E3TKRS OF OENTIS1C ACID AND THKIR TOXICITIKS

DISSERTATIONPresented in Partial Fulfillment of the Requirements

for the Degree Doctor of Philosophy in the Graduate Sohool of The Ohio State

University

ByJ. FRANK NASH, B.S.

The Ohio State University 1052

Approved by:

Adviser

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TABLK OF CONTENTSPage

INTRODUCTION......................................... 1DISCUSSION OF THE LITERATURE......................... 3EXPERIMENTAL........................................ 11N^w Gentlalo Acid Esters............................ 22

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Die thy lam lno ethyl Oentlsate Hydrochloride.......... 22i

a. Preparation................................... 22b. Properties.................................... 27

^)lethylaminoethyl 5-Aoetyl-Oentiaate Hydrochloride.. 28a. Preparation................................... 28b. Properties.................................... 32

pie thylamlnoethyl 5-Methoxy-Gentisate Hydrochloride. 33a. Preparation................................... 33b. Properties.................................... 35i

pie thylaminoethyl 5-Bensoy1-Gentiaate Hydrochloride. 36i a. Preparation................................... 36b. Properties.................................... 38

)le thylaminopropyl Oentlsate Hydrochloride......... 39a. Preparation................................... 39b. Properties.................................... 41

tiethylaminopropyl 5-Aoety1-Gentiaate Hydrochloride. 42! a. Preparation................................... 42b. Properties.................................... 43

piethylaminopropyl 5-Methoxy-0entlsate Hydrochloride 44a* Preparation................................... 44

i| b. Properties.................................... 45

80945<i

illPag®

Die thylaminopropyl 5-benzoyl-Gentlsa te Hydrochloride 46a. Preparation.................................... 46ba Properties..................................... 47

5-PhenylaoetyJ^Gentisic A d d ....................... 48aa Preparation................................... 48b« Properties.................................... 50

5-Paranfcrobentoy1-Qentisle Acid.................... 51a« Preparation................................... 51b a Properties.................................... 52

5-Anisyl^Oentislo Acid............................. 53a. Preparation................................... 53b. Properties.................................... 55

5-Aoe tylsalioyl**Gentiaio A d d ...................... 56a. Preparation................................... 56b a Properties.................................... 57

5,&'-Sueolnyl*>Dlgentlalo Acid...................... 58a# Preparation................................... 58b a Propertlea.................................... 60

Proof of the Structure of the Synthesised Esters bythe Analyses of their Infrared Speotra.............. 61

Infrared Speotra and Interpretation.............. 65Toxicity Teats...................................... 79

Toxicity of Carboxyllo Aoid Esters of Gentialc A d d 81 Toxicity of Phenolic Eaters of Gentislc Aoid...... 87

SUMIAKY.............................................. 93COW3UJ3IOMS........................................... 103BIBLIOGRAPHY.......................................... 108ATTTORT (MR A PHY ................... 112

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INDEX TO T ABIES Tablet Page

I. Wave Length of absorption by functional groupsIn the Infrared speotra....................... 65

II. Analyses of Infrared Speotra of Known Compounds 68III. Analjses of Infrared Speotra of Die t^amino-

ethanol Esters of Gentlsio Aoid............... 71IV. Analyses of Infrared Speotra of DiettxAamino­

ethanol Waters of Gentlsio Aoid 75V. Analyses of Infrared Speotra of Phenolic Esters

of Gentlsio Aoid.............................. 75VI. I2>5q of Diethylamlnoethyl Gentlsate Hydro­

chloride...................................... 81VII. U>go Diethylamlnoethyl 5-Aoetyl-Gentlsate

Hydro ohlor Ida................................. 81VIII. ID^q of Diethylamlnoethyl 5-Methoxy-Gentlsate

Hydroohlorlde ............................... 81IX. ID5Q of Die thylamlnoe thyl 5-bensoyl-Gentlsate

Hydroohlorlde................................. 82X. IDgQ of Die thylaminopropyl Gentlsate Hydro­

ohlorlde...................................... 82XI. IDgQ of Diethylaminopropyl &-Aoetyl-Gentlsate

Hydroohlorlde................................. 82XU. XD50 of Die thylaminopropyl 6-Methoxy -Gentlsate

Hydroohlorlde .... 84XIII. XD50 °f Die thylaminopropyl 5-bensoyl-Gentlsate

Hydroohlorlde................................. 84

87878788888899

101

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IDp- of 5-Phenylacetyl-aentisic Acid,... ouIO ^ of 5-Paranltrobensoyl-Gentisio AoidU>_~ of 5-Anisyl-Qentlsio Acid.........ouU)^n of 5-Aoetylaalioyl-Qentisic Aoid,.. 50IZ)gg of 5,5'-Suoolnyl-Digentlaio Acid...IDgQ of Oentlslc Aoid..................Analyses of Synthesised Compounds,.....Properties of Synthesised Compounds....

ViIHDKX TO FIGURES

Figurai Page1. Infrared Spectrum of Gentlsio Acid.............. 662* Infrared Spectrum of 5-Aoetyl-Gentisic Acid 663. Infrared Spectrum of 5-Methoxy-Oentlslc Acid.... 664. Infrared Spectrum of 5-Benaoyl-Gentlslo Acid.... 675. Infrared Speotrum of Dlaoetyl-Oentlaio Aoid 676. Infrared Speotrum of Diethylamlnoethyl Gentlsate

Hydrochloride.................................... 677. Infrared Speotrum of Diethylamlnoethyl 5-Acetyl-

Gentiaate Hydrochloride.......................... 698* Infrared Speotrum of Die thylamlnoe thyl 5-

lle thoxy-Gent lea te Hydroohlorlde.................. 699. Infrared Speotrum of Die thylamlnoe thyl 5-

Bencoyl-Gentlsate Hydroohlorlde*................. 6910* Infrared Speotrum of Diethylaminopropyl Genti-

sate Hydroohlorlde*.............................. 7011* Infrared Speotrum of Die thylaminopropyl 5-

Aoe tyl-Gentlsate................................. 7012* Infrared Speotrum of Diethylaminopropyl 5-

Methoxy-Gentlsate Hydroohlorlde*................. 7013* Infrared Speotrum of Die thylaminopropyl 5-

Bensoyl-Gentlsate Hydroohlorlde*................ 7214* Infrared Speotrum of 5-Phenylaoetyl-Gentlelo Aoid 7215* Infrared Speotrum of 5-Paranltrobensoyl-Gentlslo

Aoid............................................... 72

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Figure Page16. Infrared Speotrum of 5-Anlsyl-Gentislc Acid... 7417. Infrared Speotrum of 5-Aoetylsalloyl-Gentlslc

Aoid........................................... 7418. Infrared Speotrum of 595'-3ucolnyl-Digentisio

Aoid........................................... 7419. Graph of U>5q of Diethylamlnoethyl Eaters of

Qentlsio Aoid and Substituted Gentlsio Aoids.. 8320. Graph of IDqq of Diethylaminopropyl Esters of

Gentlsio Aoid and Substituted Gentlsio Aolds.. 8521. Graph of I D ^ of Fhenollo Esters of Gentlsio

Aoid........................................... 8922. Graph of I D ^ of Gentlsio Aoid................ 90

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ACKNOWLEDGMENTS

I wish to acknowledge with gratitude the advice and suggestion* of Dr. Bernard V. Christensen, Dean of the College of Pharmacy, In the development of this thesis.

To Dr. Frank W, Bope I am greatly Indebted for the thought, time, and patlenoe while this thesis was developed.

I also wish to acknowledge the advise and suggestionsof Dr. John W. Nelson In the development of the toxicityprocedure.

I acknowledge the suggestions of Dr. Melvin 3. Newman, of the Chemistry Department, In the development of the ayntheaes.

A special note of appreciation is extended to Dr. Christopher Wilson, of the Chemistry Department, for use of the Infrared laboratory.

The financial aaslstanoe of the Upjohn Company, In the form of a fellowship, and of the United States Government, in the nature of Public Law 346, is responsible in a large measure for this thesis.

I wish to thank the Eli Lilly Company for the Carbonand Hydrogen analyses.

To all others who have assisted In any way, I wish to express slnoere thanks and grateful appreciation.

J. Frank NashJune, 1952

KSTRKS OF ORNTISIC ACID aND THKIF T0XICIT1KS

INThODUCTIQN

The preparation of some new eaters of 2,5-dihydroxy- bensolo acid or gentlsio aoid whloh offer possibilities of being of medicinal value as antirheumatioa, antlarthritics, or analgeslos was suggested by several factors. First, in spite of the wide attention given to adrenal cortical hor­mones, there is still a great need for good medicinal agents for the treatment and symptomatic relief of rhuematic and arthritic disorders. Since treatment of these disorders is usually of long duration, it is desirable to use medl- olnal agents of low toxicity, and also agents that will produce a minimum of alteration of normal physiologic functions* Compounds suoh as the sallcy~„ates have been used in this oapaolty for many years.

A second factor suggesting this work is the Increasing use of sodium gentlsate In the treatment of rheumatic condi­tions and as an analgetic.

Still another factor suggesting preparation of some esters of gentlsio a d d is the apparent need for an efficient non-addicting analgeslo stronger than aspirin yet weaker than the opiates.

The groups chosen to be coupled with gentlsio acid were groups of established pharmacological activity. Diethylam­lnoe thanol has pronounced analgeslo effect without any toxic

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side-aotions when injected intravenously (1). Also, diethylamlnoethanol appears to have analgesic properties when given orally or intramuscularly (2). This grouping is also prominent in local anesthetios, antispasraodics, and antihistaminios. Diethylaminopropanol was used to determine the effect of an additional oarbon in the alcohol.

Aoetylsalloyllc and suooinlc acids are both analgesics of proven medicinal value.

Phenylaoetlc aoid esters have proven antlspasmodlc activity and phenylalkylamlnes are analgesic (3).

Paranitrobensolc aoid and more particularly its reduc­tion product, paraamlnobenzolo aoid, has a variety o', pharmocologioal properties among which are local anesthetlo and anti-infective effects.

Anisic sold or parsmethoxybensolo acid is reported to have antirheumatio and local anesthetic activity (4).

Aoetyl, methoxy and bensoyl groups were substituted in the oarboxylio aoid esters because of the proven value of these functions in enhancing the analgeslo value of phenolic compounds.

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DISCUSSION OF THB LITERATURE

Although salicylic aoid and aodium aalloylate have been uaad In the treatment of rheumatic conditiona alnoe 1376 and 1877, respectively (6), aalloyllo aoid'a oxidation product, gentlalo add, remained unknown medio in ally until September, 1048 when Meyer and Ragen reported the uae of aodlum gentlaate for the relief of rheumatic aymptoma (6 ). Since then, numeroua reporta from the Western Hemisphere have substanti­ated the original belief of Meyer and Ragen.

The specific effeot of salicylates upon the activity ofhyaluronldase and aalloylate action In rheumatic conditions date back to the work of Ouerra In 1946 (7) (8). Ouerrabelievea rheumatlo fever to be a disease of the mesenohymatissues. During the evolution of the disease, whether of baoterlal or endogenous origin, permeability ohangea take plaoe pointing to Interrelationship of several characteris­tics of rheumatism and the spreading faotors of connective tissue.

The Importance of the spreading effeot of hyaluronldase on connective tissue Is based on the observation of Meyer and Palsnr (9), who pointed out that hyaluronic aoid Is the principal substrate of connective tissue and mucoid struc­tures, Connective tissue and mucoid structures compose almost entirely the regions affeoted by rheumatism, such as articulations and synovial fluid.

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Durin-Reynals (10), MoClean (11), Kandall and associates (12), and especially Crowley (13), have observed that several aioroorganlsas Including more than 200 strains of hemolytic streptoool produce or possess hyaluronldase.

Ouerra showed that hyaluronldase of bacterial origin or froa teatloular extract, in ljt dilutions, increased the spread of Bvane Blue In humans and India Ink In rabbits. The spreading resulted froa ensyaatlo aotlvlty which hydrolysed the hyaluronlo sold present* The viscosity was deoreased and, therefore, the passage of liquids, exudates and patho- genlo aloroorganlsas was favored (7).

Sodlua aalloylate adalnistered orally or Intravenously In dosage of 0*072 g./kg* oaused 57jt reduotion of skin dif­fusion effeot of hyaluronldase in rabbits, compared to saline controls* A dose of 0*10 g*/Kg*, by the same routes of adalnlstratlon, oaused 60£ reduotion of diffusion effeot. Moreover, huaans who had contaoted rheumatic fever, upon lntradermal injection of a mixture of hyaluronldase with ■vans Blue gave unique reaotlons with enormous diffusion of the dye and looal edema that some times oooupled the era injeoted* Salloylates in these oases inhibited the nativity of the ensyae and reduoed Its spreading effeot in connective tissue* Ouerra, therefore believed that the aotlvlty of the salloylates was due to the Inhibition of the aotlvlty of hyaluronldase (8).

It has been deaonstrated that salicylates do not inhibit

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the aotlvlty of hyaluronldase In as low concentrations In vitro as they do In vivo (6), (14), (15), (16), (17),Swyer (17), by using the visooslaetrlo teohnlque In vitro, recorded no dlreot Inhibitory effeot on the aotlvlty of hyaluronldase by sodium aalloylate or aoetylsalloyllo aoid exoept at relatively high concentrations, l.e, greater than 3$ and 0,539t, respectively. Such Inhibition at these con­centrations Is apparently due to lowering of pH and. In the oaam of sodium aalloylate, to the Increased oonoentratlon of salt, Swyer believed the results obtained by Ouerra were due to the presenoe of histamine or some substance of similar action, and that sodium salicylate, by Inhibiting the activity of the substance reduced the apparent spreading effeot caused by hyaluronldase,

lfeyer and Ragen (6 ), (13) claimed that the Inhibitionof hyaluronldase activity was due to the fact that salloy­lates are oonverted to gentlsio aoid which acts as the true inhibitor. The seml-qulnone formed condenses in an Irrever­sible manner with the enzyme, Salloylates are known to yield from 4 to 6^ gentlsio aoid (5), (19), (20), Thissubstantiates the statement that 2,5 bensoquinone oarboxylio aoid Inhibits the aotlvlty of hyaluronldase (21),

However, Roseman et al (22) showed pure analytical gentlsio aoid to be devoid of hyaluronldase Inhibitory activity. The crude gentlsio acid as well as pure gentlsio aoid treated with alkali in the presenoe of air did show

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aotlvlty. Similar results were obtained with homogentlslo aoid. Therefore, It was concluded that the aotlvlty of gentlsio aoid was probably due to Impurities in the product which have been formed by oxidation. The work of Lowenthal and Gagnon (23) indicated the same laok of Inhibitory aotlv­lty of gentlalo aoid.

A Hating of products that inhibit the aotlvlty of hyaluronldase shows the aotlvlty of phenol substituted produo te inoreases with inorease In length of the hydro- oarbon ohain. The salloylates and gentlsates were oonsldered inactive in vitro (24).

Guerra also showed that glucuronic aoid excretion inoreases in the urine, upon lnjeotlon of salloylates. How­ever, gentlalo aoid diminishes the exoretlon of this product. Slnoe hyaluronic aoid Is composed of D-gluo os amine, gluouronic aoid and N-aoetylgluoosamlne, the action of gentlsio aoid ap­pears to be desirable in respect to exoretlon (25).

Another oause of rheumatoid arthritis has been thought to be hormone imbalanoe (19). The salloylates may stimulate the adrenal oortex oauslng liberation of oortlooid substanoe. Adrenal oortloal stimulation oan be demonstrated in rata and guinea pigs by an Inorease in exoretlon of urinary 12 ketosterolds and a deorease In olroulatlng eosinophils (26). However, a deorease In olroulatlng eosinophils oould not be found after administration of salloylates or gentlsates to

7

four medloal students. Thlebst et al, (27) trying to corre­late rheumatlo arthritis and permeability of the synovial meatorane showed sodium aalloylate retards permeability more than sodium gentlsate, The retardation was based upon the Injeetlon of phenglphthaleln Into the lntrartloule and then raoordlng the time for appearanoe of urinary coloration. The retardation has a favorable action In rheumatle arthritis.The meohanlam of aotlon was oonoluded to be oonneoted with the function of the suprarenal oortex.

Slnoe 1948 there has been extensive work done in the United States* France, Britlan, and Italy In regard to the use of gentlsates for rheumatlo conditions and analgesia.In rheumatlo fever* Meyer and Ragen (6) report uniformly good results using sodium gentlsate therapy. The administration of sodium gentlsate was followed by disappearance of pain, alleviation of swelling and heat In joints, lowering of temperature to normal and by the falling of the sedimentation rate to normal. In siost oases the disease was alleviated for five to ten months after removal of sodium gentlsate therapy. However* even In those oases where the disease returned in several days* It oould again be relieved by administration of sodium gentlsate, Schaefer et al (28) and others (29) also obtained good therapeutic results w2th sodium gentlsate In rheumatlo fever,

Meyer and Ragen (6 ) also used gentlsates to treat patients afflloted with rheumatoid arthritis. Results were favorable.

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Came 11 n, et al (5) as wall as Ory (50) and others (51) working with large groups of patients obtained encouraging results In rheumatoid arthritis and Boulllard disease. Ory also reported sodium gentlsate to be valuable in gout.

Bat ter wan, et al (32) studied the analgeslo effects of sodium gentlsates In approximately 100 oases. Control of pain regardless of severity, diagnosis, or dosage adminis­tration for hospitalised patients was obtained in 7 of the trials, without a single oase of toxlolty. Satisfactory analgesia ooourred in 57)C of the ambulatory patients. Un­toward reactions were noted In 11$£ of the ambulatory patients, but this was within the experimental range of placebos.

31noe the aotlvlty of sodium gentlsate Is In the same range as that of salicylates all the reports on aotlvlty are relative te that of sodium salicylate. According to the literature the gentlsates offer the following advantages over the salloylates In the above diseases. JL. Sodium gentlsate removes one step in the metabolic process. Salloylates are oxidised to gentlsates before they are ef- feotlve, so by giving sodium gentlsate we give the active drug. 2, Sodium gentlsate Is not as Irritating as the salloylates to the gastrlo mucosa. The gastric system ap­pears to be able to take In an unlimited amount. £. Intra­venous Injection causes no sclerosis; muscular tolerance Is good, and the renal functions are not affeoted. No oasehas yet been reported showing the development of tinnitus or

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signs of salloyllam resulting fron use of sodium gentlsate.No aural symptoms or inoreased prothrombin time has been reported. 5. There are no Indications that sodium gentlsate oauses sMthemogloblnemla or liver damage. £. The inorease In urinary gluouronlo aoid exoretlon observed with aalloylate Ingestion does not ooour elth sodium gentlsate.

In spite of nmwrous reports to the contrary, Caprettl and Ardulnl (55) report gentlsate therapy to be less effec­tive than aalloylate regime in the treatment of Boulllard disease.

Although reoently gentlsio aoid has been reported to disappear from the body by urinary exoretlon and metabolism to unknown produots (19) (20) as early as 1895 Llkhatsohaff (54) reported a urinary Inorease of ethylsulfurio aoid after administration of gentlsio sold to dogs. The same result was obtained by Staub and associates (55) In 1932.

The only extensive report on the toxlolty and pharma­cology of gentlsio sold was by Oinoulhlao and associates (56). The report lnoludea the oomparlson of sodium gentlsate and sodium aalloylate. The U>6q for rats by intraperltloneal injection was 5*1 g./Kg. and 0.88 g./Kg respectively.Orally, the I D ^ for sodium gentlsate oould not be determined beoause the drug was well tolerated even at the dosage level of 5 g./Kg. However, the oral ID53 for sodium aalloylate was found to be 2.02 g./Kg.

The two drugs were screened for several activities.

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lm Neither drug showed noticeable action on smooth muaols fibers* 2, Sodium gentisste showed little or no hyperten- aIts aotlvlty exiept at high dosage levels, but sodium salioylate gave response of higher nature and longeriduration. S. Neither drug exhibited notable antlhistamlnlc aotion. 4. Both drugs showed anti-edema activity but sodium aalloylate was the more active. £. A alight inorease of bile flow was produced by both drugs in doses of 100 mg./Kg. <5. The mloroblologloal aotlvlty of sodium gentlsate against several strains Of bacteria and fungi was very moderate or nil and inferior, to that of sodium salioylate in equal oon- oentratlons of liquid medium.

In spite ofithe fact that over 100 substituted acids, phenolic esters And oarboxyllo acid esters have been prepared none of these derivatives have been tested pharmacologically to determine if they have therapeutic value (37). However, in 1961 a U* S* patent was issued to Hoffer in which he pre­pared complexes of gentlsio acid with antlpyrlne, lsopropyl- antlpyrine and 4-dlmethylaalno antlpyrlne to yield compounds that possessed a^Dtlpyretlo and antlrheumatio aotlvlty (38). These products wfre also patented in Oermany (39).

gentlsio a d d ooomierolally available Is the sodium salt* Tht dosage fora is compressed tablets of seven grains* A disouision on tablet preparation of sodium gentlsate is given by Lauter and associate (40).

The fora of

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KXgBRIMBBTAL

Katers of gentlalo aoid or 2,5 dihydroxy bensolo aoid oan be prepared either with the oarboxylio aoid function or with the phenollo function of the compound. Betera of the amino aloohola are of the first type, whereas the esters of aolds oonfora to the latter type.

Carboxyllo aoid eaters may be synthesised as follows:lm Interaction of an Aoid Chloride and Amino Aloohol.flcoq + loccsjfeMCRJt Rco2(ciipx§ab2R »Alipietic or Aromatic

The general procedure la to reaot equlraolar portions of the aoid ohlorlde and the amino aloohol In an Inert solvent suoh as benaene. The hydroohlorlde of the ester usually preolpltates from the reaotlon mixture either as an oil or in crystalline form. If a solid, the product oan be purified by reorystsd.llsation from a suitable solvent. However, If the product la an oil, the solvent Is removed under vaouum and the salt la generally converted Into Its base by use of alkali. The aqueous solution la then extracted with ether several times. The ether la removed under vaouum, leaving the baalo eater, mienever possible the eater is distilled at reduoed pressure. The produot la then welghed, plaoed in a suitable solvent (i.e. ether or benzene) and treated with one equivalent of an anhydrous aloohollo or ethereal solution of hydrogen chloride. The use of exeess hydrogen ohlorlde, nay delay or inhibit crystallisation and result in the

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separation of an oil which remains as suoh Indefinitely.Other salts suoh as the sulfate, hydrobromlde and citrate may be prepared in a similar manner (41).

Aoids which oontaln additional funotional groups suoh as alcohols, phenols and/or amines oan be made Into aoid ohlorlde* only when these groups are protected In the usual manner (i.e. formation of aoetates or oyollc aoetals). After esterfloatlon the protective groups oan be removed.

£• Interaction of an Acid with a Chlorallcamine.

R "AKjWt* orHorensteln and Fahllohe (42) proposed this method for

syntheses of basic esters. Bqulmolar quantities of a oar- boxyllo aoid and ohloralkamlne are condensed In one of two ways. The oondensation oan be carried out by mixing the halide with a Isopropanol solution of the acid and refluxlng the mixture for several hours. The hydroohlorlde may pre­cipitate from the solvent and oan be purified by reorystal- llsatlon, many time* from the same solvent. However, If the salt Is soluble In the solvent. It is advisable to remove the solvent, liberate the * star base with alkali and proceed as under section 1,

An alternate prooedure Is to add the halide to an alco- hollo solution of the aoid, remove the aloohol under reduced pressure and heat the residue lO hours at 90° to 100°. The gummy mass obtained oan be extracted with acetone to remove

13

the unreaoted Materials and excess solvent. The salt of the ailns whloh la Insoluble remains and la filtered off. The salt oan be recrystallised from a suitable solvent.

The method Is suitable for esterlfioatlon of aolds containing reaotlve groups or esterlfIoatlon where the sold ohlorldes are Inherently unstable or dlffloult to prepare (43).

3_. Interaction of the Sodium or Silver Salt of a Carboxyllo Aoid and a Chlorallcamlne Hydroohlorlde.

RCCfcda o r Aj ♦ O (CM * g (A)* > RCo cn^ e N<*Ja + NeCJor

ft • Alifkmti* o r A reeatle

Horenstain and Fahlloke (42) reacted these materials In the absenoe of a solvent. The reaotants were heated gener­ally to 130°or 140° for several hours. The mass was ex­tras ted with a suitable solvent while hot and the compound orystalllsed fro* the solvent.

4. Reaction of an Alkyl Bster with an Amino Alcohol.ROOjtR* + *XClfcfctN(AJ!t M y * ) *CO,(CN*)* +• R'oWR> Ali'plkta Or hrom sher * MifkmiiCThe prooess oonslsts of heating the methyl or ethyl

ester of the aoid with an exoess of the dlalkylamlnoalkanol, usually In the presenoe of a small amount of sodium alkoxlde, In suoh a manner that the lower boiling aloohol distils as It Is displaced. Prolonged heating Is required, therefore many unstable baalo esters oan not be prepared In this manner (43).

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5. Reaotlon of a Halogen Derivative with an Amine.RCO»«W*X + MMWj -®— > RCOiCCJ^Sot!*R* A m e^ fc o r

The reaotlon between ohloro-and bromoalkyl esters and •nines is of United value beoause of low yield and super­iority of other nethods (44).

6. Reaotlon of a Carboxyllo Aoid with an Amino aLcohol in the Presenoe of a Catalyst.

RCCfeM + HO<C%>xN<ftfc ^CC^CCI^NCRt, + HiOR * diyJbaffc or kt+m&ic. *

The aloohol is generally added in excess to dissolve the aoid whloh is generally a solid. As oatalyst one oan employ either gaseous hydrogen ohlorlde or concentrated sulfurio aoid. The exoess aloohol is then removed under vaouun and the salt prepared as in section 1 (45).

The experimental work performed by the author demon­strated that esterlfioatlon of gentlsio acid with die- thylaninoethanol and dlethylamlnopropanol took plaoe most suooessfully and in best yields by following the seoond prooedure. However, the halogen derivative of dimethylamlno- ethanol oould not be found in the literature and the reaotlon did not yield the desired produo ts. The reaotlon is good only for tertiary amines# beoause amides or mixtures of amides and esters are obtained when secondary or primary amino sloohols are used (46).

The first prooedure was attempted by utilising gentisyl

IS

ohlorlde# but no reaulta vara obtained. The produotion of tba aoid ohlorlde of gentlalo aoid without protection of the phenollo groupa la queatlonable although ealloyl ohlorlde haa bean reported by Kopeteohnl and Ladlelaus (47), as wall aa Wolffanatain (48).

The reaotlon of aodlum gantlaate with the hydroohlorlde aalta of prlamiry, aeoondary and tertiary amino halide a did not yield eatere for any of the aminea.

Dlreot aaterIfIoatlon by Interaction of gentlalo aoid and the amino aloohol, dlaaolved In exoeaa of the amino aloohol, did not yield an eater upon addition of aeveral dropa of oonoentrated aulfurlo aoid. The other methoda of aaterifIoatlon of gentlalo aoid were not attempted.

Katera of phenollo funotlona may be prepared In the fol­lowing manner. In the general oaae# the reaotlon la between a phenol and an aoid ohlorlde. The varlationa are In the uae of aolvent and oondenalng agent.

R * AipkHc or l e m ftc

1. The Interaction of a Phenol and Aoid Chloride in Inert Solvent. Bqulmolar port Iona of aoid ohlorlde and phenol are diaaolwed In bensene or other aultable aolvent ahd enough pyridine added to oondenae with the hydrogen

4- ntoci

16

ohlorlde liberated. The solution,shaken several times with dilute hydroohlorlo aoid, removes any excess pyridine. If the product Is a solid. It may be filtered off and reerystal- llsed from a suitable solvent. However, If the product remains In solution the solvent Is generally removed under reduced pressure and the mass Is shaken well with 5$ sodium bicarbonate to remove the unreaoted phenol and the exoess acid ohlorlde. The residue is filtered and reorystallized from suitable solvent (49).

2. The Interaction of a Phenol and a A d d Chloride in Pyridine. A mixture of equlmolar portions of acid chloride and phenol are added to pyridine and the mixture heated on the steam bath from 10 to 50 minutes. When the mixture Is cool. It is placed in an Ice bath and 5 times its volume of (#» sodium bloarbonate added with constant stirring until ths product crystallises. The solid Is filtered, washed with 5% sodium bloarbonate and dried. The crude product may be re­orystallised from a suitable solvent (50).

£. The Interaction of an A d d Chloride and a Phenol in Aqueous Alkali, (Sohotten-Bauman reaotlon). Bqulmolar por­tions of the phenol and acid ohlorlde are reacted In 10)£ sodium hydroxide. The phenol Is dissolved in the alkaline solution and the acid ohlorlde added with continuous stirring. The mixture is oooled slowly to room temperature and then In an ice bath. Cold dilute hydroohlorlo a d d is added until strongly add. The preolpltate Is filtered and dried. The

17

produot oan ba purIflad by dissolving It In aoetone or aloohol and rspreolpltatlng by adding water. The process Is repeated in the earn solvents to give greater purity. However, final purification should be brought about by reorystalllsatlon from a suitable solvent(51) .

Although the first two procedures were tried repeatedly with gentlsio aoid and various aoid ohlorldea, no results oould be obtained. In the majority of oases both the gentlalo acid and the other aoid were recovered. It appears that only hydrolysis of the aoid ohlorlde took place.

The third prooedure yielded good results and aooeptable yields of the phenollo esters. Esters of salloyllo acid, 5-aoetyl-gentlslo acid, dlphenylacetic acid and die thy1- aoetlo sold oould not be obtained by this prooedure. It Is possible that the aoid chlorides of these acids were not obtained.

Baoh ester synthesised was analyzed In the following manner, when applloable, to determine its structure and element oontent.

'I. Physical Properties, i.e. miorosoopio appearance and solubility.

2. Stability In Air and Aqueous Media.£. Qualitative Elemental Content.4,. Ferrlo Chloride Color Test.£. KJeldahl Nitrogen.6, Ionlo Chlorine Titration.

18

7^ Carbon and Hydrogen*.&. Houtral Equivalent.9. Infrared Speotrum.The physloal properties of eaoh compound were determined

by examination of the product under Hie mlorosoope and determination of its relative solubility in various solvents.

The stability of the compounds in air and aqueous solu­tion was observed during several weeks.

Qualitative elemental content was determined by the proeedures of Shrlner and Fuson (52).

The phenolic oolor teat, by use of ferric chloride T.S., was determined for each compound.

KJeldahl nitrogen was determined by the micro teohnlque (53).

Ionic ohlorlne was determined by titration with standard silver nitrate, the Mohr Method (54).

Carbon and hydrogen analysis was done by Bll Lilly and Co. researoh laboratories.

Modification of the standard neutral equivalent procedure was neoessary to obtain aoourate molecular weights of the synthesised oarboxyllo acid e%ter hydrochlorides. However, the moleoular weights of the phenollo acid eaters were de­termined in the usual manner.

*Only Diethylaminoethyl 5-acetyl-gentlsate hydrochloride, diethylaminoethy1 5-bensoyl-gentlsate hydrochloride and dlethylamlnopropyl gentlsate hydrochloride, of the oarboxylic acid ester hydrochlorides.

19

The astera formed through the oarboxyllo acid group ware all tertiary amines, in the form of their hydroohlo- ridea. Upon the titration of the oompounds with atenderized carbonate free base, the basio amines were released and buffer systems were established. Therefore, no sharp break in the titration ourves could be obtained. The problem was to remove the basio amines whioh were partially or com­pletely water soluble.

The first prooedure tried was to underlay the aqueous solution of the salts with chloroform. Upon titration of the salts with base the amines passed Into the chloroform layer. Only fair results were obtained, due to the fact that the partition coefficient of the amines between water and chloroform was not large enough* The amines therefore, still exerted some effect on the pH readings. The prooedure possibly oould be improved by use of an Immiscible solvent that had a larger partition coefficient for the amines than chloroform with water, or by the determination of the parti­tion ooeffloient of the amines in water and chloroform and applying a correction to the resulting molecular weight.

k method that was found to give good results was the determination of the neutral equivalent of the base, Instead of the aoid. In this an excess of standard base was added to 36ml* of a 60jt alcoholic solution of the amine hydrochloride. The basic amine was liberated. The excess standard alkali and the amine were then titrated with standard acid to yield

20

sodium ohlorid# and ttaa salt of the amine, respectively. The entire titration was oarrled out under nitrogen to prevent air oxidation of the phenol In baalo solution and also to prevent oontaminatlon by oarbon dioxide of the air. A aharp break in the titration curve was obtained at the end point.

Oood results oan be obtained by use of 0.1 N. alkali and 0.1 N. aold. Mloroburettea are of value especially when small samples are being titrated.

Care should be exeroiaed to standardize the base and acid in 50jC aloohol which la neutral to phenolphtaleln. Therefore, although the pH readings may not be aocurate, as compared to aqueous solution, the change of pH per volume of acid will be the same. However, a blank should be obtained for 25 ml. of 50J( aloohol to determine the amount of aold needed to return the aloohol to a pH equal to the pH at the end point of a titration of a sample.

Saponifloatlon of the estera of the oarboxyllo aold was attempted In aleohollo alkali, but poor results were ob­tained. More alkali was consumed upon refluxing the ester with aleohollo alkali than by allowing the ingredients to set _ in the oold, but the consumption did not represent an even number of equivalents of alkali. It Is believed that due to the rapid oxidation of phenols, and more particularly quinones, in alkali media, good saponifloatlon numbers oan be obtained only if both phenollo groups are blooked. The author attempt­ed to make the dlaoetyl and dimethoxy derivatives of gentisio

21

aold, and found It very difficult to aoetyl&te or methylate the number 2 position of the acid* Therefore, no aaponlfloa­tlon values were obtained*

▲11 neltlng points were obtained by using the melting point bath apparatus. The thermometer was oallbrated over the m*p* range of 50° to 286° by means of eight pure com­pounds. The oorreoted m.p* refers to correction for emergent stem.

22Mew Bstera of Qentistlc Aold1

Dio thylamlnoethyl Qentlsate HydrochiorIds'*

O

CO^CttcClhL^CCVaC^

a, PreparationDiothylamlnoethyl gontlsato hydroohlorido was prepared

by the interaction of the halogen derivative of diethylamino- ethanol and gentleio aold; the Horeneteln and Pahllche method (42)•

Die thylamlnoethyl chloride waa prepared by two methods. The flret prooedure was aa follows (55):

Diethylamlnoethanol, 43 g. (0*37 mole), waa added to 50 ml, of bensene contained in a three neok, 250 ml. flask, in the hood, Thionyl chloride, 115,0 g, (0,97 mole), waa plaoed in a separatory funnel and added slowly with constant stirring to the bensene-amino aloohol mixture. The reaction was as follows}

The reaotlon was violent and strongly exothermic. After addi­tion of all the thionyl ohlorlde the product was refluxed 6 hours. The mixture was dark brown In oolor and contained

*Hew Compound.

+ acu^NCCfettj^

(UyaUfcNQfctfcoN + sock (CKjCKJj, H + SOt

*s.. pages 51-79 for proof of strueture.

23

a voluminous amount or solid. The excess thionyl ohlorlds ond the solvent were removed under reduced pressure. The rAsldu# was hAAtAd to 150°, for 2 hours st the rsduosd pros- surs, The rssldUA was then dissolved In water and the solution was filtered. The filtrate was placed In the refrig­erator for several hours. The oold solution was made strong­ly alkaline with 40JC sodium hydroxide, until the precipita­tion of a light brown material subsides. The reaotlon was as followss

The mixture was extracted with several portions of ether until only a light yellow aqueous solution remained. After removal of the solvent by evaporation, the remainder was distilled to yield a milky solution of strong anuoniaoal

at 18 am.The seoond prooedure (56) consisted In placing 72.5 g.

(0.61 mole) of thionyl chloride In a three neok flask pro­vided with a stirrer, reflux condenser and dropping funnel. The flask was placed In an Ice bath In the hood until the liquid was thoroughly oooled. Diethylaalnoethanol, 52.2 g, (0.48 mole), waa added dropwise with constant stirring to the

KfcCJUkNCfcOhCI (c*,au,Nafe<K,ci + + 40

odor, b.p. 68-70° at 30 m.Redistlllation yielded 30.0 g. (60.2^) of a dear,

colorless, strongly awionlaoal smelling liquid, b.p. 53-54°

24

oold thionyl chloride. After all the aloohol was added the produot waa stirred an additional hour at room temperature.

The entire contents of the flask was transferred to a 1000 ml. beaker ehloh oontained 250 ml* of absolute aloohol.The solution was heated to a gentle boll for 1 hour or until gases oeased to be evolved. The solution was filtered hot and plaoed in the refrigerator over night.

The preolpltate was filtered and dried. The orude yield of diethylamlnoethyl chloride hydrochloride was 52 g. (68.9%) of a light brown solid* m.p. 205.5-206,5° (Corr. 209-212°).

▲ less pure fraction was obtained by oonoentratlng the filtrate to 1/3 the original volume and recovering the pre­olpltate, The yield was 15,72 g, (20.9%) of the less pure, dark brown solid, m,p, 193.6-198,5° (Corr. 198,5-204°).

The total orude yield was 67.72 g, (89.8£).Diethylamlnoethyl ohloride hydroohloride, 30 g.

(0,17 mole)* was plaoed in a 1000 ml. beaker and 10 g, of orushed ioe added. The beaker was plaoed in an loe bath and 40 ml, of ether added. When the beaker and contents were oold* the mixture was made strongly alkaline with 40£ sodium hy droxide. The extraction was oarrled out several times. The ethereal solution of the halide was dried over drierlte for several hours. After the removal of the solvent by evapora­tion* the remaining red colored oil was distilled to yield 17,2 g. (74.1j£) of a olear* colorless* strongly ammonlacal

25

smelling liquid, b.p. 44-46° at 13 mm. (67).Burtner (68) describes a method for the liberation of

the basio halide from the hydroohlor ids by addition of flaked sodium hydroxide to the salt. The mixture mas stirred and the halide distilled under reduoed pressure. The halide was dried over potassium oarbonate. However, It was found that the yield was poorer and a less pure compound was obtained than the extraction prooedure.

Diethylamlnoe thyl chloride, 6.5 g.(0.O5 mole) was added to an alooholle solution of 7.4 g. (0.06 mole) of gentislo aold (Delta Chemloal Corporation). Heat was evolved and the solution waa shaken till oool. The aloohol was removed under reduoed pressure. The giumy residue was heated 2 hours at 80° and 10 hours longer at 100°.

Aoetone was added to the gummy residue and dissolved a portion of It, leaving a floooulant, white precipitate. The preolpltate was filtered and the dried product represented a orude yield of 9.6 g. (65.1%), m.p. 140-148.6° (Corr. 143- 181.5°).

The orude ester salt, 9.18 g., was dissolved in water and 3.66 g. of sodium oarbonate (67.2 ml. of 6% sodium oar­bonate) added. A voluminous shite, cloudy solution resulted. Three 40 ml. portions of ether were ueed to extraot the basio amine from the aqueous media. To the ethereal solution of the basle mslne was added 1.16 g. of hydrogen ohloride. (21.8 ml. of 1.4616 H. alooholio hydrogen ohloride solution). The

86

yield was 6,7 g. (45,9^) of a vhite, heavy solid, m.p.148.5-151° (Corr. 158.5-154°).

The diethy lamlnoethyl gentlsate hydrochloride waa again made baalo, extracted with ether and repreolpltated as the salt, m.p. of 150-151° (Corr. 155-154°).

An alternate prooedure desorlbed by the same authors was as follows (42)|

Oentisio aold, 5 g. (0.05 mole) was dissolved in 50 ml. of ieopropanol and to the solution was added 4.1g. (0.05 mole) of die thy lamlnoe thyl ohloride. The mixture became slightly warm upon shaking. Whan the mixture cooled. It was refluxed 8 hours. Upon cooling to room temperature, crystal­lisation ooourred. The filtered and dried produot represent­ed a orude yield of 8.8 g. (25.550 °f * white, floooulant preolpltate, m.p. 140-170° (Corr. 145-175.5°).

The eater upon reorystalllsatlon from lsobutanol yielded 1.88 g. (19.8)0 of a white, crystalline solid, m.p.151.5—158.5° (Corr. 154-155°).

The produot showed no change In melting point range after It waa dried In the Abderhalden Drying Pistol for 5 hours at 100° and 1-5 no.

37

b, PropertiesDie thy lamlnoe thyl gentlsate hydro obi or ids Is a white,

grainy powder. Colorless plates are seen under microscopic magnification when the compound is purified by the repreolp- Itatlon method. However, when re crystallised from Isobutanol It appears to be a sparkling, crystalline solid. Mlorosooplo examination revealed large, colorless plates. The compound has a sweet aromatlo smell.

The salt Is very soluble In water, absolute aloohol,95J( aloohol, dilute alcohol, butanol, hot lsobutanol and pyridine. It is partially soluble in isopropanol and In­soluble In ohloroform, carbon tetrachloride, toluene, ben- sene, ethyl aoetate, ether, acetone, petroleum ether, Skellysolve B C, dloxane and cold lsobutanol.

The solid Is stable In air. The compound is unstable In aqueous solution turning from a colorless to a brown solution In several days, and upon adding alkali, the color change takes place In several minutes.

Qualitative element analysis shows nitrogen and chlorine to be present.

The ferrlo ohloride test Is positive, (blue-purple In aqueous solution)•

Analysiss Calculates for N» 4*84; Cl, 13,6^Pounds H, 4,73, 4,64) Cl, 13,58, 18,48.

Meutral Bqulvalent. Calculated for C ^ H ^ O ^ N s 353,30 Pounds 250,92, 255,50.

88

DIBTHYL4MT MfneTWYf, 5-ACBTYL-(gNTI 3ATB HYDROCHLORIPBT*CiOjt <^Ukg<CtkC1b)A

ciscofc^^a. Preparation

Die thy lamlnoe thyl B-aoety1-gentiaate hydrochloride waa prepared by the interaotion or 5-aoetyl-gentlalo acid and die thylamlnoethyl ohloride (42).

B-aoetyl-gentlalo aold waa prepared by two prooedurea. The firat oonaiated of the interaotion of gentlalo aold with aoetlo anhydride, while the aeoond method employed aoetyl ohloride aa the aoetylatlng agent.

The flrat prooedure waa carried out aa followa (59):To 300 ml. of 8 N. a odium hydroxide, heated to 60°, waa added 30 g. (0.20 mole) of gentlalo aold. When aolutlon was ooaiplete# 39 g. (0.38 mole) of aoetlo anhydride was added and the aolutlon waa atlrred till oool. The entire flask and oontenta were plaoed in an ioe bath till oold. Cold dilute hydroohlorlo aold waa then added and a creamy, white preoipitate formed. The preolpltate waa filtered and dried. The filtrate waa made more atrongly aold with concentrated hydroohlorlo aold to Insure precipitation of all the 5-aoetyl gentlalo aold. The total yield waa 35 g. (91.8%) of a light brown aolld, m.p. 100-104.5° (Corr. 102-106.5°).

The aoetylated oompound waa reoryatalllaed from toluene

*Mew Coaipound

29

yielding 24 g. (63.0)0 of a white solid, m.p. 131.5-132*2° (Corr. 133.6-134.2°).

The equationa for the reactlone Involved in the above prooedure are aa followss

The eeoond method for the preparation of the 5-acetyl derivative ueed aoetyl ohloride (60).

Aoetyl ohloride waa prepared by dropwiee addition of 80 g. (0.58 mole) of phoaphoroua trichloride to 100 g. (1.66 mole) of glaolal aoetlo aold.

The reaction flaak waa plaoed in an ioe bath and waa oooled 1/2 hour. After heating at 60° for 1 hour the aoetyl ohloride waa dietilled from the mixture yielding 44.2 g. (35.0)0 of a oolerlea* atrongly irritant smelling liquid,b.p. 60-56°.

The aoetyl ohloride waa reaoted with gentlalo acid in baalo media, i.e. Sohotten-Bauman reaction, but very poor yield of the 6-aoetyl-gentlalo aold waa recovered. The re­action waa not lnveatlgated further beoauae of the better

+ x»faON^4

CMsCOfN*

JCAjCOCl + fcPOj

50

yield obtained by the use of aoetlo anhydride.

(0 r i 0" + C%OOC| *ss| + » W f HiO

J i j *

“ « j l T * » - u O " *NeCf

Diethylamlnoe thyl 5-acety1-gentIsio aold hydroohlorlde was prepared successfully by the same two procedures which were used to prepare diethylamlnoethyl gentlalo aold.

Bqulmolar portions of the halide and acid were heated for 2 hours at 80° and then 10 more hours at 100°, A orude produot of 49.4% yield, m.p. 108.5-119.5° (Corr. 110.5-121.5°) precipitated when the mass was mixed with acetone.

Purification of the orude produot was attempted by dis­solving the solid In water, adding an equivalent portion of sodium oarbonate, extracting several times with ether and repreoipltatlng the salt by adding alcoholic hydrogen chloride to the ether. However, an oil separated and oould be solidi­fied only by oooling. The yield was about 20?£ of a white solid, m.p. 118.5-126.5° (Corr. 120.5-128.5°).

Upon repeating the reaction, and reorystallislng the orude compound from lsopropanol, the yield was 5.9£ of a white, orystalllne solid, m.p. 153-134° (Corr. 135.3-136.5°).

Muoh better euooeas was obtained when equimolar portions of die thylamlnoethyl ohloride and 5-aoetyl-gentlslo aold were refluxed In isopropanol for 8 hours. Upon oooling the solu-

31

tlon yielded 82.8JC of * whit®, crystalline solid, m.p.106.5-130.5°(Corr* 108.5-133.0°).

The «at«r was reorystalllsed 3 timea from Isopropanol yielding 44.S£ of a whit®, needle-llke solid, m.p. 133.5- 134.5° (Corr. 136-137°).

The oompound showed no change in melting point when it was dried in the Abderhalden Drying Pistol as previously described.

b. PropertieaDie thylaminoethyl 5-aoetyl-gentlsate hydrochloride, when

reoryetelilted froei Isopropanol, appears under the miorosoope at a colorless, floooulant solid oonaieting of long, thin, oolorleae needles. To the naked eye the oompound appears as a white, floooulant, feathery powder. The ester has a sweet, aromatio smell.

The salt is very soluble in water, absolute aloohol, 95?b aloohol, ohloroform and hot isopropanol. It Is partially soluble in butanol, lsobutanol, ether and acetone, but in- soluble In ethyl aoetate, oold Isopropanol and petroleum ether.

The dry salt is stable in air, however, In aqueous solu­tion it turns brown in several days. The color ohange takes plaoo in several minutes time in alkaline media.

Qualitative element analysis shows nitrogen and ohlorlne present.

The ferrlo ohloride test Is positive.Analysiss Calculated for Cj^HgeOsNCls C, 54.30; H, 6.6B

W, 4,821 Cl, 10.69. Founds 0,54.41 ; H, 6.78 ; N, 4.21 and 4.13; Cl, 10.60 and 10.64.

Meutral equivalents Calculated for C15H21O5NS 295.33. Founds 301.51, 301.79.

33nTT m T M M1MQgnnfL 6-MKTHOXY-OBNTISATB HYDROCHLORIDE

COtCttiCUxNCCHxClWi' So* ■«

dbo^Vx'A. Preparation

Die thylanlnoe thyl 5-nethoxy-gentlsate hydrochloride was produoed by the raaotlon of diathylanlnoethyl ohlorlda with 5«wethoxy-gentlslo aold (42).

Tha 5-nethoxy derivative of gantlalo aold waa prepared by dissolving 20 g. (0.13 nole) of gentlalo aold In 100 ail, of 10£ hydroxide and adding 44,2 g. (0.35 nole) of dine thyl sulfate. The nlxture «aa ahaken 30 nlnutea during whloh there waa a atrong evolution of heat. The nlxture was re­fluxed 1 hour. The resulting aqueous nlxture waa aold and waa nade strongly alkaline with 90% aodlun hydroxide. The heterogenous nlxture was refluxed for another hour. At thla point the nlxture waa alkaline or waa nade alkaline aa before and the refluxlng repeated. The nlxture was aoldlfled with dilute hydroohlorlo aold and oooled.

A yield of 30 g. (B5.6J0 of a llgit brown, crystalline solid, n.p. 133.5-145.5° (Corr. 136-148°) was obtained (61).

The nethyl ether waa reoryatalllsed fron water to yield 15 g. (64.2JC) of a colorless, needle-like solid, n.p. 143.5- 144.5° (Corr. 146-147°).

•dew 6anpound.

34

The reaction* involved in the preparation of 5-mo thoxy-

Diethy lamlnoethyl 5-roethoxy-gentleate hydrochloride «aa formed by the reaction of the halogen derivative of the amino aloohol and gentieio aold in absolute aloohol. The alcohol was removed under reduoed pressure and the mixture heated for 2 hours at 80° and the n 10 additional hours at 100°. Upon crystallisation, from lsobutanol, a yield of 92. of a sweet smelling solid, m.p, 178-183.5° (Corr. 182*5-188°) was obtained.

Hecrystallisation of the compound from lsobutanol, 3*

times, yielded 58.0^ of a white, crystalline solid, m.p. 182.3-183.0° (Corr. 186.8-187.5°).

The compound was dried In the Abderhalden Drying Pistol as previously described and no change in the melting point waa notloed.

gentlalo aold were as follows*

4- CObbSd*

35

b.* PropertiesDie thy lamlnoe thyl 5-methoxy-gentlsate hydrochloride la a

light brown, or oolorless, oryatalllne aolld. It appears aa large, oolorleaa needles under the microscope. The compound has a sweet, aromatlo smell.

The salt Is soluble in water, absolute aloohol, 95% aloohol, dilute aloohol, chloroform, toluene and hot isobu­tanol. It is partially soluble In lsoproponal and ether, but Insoluble In aoetone and oold lsobutanol.

The amlno-ester Is stable as a solid. However, an aqueous solution is unstable, turning brown In several days. The color change Is greatly hastened in alkaline solution.

Analysis for the element content of the compound shows nitrogen and chlorine to be present.

A positive test is obtained with ferric chloride.Analysis* Calculated for ci4H22°4Ncl* N' 4.61; Cl,

11,67, Found* V. 4.46, 4,41; Cl, 11,70, 11,55.Neutral equivalent* Calculated for 267,32,

Founds 268,59, 270,31,

56

pTWTWYT^irr wmcmrvx 5-BgNZ0YL-(gMTISA,IB HYDROCHLOHIDK*

0 - 0

a ^ c ^ c v * ) .

PreparationDie tfcgOaalnoethyl 5-benmoyl-gentisate hydrochloride was

formsd by the s u m reaction as tha other oarboxyllo acid ester hydroohlorldes of gentlslo aold (42).

S-benmoyl-gentlsio aold was prepared by the Sohotten- Bauman reaotlon between bensoyl ohloride and gentlalo aold (68). The yield waa 84.2£ of a whita, floooulant solid, a.p. 166.5—171.5° (Corr. 170-175°).

Upon rsoryatallIsatIon of the compound from water, a white aolid, m.p. 172.5-174.5° (Corr. 176-178°) waa obtained.

The solid was reorystalllsed twloe by dissolving it In hot aeetone and adding water till the solution was oloudy.Upon oooling, a oryatalllne solid, m.p. of 178-179° (Corr.181.5-188.5°), preolpitated.

5-bensoyl-gentlsio aold and diethylamlnoethyl ohloride were reaoted In equlmolar portions in Isopropanol. The solu­tion was refluxed 4 hours and was oooled to yield 77*8% of a white solid, m.p. 150.5-146.5° (Corr. 153-149°).

The oowpound after 5 reoryatalliestiona from isopropanol, yielded 5.1 g. (5S.l£) of a white, floooulant solid, m.p. 155.0-154.0° (Corr. 156.5-157.5°).

*»ew Compound.

37oThe compound was dried in the vacuum oven at 100 ,

10-15 su. for 13 hours. The melting point was the same.

38

b , PropertiesDie thy lamlnoethyl 5-benzoyl-gentlsate hydrochloride la

white, floooulant powder. Under the mlorosoope, It appears to be long, thin, oolorleaa needles. The compound Is odor­less.

The salt Is soluble in water, absolute aloohol, 95% aloohol, dilute aloohol, chloroform, hot lsobutanol, hot Isopropanol and hot toluene. It Is partially soluble In ether and Insoluble in oold lsobutanol, oold isopropanol and oold toluene.

The produot Is stable in air, when dry. However, It turns brown in aqueous solution within several days. The change of color takes place within several minutes in alka­line solution.

The qualitative element content is found to be nitrogen and ohlorlne.

The phenollo teat with ferrlo ohloride is positive.Analysis* Calculated for CgQHg^OgNCl; C, 60.85; H, 6.14;

N, 3,56; Cl, 9,00. Pound* C. 60.40; H. 6.41; N, 3.4b, 3.40; Cl. 8.92, 8,95,

Neutral equivalents Calculated for ^20H23^5^: 357.39Pound* 357.10, 354,39.

59

DIKTHYLAMIMOPROPYL OBNTISATS HYDROCHLORIDE*CO*C%CHrC4«fi«*ab)J,

.O “ ”a. Preparation

Diethylaminopropyl gentieate hydroohloride was prepared by the Interaction of diethylaminopropyl chloride and gentlelo aold (42).

Die thyIanlnopropyl ohloride was prepared from the alco­hol, A aolutlon of 52.33 g. (0.43 mole) of 3-dlethylamlno, 1-propanol in 86.66 ml. of anhydrous chloroform was added, dropwlse, to a aolutlon of 96.66 g. (0.81 mole) of thionyl ohloride In 400 ml. of anhydrous chloroform. The reaction waa oontroled by oooling the flask. After the reaction had oooled, the mixture was refluxed 3 hours. The solvent and excess thionyl ohloride were then removed under reduoed pressure, leaving a brown-black residue.

The solid was plaoed in a beaker in an Ice bath. Enough ether was added to cover the solid and then aold, 40^ sodium hydroxide was added. The aqueous solution of basic amine was extraoted 3 times with ether. The light-brown ethereal ex­tract waa dried over drlerlte several hours.

After removal of the solvent, the remainder was distilled to yield 47.0 g. (78.0^) of a colorless, strongly ammonlao&l smelling liquid, b.p. 93-97° at 25 ran. (63)

An equlmolar portion of diethylaminopropyl ohloride was

♦liew Compound.

added to an laopropanol aolutlon of gentlalo aold. The solu­tion waa refluxed 6 houra, and oooled.

After evaporation of the Isopropanol, the semi-solid was nixed wltH aoetone and a yield of 37.0J6 of a white solid, n.p. 163.5-169.5° (Corr. 166.0-172°), waa obtained.

The compound was reorystalllsed from lsobutanol to yield 24.5# of a white, crystalline aolld, m.p. 171.7-172.7°(Corr. 174.2-175.2°).

The melting point remained constant when the ester was dried In the Abderhalden Drying Pistol In the same manner as previously described.

41

b. PropertiesDiethylaminopropyl gentlaate hydrochloride is colorless,

crystalline needles* Under the microscope It appears to be long, thin, colorless crystals*

The hydrochloride Is soluble in water, absolute alco­hol, 9556 aloohol, dilute aloohol, Isopropanol, chloroform and hot lsobutanol* It Is Insoluble in acetone and oold lsobutanol.

The dry produot is stable in air, but in aqueous solu­tion It darkens In several days; the time la decreased to several minutes In alkaline media.

The analysis of the ootnpound for elements shows nitrogen and chlorine*

The ferric ohloride test shows a phenol to be present.Analysis: Calculated for Cj^HggO^NCl: C, 55,35; H, 7*30;

N, 4.61; Cl, 11.68* FoundtC, 65*45 ; H, 7.42 ;N, 4.73, 4*57; Cl, 11.76, 11.84*

Neutral equivalents Calculated for 267.32.Founds 264*98, 266,01*

DIBTHYUUCHOPROPYL 6-ACBTYL-GENTI3ATB HfDROCHLORIPg*CpsC%C«^%N(CNtCH>^^ S O K

a. PreparationDie thylaalnopropyl 5-aoetyl-gentisate hydrochloride was

prepared by the Horenateln and Pahliohe reaction (42). DIethylaBilnopropyl ohloride and 5-aoetyl-gentlsio acid were prepared as previously desoribed. The aold was dissolved in isopropanol and an equlmolar portion of halide was added with oonstant shaking. The mixture was refluxed 6 hours and was oooled to room temperature. No precipitation took place. After evaporation of the Isopropanol, the guany residue yielded 35.6*6 of white solid, m.p. 152-153.5° (154.5-156°), upon addition of ethyl aoetate.

The produot was reorystalllxed 3 times from toluene to yield 20.6)6 of white, floooulant solid, m.p. 157.0-157.5° (Corr. 160.5-161°).

The melting point remained oonstant when the oompound was dried In the Abderhalden Pistol as outlined for the preceding compounds.

*New Compound

43

b . Prop#rtiesDie thylaminopr opyl 5-aoetyl-gentisate la a small white,

needle-like solid. It appears to bs long, thin, colorless oasdlss under the miorosoope. Tha solid has a sweat, aromatio odor.

Tha oast pound Is soluble In water, absolute aloohol, 95^ aloohol, dilute aloohol, ether, chloroform, acetone, and hot toluene. However, it is Insoluble in ethjl acetate and cold toluene.

The dry eolld Is stable upon exposure to air, but un­stable In aqueous solution, turning brown In several days.The baeio amine is more unstable, turning brown within 1 hour.

The compound gives positive tests for nitrogen and ohlorine.

The ferric ohlorlde test is positive.Analysis! Calculated for C^gHg^gNClt N, 4.05; Cl,

10.86. Founds N, 3.96, 4.11; Cl, 10.44, 10.26.Meutral equivalent. Calculated for C^gHggOg N: 309.36.

Pounds 310.36, 308.66.

44

DINTHYIAMI NOPROPTL S-MBTHOXY-QBNTiaA'IB HYDROCHLOBIDB*

Die thylaminopropyl 5~me thoxy- gen ti sate hydrochloride was prepared by the Interaction of diethylaminopropyl chlo­ride and &~methoxy-gentiaio acid (42).

Zquiaolar portions of the ingredients were mixed In isopropanol and refluxed 6 hours. Upon ooollng 54.7^ of a white, floooulant solid, m.p. 185.0-186.5° (Corr. 189.5- 191°), precipitated.

The produot was recrystallised from isopropanol twice to yield 4 8 . of white, floooulant solid, m.p. 184.5-185.5° (Corr. 189-189.8°).

The compound was dried in the Abderhalden Pistol as previously described. No change in melting point was notloed.

CK|0a. Preparation

W % c % « ( a b c *>, SON Rcl

*B4w Compound

45

b. PropertiesDiethylaminopropyl 5-ipethoxy-gentlsate hydrochloride

la a white, floooulant powder. It appears to be short, thin, oolorless needles under the alorosoope. It has a sweet, arosetic arose.

The compound Is soluble In water, 95# aloohol, absolute aloohol, dilute aloohol, ohloroforn and hot Isopropanol. It Is Insoluble In 00Id Isopropanol.

The salt la stable as a solid, but unstable in aqueous solution. In whloh it turns brown. The color change is facilitated by alkali.

Qualitative elemental analysis shows nitrogen and chlorine.

The ferrlo chloride color test is positive.Analysis! Calculated for C^gHg^^NCl: N( 4.41; Cl,

11.16. Pounds N, 4.38, 4.32s Cl, 11.25, 11.29.Neutral equivalents Calculated for C^HggO^Ns 281.34.

Pounds 275.81, 284.53.

46

nT^THVlAMTMQPRQPYL 5-BKNZO YL-QBNT13ATB HYPROCHXORIPK*

Diethylaminopropyl 5-bens oyl-gen tisate hydrochloride was prepared by the Horenstein and Fahllohe reaction (42).

Diethylaminopropyl chloride was added to a solution of 6-bensoyl-gentlslo acid in Isopropanol, The mixture was refluxed for 4 hours, was cooled and the solvent was evaporated. The mass yielded 31,6£ of a white solid, m.p, 127-136° (Corr, 129*5-137,5°), when mixed with ethyl aoetate*

Upon 2 reorystalllsations from toluene, the yield was 16,9j( of a white, floooulant solid, m.p* 142,7-143*7°(Corr. 146*2-146.2°).

The ester was dried In the Abderhalden Drying Pistol as previously described, and no change In melting point was notloed.

*New Compound.

COMsCfcClltNCCfcCllsttMil

±* Preparation

47

b, PropertiesDiethylamlnopropyl 5-ben*oyl-gentlsate hydrochloride

la a white, fluffy solid which appears to be small, color­less needles when viewed with the microscope.

The product Is soluble In water, absolute alcohol,93$ aloohol, ohloroform, hot toluene and acetone. It Is Insoluble in oold toluene, ether and ethyl aoetate.

The eater is stable when exposed to air, but turns brown in aqueous solution after several days, basic solu­tion hastens the oolor ohange.

Qualitative element analysis shows nitrogen and ohlorlne to be present.

The ferric ohlorlde test is positive.Analysis, Calculated for Cgi^gObNClx N, 3.43: Cl, 8.69

Pounds N, 3,58, 3,61; Cl, 8,60, 8,64.Neutral equivalent. Calculated for C21H25°5N : ^73,42

Founds 363,12, 367.56.

48

a-PHKNYIACBTYL-GBfTISIC ACIP*to*

Q c J Q

i, Preparation 5-phenylacetyl-gentlslo aoid was prepared by the inter-

aotlon of phenylaoetyl chloride and gentislo aoid. The Sohotten-Bauman type reaction was used (59). The reaction was suitable beoause of the high reactivity of the number 5 phenolic function of gentislo a d d in alkaline media and the Inertness of the number 2 phenolic funotion,

Fhenylaoetyl chloride was produced in the usual manner, i.e. Interaction of phenylaoetlo acid and thlonyl chloride. Upon removal of the excess thionyl chloride, the remainder was distilled to yield 78.0% of red colored, strongly irri­tant smelling, oily liquid, b.p. 158° at 50 mm.

Oentlslo acid, 6 g. (0.04 mole), was dissolved in 50 ml. of 10% sodium hydroxide and 5.4 g. (0.04 mole) or phenyl- acetyl chloride was added with rapid stirring. A great amount of heat was evolved and when the mixture was cooled only a slight amount of preoipitation took place. The mix­ture, however, yielded 7g. (65.9#) of a dense, light yellow preoipitate, m.p. 126.5-156.5° (Corr. 129-159°), when made acidic with dilute hydrochloric add.

•llew Compound.

49

The reactions that took place In the above preparation

The product was a mixture of white, fluffy solid and yellow, ourdy solid, m.p. 141.5-144.5° (Corr. 144-147°), when repreolpltated from an acetone solution by adding water.

The mixture yielded S g, (28.9)0 of a white, fluffy solid, m.p. 145.5-146.5° (Corr. 148-149°), when reorystal Used from water.

The compound was dried In the drying pistol In the seme manner as the other compounds. No change in melting point of compound resulted.

were as followss

50

b. Propertiea5-phenylacetyl-gentlelo aoid la a white, cotton-like

solid. Under the microscope it appears to be long, thin, oolorlese fibers. It has no noticeable odor.

The aoid is soluble in absolute aloohol, ether, aoetone, 98jC aloohol and chloroform. It is partially soluble in hot water, but insoluble in oold water.

The product is stable on exposure to air, but will turn brown in water when solubilized by baaio sodium salts.

The phenolic ester gives a strong blue oolor when ferrio ohlorlde is added to an aloohollc solution.

Analysis! Calculated for Cj^H^Og: C, 66.17; H, 4.44. Founds C, 66.21; H. 4.51.

Veutral equivalent. Calculated for C^sH^Ogt 272,25. Founds 272.96, 273.78.

51

6-PARAMITROBgMZOYL-qKirnSIC ACID»

a. FwpnAtlon 8-paranltrobensoyl-gentlslo aoid was prepared by the

Sohotten-Bauaan reaction between paranltrobensoyl ohlorlde and gentlelo aoid (69).

Paranltrobensoyl ohlorlde was obtained In the usual nanner, i.e. lnteraotlon of the aoid and thlonyl ohlorlde (65). The thlonyl ohlorlde was evaporated and the residue recrystallised fron toluene to yield 66. t£ of a yellow, needle*like solid, a.p, 75,0-73.0° (Corr. 74,2-75,1°).

The aoid ohlorlde and gent Is lo aoid were reaoted as stated to yield 79.9JC of a yellow solid, a.p. 210-220,5° (Corr, 815,8 826.3°).

The crude produot was reorystalllsed froa dilute alcohol, 5 times, to yield 39, of a llgit, yellow, cry­stalline solid, a.p. 886-888•5°d, (Corr, 831.8-234.3°d.). Pinal purification by reerystalllsatlon froa boiling wateryielded 38.0JC of a light yellow, fluffy solid, a.p, 229-

f'830,6 d, (Corr, 834.8-836,3°d.).

There was no change In melting point range upon drying the produot In the Abderhalden pistol In the manner described for the other ooapounde,

•kew compound,

52

to. Properties 5«pavanitrobenzoyl-gentlsio aoid la a light yellow,

floooulant solid. Whan viewed under the microscope, It appears to toe light yellow needles.

The product is soluble in lsobutanol, ethyl acetate, hot dilute aloohol, ether and acetone. It is partially soluble in hot water, absolute aloohol and 95% alcohol, tout insoluble in water, cold dilute aloohol, chloroform and toluene.

The solid is stable In air, but In alkaline aqueous solution it rapidly turns brown.

Element analysis shows nitrogen.The sold shows a strong ferrlo ohlorlde test.Analysis* Calculated for Ci4Hg O7N: C, 55.46; H, 2.99.

Pounds C, 55.45; H. 2.97.Heutral equivalent. Calculated for C^HgO^N; 505.20.

Founds 505.57, 502.80.

55

S-AHISYL-OBNTISIC ACID*COdl

a. Preparation5-anlsyl-gentislo aoid formed when anlayl chloride

was added to an alkaline solution of gentlslc acid (59).The anlelo aoid wae prepared from me thy1-parahydroxy­

benzoate or methyl paraben. The methylation using methyl sulfate has been deaoribed previously when the 5-methoxy- oarboxyllo aoid esters were discussed (61).

The yield was 70# of a white, needle-like solid, m.p* 155-175° (Corr. 158.5-179.0°).

The orude produot was reoryatalllsed twice from water to yield 50.5# of a white, needle-like solid, m.p. 184.5- 186° (Corr. 189-190.5°).

Anlayl ohlorlde was prepared by the lnteraotlon of thlonyl ohlorlde and anisic aoid. Upon removal of the thlonyl ohlorlde, the remainder was distilled to yield 74.2 > of a oolorless. Irritant smelling, oily liquid, b.p. 129- 151° it 8 m .

When the Sohotten-Bauman type reaotlon was carried out between anisyl ohlorlde and gentlslo aoid a yield of 83.7# of light gray solid, m.p. 175-205° (Corr. 178.5-210°), was obtained.

-wKew Compound.

54

The product waa purified by diesolving it alternately In hot aoetone and hot aloohol, then adding hot eater till the solution became oloudy. After repeating the purifica­tion 3 times a yield of 66,6£ of a white, floooulant solid, jn,p. 308,4-209,5° (Corr. 213.9-215.0°), was obtained.

When the solid was dried in the Abderhalden Drying Pistol as desorlbed previously, no ohange in melting point took plaoe.

55

b. Properties5-anisyl-gentisic aoid Is a light, white solid. Under

the microscope it appears as bundles of dense, fine, color­less needles. It is odorless.

The product is soluble in absolute aloohol, chloroform, ether, 98% aloohol and acetone. It is partially soluble in hot water and Insoluble in cold water.

The aoid is stable as a solid, but unstable when plaoed in solution by alkaline solubiliser.

The compound gives a positive ferric chloride test.Analysis! Calculated for ci5Hi2°6s C' 62«50* H* 4.19

Pounds C, 62.45; H, 4.54.Neutral equivalent. Calculated for 288.25

Founds 289.25, 291.76.

66

6-ACBTYLS^Er nvT^-flHNTISIC ACID»

V U ja. Preparation

6<-aee.tylaalioyl-gen tisio aoid waa prepared by the Interaction of aoetylaalioyl ohlorlde and gentisic aoid (59) *

Aoetylsalloyllo aoid was treated with thlonyl ohlorlde and yielded 62,3?£ of a colorless, strongly Irritating liquid, b.p, 135,5-136,5 at 8 mm. The liquid solidified to a white powder In several days (65).

Oentiala aoid waa dlaaolved In 10^ aqueous sodium hydroxide and aoetylaalioyl ohlorlde added dropwlse with oonatant stirring. Upon cooling, the cloudy solution was made aoid with dilute hydroohlorlo aoid. The yield was 73, of a light brown aolid, m.p, 163,5-168.5° (Corr, 167-172°).

The orude product was reorystalllsed twloe from an aoetone-water mixture and onoe from a aloohol-watar mixture to yield 16,1^ of a light gray solid, m.p, 203.5-204,7° (Corr, 2O9°-210.2°)#

The m,p« remained constant when the product was dried in the Abderhalden Pistol like the other compounds,

*Hew Cor pound.

57

b. Properties5-aoetylsalloyl-gentlslo aoid la a white or light gray,

fine, floooulant powder* Under the miorosoope email hair- like sheavea of oolorlese needles are seen.

The aoid is soluble in absolute aloohol, 9P# alcohol, ether, isobutanol, aoetone, and glaoial aoetio aoid. How­ever, it 1s insoluble In water,chloroform and dilute aoetio aoid.

The product Is stable in air, but turns dark in aqueous alkaline solution*

A strong positive test for phenols is obtained by use of ferrio ohlorlde*

Analysis! Calculated for C16H1207s C, 60.76; H, 3.83, Founds C, 61,74, 61*53; H, 3*92, 3*86.

Neutral equivalent. Calculated for C1QH1207: 316.26.Pounds 289*68, 281*11.

58

&. 51 -3UCCINYI*»DIQBNTI3IC ACID*

a. Preparation&,5V-suoolnyl-digentlalo aoid waa prepared, aa were

all the phenolic eatera, by the Schotten-Bauman reaction (59).Suoolnyl ohlorlde waa prepared by the following pro­

cedure (66),Suoolnlo aoid, 20 g* (0.17 mole), and 35.4 g. (0.17

jK»le) of phoaphoroua pentachloride were placed in a flask provided with an air condenser in a well ventilated hood.The mixture was heated, In an oil bath, alowly to 125°.The mixture flrat liquified and then formed a blaok maaa.The heating waa oontlnued 20 hours. Upon cooling, phos­phorous oxychloride was removed and the residue distilled to yield S0m0% of a light yellow-green, Irritant smelling liquid, b.p. 84° at 8 mm.

The following reaction represents the preparation of auooinyl ohlorlde.

+ pa, J1H 4 + Poq, + H,oSuoclnyl ohlorlde and gentlaic acid when reacted In the

ratio of 1 s 2, yielded 27.4# of a light brown aolld, m.p.4^ew compound.

CUtOO*

59

210-215°d. (Corr. 215.6-220.5°d).The orude product was rearystalllzed.3 times.from a

aoetona-watar mixture to yield 12.4% of a light brown solid, a.p. 227-229°d, (Corr. 254-256°d.).

Final reoryatalllsatlon from hot alcohol-hot water yielded 10.1% of a light brown or white solid, m.p. 230- 231.5°d. (Corr. 237-238.5°d.).

The solid maintained the same melting point after drying in the Abderhalden Drying Pistol as previously des­cribed.

60

b. Properties5,6*-suocinyl-digentiaio aoid Is a very light brown or

white solid* It appears to be small, colorless fibers under microscopic magnification*

The product is soluble in acetone, absolute aloohol,95^ aloohol and pyridine. It is partially soluble in ether, but insoluble in chloroform, water, benzene and toluene*

The acid is stable on exposure to air, but unstable in aqueous alkali, turning brown in a short time*

The ferric chloride test is positive.Analyslst Calculated for C10H14O1QS C, 55.39; H, 3.62.

Pounds C, 55*20; H, 3*82*Neutral equivalents Calculated for c xbH14°10S 390.29

Pounds 587,59, 389,14*

9

61

Proof of Structure of the Synthesised Eaters by the Analyses of Their Infrared Spectra.

In any ohemloal synthesis It la naoaaaary to prove the structure of tha oompound prepared. Quantitative analysis of tha element content of a oompound and the determination of other phyeloal constants, I.e. molecular weight, of the product establishes only Its empirical formula. The possi­bility atij.1 exists that the oompound may be an laomer of the desired synthetlo. By uae of infrared analysis the funotlonal groups of the compound can be determined. The •finger print* of the oompound la said to have been obtained. The Infrared spectra of the compounds prepared have been utilised In this way to determine their struoture.

An atom may absorb energy In the form of heat or light by converting the Incident energy into a form of potential energy. This Is aooompllshed by an electron "jumping" from a low-energy level to a higher energy level. This makes up the absorption speotrum of an atomic vapor shioh generally oonalats of a few dark lines.

However, there are three ways In which discrete amounts of energy may be absorbed by molecular vapor. The first is the electron jump. In a molecule the possible eleotronlo energy levels are modified by the presence of several atoms, therefore energy may be absorbed by converting It into en­ergy of vibration or rotation. In general, the energy in­

62

volved in the change of energy level* of an eleotron la greater than that Involved in vibration or rotation. There­fore, in a moleoule, the absorption line corresponding to a single eleotronio energy change is found to be spread out into a band of many fine lines because of the many pos­sible values of vibrational and rotational energy changes.

This condition holds true in general for a molecular vapor or gas. If this gas is compressed, however, it is observed that the fine lines of the band spectrum coalesce beoause of the effeots of crowding the molecules together. Finally, in a liquid, all the fine structure will became simply a region of absorption. Such an absorption region may represent only a single eleotron transition, modified by vibrational and rotational effeots.

Infrared and Raman spectra usually Involve energy trans­itions due to vibration and rotation alone. It is thus possible to study the dynamics of the moleoule Itself. (67).

The Infrared speotrophotome ter is used for quantitative measurements of concentration and even for automatic con­trol and recording. In addition, it is a valuable tool for deteotlng and identifying small amounts of impurities and for the elucidation of the structure of moleoules. (63).

The establishment of molecular struoture has an empir­ical basis and rests on the observation that oertaln comb1nations of a tans, frequently in complex moleoules, may be associated with oertaln absorption bands in the

infrafed of the** substances, If the band# produced by eaoh atomio group were Invariant and unique in their posi­tion in the apeotra of all substances containing the group it would be imnedlately possible to determine the presence or absenoe of any group in a given moleoule by the measure­ment of its Infrared spectrum. However, eaoh oompound possesses its own speotrum, The other structures adjacent to a given group influence Its electronic and spatial con­figurations, In addition, the masses of the other atoms are Involved in the vibration change from moleoule to mole­cule, As a result, a given group may show somewhat different values for its absorption bands In the spectra of different moleoules which oontaln it. Further, the bands due to one group in one compound may have the same wave length value as an entirely different structural group in another oom­pound, As a result, some degree of uncertainty may attach Itself to the Interpretation of bands suspected of emanating from any particular group,

A complete teat of a given moleoular structure based on Infrared data would result only if a precise mathematical computation would predict from the struoture the spectrum actually observed. Such proof la possible only in simple moleoules. It has been achieved for a number of such mole­oules where the Intramolecular linkages were well known and only the orientation of the bonds and the interatomic dis­tances were unknown.

04

The above conditions were never fulfilled for complex moleoules* The absorption speotrum and limited ohemical data are the only souroes of Information available. The Intramolecular distances are unknown, and there Is little or no basis for computation. Bven when the linkages are known* the complexity of the moleoules defies such an ap­proach. Under these conditions it is neoessary to resort to empiricism and to draw conclusions about structures by correlations between bands which occur regularly with re- ourrlng structural features throughout a series of com­pounds.

The Interpretation of the speotrum, based upon com­parison with the speotrum of compounds containing similar known functional groups* In combination with other physical and chemical evidence may furnish a guide sufficient for the identification of the structure of the oompound. (69).

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Figure 1Infrared Speotrum of Qentialo Aoid

Figure 2Infrared Speotrum of 5-Aoetyl-Gentiaio Aoid

hj

*• •»»

Figure SInfrared Speotrum of 5-Methoxy-Oentlaic Aoid

67

M • * " *

Figure 4Infrared Speotrum of 5-Benzoyl-Gentlaic Acid

.R

jinrli'iil; i9iin::fisxR::7;:ss;sss:s»nnK8Figure 5

Infrared Speotrum of Dlaoetyl-Gentialo Acid

isniusssaimntww

Figure 6Infrared Speotrum of DlefchylAminoethyl-Gentlaate

Hydroohlorlde

68

FigureNumber

2

Table II.Analyses of Infrared Speotra of Known Compounda

KnownCompound

% Functional U.laximum ofSolvent Conoen- Groupa Absorption

tration by Functional Groups

in h

GentlaioAcid

5-Aoetyl- Gentisio Ac Id

CH^OCHg 10

CHClj

W TrTTSRenoT^^T?^^Aromatic C-H 3.09C«0 in acid 5,95C*C In benzene 6.14CgHg 6.75Aromatic C-H 3.30C*0 in ester 5.69C^> in acid 5.97C«C in benzene 6.12C6H5 6.72

5-MethOxy- Gentisio Acid

CHC1,Aromatic C-H 3.34

2.5 C*0 in acid 5.98C*C In benzene 6.15C6H5 6.71

6-Bensoy1- Gentlsio Acid

CHC1,Aromatic C-H 3.35C*0 In ester 5.76C«0 in acid 5.93C»C in benzene 6.12C6H5 6.73

Dlaoetyl-GentlsloAoid

CHC1,Aromatic C-H 3.32C*0 in ester 5.67

5 C*0 in aoid 5.87C*C in benzene 6.15C & 6.72

(41 *»• If#r I- I * « P « « * 1, »•*" «* **I Itt r*

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Figure 7Infrared. 3peotruei of Dlethylamlnoe thyl 5-Ace tyl-Gentisate

Hydrochloride

I:-

• •k *n it»9 « * * ■ * > - ■. •• 4 » »* *• (■•tfv* I” •■ l .•»#*»;.**** * & I* MB H - <l»” < (• M *> • * » ■ !» li»« * ■■:'*••t i • V919S ii • . iiiai r.a n.i • • i i i i attm sn i

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ic: : : i ::::33 i i i i i ISHHSm iS IK II

Figure 8Infrared Speotrun of Diethylamlnoethyl 5-Methoxy-Gentleate

Hydroohloride

* M*«* mmm«»* * e * *ft «t * # a * 111K 4 a v

#■•1 a e• ffii , -*- m * * - -. «t A ft * - • « » •

t « L b «• I 1 * » * » * im* t i f f i l l

- : : : : s s iiii

Figure 0Infrared 3peotrun of Diethylamlnoethyl 5-Bensoyl-Gentisate

Hydrochloride

Figure 10Infrared Spe otrua of DIethylaainopropyl Qentisate Hydro­

chloride

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■

Figure 11Infrared Speotrua of DIethylaainopropyl 5-Acetyl-Gentieate

HydroohlorIde

:f3 Ml'. .'W*

rr5*"* f r 4

j- - fin » w •siic c ish ii

::aiUI»IIIKSiialSi»■■■■■■•*••■*«■* iifim kw mm*•««4-tti •■■■

s s s H iiin ie

Figure 18Infrared Speotrua of DIethylaainopropyl 5-Methoxy-Gentieate

HydroohlorIde

71Table III.

Analyses of Infrared Spectra of Diethylamlnoethanol Esters _____________________ of Qentislo Aoid

Figure Synthetla Number Compound

6

% Con­centre- Functional

Solvent tlon Croups

Ulilhyl-1 1'amlnoe thylQentisateHydrochloride

AMaximum of Absorption by function­al group s

In aAromatic C-H 3*42

Nujol --- C=0 In ester 5.92C*C in benzene 6,08C6H5 6.72

8

Die thyl­amlnoe thyl 5-Aoetyl- CHC1.Qentisate *Hydrochloride

Die thyl­amlnoe thyl 5-Methoxy- Qentisate CHCl^Hydroohlor ide

OH In phenol 3.10Aromatic C-H 3.39

lO HC1 4.21C»0 In ester 5.66,

5.89C=C in benzene 6.12C6H5 6.73

OH In phenol 3.08Aromatic C-H 3.40

3 HC1 4.30C*0 in ester 5.93C=C in benzene 6.15C6H5 6.69

Die thyl­amlnoe thyl 5-Bens oyl- CHC1,Qentisate ^Hydroohloride

OH In phenol 3.08Aromatic C-H 3.39

10 HC1 4.25C*=0 in ester 5.76,

5. 90C=C in benzene 6.11C6Hs 6.70

Figure 15Infrared Spectrum of Diethylamlnopropyl S-tJenaoyl-Gentiaate

Hydrochloride

itfiiu:

wi mmmrni::::::xici

Figure 14Infrared Spectrum of 5~Fhenylaoetyl-Gentlaic Acid

Figure 15Infrared 3peotrum of' 6-Paranitrobenroyl-Oentialo Acid

73Table IV.

Analyses of Infrared Speotra of Diethylam1 no propanol Eatersof Oentleio Aoid------------------------------ --- -------------------iMAXlUUm of

% Con- AbsorptionFigure Synthetio centra- Functional by fUfOc-Kueiber Compound Solvent tion Croups tional

groups in

Die thyl- 10 aalnopropyl Centisate Hydrochloride

Aromatic C-H 3.19Nujol — - C*0 in ester 6,00

C“C in benzene 6.13C 0Hg 6.75

Die thyl- aminopropyl

11 5-Aoetyl- Cen tisate Hydrochloride

chci3

Aromatic C-H HC1C*K) In ester C*C in benzene

3.354.225.67,5.926.126.72

12

Die thyl- amlnopiepyl 5—methoxy gen tisate Hydrochloride

CHC1, 10

OH in phenol 3.10Aromatio-C-H 3.40HC1 4.18C««0 in ester 5.94C-C in benzene 6.15C6H5 6.68

Diethyl- amlnopropyl

13 5-Benzoyl- Gen tisate Hydrochloride

CHC1.OH in phe no1 Aromatio C-H

10 HC1C«>0 in eaterC»C in benzenec6h5

3.083.394.235.76,5.926.126.72

74■ -I ■ **\

AKiiSi -I *3*2*3*" r u i tu ml»4* (g* -* r. «•« s . 5 .» •* r. - ■.»**. mm, ■•■■ISIMHi

.i . m H l i n’»*» tKHMIBI --t-fmm '>-iaaa|a• * -t a a a l s' <<taa>am t::ism* *

Figure 16Infrared 3peotrum of 5-Ani*yI-Gentisio Aoid

Figure 17Infrared Spectrum of 5-Aoetylaalioyl-Gentialo Aoid

* * * ■ W WB BttAflfl

«*aap a *3

Figure 18Infrared Spectrum of 6,5* Succinyl-DIgentiaio Acid

75

Analysts of InfraredTable V

Spectra of Ac Id

Fbencilc listers of Gentlsic

FigureHumber

SyntheticCompound

% Con-oentra- Functional

Solvent tlon GroupsAMaximura of Absorption by func­tional groups in

H5-Phenyl- OH in phenol 2.93

1 A aoetyl- CHC1, 10 Aromatic C-H 3.32Gentlelo C*0 In eater 5.67Aoid C«0 In aold 5.98

C*C in benzene 6.12C6h5 6.71

5-Paranitro- Sat- Aromatic C-H 3.35benzoyl- ur- C*0 in ester 5.72

15 Gontisic CH3OCH3 ated C*0 in aold 5.92Aoid C*C In benzene 6.15

Nitro 6.48

5-Anley1- Aromatic C-H 3.36aentlslo CHC1-* 5 C*0 In ester 5.79

16 Aoid C*0 In aold 5.95C»C In benzene 6.20C6«6 6.62,

6.755-Aoetyl- Aromatic C-H 3.35salloyl CHC1, Sat- C*0 In aold 5.95

17 Gentiaio 3 ur- C*C in benzene 6.15Aoid ated C6«5 6.72

5,5f-Suoolnyl- 18 Digentlalo Nujol

Ao IdC«0 In ester 5*65C«0 In aoid 5.96C«C In benzene 6*09

76

In figures 1 through 5 the infrared spectra for known compounds ere shown. The two main features that are out­standing in each spectrum are the low transmissions in the 3.0 to 4.0 micron range and the 5.6 to 6.0 micron range.In figure 1, the strong absorptions in these regions, indi­cates clearly a phenolic group or groups and one carboxyl!o aold function, respectively. However. In figures 2 through

, 5 where the 5-phenolic function has a substituted group onIt, the nature of the absorption is markedly changed. Spe- olal attention Is called to figure 4 because it is a disub- stltuted phenolic ester. However, here the absorption in the 5.0 to 4.0 micron range appears very similar to that of figures 2,3,and 5. This seems to indicate the absorption pattern change is due to the substitution in the number five position of gentlslc aold.

In the region of 5.6 to 6.0 microns of figures 2 and 5 two absorption "humps* are prominent. One represents the ester on the number five phenolic group and the other shows the oarboxyllo sold function. In figure 4, this region la nearly solid Indicating very strong absorption due to the esters and the oarboxyllo aold groups. Figure 3 shows only the acid function,but the ether does not show clearly because each spectrum has strong absorption in the 8.95 to 9.15 nioron region.

Since these two regions, i.e. 3.0 to 4.0 micron and 5.6 to 6.0 micron, are the only ones of value for the lnterpre-

77

tatlon of the known compounds spectra, the unknowns can be evaluated only in these regions.

Figures 6 to 13, inclusive, represent the oarboxyllo aoid ester hydrochlorides synthesised. In each speotrum noticeable change of absorption has taken plaoe In the 3.0 to 4,0 micron region, from that of gentlslo acid. It seems to Indicate from these tracings that any substitution on the three functional groups greatly decreases the absorp­tion in the 3.0 to 4.0 micron region.

Figure 6 shows strong absorption at 3,40-3.50, 6.73- 6.88 and 7.22 microns. The strong absorptions at these wave lengths are due to nujol and are prominent In figures 10 and 18 also.

The “carbonyl* region shows exactly what one would pre­dict from a look at the proposed structure of the synthelzed compounds. Figures 6, 8, 10, and 12 show only one absorp­tion maximum in the 5.6 to 6.0 micron region. This repre­sents the oarboxyllo aoid ester group of these compounds. However, diethylamlnoethyl 5-acetyl-gentisate hydrochloride, dlethylamlnoethyl 5-benzoyl-gentisate hydroohloride, dle- thylaminopropyl 5-aoetyl-gentisate hydrochloride and dle- thylamlnopropyl 5-bensoyl-gentisate hydrochloride (figures 7, 9, 11, and 13 respectively) show two absorption maxima in this range. One peak is due to the ester on number five phenollo group and the other Is due to the oarboxyllo acid ester.

78

Figures 14 to 18, inclusive, represent esters of the number five phenolio hydroxy of gentisic aoid.

In eaoh of these compounds the spectrum shows a d e ­creased absorption in the 3,0 to 4,0 micron region, from that of the gentisic acid spectra.

The region of 5,6 to 6,0 micron show two strong absorp­tion bands in each speotrum with the exception of Figure 17. The first "hump" represents the ester linkage at the number five position of gentisic aold and the second represents the oarboxyllo acid function.

Figure 15 also shows the nitro group peak at a wave length of 6,48 micron.

Figure 17, 5-aoetylsalloyl-gentislc acid shows only one carbonyl group, which is located at 5.95 microns. This in­dicates no esterifloatlon at the number 5 hydroxyl position of gentisic aold. However, the absorption spectrum of the compound la greatly different from that of gentisic acid especially in the 3,0 to 4,0 micron range. Therefore, al­though the speotrum indicates no phenolic ester, it is b e ­lieved that Figure 17 represents the speotrum of a new gen-

tisio aoid derivative.From the analyses of the spectra, the facts indicate

that the reaction between dialkylamlnoalkyl ohlorldes and gentisic aoid yielded dlalkylamlnoalkyl gentisate hydro­chlorides, and the reaotlon between gentisic aoid and the aold ohlorldes of the speolfled acids yielded the 5-phenolle

esters of gentisic aold.

79

T o x l o l t y TestaE v e r y compound thought to be of m e d i c i n a l value must

be screened pharmacologically to determine its activity.One of the first and m o s t important steps in this process is the d e t e r mination of its toxicity. Jhe usual m ethod of e x p r e s s i n g toxlolty is in terms of the dose that kills 50>°of a large group of animals, or the ii> . The term I D50

the abbreviation for "lethal dose to 50^". The term applies for acute or chronic toxlolty tests. The aouto toxicity refers generally to deaths that occur within 24-48 hours after a d m i n i s t r a t i o n of the drug. Chronic toxicity studies extend over a period of weeks, followed by autopsy of the animal to determine w h a t tissues and organs,, if any, are affected.

The oarboxylic acid eaters bei n g w a t e r soluble were a d ­m i n i s t e r e d Intravenously.

The phenollo esters were prepared as follows and a d ­minist e r e d Intravenously. The m a t erial was d i ssolved in alcohol, 3# T w een 80 was added, and the mixture was added to w a t e r w h i c h contained not more than 5% acacia. The totalalcohol content was not more than 5%.

Propylene g lycol and polyethylene glyool d issolved the phenolic esters, but b o t h solvents were toxic when adm i n i s ­tered In doses as low as 0.1 ml. per mouse, hence, could not be used.

The technique used in these tests was as follows:

80

1 # Swiss mice from the same source of supply, weighing from 10 g* to 28 g. were used. They were In good health and had been kept under observation for at least 3 days prior to their use. They were housed in standard cages in a room r e l a t i v e l y oonstant in so far as temperature and hu m i d i t y are oonoerned. Their diet consisted of an unlimit­ed ration of water and Purina Dog Checkers.

2. The solutions were Injected b y means of a 1 ml. tuberculin syringe and a 25 guage, 1 inch needle.

3* A l l doses administered were on a mg./Kg. basis.4* The mice were starved 24 hours to standardize their

metabolism.5, The solutions were given slowly into the tail veins

of the mouse.6 . In e a c h group of esters, i.e. carboxylic acid and

and phenolic, it was neoess&ry to first find the toxicity range. The other compounds in a group wore found to fall

w i t h i n this toxic range.7 # The essential requirements for B e h r e n ’s method of

statistical evalua t i o n were m e t so the la reported andgraphs d r a w n using this Integrated data (70).

81

T o x i c i t y of C a r b o x y l i c A c i d E a t e r s of G e n t l a l c Acid

T a b l e VIL D b o of D i e t h y l a m l n o e t h y l Q e n t l a a t e H y d r o c h l o r i d e______

E x p e r i m e n t a l D a t a I n t e g r a t e d DataNo* of D o a e % %a n i m a l a m g / K g Ali v e D e a d M o r t a l i t y Alive D e a d M o r t a l i t y

12 10 12 0 0.00 31 0 0.0012 30 11 1 8.35 19 1 5.0012 50 6 6 5 0 . 0 0 0 7 26 .6712 7 0 2 10 83.45 2 17 89.4712 JO 0 12 100.00 0 20 100.00

“ >50 e q u a l a 5 1 , 2 m g / K g .

Table VIIIi>5Q of D ie thylamlnoe thyl 5-Ace tyl-Oentlaate H y d r o c h l o r i d e E x p e r i m e n t a l D a t a I n t e g r a t e d DataNo. of D o a e ^anlm a l a m u / k g A l i v e D e a d M o r t a l i t y Alive D e a d M o r t a l i t y

12 3 0 12 0 0.00 31 0 0.0012 40 10 2 16.67 19 2 9.5312 50 4 8 66.67 9 10 52.6312 60 5 7 58.33 5 17 7 3 . 9 212 7 0 0 12 100.00 0 29 100.00

I D50 o q u a l a 5 1 . 5 m g / k g .

T a b l e VIII^ 8 0 of D i e t h y l a n i m o e t h y l 5 - M e t h o x y - o e n t l a a t e H y d r o c h l o r i d e E x p e r i m e n t a l D a t a I n t e g r a t e d DataNo. of anima la

Doae1*sl/¥Lr Alive D e a d

%Mor tality Aliy?.D e a d

>Mortality12 lO 12 0 0 .00 32 0 0.0012 20 11 1 8.35 20 1 4.7612 30 5 7 58.33 9 8 47.0612 40 4 8 66. 6 7 4 16 80.0012 50 0 12 100.00 0 28 100.00

ID50 *quala 32 .2 rag. /Kg.

82Table IX

I D k _ of D i e t h y l a m l n o e t h y 1 5 - B e n z o y l - G e n t i s a t e Hyd r o c h l o r i d eD U ___ _____E x p e r i m e n t a l D a t a I n t e g r a t e d DataNo. of D o a e % /0a n l m a l ^ j n g ^ ^ ^ A l ^ ^ j J e a d J l o r ^ a l j ^ ^ ^ ^ A k l ^ / e ^ e a d ^ J o i M f c a l ^ ^

12 10 12 O 0.00 25 0 0.0012 30 9 3 2 5 . 0 0 13 aw 18. 7512 50 4 8 66.67 4 11 73.3312 7 0 0 12 100.00 0 23 100.uO

£ D50 e q u a l s 41.3 mg.

T a b l e XU >5Q of D i e t h y l a m i n o p r o p y l Q e n t i s a t e Hyd r o c h l o r i d e

E x p e r i m e n t a l D a t a In tegrated DataNo. of Doae % 'uranlmala m g / K g Alive De ad M o r tall ty Alive Dead M o r t a l i t y

12 3 0 12 0 0.00 23 0 0.0012 50 8 4 33.33 11 4 26.6712 70 3 9 7 5 . 0 0 rt w 13 81.2512 90 0 12 100.00 0 25 100.00

I D g o e q uals 58.5 mg.

T a b l e XII D50 of D i e t h y l a m l n o p r o p y l 5 - A c e t y l - G e n t l a a t e H y d r o c h l o r i d e

E x p e r i m e n t a l D a t a In tegra ted D a t aNo. of Doae a/*a n l m a l a m g / K g A l i v e D e a d M o r t a l i ty Alive D e a d M o r t a l i t y

12 30 12 0 0.00 23 0 0 .0012 40 5 7 58.33 11 7 38.8812 50 4 8 66.67 6 15 7 1 . 4 312 60 2 10 8 3 .33 2 25 92.5912 7 0 0 12 100.00 0 37 100.00

I D b o e q u a l a 4 3 . 8 mg. Ag.

83

fiIi

Figure 19Gr aph of I D50 Diethylamlnoethyl Eaters of Gentisic Acid

and Substituted Gentisic Acids© D i e t h ylamlnoethyl Gentlaate Hydrochloride A D i e t h ylamlnoethyl 5-Aoetyl-Gentisate Hydrochloride 4 D i e t h ylamlnoethyl 5-Methoxy-Gentisate Hydrochloride X D i e t h ylamlnoethyl 5-Benzoyl-Gentisate Hydrochloride

64Table XII

II>50 of D i e t h y l a m i n o p r o p y l 5-Methoxy-Gentieata HydrochlorideE x p e r i m e n t a l DataNo* of Doae A n l m a l a m g / K g Alive

%Dead Mortality

Integrated

Alive Dead

Da tac/°

Mortality

12 10 12 O 0.00 22 0 0.0012 20 7 5 41.67 10 5 33.3312 30 2 10 83.33 3 15 83.3312 40 1 11 91.67 1 26 96.2912 50 0 12 100.00 0 38 100.00

£ D&0 equals 22.0 mg. /Kg.

Table XIIIU >50 of Die tiiylamlnopropyl 5-Benzoyl -Gentla&te Hydrochloride

Ex p e r i m e n t a l Data Integrated DataNo, of Doae %Anl m a l a m g / K g Alive D e a d Mortality Allvo Dead Mor t a l i t y

12 20 12 0 0.00 31 0 0.0012 25 11 1 8.33 19 1 5.0012 30 6 6 50.00 Q 7 46.6712 35 2 10 83.33 2 17 89.4712 40 0 12 100.00 0 29 100.00

I°50 oquala 30*2 nig./Kg.

85

Figure 20G r a p h of I D50 of D i e t h y l a m l n o p r o p y l E a t e r s of Gentisic Acid

and S u b s t i t u t e d Gentisic A c i d s

© D ie t h y l a m i n o p r o p y l Qentisate H y d r o c h l o r i d e .A Die t h y l a m i n o p r o p y l 5 - A c e t y 1 - G e n t i s a t e H y d r o c h l o r i d e + D i e t h y l a m i n o p r o p y l 5 - M e t h o x y - G e n t i s a t e H y d r o c h l o r i d e X D i e t h y l a m i n o p r o p y l 5-B e n z o y l - G e n t i s a t e H y d r o c h l o r i d e

86

The least toxlo compound of the oarboxyllo acid ester h y d r o c h l o r i d e • was diethylaminopropyl gentisate h y d r o c h l ­oride with a 12>5o of 58.5 mg./Kg. and the moat toxic com­pound was d i e t h y l a minopropyl 5-methoxy-gentlaate h y d r o c h l ­

oride w i t h a IZ>5q of 22*0 ag. /Kg.Generally, the diethylamlnopropanol eater h y d r o chlorides

are more toxlo than the d i ethylamlnoethanol counter-parta. However, the first meofcers of the series arc reversed In this reapsot. These oompounda follow the general pattern of Inoreased oarbon ohaln lnoreases toxicity.

The m e t h o x y group shows i t ’s noted toxicity b y b e ing the m o s t toxlo ester In b o t h die die thylamlnoe thanol and d iethy l a m l n o p r o p a n o l aeries.

The 5-benzoyl and 5-aoetyl derivatives of the amino- aloohol e s t e r h y d r oohlorIdes are less toxic than the 5 - methoxy d e r i v a t i v e .

A p p a r e n t l y the toxlolty of these compounds is due prin­c i p a l l y to die amino aloohols because the parent compounds

are relati v e l y non-toxlo.The mloe, that died, went into convulsions and died

w i t h i n a m inute after the oompound was injected. No deaths ooourred after 5 minutes from the time of injection.

87T o x l o l t y of Phenollo Matera of Gentisic Acid

Table X I VIDgQ of 5 - P h e n ylaootlo-Oentlelo A o l d

E x p e r i m e n t a l D a t a I n t e g r a t e d DataNo. of Doae % 7°anlmala m g / K * Alive D e a d Mortality Alive D e a d M o r t a l i t y

12 160 12 0 0.00 31 0 0 .0012 1 8 0 9 3 25.00 19 3 14.2912 200 6 6 - 50.00 10 9 47.3712 220 4 8 66.67 4 17 80. 9612 240 0 12 100.00 0 29 100.00

U>5Q equals 201.2 m g ./Kg,

Table XVU>50 of 5-Paranltrobensoyl- Gentialc Aoid

B x p e r l m e n t a l D a t a I n t e g r a t e d DataNo. of Doae % %anl m a l a mg/fot Alive Dead M ortality Alive Dead Mortality

12 SO 12 0 0.00 39 0 0.0012 SO 9 3 25.00 27 3 10.0012 7 0 9 3 25.00 18 6 25.0012 90 7 5 41.67 9 11 55.0012 110 2 10 83.33 o** 21 92.1712 ISO 0 12 100.00 0 33 100.00

II>50 equals 85 • 5 mg. Ag.

T a ble XVIU>50 of 5 - A n l syl-Gentlaio Aold

E x p e r i m e n t a l D a t a I n t e g r a t e d DataNo. of Doaa % %anlmala Alive Dead M o r t a l i t y Alive D e a d M o r t a l i t y

12 20 12 0 0.00 36 0 0.0012 3 0 10 2 16.67 24 2 7 . 6 912 40 5 7 58.33 14 9 39.1312 50 6 6 50.00 9 15 62.5012 6 0 3 9 75.00 3 24 88.8812 7 0 0 12 100.00 0 36 100.00

I DS0 aquala 44 .9 mg. Ag.

88

TAB IK XVIIIDcq of 5-Aoetylsalloyl-Gentisic Aold

Experimental Data Integrated DataMo, of animals

DoseAlive Dead %Mortality Alive

%Mortalit:

12 10 12 0 0,00 33 0 0.0012 20 9 5 25,00 21 3 12.5012 30 7 5 41.67 12 8 40.0012 40 4 8 66.67 5 16 76.1912 50 1 11 91.67 1 27 96.4312 60 0 12 100.00 0 39 100.00

ID^o *4uals 32• 4 mg. /Kg.

TABLE. XVIIIIDgo of 5,5'-3uoolnyl-Digentlsio Aoid

Experimental Data Integrated DataNo. of Doaa anlmala rng/K^ A^v^Dea^Mo^a^Llt^^^^^^^^ea^JJortalitT^

12 100 12 0 0.00 26 0 0.0012 130 8 4 33.33 16 4 20.0012 160 6 6 50.00 8 10 55.5512 190 2 10 93.33 2 20 90. 9112 220 0 12 100.00 0 32 100.00

IDgo •V*als 155.0 mg./Kg,

TABIS XIX IDgo of dentIsle Aold

Experimental Data Integrated DataMo, of Doae % %anlma^^^jg^^^^i^llve^Dead^2lortaLlty__ Alive Dead Mortality

12 320 12 0 0.00 38 0 0.0012 340 10 2 16.67 26 2 7.1512 360 8 4 33.33 16 6 27.2712 380 6 6 50.00 8 12 60.0012 400 2 188:88 2 22 91.5812 420 0 12 0 34 100.00

IDg0 equals 374,0 mg./kg.

89

m

Figaro 81Qrapti of U>5Q of Fhonollo Bitara of Gontlolo Aold

8 5-Ptaonyloootyl-Gontioio Aold A 5-Poronltrobonftoyl-Oontlolo Aold + 6»Aaloyl-Ooatlolo Aold* 6-Aootyloolloyl-OontiBlo Aold• 6,ft'-Suooinyl-Dlgontlolo Aold

00

Ii

i

I

VI

Figure 22 Gruph of IDgo of Gentisic Aold

91

The phenolic eaters vary In toxicity from 32.4 mg./Kg. for 5-aoetylsalicyl gentisic acid to 201.2 rag./Kg. for 5- phenylaoetlo-gentislo aold.

The increase in toxicity of these compounds over that of gentisic acid m a y be attlrubuted to the inherent toxicity of substituted functional group, i.e. paranitrobenzcyl and anisyl. However, the increased acidity of the compound may also be partially responsible for Its increased toxicity.

Groups on the benzene ring of the substituting function appears to Increase the toxicity of the compound.

The phenolic enters caused convulsions before death in the mloe killed. However, the action of these compounds was slower, generally requiring several minutes to kill the ani­mal injected.

Since ID,.,-, for gentisic acid was found to be 374.0 oOmg./Kg., intravenously, and the ID50 reported for sodium gentlsate was 3.1 mg.Atg., intraperitioneal (36), the tox­lolty of the esters synthesized is considerably greater than gentisic acid or its sodium salt. The toxicity of these compounds la also greater than that reported for sodium salicylate (i.e. 0.88 gm/Kg., lntraperltioneal) (36). These reported values were determined on rats and seem high com­pared to that which was found for gentisic acid intravenously.

Generally, the toxlolty of phenolic esters is lower than that of the carboxylic acid esters. This m a y be due to the

92

f a c t that the f o r m e r types were given in suspensions whe r e a s the l a t t e r were aqueous solutions.

Due to the h i g h e r toxlolty of the p r e p a r e d products, they w i l l need to have c o n s i d e r a b l y more a c t i v i t y than g e n t l a l o a o i d to be of greater value m e d i c i n a l l y .

No ohronic toxicity studies of these compounds wor© made, b u t f r o m o b s e r v i n g the animals i n j e c t e d on a given day, it w a s noted that no d eaths o c c u r r e d a f t e r 5 to 10

m i n u t e s f r o m the time of injection.If these compounds have analgesic value, they w o uld

p r o b a b l y b e a d m i n i s t e r e d orally, i.e. as tablet or powder. O r a l toxicity is m u c h l o w e r than intravenous tonicity so the therapeutic index of the compound m a y be b e l t e r than the results show.

r 93

Summary

A t t e m p t s were made to synthesize several amino alcohol ester h y d r o c h l o r i d e s of gentlslc acid. From the attempts eight new compounds were formed. It w as found that the r e ­actions b e t w e e n the chlorides of the alcohols and the free acids gave the best yields and apparently the most nearly pure products. It was also observed that in the case of the successful reactions the intermediate halides had been r e ­ported In the literature, w h e r e a s in the cases where unre- ported intermediates had to be used, the yields were poor and the products of d o u btful purity.

The five phenolic eaters were prepared successfully by the S o h o t t e n - B a u m a n reaction. The yields were good and the products r e l a t i v e l y pure. The acid chlorides were prepared by the use of thionyl chloride and the properties of these acid c h lorides w e r e found to be of the same nature as liter­ature r e p orted values.

Th e reaction b e t w e e n the acid chlorides and ^entiaic acid, usi n g pyridine or dime t h y l a n 1line as a condensing medium, was not successful. It Is believed that the pyridine or d i m e t h y l a n l l l n e was not strong e nough to remove the h y d rogen f r o m the phenolic hydroxide and the chlorine from the acid chloride.

T o prove the site at w h i c h e s t c r ifIcation took place on gentlslc acid, w h e n the acid and the halide or acid chloride

94

were reacted, two qualitative procedures and one quantitative technique were utilized. The first qualitative test was the ferric chloride color teat. The test yields a dark blue coloration for orthohydroxyl carboxylic acids, but a nega­tive test w i t h metahydroxyl oarboxylio acids and substituted orthohydroxyl carboxylic acid esters. Therefore, since all the esters give a positive test, it is indicative that the number 2 hydroxyl group of gentisic acid was free.

The second qualitative proof of the point of esterifi- oation was the determination and analysis of the infrared spectrum of the compound. B y comparing the spectrum of the synthesised compound to that of known compounds, it is easy to see whether one or two carbonyl functions are present.Gentisic acid shows one carbonyl and carboxylic acid esters of gentlslc acid also show only one carbonyl function when the number 5 hydroxyl is free or is blocked by an ether linkage. However, for thoss carboxylic acid esters that also have acetyl or benzoyl function in number 5 postion of gcn- tisie acid, two carbonyl groups are located on each spectrum. All the spectra of the phenolic esters show two carbonyl functions present, one is due to the carboxylic acid group of gentlslc acid and the other is due to the ester at number 5 position of the acid.

The quantitative proof of esterif ioa tion was de tornilr.ed b y the neutral equivalent procedure. The phenolic esters were titrated directly with base and the carboxylic acid

95

eater hydrochlorides were titrated w i t h base, liberating the amine and the amine was again converted to the salt b y aoidic titration. The molecular weights are good for the phenolic esters exoept for 5-aoetylsalicyl-gentiaic acid.The mol e c u l a r w e ight determinations for the carboxylic acid ester hydroohlorides range f r o m good to satisfactory. B e t ­ter results could have b e e n obtained If a larger sample of synthetic compound and microburettes had been used.

The unreactive nature of the number 2 hydroxyl group of gentlslc acid is attributed to strong hydrogen bon d i n g b e t w e e n the hydrogen of the h y d r o x y l gro u p and the carbonyl of the acid group, and to the electron effect. I.e. the number 5 h y d r o x y l group supplies electrons to the ring e s ­pecially ortho and para, thus hindering the removal of the hydrogen of the number 2 hydroxyl. However, the latter e f ­fect m a y be offset b y the m e t a directing carboxylic acid function ortho to the h y d r o x y l group in question.

The only synthesized compound the formula of which Is In doubt is that of 5-aoetylsalioyl-gentisic acid. The two outstanding discrepancies between the proposed and the a c t ­ual structure of the compound are the m o l e c u l a r weight and the Infrared spectrum. The carbon and hydrogen analysis also shows the found carbon to be about 1% higher than the calculated amount for 5-acetylsalicyl-gentiaic acid. How­ever, the author believes the compound to be a new synthetic derivative of gentlslc acid. This Is supported by the faots

96

that the infrared spectrum, neutral equivalent anti carbon and hydrogen analysis are all very different from those of the parent compounds. The following m a y explain the dis- orepanciesx The produot was prepared b y the Interaction of aoetylaalloyl chloride .nd gentlslc acid In basic media.The a d d chloride was prepared as previously reported in the literature. The acid chloride produced had the accepted physical properties. It is known that acetyl groups are m o s t easily hydrolysised in basic solution. Therefore, the possibility exists that either before or after the desired esterifioatlon took place, hydrolysis of the acetyl group of acetylsalioyl chloride was brought about. The compound produced, therefore, may be 5-aalicyl-gentisio acid. The molecular weight of the compound, i.e. 274,22 g,, is more nearly equal to the found molecular weight, 285,2 3 g , , than is the molecular weight of 5-aoetylsalicyl-gentlslc acid, i.e, 516,26 g. The carbon m d hydrogen analysis is also more nearly oorreots Calculated for C ^ H ^ o ^ e 3 c * 61.51;H, 3.63* Founds C, 61,74, 61,53; H, 3,92, 3,86. The a s s u m p ­tion that the n e w compound is actually 5 - salicyl-gentisio a d d also m a y explain w h y only one carbonyl function shows on the Infrared spectrum of the compound. It may be that the hydrogen b o n d i n g between the hydroxyl function of the salloyl r a d i o a l and the carbonyl function of the ester group is too strong to be detected. Therefore, only the carboxylic f unction of gentlslc acid shows in the spectrum.

97

The relative high toxicity of the carboxylic acid eater hydroohlorides appears to be primarily due to the amino aloohol portion of the molecules* However, the groups In number 5 position of gentlslo acid do exert some influ­ence in theae eaters, especially the methoxy group. The m e t h o x y group la noted for Its toxic nature and both d e r i v ­atives containing this grouping are the most toxic of their a e r i e s .

The Increased length of chain, i.e. ethyl to propyl, of the aloohol, also Increases toxicity as one would expect,

The toxicity increase of the phenolic esters over that of gentlslc acid may be because of the increased acid strength of the compound. The increased acidity is brought about b y the re placemen t of the ortho-para directing hydroxyl group by the xneta directing ester function. The functional groups added also play their part because the nitro and m e t h o x y containing compounds are the most toxic of the series. The 5-aoetylsalicyl-gentiaic aoid appeared to be much more toxlo than one would predict. This uncertainty again indi­cates that the proposed structure m a y be incorrect.

A l l the esters of gsntisio acid were prepared with the hope of producing new and better drugs. The syntheses were planned from the point of view of combining two medicinally active nuclei to form a compound more active than a physical mixture of the compounds. Since the reacting compounds have analgetic and antlrheumatio activity, it is believed that

98

the synthesised compounds should have a c t i v i t y of the same nature. The pos s i b i l i t y also exists that the carboxylic sold e s t e r hydrochlorides w i l l have local anesthetic and/or antlspesmodio aotivity. The phenolic esters m a y also be b a o t e r l o l d a l In nature.

The value of these oompounds w i l l have to be determined b y p h a r m a c o l o g i c a l testing before the true m e d i c i n a l value of the synthetics can be stated. If a o t ivity is shown b y the oarboxylio aold esters. It Is suggested that other active compounds m a y be prepared b y a l t e r i n g the amlno alcohol ahaln, as In the case of local anesthetics, and r e placing the tertiary amine w i t h primary and secondary amines.

If the p h e n o l i c esters show m e d i c i n a l value, an u n l i m ­ited number of esters oan be p r e pared b y coupling of other s m d l o l n a l l y active groups to gentlslc a d d .

Also, the number 2 hydroxyl position of gentlslc acid should be a t t a c k e d b y other procedures to attempt s u b stitu­tion In this p o s ition of gentlslc aold.

The analyses a n d toxicity of the synthesise eaters of gentl s l c aold are listed in Table XX. Tab l e XXI Includes the physloal, properties, the stability studies, a n d the ooloratlon teat w i t h ferric chloride for the esters.

99

TAB IE ] ANALYSES OF SY

lama of Compound (ftlplrloil Formlt) Structural FormulaIBByTBnSBylPQastlaataM m h l w i d i(Ol0l«O&4Kl)Dla IhrtiilaN thjl Aaa ayl-Oa at iaa ta Hydrochloride (C^^lgO^KOl)Dia tfe/lamlnae thyl 6*M» thoxy*>Oa n 11 aa ta Mvoihlorlla

Dla thy lam la oe thyl 6-Bansarlr Qaatlaata Hydraahlarlda

Dla thylaartaopropyl QaatlaataHydreohloride(014IW V ,C1>Dla (kilaaiMpropyl 5-*Aaa»yl-Qentleate Hydrochloride (0lfH1406»Cl)Dla tteltti&nipnpTl 5-Wethoxy-Oentieato

Hydreohloridet,u V i * I»

JgWWfci

H O O T ‘

Molaoular Weight N la grama

Calonlafd* Found* C, 253*50 852*11

295.S3

f « Ac%» C r

jMrO*iM<a%0|357,39

267,32

309.35

w O “281.34

301.65

**Nj|gc*«Wi 267,32 269.35

355.75

265.50

309.46

280.17

TAB 18 XX£3 OF SYNTHBSiaO) COMPOUNDSlght Nitrogen Content Chlorine Content Carbon & Hydrogen ^

In per oent in per oent Content in per oent 50. Fouff* Found* Oloul.fd Found* C.laul.fd Faund ma./Za.53.11 4^84 4.80 12.60 12.61 - ^ 7 ------ 51.2

11,66 4,22 4.17 10,69 10.62 C, 54.30 C# 54.41 51.5H, 6.68 H, 6.78

39.36 4.61 4.70 11,68 11.62 32.2

-5*76 3.66 3.42 9.00 8.94 C, 60.85 C, 60.40 41.7H, 6.14 H, 6.41

>5.60 4.61 4.65 11.68 11.80 C, 65,35 C, 55.45 58.5H, 7.30 H, 7.42

9.46 4.05 4.04 10.25 10.34 44.5

5.17 4.41 4.35 11.16 11.27 22.1

100

XX

Dlothylaalnopropyl C40U3bCH.mciiiCJ(>5-Bonioyl-0«ntl8ato ^ ^ y l | i r *373.42 366. MHydrochloride

J A C W iCM,*

Q c o C r ^

joa

Q c * j £ r

5* Phenylaoetyl*Gentielo Add § \ r u.rrJL I 272,25 273.38(Ci8H18°5)

5-Par anitrohemoyl- * >N Q 19Gentlaio Add " 303.20 304.19' OQ*Mf 7C0f(C14Hg07N)

5-Aniayl-Gentieio 208.25 290.50<ci5Bi20e> c ^ c a k j

6-Aoatylaalloyl-Oentiaio Aold j v T l L 1 316.26 285.23

L / c<

8(0?f S r i;U 390,29 388.37

Oartlalo Aold (c7BeOi)

jam

- c O "*AY*raga of too valuaa#Wolaoular Wolght Calculated for Free Amine,

TABLE XX (COtfT'D)

5.34 5.43 3.60 8.69 3,62

3.38 C, 66.17 H, 4.44

C, 66.21 II, 4,51

4.19 C, 55.46 H, 2.99

C, 55.43 K, 2.97

D.50 C, 62.50 H, 4.19

C, 62.43 H, 4.54

5.23 G, 60.76H, 3.83

C, 61.65# H, 3.39#

8.37 C, 55.39 H, 3.62

C, 55.20 H, 5.82

30.3

201.3

83.5

45.0

32.4

155.0

374.0

101

TABI£ XXI ____________________ Properties of Synthesized Compounds

SolubilityName of Hlorosooplo Alco- Ace- Chlo-CoigBOttad m,p»# Appearance Water hoi Kther tone roforrrBl#tttyl3 iI5 o o t n y r i 5 0 - ^ " B u o I o r T e » iy !"* ' ........ .

Oenfcisate 151° Plates s a 1 1 1Hydrochloride

Dlethylamlnoe thyl 155,5- Colorless5-Acetyl-Qentlsa te 134*5° Needles s s s i s s l s s

HydroohlorideDiethylamlnoethyl 182,3- Colorless5-Methoxy-Oenti- 183,0° NeedlNeedles s s ol s

sateHydrochloride

Diethylamlnoethyl 153- Colorless s s si s s5-Bensoyl-Oentl- 154° Needles

sate Hydrochloride

Dlethylamlnopropyl 171,5 ColorlessOentlaate 172.5° Plates s a i l

HydroohlorideDlethylamlnopropyl 157- Colorless5-Acetyl-Qentlsate 157,5° Needles s s s sHydrochloride

Dlethylamlnopropyl 184,5- Colorless5»Methoxy-Oentl- 185-3° Needles s s 1 si s

sate HydroohlorideDlethlaminopropyl 142,7- Colorless5-Benaoyl-Qenti- 143,7° Needles s a l s

sateHydroohloride

5-Plenylaoetyl- 145-5- ColorlessCent!sic Aold 146,5° Fibers 1 s s s

TABIB XXISynthealaad Compounds________________________________________Solubility Stability# F e r r i c #Aloo- Ace- Chlo- Chloride

• 1 1 1 + - +

a al a al a s - +

9 si a 1 s + - +

• s i a a a + ♦

a l i a + - +

a a a a + - +

a i al a s + - +

a 1 a a + - +

a a a a + -b" +

102

TAI3LK XXI (CONT'D)

S-Paranltrobensoyt 229- YellowGentlslc Aold 230.5 d. Needles 1 si 3 3

5-Anifyl-Gentisio 209.4- Colorless Aold 209.5° Needles s 3 s

5-Acetylsallcy> 203.5- Gentlslc Aold 204.7

Sheavesof

Colorle ss Needles

5,5'-Suoclnyl* 230- Colorless s si a a 3Gentlslc Aold 231,5°d. Fibers

• Uncorrected

f + equals positive; - equals negative

■ b equals basic.

TAB IS XXI (CONT*D)

1 a l 8 a s -b"

a s -b"

a a -b"

a a l a s - b 1

103

CONCLUSIONS

I* Thirteen new esters of gontisic acid wore prepared, eight of vrhioh ere oarboxyllo aold esters and five are phenolic esters.

A. The carboxylic acid ester hydrochlorides.(1) The oarhoxylic acid esters were prepared in

good yield, by the interaction of the chloride of tertiary amino alcohols and gentlslc acid or substi­tuted gentlslc acids.

(2) The physical properties of the oompounds, i.e. melting point, microscopic appearance and solubility,

were determined.(3) The carboxylic acid esters are salts, and

therefore, they are readily soluble in water. However, aqueous solutions of the salts must be buffered so the solution is acid to insure stability.

(4) Qualitative and quantitative nitrogen and chlorine content of each oarboxyllo aold ester salt was determined.

(5) All the oarboxyllo acid ester hydrochlorides

gave a positive ferric ohlorlde test.(6) Diethylamlnoethyl 5-acetyl-gentisate hydrochlo­

ride, diethylamlnoethyl 5-benzoyl-gentisate hydrochlo­ride and dlethylamlnopropyl gentisate hydrochloride were analyslzed for oarbon and hydrogen.

104

(7) A m o d i f i e s t l o n of the u s u a l proceduro for d e ­t e r m i n a t i o n of n e u t r a l e q u i v a l e n t s of the carbo x y l i c a c i d e a t e r h y d r o c h l o r i d e was e m p l o y e d . The procedure w as to l i b erate the amine b y a d d i t i o n of e x c e s s s t a n ­d a r d b a s e and then t i t r a t e the e x c e s s base and the a m i n e w i t h s t a n d a r d acid.B. P h e n o l i c eaters.

(1) T he n u m b e r 2 h y d r o x y l group of g e n t i s i c a c i d w as f o u n d to be v e r y d i f f i c u l t to r ^ act with, if not e n t i r e l y inert; w h e r e a s , the n u m b e r 5 h y d r o x y l g r o u p was v e r y r e a c t i v e .

(2) T h e p h e n o l i c e s t e r s w e r e p r e p a r e d b y the I n ­t e r a c t i o n of the acid c h l o r i d e and the v a r i o u s aci d s and the n u m b e r 5 h y d r o x y l g r o u p of g e n t i s i c acid in a l k a l i n e media, i.e. S c h o t t e n - B a u m a n reaction.

(3) The p h y s i c a l p r o p e r t i e s of the compounds, i.e. m e l t i n g p oint, m i c r o s c o p i c a p p e a r a n c e and solu b i l i t y , w e r e d e t e r m i n e d .

(4) T h e p h e n o l i c e s t e r s c a n be m a d e p a r t i a l l y s o l u b l e b y m a k i n g the s o d i u m salt of the g e n t i s i c acid p o r t i o n of the m o l e c u l e . A l t h o u g h the salts may be u n s t a b l e in solution, if In t a b l e t form, t h e y w o u l d f a v o r a b s o r p t i o n o f the compound.

(5) A l l the p h e n o l i c e s t e r s gave a p o s i t i v e f e r r i c c h l o r i d e test.

105

(6) S - V a r a n i t r o b e n z o y l - g e n t i a 1 c a c i d gave a p o s i ­tive test for nitrogen.

(7) T he phenolic e s t e r s were analysized <’or carbon a nd hydrogen.

(8) The n e u t r a l e q u i v a l e n t s of the phenolic esters were d e t e r m i n e d in the u s u a l manner.

(9) The average equivalent of 5 - a c e t y l a a l i c y l - g e n t i s i c acid w a s found to be 30 g. too low. T h e r e ­fore* doubt e x i s t s as to w h e t h e r this is the correct f o r m u l a of the compound.

II. The Infrared s p e c t r a of the new e s t e r s of gentlslc a c i d were a n a l y s i z e d to prove the structure of the compounds.

A. The carboxylic acid ester h y d r o c h l o r i d e s .(1) D i e t h y l a m l n o e t h y l gentisate h y d r o c h l o r i d e ,

d i e t h y l a m l n o e t h y l 5 - m e t h o x y - g e n t l s a t e h y d r o c h l o r i d e and the d l e t h y l a m l n o p r o p y l c o u n t e r - p a r t3 3how o n l y one o a r b o n y l f u n c t i o n in the spectra. T h i s i n dicated ea- t e r l f i o a t i o n through the carboxylic acid of g e n t l s l c

aold.(2) Die thylaminoethyl 5 - a c e t y l - g e n t l s a t e h y d r o ­

chloride* d i e t h y l a m l n o e t h y l - 5 - b e n ^ o y l - g e n t isate h y d r o ­chloride and the d l e t h y l a m l n o p r o p y l c o u n t e r - p a r t s show two o a r b o n y l f u n c t i o n ® in their spectra. T h i s indicates e s t e r i f l e a t i o n through the c a r b o x y l i c aold a n d the n u m ­

b e r 5 h y d r o x y l of gentlslc aold.

106

B. The phenolic esters.(1) All five phenolic esters show two carbonyl

functions in their spectra. This Indicates that the carboxylic acid of gentlslc acid Is free and that the point of esterlfloatlon Is at the number 6 hydroxyl group of gentlslc acid.

(2) The spectrum of 5-acetysalicyl-g«ntlsic acid shows only ono oarbonyl function. Therefore, doubt exists as to whether the proposed formula is the true nature of the compound.

H I . The H>5q of each new ester of gentlslc acid was de ­termined by Intravenous injection of the compounds Into white mice.

A. The carboxylic acid oster hydrochlorides.(1) The most toxic of the carboxylic acid ester

hydrochlorides Is 58.5 mg./Kg. on mice whereas the least toxic is 22,1 mg./Kg.

(2) Die thyleuninoe thyl 5-me thoxy-gentisa te hydro­chloride is the most toxic compound of the diethyl­amlnoe thanol esters and dlethylamlnopropyl 5-methoxy- gentisate hydrochloride is the moat toxic compound of the dlethylamlnopropyl esters. The latter Is the most toxio compound of all the carboxylic acid ester hydro­ohlor ides.

Diethylamlnoethyl 5-acetyl-gentisate hydrochloride is the least toxic compound of the diethylamlnoethyl

107

eaters and dlethylamlnopropyl gentlaate hydrochloride la the least toxic oompound of the dlethylamlnopropyl esters. The latter is the least toxic c nn pound of the oarboxyllo acid ester hydrochlorides.

5-aoetyl, 5-me t h o x y and 5-benzoyl gentlslc acid eaters of d i e t h y l a m inopropanox hydrochloride are more toxlo oompounds than the diethylamlnoethanol deriva- tie a of the same acid.B. The phenolic esters.

(1) Ihe phenolic esters mi n i m u m and maximum toxicity varies from 201.3 mg. ACg. to 32.4 mg.''Kg., respectively on mice.

5-phenylacetyl-gentlsl c aold Is the least toxlo phenolic ester and 5-acetylasalicyl-genti sic acid is the most toxic.

S u bstitution on the benzene ring of the phenolic esters seems to greatly Increase the toxicity of this group of compounds.

108

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AC,Obi OGHAFHY

I, J. Frank Nash, was born In Ea3t Canton, Ohio, "ay 19, 1924. I reoelved my secondary school education In the public schools of the city of Fast Canton, Ohio and Osna- burg Township. My undergraduate training was obtained at Ohio State University, from which I received the degree of Bachelor of Science in 194t30 In 1949 I was appointed Upjohn Fellow in Pharmaceutical Chemistry, College of Pharmacy, Ohio State University, I held this fellowship for a period of three years while completing the require­ments for the degree Doctor of Philosophy.