Synthesis and biological evaluation of some substituted-2-n-(5-chloro-2- methoxy-4-methylphenylsulphonyl) glutamic acid derivatives against prostate cancer cell line PC3

Article abstract of DOI:10.1248/cpb.c12-00881

New series of substituted glutamine 5a-l and glutamic acid diamides, diureide and dihydrazide 7a-e were synthesized from parent glutamic acid compound 3 and evaluated for their cytotoxic activity against tumor cell line PC3 (prostate cancer cell line). Most of the tested compounds exploited potent growth inhibitory activity with IC₅₀ values ranging 0.034-3.97 μm. Particularly, compounds 5a, 3, 5j, 5b, 7c, 7e, 5l, and 5k exhibited superior potency (IC₅₀=0.034, 0.04, 0.05, 0.074, 0.25, 0.4, 0.49, 0.522 μm, respectively) to the reference drug Doxorubicin (IC₅₀=0.63 μm), while compound 7b showed IC₅₀, 0.71 μm, comparable to that of Doxorubicin. In summary, the newly synthesized compounds provided promising new lead for the future design and development of glutamine and glutamic acid derivatives as novel antitumor agents. The quantitative structure-activity relationship (QSAR) study was applied to find a mathematical correlation between the structures of compounds and their activity against PC3 cell line expressed as IC₅₀ values.

Full text of DOI:10.1248/cpb.c12-00881

212
Regular Article
Chem. Pharm. Bull. 61(2) 212–221 (2013)
Vol. 61, No. 2
Synthesis and Biological Evaluation of Some Substituted-2-N-(5-chloro-
2-methoxy-4-methylphenylsulphonyl) Glutamic Acid Derivatives against
Prostate Cancer Cell Line PC3
Ghaneya Sayed Hassan and Doaa Ezzat Abdel Rahman*
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University; Cairo 11562, Egypt.
Received October 7, 2012; accepted November 1, 2012
New series of substituted glutamine 5a–l and glutamic acid diamides, diureide and dihydrazide 7a–e
were synthesized from parent glutamic acid compound 3 and evaluated for their cytotoxic activity against
tumor cell line PC3 (prostate cancer cell line). Most of the tested compounds exploited potent growth in-
hibitory activity with IC50 values ranging 0.034–3.97µm. Particularly, compounds 5a, 3, 5j, 5b, 7c, 7e, 5l,
and 5k exhibited superior potency (IC50=0.034, 0.04, 0.05, 0.074, 0.25, 0.4, 0.49, 0.522µm, respectively) to
the reference drug Doxorubicin (IC50=0.63µm), while compound 7b showed IC50, 0.71µm, comparable to that
of Doxorubicin. In summary, the newly synthesized compounds provided promising new lead for the future
design and development of glutamine and glutamic acid derivatives as novel antitumor agents. The quantita-
tive structure–activity relationship (QSAR) study was applied to find a mathematical correlation between the
structures of compounds and their activity against PC3 cell line expressed as IC50 values.
Key words glutametergic system; glutamic acid; glutamine; prostate cancer (PC); PC3 cell line
Prostate cancer (PC) is the most commonly diagnosed can- proliferation of neoplastic cells. This was attributed to obtain
cer among men around the world and it is considered the sec- l-glutamic acid as product of hydrolysis of different types of
ond most common cause of cancer-related deaths.1,2) Human malignant tumors.6,7) Therefore, l-glutamic acid and glutamine
PC cell lines have been classified into androgen-dependant gained great importance in formation and proliferation of dif-
line established from metastatic PC in the lymph-node such ferent PC cell lines.
as LNCaP cell line and androgen-independent line established
Furthermore, glutamate carboxypeptidase II (GCPII),
from a patient with bone or brain metastatic PC such as PC3 a membrane bound cell surface peptidase, is identical to
cell line or DU145 cell line respectively.3) However, after PC prostate-specific membrane antigen (PSMA). PSMA is a
makes metastases, chemotherapy plays an extremely impor- putative class II transmembranous glycoprotein and function-
tant role. With the pass of the time, PC cell lines become ally serves as transporter or binding protein or has hydrolytic
resistant to the current antiprostate cancer drugs. Therefore, activity.8–11) Isolated membrane fractions from human PC cell
there is a necessity to develop new antiprostate cancer agents lines (LNCaP, PC3 and DU145) can be tested for hydrolase
with the ability to be active against several PC cell lines.2)
activity. Unlike LNCaP cell membranes, which highly express
Glutamine (Gln) is the most abundant free amino acid in PSMA, membranes isolated from other human prostate adeno-
the human body, it is essential for the growth of normal and carcinoma cells (PC3 and DU145) did not exhibit comparable
neoplastic cells.4) A continuous supply of glutamine may be hydrolase activity.11) PSMA serves as an attractive target for
necessary for cancerous cell growth since cancer has been cancer immunotherapy by virtue of its abundant and restricted
described as a nitrogen trap.4,5) In addition, l-glutamic acid expression on the surface of prostate carcinoma and it is con-
provided an important role in formation and uncontrolled sidered as a tumor marker for fast and reliable diagnosis of
Fig. 1. Representative Inhibitors of GCPII and Anti-tumor Active Compounds
The authors declare no conflict of interest.
© 2013 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: doaaezzat2004@yahoo.com

February 2013
213
Reagents and conditions: (i) ClSO3H, 0°C; (ii) l-(+)-glutamic acid 2m NaOH, 70°C, 1h; (iii) conc. HCl; (iv) CH3COCl, 5°C, 1h, reflux 2h, stir room temperature 24h;
(v) R-NH2, stir room temperature 24h; (vi) 6m HCl.
Chart 1
PC.10,12) There are a number of GCPII inhibitors, characterized consumption and lactate production but did not induce apopto-
by a functional group connected to either a glutaryl moiety sis.21,22) In addition, Gln deprivations modulate directionality
or the glutamyl amino group, which either terminates the and motility to inhibit invasion in PC3 cells.23)
structure or serves as a linker to another molecular fragment.
GCPII inhibitors include 2-PMPA (A), GPI-18431 (B), DCIBzl (Fig. 1) were prepared and biologically evaluated against
(C) and N-substituted glutamyl sulfonamides (D) (Fig. 1). Ehrlich ascites carcinoma. Results revailed that, introduction
Substituted benzenesulphonylglutamic acid analogs (E)
In most inhibitors, glutarate/glutamate moiety appears of methoxy group to benzene nucleus of the disubstituted
to occupy the specificity pocket of GCPII while phospho- glutamic acid analogs resulted in marked improvement in
nate, phosphinate, urea or sulfonamide moiety ligates to the tumor weight inhibition compared to the chloro, methyl and
catalytic zinc ion at the active site.12–15) Structure–activity unsubstituted benzene ring analogs having similar amino
relationship for the glutamate binding site revealed that both substituents24) while unsubstituted ring analogs showed activ-
carboxylic acid groups of glutamate moiety should be free. Es- ity with different amino substituents.25) In case of glutamine
terification of terminal carboxylic group reduces the activity analogs presence of chloro, nitro, methyl and ethyl substituent
and of both carboxylic groups drastically reduces activity.12)
provided more reactive compounds compared with un substi-
Recently, there is increasing evidence to suggest that the tuted benzene ring analog.26,27)
glutamatergic system (Glu system) may be involved in cancer
Based on these, we report the synthesis of some substituted-
biology. The Glu system comprises the Glu receptors (GluRs), 2-N-(5-chloro-2-methoxy-4-methylphenylsulphonyl) glutamic
the Glu transporters (GluTs) and glutamine synthetase (GS). acid derivatives and the antitumor evaluation of the prepared
The expression of Glu system components were assessed in compounds against PC3 cell line. Synthesis of substituted-
human androgen-independent PC3 and androgen dependent 2-N-(5-chloro-2-methoxy-4-methylphenylsulphonyl) glutamic
LNCaP PC cells.16) Therefore, due to detection of the expres- acid derivatives was proposed to study the effect of presence
sion of mGLuR5 (metabotropic GluRs, mGluRs promote of chloro, methyl and methoxy substituents all together on
intracellular signal transduction pathways via activation of G activity. Compounds 5a–l, were designed as structural analogs
proteins)16,17) and GS (the metabolism of Glu by GS completes of glutamine, the most reactive amino acid especially in the
the Glu system, controlling intracellular glutamate signaling tumor cells, while compounds 7a–e were designed as disub-
pathways by the conversion of glutamate to glutamine)16,18–20) stituted glutamic acid analogs. PC3 cell line was selected
at the protein level in both PC3 and LNCaP cells, it is possible for biological antitumor evaluation for many reasons. First,
that PC3 and LNCaP prostate cancer cells possess ion-depen- structure of the prepared compounds is similar with glutamate
dent channel activity and intracellular G protein-dependent and glutamine. Second, PC3 cells do not express PSMA, to
signaling pathways controlled by glutamate. The possible im- exclude the effect on PSMA as the prepared compounds have
portance of the Glu system in PC cells can be foreseen based one carboxylic group or both carboxylic groups substituted
on its relationship to prostate-specific membrane antigen and expected to be inactive as PSMA inhibitor. Quantitative
(PSMA) expression, expression of PSMA in transfected PC3 structure–activity relationship (QSAR) study was also per-
cells (PC3 cells do not express PSMA) reduced the invasion formed for understanding and validating the cytotoxic activi-
potential of PC3 cells, suggesting that this reduction in the ties.
invasion capability was due to PSMA expression and not to
intrinsic properties of transfected PC cell lines.16)
Results and Discussion
Moreover, relative specific amino acid dependency is one of
Chemistry The procedure for the preparation of targeted
the metabolic abnormalities of cancer cells, and restriction of compounds 5a–l and 7a–e were summarized in (Charts 1, 2).
specific amino acids may induce apoptosis of PC cells. Spe- 5-Chloro-2-methoxy-4-methylbenzene-1-sulphonyl chloride 2
cific amino acid dependency differentially regulates glucose (Chart 1) was prepared by chlorosulphonation28) of 1-chloro-
metabolism, oxidation-reduction reactions of mitochondria and 4-methoxy-2-methylbenzene 1 with chlorosulphonic acid and
mitochondrial damage in DU145 and PC3 cell lines.21) Restric- melting point of the obtained product was as reported.29)
tion of Gln in PC3 cell lines inhibits ATP synthesis, induces With the application of Schotten–Baumann reaction,30)
generation of reactive oxygen species (ROS), reduces glucose 2-N-(5-chloro-2-methoxy-4-methylphenylsulphonyl) glutamic

214
Vol. 61, No. 2
Reagents and conditions: (i) diethyl glutamate, triethylamine, dry benzene, 24h; (ii) R-NH2, ethanol, reflux 16h.
Chart 2
acid 3 (Chart 1) was prepared by one step condensation of of the two carboxylic OH protons. Mass spectra showed mo-
5-chloro-2-methoxy-4-methylbenzene-1-sulphonyl chloride 2 lecular ion peak.
with solution of l-glutamic acid in 2m NaOH. Structure of
N1,N5-Bis substituted-2-N-(5-chloro-2-methoxy-4-methyl-
compound 3 was confirmed on the bases of elemental analy- phenylsulphonyl) glutamic acid diamides 7a–c, diureide 7d
sis and spectroscopic data (IR, 1H-NMR, MS). IR spectra and dihydrazide 7e, (Chart 2) were achieved by refluxing
showed NH and OH carboxylic stretching bands at 3425cm−1 compound 6 with appropriate amino compounds in ethanol.
and 2943, 2849cm−1, respectively, two carboxylic carbonyls at The proposed structure of 7a–e was confirmed by micro
1
1728, 1670cm−1 and SO2 group at 1369, 1118cm−1. H-NMR analysis and spectroscopic data. IR spectra showed additional
spectra showed the aliphatic protons of glutamic acid in addi- NH bands at 3448–3101 and disappearance of OH carboxylic
1
tion to methyl and methoxy protons at the region δ=2.05, 2.31, stretching bands. H-NMR spectra showed absence of ethyl
3.71, 3.75ppm, aromatic protons at 6.98 and 7.61ppm. The NH protons and appearance of additional aromatic protons at δ=
and two carboxylic OH protons appeared at δ=9.71, 10.52, 6.81–7.84ppm (except for 7a, 7d) and additional NH at δ=
10.93ppm, exchanged with D2O. Mass spectra showed molec- 3.93–10.10ppm, exchanged with D2O. The 13C-NMR spectra
ular ion peak. Cyclization of compound 3 with acetyl chloride for compound 7e showed bands at δ: 19.79, 20.04, 36.00, 55.78,
provided 1-(5-chloro-2-methoxy-4-methylphenylsulphonyl)-5- 56.49, 172.00 and 176.00 for CH3, CH2CH2CO, CH2CO, CH,
oxopyrrolidine-2-carboxylic acid 4 (Chart 1). Disappearance OCH3, and 2 CONH, respectively.
1
of NH group from IR and H-NMR spectra proved structure
of compound 4.
Antitumor Screening All newly synthesized compounds
were evaluated for their cytotoxicity against PC3 (prostate
The oxopyrrolidine-2-carboxylic acid
4
was reacted cancer cell line) by Skehan’s method31) as they may be good
with appropriate amino derivative and gave 2-N-(5-chloro- predictors of clinically useful drugs. In this protocol, cell
2-methoxy-4-methylphenylsulphonyl)-N5-substituted gluta- line was inoculated and incubated in plate for 24h. Test com-
mine 5a–l (Chart 1). The structure assignment for the pre- pounds were then added with different concentrations (0.001,
pared 5a–l was deduced by elemental and spectral analysis. 0.01, 0.1, 1, 10, 100µm) and incubated for 48h. Surviving
IR spectra showed additional NH bands at 3587–3120. curves were plotted as a relation between concentration and
1H-NMR spectra of the showed additional aromatic protons the surviving fraction to calculate IC50 (concentration that
at δ=6.42–8.10ppm (except for 5a, 5h). 13C-NMR spectra for reduce the surviving fraction to 50%)32) using known drug
compounds 5d and 5j showed bands at δ: 19.70–19.72, 26.00, Doxorubicin (Dox) as a positive control (Table 1).
31.00–36.00, 55.80–55.83, 171.10–174.00 and 175.00–176.00 for
CH3, CH2CH2CO, CH2CO, CH and OCH3, CONH, and COOH pounds exhibited promising activity where IC50 ranged from
respectively. 0.034–3.97µm for compounds 3, 5a, 5b, 5e, 5i, 5j, 5k, 5l, 7a,
Attempts to prepare the disubstituted compounds were 7b, 7c, 7d and 7e.
It was interesting to notice that, most of the tested com-
done via different methods. Reaction of compound 3 with
Parent intermediate compound 2-N-(5-chloro-2-methoxy-
different aniline derivatives using phosphorus trichloride in 4-methylphenylsulphonyl) glutamic acid 3 exhibited potent
chlorobenzene or preparation of the acid dichloride deriva- cytotoxic activity where the IC50 0.04µm. Replacement of one
tive of compound 3 followed by reaction with different amino carboxylic function with amide, anilide, uride or hydrazide
compounds gave the monosubstituted compounds 5b–f and function resulted in glutamine derivative with change in activ-
5b,h,i respectively, supported by the measured melting points ity to more potent or potent derivatives and some were less
and IR spectral data.
2-N-(5-Chloro-2-methoxy-4-methylphenylsulphonyl) glu-
active.
Glutamine derivative 5a was very promising and more
tamic acid diethyl ester 6, (Chart 2) was prepared by reaction potent than the parent compound (IC50, 0.034µm). Anilide
of the sulphonyl chloride derivative 2 and diethyl glutamate. derivative 5b showed more potency than substituted anilide
IR spectra showed NH at 3441cm−1, absence of OH carboxylic derivative 5e as substitution with methoxy group at position 4
stretching band, two ester carbonyls at 1720, 1670cm−1 and slightly affect activity where IC50, 0.074 and 3.97µm, respec-
1
SO2 group at 1377, 1172cm−1. H-NMR spectra showed the ali- tively. Substitution at position 4 of the anilide with methyl or
phatic protons of glutamic acid in addition to methyl, methoxy chloro or sulphonamido group reduced activity. Heteroaro-
and ethyl protons at the region δ=1.20, 1.40, 2.30, 2.36, 3.09, matic anilide and uride substitution greatly reduced activity.
3.64–3.80ppm, aromatic protons at 6.96 and 7.59ppm. The
Hydrazide derivatives 5i–l showed good activity as IC50,
NH appeared at δ=9.75ppm, exchanged with D2O and absence ranging from 0.05–3.23µm where the chloro substitution at

February 2013
215
Table 1. IC50 of Tested Compounds for Antitumor Screening against
PC3 (Prostate Cancer Cell Line)
sized glutamic acid derivatives, QSAR study was undertaken.
Descriptors of the molecular modeling software, Molecular
Operating Environment (MOE version 2008.10.2),33) were
used. The structural descriptors used in the generation of
these models include; Kier molecular flexibility index (Flex),
Water accessible surface area (ASA), the ionization potential
(IP or mndo_IP), Value of the potential energy (E) and van
der Waals component of the potential energy (E_vdw) as
shown in Table 2.
To test the best structural predictors for activity, stepwise
linear regression analysis (SLRA) technique was used.
The preliminary regression analysis has indicated that out
of the 18 tested compounds, four compounds (5d, f–h) were
outliers. These outliers were recognized by having high pre-
liminary Z-score values (>2.5) against the selected descrip-
tors.34) For better statistical results these compounds were
deleted from further regression procedure.
Cpd. No.
IC50 (µm)
3
0.04
0.034
0.074
17.4
22.03
3.97
17.9
76.7
33.8
3.23
0.05
0.522
0.49
1.07
0.71
0.25
2.18
0.4
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
7a
7b
7c
7d
7e
Dox
For the current dataset of 14 compounds, the QSAR model
development was restricted to a maximum of three variables
as one should select one parameter for five compounds data
set (5:1 for compounds: descriptor).
The simple linear regression analysis yielded one statisti-
cally significant correlation (Eq. 1), that was the correlation
0.63
position 4 of phenylhydrazide was most active one among between the activity and the ionization potential (IP) of the
substituted hydrazide derivatives and the unsubstituted deriva- tested compounds.
tive was the least active one which explore the importance of
substitution among these active compounds.
Replacement of both carboxylic functions with diamide,
IC50 =69.9( 21.2) − 7.15( 2.24) IP
n=14, Se =3.49, r =0.678, r2 =0.460, F =10.22
(1)
diuride and dihydrazide function 7a–e resulted in active com- n: Number of compounds. Se: Standard error of estimation.
pounds with IC50, ranging from 0.25–2.18µm. It was noticed r: Simple (multiple) correlation coefficient. r2: Fraction of the
that diamide 7a and dianilide 7b were active compounds but variance. F: Fisher’s statistics.
activity decreased 30 and 10 folds from the amide 5a and
Stepwise regression analyses using different combinations
anilide 5b, respectively. Dianilide substituted with sulphon- of IP and other structural descriptors resulted into bi-paramet-
amido group 7c and diuride 7d were active irrespective that ric models including: IP, E; IP, E_vdw; IP, ASA and IP, Flex
monosubstituted derivatives 5f and 5h had reduced activity. which show better statistics than the mono-parametric model
Dihydrazide 7e showed increased activity 8 folds than the discussed above.
monosubstituted one 5i.
Trials were made to correlate three combined descriptors
Compounds 3, 5a, 5b, 5j, 5k, 5l, 7c, 7e were found to be with the biological activity aiming to improve the statistical
more active than Dox while compounds 7a and 7b were nearly parameters of the obtained models. Two tri-parametric models
active as Dox.
were obtained from stepwise regression analyses (Models 1
QSAR Study In an attempt to correlate the antiprolifera- and 2).
tive activity with the structure conformation of the synthe-
Table 2. The Molecular Descriptor Values of the Studied Compounds
Descriptors
Cpd. No.
Kierflex
ASA
IP
E
E_vdw
3
7.2453
6.9065
7.4378
7.6659
8.2508
7.7329
8.7610
8.5475
9.5130
6.5811
7.8414
11.2955
7.7464
8.4134
568.8266
575.6415
689.5095
708.0336
714.5831
719.5165
741.2696
748.0812
767.4078
572.9178
792.5010
917.0554
655.1626
789.6392
10.1247
10.0520
9.5634
8.8231
9.4067
9.2009
9.2893
9.2917
9.8423
9.8209
9.0720
9.7339
9.6383
9.1544
4.6182
8.3250
35.3791
36.0493
53.3627
53.3364
56.6556
53.0716
52.2035
57.5350
56.1462
39.0610
71.0572
74.2773
43.4123
66.7716
5a
5b
5c
5e
5i
5j
5k
5l
7a
7b
7c
7d
7e
38.7099
36.9720
44.3930
54.2075
52.5998
61.9106
23.7034
7.1049
65.3133
6.6719
−42.5792
94.1508

216
Vol. 61, No. 2
value of Flex suggested that the decrease in ionization poten-
tial and increase in Kier molecular flexibility index lead to
enhancement of activity. This was in good agreement with the
obtained experimental data, for the most active compounds 3
and 5a, slight decrease in ionization potential and increase in
Kier molecular flexibility index (Table 2) led to comparable
activity IC50 0.04 and 0.034µm, respectively. In case of com-
pounds 5b and 5j decrease in ionization potential and increase
in Kier molecular flexibility index while increase in Water ac-
cessible surface area (ASA) (Table 2) led to slight decrease in
activity IC50 0.074 and 0.05µm, respectively.
IC50 =121.4( 22.4) 11.7( 2.2) IP
 0.06( 0.02) E  0.11( 0.07) E_vdw
n=14, Se =2.631, r =0.862, r2 =0.743, F =9.667
(Model 1)
IC50 =150.3( 28.6) 13.9( 2.7) IP
 0.08( 0.02) ASA+4.8( 1.8) Flex
n=14, Se =2.604, r =0.865, r2 =0.749, F =9.940
(Model 2)
Fraction of the variance (r2): Represent the goodness of fit.
The value of r2 may vary between 0 and 1, when multiplied
Conclusion
According to cytotoxic activity of tested compounds against
by 100 gives explained variance in biological activity, where 1 PC3 (prostate cancer cell line), it is apparent from the results
means a perfect model explaining 100% of the variance in the that:
data, and 0 means a model without any explanatory power. It
Parent intermediate compound with both free carboxylic
has already been suggested that the only QSAR model having groups 3 exhibited promising activity.
r2>0.6 will be considered for validation.35) The value of r2 for
Models 1 and 2 were 0.743 and 0.749 respectively.
Some monosubstituted glutamic acid derivatives as amide,
anilide and hydrazide showed promising activity. Substitution
The observed activities (Obs. IC50) together with the pre- of anilide derivative reduced activity while heteroaromatic
dicted activities (Pred. IC50) for the tested compounds calcu- anilide and uride were inactive.
lated using multi-linear regression (MLR) are listed for Model
2 in Table 3.
Disubstituted glutamic acid derivatives as diamide, diani-
lide, diuride and dihydrazide showed good activity.
From Model 1 equation, cytotoxic activity was negatively
Compound with carboxylic groups free, compounds with
correlated with IP, E and E_vdw. The high coefficient value of monosubstituted carboxylic group and compounds with disub-
IP and the comparatively lower value of E_vdw suggested that stituted carboxylic groups showed promising activity. These
the decrease in ionization potential and van der Waals compo- results supported the suggestion that these compounds are not
nent of the potential energy lead to enhancement of activity. active as PSMA inhibitors as PC3 do not express PSMA11)
This was in good agreement with the obtained experimental and structure–activity relationship study of PSMA inhibitors12)
data, for the most active compounds 3 and 5a, slight decrease supported decreased activity of monosubstituted compounds
in ionization potential and slight increase in van der Waals and inactivity of disubstituted compounds relative to parent
component of the potential energy for compound 5a (Table compound with both carboxylic groups free.
2) led to comparable activity 0.04 and 0.034µm, respectively.
Most active compounds had structure similarity with glu-
In case of compounds 5b and 5j decrease in their ionization tamine 5a, IC50, 0.034µm (34 nm), 5j, IC50, 0.05µm (50 nm),
potential and increase in van der Waals component of the 5b, IC50, 0.074µm (74 nm) and glutamic acid 3, IC50, 0.04µm
potential energy (Table 2) led to slight decrease in activity as (40 nm). It may be suggested that, these compounds may act
IC50 increased, 0.074 and 0.05µm, respectively.
through inhibition of GS, antagonistic effect on mGluR5 (as
From Model 2 equation, cytotoxic activity was negatively both of them are expressed in PC3) or may cause restriction of
correlated with IP, ASA and positively correlated with Flex. Gln amino acid that result in several consequent effects.
The high coefficient value of IP and the comparatively lower
Briefly, the glutamic acid derivatives serve as promising
Table 3. The Observed Activities for PC3 Cell Line (Obs. IC50) Together with the Predicted Activities (Pred. IC50) for the Tested Compounds Calculated
Using Multi-linear Regression (MLR) for (Model 3)
MLR validation
Cpd. No.
Obs. IC50
Pred. IC50
Residual
Z-Score
3
0.0400
0.0340
0.0740
17.4000
3.9700
3.2300
0.0500
0.5220
0.4900
1.0700
0.7100
0.2500
2.1800
0.4000
0.0281
−1.1047
−0.6888
13.4354
3.4769
3.4821
5.4647
3.8902
−0.6355
0.7576
0.0119
1.1387
0.7628
3.9646
0.4931
−0.2521
−5.4147
−3.3682
1.1255
0.3124
0.5718
0.0054
0.5134
0.3439
1.7876
0.2223
0.1137
2.4414
1.5187
0.5075
0.1408
0.2578
1.1185
0.1296
0.6939
5a
5b
5c
5e
5i
5j
5k
5l
7a
7b
7c
7d
7e
0.1382
−2.2307
2.4675
2.4807
−0.2875
−1.5390
1.9390

February 2013
217
molecule for subsequent molecular modification in the search (10mmol) of compound 3, acetyl chloride (10mL) was added
for novel anti-tumors. Furthermore, the result of the QSAR slowly with stirring at 5°C then refluxed on steam bath for 2h.
studies performed made clear that ionization potential of a The reaction mass was poured onto crushed ice slowly with
molecule is the key for antiproliferative activity.
continuous stirring. It was kept cold overnight when the semi-
solid mass solidified. It was filtered, dried and crystallized
from ethanol. Yield 85%. mp >300°C. 1H-NMR (DMSO-
Experimental
Chemistry Melting points were determined by open cap- d6) δ: 2.26–2.36 (5H, m, COCH2CH2CHCOOH and CH3),
illary tube method using Gallen Kamp melting point apparatus 3.33 (2H, t, J=13.4Hz, COCH2CH2CHCOOH), 3.73 (4H, t,
MFB-595-010M (Gallen Kamp, London, England) and were J=11.8Hz, CH and OCH3), 6.96 (1H, s, H-3 Ar), 7.59 (1H, s,
uncorrected. Microanalysis was carried out at The Regional H-6 Ar), 11.00 (1H, s, COOH exch. D2O). IR (KBr) cm−1: 3085
Center for Mycology and Biotechnology, Al-Azhar University (CH Ar), 2981 (CH aliphatic), 2939, 2850 (OH carboxylic),
and at Organic Microanalyses Section, Central Laboratory, 1774, 1735 (2 C=O), 1597, 1562 (NH, C=C), 1369, 1118 (SO2).
National Research Center. Infrared Spectra were recorded as MS m/z: 344 (M+−3). Anal. Calcd for C13H14ClNO6S (347.77):
potassium bromide discs on Schimadzu FT-IR 8400S spectro- C, 44.90; H, 4.06; N, 4.03. Found: C, 45.13; H, 4.19; N, 4.38.
photometer (Shimadzu, Kyoto, Japan) and expressed in wave
General Procedure for Synthesis of 2-N-(5-Chloro-2-me-
number (cm−1). The NMR spectra were recorded on a Varian thoxy-4-methylphenylsulphonyl)-N5-substituted Glutamine
1
Mercury VX-300 NMR spectrometer. H spectra were run at (5a–l), Chart 1 To a suspension of 4 (3.47g, 10mmol) in
300MHz and 13C spectra were run at 75.46MHz in deuter- water (30mL), the appropriate amino derivative (10mmol) was
ated chloroform (CDCl3) or dimethylsulphoxide (DMSO-d6). added and stirred at room temperature for 24h. Water (30mL)
Chemical shifts are quoted in δ as parts per million (ppm) was added, cooled and acidified with 6m HCl to pH 3.0. The
downfield from tetramethylsilane (TMS) as internal standard. precipitate obtained was filtered, washed with water and dried.
Mass spectra were recorded using Hewlett Packard Var- The crude mass was crystallized from the appropriate solvent
ian (Varian, Polo, U.S.A.) and Shimadzu Gas Chromatograph to yield 60–85%.
Mass Spectrometer-QP 1000 EX (Shimadzu, Kyoto, Japan).
2-N-(5-Chloro-2-methoxy-4-methylphenylsulphonyl) Gluta-
TLC were carried out using Art.DC-Plastikfolien, Kieselgel mine (5a): General procedure was adopted to prepare 5a
60 F254 sheets (Merck, Darmstadt, Germany), the developing employing 4 (3.47g, 10mmol) in water (30mL), and liquor
solvents were benzene–methanol (4:1) and the spots were vi- ammonia (15mL, 33%). After the reaction is complete, the ex-
sualized at 366, 254nm by UV Vilber Lourmat 77202 (Vilber, cess ammonia was evaporated and water (30mL) was added,
Marne La Vallee, France).
cooled and acidified with 6m HCl to pH 3.0 to yield 65%. The
5-Chloro-2-methoxy-4-methylbenzene-1-sulphonyl Chlo- crude mass was crystallized from ethanol. mp 295–296°C.
ride (2), Chart 1 1-Chloro-4-methoxy-2-methylbenzene 1 1H-NMR (DMSO-d6) δ: 1.15 (2H, q, J=6.3Hz, CH2CH2CO),
(1.56g, 10mmol) was added to chlorosulphonic acid (5.8g, 2.39 (5H, t, J=7.9Hz, CH3 and CH2CO), 3.66 (4H, t, J=7.9Hz,
50mmol), magnetically stirred at 0°C, over a period of 30min. CH and OCH3), 7.21 (1H, s, H-3 Ar), 7.85 (1H, s, H-6 Ar),
The stirring was continued for further 1h at room tempera- 8.10 (3H, s, NH and NH2 exch. D2O), 11.0 (1H, s, COOH exch.
ture then the mixture poured onto crushed ice. The obtained D2O). IR (KBr) cm−1: 3533, 3240 (NH2, NH), 3059 (CH Ar),
solid was filtered, dried and crystallized from methanol. mp 2927, 2858 (OH carboxylic), 1735, 1665 (2 C=O), 1600, 1562
126–128°C (as reported 126–128°C).29) IR (KBr) cm−1: 3425 (NH, C=C), 1373, 1180 (SO2). MS m/z: 364 (M+). Anal. Calcd
(NH), 3109 (CH Ar), 2985, 2951 (CH aliphatic), 1597, 1554 for C13H17ClN2O6S (364.80): C, 42.80; H, 4.70; N, 7.68. Found:
(NH, C=C), 1377, 1172 (SO2).
2-N-(5-Chloro-2-methoxy-4-methylphenylsulphonyl)
C, 43.11; H, 4.82; N, 8.04.
2-N-(5-Chloro-2-methoxy-4-methylphenylsulphonyl)-N5-
Glutamic Acid (3), Chart 1 To l-(+)-glutamic acid (1.47g, phenylglutamine (5b): Yield 65%. The crude mass was crys-
1
10mmol) dissolved in 2m NaOH (5mL), 5-chloro-2-methoxy- tallized from ethanol. mp 251–254°C. H-NMR (DMSO-d6)
4-methylbenzene-1-sulphonyl chloride 2 (2.55g, 10mmol) was δ: 2.25–2.35 (5H, m, CH2CH2CO and CH3), 2.38 (2H, t,
added slowly with constant stirring, maintaining the internal J=10.2Hz, CH2CO), 3.65 (1H, t, J=5.7Hz, CH), 3.72 (3H, s,
temperature at 70°C. The reaction was continued until a OCH3), 5.65 (1H, s, NH exch. D2O), 6.96–7.84 (7H, m, Ar
clear homogeneous solution resulted. After the reaction was H and H-3,6 Ar), 7.79 (1H, s, NH exch. D2O), 10.63 (1H, s,
complete as showed by TLC, it was allowed to cool, acidified COOH exch. D2O). IR (KBr) cm−1: 3437 (2 NH), 3105 (CH
to pH 3.0 with HCl, saturated with NaCl. The obtained solid Ar), 2960 (CH aliphatic), 2920, 2642 (OH carboxylic), 1700,
product 3 was filtered, dried and finally crystallized from 1660 (2 C=O), 1600, 1546 (NH, C=C), 1373, 1168 (SO2). MS
1
ethyl acetate-benzene. Yield 80%. mp 254–255°C. H-NMR m/z: 442 (M++2). Anal. Calcd for C19H21ClN2O6S (440.90): C,
(DMSO-d6) δ: 2.05 (2H, q, J=6.5Hz, CH2CH2CO), 2.31 (5H, 51.76; H, 4.80; N, 6.35. Found: C, 52.13; H, 4.97; N, 6.49.
t, J=9.7Hz, CH2CO and CH3), 3.71 (1H, t, J=9.3Hz, CH), 3.75
2-N-(5-Chloro-2-methoxy-4-methylphenylsulphonyl)-N5-(4-
(3H, s, OCH3), 6.98 (1H, s, H-3 Ar), 7.61 (1H, s, H-6 Ar), 9.71 methyphenyl) Glutamine (5c): Yield 74%. The crude mass was
1
(1H, s, NH exch. D2O), 10.52 (1H, s, COOH exch. D2O), 10.93 crystallized from ethanol. mp 248–249°C. H-NMR (DMSO-
(1H, s, COOH exch. D2O). IR (KBr) cm−1: 3425 (NH), 3085 d6) δ: 2.27–2.35 (8H, m, CH2CH2CO and 2×CH3), 2.38 (2H,
(CH Ar), 2981 (CH aliphatic), 2943, 2854 (OH carboxylic), t, J=10.5Hz, CH2CO), 3.65 (1H, t, J=6.9Hz, CH), 3.72 (3H,
1728, 1670 (2 C=O), 1597, 1562 (NH, C=C), 1369, 1118 (SO2). s, OCH3), 6.96–7.84 (6H, m, Ar H and H-3,6 Ar), 9.74 (3H,
MS m/z: 365 (M+). Anal. Calcd for C13H16ClNO7S (365.79): C, brs, 2×NH and COOH exch. D2O). IR (KBr) cm−1: 3425 (2
42.69; H, 4.41; N, 3.83. Found: C, 42.82; H, 4.38; N, 4.11.
1-(5-Chloro-2-methoxy-4-methylphenylsulphonyl)-5-oxo- carboxylic), 1700, 1670 (2 C=O), 1600, 1560, 1540 (NH, C=
pyrrolidine-2-carboxylic Acid (4), Chart
To 3.65g C), 1373, 1165 (SO2). MS m/z: 456 (M++2). Anal. Calcd for
NH), 3101 (CH Ar), 2960 (CH aliphatic), 2924, 2642 (OH
1

218
Vol. 61, No. 2
C20H23ClN2O6S (454.92): C, 52.80; H, 5.10; N, 6.16. Found: C,
53.14; H, 5.28; N, 6.29.
N5-(Aminocarbonyl)-2-N-(5-chloro-2-methoxy-4-methyl-
phenylsulphonyl) Glutamine (5h): Yield 73%. The crude
2-N-(5-Chloro-2-methoxy-4-methylphenylsulphonyl)-N5-(4- mass was crystallized from ethanol. mp 270–272°C. H-NMR
chlorophenyl) Glutamine (5d): Yield 75%. The crude mass (DMSO-d6, D2O) δ: 2.20–2.30 (5H, m, CH2CH2CO and CH3),
was crystallized from ethanol. mp 236–237°C. 1H-NMR 3.71 (2H, t, J=7.9Hz, CH2CO), 4.07 (4H, t, J=7.9Hz, CH and
(DMSO-d6) δ: 2.27–2.38 (5H, m, CH2CH2CO and CH3), 2.60 OCH3), 6.96 (1H, s, H-3 Ar), 7.56 (1H, s, H-6 Ar). IR (KBr)
(2H, t, J=7.3Hz, CH2CO), 3.67 (1H, t, J=7.4Hz, CH), 3.71 cm−1: 3390, 3305, 3217, 3120 (NH2, 2 NH), 3008 (CH Ar),
(3H, s, OCH3), 5.63 (1H, s, NH exch. D2O), 6.98 (1H, s, H-3 2954 (CH aliphatic), 2854, 2546 (OH carboxylic), 1705 (3 C=
Ar), 7.16 (2H, dd, J=11.1, 3.0Hz, H-2′,6′ Ar), 7.39 (2H, dd, O), 1600, 1570 (NH, C=C), 1373, 1145 (SO2). MS m/z: 407
J=11.1, 3.0Hz, H-3′,5′ Ar), 7.60 (1H, s, H-6 Ar), 8.40 (1H, brs, (M+). Anal. Calcd for C14H18ClN3O7S (407.83): C, 41.23; H,
NH exch. D2O), 10.20 (1H, s, COOH exch. D2O). 13C-NMR 4.45; N, 10.30. Found: C, 41.10; H, 4.45; N, 10.10.
1
(DMSO-d6) δ: 19.70 (CH3), 26.00 (CH2CH2CO), 31.00
2-N-(5-Chloro-2-methoxy-4-methylphenylsulphonyl)-N5-
(CH2CO), 55.80 (CH and OCH3), 114.81 (C-3), 122.29 (C-2′,6′), phenylglutamic Acid Hydrazide (5i): Yield 80%. The crude
1
122.78 (C-1), 128.27 (C-5) 128.60 (C-6), 129.35 (C-3′,5′), mass was crystallized from ethanol. mp 262–263°C. H-NMR
134.35 (C-4′), 135.69 (C-1′), 137.56 (C-4), 154.74 (C-2), 171.10 (CDCl3) δ: 1.83–2.0 (7H, m, CH2CH2CO and CH3), 3.44 (1H,
(CONH), 175.00 (COOH). IR (KBr) cm−1: 3433 (2 NH), t, J=10.2Hz, CH), 3.53 (3H, s, OCH3), 5.0 (1H, s, NH exch.
3101 (CH Ar), 2940 (CH aliphatic), 2924, 2638 (OH carbox- D2O), 6.23 (1H, s, NH exch. D2O), 6.42–7.42 (7H, m, Ar H
ylic), 1680, 1640 (2 C=O), 1597, 1550 (NH, C=C), 1373, 1149 and H-3,6 Ar), 8.10 (1H, s, NH exch. D2O), 9.50 (1H, s, COOH
(SO2). MS m/z: 475 (M+). Anal. Calcd for C19H20Cl2N2O6S exch. D2O). IR (KBr) cm−1: 3290 (3 NH), 2993 (CH aliphatic),
(475.34): C, 48.01; H, 4.24; N, 5.89. Found: C, 48.36; H, 4.45; 2951, 2735 (OH carboxylic), 1710, 1630 (2 C=O), 1604, 1546
N, 5.98.
(NH, C=C), 1369, 1176 (SO2). MS m/z: 455 (M+). Anal. Calcd
2-N-(5-Chloro-2-methoxy-4-methylphenylsulphonyl)-N5-(4- for C19H22ClN3O6S (455.91): C, 50.05; H, 4.86; N, 9.22. Found:
methoxyphenyl) Glutamine (5e): Yield 67%. The crude mass C, 50.28; H, 4.70; N, 9.20.
was crystallized from ethanol. mp 210–211°C. 1H-NMR
2-N-(5-Chloro-2-methoxy-4-methylphenylsulphonyl)-N5-(4-
(DMSO-d6) δ: 2.26–2.36 (5H, m, CH2CH2CO and CH3), chlorophenyl) Glutamic Acid Hydrazide (5j): Yield 85%. The
2.38 (2H, t, J=7.9Hz, CH2CO), 3.65 (4H, t, J=11.8Hz, CH crude mass was crystallized from ethanol. mp 248–250°C.
and OCH3), 3.72 (3H, s, OCH3), 5.65 (1H, s, NH exch. D2O), 1H-NMR (DMSO-d6) δ: 2.25–2.35 (7H, m, CH2CH2CO and
6.68–6.80 (4H, m, Ar H), 6.96 (1H, s, H-3 Ar), 7.59 (1H, s, CH3), 3.73 (4H, t, J=7.3Hz, CH and OCH3), 6.92 (1H, s, H-3
H-6 Ar), 7.73 (1H, s, NH exch. D2O), 10.20 (1H, s, COOH Ar), 6.95 (2H, dd, J=12.3, 3.0Hz, H-2′,6′ Ar), 7.22 (2H, dd,
exch. D2O). IR (KBr) cm−1: 3450, 3410 (2 NH), 3100 (CH Ar), J=12.3, 3.0Hz, H-3′,5′ Ar), 7.61 (1H, s, H-6 Ar), 8.31 (1H, brs,
2924, 2638 (OH carboxylic), 1700, 1680 (2 C=O), 1620, 1597, NH exch. D2O), 10.01 (3H, s, 2×NH and COOH exch. D2O).
1570 (NH, C=C), 1380, 1165 (SO2). MS m/z: 467 (M+−3). 13C-NMR (DMSO-d6) δ: 19.72 (CH3), 26.00 (CH2CH2CO),
Anal. Calcd for C20H23ClN2O7S (470.92): C, 51.01; H, 4.92; N, 36.00 (CH2CO), 55.83 (CH and OCH3), 114.80 (C-3), 115.96
5.95. Found: C, 51.23; H, 5.19; N, 6.18.
(C-2′,6′), 122.76 (C-1), 125.19 (C-4′), 128.30 (C-5), 128.79
N5-[4-(Aminosulphonyl)phenyl]-2-N-(5-chloro-2-methoxy-4- (C-3′,5′), 134.54 (C-6), 137.47 (C-4), 144.37 (C-1′), 154.79
methylphenylsulphonyl) Glutamine (5f): Yield 60%. The crude (C-2), 174.00 (CONH), 176.00 (COOH). IR (KBr) cm−1:
mass was crystallized from ethanol. mp 209–210°C. H-NMR 3282 (3 NH), 2993 (CH aliphatic), 2931, 2735 (OH carbox-
1
(DMSO-d6) δ: 1.20 (2H, q, J=5.5Hz, CH2CH2CO), 2.39 (3H, ylic), 1700, 1630 (2 C=O), 1597, 1546 (NH, C=C), 1369, 1170
s, CH3), 2.79 (2H, t, J=6.8Hz, CH2CO), 3.53 (1H, t, J=5.5Hz, (SO2). MS m/z: 491 (M++1). Anal. Calcd for C19H21Cl2N3O6S
CH), 3.65 (3H, s, OCH3), 5.77 (2H, brs, 2×NH exch. D2O), (490.36): C, 46.54; H, 4.32; N, 8.57. Found: C, 46.74; H, 4.41;
6.58 (2H, d, J=8.1Hz, H-2′,6′ Ar), 6.85 (2H, brs, NH2 exch. N, 8.86.
D2O), 7.20 (1H, s, H-3 Ar), 7.45 (2H, d, J=7.8Hz, H-3′,5′ Ar),
2-N-(5-Chloro-2-methoxy-4-methylphenylsulphonyl)-N5-(4-
7.85 (1H, s, H-6 Ar), 9.60 (1H, brs, COOH exch. D2O). IR methoxyphenyl) Glutamic Acid Hydrazide (5k): Yield 85%.
(KBr) cm−1: 3464, 3379, 3340, 3248 (NH2, 2 NH), 3101 (CH The crude mass was crystallized from ethanol. mp 186–187°C.
Ar), 2940 (CH aliphatic), 2920, 2850 (OH carboxylic), 1680, 1H-NMR (DMSO-d6) δ: 2.25–2.35 (5H, m, CH2CH2CO and
1660 (2 C=O), 1620, 1597, 1560 (NH, C=C), 1311, 1149 (SO2). CH3), 2.56 (2H, t, J=7.9Hz, CH2CO), 3.65–3.80 (7H, m, CH
MS m/z: 519 (M+). Anal. Calcd for C19H22ClN3O8S2 (519.98): and 2xOCH3), 6.88–6.95 (5H, m, Ar H and H-3 Ar), 7.59 (1H,
C, 43.89; H, 4.26; N, 8.08. Found: C, 44.19; H, 4.60; N, 8.23.
s, H-6 Ar), 9.75 (4H, s, 3×NH and COOH exch. D2O). IR
2-N-(5-Chloro-2-methoxy-4-methylphenylsulphonyl)-N5- (KBr) cm−1: 3444, 3421, 3271 (3 NH), 3060 (CH Ar), 2997
(pyridin-4-yl) Glutamine (5g): Yield 80%. The crude mass (CH aliphatic), 2927, 2731 (OH carboxylic), 1680, 1660 (2 C=
was crystallized from acetone. mp 251–252°C. 1H-NMR O), 1620, 1593, 1525 (NH, C=C), 1369, 1176 (SO2). MS m/z:
(DMSO-d6) δ: 2.20–2.35 (5H, m, CH2CH2CO and CH3), 2.60 485 (M+). Anal. Calcd for C20H24ClN3O7S (485.94): C, 49.43;
(2H, t, J=8.6Hz, CH2CO), 3.71 (4H, t, J=8.6Hz, CH and H, 4.98; N, 8.65. Found: C, 49.78; H, 5.17; N, 8.92.
OCH3), 6.79 (2H, d, J=7.8Hz, H-2′,6′ Ar), 6.96 (1H, s, H-3
N5-[4-(Aminosulphonyl)phenyl]-2-N-(5-chloro-2-methoxy-4-
Ar), 7.59 (1H, s, H-6 Ar), 8.02 (2H, s, 2×NH exch. D2O), 8.10 methylphenylsulphonyl) Glutamic Acid Hydrazide (5l):
(2H, d, J=7.2Hz, H-3′,5′ Ar), 13.00 (1H, brs, COOH exch. Yield 61%. The crude mass was crystallized from ethanol.
D2O). IR (KBr) cm−1: 3390, 3209 (2 NH), 3105 (CH Ar), 2962 mp 241–243°C. 1H-NMR (DMSO-d6) δ: 2.25–2.35 (5H, m,
(CH aliphatic), 2939, 2796 (OH carboxylic), 1662, 1612 (2 C= CH2CH2CO and CH3), 2.38 (2H, t, J=6.8Hz, CH2CO), 3.73
O), 1600, 1535 (NH, C=C), 1369, 1184 (SO2). MS m/z: 441 (4H, t, J=9.0Hz, CH and OCH3), 6.96 (1H, s, H-3 Ar), 6.98
(M+). Anal. Calcd for C18H20ClN3O6S (441.89): C, 48.92; H, (2H, d, J=9.0Hz, H-2′,6′ Ar), 7.21 (2H, brs, NH2, exch. D2O),
4.56; N, 9.51. Found: C, 49.07; H, 4.63; N, 9.85.
7.59 (1H, s, H-6 Ar), 7.72 (2H, d, J=8.7Hz, H-3′,5′ Ar,), 8.70

February 2013
219
(1H, s, NH exch. D2O), 10.09 (3H, brs, 2×NH and COOH OCH3), 5.40 (5H, broad, NH and 2×NH2, exch. D2O), 6.81
exch. D2O). IR (KBr) cm−1: 3325, 3294, 3236 (NH2, 3 NH), (5H, d, J=8.1Hz, 2×H-2′,6′ Ar and NH exch. D2O), 6.95 (1H,
3060 (CH Ar), 2960 (CH aliphatic), 2927, 2735 (OH carbox- s, H-3 Ar), 7.55 (5H, d, J=8.1Hz, 2×H-3′,5′ Ar and NH exch.
ylic), 1680, 1660 (2 C=O), 1597, 1543 (NH, C=C), 1346, 1172 D2O), 7.57 (1H, s, H-6 Ar). IR (KBr) cm−1: 3350, 3250, 3101
(SO2). MS m/z: 534 (M+). Anal. Calcd for C19H23ClN4O8S2 (2NH2, 3 NH), 3078 (CH Ar), 2916 (CH aliphatic), 1680, 1670
(534.99): C, 42.66; H, 4.33; N, 10.47. Found: C, 43.08; H, 4.68; (2 C=O), 1597, 1558 (NH, C=C), 1311, 1149 (SO2). MS m/z:
N, 10.73.
2-N-(5-Chloro-2-methoxy-4-methylphenylsulphonyl) H, 4.19; N, 10.39. Found: C, 44.69; H, 4.10; N, 10.57.
Glutamic Acid Diethyl Ester (6), Chart 2 A mixture of
2-N-(5-Chloro-2-methoxy-4-methylphenylsulphonyl)-N1,N5-
674 (M+). Anal. Calcd for C25H28ClN5O9S3 (674.17): C, 44.54;
sulphonyl chloride derivative 2 (2.55g, 10mmol) and diethyl glutamic Acid Diuride (7d): Yield 75%. The crude mass was
1
glutamate (2.03g, 10mmol) in dry benzene (50mL) and tri- crystallized from ethanol. mp 281–283°C. H-NMR (DMSO-
ethylamine (few drops) was refluxed for 24h. The solvent was d6, D2O) δ: 1.16 (2H, q, J=7.9Hz, CH2CH2CO), 2.38 (3H, s,
evaporated and residue was triturated with water. The ob- CH3), 2.80 (2H, t, J=7.9Hz, CH2CO), 3.64 (4H, t, J=11.8Hz,
tained solid product was filtered, dried and crystallized from CH and OCH3), 7.20 (1H, s, H-3 Ar), 7.84 (1H, s, H-6 Ar). IR
1
ethanol. Yield 85%. mp 136–138°C. H-NMR (DMSO-d6) δ: (KBr) cm−1: 3406, 3275, 3213, 3101 (2NH2, 3 NH), 3078 (CH
1.20 (2H, q, J=7.5Hz, CH2CH2CO), 1.40 (6H, t, J=7.9Hz, Ar), 2951 (CH aliphatic), 1710, 1700, 1680 (4 C=O), 1597,
2×CH2CH3), 2.30 (3H, s, CH3), 2.36 (2H, t, J=11.8Hz, 1562 (NH, C=C), 1311, 1149 (SO2). MS m/z: 449 (M+). Anal.
CH2CO), 3.09 (1H, t, J=7.9Hz, CH), 3.64–3.80 (7H, m, OCH3 Calcd for C15H20ClN5O7S (449.87): C, 40.05; H, 4.48; N, 15.57.
and 2×CH2CH3), 6.96 (1H, s, H-3 Ar), 7.59 (1H, s, H-6 Ar), Found: C, 40.23; H, 4.51; N, 15.78.
9.75 (1H, s, NH exch. D2O). IR (KBr) cm−1: 3441 (NH), 3109
N1,N5-Bis Phenyl-2-N-(5-chloro-2-methoxy-4-methylphenyl-
(CH Ar), 2985, 2947, 2927 (CH aliphatic), 1720, 1670 (2 C=O), sulphonyl) Glutamic Acid Dihydrazide (7e): Yield 70%. The
1597, 1562 (NH, C=C), 1377, 1172 (SO2). MS m/z: 421 (M+). crude mass was crystallized from ethanol. mp 220–223°C.
Anal. Calcd for C17H24ClNO7S (421.89): C, 48.40; H, 5.73; N, 1H-NMR (DMSO-d6) δ: 2.28–2.39 (7H, m, CH2CH2CO and
3.32. Found: C, 48.56; H, 5.88; N, 3.71.
CH3), 3.72 (1H, t, J=7.5Hz, CH), 3.79 (3H, s, OCH3), 3.93
General Precedure for Synthesis of N1,N5-Bis Substi- (2H, s, 2×NH, exch. D2O), 6.42 (2H, brs, 2×NH, exch.
tuted-2-N-(5-chloro-2-methoxy-4-methylphenylsulphonyl) D2O), 6.81–7.84 (12H, m, Ar H, H-3,6 Ar), 9.31 (1H, s, NH
Glutamic Acid Diamides (7a–c), Diureide (7d) and Dihy- exch. D2O). 13C-NMR (DMSO-d6) δ: 19.79 (CH3), 20.04
drazide (7e), Chart 2 A mixture of diester derivative 6 (CH2CH2CO), 36.0 (CH2CO), 55.78 (CH), 56.49 (OCH3),
(4.21g, 10mmol), liquor ammonia, aniline, 4-aminobenzene- 112.51 (C-3), 113.57 (2×C-2′,6′), 114.35 (C-1), 120.20
sulphonamide, urea or phenylhydrazine (20mmol) in abso- (2×C-4′), 123.55 (C-5), 124.43 (C-6), 128.39 (C-4), 128.76
lute ethanol (50mL) was refluxed for 16h. The solvent was (2×C-3′,5′), 147.04 (2×C-1′), 155.15 (C-2), 172.00 (CONH),
evaporated under reduced pressure and water was added. The 176.00 (CONH). IR (KBr) cm−1: 3441, 3290, 3201 (5 NH),
separated solid was filtered and dried. The crude mass was 3016 (CH Ar), 2924 (CH aliphatic), 1640, 1620 (2 C=O), 1604,
crystallized from the appropriate solvent to yield 70–80%.
1562 (NH, C=C), 1369, 1161 (SO2). MS m/z: 545 (M+−1).
2-N-(5-Chloro-2-methoxy-4-methylphenylsulphonyl)
Glu- Anal. Calcd for C25H28ClN5O5S (546.04): C, 54.99; H, 5.17; N,
tamic Acid Diamide (7a): Yield 78%. The crude mass was 12.83. Found: C, 55.21; H, 5.32; N, 13.14.
1
crystallized from ethanol. mp 268–270°C. H-NMR (DMSO-
Antitumor Screening All newly synthesized compounds
d6) δ: 2.26–2.40 (5H, m, CH2CH2CO and CH3), 2.61 (2H, t, were tested against the tumor cell line PC3 (prostate cancer
J=11.8Hz, CH2CO), 3.69 (1H, t, J=7.9Hz, CH), 3.74 (3H, s, cell line) at Department of Pharmacology and Toxicology,
OCH3), 6.97 (1H, s, H-3 Ar), 7.20 (5H, broad, NH and 2×NH2 Faculty of Pharmacy, Ain Shams University using the Sulfo
exch. D2O), 7.59 (1H, s, H-6 Ar). IR (KBr) cm−1: 3448, 3425, Rhodamine B stain (SRB) assay by the method of Skehan et
3170 (2NH2, NH), 3051 (CH Ar), 2985 (CH aliphatic), 1670, al.31)
1640 (2 C=O), 1597, 1562 (NH, C=C), 1369, 1118 (SO2). MS
Procedure Cells were plated in 96-multiwell plate
m/z: 363 (M+). Anal. Calcd for C13H18ClN3O5S (363.82): C, (104 cells/well) for 24h before treatment with the compounds
42.92; H, 4.99; N, 11.55. Found: C, 43.18; H, 5.17; N, 11.69.
to allow attachment of cell to the wall of the plate. Test com-
N1,N5-Bis Phenyl-2-N-(5-chloro-2-methoxy-4-methylphenyl- pounds were dissolved in DMSO and diluted to the appropri-
sulphonyl) Glutamic Acid Diamide (7b): Yield 80%. The crude ate volume. Different concentrations of the compound under
1
mass was crystallized from ethanol. mp 201–202°C. H-NMR test (0.001, 0.01, 0.1, 1, 10, 100µm) were added to the cell
(DMSO-d6) δ: 2.28–2.44 (7H, m, CH2CH2CO and CH3), 3.64 monolayer. Triplicate wells were prepared for each individual
(1H, t, J=9.0Hz, CH), 3.86 (3H, s, OCH3), 6.95–7.84 (12H, m, dose. Monolayer cells were incubated with the compounds for
Ar H and H-3,6 Ar), 10.10 (3H, s, 3×NH exch. D2O). IR (KBr) 48h at 37°C and in atmosphere of 5% CO2. After 48h, cells
cm−1: 3263 (3NH), 3080 (CH Ar), 2978 (CH aliphatic), 1680, were fixed, washed and stained for 30min with 0.4% (w/v)
1670 (2 C=O), 1597, 1562 (NH, C=C), 1369, 1161 (SO2). MS Sulfo Rhodamine B dissolved in 1% acetic acid. Excess stain
m/z: 517 (M++1). Anal. Calcd for C25H26ClN3O5S (516.01): C, was washed with acetic acid and attached stain was recovered
58.19; H, 5.08; N, 8.14. Found: C, 58.41; H, 5.09; N, 8.43.
with Tris ethylenediaminetetraacetic acid (Tris EDTA) buffer.
N1,N5-Bis [4-(Aminosulphonyl)phenyl]-2-N-(5-chloro-2- Colour intensity was measured in an enzyme-linked immu-
methoxy-4-methylphenylsulphonyl) Glutamic Acid Diamide nosorbent assay (ELISA) reader. The relation between the
(7c): Yield 78%. The crude mass was crystallized from surviving fraction and drug concentration was plotted to get
ethanol. mp 248–249°C. 1H-NMR (DMSO-d6) δ: 1.10 (2H, the survival curve of each tumor cell line after the specified
q, J=7.9Hz, CH2CH2CO), 2.30 (3H, s, CH3), 2.60 (2H, t, compound. IC50 and R fraction of the tested compounds were
J=9.0Hz, CH2CO), 3.44 (1H, t, J=9.0Hz, CH), 3.65 (3H, s, illustrated in Table 1.

220
Vol. 61, No. 2
The dose response curve of compounds was analyzed using together with the predicted activities (Pred. IC50) for the tested
Emax model.
compounds calculated using multi-linear regression (MLR)
are listed (Table 3). All compounds showed very good results
with Z-scores not exceed the value of 2.5 indicating excellent
predictive ability of the model.
[D]m
Kdm +[D]m




% Cell viability=(100− R)× 1−
+R






Outliers Compounds 5d, 5f, 5g, 5h were considered as
Where R is the residual unaffected fraction (the resistance outliers as they were unable to fit in a QSAR model. Separat-
fraction), [D] is the drug concentration used, Kd is the drug ing these outliers from the main data set and formulating an-
concentration that produces a 50% reduction of the maxi- other QSAR can resolve the problem. Outliers may be acting
mum inhibition rate and m is a Hill-type coefficient. IC50 was by a different mechanism.
defined as the drug concentration required to reduce fluores-
cence to 50% of that of the control (i.e., Kd=IC50 when R=0
Acknowledgment Authors are thankful to drug design
laboratory, Pharmaceutical Chemistry Department, Faculty of
and Emax=100−R).32)
QSAR. Computational Method All the computational Pharmacy, Assiut University, for assistance in performing the
works were performed on Molecular Operating Environment QSAR study.
software (MOE version 2008.10.2).33) The structures of 18
compounds used as training set were sketched using molecular
builder of MOE and each structure was subjected to energy
minimization up to 0.01kcal/molÅ using the MMFF94x force
field. Optimization methods were used followed by conforma-
tional search of each energy-minimized structure. The most
stable conformer of each structure was selected and saved
into database to generate the common descriptors. QuaSAR
descriptor module of MOE was used to calculate descriptors
for each molecule. The probability density functions used are
Gaussian. The root mean square deviation (RMSD) tolerance
was set to 0.5Å. Regression analysis was performed using
PC3 IC50 as dependent factor and the calculated descriptors as
predictable variables.
In this study, the pool of descriptors was optimized using
principal components analysis (PCA). The optimization start-
ed with the reduction in the number of molecular descriptors
by the determination of the highly inter-correlated descrip-
tor pairs and only one from each pair was selected; then the
descriptors with insignificant variance through the data set
were also rejected. QSAR model was then constructed after
ensuring reasonable correlation of cytotoxic activity with the
individual descriptors and minimum inter-correlation among
the descriptors used in the derived model. The quality of the
model was assessed using the statistical parameter r2.
Molecular Descriptors Flex: Kier molecular flexibility
index (Kier and Hall Connectivity and Kappa Shape Indices):
calculated using (KierA1) (KierA2)/n.
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