percent s u l f u r i c acid, yielding formaldehyde (12,52) which can be determined with chromotropic acid. The

432

PR I MI DONE

Br i t i sh Pharmacopoeia i d e n t i f i c a t i o n test f o r primidone i s the appearance of a pinkish-blue color on heating with chromotropic acid solut ion (53).

6.4 Polarography

Bozsai and Vastagh (54) determined primidone by polarography a f t e r n i t r a t i n g i t w i t h a su l fu r i c -n i t r i c acid mixture. The phenyl group nitrates t o form the 3'-nitrophenyl der ivat ive (a) , which i s reducible at the dropping mercury electrode. t a b l e t s containing primidone, as w e l l as t o the raw material. vo l t VS. the saturated calomel electrode.

The method was applied t o

The observed half-wave po ten t i a l was -0.17

6.5 T i t r a t i o n

Kalinowska e t a1 ( 5 5 ) t i t r a t e d primidone, i n an acetone-water solut ion containing sodium hydroxide, with s i l v e r n i t r a t e . The endpoint was a pe r s i s t en t t u rb id i ty , caused by p rec ip i t a t ion of t he d i - s i l ve r salt of t h e drug ( the mono-silver salt was soluble).

6.6 O t h e r Analytical Tests

Barysheva ( 5 6 ) found t h a t primidone showed a blue luminescence i n the u l t r av io l e t . Cooper (57) lists t h e colors formed by many drugs with p-dimethylaminobenzaldehyde; primidone forms a primrose color. Ammonia is evolved when primidone i s heated with anhydrous sodium carbonate (17). rapid color test f o r detect ing small amounts of pheno- b a r b i t a l or 5-phenyl-5-ethylthiobarbiturhc acid i n primidone. Kassau (59), Kuhnert-Brandstatter e t a1 ( 4 ) , and Penprase and Biles (7) describe microscopic t e s t s f o r primidone. Shamotienko (60) developed a color t e s t f o r primidone, i n which the same is boiled with chloramine, copper s u l f a t e , and sodium hydrofide; primidone causes a reddish-violet color t o develop. Lobanov and Gorbunova (61) determined primidone in t e r f e romet r i ca l ly i n t a b l e t s and powders.

Haug and Panescu (5s) describe a

433

RAYMOND D. DALEY

7 . References

1. 2.

3.

4. 5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17

18 . 19.

20.

Drug Standards 2, 101-2 (1957). A. L. Hayden, 0. R. Sammul, G. B. Selzer, and J. Carol, J. Ass. Offic. Anal. Chem. u, 797-900 ( 1962 G. R. Bedford, Imperial Chemical Industr ies Limited, personal gommunication. M. Kuhnert-Brandstatter, A. Kofler, R. Hoffman, and H.-C. R h i , Sci. Pharm. 2, 205-30 (1965). R. A. Locock and R. T. Coutts, Org. Mass Spectrom. 2, 735-45 (1970). B. R. Brewster, Imperial Chemical Industr ies Limited, personal communication. W. G. Penprase and J. A. Biles, J. Am. Pharm. ASSOC. 523-8 (195E). J. Roy, M. Gadret, and C. Bregere, Bull. SOC. Pharm. Bordeaux 103, 3-8 (1964). W. R. &on, H. C. Carrington, and C. H. Vasey, U. S. Patent 2,576,279 (1951); C.A. 4.6, 616I.h. W. R. Boon, H. C. Carrington, and C. H. Vasey, Patent 2,578,847 (1951); C. A. 46, 61621. C. H. Vasey, U. S. Patent 2,676,176 (1954);

W. R. Boon, H. C. Carrington, N. Greenhalgh, and C. H. Vasey, J. Chem. SOC. m, 3263-72. E. London and C. H. Vasey, Brit. Patent 710,269

W. R. Boon, N. Greenhalgh, E. London, and C. H. Vasey, B r i t . Patent 709, 808 (1954); C.A. 49, 11027 c . W. P. Seebode and C.H. Vasey, B r i t . Patent 919,703 (1963); C.A. 2, 7540a. M. T. Bush and E. Helnan, Life Sci. &, 403-9 ( 1965 Primidone monographs, United S ta tes Pharmacopeia, 18th revision, Mack Print ing Co. , Easton, Pa.,

V. I. Nekrasov, Aptech. Delo 12 (3), 37-41 (1966); C.A. &, 8676a. V. I. Nekrasov, Tr. 1 [Pervogol Mosk. Med. Inst . - 61, 257-9 (1968); C.A. 71, 94823m. J. Wysokowski and W. Rewerski, Diss. Pharm. Pharmacol. 20 (4), 369-72 (1968).

C.A. 19, 11027g.

(1954); C.A. 50, 2687h.

1970, pp. 538-40.

434

PRIM1 DONE

21.

22.

23.

24.

2 5.

37.

38. 39.

G. Knoll and L. Trampau, Ger. (East) Patent 43, 699 (1966); C.A. a, 19638g. C. H. Vasey, B r i t . Patent 710,267 (1954); C. A. I!&, l4816f. J. M. Thorp, Congr. Int. Biochirn. Resumes Commun. 3rd (Brussels) m, 132; C.A. 50, l4127a. J. M. Fuj imto , W. H. Mason, and M. Murphy, J. Pharmacol. Exp. Ther. C. A. &, 48068j. W. Rusiecki, M. Henneberg, and B. Ostrowska, Diss. Pharm. Pharmacol. 18 (3) , 299-304 (1966); C.A. 66, 9578~. J. W. Huisman, Pharm. Weekbl. (27), 799-802 (1969). G. L. Plaa, J. M. Fujimoto, and C. H. Hine. J. Am. Med. ASS. 168, 1769-70 (1958). J. Bogan and H. Smith, J. Pharm. Pharmacol. 20 (11, 66-7 (1968). H.-H. Frey and I. Hahn, Arch. Int . Pharmacodyn. Ther. 128, 281-9 (1960); C.A. f?zI l0693d. J. W. Huisman, Pharm. Weekbl. (20), 573-600 (1968); C.A. 9, 427362. T. C. Butler and W. J. Waddell, Proc. SOC. Fkp. Biol. Med. 2, 544-6 (1956). C. E. Pippenger and H. W. Gi l len , Clin. Chem. 15 (71, 582-90 (1969). M. A. Ehrenson, P. Jones, and B. Darcey, Clin. Chem. 16 (2) , 107-10 (1970). W. FGrst, H. Lauterbach, K.H. Daute, and E, Klust, Zentralbl. Pharm. loq, (9) , 907-12 (1970). H. J. Kupferberg, Clin. Chim. Acta 3, 2E3-8

C. Gardner-Thorpe, M. J. Parsonage, P. F. Smethurst, and C. Toothi l l , Clin. Chim. Acta 36, 223-30 (1972 ) H. F. Proelss and H. J. Lohmann, Clin. Chem. us 222-8 (1971) H. J. Kupferberg, J. Pharm. Sci. 61, 284-6 (1972). E. C. C. Clarke, Ed., T s o l a t i o n and Ident i f ica- ion of Drugs", The Pharmaceutical Press, London,

(2), 379-88 (1968);

(1970).

1969.

RAYMOND D. DALEY

40.

41.

42. 43.

44.

45. 46.

47.

48.

49.

50.

51.

52

53 . 54.

55.

56.

57. 58.

59. 60.

61.

62.

Toseland J. G ve d D. J. Berry, Clin, Acta 2, '321-6~19?2~ Van Meter, H. S. Buckmaster, and Shelley, Clin. Chem. 16, 135-8 (1970). Van Meter, Clin. Chem. x, 460 (1971).

D, Sampaon, I. Harasymiv, Bnd W. J. Hensley, Clin.

B. B. Gallagher and I. P. Baumel, i n "Antiepileptic Druge", D. M. Woodbury, J. K. Penry, and R. P. Schmidt, Fds., Raven Press , New York, 1972, pp. 353-6. J. W. Huisman, Clin. Chim. Acta lJ, 323-8 (1966). C. Gardner-Thorpc, M. J. Parsonage, and C. Toothi l l , CUn. Chim. Acta 22, 39-47 (1971). C. E. Pippenger, J. E. Scot t , and H. W. Gillen, Clin. Chem. 2, 255-60 (1969). J. Yamamoto, H. Suzuki, and S. Okuo, Yakuzaigaku 3 (4), 23-6 (1965); L A . Q, 18428d. L. K. Garretson and P. G. Dayton, CUn. Pharmacol. Ther. 11, 674-9 (1970). W. A. D i l l , A. Kaeenko, L. Wolf, and A. J. Clazko, J. Pharmacol. Exp. Ther. 118, 270-9 (1956). J. Wysokawski and W. Rewerski, Dies. Pharm. PharmacOl. 20, 369-72 (1968); C.A. 70, 1031%. A. Spinks and W. S. Waring, Progr. Med. Chem. 2, 286 (1963).

ch-0 & 382-5 (1971).

Bri t i sh Pharmacopeia 1968, The Pharmaceutical Press, London, p. 807. G. Bozsai and G. Vastagh, Pharm. Zentralhalle 103,

Z. E. Kalinowska, L. Podkowska, and J. Mieszczakowska, Farm. Polaka 14, 329-31 (1963) ;

A. I. Barysheva, Aptech. Delo 2 (4), 78-80

P. Cooper, Pharm. J. lJJ, 495-6 (1956). U. Haug and I. Panescu, P h a m z i e l8, 340-1

E. Kasaau, Deut. Apoth. Ztg. 10 , 613-6 (1964). G. D. Shamotienko, Farm. Zh. 74 Kiev) 22 (4), 53-4 (1968); C.A. 70, l4468r. V. I. Lobanov and L. F. Gorbunova, Farm. Zh. (Kiev) 26 (31, 38-41 (1971). H. V. S t ree t , J. Chromatog. Q, 358-66 (1969).

403-8 (1964); C.A. 6l, 10537h.

C.A. 60, 10478g.

(1964); C.A. &, 15157go

( 1963

436

PRIM1 DONE

ACKNOWLEM;MENTS

The writer wishes to thank Dr. B. T. Kho of Ayerst Laboratories, Inc., for his suggestions f o r improvement of the manuscript, and Dr. G. R. Bedford and Dr. B. R. Brewster of Imperial Chemical Industries Limited for their mass spectral and nuclear magnetic resonance information.

PROPIOMAZINE HYDROCHLORIDE

Kathleen B. Cvomhie mid Leo I=. Culler1

43Y

K. B. CROMBIE AND L. F. CULLEN

C0NTE;NTS

1. Description 1.1 N a m e , Formula, Molecular Weight 1.2 Appearance, Color, Odor

2 .1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectra 2.3 Ultraviolet Spectra 2.4 Mass Spectra 2.5 Melting Range 2.6 Mff e r e n t i a l Thermal Analysis 2.7 Solubi l i ty 2.8 Crystal Properties 2.9 Ionizat ion Constant, pKa

2 . Physical Properties

3. Synthesis 4. S t a b i l i t y - Degradation 5. Drug Metabolic Froducts 6. Ident i f ica t ion 7. Methods of Analysis

7.1 Elemental Analysis 7.2 Ultraviolet Spectrophotometric Analysis 7.3 Colorimetric Analysis

7.31 Palladium Chloride 7.32 p-Benzoquinone

7.h Polarographic Analysis 7 .5 Ti t r imetr ic Analysis

7.51 Sodium Lauryl Sulfate 7.52 Ceric Sulfate 7.53 Perchloric 4cid

7.6 Chromatographic Analysis 7.61 Thin Layer Chromatography 7.62 Paper Chromatopaphy 7 -63 Gas Chromatography

8. References

440


1. Descript ion - 1.1 Name, Formula, Molecular Weight

Propiomazine hydrochlor ide i s des igna ted by t h e fol lowing chemical names : :lo-( 2-dimethyl&nopropyl) - 2 -pr opionylp hen o th i az i ne h d r oc h l o r i d e l , and propi onyl-

Chemical Abs t rac ts , the compound i s l i s t e d under t h e heading: 1-[lo-12-( dimethylamino) propyl ] phenothiazin- 2-yl]-1-propanone monohydrochloride. The most commonly used t r a d e name i s Largon Hydrochloride. The empi r i ca l formula is C20H2~N20S.ACl with a molecular weight of

promethazine hydrochloride 3; . In the s u b j e c t i n d i c e s of

3-76 a 9 5 0 CH3

CH2-kHN (CH, ) z I

1.2 Amearance. Color. Odor

Propiomazine h drochlor ide i s a yellow, prac- t i c a l l y odor less powder if . 2 . Phys ica l P rope r t i e s

2.1 I n f r a r e d Spec t ra The i n f r a r e d spectrum of propiomazine hydrochlor-

ide ( N .F, Reference Standard m a t e r i a l ) , presented i n Fig- u re 1, has been run i n a KBr p e l l e t . t i o n band assignments have bee Wavelenpth of Plbsorption (cm .-')

The fa l lowing absorp- made.3

Vibrat ion Mode

2580 and 21150 NH' s t r e t c h i n g 2920 CH s t r e t c h i n g

1690 C = O s t r e k h i n g (aryl

15'90 ketone )

aromatic C=C in-plane v i b r a t i o n

continued . . . . 44 1

P P N

F i g u r e 1. I. R. Spec t rum of Propiomazine H y d r o c h l o r i d e (N. F. R e f e r e n c e S t a n d a r d M a t e r i a l ) - 1% KBr I n s t r u m e n t : P e r k i n E l m e r Model 2 1

0 9 0 H E rn D 2 0

r rn z


2.1 In f r a red Spectra (cont inued) Wavelength of Absorption (cm.-l) Wbra t ion Mode

l460 C-CH, deformation -CH,- deformation N-CH, deformation aromatic #J s t z e t c h i n p C-S s t r e t c h i n g aromatic CH out-of-

1325 1055

890 and 867 plane deformation ( 1 , 2 , h - t r i subs t i t v + e d benzene r ing )

plane deformation ( or tho -d i subs t i t u t ed benzene r i n g )

752 aromatLc Ch out-of-

2.2 Nuclear Magnetic Resonance Spec t ra The nuclear magnet.ic resonance (NMR) spectrum

(Figure 2 ) was obtained by prepar ing a sa tu ra t ed s o l u t i o n of propiomazine hydrochlor ide ( N .F. Reference Standard ma te r i a l ) i n deuterochloroform containing te t ramethyl - s i l a n e as the i n t e r n a l s tandard . The only exchangeab1.e proton is t h e hydrogen a s soc ia t ed w i t h HC1. The f o low- i n g NMR proton s p e c t r a l assignments have been made. ; Chemical S h i f t ( ppm. ) Proton

c) 1.20 I! - CH2-% t r i p l e t 1 6 7 CH-C& doublet 2.91 N-( CH, )a s i n g l e t

3.10 I-a&CH3 qu ar t.e t, 3.50 t o 4.40 N-CP, -CH --- 4.97 N-CHZ-CH ---

7.42 and 7.75 aroma t,Z -- - 2.3 U l t r a v i o l e t Spec t ra

Tompsett reportedAmax. at. 242 m u and 273 m u f o r propiomazine hydrochlor ide i n 1 N hydrochlor ic acid.5 Propiomazine hydrochlor ide (N.F. Seference Standard ma te r i a l ) when scanned between 350 mri '2nd 220 pi exhilli t ed hmax. a t 242 mu ( € 4 7 . 5 ) and 273 mu ( E - 4 5 . 2 ) . This spectrum is shown i n Figure 3.

443

P P P

F i g u r e 2 . NMR Spect rum of Propiomazine H y d r o c h l o r i d e (N. F. R e f e r e n c e S t a n d a r d M a t e r i a l ) S o l v e n t : d e u t e r o c h l o r o f o r m , i n t e r n a l s t a n d a r d : t e t r a m e t h y l s i l a n e I n s t r u m e n t : Jeolco Model C - 6 0 HL

0 a 0 z rn

I < D a 0 0 I r

Figure 3. UV Spectrum of Propiomazine Hydrochloride (N. F. Reference Standard Material) Solvent: 1N hydrochloric acid Instrument: Cary Model 14

K. 6. CROMBIE AND L. F. CULLEN

2.4 Mass ,Spectrum The mass spectrum of propiomazine hydrochloride

(N .F. Reference Standard material) was obtained by d i r e c t inser t ion of the

and the ionizing electron beam energy was 60 eV. resolut ion data was compiled and tabulated with the a id of an on-line m)P-8 Dig i ta l Computer.

and the high resolut ion mass spectrum assignments of t h e prominent ions a re given i n Table I.

ion of t h e free base a t m/e 340.1622. The first prominent fragment i s a t mass 269.0854 which corresponds t o the loss

N, supported by the fragment a t 71.0736. This metast ckHS b e ion y ie lds a protonated frag- of the metastable ion ,

ment a t mass 72.0806 which i s the most abundant peak i n the spectrum. Documented f i s s i o n from the molecular ion of the side-chain C-C b o n d 4 t o the phenothiazine r ing nitrogen generates the metastzble 10-methylene-phenothiazinyl ion a t m/e 268.0807 which loses the s u l f u r group t o form t h e m

the charged ion 2-propionyl-phenothiazin-10-yl r e s u l t i n g from the cleavage of the e n t i r e s ide chain attached to the 10-position r ing nit.rogen.

In the p a r t i a l fragmentation of the 2-substitu- en t , an ion of mass 225.0261 with the formula CUH~ONS is observed which corresponds t o the loss of C2Hs from the fragment a t 25J-1.0627. Cleavage of the e n t i r e 2-substitu- en t from 2-propionyl-phenothiazin-10-yl y i e l d s a charged

ample i n t o an Ms-902 double focusing mass spectrometer 3 . The ion source temperature was 20OOC.

The high

Results a re presented a s a bar graph (Figure 4 )

Propiomazine hydrochloride gives a molecular

C OC H, C H3 \

ion of m/e 236.1107 F, . A peak a t 254.0627 is indica t ive of

a t d e 197.0284.

446

F i g u r e 4 . Mass Spec t rum of Propiomazine H y d r o c h l o r i d e (N.F. R e f e r e n c e S t a n d a r d M a t e r i a l ) I n s t r u m e n t : AEI-Model MS 902


TAELE I

High Resolution Mass Spectrum Assignments of Propiomazine HC1

Measured Mass

340.1622

281 .0891

269.0854

268.0807

25’4.062 7

236 ~ 1 0 7

225.0261

19 7.0284

72.0806

7 1 -0736

Calculated Mass

340 .1608

281.0873

269.0874

268.0796

254.0639

236 .ii@

225.0247

197.0298

72.0813

71 00734

448


2.5 Melting Range The fol lowing melt ing p o i n t temperatures ( "C.)

have been obtained on propiomazine hydrochloride ( N .F. Reference Standard m a t e r i a l ) employlng t h e U .S:P .,6VIII, Class 1, conditions:9 201-201, with decomposition .

2.6 D i f f e r e n t i a l Thermal Analysis The d i f f e r e n t i a l thermal a n a l y s i s (DTA) curve of

propiomazine hydrochloride (N .F. Reference Standard mater- i a l ) run from room temperature t o t h e melt ing po in t ex- h i b i t s no endothems o r exotherms o the r than an endotherm a t 199" - 203OC. assoc ia t ed with t h e m e l t . 1 ° Since the compound melts with decomposition, t h i s endotherm i s no t wel l def ined , The DTA curve was run on a Dupont 900 DTA us ing a micro c e l l and a hea t ing r a t e of 5"C./rnin.

2,7 S o l u b i l 9 The fol lowing approximate s o l u b i l i t y da t a have

been determi ed f o r propiomazine hydrochloride a t room temperature : ,11

water: greater than 1 g./ml. i s o t o n i c sodium ch lo r ide so lu t ion :

95% ethanol : 100 mg./ml. chloroform: 400 mg ./ml. acetone: 10 mg./ml. benzene: less than 0.1 mg./ml. ether: less than 0.1 mg./ml.

2.8 Crystal P rope r t i e s

p rea t e r than lg ./ m l .

The X-ray powder d i f f r a c t i o n p a t t e r n of propiomazine hydrochloride ( N .F. Reference Standard m a t e r i a l ) obtained with a P h i l i p s d i f f rac tometer using Cu K d . r ad i - a t i o n l o i s shown i n F igure 5 . f o r t he d i f f r a c t i o n p a t t e r n shown i n Figure 5 are given i n Table 11.

10 The calcl i la ted d-spacings

449

P !A 0

1 w

yo

W

W

70

60

so I

40

so

90

a0

70

loo

Pmrrn No.: 236 m: 9/I5/70 Wr.: N.D.A

Sarapk: P m p i a n u i n HCI

Sourer: N.F. Ralerence Sbmrd Molerial

VO

ma TUD~ tw; cu volt: pow MA 24

Sun 4 W : 1Ymin. C.P.S.: Xt@

c u d crpw Mono.: Yes @in: 1211

P e r m i Sum.: 0

DMcta: Prap. K.V.: 1.6 70

Time Const.: 2

w

so

40

ao

0 0 D a

F i g u r e 5. X-Ray D i f f r a c t i o n P a t t e r n o f P r o p i o m a z i n e H y d r o c h l o r i d e (N.F. R e f e r e n c e S t a n d a r d M a t e r i a l ) R a d i a t i o n : C u K a I n s t r u m e n t : Nore lco P h i l i p s D i f f r a c t o m e t e r

0 n 0 z F rn P z O r n


TABLE I1

ttdtt Spacings for Propiomazine l-@drochloride

- 28 - d ( G

6 ..5' 13.60

13.1 6.76 14 07 6.02 15.0 5 .91 18 ..5 4.79" 19 -6.5 4.52* 2 1 .o 4.23* 22.5 3.959 23.05 3.86 23.7 3 *75 24.25 3 -67 24.6 3.62 26.3 3.39 27-25; 3 2 7 28 .S 3 -13

9 08 9 -03

29 .O 3 -08 30.2 2.96

32 *25 2.78 3L .6 2 059 3.5.8 2.51 36.3 2 047

30.7 2 .91

*Most intense peaks

d=( interplanar dis tance) 2 s i n 6 n h

45 1


2.9 Ion iza t ion Constant, pKa (Apparent) The PKa f o r propiomazine hydrochloride has been

determined po ten t iome t r i ca l ly togbe- 6.6 by aqueous t i t ra - t i o n wi th 0.W sodium hydroxide.

3 . Synthesis

prepa a t i o n of propiomazine hydrochloride. Farbenfabriken Bayerr2 prepared propiomazine by r e a c t i n g 2-dimethylaminopropyl ch lo r ide with 2-prapionylphenothiazine i n t h e presence of sodium amide as the condensing agent , and subsequent ly convert ing t h e base form t o t h e hydrochloride (See Figure 6 )

sements Clin-RylaB which i s based on t h e decarboxylat ion by heat ing of t h e aminoalkyl ester of t h e N-carboxy- phenothiazine, S p e c i f i c a l l y , 2-propionylphenothiazine i s converted i n t o i ts N-carbonyl ch lor ide de r iva t ive by reac- t i n g wi th phosgene. dimethylamino-1-propanol y i e l d s t h e ester hydrochlor ide, which is decarboxylated by hea t ing (See Figure 7) .

Two bas i c syn the t i c r o u t e s have been repor ted f o r t h e

An al te ate syn thes i s was developed by E tab l i s -

Reaction of t h e ch lo r ide wi th 2-

k . S t a b i l i t y - Degradation

s o l i d state14 and i n buf fered aqueous s o l u t i o n s exposed t o long term acce le ra t ed thermal condi t ions which are pro- t e c t e d from W l i g h t and contain a s i t a b l e an t iox idan t system t o exclude d isso lved oxyizen.l3 It i s common for t h e photocatalyzed oxida t ive decomposition of s t r u c t u r a l l y r e l a t e d phenothiazine d e r i v a t i v fg-58 proceed by a semiquinone free r a d i c a l mechanism. J. Kofoed et.a1?]-,*2 oxidized propiomazine t o t h e corresponding .$oxide or s u l - foxide by r e a c t i n g the compound wi th hydrogen peroxide a t 60°C. Ekposure of aqueous s o l u t i o n s of propiomazine hydrochlor ide t o more severe thermal condi t ions has r e s u l t e d i n t h e cleavage of t h e compound t o form 2-propi n y l pheno- t h i a z i n e as an oxida t ive decomposition product .'3 A s i m - i l a r cleavage r eac t ion was observed under acce lera ted thermal condi t ions f o r aqueous so lu t ions of a somewhat s t r u c t u r a l l y similar phenothiazine d e r i v a t i v e , prometha- z ine hydrochloride, r e s u l t i n g i n t h e f methylphenothiazine and phenothiazine .

Propiomazine hydrochlor ide i s very s t a b l e i n both t h e

Of lo-

452

PROPlOMAZlNE HYDROCHLORIDE

H~C- CH-CH, CI (1) condense wi th NaNH ( 2 ) converted t o H C 1 '

Fig. 6 -- l a r b e n f a b r i k e n Bayer Synthesis of Propiomazine H C 1

453

K. 6

. CR

OM

EIE

AN

D L

. F. C

UL

LE

N

&

D? Y u I +

i? I 8

Y 0 --ri

a

(d

s F 0 P

0

0) a

f;r (1

rl u

X

N

B 0 k 0

rl V

454


5 . Drug Metabolic Products

r a t i o n , Robinson26 charac te r ized t h e predominant b io t r ans - formation product as the aromatic r i n g hydroxylated metabo- l i t e . The metabolic product r e s u l t i n g from dea lky la t ion of the 10-d ia lkylminoalkyl s u b s t i t u e n t , which i s a characteristic metabol i te of most b i o l o g i c a l l y active phenoth i az ine de r iva t ives , was n o t de tec ted i n t h i s s tudy.

6. I d e n t i f i c a t i o n hopiomazine hydrochloride can be i d e n t i f i e d by vir-

t u e of i t s c h a r a c t e r i s t i c I R , UV, and X-ray s p e c t r a (See 2.1, 2.3, and 2.8). on the formation of t h e maleate sa l t of propiomazine and measurement of t h e c h a r a c t e r i s t i c mel t ing po in t (157' - 165°C.) of t h e maleate sa l t form. t a l l i n e de r iva t ive27 and co lo r i d e n t i f i c a t i o n t e s t s 2 8 have been repor ted t o d e t e c t and d i f f e r e n t i a t e microgram q u a n t i t i e s of propiomazine from o t h e r s t r u c t u r a l l y r e l a t e d phenothiazine de r iva t ives .

On incubat ion of propiomazine wi th a r a t l i v e r prepa-

The N.F. XIII' descr ibes a tes t based

A series of microcrys-

7. Methods of Analysis ? .1 Elemental Analysis*

Element % Theory % Determinedlo

C H N S c1

63 -73 63 059 6.68 6.62 7 -43 7 . 2 1 8 .51 8.27 9 -40 9.48

9 Propiomazine HC1, N.F. Reference Standard material

7.2 U l t r a v i o l e t Spectrophotometric Analysis The u l t r a v i o l e t absorp t ion maximum of prapioma-

zine hydrochloride a t about 240 mu. i p aqueous s o l u t i o n s has been u t i l i z e d f o r a s say purposes . Determination of t h e a b s o r p t i v i t y value i s use fu l f o r a s say of raw material and f o r formulat ions af ter the propiomazine has been i s o - lated. Propiomazine hydrochloride could poss ib ly be sepa- r a t e d from i n t e r f e r i n g formulat ion components by an ex- t r a c t i o n of t h e base form w i t h orcanic so lven t s from an a l k a l i n e aqueous so lu t ion .

455


Tompse t t 5 quan t i t a t ive ly determined p r opiomazine i n b io loe ica l mater ia l by comparing i ts absorbance t o t h a t of reference standard mater ia l i n lN hydrochloric acid a f t e r ex t r ac t ing the propiomazine from an alkal ine aqueous phase with chlorof om.

7.3 Colorimetric Analysis 7.31 Palladium Chloride

Modifications of t he yRlladium-phenothiazine der ivat ive complex procedure of Ryan have been applied t o the quan t i t a t ve analysis of propiomazine hydrochloride successfully. 1~1$ The colorimetric procedure i s based on the react ion of palladium with propiomazine i n an aqueous solut ion buffered a t about pH 3 t o form a colored complex which is spectrophotometrically measured a t 465 mp. t h i s complex formation i s based on an e l ec t ron t r a n s f e r from the s u l f u r moiety t o the palladium ions, the procedure provides a method t o assay propiomazine i n the presence of i t s corresponding sulf oxide oxidative decompo- s i t i o n product.

Since

7.32 p-Benzoquinone A color r eac t ion i s obtained by the act ion

of p-benzoquinone i n a s t rongly acid medium o r propioma- ~ i n e * ~ . measurement of i t s absorption i n the v i s i b l e region. T h i s redox react ion i s based on the formation of t h e ca t ion ic r a d i c a l of propiomazine with p-benzoquinone ac t ing a s an electron acceptor.

A s t ab le colored species results which al-lows

7.4 Polarographic :lnalysis Progiomazine hvdrochloride exh ib i t s an anodic

wave a t +O.Ov i n qcidic a ueous s o l u t i o n a t a concentra-

electrode vs. a caloinel rer'arence electrode, s c i t a b l e f o r a quan t i t a t ive assay30, The wave corresponds t o the oxidat ion of t he su l fu r i n the phenothiazine nucleus t o the s u Lf oxide3I.

t i o n range of lo-' t o '10- 3 1; using a s t a t iona ry platinum

7.5 Titrirnetric Analvsis ~ 7.51 Podium La&l SuJfgte

Pe l l e r in e t . a l . J L d 3 developed a semi- micro, heterogeneous p h a q i d i c aqueous-chlorof orm)


t i t r i m e t r i c procedure f o r propiomazine and r e l a t e d pheno- t h i a z i n e de r iva t ives a t the 0.0smM l e v e l employing 0.01M sodium l a u r y l s u l f a t e as the t i t r a n t and methyl yellow as the i n d i c a t o r . The au thors demonstrate t h a t common tab- l e t exc ip i en t s do no t i n t e r f e r e i n the assay.

7.52 Ceric S u l f a t e A spectro3hotometri.c t i t r a t i o n procedure

employing 0.02N c e r i c s u l f a t e as t h e t i t r a n t , used t o assay a v a r i e t y of phenothiazine d e r i v a t i v e s and t h e i r dosage f o r m , can be appl ied t o t h e q u a n t i t a t i v e ana lys i s of propiomazine hydrochloride3”. I n i t i a l l y , a colored semi-quinone in te rmedia te i s formed which r ep resen t s t he first s t age i n the oxidat ion. Upon l o s s of a second e l e c t r o n , t he s o l u t i o n becomes c o l o r l e s s as a resul t of t he formation of t he su l foxide d e r i v a t i v e of t h e phenoth i az ine . t h i az ine under t h e con i t i o n s o f t h e t i t r a t , i o n a r e descr ibed

excess c e r i c i on remains i n s o l u t i o n and i s charac te r ized by an inc rease i n absorbance, spec t rophotometr ica l ly measured a t 1120 mu, which i s d i r e c t l y p ropor t iona l t o the c e r i c i on concentrat ion. Employing a imilar t i t r i m e t r i c

changes i n o p t i c a l d e n s i t y of t he r eac t ion s o l u t i o n a t 270 mu, t h e wavelenpth of m a x i m u m absorbance f o r t h e in te rmedia te semi-quinone f ree r a d i c a l .

This mechanism f o r t h e oxidat ion of t h e pheno-

by Merkle and Discher3 2 . The endpoint i s ascr ibed when

procedure, Chatten, Locock and Krause 3E measured the

7.53 Pe rch lo r i c Acid The non-aqueous poten t iomet r ic t i t r a t i o n of

m i n e hydrochloride salts, introduced by Pifer-Xoll ish37 and used i n the assay of var ious phenothiazine der iva t ive$ can be appl ied t o the determinat ion o f propiomazine hydroch lor ide . The procedure involves i n i t i a l r e a c t i o n o f t h e m i n e hydrochloride group wi th mercuric a c e t a t e i n an a c e t i c a c i d medium. The ha l ide i s t i e d up as undissoci- a t ed HgCl, and t h e a c e t a t e i on l i b e r a t e d can be t i t r a t e d as a base w i t h pe rch lo r i c ac id . h e r c u r i c a c e t a t e i s es- s e n t i a l l y undissoc ia ted i n a c e t i c ac id and t h e excess , t he re f ore , does not i n t e r f e r e .

45 7


7.6 Chromatographic Analysis Chromatographic met.hods can be used q u a l i t a t i v e l y

f o r i d e n t i f i c a t i o n and q u a n t i t a t i v e l y f o r determinat ion of p u r i t y and s t a b i l i t y of propiomazine hydrochloride.

7.61 Thin Layer Chromatographz The va r ious e luan t and adsorbent systems

used f o r t h i n layer chromatography of propiomazine hydroch lo r ide are given i n Table 111. The v i s u a l i z a t i o n techniques used f o r the de t ec t ion of propiomazine are a l s o included i n Table 111.

7.62 Paper Chromatography Many paper chromatographic me t.hods have

been repor ted i n t h e l i terature t o permit the i s o l a t i o n and de tec t ion of propiomazine hydrochloride. A summary of t he ava i l ab le d a t a i s recorded i n Table TV.

7.63 Gas Chromatography ProDiomazine hvdrochloride has been chroma-

tographed a t an opera t ing t e 6 e r a t u r e of 225'C. on a 2.9% SE-30 Chromosorb W column and a $ XE-60 s i l i c o n e n i t r i l e polymer on Chromosorb W Employing a 3% SEE-30 I l ia toport S column operated a t a temperature of 24OoC., it i s poss ib l e t o sepa ra t e propiomazine hydroch3 or ide &rom i t s degradat ion product , 2-p~f)J~ ny l phenothiazine.

Kofoed, et.al., hromatographed propio- masine, as w e l l a s i t s su l foxide , on a 3% ,SE-30 Gas Chrom Q column a t tem eratures of 210'C. and 25'0'C.

cedure f o r propiomazine and r e l a t e d phenothiazines based on examination of t he c h a r a c t e r i s t i c gas chromatographic p a t t e r n of i t s pyro lys i s products.

Fontan, J a i n , and Kirk3 8 have developed an i d e n t i f i c a t i o n pro-

458


Table I11

Thin Layer ChromatoRraphic Systems f o r Propiomazine HC1

Solvent V i sua l i za t ion System Adsorbent Technique % Reference

A S i l i c a Gel G A, B 0.52 39

B S i l i c a Gel G A* B 0.66 39 ,

39 C S i l i c a Gel G B ---- D S i l i c a Gel G C,D 0.77 5 , 21

with Fluorescent I n d i c a t o r

E S i l i c a Gel E wi th 0 . 1 N NaOH

F S i l i c a G e l E wi th 0 . 1 N NaOH

F S i l i c a Gel E wi th 0 . 1 N KHSO,

G S i l i c a Gel E wi th 0.1N NaOH

H S i l i c a Gel B,C* with 0.1N D, E NaOH o r 0 . 1 N KHSO,

I S i l i c a Gel E wi th 0.1N KHSO,

0.192 LO

0. LO Ll, L2

0.26 141, 112

continued.. . . 459


Table 111 (continued)

Thin Layer ChromatoRraphic Systems for Propiomazine HC1

Solvent Visualization System Adsorbent Technique

J Silica Gel C G with F luo.re sc ent Indicator

K Silica Gel G A,C,F, G,H,I* J

L Silica Gel G A,C,F, with 0.1N G,H$I, N aOH J

M Silica Gel G C with Fluorescent Indic at o r

N Silica Gel C,K, L, M

0 Silica Gel C

P Silica Gel C

Q Silica Gel C

Solvent Systems

A Cyc lohexane : diethylamine ( 9 : 1)

B Methanol: NH, OH (100:1.5)

C Ch1oroform:diethylamine ( 9 : 1)

D O.!! M ammonium acetate in 80% methanol

Reference

h 7

LO

LO, Lh

h 5

26

46

L 6

h6

E Cyclohexane: benzene:diethylamine (75:15:10) continued....

460


Table I11 (continued)

Thin Layer Chromatographic Systems for Propiomazine HC1

Solvent Systems (concluded)

F Methanol

G Acetone

H Methyl acetate

I 95% Ethanol

J pyridine:methanol (50 :50 )

K Benzene:ethanol: NH,OH (95:15:5)

L Ch1oroform:methanol (90:lO)

M Benzene:acetone: NH,OH (37:7:1)

N Chloroform : ethanol: NH, OH (80 : 20: 1)

0 To1uene:dimethylformamide (100: 25)

P Toluene : methanol : dimet hylf ormamide ( h 5 : 30 : 20)

Q Acetone :methanol: triethanolamine (100 : 100: 3)

Visualization Techniques

A Acid Ferric Chloride [FeClJ in 20% HC10,: 50% HN4 ( 1*5: 501

B Potassium Iodoplatinate Reagent

C Ultraviolet Light

D Dragendorff Reagent continued....

46 1


Table I11 (concluded)

Thin Layer Chromatographic Systems for Propiomazine HC1

Visualization Techniques (concluded)

1% I, in methanol

Folin-Ciocalteau Reagent

Madelin Reagent

Cinnamaldehyde

Ehrlich Reagent [p-dimethylaminobenzaldehyde in acetic acid - phosphoric acid

Furf ural

Sulfuric Acid Spray

Hydrobromic Acid

Sodium Metaperiodate

Table IV

Paper Chromatographic Systems for Propiomazine H C 1

Solvent Paper System Paper Treatment

A Whatman No. 1 Buffered i n 5% sodium dihydrogen c i t ra t e

B Whatman No. 1 Impregnated with or No. 3 10% t r i b u t y r i n i n

acetone P 0. W

C Whatman No. 1 Impregnated with o r No. 3 lU% t r i b u t y r i n i n

acetone

Solvent Systems

A 0.02 M C i t r i c Acid i n 87% n-butanol

B Acetate buffer (pH L . 6 )

C Phosphate buffer (pH 7 . L )

Visual izat ion 2 Technique Ref erence

0.77 A*B*C 28, ‘7, h 8

.. I r 0 z 0,

Visual izat ion Technique

A Ul t rav io l e t Light

B Potassium Iodoplat inate Reagent

C Bromocresol Green Spray


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Rahway, N.J.

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11. J. A. Lash, Wyeth LaborRtories, Inc., Personal

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J. Am. Chem. SOc.9 63, 351 (19h1). 20. A. Felmeister and C. Discher, J. Pharm. Sci., 53,

464


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465

K . 8 . CROMBlE AND L. F. CULLEN

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(1951t ).

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466

SULFAMETHOXAZOLE

Briicc C. Rudj, and Bernard Z. Scnkowski

467

8. C. RUDY AND 6. Z. SENKOWSKI

INDEX

Analy t ica l P r o f i l e - Sulfamethoxazole

1. Descr ipt ion 1.1 Name, Formula, Molecular Weight 1 . 2 Appearance, Color, Odor

2 , Phys ica l P r o p e r t i e s 2 . 1 I n f r a r e d Spectrum 2 . 2 Nuclear Magnetic Resonance Spectrum 2 .3 U l t r a v i o l e t Spectrum 2 . 4 Fluorescence Spectrum 2.5 Mass Spectrum 2.6 Opt ica l Rotat ion 2.7 Melting Range 2 .8 Differential Scanning Calor imetry 2 .9 Thermal Gravimetr ic Analysis 2.10 S o l u b i l i t y 2 - 1 1 X-ray Crys t a l P rope r t i e s 2 . 1 2 Dissoc ia t ion Constant

3. Synthes is



6. Methods of Analysis 6 . 1 Elemental Analysis 6 .2 Phase S o l u b i l i t y Analysis 6 . 3 Thin Layer Chromatography 6.4 Direc t Spectrophotometr ic Analysis 6 .5 Color imet r ic Analysis 6 .6 F luo r ime t r i c Analysis 6 .7 T i t r i m e t r i c Analysis

7 . References

468

SULFAMETHOXAZOLE


1.1 Name, Formula, Molecular Weight Su l f ame thoxazo le i s N ' - ( 5 -me thy l -3 - i soxazo ly l )

s u l f a n i 1 ami de .

O H II I

S-Nq- ' 0

I; c1 OH1 1 N 3 0 3 S Molecular Weight : 253.28

1 . 2 Appearance, Co lo r , Odor Su l f ame thoxazo le occur s a s a w h i t e t o s l i g h t l y

o f f - w h i t e , p r a c t i c a l l y o d o r l e s s , c r y s t a l l i n e powder


2 . 1 I n f r a r e d Spectrum The i n f r a r e d spectrum o f r e f e r e n c e s t a n d a r d

su l f ame thoxazo le i s p r e s e n t e d i n F igu re 1 ( 1 ) . The spec t rum was measured w i t h a Perkin-Elmer 621 S p e c t r o - photometer i n a K B r p e l l e t c o n t a i n i n g 0 . 9 mg s u l f a - methoxazole/300 mg o f K B r . Tab le I l i s t s t h e a s s ignmen t s f o r the c h a r a c t e r i s t i c bands i n t h e i n f r a r e d spec t rum ( 1 ) .

TABLE I

I n f r a r e d Ass i gnnient s f o r S u l famet hoxazo 1 e

Frequency (cm-l) C h a r a c t e r i s t i c o f 3469 and 3379 NH, s t r e t c h 3300 NI i s t re tch 1616 Combination NH2 de fo rma t ion and

1595 and 1500 Aromatic C = C s t r e t c h 1313 - 1303 Asymmetric SO2 s t r e t c h 1155 - 114.3 Symmetric SO2 s t r e t c h 828 'I'wo a d j a c e n t 11's on benzene r i n g

i s o x a z o l e r i n g s t r e t c h

469

6. C. RU

DY

AN

D 6. 2. S

EN

KO

WS

KI

470

SULFAMETHOXAZOLE

2 . 2 Nuc lea r Magnetic Resonance Spectrum The spectrum shown i n F igu re 2 was o b t a i n e d on a

J e o l 60 MHz NMR by d i s s o l v i n g 53.6 mg o f r e f e r e n c e s t a n d a r d su l f ame thoxazo le i n 0 . 5 m l o f DMSO-d6 c o n t a i n i n g t e t r a - m e t h y l s i l a n e a s an i n t e r n a l r e f e r e n c e ( 2 ) . The s p e c t r a l ass ignments are given i n Tab le I1 ( 2 ) . I n o r d e r t o e s t a b l i s h t h e chemical s h i f t f o r t h e s i n g l e p r o t o n (H ) on t h e i s o x a z o l e r i n g which appea r s a s a s h o u l d e r on t h e b roade r p r imary amine p r o t o n s peak, i t was n e c e s s a r y t o s h i f t t h e amine p r o t o n s peak t o a n o t h e r p l a c e i n t h e spectrum. Th i s was accomplished by adding 8 ~1 o f D C 1 t o form t h e q u a t e r n a r y s a l t . This caused t h e broad s i n g l e t t o s h i f t t o 5 .62 ppm and i s o l a t e d a s i n g l e t a t 6 .10 ppm a s s i g n a b l e t o t h e s i n g l e p ro ton (Hb) on t h e i s o x a z o l e r i n g (F igu re 2 - I n s e r t ) .

b

TABLE I 1

NMR Assignments f o r Sulfamethoxazole

Chemical S h i f t S t r u c t u r e P ro tons PPm Mu 1 t i p 1 i c i t y

f 11.00 s (b)

S = s h a r p s i n g l e t ; S ( b ) = broad s i n g l e t ; d = d o u b l e t ; J = s p l i t t i n g c o n s t a n t i n Hz

2 . 3 U l t r a v i o l e t Spectrum When t h e UV spectrum o f su l f ame thoxazo le was

scanned between 350 t o 215 nm, one maximum and one minimum were obse rved . The maximum i s l o c a t e d a t 256-257 nm ( ~ = 1 . 7 2 x l o 4 ) and t h e minimum a t 224-225 nm. The spectrum shown i n F igu re 3 was o b t a i n e d from a s o l u t i o n o f 1 .025 mg sulfamethoxazole/100 ml o f pH 7 .5 phosphate b u f f e r ( 3 ) . This spectrum i s t h e same as ones o b t a i n e d u s i n g 0.1N NaOfI as t h e s o l v e n t ( 4 ) .

471

Figure 2

NMR Spectrum o f Sulfamethoxazole

I n s e r t : Spectrum i n Region o f 5 t o 8 ppm Af te r Addit ion o f DC1 t o Sulfamethoxazole

r I

m 0 n C 0

SULFAMETHOXAZOLE

.8

Figure 3

UV Spectrum of S u l famethoxazole

-

-

0

NANOMETERS

473

6 . C. R U D Y AND B. 2. SENKOWSKI

2.4 Fluorescence Spectrum The e x c i t a t i o n and emission s p e c t r a f o r s u l f a -

methoxazole (1 mg/ml of methanol) a r e shown i n Figure 4 (5). One maximum appears in t h e e x c i t a t i o n spectrum a t 314 nm and one maximum i n t h e emission spectrum a t 338 nm.

2 .5 Mass Spectrum The mass spectrum of sulfamethoxazole was ob-

t a i n e d us ing a CEC 21-110 mass spec t rometer with an ion- i z i n g energy of 70 ev. meter was analyzed and presented i n t h e form of a b a r graph, shown i n F igure 5, by a Varian 100 MS dedica ted computer system (6 ) . The peaks a t m/e 92, 108, 140, and

H2N-C6H4-S02, r e spec t ive ly . The peak m/e 189 i s due t o t h e lo s s of SO;! from t h e pa ren t and t h e peak a t m/e 174 i s due t o t h e l o s s o f both SO2 and CH3 from t h e pa ren t . Loss o f SO2 i s a t y p i c a l rearrangement r e a c t i o n wi th sulfonamides. The s t r o n g M+2 peak at 255 may be t h e 34S i so tope peak, however, t h i s peak is not expected t o be so i n t e n s e ( 6 ) .

The output from t h e mass spec t ro -

156 correspond t o H ~ N - C ~ H L , , H2N-CgHq-0, H;!N-CgHq-SO, and

2.6 Opt ica l Rota t ion Sulfamethoxazole e x h i b i t s no o p t i c a l a c t i v i t y .

2.7 Melting Range The melt ing range r epor t ed i n NF XI11 f o r s u l f a -

methoxazole i s 170' t o 173O us ing the c l a s s I procedure (7).

2 .8 D i f f e r e n t i a l Scanning Calor imetry (DSC) The DSC scan f o r sulfamethoxazole i s shown i n

Figure 6. temperature program was 10°/minute. The AHf was found t o be 7.5 kcal/mole. The endotherm observed a t 204OC i s a t t r i b u t e d t o t h e decomposition of t h e sulfamethoxazole (8).

A mel t ing endotherm i s observed a t 172' when t h e

2.9 Thermogravimetric Analysis (TGA) A thermal g rav ime t r i c ana lys i s performed on

sulfamethoxazole exh ib i t ed no l o s s of weight when it was heated t o 105OC a t a ra te o f 10°/min ( 8 ) .

2.10 S o l u b i l i t y The s o l u b i l i t y d a t a f o r sulfamethoxazole obta ined

a t 25OC are given i n Table I11 (9).

474

SULFAMETHOXAZOLE

Figure 4

Fluorescence Spectrum of Sulfamethoxazole

300 400

NANOMETERS

475


a, rl 0 N (d x 0

I I I I 1 I I I I I I I 1 I I I s

01

0 P

c E

0 s

0 c?

8

0 m

3

0 P

a 0 0 0 0 0 0 0 0 0 0 r n m + @ o e m w -

A l I S N 3 1 N I 3 A l l V l 3 U

476

SULFAMETHOXAZOLE

Figure 6

DSC Curve f o r Sulfamethoxazole

I I I AHf = 7.5 kcal/rnole

I I I I I I I I AHf = 7 5 kcal/rnole

I

I I 140 160 180 200

TEMPERATURE O C

477


TABLE I11

S o l u b i l i t y o f Sulfamethoxazole i n Di f f e ren t Solvents

Solvent

3A a lcohol benzene chloroform 95% e thanol e t h y l e t h e r i s o p rop an o 1 me t h ano 1 petroleum e t h e r 30" 0.1N NaOH water

,60"


30.6 0 . 5 2 . 3

37.8 2.7 8 .8

90.3 0 . 2

16.0 0 . 5

2 . 1 1 X-ray Crys t a l P rope r t i e s The x-ray powder d i f f r a c t i o n p a t t e r n of s u l f a -

methoxazole i s presented i n Table IV (10) . The i n s t r u - mental condi t ions a r e given below.

Instrument Condit ions :


Tube t a r g e t : Copper C

Radia t ion : Cu Ka = 1.542 A Opt ics : 0.1" Detector s l i t

Generator : 50KV-12-1/2 MA

M . R . S o l l e r s l i t 3" Beam s l i t 0.0007 inch N i f i l t e r 4" t ake o f f angle scan a t 0.2' 20 p e r minute

8 .7 f i n e Sealed propor t iona l counter tube and DC vo l t age a t p l a t e a u Pulse he ight s e l e c t i o n EL-5 v o l t s Eu - o u t , Rate meter T . C . 4 2000 C/S f u l l s c a l e

Goniometer: Detec tor : Amplif ier gain - 16 coarse ,

Recorder: Chart speed - 1 i n . / 5 min. Samples : Prepared by gr inding a t room temp.

47 8

SULFAMETHOXAZOLE

TABLE IV

X-ray Powder D i f f r a c t i o n P a t t e r n o f Su l f ame thoxazo le

20 ~

6.86 '11.02 12.32 13.74 16.70 1 7 . 0 3 17 .48 18.12 18 .56 19.62 20.68 21.56 22.39 23.28 23.86 24.68 26.20 26.44 27.40 28.76 29.22

0

d(A) *

12 .90 8 . 0 3 7 . 1 8 6.44 5 . 3 1 5 . 2 1 5 . 0 7 4 . 9 0 4 . 7 8 4 .52 4 .29 4 .12 3 .97 3 .82 3 . 7 3 3 .61 3 .40 3 .37 3 .25 3 .10 3 .06

* * 1/10

2 10 43

5 7

1 2 50

4 16 20 43 40 24

4 100

15 5 5

38 17

5

20

30.06 3 2 . 2 2 33.37 34.50 34.92 36.42 36.95 37 .68 38.25 38.80 39.52 40 .71 41.70 43 .32 43.68 45.10 45.80 46.38 49 .00 50 .72

0

d(A) *

2.92 2.78 2 .69 2.60 2.57 2 . 4 7 2 .43 2 .39 2 .35 2.32 2.28 2 . 2 2 2 . 1 7 2 .09 2.07 2 . 0 1 1 . 9 8 1 .96 1.86 1 .80

* * I / p

2 15

5 5 8 1 4 4 1 1 3 1 4 4 3 4 2 2 3 2

nX 2 S i n 0 * d = ( i n t e r p l a n a r d i s t a n c e )

** I l I 0 = r e l a t i v e i n t e n s i t y (based on h i g h e s t i n t e n s i t y o f 1 . 0 0 )

2 . 1 2 D i s s o c i a t i o n Cons tan t The pKa f o r s u l f a m e t h o x a z o l e has been de te rmined

s p e c t r o p h o t o i n e t r i c a l l y t o be 5 . 5 5 + 0 . 0 5 and by t i t r a t i o n o f s u l f a m e t h o x a z o l e i n an e x c e s s 07 0.1N H C 1 t o be 5 . 6 3 + 0 . 0 3 (11). These v a l u e s a g r e e well w i t h t h e pKa v a l u e o f 5 . 6 0 a t 25'C r e p o r t e d by Nakagaki , Toga, and Terada ( 1 2 ) . -

419

B. C. RUDY AND 6. Z . SENKOWSKI

3. Synthesis Sulfamethoxazole i s prepared by the r eac t ion scheme

shown i n Figure 7. with N-acetyl -p-amino-benzene su l fonyl ch lo r ide . The ace ty l group i s then cleaved t o y i e l d sulfamethoxazole (11).

5-methyl-3-aminoisoxazole i s reac ted


and i n water were re f luxed f o r one hour , no decomposition was observed by t h i n - l a y e r chromatography (1 ) . When sulfamethoxazole i s re f luxed i n 0 . 4 N H C 1 , t h e molecule f i rs t cleaves t o y i e l d s u l f a n i l i c a c i d and 5-methyl-3-amino- i soxazole ( 1 , l l ) and on extended hea t ing i n t h e H C 1 s o l u t i o n , 3 a d d i t i o n a l d i azo t i zab le products de t ec t ab le by paper chromatography a r e formed (11) . Pure sulfamethoxazole was found t o be s t a b l e when subjec ted t o a l l O ° C tempera- t u r e f o r 5 days (11) .

When 10% so lu t ions o f sulfamethoxazole i n 0 . 4 N NaOH

5 . Drug Metabolic Products Sulfamethoxazole i s metabolized t o i t s N4-acetyl

d e r i v a t i v e which i s t h e major form found i n human u r ine (13,14). which have not been completely i d e n t i f i e d a r e a l s o present i n l e s s e r q u a n t i t i e s i n human u r ine (13). In human blood t h e sulfamethoxazole e x i s t s almost e n t i r e l y as t h e i n t a c t drug (13) .

The i n t a c t drug along with t h r e e o t h e r metabol i tes


6 . 1 Elemental Analysis The r e s u l t s from the elemental ana lys i s a r e

l i s t e d i n Table V (15) .

TABLE V

Elemental Analysis of Sulfamethoxazole

E 1 ement % Theory % Found

C 47.43 47.28 H 4.38 4.38 N 16.60 16.84 S 12.65 1 2 . 7 4

480

SU

LF

AM

ET

HO

XA

ZO

LE

I

r. a, k

& .d

L

2

rn

Q

0

a, r: L

)

c x rn

x z 1

I

0

O= u*

I +

48 I


6.2 Phase S o l u b i l i t y Analysis Phase s o l u b i l i t y ana lys i s i s c a r r i e d out us ing

isopropanol as t h e so lven t . Figure 8 which a l s o l i s t s t h e condi t ions under which t h e a n a l y s i s was c a r r i e d out (9) .

A t y p i c a l example i s shown i n

6 . 3 Thin Layer Chromatographic Analysis The fol lowing TLC procedure i s u s e f u l f o r

s epa ra t ing sulfamethoxazole from s u l f a n i l i c a c i d and su l fan i lamide (16) . Using s i l i c a ge l G p l a t e s and a so lvent made from a mixture o f a l coho l , n-heptane, ch lo ro - form, and g l a c i a l a c e t i c a c i d (25:25:25:7), spo t 0 . 1 mg o f sulfamethoxazole d i s so lved i n ammoniacol methanol and s u b j e c t t o ascending chromatography. Af te r development o f a t l e a s t 1 2 cm, t h e p l a t e i s a i r d r i e d and sprayed with Modified E h r l i c h ' s Reagent (100 mg p-dimethylaminobenzaldehyde i n 1 m l concent ra ted hydrochlor ic ac id d i l u t e d t o 100 m l wi th 3 A a l c o h o l ) . The approximate R va lues are: f

S u l f a n i l i c Acid 0 .1 Sul f an i 1 ami de 0 .5 Sul fame thoxazo l e 0 .7

A so lven t system which can be used t o s e p a r a t e sulfamethoxazole from i t s N4-acetyl metabol i te i s ch loroform:heptane:alcohol ( 1 : l : l ) . Following t h e same b a s i c procedure a s above t h e Rf value f o r sulfamethoxazole i s 0.75 and f o r t h e N 2 - a c e t y l d e r i v a t i v e 0 .42 (3).

6.4 Direc t Spectrophotometr ic Analysis

methoxazole may be c a r r i e d out us ing 0.1N NaOH as t h e s o l - vent . In t h i s so lven t Beer 's Law holds between 0 .26 ugm/ml t o 26 pgm/ml (1 x M t o 1 x M) (17) .

Direct spectrophotometr ic ana lys i s o f s u l f a -

6 .5 Color imet r ic Analysis The co lo r ime t r i c a n a l y s i s of sulfamethoxazole i s

accomplished by d i a z o t i z a t i o n and coupl ing with N-(1- Naph thy1 e t hylenediamine d i hydrochl o r i d e (Brat ton-Mars ha1 1 Reagent). The r e s u l t a n t r ed c o l o r i s measured a t 540 nm 08)

6.6 F luor imet r ic Analysis Fluorescence a n a l y s i s has been used t o determine

t h e concent ra t ion o f sulfamethoxazole i n blood (19) . For

482

Figure 8

P Tx. w

I / PHASE SOLUBILITY ANALYSIS I SAMPLE SULFAMETHOXAZOLE SOLVENT: ISOPROPANOL

EQUILIBRATION: 20 HRS. at 25" C EXTRAPOLATED SOLUBILITY:

SLOPE: 0.1%

10.8 mg/g of SOLVENT

-. " 0 25 50 75 100


E. C. RUDY AND E. 2. SENKOWSKI

t h i s purpose d e t e c t a b l e l e v e l s i n t h e range o f 1 p g p e r m l o f blood a r e needed. In o rde r t o achieve t h i s s e n s i t i v i t y it i s necessary t o make a f luo rescen t d e r i v a t i v e o f s u l f a - methoxazole. One such d e r i v a t i v e i s the h igh ly f luo rescen t “Dansylsul fonamide” which i s formed by t h e r e a c t i o n shown i n Figure 9 . The f luorescence y i e l d o f t h i s compound is s u f f i c i e n t t o measure about 1 pg o f sulfamethoxazole p e r m l o f blood. An even more s e n s i t i v e d e r i v a t i v e i s formed when sulfamethoxazole i s r eac t ed wi th 4,s-methylene dioxy phthaldehyde (Figure 9 ) . The s e n s i t i v i t y l i m i t f o r t h i s d e r i v a t i v e i s from 1 x l o - * t o 1 x mole/ml ( 2 0 ) .

6 . 7 T i t r i m e t r i c Analysis The po ten t iome t r i c t i t r a t i o n with sodium n i t r i t e

i s t h e method of choice t o assay sulfamethoxazole (16) . The d r i ed sample i s d i s so lved i n g l a c i a l a c e t i c a c i d and water ( 1 : 2 ) . Hydrochlor ic ac id i s added, t h e s o l u t i o n cooled t o 15’C, and immediately t i t r a t e d with 0.1M sodium n i t r i t e . using calomel and plat inum e l e c t r o d e s . sodium n i t r i t e i s equiva len t t o 25.33 mg of s u l f a - methoxazole.

The end po in t i s determined po ten t iome t r i ca l ly Each m l o f 0.1M

sulfamethoxazole can a l s o be t i t r a t e d i n dimethylformamide w i t h 0 .1N L i O C H 3 us ing thymol b lue as t h e i n d i c a t o r ( 1 7 ) . replaced by t h e l i t h ium ion . Each m l o f 0.1N l i t h ium methoxide i s equiva len t t o 25.33 mg of sulfamethoxazole .

The pro ton on t h e sulfonamide func t ion is

484

SU

LF

AM

ET

HO

XA

ZO

LE

a,

0

M

N

a,

m

ka

,

.d

k

a, a

a, 0

F: a, u m a, k

0

3

U

4

I" V

0-m

-0

Q 2 2

Y

Q f +

4x5


8. References

1.

2 .

3.

4 .

5 .

6 .

7. 8 .

9.

10.

11.

1 2 .

13.

1 4 .

15.

16. 1 7 .

18.

19.

20 a

Hawrylyshyn, M. Hoffmann-La Roche I n c . , Personal Communication. Johnson, J . H . , Hoffmann-La Roche Inc . , Personal Communication. Sokoloff , H . K . , Hoffmann-La Roche Inc . , Personal Communication. F ischer , I . , Hoffmann-La Roche Inc . , Personal Communication. Boatman, J . , Hoffmann-La Roche Inc . , Personal Communication. Benz, W . , Hoffmann-La Roche I n c . , Personal Communi c a t ion . National Formulary XIII, 676 and 815, (1970). Donahue, J . , Hoffmann-La Roche I n c . , Personal Communication. MacMullan, E . , Hoffmann-La Roche I n c . , Personal Communication. Hagel, R . B . , Hoffmann-La Roche I n c . , Personal Communication. Schmidli , B . , Hoffmann-La Roche Inc. , Unpublished Data. Nakagaki, M . , Koga, N . , and Terada, H . , J . Pharm. Sac. Japan, - 8 3 , 586 (1963). Koechlin, B . , and Tvron. G . , Hoffmann-La Roche Inc . Unpublished Data, Brandman, O . , and Engelberg, R . , Current Therap. Res., 2 , 364, (1960). Scheid i , F . , Hoffmann-La Roche Inc . , Personal Communication. National FormuZary XIII, 677, (1970). Bandong, L . , Hoffmann-La Roche Inc . , Personal Communcation. Brat ton, A. C . , and Marshal l , F. K . , J . B i o l . Chem. 128, 537 (1939). de S i l v a , J . A . F . , and D'Arconte, L . , J . Forensic. S c i . , - 14 , 184 (1969). Amano, T . , J . Pharm. Soe. Japan, - 85, 1049 (1965).

-

486

SULFISOXAZOLE

Bri1c.e C. RiiL/j* arid Berriurd Z. Scnkowshi

487


INDEX

Analytical Profile - Sulfisoxazole

1. Description

1.1 Name, Formula, Molecular Weight 1 . 2 Appearance, Color, Odor


2 .1 2 . 2 2 .3 2.4 2 .5 2.6 2.7 2 .8 2 .9 2.10 2 . 1 1 2.12

Infrared Spectrum Nuclear Magnetic Resonance Spectrum Ultraviolet Spectrum Fluorescence Spectrum Mass Spectrum Optical Rotation Melting Range Differential Scanning Calorimetry Thermal Gravimetric Analysis S olub il i ty X-ray Crystal Properties Dissociation Constant

3 . Synthesis

4. Stability Degradation

5. Drug Metabolic Products and Pharmacokinetics


6 . 1 Elemental Analysis 6 .2 Phase Solubility Analysis 6 .3 Thin Layer Chromatographic Analysis 6 .4 Direct Spectrophotometric Analysis 6 . 5 Colorimetric Analysis 6.6 Fluorimetric Analysis 6.7 Titrimetric Analysis

7 . References

488

SULFISOXAZOLE


1.1 N a m e , Formula, Molecular Weight S u l f i s o x a z o l e is N~-(3,4-dimethyl-5-isoxazolyl)

s u l f a n i l a m i d e .

O H

Molecular Weight: 267.31 '1 1H13N303S

1 . 2 Appearance, Co lo r , Odor S u l f i s o x a z o l e o c c u r s as a w h i t e t o s l i g h t l y

y e l l o w i s h , o d o r l e s s , c r y s t a l l i n e powder.


2 . 1 I n f r a r e d Spectrum The i n f r a r e d spec t rum of s u l f i s o x a z o l e is p re -

s e n t e d i n F i g u r e 1 (1). The spec t rum w a s measured w i t h a Perkin-Elmer 621 Spec t ropho tomete r i n a K B r p e l l e t con- t a i n i n g 1 .0 mg s u l f i s o x a z o l e / 3 0 0 mg o f K B r . Tab le I l is ts t h e a s s ignmen t s f o r t h e c h a r a c t e r i s t i c bands i n t h e i n f r a - r ed spectrum (1).

Tab le I

I n f r a r e d Assignments f o r S u l f i s o x a z o l e

Frequency (cm-1) C h a r a c t e r i s t i c of

3485 and 3380

1629 1595 and 1503 1343 and 1160

840

Asymmetric and symmetr ic

NH2 de fo rma t ion v i b r a t i o n s Aromatic C=C s t r e t c h Asymmetric and symmetric

Two a d j a c e n t H's on phenyl

NH s t r e t c h

SO2 s t r e t c h

r i n g

489


al rl 0 N rd x 0 m d CCI rl 1 v)

w 0

f4 CI U a, a cn a a, f4 rd k w c H

(u (u-

s- 2-

cu--

0- a--

OD-

b- -

Q--

m--

e--

- m- -

0 %

s 0

0 0 OD

0 Q -

0

8

e E

2

8 s3

0

0 0

0

(u

0

m

0 0 0 *

490

SULFISOXAZOLE

2 .2

J e o l c o 60 0 . 5 m l of

Nuclear Magnetic Resonance Spectrum (NMR) The spec t rum shown i n F i g u r e 2 w a s o b t a i n e d on a MHz NMR by d i s s o l v i n g 60.9 mg o f s u l f i s o x a z o l e i n DMSO-db c o n t a i n i n g t e t r a m e t h y l s i l a n e as a n i n t e r -

n a l r e f e r e n c e . Tab le I1 ( 2 ) .

The s p e c t r a l a s s ignmen t s a re g i v e n i n

Tab le I1

NMR Assignments f o r S u l f i s o x a z o l e

Chemical Type o f P r o t o n s S h i f t Fpm M u l t i p l i c i t y

CH3 on no. 4 i s o x a z o l e carbon 1 . 6 3 Sharp s i n g l e t CH3 on no. 3 i s o x a z o l e ca rbon 2 .05 Sharp s i n g l e t NH2 on benzene r i n g s 6 . 1 Broad s i n g l e t A r o n a t i c p r o t o n s o r t h o t o

Aromatic p r o t o n s o r t h o t o

NH a d j a c e n t t o i s o x a z o l e r i n g '1.10.5 Broad s i n g l e t

amine moiety 6.58 Doublet (J=9Hz)

su l fonamide moiety 7.42 Doublet ( J=9Hz)

2 . 3 U l t r a v i o l e t Spectrum ( U V ) When t h e UV spec t rum of s u l f i s o x a z o l e w a s scanned

from 350 t o 205 nm, one maximum and one minimum were observed. The maximum i s l o c a t e d a t 253 nm ( E = 2 . 1 x l o 4 ) and t h e minimum a t 222 nm. The spec t rum shown i n F i g u r e 3 w a s o b t a i n e d from a s o l u t i o n of 0 .9998 mg s u l f i s o x a z o l e / 100 ml of pH 7 . 5 phospha te b u f f e r ( 3 ) .

2 . 4 F luo rescence Spectrum The e x c i t a t i o n and emiss ion s p e c t r a f o r s u l f i s o x -

a z o l e (1 mg/ml o f m e t h a n o 1 ) a r e shown i n F i g u r e 4 ( 4 ) . One maximum a p p e a r s i n t h e e x c i t a t i o n spec t rum a t 319 nm and one maximum i n t h e emis s ion spec t rum a t 336 nm.

2.5 Mass Spectrum The mass s p e c t r a l d a t a f o r s u l f i s o x a z o l e w a s ob-

t a i n e d u s i n g a CEC 21-110 mass s p e c t r o m e t e r w i t h an i o n i z - i n g energy of 70 ev. An o n - l i n e computer, Var i an Data Systems 100 MS, w a s used t o c a l c u l a t e t h e masses and com- p a r e t h e i n t e n s i t i e s t o t h e b a s e peak. The mass spec t rum o b t a i n e d from t h i s computer a n a l y s i s is shown i n F i g u r e 5

3 Y l

B. C

. RU

DY

AN

D B

. 2. S

EN

KO

WS

KI

4Y2

SULFISOXAZOLE

1.0

.0

.6 w u z

CL: 8 a m a .4

.2

0

Figure 3

UV Spectrum of Sulfisoxazole

r

NANOMETERS

4Y3


494

SULF ISOXAZO LE

I I I I I 1 I I I 0 m N

0 W N

0 * N

0 N N

0

R

0 m

0

\

I 0 f

B

0 0

P

0 W

0

495

€3. C. RUDY AND E. Z . SENKOWSKI

(5 ) . The peaks a t m / e 92, 108, 140, 156, and 172 c o r r e - spond t o H2N-CgH4, H2N-C&-0, HzN-C~H~-SO, H2N-Cf,H4-S02, and H ~ N - C ~ H ~ - S O Z - N H ~ , r e s p e c t i v e l y . The peak m / e 203 i s due t o t h e l o s s of SO2 from t h e p a r e n t and t h e peak a t rn/e 198 i s due t o t h e l o s s of C4H7N from t h e p a r e n t . The l o s s o f SO2 is a t y p i c a l rearrangement r e a c t i o n w i t h s u l f o n - amides ( 5 ) .

2.6 O p t i c a l R o t a t i o n S u l f i s o x a z o l e e x h i b i t s no o p t i c a l a c t i v i t y .

2.7 Mel t ing Range The m e l t i n g r ange r e p o r t e d i n USP X V I I I f o r

s u l f i s o x a z o l e is 1940 t o 199OC u s i n g t h e class Ia p rocedure ( 6 ) .

2 .8 D i f f e r e n t i a l Scanning Ca lo r ime t ry (DSC)

m e l t i n g r e g i o n depend g r e a t l y on t h e p r e v i o u s the rma l h i s t o r y o f t h e sample. When a DSC s c a n f o r s u l f i s o x a z o l e w a s o b t a i n e d u s i n g a h e a t i n g ra te of 10°C/minute, a m e l t i n g endotherm s t a r t i n g a t 191.6OC and a decomposi t ion exotherm s t a r t i n g a t 201.7OC were observed ( F i g u r e 6 ) . Because o f t h e sample i n s t a b i l i t y i n t h e m e l t i n g r e g i o n , t h e AHf w a s no t o b t a i n e d ( 7 ) .

The the rma l p r o p e r t i e s of s u l f i s o x a z o l e i n t h e

2.9 Thermogravimetr ic Ana lys i s (TGA) No we igh t l o s s was observed by TGA between

ambient and 225OC when t h e sample was h e a t e d a t 10°C/minute. A weight l o s s began abou t 225OC and amounted t o abou t 60% of t h e sample weight a t 475OC ( 7 ) .

2.10 S o l u b i l i t y The s o l u b i l i t y d a t a f o r s u l f i s o x a z o l e o b t a i n e d

a t 25OC are g i v e n i n Tab le I11 (8).

Table I11

S o l u b i l i t y o f S u l f i s o x a z o l e i n D i f f e r e n t S o l v e n t s

So lven t So lub il i t y (mg / m l )

3A a l c o h o l benzene

1 8 . 6 2 . 1

496

SULFISOXAZOLE

Figure 6

DSC Curve f o r Sulfisoxazole

I I I I

E x o


c h l o r o f o m 2.8 95% e t h a n o l 10 .5 e t h y l e t h e r 2 .5 methanol 14 .9 pe t ro l eum e t h e r 30°-60' 1 . 4 2-propanol 8 .6 w a t e r 0.2

2 . 1 1 X-ray C r y s t a l P r o p e r t i e s The x-ray powder d i f f r a c t i o n p a t t e r n of s u l f i s -

oxazo le i s p r e s e n t e d i n Tab le I V ( 9 ) . The i n s t r u m e n t a l c o n d i t i o n s are g i v e n below.

Ins t rumen t Cond i t ions :

Gene ra l Electr ic Model XRD-6 Spec t rogon iomete r

Genera to r : 50KV-12-112 MA Tube t a r g e t : Copper R a d i a t i o n : Cu J&= 1.542 8 o p t i c s : 0.1' D e t e c t o r s l i t

M.R. S o l l e r s l i t 3' B e a m s l i t 0.0007 i n c h Ni f i l t e r 40 t a k e o f f a n g l e

Goniometer: Scan a t 0.2O 20 p e r minu te D e t e c t o r : A m p l i f i e r g a i n - 1 6 c o u r s e ,

8.7 f i n e S e a l e d p r o p o r t i o n a l c o u n t e r



Rate meter T . C . 4 2000 CIS f u l l scale


Recorder: Cha r t speed - 1 i n c h p e r 5 minu tes

Samples p repa red by g r i n d i n g a t room t empera tu re .

498

SULFISOXAZOLE

Table I V

X-ray Powder D i f f r a c t i o n P a t t e r n of S u l f i s o x a z o l e

28 - 12.09 1 3 . 2 3 1 4 . 2 8 1 5 . 2 2 1 6 . 3 2 16 .94 1 7 . 4 0 18 .56 19 .48 20.26 20 .64 21 .76 22 .86 23.46 23 .74 24 .45 25 .68 26 .62 2 7 . 1 8 2 7 . 8 3 28.26 29 .52 30 .14 30 .86 31.36 31 .74 3 2 . 2 0 33 .00 3 3 . 2 0

d (8) * 7 . 3 2 6 .69 6 .20 5 . 8 2 5 . 4 3 5 . 2 3 5 . 1 0 4 .78 4 .56 4 .38 4 . 3 0 4 . 0 8 3 .89 3.79 3 . 7 5 3.64 3.47 3 . 3 5 3.28 3 . 2 1 3.16 3 . 0 3 2.97 2 . 9 0 2 . 8 5 2 .82 2.78 2 . 7 1 2 .70

I/I ** 0

100 3 1

6 26

4 1 5 4 1 14 1 4 21 36 1 3 96

8 5

19 8

65 1 4

9 2

1 3 5 2 3 2 2 6 6

28

33.76 3 4 . 3 2 3 5 . 0 0 35.26 3 6 . 0 4 36 .52 3 7 . 8 2 3 8 . 0 6 38.44 39.16 39 .78 4 0 . 6 2 42 .12 4 3 . 5 6 4 3 . 9 4 44 .66 45 .30 4 5 . 9 0 4 6 . 4 0 47 .02 47.99 4 8 . 7 6 4 9 . 0 0 4 9 . 7 0 50.14 5 0 . 7 3

-

* nX d - ( i n t e r p l a n a r d i s t a n c e ) 2 S i n 0

d (2) * 2 . 6 5 2 . 6 1 2.56 2 . 5 5 2 .49 2.46 2 . 3 8 2 .36 2 . 3 4 2.30 2.27 2.22 2 . 1 5 2 . 0 8 2 .06 2 .03 2.00 1 . 9 8 1 . 9 6 1 . 9 3 1 . 9 0 1 . 8 7 1 . 8 6 1 . 8 3 1 . 8 2 1 . 8 0

I/&)**

6 6 1 1 6 5 2 2 2 7 3 4 7 3 1 4 1 1 2 4 3 2 1 1 1 1

* >k = r e l a t i v e i n t e n s i t y (based on h i g h e s t i n t e n s i t y

of 1.00) %, 2 . 1 2 D i s s o c i a t i o n Constant

The pka f o r s u l f i s o x a z o l e has been determined s p e c t r o p h o t o m e t r i c a l l y (11) and by t h e t i t r a t i o n o f


s u l f i s o x a z o l e i n an e x c e s s of 0.1N NaOH w i t h 0.1N H C 1 t o b e 5.0 (12 ,13 ) .

3 . S y n t h e s i s S u l f i s o x a z o l e may be p repa red by t h e r e a c t i o n scheme

shown i n F i g u r e 7 . 3,4-Dimethyl-5-aminoisoxazole is re- a c t e d w i t h N-acetyl-p-aminobenzene s u l f o n y l c h l o r i d e . The a c e t y l group i s then c l eaved t o y i e l d s u l f i s o x a z o l e (10).

4. S t a b i l i t y Degradat ion When 2% s o l u t i o n s of s u l f i s o x a z o l e are p repa red i n

e thano1:water ( 1 : l ) and ethanol :O. lN NaOH (1:l) and re- f luxed f o r one hour , no decomposi t ion was observed by t h i n - l a y e r chromatography ( 3 ) . When a 2% s o l u t i o n of s u l f i s - oxazo le i n e thanol :O. lN H C 1 (1:l) w a s r e f l u x e d f o r one hour s l i g h t decomposi t ion t o 3,4-dimethyl-5-aminoisoxazole and s u l f a n i l a m i d e w a s observed ( 3 ) . Pure s u l f i s o x a z o l e h a s been found t o be s t a b l e when s u b j e c t e d t o a t empera tu re of 105OC f o r 5 days ( 3 ) . I n t a b l e t form, s u l f i s o x a z o l e i s s t a b l e f o r 5 y e a r s (14).

5. Drug Metabo l i c P roduc t s and Pharmacokinet ics

t i v e which is t h e major m e t a b o l i t e found i n human u r i n e . S u l f i s o x a z o l e i s u l t i m a t e l y e l i m i n a t e d from t h e body s o l e l y by means of u r i n a r y e x c r e t i o n w i t h a mean of 54 p e r c e n t of t h e dose e x c r e t e d as t h e ' ' f r ee" d rug and t h e remainder as t h e Nq-acetylated b i o t r a n s f o r m a t i o n product (15).

A pharmacok ine t i c p r o f i l e of s u l f i s o x a z o l e was d e t e r - mined from d a t a o b t a i n e d from I.V. a d m i n i s t r a t i o n of t h e drug i n humans. The d a t a sugges t ed t h e u s e of a two com- partment open system model (16) . The e x c e l l e n t agreement between t h e s i m u l a t e d and expe r imen ta l d a t a r e f l e c t s t h e r e l i a b i l i t y of t h e assumption of psuedo f i r s t o r d e r k i n e t i c s f o r a l l p r o c e s s e s (15) .

S u l f i s o x a z o l e i s metabol ized t o i t s N4-acetyl d e r i v a -

6 . Methods of Ana lys i s

6 . 1 Elemental Ana lys i s The r e s u l t s from t h e e l emen ta l a n a l y s i s are

l i s t e d i n Table V ( 1 7 ) .

5 00

Figure 7

Synthes is of Sulfisoxazole

s 0 + C H 3 C - N H o ! - N B O Y C H 3 C - N H a - C I 4-

0 H3C CH3 0 N-acetyl-p-amino-benzene 3,4-dimethy-5- H& CH3

sulfonyl chloride aminoisoxazole 5- (p-acetylsulfanilamido) - 3.4- d imethyl-isoxazole

m C r n v) 0 X

0 r rn

-

Zl

sulfisoxazole


Table V

Elemental Ana lys i s of Su l f i s o x a z o l e


C 49.43 49 .32 H 4.90 4 . 8 1 N 1 5 . 7 2 15 .74 S 12.00 12.07

6 . 2 Phase S o l u b i l i t y Ana lys i s Phase s o l u b i l i t y a n a l y s i s may be c a r r i e d o u t

u s i n g e t h y l a c e t a t e a s t h e s o l v e n t . A t y p i c a l example i s shown i n F i g u r e 8 which a l s o l i s t s t h e c o n d i t i o n s under which t h e a n a l y s i s was c a r r i e d o u t ( 8 ) .

6 .3 Thin Layer Chromatographic Ana lys i s (TLC) TLC systems have been pub l i shed which can b e used

t o s e p a r a t e s u l f i s o x a z o l e from i t s m e t a b o l i t e s and o t h e r sulfonamides. I n t h e system r e p o r t e d by Karp i t s chka (18) t h e sample i s s p o t t e d on a K i e s e l g e l G p l a t e and developed i n t h e s o l v e n t , ch1oroform:n-butano1:petroleum e t h e r (1:l:l). S u l f i s o x a z o l e h a s a Rf o f 0 . 5 1 . The TLC system r e p o r t e d by Woll ish e t a l . ( 1 9 ) u t i l i z e s a S i l i c a G e l G p l a t e and chloroform: heptane: e t h a n o l (1: 1:l) a s t h e deve lop ing s o l v e n t . of 0 .7 .

I n t h i s system s u l f i s o x a z o l e has a Rf

6 . 4 Direct Spec t ropho tomet r i c Anal* Direct spec t ropho tomet ry i s n o t o f t e n used f o r

t h e a n a l y s i s of s u l f i s o x a z o l e because o t h e r su l fonamides tend t o i n t e r f e r e . However, i n t h e absence o f any i n t e r - f e r e n c e s , t h e Amax a t 253 nm i n pH 7 . 5 phosphate b u f f e r may be used f o r d i r e c t s p e c t r o p h o t o m e t r i c a n a l y s i s .

6.5 C o l o r i m e t r i c Ana lys i s The c o l o r i m e t r i c a n a l y s i s of s u l f i s o x a z o l e may

be accomplished by d i a z o t i z a t i o n and coup l ing w i t h N-(1- naphthy1)-ethylenediamine d i h y d r o c h l o r i d e (Brat ton-Marshal l Reagent) . The r e s u l t a n t p u r p l e c o l o r i s measured a t abou t 550 nm (20) .

502

SU L F ISOXAZO LE

F i g u r e 8

SAMPLE S U L F I S O X A Z O L E

S O L V E N T E T H Y L ACETATE

S L O P E I 0 40%

E O U l L l B R A T l O N 2 0 H O U R S 0 1 25OC

E X T R A P O L A T E D S O L U B I L I T Y 14 5 m g / g o f E T H Y L -

-

A C E T A T E

-

I- z W > -I 0 In rn

w I- 3 -I 0 fn

. r

rn E

z 0 k fn 0 a x 0 u z

I- 3 J 0 lo

0

6

4 'v 0 2 5 5 0 7 5 I00

S Y S T E M C O M P O S I T I O N m g o f S A M P L E p e r g S O L V E N T

503

8 . C. RUDY AND B. 2. SENKOWSKI

6.6 F l u o r i m e t r i c Analys is F luorescence a n a l y s i s h a s been used t o d e t e r -

mine t h e c o n c e n t r a t i o n of s u l f i s o x a z o l e i n plasma (21) . For t h i s purpose d e t e c t a b l e l e v e l s i n t h e range of 1 ug p e r m l of plasma are needed. I n o r d e r t o a c h i e v e t h i s s e n s i t i v i t y i t i s necessary t o make a f l u o r e s c e n t d e r i v a - t i v e of s u l f i s o x a z o l e . One such d e r i v a t i v e i s t h e h i g h l y f l u o r e s c e n t "Dansylsulfonamide" which i s formed by t h e r e a c t i o n shown i n F i g u r e 9 . The f l u o r e s c e n c e y i e l d of t h i s compound i s s u f f i c i e n t t o measure about 1 ug of s u l f i s o x - a z o l e p e r m l of plasma. An even more s e n s i t i v e d e r i v a t i v e i s formed when s u l f i s o x a z o l e i s r e a c t e d w i t h 4,5-methylene- dioxy-phthalaldehyde t o form a phtha l imidine d e r i v a t i v e (F igure 9 ) . from 1 x 10-8 t o 1 x 10-10 mole/ml (22) .

The s e n s i t i v i t y l i m i t f o r t h i s d e r i v a t i v e is

6.7 T i t r i m e t r i c Analysis The t i t r a t i o n w i t h sodium methoxide i s t h e

method of c h o i c e t o a s s a y s u l f i s o x a z o l e ( 6 ) . The sample i s d i s s o l v e d i n dimethylformamide, thymol b l u e i s added, and t h e t i t r a t i o n w i t h 0.1N NaOCH3 t o a b l u e end-point is c a r r i e d o u t . A b lank d e t e r m i n a t i o n is performed and any necessary c o r r e c t i o n made. e q u i v a l e n t t o 26.73 mg of s u l f i s o x a z o l e .

Each ml of 0.1N NaOCH3 i s

5 04

Figure 9

N(CH,),

@8 o=s=o

CI Dansyl - Reogent

Fluorescence Derivatives of Sulfisoxazole

C H ~ & N H ~ ~ - ~ ! J . Q 0 O H

\ I H3C CH3

N4-wetyl metotolite Y(CH3)2

\ +

lm. 4 N HCI c

c ,

Dansyl- Sulfonomide Ex 3555/Em 485 m

phthalimidine derivative EX 330/Em 400 nm


9. References

1.

2 .

3.

4.

5.

6.

7.

8.

9.

10.

11.

12 * 13.

14.

15.

16.

17.

18. 19.

20.

21.

22.

Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal Communication. Johnson, J.H., Hoffmann-La Roche Inc., Personal Communication. Rubia, L.B., Hoffmann-La Roche Inc., Personal Communication. Boatman, J., Hoffmann-La Roche Inc., Personal Communication. Benz, W., Hoffmann-La Roche Inc., Personal Communication. United S t a t e s Pharmacopeia XVIII, 699 and 935 (1970). Moros, S., Hoffmann-La Roche Inc., Personal Communication. MacMullan, E. , Hoffmann-La Roche Inc. , Personal Communication. Hagel, R. B. , Hof fmann-La Roche Inc . , Personal Communication. Hoffer, M., Hoffmann-La Roche Inc., Personal Communication. Motchane, A., Hoffmann-La Roche Inc. , Unpublished Data. Rieder, J., Amzeimit te l -Forsch, 13, 81 (1963). Nakagaki, M., Koga, N., and Terada, H., J . Pharm. Soc. Japan, 83, 586 (1963). Clyburn, T., Hoffmann-La Roche Inc., Personal Communication. Kaplan, S.A., Weinfeld, R.E., Abruzzo, C.W., and Lewis, M., J . Pharm. S c i . , 61, 773 (1972). Riegelman, S., Loo, J.C.K., and Rowland, M., J . Pharm. S c i . , 57, 117 (1969). Scheidl, F., Hoffmann-La Roche Inc., Personal Communication. Karpitschka, N., Mikrachim. Acta, 1963, 157. Wollish, E.G., Schmall, M., and Hawrylyshyn, M., Anal. Chem., 33, 1138 (1961). Bratton, A . C . , and Marshall, F.K., J . B i o l . Chem., - 128, 537 (1939). deSilva, J.A.F., and D'Arconte, L., J . Forensic. S c i . , 14, 184 (1969). Amano, T., J . Pharm. Soc. Japan, 85, 1049 (1965).

506

TRICLOBISONIUM CHLORIDE

6. C. RUDY AND 6 . 2. SENKOWSKI

I N D E X

Analy t ica l P r o f i l e - Triclobisonium Chloride

1. Descr ip t ion 1.1 Name, Formula, Molecular Weight 1 . 2 Appearance, Color , Odor

2 . Phys ica l P rope r t i e s 2 . 1 I n f r a r e d Spectrum 2 . 2 Nuclear Magnetic Resonance Spectrum 2 .3 U l t r a v i o l e t Spectrum 2 . 4 Fluorescence Spectrum 2 .5 Mass Spectrum 2.6 Op t i ca l Rotat ion 2 . 7 Melting Range 2.8 D i f f e r e n t i a l Scanning Calorimetry 2.9 S o l u b i l i t y 2.10 X-ray Crys t a l P rope r t i e s

3 . Synthes is



6 . Methods o f Analysis 6 . 1 Elemental Analysis 6.2 Thin Layer Chromatographic Analysis 6 . 3 Color imet r ic Analysis 6 . 4 T i t r i m e t r i c Analysis

7 . References

508

TR I CLOBISON I UM CH LOR I DE

1. D e s c r i u t i o n

1.1 Name, Formula, Molecular Weight T r i c l o b i s o n i u m c h l o r i d e i s hexamethylenebis

[ dimet hy 1 [ 1 -met hy 1 - 3 - (2 , 2 ,6 - t rime t hy 1 cyc lohexy l ) propy 1 1 ammonium] d i c h l o r i d e .

Molecular Weight: 605.91 C36H74C12N2

1 . 2 Appearance, C o l o r , Odor T r i c l o b i s o n i u m c h l o r i d e o c c u r s as a w h i t e o r

n e a r l y w h i t e , p r a c t i c a l l y o d o r l e s s , c r y s t a l l i n e powder.


2 . 1 I n f r a r e d Spectrum The i n f r a r e d spectrum o f t r i c l o b i s o n i u m c h l o r i d e

i s p r e s e n t e d i n F igu re 1.. a Perkin-Elmer 621 Spec t ropho tomete r on a K B r p e l l e t c o n t a i n i n g 1 . 5 mg t r i c l o b i s o n i u m c h l o r i d e / 3 0 0 mg o f KBr. The bands a t 2960 and 2860 cm-I a r e a t t r i b u t e d t o a l i p h a t i c C-H s t r e t c h i n g and t h e bands a t 1474 and 1382 cm-l t o C-ti de fo rma t ion v i b r a t i o n s (1).

The spec t rum was measured w i t h

2 . 2 Nuc lea r Magnetic Resonance Spectrum (NMR) The spec t rum shown i n F igu re 2 was o b t a i n e d on

a J e o l 60 blHz NMR by d i s s o l v i n g 5 7 . 2 mg o f t r i c l o b i s o n i u m c h l o r i d e i n 0 . 5 m l o f CDC13 c o n t a i n i n g t e t r a m e t h y l s i l a n e as an i n t e r n a l r e f e r e n c e ( 2 ) . Due t o t h e many n e a r l y e q u i v a l e n t p r o t o n s i n t h i s mo lecu le , t h e NMK spec t rum c o n s i s t s o f broad bands i n t h e 0 t o 4 ppm r e g i o n . The s p e c t r a l a s s ignmen t s a r e g iven i n Tab le I ( 2 ) .

509

0. C. RUDY A N D 0 . 2. SENKOWSKI

a al k cd h cu c H

(u (u- - ao- m-

y--

0- u) z a-- 0 c 0 *- z

r---

I I-

W -J W

s w-- 2 ? m--

w--

-

m- -

7

0

0 0

33NVl l IWSNVU %

510

TR ICLOBlSONlUM CHLORIDE

Figure 2

NMR Spectrum of Triclobisonium Chlor ide

lo0 2 0 I BC I20

511


Tab le I

NMR Assignments f o r T r i c l o b i s o n i um C h l o r i d e

Type P ro ton Approx. Chemical S h i f t (ppm)

CH3)s on r i n g s 0.80-0.95

CH3's on c h a i n 1 .47

and c h a i n (no t a d j a c e n t t o n i t r o g e n s )

CH3's on n i t r o g e n s 3 .30

t o n i t r o g e n s

C H 2 I s & C H ' s i n r i n g s 1.05-2.30

C H 2 I s 6 C H I S a d j a c e n t 3 .45-3.90

2 . 3

minima i n

2 . 4

U l t r a v i o l e t Spectrum (UV) T r i c l o b i s o n i u m c h l o r i d e e x h i b i t s no maxima o r an aqueous s o l u t i o n (1 mg/ml) from 700 t o 210 nm.

F luo rescence Spectrum T r i c l o b i s o n i u m c h l o r i d e e x h i b i t s no f l u o r e s c e n c e

when i r r a d i a t e d w i t h w h i t e l i g h t . (3)

2.5 Mass Spectrum The mass spectrum o f t r i c l o b i s o n i u m c h l o r i d e

shown i n F igu re 3 was o b t a i n e d from a C E C 21-110 mass s p e c t r o m e t e r w i t h an i o n i z i n g ene rgy o f 70 eV ( 4 ) .

Weak peaks were obse rved up t o m/e 616 i n t h e low r e s o l u t i o n spectrum. S i n c e q u a t e r n a r y ammonium s a l t s are n o t s u f f i c i e n t l y v o l a t i l e , t h e mass spectrum o b t a i n e d i s p r i m a r i l y t h a t o f t h e the rma l d e g r a d a t i o n p r o d u c t s . In t h i s c a s e t h e main p r o d u c t observed had a molecu la r weight o f 504, C34H68N2 by h i g h r e s o l u t i o n a n a l y s i s , which co r re sponds t o t r i c l o b i s o n i u m c h l o r i d e minus 2 C H 3 C l ' s . The low r e s o l u t i o n spectrum a l s o c o n t a i n e d a peak a t m/e 518 p robab ly due t o t h e l o s s o f H C 1 p l u s CH3C1. component o f molecu la r weight 504 g i v e s r i s e t o t h e b a s e peak a t m/e 351 and a l s o m/e 225 v i a t h e u s u a l c l eavage B t o t h e amino group. p robab ly a fragment from t h e c e n t e r p a r t o f t h e molecule

The

The peak a t m/e 186, C11H26N2, i s

(4 ) *

512

TR

lCL

OB

lSO

NlU

M C

HL

OR

IDE

0

E

3

513


2.6 Opt ica l Rotat ion Triclobisonium c h l o r i d e e x h i b i t s no o p t i c a l

a c t i v i t y .

2 . 7 Melting Range Triclobisonium ch lo r ide decomposes on mel t ing .

The decomposition range depends on t h e r a t e of hea t ing . When t h e NF Class I procedure i s used ( 5 ) , t r i c lob i son ium ch lo r ide melts w i t h decomposition between 243 and 253'C.

2 . 8 D i f f e r e n t i a l Scanning Calor imetry (DSC) The DSC scan f o r t r i c lob i son ium ch lo r ide , shown

i n Figure 4 , was obta ined using a Perkin-Elmer DSC-1B Calor imeter . The sample pan contained a small hole i n t h e top t o allow t h e decomposition products formed t o escape. With a temperature program of 10"C/min., an endotherm was observed s t a r t i n g a t 253.4'C which is due t o t h e melt and/ o r decomposition of t h e molecule ( 6 ) . Due t o t h e n a t u r e o f t h i s peak no AHf was ca l cu la t ed .

2 . 9 S o l u b i l i t y The s o l u b i l i t y da t a f o r t r i c lob i son ium c h l o r i d e

obtained a t 25'C a r e given i n Table I1 (7) .

Table I 1

S o l u b i l i t y of Triclobisonium Chloride i n Di f f e ren t Solvents


3A a lcohol benzene ch l o r o form 95% e thanol e t h y l e t h e r met hano 1 petroleum e t h e r

(30'-60') 2 -propano1 water

> 5.0 x lo2 1 .3 x 10 4 . 2 x 10;

1.6 x 10

5.0 x 10

4 . 1 x 10;

> 5.0 x

> 5.0 x lo!

> 5.0 x 10

514

TR

ICL

OB

ISO

NIU

M C

HL

OR

IDE

s 8 0

R Y

:$ 3 W

(L

03

(L

W a

+

0

x 0

UJ

N

0

a, N

B. C. RUDY AND 6. 2. SENKOWSKI

2.10 X-ray Crys t a l P rope r t i e s The x-ray powder d i f f r a c t i o n p a t t e r n o f t r i-

clobisonium c h l o r i d e is presented i n Table I11 (8 ) . The ins t rumenta l cond i t ions are given below.



Goniometer Detect o r :

Generator : 50 KV-12-$ MA Tube t a r g e t : Copper 0

Radiat ion : Cu K = 1.542 A Optics : 0 . 1 " Detector s l i t

M.R. S o l l e r s l i t 3' Beam s l i t 0.0007 inch N i f i l t e r 4" t ake o f f angle Scan a t 0.2 ' 20 pe r minute Amplif ier gain - 16 coa r se , 8 . 7 f i n e Sealed propor t iona l counter tube and DC vo l t age a t p 1 a t eau Pulse he ight s e l e c t i o n EL-5 v o l t s ; EU - out Rate meter T . C . 4 2000 C/S f u l l s c a l e Chart speed - 1 inch/5 min. Recorder:

Samples prepared by gr inding a t room tempera ture .

TABLE I11

X-ray Powder Di f f r ac t ion Pa t t e rn Triclobisonium Chloride

* * - 20 d ( i ) * I / I 0

3 .98 2 2 . 2 39 4.44 1 9 . 9 75

12.44 7 . 1 2 19 1 2 - 9 2 6.85 10 13.52 6 .55 25 14.74 6 . 0 1 100

20 __

23.78 24.46 26.18 26.38 27.14 28 .00

5 16

0 * * d(A)* UI,

3.74 19 3.64 17 3.40 6 3.38 2 2 3.29 2 2 3.19 15

TRlCLOBlSONlUM C H L O R I D E


X-ray Powder D i f f r a c t i o n P a t t e r n T r i c l o b i sonium C h 1 o r i de

20 ~

15.40 15 .94 16.56 17.16 17.78 19.92 20. bO 21.60 22.00 23.36

0

d(A) *

5 . 7 5 5 . 5 6 5 , 3 5 5 . 1 7 4 . 9 9 4 . 4 6 4 .31 4 . 1 1 4 .04 3.81

* * 1/10

6 28

6 14 67 18

4 1 3 19 25

20 -

28.62 29.42 30.56 31.04 32.10 32.76 34.96 35.74 41 .16 45.12

0

d(A) *

3 .12 3 .04 2 . 9 3 2 .88 2 .79 2 . 7 3 2 . 5 7 2 . 5 1 2 .19 2 . 0 1

* * 1/10

19 28 11 15 11 1 3

8 8 8 8

n i * d = ( i n t e r p l a n a r d i s t a n c e )

** 2 S i n 0

I / I , = r e l a t i v e i n t e n s i t y (based on h i g h e s t i n t e n s i t y o f 1 . 0 0 )

3 . S y n t h e s i s T r i c l o b i s o n i u m c h l o r i d e may be p r e p a r e d by t h e r e a c t i o n

scheme shown i n F igu re 5 . s y n t h e s i s o f s e v e r a l symmetr ical b i s - q u a t e r n a r y amines d e r i v e d from (3-ionone has been p u b l i s h e d by T e i t e l ( 9 ) .

A comprehensive review o f t h e

4 . S t a b i l i t y Degradat ion T r i c l o b i s o n i u m c h l o r i d e was found t o be s t a b l e t o l i g h t -

and oxygen i n bo th s l i g h t l y a c i d i c (plt 3-7) and s l i g h t l y b a s i c (pH 7-9) s o l u t i o n s . In s t r o n g l y a l k a l i n e s o l u t i o n s t h e compound decomposes t o y i e l d t e r t i a r y amines (10 ) . I n t h e cream o r o i n t m e n t , t h e t r i c l o b i s o n i u m ch1,oride i s s t a b l e f o r a minimum o f 4 y e a r s . The b u l k s u b s t a n c e i s s t a b l e when s t o r e d a t room t e m p e r a t u r e unde r anhydrous c o n d i t i o n s . Decomposition o c c u r s when it i s s u b j e c t e d t o e l e v a t e d t e m p e r a t u r e s as can be d e t e c t e d by d i s - c o l o r a t i o n and odor o f t h e powder.


f o r the t r e a t m e n t and /o r p r e v e n t i o n o f l o c a l i n f e c t i o n s . S i n c e i t i s n o t a d m i n i s t e r e d i n t e r n a 1 1 y , t ri c 1 o b i s o n i um

T r i c l o b i s o n i u m c h l o r i d e i s an a n t i b a c t e r i a l a g e n t used

517

Figure 5

Synthesis o f Triclobisonium Chloride (9)

[email protected];0 Ammonia "3z CH CHNHZ Formic Acid

Fo mialdehyde 2 1

H (Raney Ni) ' a 3

"3

B-ionone m 0

TRlCLOElSONl UM CHLORIDE

c h l o r i d e i s n o t me tabo l i zed by t h e body.

6 . Methods o f Ana lys i s

6 . 1

i n Tab le

E 1 ement a1 Ana lys i s The r e s u l t s from t h e e l e m e n t a l a n a l y s i s a r e l i s t e d IV (11 ) .

TABLE IV

Elemental Ana lys i s o f T r i c l o b i s o n i u m C h l o r i d e

E 1 emen t % Theory % Found

C 71.36 69.29 H 12.31 12.30 N 4.62 4 . 5 7 c1 11.70 11.46

6 . 2 Thin Layer Chromatographic A n a l y s i s (TLC) The f o l l o w i n g TLC p rocedure may be used t o i d e n t i -

f y t r i c l o b i s o n i u m c h l o r i d e . Spot 500 mcg o f t r i c l o b i s o n i u m c h l o r i d e which was d i s s o l v e d i n methanol on a s i l i c a g e l G p l a t e and s u b j e c t i t t o a scend ing chromatography i n a p a p e r l i n e d , s a t u r a t e d t a n k u s i n g a mix tu re of a c e t o n e : 5 % K C 1 : benzene (80:40:1) as t h e s o l v e n t . A f t e r deve lop ing t h e p l a t e a t l eas t 15 cm, i t i s a i r d r i e d and sp rayed w i t h iod ine -mod i f i ed Dragendorff r e a g e n t t o v i s u a l i z e t h e s p o t . The t r i c l o b i s o n i u m c h l o r i d e w i l l a p p e a r as a brown s p o t about Rf 0.5 ( 1 2 ) .

6 . 3 C o l o r i m e t r i c A n a l y s i s

6 .31 Ion P a i r E x t r a c t i o n o f Bromocresol Green L'omDlex After d i s s o l u t i o n o f t h e t r i c l o b i s o n i u m

c h l o r i d e cream, t h e t r i c l o b i s o n i u m c h l o r i d e i s complexed w i t h bromocresol g reen and t h i s complex i s e x t r a c t e d i n t o a chloroform s o l u t i o n . The absorbance o f t h i s s o l u t i o n , as wel l as t h e abso rbance o f t h e complex s i m i l a r l y p r e - pa red from r e f e r e n c e s t a n d a r d t r i c l o b i s o n i u m , i s r e a d a t 630 nm and t h e amount of t r i c l o b i s o n i u m c h l o r i d e p r e s e n t i n t h e cream i s c a l c u l a t e d ( 1 3 ) .


6.32 Ion-Pair Ext rac t ion of Bromothymol Blue Comp 1 ex For t he t r ic lobisonium ch lo r ide ointment

bromothymol b lue is used t o form t h e ion -pa i r complex. procedure i s similar t o t h a t €or t h e cream except poly- e thylene g lycol 4000 and polyethylene glycol 400 a r e added t o t h e re ference s tandard t ri c l o b i s onium ch lo r ide s o l u t i o n and a l s o t o a blank. Af te r e x t r a c t i o n of t h e complex from a bas i c aqueous s o l u t i o n , t h e absorbance i s read a t t h e 384 nm maximum f o r t h e sample, s t anda rd , and t h e blank. These values a r e used t o c a l c u l a t e t h e amount o f t r i - clobisonium i n t h e ointment (13) .

6 . 4 T i t r i m e t r i c Analysis

The

The non-aqueous t i t r a t i o n as descr ibed i n t h e NF XI11 i s t h e method o f choice f o r t h e ana lys i s o f bulk t r ic lobisonium ch lo r ide (13). An accura te ly weighed 1 gm sample i s d isso lved i n 80 m l o f g l a c i a l a c e t i c ac id . F i f teen mi l l i l i t e r s of mercuric a c e t a t e and 4 drops of c r y s t a l v i o l e t a r e added and t h e t r ic lobisonium ch lo r ide i s t i t r a t e d t o a blue-green end-point with 0 .1N H C 1 0 4 i n g l a c i a l a c e t i c a c i d . A blank t i t r a t i o n is performed and any necessary co r rec t ion made. Each m l o f 0.1N H C 1 0 4 i s equivalent t o 30.30 mg o f C36H74C12N2 (13) .

520

TR ICLOBISON I UM CHLORIDE

7 . Keferences

1.

2 .

3 .

4 .

5 . 6 .

7 .

8.

9 . 10 .

11.

1 2 .

13.

Hawrylyshyn, M . , Hoffmann-La Roche I n c . , P e r s o n a l Communication. Johnson , J . H . , fioffmann-La Roche I n c . , P e r s o n a l Communication. Boatman, J . , Hoffmann-La Roche Inc. P e r s o n a l Communication. Benz, W . , Hoffmann-La Roche I n c . , P e r s o n a l Commun i c a t i on . National Forniulary X I I I y 815 (1970) . bloros, S . , Hoffmann-La Koche I n c . , P e r s o n a l Communication. MacMullan, E., 1-Ioffniann-La Roche I n c . , P e r s o n a l Commun i c a t i o n . Hage l , R . B . , Hoffniann-La Koche I n c . , P e r s o n a l Communication. T e i t e l , S . , J . Med. Chem., 6 , 780 (1963) . T e i t e l , S . , Iloffmann-La floche Inc. , Unpublished Data. S c l i e i d l , F . , Iloffmann-La Roche I n c . , Pe r sona l Communication. Bandong, L . , Iloffmann-La Roche l n c . , P e r s o n a l Communication. National FormuZary XIII , 722-723 (1970) .

52. I

TRIFLUPROMAZINE HYDROCHLORIDE

Kluiis Florcp

513

KLAUS FLOREY

1.

2.

3.

4.

5.

6 ,

7 .

8.

9.

TABLE OF CONTENTS Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor.

Physical Properties 2.1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 pK, pH 2.6 Melting Range 2.7 Differential Thermal Analysis 2.8 Thermogravimetric Analysis 2.9 Solubility 2.10 Crystal Properties

Synthesis

Stability - Degradation Drug Metabolic Products

Methods of Analysis 6.1 Elemental Analysis 6.2 Gravimetric Analysis 6.3 Nonaqueous Titration 6.4 Electroanalysis 6.5 Titrimetric Analysis 6.6 Spectrophotometric Analysis 6.7 Spectroflurometric Analysis 6.8 Electrophoresis 6.9 Chromatographic Analysis

6.91 Paper 6.92 Thin-Layer 6.93 Gas-Liquid

Identification and Determination in Body Fluids and Tissues

Miscellaneous

References

TR I FLUPROMAZINE HYDROCHLORIDE

1. Description

1.1 Name, Formula, Molecular Weight

Triflupromazine Hydrochloride is lO-L?-(Dimethylamino)-propy&7-2- trifluromethylphenothiazine hydrochloride; also 2-firifluoromethy~ -10- ( y -dimethylaminopropyl) phenothiazine: S o 4703.

CH2-CH2-CH2-N (CH3) 2 I

Cl8H19F3N2S. HC1 Mol. wt. 388.89

1.2 Appearance, Color, odor.

Triflupromazine hydrochloride occurs as a white to pale tan, crystalline powder, with a slight characteristic odor.


2.1 Infrared Spectra

The infrared spectra of Squibb Standard # 48244-003 are presented in figures 1 and 2.l spectrum of the free base and a dis- cussion of spectra-structure

The infrared

x r

Figure 1. I. R. Spectrum of Trif lupromazine Hydrochloride. Squibb Standard #48241-003. Mineral oil mull. Instrument: Perkin-Elmer 21.

D C cn n r 0 D rn <

Figure 2. I . R . Spectrum of Triflupromazine Hydrochloride. Squibb Standard #48241-003 in KBr pellet from methanol. Instrument: Perkin-Elmer 21.

-I 9 n r

5 n 0 z 9 N z rn I <

0 rn

KLAUS FLOREY

c o r r e l a t i o n of h e n o t h i a z i n e s have been p r e s e n t e d . f l y 3 The s p e c t r a p u b l i s h e d by Sammul, e t a14 a r e i n e s s e n t i a l ag reemen t w i t h t h o s e p r e s e n t e d h e r e .

2 . 2 Nuclear Magnet ic Resonance Spectrum

The NMR spec t rum of t r i f l u p r o m a z i n e h y d r o c h l o r i d e i s p r e s e n t e d i n F i g u r e 3. 67 r e sonances a r e s e e n a s two s i n g l e t s . The methylene p r o t o n r e s o n a n c e s of t h e two me thy l g roups on t h e p r o t o n a t e d n i t r o g e n appea r a s two se t s of t r i p l e t s because of r o t a t i o n a l non-equiva lence . The f o l l o w i n g a s s ignmen t s i n t a u v a l u e s a r e c o n s i s t e n t w i t h t h e s t r u c t u r e :

The N-methyl p r o t o n

N C H 3 7 . 3 5 ( s ) 7 .27 ( s ) CH2-CH 2- CH2- N

I .CH3

\ / F:Hcl

Aromatic p r o t o n s : 3.2- 2 . 5 z:

The NMR spec t rum o f t h e f r e e b a s e h a s been p r e s e n t e d and d i s c u s s e d .

528

1 I I 1 I 1 I I I I . . . , . , , , I , , , , , , , I . , , . , I . . . , . I . , . . I .

0 WM ,, 7 0 6 0 5 0 .o 3 0 20 I 0 . i

#I>

Fig. 3 NMR Spectrum of Triflupromazine Hydrochloride (Squibb Batch # 48244-003 in C D C 1 3 , containing tetramethylsilane as an internal reference. Instrument: Varian A-60.

KLAUS FLOREY

2 . 3 U l t r a v i o l e t Spectrum

The f o l l o w i n g U . V . d a t a h a v e been r e c o r d e d :

1 1. i n methanol ( I n s t r u m e n t Cary 15)

max 260 nm E1% 869 1 c m

rnax 308 nm E1% 92 .8 1 c m

3 2 . i n 95% e t h a n o l ( I n s t r u m e n t Cary 1 4 )

max 258 nm f 34,000 max 308 nm 3 ,700 min 224 nm 10 ,000 min 280 nm 1 ,600

2 .4 Mass Spectrum

The low r e s o l u t i o n mass spec t rum o f t r i f l u p r o m a z i n e i s p r e s e n t e d i n f i g u r e 4.

5 The f o l l o w i n g f r a g m e n t a t i o n p a t t e r n p r e s e n t e d i n f i g u r e 5 i s c o n s i s t e n t w i t h t h e mass spec t rum and i n agreement w i t h t h e r e s u l t s o f an independen t i n v e s t i g a t i o n . 6

530

TR

IFL

UP

RO

MA

ZIN

E H

YD

RO

CH

LO

RID

E

hlISN31NI 3

AIlV

l3tl

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a, r: -d

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m

k a

?

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W

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k

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W

0

E

3

k

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C

nN

m

o

dm

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4J

dr:

oa

, W

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m

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AU

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sp

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B 0

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m

co d

NN

x

2-u aJ

8 -2 u2 I

Y

N

aJ

\

E

532

TR I F LUPROMAZINE HYDROCHLORIDE

2 . 5 p K , pH

The pKa o f t h e h y d r o c h l o r i d e was d e t e r m i n e d t o be a p p r o x i m a t e l y 6. 57 t h e pKb o f t h e f r e e b a s e t o be 4 . 5 9 . '8 The pH o f a 2% s o l u t i o n of a r e c e n t s t a n d a r d was f o u n d t o be 4 . 7 . l The pKa o f t h e f r e e b a s e was d e t e r m i n e d t o be 9 . 2 . 9

2 . 6 M e l t i n g Range

The f o l l o w i n g m e l t i n g ( d e c o m p o s i t i o n ) r a n g e s h a v e b e e n r e p o r t e d ( O C ) .

173-174 ( R e c r y s t a l l i z e d from d r y c h l o r o b e n z e n e a n d a c e t o n i t r i 1 e ) l O

1 7 6- 17 7

2 . 7 D i f f e r e n t i a l Thermal A n a l y s i s

D i f f e r e n t i a l Thermal A n a l y s i s o f S q u i b b B a t c h AR-IX-113E showed an e n d o t h e r m a t 174OC.

2 . 8 T h e r m o g r a v i m e t r i c A n a l y s i s

T h e r m o g r a v i m e t r i c a n a l y s i s o f S q u i b b B a t c h AR-IX-113E g a v e a w e i g h t l o s s of 0.1%.

533

KLAUS FLOREY

2.9 Solubility

Triflupromazine hydrochloride is very water soluble and hygroscopic. 12

solubility in

water: >lg/ l ml ethanol: >lg/ l ml

chlorotorm: 0.6g/l ml ether: insoluble

The solubility of the free base ir water was determined to be 5

2.10 Crystal Properties

For forensic identification a picture of triflupromazine crystals from 95% ethanol has been published with those of other phenothiazine~l~.

An X-ray powder diffraction pattern of triflupromazine hydrochloride is presented in table I. 14

534

TRIFLUPROMAZINE HYDROCHLORIDE

T a b l e I

d (g) * 1/100**

1 4 . 7 5 50 8 . 0 5 50 7 . 0 5 35 5 . 0 5 60 4 . 7 5 100 4 . 5 0 25 4 . 2 5 1 5 4 . 1 0 8 0 3 .85 4 0 3 . 7 5 40 3 . 5 5 40 3 . 4 5 10 3 . 3 0 10 3 . 2 0 1 5

0 * d = i n t e r p l a n a r d i s t a n c e ( A ) * * Based on h i g h e s t i n t e n s i t y of

100 Cu k a r a d i a t i o n ; = 1 , 5 4 1 8 x

The X-ray powder d i f f r a c t i o n p a t t e r n of t h e p i c r a t e h a s b e e n u s e d f o r i d e n t i f i c a t i o n i n f o r m u l a t i o n s . 1 4

3. S y n t h e s i s T r i f l u p r o m a z i n e i s p r e p a r e d by a d d i t i o n o f t h e d i m e t h y l a m i n o p r o p y l s i d e c h a i n t o t h e p h e n o t h i a z i n e n u c l e u s (11) lo, o r i t s N - a c e t y l d e r i v a t i v e 6 5 (111) a n d c o n v e r s i o n o f t h e b a s e t o t h e h y d r o c h l o r i d e ( I ) . D i a z o t i z a t i o n of the 7-aminoanalogue ( I V ) a l s o y i e l d s I. 1 5

4 . S t a b i l i t y - D e g r a d a t i o n

T r i f l u p r o m a z i n e h y d r o c h l o r i d e i s h y g r o s c o p i c . When m o i s t u r e i s e x c l u d e d i t i s s t a b l e i n t h e

535

KLAUS FLOREY

I I R = H I11 R = COCH3

IV

I

YH2-CH2-CHZ-N-CH3 CH*-CH2-CH2-N(CH3) 2 H I

V VIII

CH -CH2-CH2-N ( CH3) 1 2

VI R = OH;R',Rlt = H VII R' o r R' = OH;R=H

Fig. 6 Synthetic and Degradative Pathways f o r Triflupromazine Hydrochloride.

536

TR I FLUPROMAZI NE HYDROCHLORIDE

crystalline state, but discolors rapid1 when exposed to ultraviolet radiation. 6 g

This discoloration also occurs on U.V. radiation of aqueous solutions with spectral changes. 66 It is undoubtedly due to radical formation and subsequent disproportionation (see below). Thin-layer chromatographic spots on silica gel are oxidized to the sulphoxide (VIII fig. 6) when exposed to air for 48-72 hours. l7 hastened this process. 17 proceeds through the colored semiquinone free radical (cf. 18). In phenothiazines generally these radicals undergo disproportionation. The products of oxidation are the sulfoxide or the 3-(or 7-) hydroxy derivatives. However, formation of the latter is severely inhibited in 10-substituted phenothiazines. l8 so far there is no evidence for the degradative formation of 7-hydroxy-triflupromazine (VI fig, 6) which has been identified as a metabolic product’’ (see Section 5). The Electron Spin Resonance spectrum of the radical has been measured and presented 21 and was found to differ from the spectrum of the parent compound in the lack of hyper fine structure. The second order decay rate constant of the semiquinone radical of triflupromazine was found to be 870 (l/mol/min) as measured by ESR.

Hydrogen peroxide Oxidation also

18,20,

18


When triflupromazine was incubated with certain liver preparation in vitro, evidence was obtained €or conversion to N-demethyl- triflupromazine (V fig. 6) , 7-hydroxytri- flupromazine (V~),traces of triflupromazine

537

KLAUS FLOREY

s u l p h o x i d e ( V I I I ) and p o s s i b l 3- o r 8- h y d r o x y t r i f l u p r o m a z i n e ( V I I ) . 419

6. Methods o f A n a l y s i s

6 . 1 E lemen ta l A n a l y s i s :

Element % C a l c .

c18 H2 0

55 .59 5.18

~~

c1 9 .12 F3 14 .66

S 8 . 2 4 N 2 7 . 2 1

6 . 2 G r a v i m e t r i c A n a l y s i s

The p r e c i p i t a t e w i t h

% Found Ref. 10 Ref . 11

55 .70 55 .33 5 .22 5 . 3 3

s i 1 i c o t u n g s t i c a c i d h a s been u s e d f o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n o f t r i f l u p r o m a z i n e h y d r o c h l o r i d e i n t a b l e t s . 2 2

6 . 3 Nonaqueous T i t r a t i o n

The nonaqueous t i t r a t i o n of t r i f l u p r o m a - z i n e h y d r o c h l o r i d e i n a c e t o n e w i t h p e r c h l o r i c a c i d i n d ioxane a s t i t r a n t and d i m e t h y l ye l low a s i n d i c a t o r h a s been d e s c r i b e d . 2 2 I t i s s u i t a b l e f o r f o r m u l a t i o n c o n t e n t a s s a y s . T i t r a t i o n w i t h p e r c h l o r i c a c i d was employed t o de t e rmine t h e h a l f n e u t r a l i z a t i o n p o t e n t i a l s (E-1 /2 ) w i t h a F i s h e r t i t r i m e t e r i n s e v e r a l o r g a n i c s o l v e n t s . The f o l l o w i n g E-1/2 v a l u e s ( i n mv) were found: a c e t i c a c i d 234, a c e t o n e 455, a c e t o n i t r i l e 424, i s o p r o p a n o l 445, n i t r o m e t h a n e 314.

8

538

TRI F LUPROMAZI NE HYDROCHLORIDE

6.4 Electroanalysis

Controlled potential electrolysis has been applied for the coulometric quantitative determination of triflupromazine and other phenothiazines. Depending on the oxidation potential (anode potential with respect to S.E.C. (standard calomel electrode)) and acid strength triflupromazine was oxidized to the free radical (Ox. Pot. + 0 . 6 5 ; 12N H2SO4) or the sulfoxide (Ox. Pot. + 1.05; 1 2 N H2SO4). The electro- chemistry of triflupromazine in acetonitrile has also been studied. 24

Polarographic evidence has been presented to prove the complexation of of triflupromazine hydrochloride with oxygen.

23

25

6.5 Titrimetric Analysis

A titrimetric method has been reported in which triflupromazine hydrochloride was titrated visually to a colorless endpoint with ceric sulfate. 26 The reaction proceeds through forming a red-colored semiquinone free radical (420 n m ) followed by formation of the colorless sulfoxide derivative. The method can be applied to pharmaceutical dosage forms.

6.6 Spectrophotometric Analysis

The ultraviolet absorption maximum at 255nm has been used for a quantitative assay of triflupromazine hydrochloride in formulations.

539

KLAUS FLOREY

6 . 7 S p e c t r o f l u r o m e t r i c A n a l y s i s

T r i f l u p r o m a z i n e h y d r o c h l o r i d e , l i k e o t h e r p h e n o t h i a z i n e d r u g s e x h i b i t s f l u o r e s c e n c e . T h i s phenomenon was f i r s t e x p l o r e d i n 0.2N s u l f u r i c a c i d . The f l u o r e s c e n c e peak was found i n t h e r a n g e o f 450 t o 475 nm, s h i f t i n g t o s l i g h t l y lower wavelength upon o x i d a t i o n w i t h po ta s s ium permanganate . A s l i t t l e a s 0 . 0 1 t o 0 .05 wg o f t h e p u r e s u b s t a n c e p e r m l . o f 0.2N s u l f u r i c a c i d c o u l d be d e t e c t e d . Due t o i n t e r f e r e n c e b y o t h e r s u b s t a n c e s , t h e s e n s i t i v i t y i n b i o l o g i c a l f l u i d s was lower ( a b o u t 0 . 8 wg/ml of f l u i d ) .

27

I n c o n c e n t r a t e d s u l f u r i c a c i d , t h e a c t i v a t i o n maximum was found a t 325 nm and t h e f l u o r e s c e n c e maximum a t 500 nm. 28 0 .05 pg/ml. I n 50% a c e t i c a c i d o x i d a t i o n t o t h e s u l p h o x i d e w i t h 30% hydrogen p e r o x i d e gave an a c t i v a t i o n maximum a t 350 m w and a f l u o r e s c e n c e maximum o f 410 mp.29

The l i m i t o f s e n s i t i v i t y w a s

6 . 8 E l e c t r o p h o r e s i s

E l e c t r o p h o r e s i s i n v a r i o u s b u f f e r s , p roposed b y Wernurn3' was c a r r i e d o u t on t r i f lupromazine h y d r o c h l o r i d e and s e v e r a l o t h e r p h e n o t h i a z i n e t ranqu i 11 i z e r s . 31 a p p a r a t u s , Whatman 3MM pape r 30 c m i n w i d t h , and a p o t e n t i a l d i f f e r e n c e of 500 t o 800 v o l t was used . D e t e c t i o n o f s p o t s was c a r r i e d o u t w i t h a 40% s u l f u r i c a c i d s p r a y (o range c o l o r ) o r

A Gordon-Misco

540

TR I F LUPROMAZINE HYDROCHLORIDE

f l u o r e s c e n c e unde r U. V. l i g h t . For t r i f l u p r o m a z i n e h y d r o c h l o r i d e , a n o d i c m i g r a t i o n ( c m ) a f t e r 40 t o 60 minu tes was:

PH 3 . 3 4 . 7 6 . 0 7 . 2 8 . 0 9 . 3 M i g r a t i o n 5 . 9 4 . 8 5 . 3 5 . 5 4 . 6 1.8

6 . 9 Chromatographic A n a l y s i s

6 . 9 1 Paper

The f o l l o w i n g pape r chromato- g r a p h i c sys tem have been r e p o r t e d .

Rf Ref . S o l v e n t sys tems: -

1 N sodium fo rma te 0 . 4 1 3 1 1 N sodium fo rma te 90/n-propanol 1 0 0 .50 3 1 1 N sodium f o r m a t e 90/1N ammonia 10 0 .12 31 1 N sodium f o r m a t e 97,/90% 0 . 5 9 31

1 N sodium a c e t a t e 0 . 3 5 31 1N sodium a c e t a t e 90/n-propanol 1 0 0 . 5 9 3 1 10% sodium c h l o r i d e

92/n-propanol 8 0 . 76 31 "Reverse Phase" : Paper impregnated w i t h g l y c e r o l t r i b u t y r a t e ,

fo rmic a c i d 3

32 0.2M sodium ace ta t e /HCl pH 4 .58 ----

D e t e c t i o n sys tems:

40% H2SO4 s p r a y C o l o r Ref . F l u o r e s c e n c e under U. V. l i g h t Orange 31 ,32 I o d o p l a t i n a t e B l u i s h Yellow 3 1

Dark 32

53 1

KLAUS FLOREY

6.92 Thin-Layer The following thin-layer chromatographic systems have been

Absorbent Solvent System

Silica G e l

I1

11

I 1

11

1 1

II

11

tert. butyl alcohol 90%/lN ammonia 10% n-proianol 88%/lg ammonia 12% ether, sat. with water 70% methanol/water 30% 85% n-propanol/water 15% n-butanol sat. with 1N - ammonia benzene-dioxane-aq. ammonia ( 60 : 3 5 : 5 ) ethanol-acetic acid-water(50:30:20) methanol-butano1(60:40) cyclohexane-diethylamine (9 : l ) chloroform-hexane(50:50) chloroform-methanol (50 : 50) ch lor o f orm-me th a no 1

acetone methanol-ammonia(98:2) chloroform-me thanol- ammonia (80:20:1) cyclohexane-benzene- diethylamine (75:15:10)

(90 : 10)

reported:

Rf

0. 36

0.50

0.48 0.22 0.22 0. 72

0.95

0.79

0.40 0.76

0.48 0.86

0.54

0.38 0.60 0.84

0.57

Ref.

31

31

31 31 31 31

33

33

33 34

35 35

36

36 36 19+

37

542

TRI FLUPROMAZINE HYDROCHLORIDE

A b s o r b e n t

* S i l i c a G e l

I I * l l *

11 *

, I * * I,**

11

11

I1

S o l v e n t Sys tem

c y c l o h e x a n e - b e n z e n e - d i e t h y l a m i n e ( 7 5 : 1 5 : 2 0 ) m e t h a n o l a c e t o n e b e n z e n e - e t h a n o l - ammonia ( 9 5 : 1 5 : 5 ) m e t h a n o l 95% e t h a n o l 15% a q . ammonium a c e t ate-me t h a n o 1 ( 2 0 :80) n - p r o p a n o l - w a t e r - a c e t i c a c i d p y r i d i n e - p e t . e t h e r - m e t h a n o l ( 1 : 4 , 5 : 0 . 1 ) a c e t o n e - e t h y l a c e t a te- e t h a n o l 5:4:1 s a t . w i t h ammonium l a c t a t e b u f f e r p H 7.

R e f . - Rf

0 . 6 0 36

0 . 5 2 37 0 . 4 8 37 0 . 7 4 36

0 . 4 8 37 0 . 3 1 37 0 . 6 9 17***

0 . 2 8 19++

0. 54 38

0 . 3 0 39

* p r e p a r e d ** p r e p a r e d

w i t h 0.1M KOH w i t h 0.1M NaHS03

J

*** I n t h i s s y s t e m , t r i f l u p r o m a z i n e s u l f o x i d e h a d

+ I n t h i s s y s t e m , t r i f l u p r o m a z i n e s u l f o x i d e h a d

++ T r i f l u p r o m a z i n e s u l f o x i d e h a d an Rf o f 0 . 1 0 .

an Rf o f 0 .50 .

a n Rf o f 0 . 7 0 ,

G e n e r a l l y , i t i s i m p o r t a n t t o p r o t e c t p l a t e s f rom l i g h t d u r i n g s p o t t i n g a n d d e v e l o p m e n t .

543

The f o l l o w i n g d e t e c t i o n sys t ems have been used :

Sys t e m F l u o r e s c e n c e unde r U. V. lamp 40% s u l f u r i c a c i d s p r a y Potass ium i o d o p l a t i n a t e 5% f e r r i c c h l o r i d e , 20% p e r c h l o r i c

50% n i t r i c a c i d (5:45:50). (FPN;

I o d i n e 1% po tas s ium permanganate Dragendor f f r e a g e n t 5% p-dimethylaminobenzaldehyde i n

1 8 N - s u l f u r i c a c i d F u r f u r a l r e a g e n t F o l i n - C i o c a l t e a u r e a g e n t 1% ammonium v a n a d a t e i n 10 ml .conc .

5% c innamic a ldehyde and 5% H C 1

S u l f u r i c ac id- formaldehyde 50% aq . s u l f u r i c a c i d i n e t h a n o l (4 : 1)

a c i d

( F o r r e s t r e a g e n t )

s u l f u r i c a c i d

i n e t h a n o l

C o l o r R e a c t i o n R e f v i o l e t 3 1 , 3 6 , 1 9 , 1 7 , 3 5 o r a n g e 4 2 , 3 1

5 2 , 1 7 f l e s h Or

p i n k

cameo p i n k cameo

f l e s h

cameo

o r a n g e

3 6 , 3 4 , 3 7 37

37

36 36 36

3 6 , 3 7

37 39 4 2 , 3 1

ii r D C

n v)

r

TRI F LUPROMAZINE HYDROCHLORIDE

6.93 Gas-liquid

Triflupromazine was well separated from other phenothiazine tranquilizers on a 80-100 mesh Gas-Chrom S glass column coated with 2% SE-30. Retention time relative to chlorpromaz’ne at a column temp. of 2 0 5 O was 0.46. Similar results were obtained on a 60-80 mesh Diatoport copper column coated with 5% ~ ~ - 3 0 . ~ ~ 9 ~ ~ flupromazine was separated from its sulphoxide. l7 a 100-120 mesh silanized Gas-Chrom P glass column coated with 1% Hi-Eff-8B (cyclohexanedimethanol succinate) . 43 The identification of triflupromazine and other phenothiazine tranquilizers by gas chromatography of their pyrolysis products has also been

40 .

In the same system tri-

Another system used was

reported. 44

Identification and Determination in Body Fluids and Tissues

Many of the references cited in previous sections concern methods devised to identify and determine triflupromazine hydrochloride and other phenothiazines for pharmacological, toxicological and forensic purposes (cf. 45). These methods can be classified as follows:

535

KLAUS FLOAEY

References

Color reaction: Separation schemes: Spectrofluorometry: Electrophoresis : Paper chromatography: Thin-layer

chromatography:

G a s - 1 iquid chroma to - graphy:

X-ray diffraction bands :

Micro crystalline identification:

Photomicrography:

46,47,48,49,50,51,52,53,54 55,50,51,52 27,29,28,56 31 31,32

50,33,31,13,36,19,37,35, 39,68

17,43,69

61,15

57,58 13

8. Miscellaneous

The following parameters of triflupromazine hydrochloride and other phenathiazine have been studied: Adsorption by solids such as talc, kaolin and activated charcoal59, surface activity at the air-solution interface60361, oil-water partitioning62, photo induced interaction with a lecithin monomolecular film63, and interaction with bovine serum albumin64.

546

9.

TRlF LUPROMAZINE HYDROCHLORIDE

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3.

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9.

10.

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12. 13.

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15.

16. 17.

18.

G. A. Brewer, private communication. W. E. Thompson, R. J. Warren, J, B. Eisdorfer and J. E. Zarembo, J. Pharm. Sci. 54, 1819 (1965). R. J. Warren, J. B. Eisdorfer, W. E. Thompson and J. E. Zarembo, J. Pharm. Sci. 55, 144 (1966). 0. R. Sammul, W. L. Brannon and A. L. Hayden, J. Ass. Offic. Anal. Chem. - 47, 918 (1964). A. I . Cohen, private communication. J. N. T. Gilbert and B. J. Millard, Org. Mass Spectrum. 2, 17 (1969). F. Russo-Alesi, private communica tion. L. G. Chatten and L. E. Harris, Anal. Chem. - 34, 1495 (1962). A. L. Green, J. Pharm., Pharmacol. 9, 10 (1967). H. L. Yale, F. Sowinski and J. Bernstein, J. Am. Chem. SOC. 79, 4375 (1957). P. N. Craig, E. D. Nodiff, T. T. Lafferty and G. E. Ullyot, J. 0rg.Chem. - 22, 709 (1957) .

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547

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548

TR I FLUPROMAZINE HYDROCHLORIDE

39.

4 0 .

41.

4 2 .

43 .

44.

4 5 ,

46.

47.

48.

49.

50. 51.

52.

53.

54.

55.

56.

2 . M a r g a s i n s k i , R. D a n i e l a k , T. Pomazanska a n d H. R a f a l o w s k a , A c t a . P o l o n . Pharm. 21, 5 ( 1 9 6 4 ) , C . A . -3 62 89419 ( 1 9 6 5 ) . M. W. A n d e r s a n d S . J. Manner ing , J . Chromatog. z, 258 ( 1 9 6 2 ) . H . F. M a r t i n , J. L. D r i s c o l l ard B. L. Gudzinowicz , Anal . Chem. 35, 1 9 0 1 ( 1 9 6 3 ) . B. J. Gudzinowicz , J. o f Gas. Chromat . 1966 110. N. C . C a i n and P. L. K i r k , Microchem. J . - 1 2 , 256 ( 1 9 6 7 ) . C. R. F o n t a n , N. C . J a i n a n d P. L. K i r k , Microchim. A c t a - 9 1964 326. J. S u n s h i n e , Handbook of A n a l y t i c a l T o x i c o l o g y . The Chemica l Rubber C o . ( 1 9 6 9 ) .

D. K. Yung and M. P e r n a r o w s k i , J .Pharm. S c i . 52, 365 ( 1 9 6 3 ) . I. S . F o r r e s t a n d A . S . Mason, Am. J. P s y c h i a t . 115, 1114 ( 1 9 5 9 ) . J. J. Heyman, M. Almudevar a n d S . M e r l i s s Am. J. P s y c h i a t . 116, 256 ( 1 9 5 9 ) . H. Kadin , J . Assoc. O f f i c . Agr. C h e m i s t s 44, 751 ( 1 9 6 1 ) . M. P i n k a s o v a , Cesk. Farm. 17, 1 8 1 ( 1 9 6 8 ) . B. G o t h e l f and A . G . Karczmar, I n t e r n . J. Neuropharmacol . 2, 95 ( 1 9 6 3 ) . P. Haux and S . N a t e l s o n , Am. J. C l i n . P a t h . , 53, 77 ( 1 9 7 0 ) . J . H. Heyman, B. Bayne a n d S . M e r l i s , Am. J. P s y c h i a t . 116, 1108 ( 1 9 6 0 ) . P . R a j e s w a r a n and P. L . K i r k , B u l l e t i n on N a r c o t i c s 3, No. 3 , 1 (1961)C. A . - 56, 11705b ( 1 9 6 2 ) . S . L. T o m p s e t t , A c t a p h a r m a c o l . e t . t o x i c o l . 2, 303 ( 1 9 6 8 ) . M . A . Mahju a n d R . P . M a i k e l , Biochem.

-7

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57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

Pharmacol. 18, 2701. C. N. Andres , J. A s s . O f f i c . Anal. Chem. L 51,1020 (1968) and i b i d 53,834 (1970) . C. Korczak-Fabierk iewicz and G. H. W. Lucas, Proc. Can. SOC. F o r e n s i c S c i . 4, 223 (1965) C. A . 63, 12972g (1965) .

D. L. So rby , E. M. P l e i n and J. D. Beumaman, J. Pharm. S c i . 55, 785(1966) . P. M. Seeman and H. S . B i a l y , Biochem. Pharmacol . , l2, 1181 (1963). G. Z o g r a f i and I. Zarenda, Biochem. Pharmacol. IS, 591 (1966) . K. S. Murthy and G. Z o g r a f i , J. Pharm. S c i . , 59, 1281 (1970) . A. F e l m e i s t e r and R. Schaubman, J .Pharm. S c i . 58, 64 (1969) . J. K r i e g l s t e i n and G. Kuschinsky, Naunyn-Schmiedebergs Arch, Pharmakol. Exp. Pa th . 1969, 262. J. Schwar t z , U. S . P a t e n t 2,997,468 (1958) .

N. H. Coy, C. M. F a i r c h i l d , B.T. Keeler , p r i v a t e communication. A. I . Cohen and G . M. J e n n i n g s , p r i v a t e communication. C. Korczak-Fabierk iewicz , C. Cimbura, J. Chromatog. 53, 413 (1970) . B. S. F i n k l e , E. J. Cherry and D. M. T a y l o r , J. Chromatog. S c i . - 9, 393 (1971) .

550

TRIMETHOBENZAMIDE HYDROCHLORIDE

Kenneth W . Blessel, Bruce C. Rudy , and Bernard Z. Senkowski

5 5 1

K. W. BLESSEL. 8 . C. RUDY, AND 8. 2. SENKOWSKI

INDEX

A n a l y t i c a l P r o f i l e - Trimethobenzamide Hydroch lo r ide

1. D e s c r i p t i o n 1.1 Name, Formula, Molecu la r Weight 1 . 2 Appearance, Co lo r , Odor

2. P h y s i c a l P r o p e r t i e s 2 . 1 I n f r a r e d Spectrum 2.2 Nuclear Magnet ic Resonance Spectrum 2.3 U l t r a v i o l e t Spectrum 2.4 F luo rescence Spectrum 2 .5 Mass Spectrum 2.6 O p t i c a l R o t a t i o n 2 . 7 M e l t i n g Range 2 .8 D i f f e r e n t i a l Scanning C a l o r i m e t r y 2 . 9 Thermogravimetr ic A n a l y s i s 2.10 S o l u b i l i t i e s 2 . 1 1 X-ray C r y s t a l P r o p e r t i e s 2.12 D i s s o c i a t i o n Constant


4 . S t a b i l i t y Degrada t ion


6. Methods of A n a l y s i s 6 . 1 Elemental Ana lys i s 6.2 Phase S o l u b i l i t y A n a l y s i s 6 .3 Thin Layer Chromatographic Ana lys i s 6.4 Direct S p e c t r o p h o t o m e t r i c Ana lys i s 6 .5 F luo rescence Ana lys i s 6 . 6 Non-Aqueous T i t r a t i o n

7. Acknowledgement

8. Refe rences

552

TRIMETHOBENZAMI DE HYDROCHLORIDE

1. Description

1.1 Name, Formula, Molecular Weight Trimethobenzamide hydrochloride is N-[p- [ 2- (di-

methyl amino)ethoxy]benzyl]-3,4,5-trimethoxybenzamide monohydrochloride.

/ H3C0

HCI

C21H28N205 HC1 Molecular Weight: 424.93

1.2 Appearance, Color, Odor Trimethobenzamide hydrochloride is a white

crystalline powder with a slight phenolic odor.


2.1 Infrared Spectrum The infrared spectrum of a sample of reference

standard trimethobenzamide hydrochloride is shown in Figure 1 (1). The spectrum was recorded on a sample of 1.0 mg of trimethobenzamide hydrochloride dispersed in 300 mg of KBr, using a Perkin Elmer 621 Spectrophotometer. The following assignments have been made (1):

Band Assignment 3308 cm-1 N-H stretch 2500 cm-1 region

1626 cm-l C=O stretch 1580 and 1496 cm-1 846 cm-l

Characteristic of hydrochloride of tertiary amine

aromatic rings 2 adjacent hydrogens on phenyl ring

2.2 Nuclear Magnetic Resonance Spectrum (NMR) The NMR spectrum of a sample of trimethobenzamide

hydrochloride is shown in Figure 2 (2). The spectrum was obtained by dissolving 57.2 mg in 0.5 ml of DMSO-d6 containing tetramethylsilane as the internal reference. The

5 5 3

K. W

. BLE

SS

EL, B

. C. RU

DY

, AN

D B

. 2. S

EN

KO

WS

KI

554

TR

IME

TH

OB

EN

ZA

MI D

E H

YD

RO

CH

LO

RID

E

7 1:

555

K. W. BLESSEL, B. C. RUDY, A N D B. Z. SENKOWSKI

s p e c t r a l a s s ignmen t s a r e g i v e n i n Tab le I ( 2 ) .

Table I

NMX Spectral Assignments f o r Trimethobenzamide Hvdrochloride*

No. Chemical Protons of Each Shift (ppm) Multiplicity Coupli,ig Constant

N, N-dimethyl

6-carbon of amino ethoxy group

4'-methoxy

3' and 5' methoxy

benzyl hydrogens on C1

a-carbon of amino ethoxy group

C2 and c6 hydrogens

C3 and C5 hydrogens

c 2 ~ and c6' hydrogens

amido hydrogen

hydrogen of hydrochloride

6

2

2

1

1

2.83

3.48

3.73

3.85

4.36

approx. 4.3

6.95

7.32

7.31

approx. 9.2

approx. 11.0

S

= 5Hz J~ -n

t B c r

S

S

d

d

d

S

t

S

(broad)

J ~ 6 - ~ J , J ~ 2 - ~ 4 = 8Hz

J ~ 3 - ~ 6 , J ~ 4 - ~ 2 = 8Hz

*An arbitrary set of numbers was assigned to the atoms in the structure to simplify the presentation of the data.

2.3 U l t r a v i o l e t Spectrum The u l t r a v i o l e t spectrum of t r imethobenzamide

h y d r o c h l o r i d e i s shown i n F i g u r e 3 ( 3 ) . The spectrum e x h i b i t s two maxima and one minimum i n t h e r a n g e of 200- 400 nm. The maxima are l o c a t e d a t 212 nm ( E = 4 . 7 x l o 4 )

556


Figure 3

Ultraviolet Spectrum of Trimethobenzamide Hydrochloride

. 5

.4

. 3 W 0 2 a m

E * v) m a

. I

N A N O M E T E R S

5 5 1

K. W. BLESSEL, B. C. RUDY, A N D 6 . Z . SENKOWSKI

4 and 258 nm (E = 1 . 2 x 10 ), and t h e minimum i s a t 238-239 nm. The c o n c e n t r a t i o n of tr imethobenzamide h y d r o c h l o r i d e was 0.404 mg/100 ml of 0.1N H C 1 .

2.4 F luo rescence Spectrum The e x c i t a t i o n and emiss ion s p e c t r a of a sample

of r e f e r e n c e s t a n d a r d t r imethobenzamide h y d r o c h l o r i d e are shown i n F i g u r e 4 ( 4 ) . The s p e c t r a were reco rded on methanol s o l u t i o n s of t r imethobenzamide h y d r o c h l o r i d e ( 1 mg/ml) u s i n g a Farand MK-1 r e c o r d i n g s p e c t r o f l u o r o m e t e r . E x c i t a t i o n a t 310 nm produced an emis s ion spectrum hav ing a maximum a t 341 nm.

2.5 Mass Spectrum The low r e s o l u t i o n mass spectrum of t r ime tho-

benzamide is shown i n F i g u r e 5 ( 5 ) . The spectrum w a s ob- t a i n e d u s i n g a CEC 21-110 s p e c t r o m e t e r w i t h a n i o n i z i n g energy of 70eV which w a s i n t e r f a c e d w i t h a Varian d a t a system 100MS. The d a t a system accep ted t h e o u t p u t of t h e s p e c t r o m e t e r , c a l c u l a t e d t h e masses, compared t h e i r i n t e n s i t i e s w i t h t h a t o f t h e b a s e peak and p l o t t e d t h e r e l a t i v e i n t e n s i t i e s a s l i n e s of v a r i o u s h e i g h t s .

The two predominant c l eavage p r o d u c t s occur at m/e 58, which co r re sponds t o CH2N(CH3)2, and m / e 195, corresponding t o t h e t r imethoxybenzoyl moiety. Other c h a r a c t e r i s t i c masses appea r ing i n t h e spectrum are m / e 330, which corresponds t o t h e l o s s of CH2N(CH3)2 from t h e molecu la r i o n , and m / e 317 co r re spond ing t o t h e l o s s o f C4H9N from t h e p a r e n t mass. The h i g h r e s o l u t i o n spectrum f u l l y confirmed t h e r e s u l t s of t h e low r e s o l u t i o n scan ( 5 ) .

The molecu la r i o n was a t m/e 388 ( f r e e b a s e ) .

2.6 O p t i c a l R o t a t i o n Trimethobenzamide h y d r o c h l o r i d e e x h i b i t s no

o p t i c a l a c t i v i t y .

2.7 Mel t ing Range The m e l t i n g range r e p o r t e d i n NF XI11 i s 187-

190°C when a Class I p rocedure is used (6 ) .

2.8 D i f f e r e n t i a l Scanning Ca lo r ime t ry (DSC) The DSC curve f o r a sample of r e f e r e n c e s t a n d a r d

trimethobenzamide h y d r o c h l o r i d e i s shown i n F i g u r e 6. The in s t rumen t used w a s a P e r k i n E l m e r DSC-1B u s i n g a

5 5 8

250


F i g u r e 4

Emission and E x c i t a t i o n S p e c t r a of Trimethobenzamide Hydroch lo r ide

I I \ Soectrum

300 350 400

NANOMETERS

450 500

559

K. W

. BLE

SS

EL, B

. C. R

UD

Y. A

ND

E. 2

. SE

NK

OW

SK

I

ii

r~

ii

ii

i

0

* 0

c)

0

(0

0

% 0

" 0

n

0

N

0

N

0

N

0

N

N

m

Hi-

0

'D 3

0 a ? a u a ? a > !

560


Figure 6

DSC Curve for Trimethobenzamide Hydrochloride

I 1 I I I I A H f = I1 0 k c a l / m o l e

56 I

K. W. BLESSEL, B. C. RUDY, AND B. 2 . SENKOWSKI

temperature program of 10°C/min. t h e mel t ing endotherm is 185.8OC w i t h t h e peak a t 188.8OC. A l l temperatures are c o r r e c t e d . The AHf was c a l c u l a t e d t o b e 11.0 Kcal/mole f o r t h e mel t ing endotherm ( 7 ) .

The e x t r a p o l a t e d o n s e t of

2.9 Thermogravimetric Analysis (TGA) The TGA s c a n of tr imethobenzamide hydrochlor ide

showed no s i g n i f i c a n t l o s s of weight up t o 23O0C a t a h e a t - i n g rate o f 10°C/min. se rved between 230-5OO0C d u r i n g which range 80% of t h e sample weight was l o s t ( 7 ) .

A cont inuous l o s s o f weight w a s ob-

2.10 S o l u b i l i t y The s o l u b i l i t y d a t a f o r a sample of r e f e r e n c e

s t a n d a r d t r inethobenzamide h y d r o c h l o r i d e a t 25OC i s given i n Table I1 ( 8 ) .

Table I1

Solvent

Petroleum E t h e r

Die thyl E t h e r Water 2-Propanol 3A Alcohol Chloroform 95% Ethanol Benzene Methanol

(30'-60')

Solub il i t y (mg / m l )

0 . 5

0 . 8

1 . 7 1 4 . 1 1 9 . 3 53.8

1 . 2 139.4

>500,

2.11 X-ray C r y s t a l P r o p e r t i e s The x-ray powder d i f f r a c t i o n d a t a o b t a i n e d f o r a

sample of r e f e r e n c e s t a n d a r d trimethobenzamide hydrochlor ide are given i n Table I11 (9) . The o p e r a t i n g c o n d i t i o n s of t h e ins t rument a r e given below.

Ins t rumenta l Condi t ions:


Genera tor : 50 KV, 12-112 MA Tube t a r g e t : Copper

5 62

T R I M E T H O B E N Z A M I D E H Y D R O C H L O R I D E

R a d i a t i o n : o p t i c s : 0.1' D e t e c t o r s l i t

Cu K, = 1.542 2

M.R. S o l l e r s l i t 3' B e a m s l i t 0.0007" Ni f i l t e r 4' t a k e o f f a n g l e

Goniometer: Scan a t 0.2O 28 p e r minute D e tec t o r : A m p l i f i e r g a i n - 16 c o a r s e ,

8.7 f i n e S e a l e d p r o p o r t i o n a l c o u n t e r t u b e and DC v o l t a g e a t p l a t e a u P u l s e h e i g h t s e l e c t i o n EL - 5 v o l t s ; Eu - Out Rate meter T . C . 4 2000 C I S f u l l scale Chart speed 1 i n c h p e r 5 minutes P r e p a r e d by g r i n d i n g a t room t empe r a t u r e

R e co r d e r :

Samples :

Tab le I11

I n t e r p l a n a r Spac ings from Powder D i f f r a c t i o n Data f o r Trimethobenzamide Hydroch lo r ide

1 2 -------a 28 dl(Ao) 1/1 - - 28 d(Ao> I/I,

4.86 12 .24 14.26 14 .58 15 .13 15 .70 16.64 17 .55 17 .86

18 .2 30 7.23 6 6 . 2 1 75 6 . 0 8 35 5.86 100 5.64 19 5 .33 64 5.05 1 2 4.97 12

31.13 2.87 8 31.30 2.86 7 31.75 2.82 1 9 32.50 2.75 29 34.44 2.60 18 35.30 2.54 3 35.56 2.52 3 36.00 2.49 4 36.96 2.43 2

n h 2 S i n e 'd = ( i n t e r p l a n a r d i s t a n c e )

111, = r e l a t i v e i n t e n s i t y (based on h i g h e s t 2

i n t e n s i t y o f 100)

563

K. W. BLESSEL, B. C. RUDY, AND B. Z . SENKOWSKI

1 8 . 3 6 1 9 . 1 8 19.49 20 .20 2 0 . 6 8 20 .96 2 1 . 3 8 22.27 2 2 . 6 3 23.03 2 3 . 5 3 24 .66 25 .02 25 .62 26 .12 2 6 . 4 8 2 7 . 0 8 27 .86 28.16 28.96 29.96 30.56

4 . 8 3 4 . 6 3 4 . 5 5 4 . 4 0 4 . 2 9 4.24 4.16 3.99 3 .93 3.86 3 .78 3 .61 3 .56 3 . 4 8 3 . 4 1 3.37 3.29 3 . 2 0 3.17 3.08 2 .98 2 .93

3 37 - 4 6 4 37.96

25 38.36 36 38 .94 1 8 39 .50 1 4 39 .85 4 5 4 0 . 4 6 6 3 4 1 . 0 0 34 4 1 . 4 8 6 8 4 1 . 9 6 35 4 2 . 8 2 4 5 4 3 . 7 2 16 45 .00 31 4 5 . 3 0 57 4 5 . 7 6 32 4 6 . 1 8 55 4 6 . 7 2 10 47.28

5 4 7 . 5 4 1 6 48.46 3 1 4 9 . 4 8

8 51 .15

2.40 2 2.37 5 2.35 3 2 .31 7 2.28 9 2 .26 5 2 .23 6 2 .20 4 2.18 7 2.15 3 2 .11 7 2.07 8 2 .01 4 2 .00 4 1 . 9 8 3 1 .97 4 1 .94 4 1 . 9 2 6 1 . 9 1 4 1 . 8 8 4 1 . 8 4 3 1 . 7 9 7

2.12 D i s s o c i a t i o n Constant The a p p a r e n t pKa of t r imethobenzamide hydro-

c h l o r i d e was determined by a p o t e n t i o m e t r i c t i t r a t i o n i n aqueous s o l u t i o n w i t h 0.02M KOH. The v a l u e o b t a i n e d w a s 8.27 - + 0 . 0 3 (10).

3 . S y n t h e s i s The r e a c t i o n sequence shown i n F i g u r e 7 w a s used t o

produce 1 k - l a b e l l e d t r imethobenzamide h y d r o c h l o r i d e and i s a l s o one of t h e s y n t h e t i c r o u t e s t o produce t h i s material(l2).

4 . S t a b i l i t y Degradat ion Trimethobenzamide h y d r o c h l o r i d e h a s been shown t o be

s t a b l e when h e a t e d a t 100°C i n a c i d , a l k a l i n e o r n e u t r a l s o l u t i o n ( 1 2 ) . I t was a l so found t o be s t a b l e when s t o r e d a t room t empera tu re € o r a pe r iod i n excess o f f i v e y e a r s ( 1 3 ) .


c h l o r i d e i n dogs h a s been c a r r i e d o u t u t i l i z i n g a A s t u d y of t h e metabolism of tr imethobenzamide hydro-

5 64


Figure 7

Synthesis of 1 4 C - label led Trimethobenzamide Hydrochloride

CH \ 3

cn

C H 3 /N-CH2-CH2-

' i -CH , 2 2 -CH -0 0 C 0 C l < S O C I ?

I CH 3 I

I CH 3 0 q O C H l

CH \ 3

C H I

565

K. W. BLESSEL, B. C. RUDY, A N D B. 2. SENKOWSKI

14C-labelled sample of the material (14). that approximately 30% of the dose was excreted intact while biotransformation of part of the remainder gave the mono-N-demethylated and the N-oxide analogs of trimethobenzamide hydrochloride as shown in Figure 8 (14).

It was found


6.1 Elemental Analysis The results of an elemental analysis of a refer-

ence standard sample of trimethobenzamide hydrochloride are presented in Table IV (15 ) .

Table IV


C 59.36 59.28 H 6.88 6.80 N 6.59 6.66 c1 8.34 8.34

6.2 Phase Solubility Analysis Phase solubility analyses have been carried out

for trimethobenzamide hydrochloride. in Figure 9. The solvent used was absolute ethanol and the equilibration time was 45 hours at 25OC ( 8 ) .

An example is shown

6 .3 Thin Layer Chromatographic Analysis A thin layer chromatographic method for the

separation of trimethobenzamide hydrochloride from its possible metabolites has been developed. The adsorbent wxi silira gel G and the solvent system was ethyl acetate:ethanol: NH40H (90: 10: 5 ) . for 1 5 cm . with potassium iodoplatinate detecting reagent (16). The approximate Rf values are given in Table V below.

The solvent front is allowed to ascend The plate is then air dried and sprayed

Table V

R -f Compound

Trimethobenzamide hydrochloride 0.9 mono-N-desmethyl metabolite 0.7 d i-N-desme thy1 met abo 1 i te 0.6

566

Figure 8

Metabolic Products of Trimethobenzamide Hydrochloride

Trimethobenzamide

N-desnothyl analog N-oxide analog

F i g u r e 9

14 1 1 1 1 I I I I 1 , I I 1 I 7

- -

h " V

- - n v v v

- -

P H A S E S O L U B I L I T Y A N A L Y S I S - SAMPLE . T R I M E T H O B E N Z A M I D E HYDROCHLORIDE - S O L V E N T : A B S O L U T E E T H A N O L

E O U l L l B R A T l O N : 4 5 HOURS a t 25'C - E X T R A P O L A T E D S O L U B I L I T Y : 10.96 m g / g of -

S L O P E . 0.18 % -

E T H A N O L - - -

I I I I I I I I I I I I I 1 I I J

c

m 0 n C 0 <


6 . 4 D i r e c t Spec t ropho tomet r i c Ana lys i s Trimethobenzamide h y d r o c h l o r i d e may be determined

d i r e c t l y i n c a p s u l e s by u t i l i z i n g t h e p rocedure which f o l - lows.

An amount of c a p s u l e powder e q u i v a l e n t t o approx- i m a t e l y 50 mg of tr imethobenzamide h y d r o c h l o r i d e is weighed i n t o a 100 m l vo lumet r i c f l a s k . The sample i s d i s s o l v e d and d i l u t e d t o volume w i t h 0.1N H C 1 , a f t e r which i t i s f i l - t e r e d . A 4 ml a l i q u o t of t h e f i l t r a t e i s t r a n s f e r r e d t o a 100 m l v o l u m e t r i c f l a s k and d i l u t e d t o volume w i t h 0.1N H a . The absorbance o f t h e s o l u t i o n i s measured a t 258 nm u s i n g 0.1N H C 1 as a r e f e r e n c e . The amount of tr imethobenzamide h y d r o c h l o r i d e i s c a l c u l a t e d by comparison t o a sample of t h e r e f e r e n c e s t a n d a r d measured i n a s i m i l a r manner ( 6 ) .

6 . 5 Fluorescence Analysis Trimethobenzamide h y d r o c h l o r i d e may be d e t e r -

mined by way o f t h e c h a r a c t e r i s t i c f l u o r e s c e n c e of t r i - methoxybenzoic a c i d which i s a p roduc t of h y d r o l y s i s .

f o r 1 7 h o u r s . The l i b e r a t e d t r imethoxybenzoic a c i d i s ex- t r a c t e d i n t o e t h e r , t h e e t h e r evapora t ed and t h e r e s i d u e t aken up i n a pH 2 b u f f e r . The f l u o r e s c e n c e i s measured a t 370 nm and 100 nm u s i n g an a c t i v a t i n g wavelength of 290 nm. The method i s a p p l i c a b l e t o t h e d e t e r m i n a t i o n o f tr imethobenzamide h y d r o c h l o r i d e i n u r i n e and b lood a f t e r e x t r a c t i o n of t h e m a t e r i a l i n t o chloroform ( 1 7 ) .

The h y d r o l y s i s i s maximal u s i n g 1.5N H C 1 a t 100°C

6 . 6 Non-Aqueous T i t r a t i o n The non-aqueous t i t r a t i o n of tr imethobenzamide

h y d r o c h l o r i d e w i t h p e r c h l o r i c a c i d a s d e s c r i b e d i n NF X I 1 1 i s t h e method of c h o i c e f o r t h e a n a l y s i s of t h e bu lk m a t e r i a l . The sample i s d i s s o l v e d i n g l a c i a l a c e t i c a c i d w i t h t h e subsequent a d d i t i o n of mercu r i c a c e t a t e T . S . and c r y s t a l v i o l e t i n d i c a t o r . The t i t r a t i o n i s performed w i t h 0 . 1 N H C 1 0 4 i n g l a c i a l ace t ic a c i d . Each m l of O . 1 N HC104 i s e q u i v a l e n t t o 42.49 mg of thri.methobenzamide h y d m c h l o r i d e ( 6 ) .

7 . Acknowlegement The a u t h o r s wish t o acknowledge t h e Research Records

Department of Hoffmann-La Roche I n c . f o r t h e i r h e l p i n t h e l i t e r a t u r e s e a r c h f o r t h i s a n a l y t i c a l p r o f i l e .

569

K. W. BLESSEL, 6. C. RUDY, A N D 6. 2. SENKOWSKI

8. References

1.

2.

3.

4.

5.

6. 7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal Communication. Johnson, J. H., Hoffmann-La Roche Inc., Personal Communication. Rudy, B., Hoffmann-La Roclie Inc., Personal Communication. Boatman, J. , Hoffmann-La Roche Inc., Personal Communication. Benz, W., Hoffmann-La Roche Inc., Personal Communication. The NationaZ FormuZary XIII, pp. 737-740 (1970). Moros, S. , Ho f fmann-La Roche Inc, , Personal Communication. MacMullan, E . , Hoffmann-La Roche Inc., Personal Communication. Hagel, R., Hoffmann-La Roche Inc., Personal Communication. Motchane, A . , Hoffmann-La Roche Inc., Personal Communication. Wineholt, R. L., Johnson, J. D., Heck, P. J. and Kaegi, H. H., Journal of LabeZZed Compounds, 5, 53 (1970). Schmall, M., Hoffmann-La Roche Inc., Unpublished Data. Clyburn, T. , Hof fmann-La Roche Inc., Unpublished Data. Schwartz, M. A . et al., Hoffmann-La Roche Inc., Unpublished Data. Scheidl, F., Hoffmann-La Roche Inc., Personal Communication. Koechlin, B., Hoffmann-La Roche Inc. , Personal Communication. Koechlin, B., Hoffmann-La Roche Inc., Unpublished Data.

5 70

ADDENDA

Tr iamc ino lone :

6 . 6 . T i t r i m e t r i c D e t e r m i n a t i o n w i t h Lead ( I V ) - a c e t a t e a l s o i n t h e p r e s e n c e o f

T r i amc ino lone a c e t o n i d e : E . C s i z e r , S . Giir6g and T. Szen , Microchimica Acta 1970, 996.

/

f

7. I d e n t i f i c a t i o n by GLC: B. S . F i n k l e , E . J. Cher ry and D. M. T a y l o r , J. Chromatog. S c i . 9 , 393 ( 1 9 7 1 ) . Plasma l e v e l s : M. Kusama, N. S a k a u c h i , S . Kumaoka Metab. C l i n . Exp. 2 0 , 590 (1971) .

T r i amc ino lone Ace ton ide : 6 .53 . P a r t i t i o n c o e f f i c i e n t € o r n-hexane-

ch loroform-dioxane w a t e r (90:10:40:5) s y s t e m : D. J. Weber, T . R. Enna l s and H. Mitchne r J. Pharm. S c i . 61, 689 ( 1 9 7 2 ) .

7. Plasma l e v e l s : M. Kusama, N. S a k a u c h i , S. Kumaoka Metab. C l i n . Exp. 20, 590 ( 1 9 7 1 ) .

57 1

ERRATA

A c e t o h e x a m i d e V o l . 1 p. 7 F i g . 4 TGA s p e c t r u m n 3 t DTA s p e c t r u m .

F i g . 5 DTA s p e c t r u m n o t TGA s p e c t r u m ,

E r y t h r omyc i n E s t o 1 a t e V a l . I T?. 1 0 3 E r y t h r o m y c i n n o t E s t r o m y c i n ( l e g e n S u n d e r s t r u c t u r e ) .

Ha l o t h a n e p. 1 2 8 l i n e 4 : Change formlala to : ~ - _ -

lOgl0P= 6 . 8 5 1 3 - 1 0 8 2 . 4 9 5 t + 2 2 2 . 4 4

Leva t e r e n o 1 B i t a r t r a t e V o l . I p . 1 5 3 , l i n e 8: l o n g n o t l o n e

l i n e 12-14: r e v e r s e t o na = 1 . 5 3 1 2 0 . 0 0 2 nB = 1 . 5 7 7 2 0.002 ny = 1 . 6 2 0 ? 0 . 0 0 2

1 5 3 ( 1 7 ) : not 153 ( 7 ) l i n e 2 2 : d e l e t e f i r s t 1 6 9 l i n e 24-26: I n s t r u m e n t u s e d : JEOL Model IMS015SC. E i e c t r o n beam 75 e . v .

p . 159, l i n e 13: d e l e t e f i r s t 1 6 9 ; also

p. 171, l i n e 6 : R. K. K u l l n i g

N o r t r i p t y l i n e H y d r o c h l o r i d e V o l . I p . 2 4 3 , l i n e 9 a n d 10:

Bu0H-I.5%HCO2H - H 2 0 (12:1:7)

573

CUMULATIVE INDEX

Acetohexamide, 1, 1; 2, 573 Ampicillin, 2, 1 Chlorprothixene, 2, 63 Chloral Hydrate, 2, 85 Chlordiazepoxide, 1, 15 Chlordiazepoxide Hydrochloride, I , 39 Clidinium Bromide, 2, 145 Cycloserine, 1, 53 Cyclothiazide, 1 , 66 Dexamethasone, 2 , 163 Diazepam, 1, 79 Dioctyl Sodium Sulfosuccinate, 2 , 199 Erythromycin Estolate, 1 , 101; 2, 573 Fluorouracil, 2 , 22 1 Fluphenazine Enanthate, 2 ,245 Fluphenazine Hydrochloride, 2, 263 Halothane, 1 , 119; 2 , 5 7 3 Isocarboxazid, 2 , 295 Isopropamide, 2 , 3 15 Levallorphan Tartrate, 2, 339 Levaterenol Bitartrate, 1, 149; 2, 573 Meperidine Hydrochloride, 1 , 175 Meprobamate, I , 209

Methyprylon, 2, 363 Nortriptyline Hydrochloride, 1 ,233 ; 2 ,

Phenelzine Sulfate, 2, 383 Potassium Phenoxymethyl Penicillin,

Primidone, 2, 409 Propiomazine Hydrochloride, 2 , 4 3 9 Propoxyphene Hydrochloride, 1,301 Sodium Cephalothin, I , 319 Sodium Secobarbital, 1, 343 Sulfamethoxazole, 2 , 4 6 7 Sulfisoxazole, 2, 487 Triamcinolone, 1 ,367 ;2 ,571 Triamcinolone Acetonide, 1,397;2,571 Triamcinolone Diacetate, I , 423 Triclobisonium Chloride, 2, 507 Triflupromazine Hydrochloride, 2 , 523 Trimethobenzamide Hydrochloride, 2 ,

Vinblastine Sulfate, 1 , 4 4 3 Vincristine Sulfate, 1 , 4 6 3

573

1, 249

55 1

575

Analytical Profiles of Drug Sub stances Volume 2 Edited by Contributing Editors

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