Design, Synthesis and Antitumor Activity of Novel D-Glucuronic Acid Derivatives

1573-4064/11 $58.00+.00 © 2011 Bentham Science Publishers

Design, Synthesis and Antitumor Activity of Novel D-Glucuronic Acid Derivatives

Ahmed. O. H. El-Nezhawy1,2b,*

, Frady. G. Adly1, Ahmed F. Eweas

2a,b, Atef. G. Hanna

3, Yehya.

M. El-Kholy4, Shahenaz H. El-Syed

4 and Tarek B. A. El-Naggar

5

1Chemistry of Natural and Microbial Products Department, National Research Center, Dokki, Cairo, Egypt;

2aMedicinal Chemistry Department, National Research Center, Dokki, Cairo, Egypt;

b Pharmaceutical Chemistry

Department, College of Pharmacy, Taif University, KSA; 3 Chemistry of Natural Compounds Department, National

Research Center Dokki, Cairo, Egypt; 4 Chemistry Department, Faculty of Science, Helwan University, Egypt;

5 Phar-

macology Department, Faculty of Pharmacy, Complutense University, Spain

Abstract: A series of D-glucuronic acid derivatives were chemically synthesized including acetylated and deacetylated

glucuronamides, as well as N-glucuronides starting from the D-glucuronic acid itself by means of protection/deprotection,

activation and condensation protocols. Structure elucidation of all products along with optimization of the synthetic steps

is described. The synthesized compounds were evaluated for their in vitro antitumor activity against MCF-7, TK-10 and

UACC-62 cell lines. The compounds 4, 5, 7, 8, 14, 16 and 18 were the most active against TK-10 cell line. On the other

hand, the most active compounds against the MCF-7 cell line were 9, 18 and 20. However, compounds 7-10 13-15 and 17

were the most active against the UACC-62 cell line.

Keywords: D-glucuronic acid, D-glucuronamide, amide linkage, antitumor, TK-10, MCF-7, UACC-62.

INTRODUCTION

Cancer has been a major lethal threat worldwide till date. This malady is not a single ailment, but rather a collection of hundreds of different diseases as a result of uncontrolled cell proliferation. Chemotherapy is a major cancer treatment be-sides surgery and radiation therapy. The majority of the chemotherapeutic drugs that are currently in use are organic drugs or natural products. The synthesis of new compounds and testing their biological and pharmacological activities are the major goals of drug development projects. D-Glucuronic acid (D-GlcA), its mono- and polysaccharide derivatives are important natural compounds, widely distrib-uted in animals and plants [1]. Besides, synthetical D-GlcA derivatives find numerous applications in bioactive mole-cules development [2-4], in material sciences [5,6], and as surface-active agents [7-10].

A variety of natural nucleosides were isolated having a D-GlcA derivative as a carbohydrate moiety [11] (Fig. 1). Gougerotin, Bagougeramines A & B, and Blasticidin C are nucleoside antibiotics which exhibit a wide spectrum of bio-logical activities including antibacterial and antiviral activi-ties by inhibition of peptide bond formation. They also ex-hibit an antitumor activity [12]. A series of nucleosides which contain a D-GlcA ring were synthesized including purine [13], pyrimidine [14], benzimidazole [15], uracil and cytosine nucleosides [16].

*Address correspondence to this author at the Chemistry of Natural and Microbial Products Department, National Research Center, Dokki, Cairo, Egypt; Pharmaceutical Chemistry Department, College of Pharmacy, Taif University, KSA; Tel: (+966) 550840062; Fax: (+202) 3337 0931; E-mail: [email protected]

ONH HO

OH

N

NHO N

O

NH2

O

HN

R1

O

NH

R

Me

Gougerotin R = H, R1 = OH

Bagougeramine A R = H, R1 = -NHC(=NH)NH2

Bagougeramine B R = -(CH2)3NH(CH2)4NH2, R1 = -NHC(=NH)NH2

ONH

N

OHO

NO

NH2

ONH2

NMe

NH2HN

Blasticidin S

Fig. (1). Natural nucleosides containing a D-GlcA derivative.

D-GlcA mimicking aza-sugars have been isolated as well as synthesized [17-23]. 5-Aza-D-GlcA is a natural product isolated from the seeds of Baphia racemosa and found to be a specific inhibitor of human liver -D-glucuronidase [24] and, recently, Cipolla et al. [25] succeeded to prepare its bicyclic analogue (Fig. 2). Many other aza-sugars derived from D-GlcA showed potential anticancer activity [26].

Glucuronide prodrugs are widely used in Cancer Tar-geted Enzyme Prodrug Therapy. For better selectivity and efficacy with reduction in systematic toxicity of cancer che-motherapy, -glucuronidase enzyme, that are selectively

624 Medicinal Chemistry, 2011 , 7, 624-638

expressed at the tumor area, is used for conversion of rela-tively non-toxic glucuronide prodrugs into the corresponding parent cytotoxic agents [27].

As a part of our ongoing research on drug discovery, we

needed to develop a facile and rapid synthetic pathway to

new drug-like small organic carbohybrids containing a D-

GlcA moiety. Our previous research in the area of drug dis-

covery has resulted in the synthesis of a series of indole–

pyrimidine (meridianin D analogues) and phthalazinone–

amino acid conjugates. These conjugates were tested for

their antitumour activity and showed moderate activities

against caucasian breast adenocarcinoma (MCF-7) cell line.

Also, our research included benzimidazole sugar conjugates

which showed anti-inflammatory and analgesic activities

[28-31].

RESULTS AND DISCUSSION

Chemistry

Studies were initiated by acetylation of the commercially

available D-glucuronic acid with acetic anhydride and a cata-

lytic amount of iodine as an acetyl transfer reagent according

to the previously described method [32] to give the corre-

sponding 1,2,3,4-tetra-O-acetyl protected mixed anhydride 1

in 98% yield. The formation of 1 was confirmed by 1H NMR

spectroscopy. In particular, the acetoxy methyl signal that

appeared at relatively low field (2.28 ppm) indicated the ex-

istence of the COOCOCH3 group, in addition to a higher

field signals observed at 2.13 (3H), 2.05 (6H) and 2.04 ppm

(3H) which corresponds to the four O-acetyl groups.

OAcO

AcO

OAc

OAc

OO

O

OAcO

AcO

OAc

OAc

OHO

Ac2O, I2,

rt, 2 h

H2O, THF,

rt, overnight

D-GlcA 1

2

OHO

HO

OH

OHO

OH

Scheme 1. Acetylation of D-GlcA.

When compound 1 was subjected to reaction with water, it afforded the corresponding 1,2,3,4-tetra-O-acetyl- -D-glucuronic acid 2 in 99% yield (Scheme 1). The formation of 2 was confirmed by NMR in which the

1H NMR spectra

revealed the absence of the acetoxy methyl signal at 2.28 ppm and the appearance of a broad signal at 3.44 ppm corre-sponding to the carboxyl group proton [32]. Compound 2 is characterized by its stability at room temperature for long period of time without detection of any decomposition but when it was dissolved in diethyl ether and left to crystallize, the

1H NMR of the crystals formed was indicative to D-

glucuronic acid tetra-acetate having an -anomeric configu-ration (Fig. 3). In particular,

1H NMR signals for the ano-

meric proton of the -anomer was observed at 6.00 ppm (d, J1-2 = 8.1 Hz), while the corresponding signal for the -anomer appeared at 6.38 ppm (d, J1-2 = 3.5 Hz).

OAcO

AcO

AcOOAc

OHO

Fig. (3). 1,2,3,4-Tetra-O-acetyl- -D-glucuronic acid.

Fortunately, the mixed anhydride 1 was a suitable precur-sor for the synthesis of the D-glucuronamide derivatives in which:

(1) All the hydroxyl groups are protected with acetyl groups.

(2) The carboxylic acid group is activated through mixed anhydride activation.

On this basis, N-(Benzyl)- (3) and N-(Allyl)-1,2,3,4-tetra-O-acetyl- -D-glucopyranuronamide (5) were obtained by the reaction of mixed anhydride 1 with benzyl or allyl amine in anhydrous DCM. The crude reactions products were purified by extractive work-up, followed by a chromatographic puri-fication (Scheme 2).

The structural assignments for compounds 3 and 5 based on their NMR spectral data were in a good agreement and confirmed the proposed structures. In particular, for com-pound 3, the

1H NMR spectrum showed the anomeric proton

as a doublet at 5.94 ppm with spin-spin coupling constant of 8.4 Hz which corresponds to a diaxial orientation of H-1 and H-2 protons indicating a -anomeric configuration. The other four protons of the glucopyranosyl ring resonated in the 5.40–4.33 ppm region. The remaining four acetoxy groups appeared as four singlets at 2.03, 1.97, 1.91 and 1.83 ppm. The five phenyl ring protons appeared as a multiplet in the 7.26–7.17 ppm region, while the methylene protons resonated at 4.20 ppm. The splitting pattern of the methylene group was ddd (double of doublets for each methylene pro-ton) which corresponds to an ABX system and it was re-duced to a double of doublets after proton exchange experi-ment (Fig. 4). This was accounted by the presence of chiral centers in the vicinity of the methylene group which makes a ‘non-equivalence’ between the two methylene protons and in turn a diastereotopic effect will be reflected in the

1H NMR

spectrum and the CH2 group will appear as a double of dou-blets (doublet for each methylene proton). Further coupling with the neighboring amide NH proton will give the ob-served ddd pattern.

HN

HOHO

OH

OH

OHO

NHOHO

OH

OHO

OO

CH2NH2

5-Aza-D-GlcA Bicyclic analogue

Fig. (2). Iminosugars derived from D-GlcA.

Design, Synthesis and Antitumor Activity of Novel D-Glucuronic Acid Medicinal Chemistry, 2011, Vol. 7, No. 6 625

Additionally, the carbon skeleton of compound 3 was fully assigned with the aid of

13C NMR spectra and DEPT

experiment. The 13

C NMR spectrum was characterized by a signal at 91.2 ppm corresponding to C-1 of the -D-glucopyranose ring. Another four signals at 72.9, 71.9, 70.1 and 68.9 ppm were assigned to C-2, C-3, C-4 and C-5. The four signals appearing at 169.8, 169.6, 169.2 and 168.7 ppm are due to the four acetoxy carbonyl carbons, while the sig-nal at 165.8 ppm is due to the amide carbonyl carbon. The signals at 20.6 and 20.5 ppm are attributed to the acetate methyl carbons. The aglycon methylene carbon appeared at 42.9 ppm, while signals at 137.4, 128.7, 127.8 and 127.6 are due to the aglycon phenyl carbons.

On the other hand, the 1H NMR spectrum of compound 5

showed a doublet at 5.75 ppm which was assigned to H-1, and the magnitude of coupling between H-1 and H-2 (7.1 Hz) was indicative to their diaxial orientation ( -anomeric configuration). The other four protons of the glucopyranosyl ring resonated in the 5.35–4.10 ppm region. The remaining four acetoxy groups appeared as four singlets at 2.15, 2.08, 2.05 and 2.03 ppm. The allyl characteristic signals were also observed as three multiplets at ~5.83 (=CH), ~5.35 (=CH2) and 3.86 ppm (CH2).

Subsequent removal of acetyl protecting groups was found to be essential. Base catalyzed saponification of com-pounds 3 and 5 was achieved at pH 13 using 0.05 M LiOH to afford the products 4 and 6, respectively, in an equilibrium mixture of both anomers (Scheme 2). The / ratio was found to be 1:1 ratio as determined by the signal integration in

1H NMR spectra. Also, the global deprotection using

Zemplén’s conditions (catalytic amount of sodium methox-ide in methanol) was tried and it revealed the same result with approximately equal yield.

The complete removal of all acetyl groups was confirmed by NMR spectroscopy in which both

1H and

13C NMR spec-

tra revealed the absence of signals corresponding to acetyl groups, in addition to an upfield shift of the glucopyranosyl ring protons in the

1H NMR spectrum.

Next, N-(4-Bromophenyl)-1,2,3,4-tetra-O-acetyl- -D-glucopyranuronamide (7) and N-(2-Chloro-4-nitrophenyl)-1,2,3,4-tetra-O-acetyl- -D-glucopyranuronamide (8) were obtained by reaction of 4-bromoaniline or 2-chloro-4-nitroaniline with 1 in anhydrous DCM. The crude reactions products were purified by the same way described for com-pounds 3 and 5 (Scheme 3). The structural assignments of 7

OAcO

AcO

OAc

OAc

OO

O

1

OAcO

AcO

OAc

OAc

NHO

5

AllNH2, DCM,

rt, overnight

LiOH, MeOH, H2O,

THF or MeONa,

MeOH

OHO

HO

OH

OH

NHO

6

OAcO

AcO

OAc

OAc

NHO

Ph

3

BnNH2, DCM,

rt, overnight

OHO

HO

OH

OH

NHO

Ph

4

LiOH, MeOH, H2O,

THF or MeONa,

MeOH

Scheme 2.

626 Medicinal Chemistry, 2011, Vol. 7, No. 6 El-Nezhawy et al.

and 8 based on their NMR spectral data were in good agree-ment and confirmed the proposed structures.

Again, subsequent removal of acetyl protecting groups from 7 and 8 was achieved to afford the products 9 and 10,

respectively, in an equilibrium mixture of both anomers (Scheme 3). The / ratio was found to be 1:1 ratio as de-termined by the signal integration in

1H NMR spectra.

Finally, N-(Thiazol-2-yl)-1,2,3,4-tetra-O-acetyl- -D-glu-copyranuronamide (11), N-(1H-Benzimidazol-2-yl)-1,2,3,4-tetra-O-acetyl- -D-glucopyranuronamide (12) N-(Pyrazin-2-yl)-1,2,3,4-tetra-O-acetyl- -D-glucopyranuronamide (13) and N-(Pyridin-4-yl)-1,2,3,4-tetra-O-acetyl- -D-glucopyra-nuronamide (14) were also obtained by the reaction of 2-aminothiazole, 2-amino-1H-benzimidazole, 2-aminopyrazine or 4-aminopyridine with 1 in anhydrous DCM (Scheme 4).

For compound 14, the crude reaction product was puri-fied by extractive work-up, followed by chromatographic

purification to give the desired product but in a very low

yield (> 5 %). In an attempt to improve the yield, the reac-tion was repeated, and this time the work-up procedure was

modified to direct subjection of the crude reaction to column

chromatography. This modification to the work-up proce-dure highly improved the yield and gave the product in 50%

yield.

The structural assignments of 11, 12, 13 and 14 based on

their NMR spectral data were in good agreement and con-

firmed the proposed structures.

Once again, subsequent removal of acetyl protecting

groups was achieved to afford the products 15, 16, 17 and

18, respectively, in an equilibrium mixture of both anomers

(Scheme 4). The / ratio was found to be 1:1 ratio as de-

termined by the signal integration in 1H NMR spectra.

On the other hand, the reaction of the fully acetylated

mixed anhydride 1 with 4-aminouracil or its N,N-dimethyl

derivative under the previously established conditions re-

vealed no formation of the amide products (19a,b) even after

changing the reaction solvent to acetonitrile this may be to

the lower basic properties of its amino group (Scheme 5).

The attention was shifted to the synthesis of a series of N-acyl-N-aryl-D-glucopyranuronosyl amine derivatives. After

refluxing the acid 2 in DCM with thionyl chloride for 2 h,

TLC analysis of the reaction mixture identified the formation

of a material that was more mobile than the starting material

which was completely consumed. Direct treatment of the

generated acid chloride with allyl alcohol in presence of

pyridine for 1 h followed by extractive work-up furnished

the allyl ester substrate 20 in 98% yield (Scheme 6). Obvi-ously, this procedure was found to be much efficient and

simpler than the previously published [33] as:

Fig. (4). Splitting pattern of the methylene protons before and after

proton exchange.

OAcO

AcO

OAc

OAc

OO

O

1

ArNH2, DCM,

rt, overnight

LiOH,

MeOH, H2O, THF

or

MeONa, MeOH

OAcO

AcO

OAc

OAc

NHO

R1

R2

OHO

HO

OH

OH

NHO

R1

R2

7 R1 = Br, R2 = H

8 R1 = NO2, R2 = Cl

9 R1 = Br, R2 = H

10 R1 = NO2, R2 = Cl

Scheme 3.


OAcO

AcO

OAc

OAc

OO

O

1

2-aminothazole

DCM, rt, overnightO

AcOAcO

OAc

OAc

NHO

S

N

11

LiOH, MeOH, H2O,

THF or MeONa,

MeOH

OHO

HO

OH

OH

NHO

S

N

15

2-aminobenz-

imidazole

DCM, rt, overnight OAcO

AcO

OAc

OAc

NHO

N

HN

12

LiOH, MeOH, H2O,

THF or MeONa,

MeOH

OHO

HO

OH

OH

NHO

N

HN

16

2-aminopyrazine

DCM, rt, overnight OAcO

AcOOAc

OAc

NHO

NN

13

OHO

HOOH

OH

NHO

NN

17

LiOH, MeOH, H2O,

THF or MeONa,

MeOH

4-aminopyridine

DCM, rt, overnightO

AcOAcO

OAc

OAc

NHO

N

14

LiOH, MeOH, H2O,

THF or MeONa,

MeOH

OHO

HO

OH

OH

NHO

N

18

Scheme 4.

OAcO

AcO

OAc

OAc

O

N

NO

O

NH

R

R

19a R = H

19b R = Me

N

N

O

OH2N

R

R

OAcO

AcO

OAcOAc

OO

O

1

DCM, rt

overnight

Scheme 5. Reaction of anhydride 1 with aminouracil and its N,N-dimethyl derivative.


OAcO

AcO

OAc

OH

O

O

OAcO

AcO

OAc

HN

O

O

X

BnNH2,THF,

rt, overnight

H2N X

MeOH, reflux, 26 h

22 X=F only

23a,b X=Br / (1:2)

20

21

OAcO

AcO

OAc

OAc

O

O

OAcO

AcO

OAc

OAc

Cl

O

SOCl2, DMF,

DCM, reflux, 2 h

, Py

DCM, 0 oC, 30 min

OH

2

Scheme 6. Synthesis of allyl ester substrate 20 and N-glycosides.

(1) The yield was superior to that achieved.

(2) The product was obtained without the need for

chromatographic purification.

The formation of allyl ester was confirmed by the obser-vation of a multiplet in the

1H NMR spectrum integrating

one proton at 5.82 ppm (=CH), in addition to, a multiplet integrating two protons at ~5.1 ppm (=CH2) and a double of doublets integrating two protons at 4.51 ppm (CH2), which are all characteristic to the allyl group.

The structure was confirmed once again by 13

C NMR as-sisted with the DEPT experiment which reveled three new allyl signals; two for olefinic carbons at 130.8 (=CH), 119.4 (=CH2) and one for methylene carbon at 66.5 ppm (CH2) which are characteristic for the allyl group.

Regioseleactive removal of the anomeric O-acetyl group was carefully performed with benzyl amine in THF which is mild enough to leave the base sensitive allyl ester unscathed. TLC analysis of the reaction mixture, after 19 h at ambient temperature, identified the formation of a material that was less mobile than the starting material 20. After silica gel pu-rification, the C1-hemiacetal 21 was obtained in moderate yield (52%) (Scheme 6).

The 1H NMR spectrum of the product confirmed its for-

mation. In particular, it showed signals corresponding to only three acetyl protons at 2.09, 2.04 and 2.02 ppm, in addi-tion to, the upfield shift of the H-1 signal. The

1H NMR also

confirmed the retention of the previously introduced allyl group by the appearance of its characteristic signals at 5.90 (=CH), ~5.16 (=CH2) and 4.61 ppm (CH2).

The N-(substituted-phenyl)-D-glucopyranuronyl amines were achieved via the reaction of equivalent amounts of the hemiacetal 21 as a glycosyl donor with para-bromo or para-fluroaniline as glycosyl acceptors in refluxing methanol.

In the reaction with para-flouroaniline, a pure -anomer

22 was obtained after silica gel purification. The structural

assignment for 22 was based on its spectral data. In particu-

lar, the 1H NMR showed the anomeric proton as triplet at

5.39 ppm with spin-spin coupling constant corresponding to

the -configuration. The four aromatic protons of the agly-

con resonated at 6.88 and 6.61 ppm and each signal inte-

grates two protons. Also, the 13

C NMR confirmed the prod-

uct structure.

In the case of para-bromoaniline, the ring reforming pro-

ceeds on both the si- and re-faces of the imino group, conse-

quently, a mixture of - and -N-glycosides was obtained in

70% overall yield. The anomeric ratio was 1:2 as indicated

from the signal integration in the 1H NMR spectrum. Ano-

mers were separated on a reversed phase silica gel column

using H2O-MeOH (20%) as an eluant and identified.

The structural assignment for 23a and 23b were based on

their spectral data. In particular, for compound 23a, the 1H

NMR showed the anomeric proton as triplet at 5.47 ppm

with spin-spin coupling constant corresponding to the -

configuration, while for 23b, the 1H NMR showed the ano-

meric proton as triplet at 5.40 ppm with spin-spin coupling

constant corresponding to the -configuration. Additionally,

for 23a, the four aromatic protons of the aglycon resonated

at 7.31 and 6.82 ppm, while for 23b, the four aromatic pro-

tons of the aglycon resonated at 7.28 and 6.54 ppm.

The formed N-glycoside was subjected to acylation either

by reacting with acyl chlorides or by coupling with carbox-

ylic acids using DCC as a carboxylic group activator, but all

these attempts were unsuccessful. Further, methylation was

also tried using MeI in presence of pyridine, TEA and NaH

separately, but also all these attempts were unsuccessful

(Scheme 7).


OAcO

AcO

OAc

HN

O

O

X

OAcO

AcO

OAc

N

O

O

X

OAcO

AcO

OAc

N

O

O

X

Me

O

R

MeI,

base

Acid chloride,

base

or

Acid, DCC

Scheme 7. Unsuccessful acylation or methylation of the prepared N-glycosides.

Antitumor Activity

The prepared compounds were divided into two groups:

the 1st group represents the acetylated derivatives, while, the

2nd

group represents the de-acetylated derivatives and the

activities of both groups were represented in the Tables 1

and 2, respectively.

Table 1. Group I Concentrations ( M) Required to Cause Different Inhibitions

Cell Line Compound Number Inhibition Parameter

TK-10 MCF-7 UACC-62

3 GI50 18 ± 2.6 >100 32 ± 6.3

TGI >100 >100 10 ± 2.7

LC50 >100 >100 >100

5 GI50 0.21 ± 0.13 >100 >100

TGI >100 >100 >100

LC50 >100 >100 >100

7 GI50 0.82 ± 0.03 18.6 ± 5.8 0.87 ± 0.41

TGI >100 >100 >100

LC50 >100 >100 >100

8 GI50 3.8 ± 1.1 >100 3.4 ± 1

TGI >100 >100 >100

LC50 >100 >100 >100

11 GI50 89 ± 4.9 >100 >100

TGI >100 >100 >100

LC50 >100 >100 >100

12 GI50 93 ± 27 >100 80

TGI >100 >100 >100

LC50 >100 >100 >100


Table 1. contd…


TK-10 MCF-7 UACC-62

13 GI50 23 ± 6.5 98 ± 24 0.42 ± 0.50

TGI >100 >100 >100

LC50 >100 >100 >100

14 GI50 0.67 ± 0.08 >100 0.21 ± 0.03

TGI >100 >100 5.5 ± 2

LC50 >100 >100 39 ± 9

20 GI50 20 ± 3 4.5 ± 0.13 16 ± 4.1

TGI 51 ± 7 >100 57 ± 12

LC50 >100 >100 >100

23 GI50 43 ± 8 59 ± 0.63 21 ± 0.91

TGI 89 ± 20 >100 79 ± 8

LC50 >100 >100 >100

The range of doses assayed was 10 4, 10 5, 10 6, 10 7 and 10 8 M. Results are mean ± S.E.M. (n = 3).

Table 2. Group II Concentrations ( M) Required to Cause Different Inhibitions


TK-10 MCF-7 UACC-62

4 GI50 0.05 ± 0.11 >100 >100

TGI 16 ± 4 >100 >100

LC50 >100 >100 >100

6 GI50 48 ± 7 >100 50 ± 8.3

TGI >100 >100 >100

LC50 >100 >100 >100

9 GI50 17 ± 4 0.56 0.76 ± 0.11

TGI >100 >100 54 ± 10

LC50 >100 >100 >100

10 GI50 11 ± 0.90 90 2.7 ± 0.35

TGI 60 ± 12 >100 8 ± 3

LC50 90 ± 16 >100 24 ± 7.8

15 GI50 25 ± 1.5 8.2 0.049 ± 0.02

TGI >100 >100 >100

LC50 >100 >100 >100

16 GI50 5.5 ± 0.04 >100 9.4 ± 3

TGI >100 >100 >100

LC50 >100 >100 >100


Table 2. contd….


TK-10 MCF-7 UACC-62

17 GI50 20 ± 2.9 36 0.08 ± 0.03

TGI 44 ± 5.2 >100 3.1 ± 1.1

LC50 96 ± 24 >100 35 ± 7.3

18 GI50 0.23 ± 0.12 0.19 15 ± 3.6

TGI 1.3 ± 0.08 >100 >100

LC50 7.3 ± 0.17 >100 >100

The range of doses assayed was 10 4, 10 5, 10 6, 10 7 and 10 8 M. Results are mean ± S.E.M. (n = 3).

The newly synthesized compounds were examined for in

vitro activity against several human cancer cell lines, includ-ing renal adenocarcinoma (TK-10), human breast adenocar-cinoma (MCF-7) and human melanoma (UACC-62). Three response parameters (GI50, TGI, and LC50) were calculated for each cell line. The GI50 value (growth inhibitory activity) corresponds to the concentration of the compounds causing 50% decrease in net cell growth, the TGI value (cytostatic activity) is the concentration of the compounds resulting in total growth inhibition and the LC50 value (cytotoxic activ-ity) is the concentration of the compounds causing net 50% loss of initial cells at the end of the incubation period. Tables 1 and 2 lists the GI50, TGI and LC50 values obtained for ace-tylated (group I) and deacetylated (group II), respectively.

Regarding the activities of the acetylated derivatives in Table 1, compounds 7, 13, 20 and 23 showed a growth in-hibitory activity (GI50) on the three mentioned tumoural cell lines at the tested doses. However, compounds 3, 8, 12 and 14 showed only growth inhibitory effect against the TK-10 and UACC-62 cells, but did not posses any activity aganist the MCF-7 cell line. The compounds 5 and 11 only affected the renal adenocarcinoma (TK-10) cell line. The compounds 5, 7, 8 and 14 were the most cytotoxic aganist TK-10 (GI50 = 0.21, 0.82, 3.82 and 0.67 M, respectively). Moreover, the most effective compounds aganist the MCF-7 cell line were 7 and 20 (GI50 =18.55 and 4.50 M, respectively). However, compounds 7, 8, 13 and 14 were the most effective aganist the UACC-62 cell line (GI50 = 0.87, 3.40, 0.42 and 0.21 M, respectively).

The growth of TK-10 cells was totally inhibited by com-pounds 20 and 23 (TGI = 51.45 and 88.99 M, respectively). At the same time, compounds 3, 14, 20 and 23 showed a cytostatic activity (TGI) against the UACC-62 cells with TGI values of 9.97, 5.49, 56.74 and 79.33 M, respectively. However, all the tested compounds did not demonstrate any cytostatic activity (TGI) against the MCF-7 cell line. Fur-thermore, only compound 14 showed a cytotoxic activity (LC50) against MCF-7 cell line at a dose of 39.35 M.

On the other hand, regarding the activities of the deacety-lated derivatives in Table 2, compounds 9, 10, 15, 17 and 18 showed a growth inhibitory activity (GI50) aganist the three mentioned tumoural cell lines at the tested doses. However, compounds 6 and 16 showed only an inhibitory effect

against the growth (GI50) of the TK-10 and UACC-62 cells, but did not posses any activity aganist the MCF-7 cell line. The compound 4 only affected the renal adenocarcinoma (TK-10) cell line. The compounds 4, 16 and 18 were the most cytotoxic aganist TK-10 (GI50 = 0.05, 5.51 and 0.23

M, respectively). Moreover, the most effective compounds aganist the MCF-7 cell line were 9 and 18 (GI50 = 0.56 and 0.19 M, respectively). However, compounds 9, 10, 15 and

17 were the most effective on the UACC-62 cell line (GI50 = 0.76, 2.71, 0.049 and 0.08 M, respectively).

The growth of TK-10 cell line was totally inhibited by 4, 10, 17 and 18 (TGI = 16.15, 60.02, 43.56 and 1.29 M, re-spectively), while, compounds 9, 10 and 17 showed a cy-tostatic activity against the UACC-62 cells with TGI values of 54.19, 8.09 and 3.11 M, respectively. However, none the tested deacetylated compounds showed neither cytostatic nor cytotoxic activities against the MCF-7 cells.

Structure-Activity Relationship (SAR)

By observing the activity of the two tested groups of compounds, the relationship between the structure variation and the activity can be concluded.

The effect of deacetylation on the activity was estab-lished by comparing the activity of group I compounds (Ta-ble 1) with their corresponding derivatives in group II (Table 2). It was obvious that the removal of acetyl groups resulted in an enhancement in the activity of all compounds against the three tumoural cell lines with the exception of the allyl, benzyl and benzimidazolyl derivatives against MCF-7 cell line, the allyl, p-bromophenyl and 2-chloro-4-nitrophenyl derivatives against TK-10 cell line and the benzyl and pyridyl derivatives against UACC-62 cell line, in which re-moval of the acetyl groups is accompanied by reduction in potency.

The effect of separation of the aromatic ring from the

amide linkage by a methylene group can be obtained by comparing the activity of the benzyl derivative in each group

(3 or 4) with the other phenyl derivatives in the same group.

It was obvious that the activity against the tested cell lines was sufficiently reduced.

The effect of replacement of allyl group with benzyl group can be obtained by comparing the activities of the allyl


derivative (5 or 6) in any group with the benzyl derivative (3 or 4) in the same group. For the acetylated series, the activity was increased against UACC-62 cells and decreased against TK-10 cells, which is inverted in the non-acetylated series. All the allyl and benzyl derivatives were totally inactive against the MCF-7 cell line.

By comparing the activities of the benzimidazolyl deriva-tive (12 or 16) in any group and the pyranzinyl derivative (13 or 17) in the same group, the effect of expanding from a five membered ring to a six membered ring was concluded in which; the activities were efficiently increased against the three tested tumoral cell lines except for the group II against TK-10 cell line. Further, by comparing the activity of the benzimidazolyl derivative (12 or 16) in any group with the thiazolyl derivative (11 or 15) in the same group, it was ob-vious that the replacement of the NH with S atom in the five membered ring also enhances the activity in most cases against the three tumoral cell lines.

Also, during the evaluation of the derivatives prepared, it was noted that, for the six membered aromatic derivatives, the C-4 position seems to play an important role in the activ-ity in which all the synthesized phenyl derivatives having C-4 substituent (Br, NO2) exhibited high activity in most cases with the bromo substituent better than the nitro substituent. Also, the pyridyl derivatives, in which the C-4 ring carbon was replaced by a nitrogen atom, exhibit a high activity in most cases and if compared with the activity of the pyrazinyl derivatives.

CONCLUSION

A highly efficient and practical method was described for the preparation of a series of D-glucuronic acid derivatives according to standard protocols. The newly synthesized compounds were examined for in vitro activity against sev-eral human cancer cell lines, including (TK-10), (MCF-7) and (UACC-62). Compounds 4, 5, 7, 8, 14, 16 and 18 were the most active against TK-10 cell line. On the other hand, the most active compounds against the MCF-7 cell line were

9, 18 and 20. Moreover, compounds 7-10 13-15 and 17 were the most active against the UACC-62 cell line. These results reflect the importance of the newly tested compounds if the results are compared with that of the well known antineo-plastic agent, Etoposide [34].

EXPERIMENTAL SECTION

General

All starting materials and reagents were purchased from Sigma-Aldrich, BDH and Fluka and used without further purification. All solvents were either of analytical grades or dried and distilled immediately prior to use: DCM from cal-cium hydride, methanol from magnesium turnings and THF from sodium/benzophenone. All of the reactions were per-formed using oven-dried glassware. Melting points were measured on a Stuart-SMP10 melting point apparatus and are uncorrected. TLC was performed using Merck precoated Silica gel 60 F254 aluminum sheets (20 20 cm, layer thickness 0.2 mm) and Merck precoated Silica gel RP-C18 F254 aluminum sheets (20 20 cm, layer thickness 0.2 mm) and spots were visualized by UV (254 nm), KMNO4 solution

and/or charring with H2SO4-EtOH (5% v/v). Column chro-matography was carried out on Silica Gel 60 (particle size 0.063–0.200 mm, 70–230 mesh ASTM, Merck) and LiChro-prep

® RP-18 (prepacked column size B (31 2.5 cm),

0.040–0.063 mm, Merck) using the specified eluents. All reaction products were stored refrigerated under 4

oC. NMR

spectra were recorded with JEOL ECA-500, JEOL EX-270, and Varian Mercury-200BB spectrometers at room tempera-ture in solvents given. Chemical shifts were expressed in parts per million (ppm) and reported either relative to an internal tetramethylsilane standard (TMS = 0.0).or relative to solvent peaks (CDCl3 = 7.2, HOD = 4.8, DMSO-d6 = 2.5) for

1H and (CDCl3 = 77.0, MeOH = 49.0) for

13C.

Multiplicities are denoted as follows: s = singlet, d = doublet, t = triplet, q = quartet, dd = double doublet, ddd = double double doublet, m = multiplet, br = broad, apt = apparently. 13

C signals were assigned with the aid of DEPT. Coupling constants (J) were reported in Hertz (Hz).

1,2,3,4-Tetra-O-acetyl- -D-glucopyranuronic Acetic An-

hydride (1) [32]

D-Glucuronic acid (2 gm, 10.30 mmol) was suspended in acetic anhydride (30 mL) and stirred at 0

oC. Iodine (140 mg,

0.55 mmol) was added and the red solution was left to stir for 30 min at 0

oC and a further 2 h at room temperature.

Acetic anhydride was mostly removed in vacuo and the formed solid was taken up in methylene chloride (50 mL), washed with 1M Na2S2O3 (2 30 mL), dried over anhydrous Na2SO4, filtered and evaporated to afford the title compound as a white solid (4.08 gm, 98%).

1H NMR (270 MHz, CD-

Cl3) 2.04 (s, 3H, COCH3), 2.05 (s, 6H, 2 COCH3), 2.13 (s, 3H, COCH3), 2.28 (s, 3H, COCOOCH3), 4.31 (d, 1H, J5-4 = 8.7 Hz, H-5), 5.12 (apt t, 1H, H-2), 5.32 (m, 2H, H-3 and H-4 overlapping), 5.80 (d, 1H, J1-2 = 6.9 Hz, H-1).

1,2,3.4-Tetra-O-acetyl- -D-glucopyranuronic Acid (2)

[32]

1,2,3,4-Tetra-O-acetyl- -D-glucuronic acetic anhydride (1, 4.08 gm, 10.09 mmol) was dissolved in water and THF (90 mL, 1:2) and stirred overnight. The solution was concen-trated and the product was extracted into methylene chloride (3 30 mL), the combined organic layers were dried over anhydrous Na2SO4, filtered and evaporated in vacuo to give the title compound. Yield 99%; white foam;

1H NMR (270

MHz, DMSO-d6) 1.97 (s, 6H, 2 COCH3), 2.01 (s, 3H, COCH3), 2.08 (s, 3H, COCH3), 3.44 (br s, 1H, COOH), 4.52 (d, 1H, J5-4 = 8.2 Hz), 4.95 (apt t, 1H, H-2), 5.05 (apt t, 1H, H-4), 5.48 (apt t, 1H, H-3), 6.00 (d, 1H, J1-2 = 8.1 Hz, H-1).

General Procedure for Preparation of per-O-acetylated-N-substituted-1,2,3,4-tetra-O-acetyl- -D-

glucopyranuronamides

To a solution of 1,2,3,4-tetra-O-acetyl- -D-glucuronic acetic anhydride (1) in dry methylene chloride (5 mL/0.1 gm) and under argon, primary amine (1 equiv.) was added and the reaction mixture was stirred overnight at room tem-perature. The mixture was then diluted with methylene chlo-ride, transferred to a separating funnel and washed with 1M HCl, saturated NaHCO3, deionized water, dried over anhy-drous Na2SO4 and filtered. The solvent was then removed


and the residue was purified by column chromatography (1:3 EtOAc-petroleum ether) to afford the title compounds.

N-Benzyl-1,2,3,4-tetra-O-acetyl- -D-glucopyranuronamide (3)

Yield 69%; white solid; mp 115–117 oC; Rf = 0.50 (1:1

EtOAc-petroleum ether); 1H NMR (500 MHz, DMSO-d6)

1.83, 1.91, 1.97, 2.03, (4s, 12H, 4 COCH3), 4.20 (ddd, 2H, CH2), 4.33 (d, 1H, J5-4 = 10.0 Hz, H-5), 4.95 (apt t, 1H, H-2), 5.12 (apt t, 1H, H-4), 5.40 (apt t, 1H, H-3), 5.94 (d, 1H, J1-2 = 8.4 Hz, H-1), 7.17–7.26 (m, 5H, aromatic H), 8.67 (t, 1H, NH);

13C NMR (50 MHz, CDCl3) 20.5, 20.6 (4 COCH3),

42.9 (CH2), 68.9, 70.1, 71.9, 72.9 (C-2–C-5), 91.2 (C-1), 127.6 (aromatic CH), 127.8 (2 aromatic CH), 128.7 (2 aromatic CH), 137.4 (aromatic C), 165.8 (CONH), 168.7, 169.2, 169.6, 169.8, (4 COCH3).

N-Allyl-1,2,3,4-tetra-O-acetyl- -D-glucopyranuronamide

(5)


EtOAc-petroleum ether); 1

H NMR (270 MHz, CDCl3) 2.03, 2.05, 2.08, 2.15, (4s, 12H, 4 COCH3), 3.86 (m, 2H, OCH2-CH=CH2), 4.10 (d, 1H, J5-4 = 8.1 Hz, H-5), 5.09–5.35 (m, 5H, CH=CH2, H-2, H-3 and H-4), 5.75 (d, 1H, J1-2 = 7.1 Hz, H-1), 5.81–5.84 (m, 1H, CH=CH2), 6.40 (t, 1H, NH).

N-(4-Bromophenyl)-1,2,3,4-tetra-O-acetyl- -D-glucopyranuronamide (7)

Yield 63%; white solid; mp 173 oC (dec.); Rf = 0.52 (1:1


1.92, 1.97, 2.03, 2.09 (4s, 12H, 4 COCH3), 4.46 (d, 1H, J5-4 = 9.7 Hz, H-5), 5.08 (apt t, 1H, H-2), 5.26 (apt t, 1H, H-3), 5.53 (apt t, 1H, H-4), 6.06 (d, 1H, J1-2 = 8.4 Hz, H-1), 7.52 (br s, 4H, aromatic H), 10.30 (s, 1H, NH).

N-(2-Chloro-4-nitrophenyl)-1,2,3,4-tetra-O-acetyl- -D-

glucopyranuronamide (8)


EtOAc-petroleum ether); 1

H NMR (500 MHz, CDCl3)

2.02, 2.06, 2.10, 2.15 (4s, 12H, 4 COCH3), 4.28 (d, 1H, J5-4 = 9.8 Hz, H-5), 5.18 (apt t, 1H, H-2), 5.35 (m, 2H, H-3 and H-4 overlapping), 5.84 (d, 1H, J1-2 = 7.5 Hz, H-1), 8.15 (m, 1H, aromatic H), 8.29 (s, 1H, aromatic H), 8.52 (d, 1H, aro-matic H), 8.90 (s, 1H, NH).

N-(Thiazol-2-yl)-1,2,3,4-tetra-O-acetyl- -D-




1.93, 1.97, 2.03, 2.08, (4s, 12H, 4 COCH3), 4.61 (d, 1H, J5-

4 = 8.9 Hz, H-5), 5.12 (apt t, 1H, H-2), 5.34 (apt t, 1H, H-3), 5.55 (apt t, 1H, H-4), 6.08 (d, 1H, J1-2 = 8.1 Hz, H-1), 7.31 (d, 1H, aromatic H), 7.50 (d, 1H, aromatic H), 12.49 (s, 1H, NH).

N-(1H-Benzimidazol-2-yl)-1,2,3,4-tetra-O-acetyl- -D-




1.96, 1.98, 2.04, 2.10 (4s, 12H, 4 COCH3), 4.65 (d, 1H, J5-4 = 11.5 Hz, H-5), 5.10 (apt t, 1H, H-2), 5.35 (apt t, 1H, H-3), 5.56 (apt t, 1H, H-4), 6.09 (d, 1H, J1-2 = 9.7 Hz, H-1), 7.23 (s, 2H, aromatic H), 7.50 (s, 2H, aromatic H).

N-(Pyrazin-2-yl)-1,2,3,4-tetra-O-acetyl- -D-



EtOAc-petroleum ether); 1H NMR (270 MHz, CDCl3)

2.02, 2.05, 2.10, 2.15 (4s, 12H, 4 COCH3), 4.24 (d, 1H, J5-4

= 8.8 Hz, H-5), 5.15 (apt t, 1H, H-2), 5.32 (m, 2H, H-3 and H-4 overlapping), 5.82 (d, 1H, J1-2 = 8.1 Hz, H-1), 8.27 (d, 1H, aromatic H), 8.38 (d, 1H, aromatic H), 8.61 (s, 1H, NH), 9.40 (s, 1H, aromatic H).

N-(Pyridin-4-yl)-1,2,3,4-tetra-O-acetyl- -D-


To a solution of 1,2,3,4-tetra-O-acetyl- -D-glucuronic acetic anhydride (1) in dry methylene chloride (2 mL/0.1 gm) and under argon, 4-aminopyridine (1 equiv.) was added and the reaction mixture was stirred overnight at room tem-perature. The solvent was evaporated and the residue was directly purified by chromatography (3% MeOH-DCM) to give the titled compound. Yield 50%; white solid; mp 195–197

oC; Rf = 0.45 (EtOAc);

1H NMR (500 MHz, DMSO-d6)

1.88, 1.93, 1.99, 2.04 (4s, 12H, 4 COCH3), 4.55 (d, 1H, J5-4 = 9.7 Hz, H-5), 5.05 (apt t, 1H, H-2), 5.20 (apt t, 1H, H-3), 5.24 (apt t, 1H, H-4), 6.02 (d, 1H, J1-2 = 8.0 Hz, H-1), 7.54 (d, 2H, aromatic H), 8.41 (d, 2H, aromatic H), 8.43 (s, 1H, NH).

General Procedure for deprotection of N-substituted-1,2,3,4-tetra-O-acetyl- -D-glucopyranuronamides

A solution of 0.05M LiOH in 2.5:1.0:0.5 MeOH-H2O-THF (6 equiv.) was added to the glycosylamide. The solu-tion was stirred at 0

oC and the progress of the reaction was

monitored by TLC (3:1 EtOAc-MeOH). After complete de-protection, the pH of the solution was adjusted to 6.0 by the addition of Amberlite IRC-50 (H

+). The beads were filtered

off, the solvents were removed in vacuo and the residue was purified by column chromatography (MeOH-EtOAc) to give the desired compounds as an equilibrium mixture of the - and -anomers.

N-Benzyl- / -D-glucopyranuronamide (4)

A portion of the intermediate (3, 183 mg, 0.65 mmol) was deprotected as described above to give 4 as a white solid (90 mg, 78%); ( : 1:1); Rf = 0.57 (1:3 MeOH-EtOAc);

1H

NMR (500 MHz, D2O) 3.16–3.64 (6H, + H-4, + H-3 and + H-2), 4.14 (d, 1H, J5-4 = 9.2 Hz, H-5), 4.31 (s, 2H,

CH2), 4.32 (s, 2H, CH2), 4.57 (d, 1H, J5-4 = 7.7 Hz, H-5), 5.15 (d, 1H, J1-2 = 3.8 Hz, H-1), 7.20–7.27 (m, 10H,

+ aromatic H); 13

C NMR (125 MHz, D2O) 42.8, 71.5, 71.8, 75.3, 75.4, 92.5, 96.2, 127.3, 127.6, 128.9, 137.6, 170.5, 171.3.

N-Allyl- / -D-glucopyranuronamide (6)

A portion of the intermediate (5, 110 mg, 0.47 mmol)

was deprotected as described above to give 6 as a white solid


(59 mg, 92%); ( : 1:1); Rf = 0.57 (1:3 MeOH-EtOAc); 1H

NMR (500 MHz, D2O) 3.15 (apt t, 1H, H-2), 3.19 (s, 2H,

OCH2-CH=CH2), 3.37–3.58 (5H, + H-4, + H-3 and

H-2), 3.70 (s, 2H, OCH2-CH=CH2), 3.76 (d, 1H, J5-4 = 9.9

Hz, H-5), 4.09 (d, 1H, J5-4 = 9.9 Hz, H-5), 4.99–5.07 (m,

4H, + CH=CH2), 5.13 (d, 1H, J1-2 = 3.1 Hz, H-1), 5.70

(m, 2H, + CH=CH2); 13

C NMR (125 MHz, D2O) 30.4,

41.4, 71.2, 71.6, 72.5, 73.9, 92.6, 96.3, 115.8, 133.2, 170.5,

171.3.

N-(4-Bromophenyl)- / -D-glucopyranuronamide (9)

A portion of the intermediate (7, 81 mg, 0.23 mmol) was

deprotected as described above to give 9 as a white solid (49

mg, 90%); ( : 1:1); Rf = 0.53 (1:3 MeOH-EtOAc); 1H

NMR (500 MHz, D2O) 3.17–3.63 (6H, + H-4, + H-3

and + H-2), 3.86 (d, 1H, J5-4 = 9.2 Hz, H-5), 4.20 (d, 1H,

J5-4 = 9.2 Hz, H-5), 4.59 (d, 1H, J1-2 = 7.7 Hz, H-1), 5.16

(s, 1H, H-1), 7.22 (d, 4H, + aromatic H), 7.39 (d, 4H,

+ aromatic H); 13

C NMR (125 MHz, D2O) 71.1, 71.5,

71.7, 72.4, 73.8, 75.3, 75.7, 92.6, 96.3, 118.3, 123.9, 132.1, 135.3, 169.0, 169.9.

N-(2-Chloro-4-nitrophenyl)- / -D-glucopyranuronamide (10)


was deprotected as described above to give 10 as a white

solid (82 mg, 69%); ( : 1:1); Rf = 0.76 (1:3 MeOH-EtOAc); 1H NMR (500 MHz, D2O) 3.24–3.69 (6H, + H-4, +

H-3 and + H-2), 4.07 (d, 1H, J5-4 = 9.9 Hz, H-5), 4.40 (d,

1H, J5-4 = 10.0 Hz, H-5), 5.25 (d, 1H, J1-2 = 3.8 Hz, H-1),

8.01 (t, 2H, + aromatic H), 8.11 (d, 2H, + aromatic H),

8.33 (s, 2H, + aromatic H); 13

C NMR (125 MHz, MeOD)

70.7, 71.8, 72.0, 72.5, 73.2, 74.3, 74.9, 76.3, 93.1, 97.3, 121.1, 121.3, 123.0, 123.8, 124.5, 139.6, 143.8, 169.9.

N-(Thiazol-2-yl)- / -D-glucopyranuronamide (15)




H-3 and + H-2), 4.06 (d, 1H, J5-4 = 9.2 Hz, H-5), 4.39 (d,

1H, J5-4 = 9.9 Hz, H-5), 5.23 (d, 1H, J1-2 = 3.9 Hz, H-1),

7.12 (d, 2H, + aromatic H), 7.36 (d, 2H, + aromatic H); 13

C NMR (125 MHz, D2O) 71.1, 71.2, 71.3, 71.6, 72.4,

73.7, 75.1, 75.3, 92.6, 96.4, 115.1, 137.1, 158.2, 168.2, 169.8.

N-(1H-Benzimidazol-2-yl)- / -D-glucopyranuronamide

(16)




H-3 and + H-2), 3.94 (d, 1H, J5-4 = 9.9 Hz, H-5), 4.49 (d,

1H, J5-4 = 7.7 Hz, H-5), 5.10 (d, 1H, J1-2 = 3.9 Hz, H-1),

7.12 (m, 8H, + aromatic H); 13

C NMR (125 MHz, D2O)

71.4, 71.9, 72.2, 72.6, 74.0, 75.6, 76.3, 76.9, 92.2, 95.9, 110.9, 123.5, 129.1, 150.2, 168.0, 169.0.

N-(Pyrazin-2-yl)- / -D-glucopyranuronamide (17)

A portion of the intermediate (13, 170 mg, 0.63 mmol) was deprotected as described above to give 17 as a white solid (88 mg, 84%); ( : 1:1); Rf = 0.38 (1:3 MeOH-EtOAc); 1H NMR (500 MHz, D2O) 3.23–3.69 (6H, + H-4, +

H-3 and + H-2), 4.02 (d, 1H, J5-4 = 10.0 Hz, H-5), 4.34 (d, 1H, J5-4 = 9.9 Hz, H-5), 5.24 (d, 1H, J1-2 = 3.1 Hz, H-1), 8.22 (m, 2H, + aromatic H), 8.24 (s, 2H, + aromatic H), 8.97 (d, 2H, + aromatic H);

13C NMR (125 MHz,

D2O) 71.1, 71.4, 71.5, 71.7, 72.5, 73.8, 75.3, 92.6, 96.3, 137.1, 140.5, 143.1, 147.2, 169.0, 170.0.

N-(Pyridin-4-yl)- / -D-glucopyranuronamide (18)

A portion of the intermediate (14, 149 mg, 0.55 mmol) was deprotected as described above to give 18 as a white solid (61 mg, 67%); ( : 1:1); Rf = 0.32 (1:3 MeOH-EtOAc); 1H NMR (500 MHz, D2O) 3.23–3.62 (6H, + H-4, +

H-3 and + H-2), 3.96 (d, 1H, J5-4 = 9.9 Hz, H-5), 4.29 (d, 1H, J5-4 = 10.0 Hz, H-5), 5.23 (d, 1H, J1-2 = 3.9 Hz, H-1), 7.55 (d, 4H, + aromatic H), 8.33 (d, 4H, + aromatic H); 13

C NMR (125 MHz, D2O) 71.3, 71.7, 73.7, 75.3, 92.6, 96.3, 115.3, 145.8, 148.8, 169.6, 170.6.

Allyl 1,2,3,4-tetra-O-acetyl- -D-glucpyranuronate (20)

Thionyl chloride (1.6 mL, 21.92 mmol) was added to a stirred solution of 1,2,3,4-O-tetra- -D-glucuronic acid (2, 3.60 gm, 9.94 mmol) and DMF (0.4 mL, 5.17 mmol) dis-solved in dry methylene chloride (25 mL) cooled to 0

oC.

The mixture was stirred for 5 min at 0 oC and then heated to

reflux for 2 h. the solvents were then removed under reduced pressure and the resulting solid was dissolved in dry methyl-ene chloride (25 mL) under argon again and cooled to 0

oC.

Allyl alcohol (0.8 mL, 11.76 mmol) and pyridine (1.92 mL, 23.74 mmol) were added and the mixture was stirred for 30 min at 0

oC and for further 30 min at room temperature. The

mixture was then transferred to a separating funnel and washed with saturated NaHCO3 (2 100 mL), 1M HCl (2 100 mL), dried over anhydrous Na2SO4, filtered and the sol-vent was removed to give the title compound as a slightly brown solid (3.93 gm, 98%) which was used in the next step without any further purification. An analytical sample was prepared by column chromatography purification.

1H NMR

(200 MHz, CDCl3) 1.93 (3s, 9H, 3 COCH3), 2.02 (s, 3H, COCH3), 4.15 (d, 1H, J4-5 = 8.8 Hz, H-5), 4.51 (dd, 2H, OCH2-CH=CH2), 5.00–5.29 (m, 5H, H-2, H-3, H-4 and CH=CH2 overlapping), 5.70 (d, 1H, J1-2 = 7.4 Hz, H-1), 5.82 (m, 1H, CH=CH2);

13C NMR (50 MHz, CDCl3) 20.3, 20.5

(4 COCH3), 66.5 (OCH2-CH=CH2), 68.6, 69.9, 71.6, 72.6 (C-2–C-5), 91.0 (C-1), 119.4 (CH=CH2), 130.8 (CH=CH2), 166.0 (COOAll), 168.6, 169.0, 169.2, 169.7 (4 COCH3).

Allyl 2,3,4-tri-O-acetyl- -D-glucpyranuronate (21)

Allyl 1,2,3,4-tetra-O-acetyl- -D-glucuronate (20, 4 gm,

9.94 mmol) was stirred in dry THF (28 mL). Benzylamine

(2.2 mL, 20.12 mmol) was added dropwise and the solution

was left to stir overnight at room temperature. The solvent

was then removed in vacuo and the residue was purified by

column chromatography (1:3 EtOAc-petroleum ether) to

afford the hemiacetal as red oil (1.84 gm, 52%). 1H NMR


(270 MHz, CDCl3) 2.02, 2.04, 2.09 (3s, 9H, 3 COCH3),

4.61 (dd, 1H, OCH2-CH=CH2), 4.92 (d, 1H, J4-5 = 10.0 Hz,

H-5), 5.16–5.39 (m, 5H, H-2, H-3, H-4 and CH=CH2 over-

lapping), 5.56 (d, 1H, J1-2 = 4.4 Hz, H-1), 5.90 (m, 1H,

CH=CH2).

General Procedure for Preparation of N-(substituted-phenyl)-2,3,4-tri-O-acetyl-D-glucpyranuronamines

A solution of allyl 2,3,4-tri-O-acetyl-D-glucuronate (21)

and para-substituted aniline (1.5 equiv.) dissolved in anhy-

drous methanol (5 mL/0.1 gm) was refluxed for 25 h. The

solvent was then remved under diminished pressure and the

residue was purified by column chromatography (1:10

EtOAc-petroleum ether) to give the title compounds. In the

case of bromo-substituted phenyl, a mixture of / anomers

in a ratio of about 1:2 was obtained which were separated by

C-18 reversed phase chromatography (20% H2O-MeOH) and

identified, while in the case of fluoro-substituted phenyl, a

pure -anomer was obtained.

N-(4-Fluorophenyl)-2,3,4-tri-O-acetyl- -D-

glucpyranuronamine (22)

Yield 63%; mp 127 oC; white solid; Rf = 0.23 (1:4 EtO-

Ac-petroleum ether); 1

H NMR (500 MHz, CDCl3) 2.00 (s,

3H, COCH3), 2.03 (s, 6H, 2 COCH3), 4.14 (d, 1H, J4-5 =

10.0 Hz, H-5), 4.67 (m, 3H, H-2 and OCH2-CH=CH2 over-

lapping), 4.73 (apt t, 1H, H-3), 5.04 (apt t, 1H, H-4), 5.20–

5.32 (m, 3H, NH and CH=CH2 overlapping), 5.39 (apt t, 1H,

J1-2 = J1-NH = 9.2 Hz, H-1), 5.84 (m, 1H, CH=CH2), 6.61 (m,

2H, aromatic H), 6.88 (t, 2H, aromatic H); 13

C NMR (125

MHz, CDCl3) 20.6, 20.7, 20.8 (3 COCH3), 66.7 (OCH2-

CH=CH2), 69.9, 70.8, 72.3, 73.5 (C-2–C-5), 85.2 (C-1),

115.7, 115.8, 116.0 (4 aromatic CH), 119.5 (CH=CH2),

131.2 (CH, CH=CH2), 140.4 (aromatic C-NH), 157.2 (aro-

matic C-F, JC-F = 229.9 Hz), 166.6 (COOAll), 169.5, 170.0,

171.0 (3 COCH3).

N-(4-Bromophenyl)-2,3,4-tri-O-acetyl- -D-

glucpyranuronamine (23a)

Yield 21%; white solid; Rf = 0.42 (1:1 EtOAc-petroleum

ether); 1

H NMR (500 MHz, CDCl3) 2.05, 2.09, 2.14 (3s,

9H, 3 COCH3), 4.49 (m, 2H, H-5 and NH overlapping),

4.61 (m, 2H, OCH2-CH=CH2), 5.12 (m, 1H, H-2), 5.23–5.35

(m, 2H, H-3 and CH=CH2 overlapping), 5.43 (apt t, 1H, H-

4), 5.47 (apt t, 1H, J1-2 = J1-NH = 3.0 Hz, H-1), 5.90 (m, 1H,

CH=CH2), 6.82 (d, 2H, aromatic H), 7.31 (d, 2H, aromatic

H).

N-(4-Bromophenyl)-2,3,4-tri-O-acetyl- -D-

glucpyranuronamine (23b)

Yield 49%; white solid; Rf = 0.42 (1:1 EtOAc-petroleum

ether); 1

H NMR (500 MHz, CDCl3) 2.01, 2.03, 2.04 (3s,

9H, 3 COCH3), 4.14 (d, 1H, J4-5 = 9.8 Hz, H-5), 4.60 (m,

2H, OCH2-CH=CH2), 4.74–4.81 (m, 2H, H-3 and H-2 over-

lapping), 5.05 (apt t, 1H, H-4), 5.10–5.33 (m, 3H, NH and

CH=CH2 overlapping), 5.40 (apt t, 1H, J1-2 = J1-NH = 9.8 Hz,

H-1), 5.87 (m, 1H, CH=CH2), 6.54 (d, 2H, aromatic H), 7.28

(d, 2H, aromatic H).

Assay for Cytotoxic Activity: Human Cell Lines

The following three human cancer cell lines were used in these experiments: the human renal adenocarcinoma (TK-10), the human breast adenocarcinoma (MCF-7) and the hu-man melanoma (UACC-62) cell lines. The human tumour cytotoxicities were determined following protocols estab-lished by NCI [35]. TK-10, MCF-7, UACC-62 cell lines were cultured in RPMI 1640 medium (BioWhittaker

®) con-

taining 20% foetal calf serum (FCS), 2 mmol/L L-glutamine, 100 U/mL penicillin and 100 g/mL streptomycin. All cell lines were maintained at 37

oC in a 5% CO2 atmosphere with

95% humidity. Maintenance cultures were passaged weekly, and the culture medium was changed twice a week. Accord-ing to their growth profiles, the optimal plating densities of each cell line was determined (15 10

3, 5 10

3 and 100

103 cells/well for TK-10, MCF-7 and UACC-62, respec-

tively) to ensure exponential growth throughout the experi-mental period and to ensure a linear relationship between absorbance at 492 nm and cell number when analyzed by the sulphorhodamine B (SRB) assay.

Testing Procedure and Data Processing

The sulphorhodamine B (SRB) assay was used in this

study to assess growth inhibition. This colorimetric assay

estimates cell number indirectly by staining total cellular

protein with the SRB dye. For the assay, cells were detached

with 0.1% trypsin-EDTA (Sigma Chemical Co.) to make

single-cell suspensions, and viable cells were counted using

a Coulter counter and diluted with medium to give final den-

sities of 15 104, 5 10

4 and 100 10

4 cells/mL for TK-10,

MCF-7 and UACC-62, respectively. One hundred microli-

tres per well of these cell suspensions was seeded in 96-well

microtiter plates and incubated to allow for cell attachment.

After 24 h, the cells were treated with the serial concentra-

tions of the synthesized compounds. They were initially dis-

solved in an amount of 100% DMSO (40 mmol/L) and fur-

ther diluted in medium to produce five concentrations. One

hundred microlitres per well of each concentration was

added to the plates to obtain final concentration of 104, 10

5,

106, 10

7 and 10

8 M for the synthesized compounds. The

DMSO concentration for the tested dilutions was not greater

than 0.25% (v/v), the same as in solvent control wells. The

final volume in each well was 200 l. The plates were incu-

bated for 48 h.

Sulphorhodamine B Method

After incubating for 48 h, adherent cell cultures were

fixed in situ by adding 50 l of cold 50% (w/v) trichloroace-

tic acid (TCA) and incubating for 60 min at 4 oC. The super-

natant was then discarded and the plates washed five times

with deionised water and dried. One hundred microlitres of

SRB solution (0.4% w/v in 1% acetic acid) was added to

each microtiter well and the culture was incubated for 30

min at room temperature. Unbound SRB was removed by

washing five times with 1% acetic acid then the plates were

air-dried. Bound stain is solubilised with Tris buffer and the

optical densities (OD) were read on an automated spectro-

photometric plate reader at a single wavelength of 492 nm.

At the end, GI50 values (concentrations required to inhibit


cell growth by 50%), TGI (concentration resulting in total

growth inhibition) and LC50 (concentration causing 50% of

net cell killing) were calculated according to the previously

described protocols [35]. Two or three experiments were

carried out for each compound. The data are given as the mean of two or three different assays ± S.E.M.

ACKNOWLEDGEMENTS

The Authors gratefully acknowledge the financial sup-

port of the National Research Centre (NRC), Dokki, Giza,

Egypt. Also, I would like to express my special thanks to

Prof. Emilia Carretera, Pharmacology Department, Faculty

of Pharmacy, Complutense University, for her help and sup-

port concerning the biological activity tests. Further, I am

grateful in every possible way to the members of the NMR

spectroscopy unit for their continuous help regarding the

necessary NMR measurements especially Dr. Sahar Awad-

Allah Mahmoud.

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Received: March 2, 2011 Revised: June 09, 2011 Accepted: July 15, 2011

Design, Synthesis and Antitumor Activity of Novel D-Glucuronic Acid Derivatives

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Transcript of Design, Synthesis and Antitumor Activity of Novel D-Glucuronic Acid Derivatives