Synthesis, quantitative structure-activity relationship and biological evaluation of 1*3*4-oxadiazole derivatives possessing diphenylamine moiety as potential anticancer agents

Article abstract of DOI:10.1248/cpb.c12-00637

Synthesis of 2,5-disubstituted-1,3,4-oxadiazole (2a-c), 3-substituted aminomethyl-5-substituted-1,3,4- oxadiazole-2(3H)-thione (4a-m) and 2-substituted thio-5-substituted-1,3,4-oxadiazole (5a, b) had been described. All the synthesized derivatives were screened for anticancer activity against HT29 and MCF7 cancer cell lines using Sulfo-Rodamine B (SRB) standard method. Most of the tested compounds exploited potent antiproliferative activity against HT29 cancer cell line rather than MCF7 cancer cell line. Compounds 2a-c, 4f and 5a exhibited potent cytotoxicity (IC₅₀ 1.3-2.0 μm) and selectivity against HT29 cancer cell line. Quantitative structure-activity relationship (QSAR) study was applied to find a correlation between the experimental antiproliferative activities of the newly synthesized oxadiazole derivatives with their physicochemical parameter and topological index.

Full text of DOI:10.1248/cpb.c12-00637

February 2013
Regular Article
Chem. Pharm. Bull. 61(2) 151–159 (2013)
151
Synthesis, Quantitative Structure–Activity Relationship and Biological
Evaluation of 1,3,4-Oxadiazole Derivatives Possessing Diphenylamine
Moiety as Potential Anticancer Agents
Doaa Ezzat Abdel Rahman
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University; Cairo 11562, Egypt.
Received July 20, 2012; accepted October 31, 2012
Synthesis of 2,5-disubstituted-1,3,4-oxadiazole (2a–c), 3-substituted aminomethyl-5-substituted-1,3,4-
oxadiazole-2(3H)-thione (4a–m) and 2-substituted thio-5-substituted-1,3,4-oxadiazole (5a, b) had been de-
scribed. All the synthesized derivatives were screened for anticancer activity against HT29 and MCF7 cancer
cell lines using Sulfo-Rodamine B (SRB) standard method. Most of the tested compounds exploited potent
antiproliferative activity against HT29 cancer cell line rather than MCF7 cancer cell line. Compounds 2a–c,
4f and 5a exhibited potent cytotoxicity (IC50 1.3–2.0µm) and selectivity against HT29 cancer cell line. Quan-
titative structure–activity relationship (QSAR) study was applied to find a correlation between the experi-
mental antiproliferative activities of the newly synthesized oxadiazole derivatives with their physicochemical
parameter and topological index.
Key words 1,3,4-oxadiazole; Mannich base; anticancer; HT29 cell line; MCF7 cell line; quantitative struc-
ture–activity relationship study
Cancer is a group of diseases characterized by uncontrolled describes the synthesis of new series of 2,5-disubstituted-
growth and spread of abnormal cells. If the spread is not 1,3,4-oxadiazole, 2-substituted thio-5-substituted-1,3,4-oxa-
controlled, it can result in death. Cancer is the second most diazole and Mannich bases of 5-substituted-1,3,4-oxadiazole-
common cause of death in the U.S., exceeded only by heart 2(3H)-thione possessing diphenylamine moiety and evalua-
disease, accounting for nearly 1 of every 4 deaths.1) Some of tion of the anti-tumor properties of the prepared compounds
the most common cancer types, such as breast cancer, cervi- against human tumor cell lines (HT29 “colon” and MCF7
cal cancer, oral cancer and colorectal cancer have higher cure “breast” cancers). Quantitative structure–activity relationship
rates when detected early and treated according to best prac- (QSAR) analysis was performed to find a correlation between
tices.2) Colorectal cancer is the third most common cancer in the physicochemical parameter and topological index of the
both men and women.1) Adjuvant chemotherapy (anticancer studied compounds and their experimental activity. It is note-
drugs in addition to surgery or radiation) for colon cancer worthy to mention that, steric bulk and structure conformation
is equally effective.1) Excluding cancers of the skin, breast of a molecule have been shown to be useful in pharmaco-
cancer is the most frequently diagnosed cancer in women.1,3,4) kinetic profile prediction of the designed molecules. QSAR
Treatment may involve radiation therapy, chemotherapy (be- study was also performed for understanding and validating the
fore or after surgery), hormone therapy or targeted therapy.1) antiproliferative activities.
Accordingly, continued research is needed to develop new
antitumor agents.
Results and Discussion
1,3,4-Oxadiazoles are an important class of heterocyclic
Chemistry The targeted compounds 2a–c, 4a–m and
compounds and attracted great interest in medicinal chemistry. 5a,b were synthesized as depicted in Chart 1. The starting
Substituted 1,3,4-oxadiazoles display a remarkable broad spec- compound, N-phenylanthranilic acid hydrazide 135) derived
trum of biological activity such as antimicrobial,5,6) antituber- from N-phenylanthranilic acid36) and intermediate compound
cular,7) analgesic,8,9) anti-inflammatory,9–12) anticonvulsant13) 5-[2-(phenylamino)phenyl]-1,3,4-oxadiazole-2-thiol 3,37) were
and anticancer14–18) activities. 2-Mercapto-5-substituted-1,3,4- prepared according to the previously reported procedures.
oxadiazole were found to exhibit antimicrobial,6,19) anti-human
N-Phenylanthranilic acid hydrazide 1 was cyclized with
immunodeficiency virus (anti-HIV),19) antitubercular,20) pes- (substituted) benzoic acid in the presence of phosphorus oxy-
ticidal,21,22) tyrosinase inhibition23) and anticancer24–27) activi- chloride to afford 2,5-disubstituted-1,3,4-oxadiazole deriva-
ties. Moreover, Mannich bases of 1,3,4-oxadiazole derivatives tives 2a–c. Structures of compounds 2a–c were confirmed on
were reported to possess antimicrobial,28) analgesic, anti- the bases of elemental analysis and spectroscopic data (IR,
inflammatory29) and anticancer activities.30)
1H-NMR and MS). IR spectra showed only one NH stretching
Furthermore, diphenylamine moiety was proved to exert band at 3421–3290cm−1 and disappearance of carbonyl stretch-
in-vitro26) and in-vivo31,32) anti-tumor activity in addition to ing band of the parent N-phenylanthranilic acid hydrazide.
inhibition of autophosphorylation of epidermal growth factor 1H-NMR spectra showed protons of methoxy group at δ=
receptor (EGFR),26) FGF-R231) and MEK32–34) tyrosine kinase 3.82ppm in compound 2c spectrum in addition to increased
enzymes.
number of aromatic protons at δ=6.68–8.21ppm and NH
In view of the above mentioned facts and as an attempt appeared at δ=8.30–8.90ppm, exchanged with D2O for com-
to obtain new potent antitumor agents, the present work pounds 2a–c. Mass spectra showed molecular ion peak at 313,
347 and 343 for compounds 2a, 2b and 2c respectively.
5-Substituted-1,3,4-oxadiazol-2-thiol 3 underwent N-amino-
The author declares no conflict of interest.
© 2013 The Pharmaceutical Society of Japan
e-mail: doaaezzat2004@yahoo.com

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Vol. 61, No. 2
Reagents and solvents: (i) 4-(un)substituted benzoic acid–POCl3; (ii) Cs2–KOH–ethanol; (iii) CH2O–NHRR′–ethanol; (iv) RX–K2CO3–acetone.
Chart 1
methylation reaction (Mannich reaction) on treating with MCF7 (breast cancer cell line) as they may be good predic-
paraformaldehyde and primary/secondary amines to afford tors of clinically useful drugs. In this protocol, cell line was
the compounds 4a–m. Attempts to perform N-aminomethyl- inoculated and incubated in plate for 24h. Test compounds
ation via reaction with formaldehyde and primary/secondary were then added with different concentrations (0.001, 0.01, 0.1,
amines in methanol6) or ethanol30) at room temperature gave 1, 10, 100µm) and incubated for 48h. Surviving curves were
the parent compound 3, supported by the measured melting plotted as a relation between concentration and the surviving
point and IR spectral data. Therefore reaction was performed fraction to calculate IC50 (concentration that reduce the surviv-
using paraformaldehyde and primary/secondary amines in ing fraction to 50%) and R, the residual unaffected fraction
refluxing ethanol.38,39) The proposed structures of compounds (the resistance fraction)41) using known drug Doxorubicin
4a–m were confirmed by micro analysis and spectroscopic (Dox) as a positive control, Table 1.
data. IR spectra showed NH bands at 3444–3232cm−1 and
From the observed antitumor activity data, Table 1, it has
C=S stretching band at 1296–1265cm−1. H-NMR spectra of been noticed that most of the tested compounds exerted sig-
compounds 4a–m showed singlet signal at δ=4.46–5.76ppm nificant activity against HT29 “colon” cell line compared with
corresponding to CH2 protons. The 13C-NMR spectra showed MCF7 “breast” cell line and less percent of resistant fraction.
bands at δ=66.00, 163.00 and 176.54ppm for compound 4a Results indicated more sensitivity and less resistance of HT29
and 67.12, 167.61 and 176.43ppm for compound 4k corre- “colon” than MCF7 “breast” cell line towards tested com-
sponding to CH2, C-5 oxadiazole and C=S respectively, in pounds.
1
addition to signals of the remaining aromatic Cs. Mass spectra
showed molecular ion peak.
Considering the observed antitumor screening data against
MCF7 “breast” cancer cell line, only compounds 2a–c and 4c
Further, the alkylation of 3 with alkyl halide in presence of revealed pharmacological activity (IC50=3.6, 9.0, 9.5, 6.6µm,
anhydrous potassium carbonate yielded S-alkylated products respectively) but compound 2c showed high percent of resis-
5a,b. The structure assignment for the prepared compounds tant fraction (34.4%).
5a,b was deduced by elemental and spectral analysis. IR spec-
However, all tested compounds showed activity against
tra showed NH band at 3302–3290cm−1. H-NMR spectrum of HT29 “colon” cancer cell line except compounds 4a,b,e and
compound 5a showed the triplet and quartet signals of ethyl 5b. Compound 4f had the most promising antitumor proper-
protons at the region δ = 1.54 and 3.33ppm for CH3 and CH2 ties among all the tested analogs against HT29 “colon” cancer
respectively, while that of 5b showed the benzyl CH2 pro- cell line (IC50=1.3 µm). Additionally, compounds 2a–c and 5a
tons at δ=4.54ppm. The 13C-NMR spectra for compound 5b revealed also promising antitumor properties against the same
showed band at δ=36.73ppm for benzylic CH2, 162.00ppm for cell line (IC50=1.5, 1.9, 1.7, 2.0µm, respectively). Activity of
C-5 oxadiazole and 165.46ppm for C-2 oxadiazole, in addition rest of active compounds ranged from IC50=2.4–6.0µm.
1
to signals of the remaining aromatic Cs. Mass spectra showed
molecular ion peak.
Structure–activity relationship based on the observed anti-
tumor properties of the synthesized compounds against HT29
Antitumor Screening In-vitro antitumor activity for all “colon” cancer cell line, indicated that in 2,5-disubstituted-
newly synthesized compounds was performed utilizing in- 1,3,4-oxadiazole derivatives 2a–c, substitution of the 4-posi-
vitro Sulfo-Rodamine B (SRB) standard method40) against the tion of phenyl ring with either chloro or methoxy group at
tumor cell line HT29 (colon adenocarcinoma cell line) and 5-position slightly decreased activity but retain promising

February 2013
153
Table 1. IC50 and R Fraction (Resistant Fraction) of Tested Compounds Table 2. The Molecular Descriptor Values of the Studied Compounds
for Antitumor Screening against HT29 (Colon Adenocarcinoma Cell Line)
Descriptors
weinerPol
and MCF7 (Breast Cancer Cell Line)
Cpd. No.
mr
Vsur_S
HT29
MCF7
Cpd. No.
2a
2b
2c
4a
4b
4c
4d
4e
4f
4g
4h
4i
4k
5b
9.5194
10.0170
10.1787
10.3340
10.6024
10.8817
10.3350
10.9681
11.4181
11.9956
11.0388
11.4699
11.6309
10.7373
34.0000
36.0000
38.0000
36.0000
37.0000
39.0000
37.0000
38.0000
40.0000
44.0000
40.0000
40.0000
42.0000
35.0000
527.7904
587.3488
535.3326
548.4037
599.2478
649.9304
601.9377
574.9385
617.9083
621.7568
580.0325
642.4895
684.5125
534.0283
IC50 µm (R fraction) %
2a
2b
2c
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
1.5 (0.0)
1.9 (0.0)
1.7 (0.0)
12.7 (0.5)
10.8 (0.0)
2.4 (0.3)
4.9 (0.0)
10.1 (0.0)
1.3 (0.0)
3.6 (0.0)
4.9 (0.0)
2.7 (0.13)
2.97 (0.8)
3.6 (0.0)
6.0 (0.1)
4.1 (0.0)
2.0 (4.0)
22.0 (—)
0.25 (0.0)
3.6 (0.2)
9.0 (0.0)
9.5 (34.4)
12.0 (0.0)
10.8 (0.0)
6.6 (0.0)
11.0 (0.0)
54.3 (0.2)
10.3 (0.6)
64.7 (11.8)
14.9 (5.3)
22.6 (1.2)
10.3 (2.6)
153.7 (—)
12.7 (0.6)
14.0 (0.34)
13.3 (39.4)
22.0 (29.4)
0.13 (0.0)
field, divided by the square root of the mean square error of
the data set. Any compound which shows a value of Z score
higher than 2.5, during generation of a particular QSAR
model, is considered as outlier.43)
From the equation, antiproliferative activity was positively
correlated with mr and negatively correlated with weinerPol
4m
5a
5b
Dox
activity. In case of Mannich reaction with primary amine that and vsurf_S. The high coefficient value of mr and the compar-
yielded Mannich bases of 1,3,4-oxadiazole-2(3H)-thione 4e–l, atively lower value of weinerPol suggested that the increase in
only 4e with unsubstituted phenyl ring was inactive while all steric bulk and polarizability of the substituent and decrease
substituted derivatives showed activity and the methyl substi- in branching lead to enhancement of activity. In other words,
tution was the most active one. Replacement of phenyl ring increase in molar refractivity while keeping proper balance
with heterocycle such as benzothiazole produced also active with branching of this substituent and structure conformation
compound 4m. Mannich reaction with secondary amine that of the overall molecule is the key for activity. This was in
yielded Mannich bases, only N-methylpiperazine and morpho- good agreement with the obtained experimental data, where
line derivatives 4c,d were active while diethylamine or piperi- in case of compounds 2,5-disubstituted-1,3,4-oxadiazole 2a–c,
dine 4a,b were inactive. S-Alkylated derivatives at 2-position substitution of the phenyl ring showed slight increase in molar
of 1,3,4-oxadiazole 5a,b showed promising activity of ethyl refractivity value accompanied by increase in weinerPol value
derivative 5a while the benzyl derivative 5b was inactive.
leading to slight drop in activity. On the other hand, in case
QSAR Study In an attempt to correlate the antiprolifera- of Mannich bases with primary amine of 5-substituted-1,3,4-
tive activity with the structure conformation of the synthe- oxadiazole-2(3H)-thione, compounds 4f–i,k showed higher mr
sized oxadiazole derivatives, QSAR study was undertaken. value and increase in weinerPol value due to the substituent
Descriptors of the molecular modeling software, Molecular compared with parent unsubstituted inactive compound 4e but
Operating Environment (MOE version 2008.10.2),42) were with considerable balance between mr value and weinerPol
used. The QSAR model development was restricted to a maxi- value accounted for activity.
mum of three variables as one should select one parameter for
QSAR results have suggested that the steric parameter of
five compounds data set (5:1 for compounds: descriptor). The the substituents is one of the most important determinants for
structural descriptors used in the generation of these mod- the activities against colon cancers, with a major contribution
els include; Molar refractivity (mr), Weiner polarity number coming from the molar refractivity of the substituents.44)
(weinerPol) and Interaction feild area (vsurf_S) as shown in
Cross-Validations Test Cross-validations are the most
Table 2. The best derived QSAR linear model for the 14 oxa- commonly used techniques for internal validation, In the case
diazole compounds (3 compounds were considered as outliers of leave-one-out (LOO) cross-validation, each member of the
and 1 compound was used for evaluation) were presented by original data set in turn is removed and developed new QSAR
the following estimated equation:
models in order to verify the internal predictive ability of the
original QSAR model, e.g. q2 (leave-one-out)=0.58158, it was
calculated by the following Equation43)
:
HT29 IC50 =12.85973+14.47783×mr  3.40007
×weinerPol  0.05556×vsurf_S
r2 =0.72546 , q2 =0.58158
(IC50 Obs.  IC50 Pred.)2
q2 =1
(IC50 Obs. IC50 average)2
The observed IC50 was plotted against their predicted values
(MLR), Table 3, Fig. 1. The Z score method was adopted for
Statistical Diagnosis Fraction of the variance (r2): Rep-
the detection of outliers. Z score can be defined as absolute resent the goodness of fit. The value of r2 may vary between
difference between the value of the model and the activity 0 and 1, when multiplied by 100 gives explained variance in

154
Vol. 61, No. 2
Table 3. The Observed Activities for HT29 Cell Line (Obs. IC50) together with the Predicted Activities (Pred. IC50) for the Tested Compounds Calcu-
lated Using Multi-linear Regression (MLR)
MLR validation
Cpd. No.
Obs. IC50
Pred. IC50
Residual
Z-Score
2a
2b
2c
4a
4b
4c
4d
4e
4f
4g
4h
4i
4k
5b
1.5000
1.9000
1.7000
12.7000
10.8000
2.4000
4.9000
10.1000
1.3000
3.6000
4.9000
2.7000
3.6000
22.0000
5.7521
2.8461
1.2773
9.5999
7.2613
1.6883
3.2407
10.5066
7.8332
2.3799
4.4463
7.2178
0.4131
19.6375
−4.2521
−0.9461
0.4227
3.1001
3.5387
0.7117
1.6593
−0.4066
−6.5332
1.2201
1.4217
0.3163
0.1413
1.0365
1.1831
0.2379
0.5548
0.1359
2.1844
0.4079
0.1517
1.5105
1.0655
0.7899
0.4537
−4.5178
3.1869
2.3625
line was more sensitive and less resistant than MCF7 “breast”
cell line towards most of the tested compounds.
In case of 2,5-disubstituted-1,3,4-oxadiazole derivatives,
substitution of the 4-position of phenyl ring slightly decreased
activity.
In case of Mannich bases of 1,3,4-oxadiazole-2(3H)-thione
produced from reaction with primary amine, only unsubsti-
tuted phenyl ring was inactive while all substituted derivatives
showed activity and the methyl substitution was the most ac-
tive one. Replacement of phenyl ring with heterocycle (benzo-
thiazole) retained activity. Mannich bases of 1,3,4-oxadiazole-
2(3H)-thione resulted from reaction with secondary amine
showed that, N-methylpiperazine and morpholine derivatives
gave active compounds while diethylamine or piperidine gave
inactive compounds.
S-Alkylation of position 2 of 1,3,4-oxadiazole with ethyl
group exhibeted promising activity while in case of benzyl
removed activity.
Fig. 1. Correlation of Observed and Predicted IC50 Using MLR
(r2=0.72546)
biological activity, where 1 means a perfect model explaining
Briefly, 1,3,4-oxadiazole derivatives serve as promising
100% of the variance in the data, and 0 means a model with- nucleus for subsequent modification in the search for novel
out any explanatory power. It has already been suggested that anti-tumors. Furthermore, the result of the QSAR studies
the only QSAR model having r2>0.6 will be considered for performed made clear that steric bulk and structure confor-
validation.45) The value of r2 for this QSAR model is 0.72546.
mation of a molecule is the key for antiproliferative activity.
Cross-Validation Test (q2): A measure of quality of the Cytotoxic results of colon cell line screening and QSAR study
QSAR model. According to the literature, a QSAR model of the synthesized compounds in this work make them a good
must have q2>0.5 for their predictive ability.45) The value of q2 trial to discover new anticancer agents. These results urge
for this QSAR model is 0.58158.
further investigations to seek for new derivatives containing
r2–q2<0.3: This difference between r2 and q2 for a QSAR oxadiazole heterocycle, diphenylamine moiety or both in fu-
model should never be exceeded by 0.3. A large difference ture work.
between r2 and q2 suggests the following: over-fitted model,
presence of outliers or presence of irrelevant variables in the
Experimental
Chemistry Melting points were determined by open
data set.44) The value of r2–q2 for this QSAR model is 0.14388.
Model Evaluation Compound 4m was used for evalua- capillary tube method using Gallen Kamp melting point ap-
tion of the model. It showed low residual value (0.0146) as the paratus MFB-595-010M (Gallen Kamp, London, England)
predicted IC50 was 4.0854µm compared with the observed IC50 and were uncorrected. Microanalysis was carried out at The
4.1000µm.
Regional Center for Mycology and Biotechnology, Al-Azhar
University and the micro analytical center, Faculty of science,
Cairo University. Infrared Spectra were recorded as potassium
Conclusion
According to anti-proliferative inhibitory activity of tested bromide discs on Schimadzu FT-IR 8400S spectrophotom-
compounds against HT29 “colon” cell line and MCF7 “breast” eter (Shimadzu, Kyoto, Japan) and expressed in wave number
cell line, it is apparent from the results that: HT29 “colon” cell (cm−1). The NMR spectra were recorded on a Varian Mercury

February 2013
155
1
VX-300 NMR spectrometer. H spectra were run at 300MHz the completion of the reaction, as established by TLC, the
and 13C spectra were run at 75.46MHz in deuterated chloro- solvent was evaporated under reduced pressure. After cooling,
form (CDCl3) or dimethylsulphoxide (DMSO-d6). Chemical the gummy oil was extracted with chloroform and dried over
Shifts are quoted in δ as parts per million (ppm) downfield anhydrous sodium sulfate then chloroform was distilled under
from tetramethylsilane (TMS) as internal standard. Mass reduced pressure to provide the title compounds in 55–70%
spectra were recorded using Hewlett Packard Varian (Var- yield.
ian, Polo, U.S.A.) and Shimadzu Gas Chromatograph Mass
3-[(Diethylamino)methyl]-5-[2-(phenylamino)phenyl]-1,3,4-
spectrometer-QP 1000 EX at 70eV and Direct inlet unit of oxadiazole-2(3H)-thione (4a): The crude product was crys-
Shimadzu GC/MS-QP5050A at 70eV. TLC were carried out tallized from ethanol. Yield 55%, mp 132–135°C. 1H-NMR
using Art.DC-Plastikfolien, Kieselgel 60 F254 sheets (Merck, (CDCl3) δ: 1.47 (6H, t, 2×CH2CH3), 3.10 (4H, q, 2×CH2CH3),
Darmstadt, Germany), the developing solvents were benzene– 5.20 (2H, s, CH2), 6.81 (2H, d, J=7.8Hz, H-2′,6′ Ar), 6.86 (1H,
methanol (4:1) and the spots were visualized at 366, 254nm d, J=6.9Hz, H-3 Ar), 7.08 (2H, t, H-5,4′ Ar), 7.22–7.42 (3H,
by UV Vilber Lourmat 77202 (Vilber, Marne La Vallee, m, H-4,3′,5′ Ar), 7.90 (1H, d, J=6.3Hz, H-6 Ar), 8.98 (1H,
France).
s, NH exch. D2O). 13C-NMR (CDCl3) δ: 11.16 (2×CH2CH3),
General Procedure for Synthesis of 2-[2-(Phenylamino)- 42.33 (2×CH2CH3), 66.00 (NCH2), 108.28 (C-4′ NHC6H5),
phenyl]-5-(un)substituted Phenyl-1,3,4-oxadiazole (2a–c) 113.53 (C-5), 117.95 (C-3), 121.35 (C-2′,6′ NHC6H5), 122.93
A mixture of acid hydrazides 1 (0.23g, 1mmol), 4-(un)- (C-6), 127.88 (C-4), 129.22 (C-3′,5′ NHC6H5), 131.05 (C-1),
substituted benzoic acids (1mmol) and phosphorus oxy- 140.87 (C-1′ NHC6H5), 142.86 (C-2), 163.00 (C-5 oxadiazole),
chloride (2.5mmol) was refluxed at 100–110°C for 6h. The 176.54 (C=S). IR (KBr) cm−1: 3296 (NH), 3035 (CH Ar),
excess solvent was distilled off under reduced pressure and 2974, 2935, 2858 (CH aleph), 1640, 1610, 1593, 1550 (C=N,
the residue was quenched with ice cold water. The solid sepa- NH, C=C), 1280 (C=S). MS m/z: 354 (M+). Anal. Calcd for
rated was filtered, washed and dried to afford oxadiazoles in C19H22N4OS (354.47): C, 64.38; H, 6.26; N, 15.81. Found: C,
70–75% yield.
63.96; H, 6.38; N, 16.17.
2-[2-(Phenylamino)phenyl]-5-phenyl-1,3,4-oxadiazole (2a):
5-[2-(Phenylamino)phenyl]-3-[(piperidin-1-yl)methyl]-1,3,4-
The crude product was crystallized from ethanol. Yield 70%, oxadiazole-2(3H)-thione (4b): The crude product was crys-
1
1
mp 192–194°C. H-NMR (DMSO-d6) δ: 7.11 (2H, d, J=8.4Hz, tallized from ethanol. Yield 58%, mp 125–128°C. H-NMR
H-2′,6′ Ar), 7.19 (1H, d, J=6.3Hz, H-3 Ar), 7.28 (2H, t, (CDCl3) δ: 1.60–1.73 (2H, m, C-4 piperidine H), 1.83–1.93
H-5,4′ Ar), 7.37–7.60 (3H, m, H-4,3′,5′ Ar), 7.80–7.97 (4H, m, (4H, m, C-3,5 piperidine H), 3.21 (4H, t, C-2,6 piperidine H),
H-6,3″,4″,5″ Ar), 8.06 (2H, d, J=8.7Hz, H-2″,6″ Ar), 8.90 (1H, 5.00 (2H, s, CH2), 6.82 (2H, d, J=6.3Hz, H-2′,6′ Ar), 6.85
s, NH exch. D2O). IR (KBr) cm−1: 3421 (NH), 3062 (CH Ar), (1H, d, J=8.1Hz, H-3 Ar), 7.12 (2H, t, H-5,4′ Ar), 7.23–7.37
1650, 1635, 1589, 1523 (C=N, NH, C=C). MS m/z: 313 (M+). (3H, m, H-4,3′,5′ Ar), 7.88 (1H, d, J=7.5Hz, H-6 Ar), 8.73
Anal. Calcd for C20H15N3O (313.35): C, 76.66; H, 4.82; N, (1H, s, NH exch. D2O). IR (KBr) cm−1: 3344 (NH), 3010 (CH
13.41. Found: C, 76.83; H, 4.97; N, 13.47.
Ar), 2951, 2924, 2843 (CH aleph), 1662, 1614, 1599 (C=N,
5-(4-Chlorophenyl)-2-[2-(phenylamino)phenyl]-1,3,4- NH, C=C), 1280 (C=S). MS m/z: 366 (M+). Anal. Calcd for
oxadiazole (2b): The crude product was crystallized from C20H22N4OS (366.48): C, 65.55; H, 6.05; N, 15.29. Found: C,
1
ethanol. Yield 73%, mp 136–138°C. H-NMR (DMSO-d6) δ: 65.93; H, 6.12; N, 15.00.
6.98 (3H, d, J=8.4Hz, H-3,2′,6′ Ar), 7.15–7.29 (2H, m, H-5,4′
3-[(4-Methylpiperazin-1-yl)methyl]-5-[2-(phenylamino)-
Ar), 7.39–7.45 (3H, m, H-4,3′,5′ Ar), 7.56 (2H, d, J=8.1Hz, phenyl]-1,3,4-oxadiazole-2(3H)-thione (4c): The crude prod-
H-3″,5″ Ar), 7.93 (1H, d, J=8.4Hz, H-6 Ar), 8.19 (2H, d, uct was crystallized from ethanol. Yield 55%, mp 77–78°C.
J=8.4Hz, H-2″,6″ Ar), 8.30 (1H, s, NH exch. D2O). IR (KBr) 1H-NMR (CDCl3) δ: 2.42 (3H, s, CH3), 2.76–2.86 (4H, m,
cm−1: 3290 (NH), 3059 (CH Ar), 1654, 1635, 1593, 1519 (C=N, piperazine H), 3.26–3.40 (4H, m, piperazine H), 5.37 (2H,
NH, C=C). MS m/z: 347 (M+) and 349 (M++2). Anal. Calcd s, CH2), 6.84 (2H, d, J=6.3Hz, H-2′,6′ Ar), 6.89 (1H, d,
for C20H14ClN3O (347.80): C, 69.07; H, 4.06; N, 12.08. Found: J=8.1Hz, H-3 Ar), 7.09–7.18 (2H, m, H-5,4′ Ar), 7.23–7.38
C, 69.29; H, 4.13; N, 12.42.
(3H, m, H-4,3′,5′ Ar), 7.87 (1H, d, J=8.1Hz, H-6 Ar), 8.51
5-(4-Methoxyphenyl)-2-[2-(phenylamino)phenyl]-1,3,4- (1H, s, NH exch. D2O). IR (KBr) cm−1: 3290 (NH), 3035 (CH
oxadiazole (2c): The crude product was crystallized from Ar), 2950, 2924, 2854 (CH aleph), 1654, 1610, 1593 (C=N,
1
ethanol. Yield 75%, mp 171–174°C. H-NMR (DMSO-d6) δ: NH, C=C), 1280 (C=S). MS m/z: 381 (M+). Anal. Calcd for
3.82 (3H, s, OCH3), 6.68 (3H, d, J=8.7Hz, H-3,2′,6′ Ar), 6.88 C20H23N5OS (381.49): C, 62.97; H, 6.08; N, 18.36. Found: C,
(2H, t, H-5,4′ Ar), 7.04–7.41 (3H, m, H-4,3′,5′ Ar), 7.53 (2H, 63.18; H, 6.19; N, 18.27.
d, J=8.7Hz, H-3″,5″ Ar), 7.93 (1H, d, J=8.7Hz, H-6 Ar), 8.21
3-[(Morpholin4-yl)methyl]-5-[2-(phenylamino)phenyl]-1,3,4-
(2H, d, J=8.7Hz, H-2″,6″ Ar), 8.70 (1H, s, NH exch. D2O). oxadiazole-2(3H)-thione (4d): The crude product was crys-
1
IR (KBr) cm−1: 3405 (NH), 3050 (CH Ar), 2920, 2850 (CH tallized from ethanol. Yield 60%, mp 123–126°C. H-NMR
aleph), 1604, 1508 (C=N, NH, C=C). MS m/z: 343 (M+). Anal. (DMSO-d6) δ: 3.61–3.78 (4H, m, morpholine H), 4.26–4.46
Calcd for C21H17N3O2 (343.38): C, 73.45; H, 4.99; N, 12.24. (4H, m, morpholine H), 5.16 (2H, s, CH2), 6.93 (2H, d,
Found: C, 73.59; H, 5.08; N, 12.60.
J=8.4Hz, H-2′,6′ Ar), 6.97 (1H, d, J=6.6Hz, H-3 Ar),
General Procedure for Synthesis of 5-[2-(Phenylamino)- 7.04–7.23 (2H, m, H-5,4′ Ar), 7.32–7.48 (3H, m, H-4,3′,5′
phenyl]-3-[(substituted
amino)methyl]-1,3,4-oxadiazole- Ar), 7.73 (1H, d, J=6.6Hz, H-6 Ar), 8.22 (1H, s, NH exch.
2(3H)-thione (4a–m) A mixture of 5-substituted-1,3,4-oxa- D2O). IR (KBr) cm−1: 3344 (NH), 3066 (CH Ar), 2978, 2924,
diazole-2-thiol 3 (2.69g, 10mmol), paraformaldehyde (0.3g, 2870 (CH aleph), 1662, 1600, 1593, 1570 (C=N, NH, C=C),
10mmol), and primary/secondary amine (10mmol) was sus- 1296 (C=S). MS m/z: 368 (M+). Anal. Calcd for C19H20N4O2S
pended in absolute ethanol (50mL) and refluxed for 8h. After (368.45): C, 61.94; H, 5.47; N, 15.21. Found: C, 61.80; H, 5.30;

156
Vol. 61, No. 2
N, 14.90.
C21H17ClN4OS (408.90): C, 61.68; H, 4.19; N, 13.70. Found: C,
3-(Phenylaminomethyl)-5-[2-(phenylamino)phenyl]-1,3,4- 61.80; H, 4.30; N, 13.79.
oxadiazole-2(3H)-thione (4e): The crude product was crys-
3-[(4-Bromophenyl)aminomethyl]-5-[2-(phenylamino)-
tallized from ethanol. Yield 60%, mp 107–108°C. 1H-NMR phenyl]-1,3,4-oxadiazole-2(3H)-thione (4j): The crude product
(CDCl3) δ: 5.13 (2H, s, CH2), 6.54 (1H, s, NH exch. D2O), was crystallized from ethanol. Yield 60%, mp 117–118°C.
6.78–6.83 (5H, m, H-3,2′,6′,2″,6″ Ar), 7.08 (3H, t, H-5,4′,4″ Ar), 1H-NMR (CDCl3) δ: 5.10 (2H, s, CH2), 5.73 (1H, s, NH
7.17–7.33 (5H, m, H-4,3′,5′,3″,5″ Ar), 7.80 (1H, d, J=6.3Hz, exch. D2O), 6.80 (2H, d, J=9.3Hz, H-2″,6″ Ar), 7.03 (3H,
H-6 Ar), 8.18 (1H, s, NH exch. D2O). IR (KBr) cm−1: 3425, d, J=7.8Hz, H-3,2′,6′ Ar), 7.12 (2H, t, H-5,4′ Ar), 7.17–7.42
3344 (2 NH), 3032 (CH Ar), 2924, 2854 (CH aleph), 1650, (3H, m, H-4,3′,5′ Ar), 7.76 (2H, d, J=8.4Hz, H-3″,5″ Ar), 7.83
1610, 1597, 1573 (C=N, NH, C=C), 1280 (C=S). MS m/z: 374 (1H, d, J=8.4Hz, H-6 Ar), 8.00 (1H, s, NH exch. D2O). IR
(M+). Anal. Calcd for C21H18N4OS (374.46): C, 67.36; H, 4.85; (KBr) cm−1: 3421, 3346 (2 NH), 3040 (CH Ar), 2970, 2924
N, 14.96. Found: C, 67.20; H, 4.90; N, 14.80.
(CH aleph), 1654, 1597, 1573 (C=N, NH, C=C), 1276 (C=S).
3-[(4-Methylphenyl)aminomethyl]-5-[2-(phenylamino)- MS m/z: 453 (M+). Anal. Calcd for C21H17BrN4OS (453.35): C,
phenyl]-1,3,4-oxadiazole-2(3H)-thione (4f): The crude prod- 55.64; H, 3.78; N, 12.36. Found: C, 55.69; H, 3.85; N, 12.18.
uct was crystallized from ethanol. Yield 65%, mp 98–99°C.
3-[(4-Methoxyphenyl)aminomethyl]-5-[2-(phenylamino)-
1H-NMR (CDCl3) δ: 2.37 (3H, s, CH3), 5.10 (2H, s, CH2), phenyl]-1,3,4-oxadiazole-2(3H)-thione (4k): The crude product
6.83 (2H, d, J=7.8Hz, H-2″,6″ Ar), 6.94 (2H, d, J=7.8Hz, was crystallized from ethanol. Yield 67%, mp 168–170°C.
H-2′,6′ Ar), 7.02 (1H, d, J=7.8Hz, H-3 Ar), 7.21–7.52 (5H, 1H-NMR (CDCl3) δ: 3.83 (3H, s, OCH3), 5.69 (2H, s, CH2),
m, H-4,5,3′,4′,5′ Ar), 7.80 (2H, d, J=7.8Hz, H-3″,5″ Ar), 8.12 5.76 (2H, s, 2×NH), 6.95 (2H, d, J=6.3Hz, H-2″,6″ Ar), 6.99
(1H, d, J=7.8Hz, H-6 Ar), 8.40 (2H, s, 2×NH exch. D2O). IR (3H, d, J=8.7Hz, H-3,2′,6′ Ar), 7.21–7.43 (5H, m, H-4,5,3′,4′,5′
(KBr) cm−1: 3348 (2 NH), 3032 (CH Ar), 2951, 2924, 2862 Ar), 7.52 (2H, d, J=7.8Hz, H-3″,5″ Ar), 8.12 (1H, d, J=7.8Hz,
(CH aleph), 1670, 1610, 1597, 1573 (C=N, NH, C=C), 1276 H-6 Ar). 13C-NMR (CDCl3) δ: 55.47 (OCH3), 67.12 (NCH2),
(C=S). MS m/z: 387 (M+−1). Anal. Calcd for C22H20N4OS 114.52 (C-2″,6″ C6H4OCH3), 118.75 (C-3″,5″ C6H4OCH3),
(388.49): C, 68.02; H, 5.19; N, 14.42. Found: C, 68.41; H, 5.26; 122.06 (C-4′ NHC6H5), 125.60 (C-5), 126.49 (C-2′,6′
N, 14.56.
NHC6H5), 127.24 (C-3), 128.18 (C-6), 129.19 (C-4), 129.35
3-[(4-Acetylphenyl)aminomethyl]-5-[2-(phenylamino)- (C-3′,5′ NHC6H5), 132.11 (C-1), 133.73 (C-1′ NHC6H5), 145.58
phenyl]-1,3,4-oxadiazole-2(3H)-thione (4g): The crude product (C-1″ C6H4OCH3), 145.84 (C-2), 158.87 (C-4″ C6H4OCH3),
was crystallized from ethanol. Yield 63%, mp 189–190°C. 167.61 (C-5 oxadiazole), 176.43 (C=S). IR (KBr) cm−1: 3444,
1H-NMR (CDCl3) δ: 2.37 (3H, s, CH3), 5.59 (2H, s, CH2), 6.72 3421 (2 NH), 3020 (CH Ar), 2920, 2850 (CH aleph), 1678,
(2H, d, J=7.8Hz, H-2′,6′ Ar), 6.94 (1H, d, J=8.4Hz, H-3 Ar), 1593, 1570 (C=N, NH, C=C), 1288 (C=S). MS m/z: 404 (M+).
6.97 (2H, d, J=9Hz, H-2″,6″ Ar), 7.10 (2H, t, H-5,4′ Ar), 7.25 Anal. Calcd for C22H20N4O2S (404.48): C, 65.33; H, 4.98; N,
(2H, d, J=7.8Hz, H-3″,5″ Ar), 7.32–7.37 (3H, m, H-4,3′,5′ Ar), 13.85. Found: C, 65.81; H, 4.87; N, 14.08.
7.72 (1H, d, J=9Hz, H-6 Ar), 7.89 (1H, s, NH exch. D2O), 8.21
4-({5-[2-(Phenylamino)phenyl]-2-thioxo-1,3,4-oxadiazol-
(1H, s, NH exch. D2O). IR (KBr) cm−1: 3444, 3336 (2 NH), 3(2H)-yl}methylamino)benzenesulfonamide (4l): The crude
3055 (CH Ar), 2924, 2850 (CH aleph), 1740 (C=O), 1654, product was crystallized from ethanol. Yield 70%, mp
1
1610, 1593, 1570 (C=N, NH, C=C), 1265 (C=S). MS m/z: 416 220–221°C. H-NMR (DMSO-d6) δ: 5.56 (2H, s, CH2), 5.58
(M+). Anal. Calcd for C23H20N4O2S (416.50): C, 66.33; H, 4.84; (1H, s, NH exch. D2O), 6.94 (2H, d, J=7.8Hz, H-2′,6′ Ar),
N, 13.45. Found: C, 66.54; H, 4.92; N, 13.68.
7.00 (1H, d, J=6.9Hz, H-3 Ar), 7.14 (2H, d, J=8.1Hz, H-2″,6″
3-[(4-Fluorophenyl)aminomethyl]-5-[2-(phenylamino)- Ar), 7.23–7.28 (2H, m, H-5,4′ Ar), 7.33–7.42 (3H, m, H-4,3′,5′
phenyl]-1,3,4-oxadiazole-2(3H)-thione (4h): The crude product Ar), 7.57 (2H, d, J=8.7Hz, H-3″,5″ Ar), 7.72 (1H, d, J=6.6Hz,
was crystallized from ethanol. Yield 60%, mp 101–104°C. H-6 Ar), 7.80 (1H, t, NH exch. D2O), 8.25 (2H, s, NH2 exch.
1H-NMR (CDCl3) δ: 4.46 (2H, s, CH2), 6.84–6.87 (5H, m, D2O). IR (KBr) cm−1: 3363, 3344, 3298, 3232 (NH2, 2 NH),
H-3,2′,6′,2″,6″ Ar), 7.03 (2H, t, H-5,4′ Ar), 7.12–7.39 (3H, m, 3035 (CH Ar), 2924, 2850 (CH aleph), 1660, 1620, 1600, 1573
H-4,3′,5′ Ar), 7.85 (2H, d, J=8.4Hz, H-3″,5″ Ar), 8.07 (1H, d, (C=N, NH, C=C), 1315, 1149 (SO2), 1265 (C=S). MS m/z:
J=6.3Hz, H-6 Ar), 8.20 (1H, s, NH exch. D2O), 9.06 (1H, s, 454 (M++1). Anal. Calcd for C21H19N5O3S2 (453.54): C, 55.61;
NH exch. D2O). IR (KBr) cm−1: 3421, 3344 (2 NH), 3047 (CH H, 4.22; N, 15.44. Found: C, 55.92; H, 4.38; N, 15.64.
Ar), 2924, 2854 (CH aleph), 1662, 1610, 1593, 1570 (C=N,
3-[(Benzo[d]thiazol-2-yl)aminomethyl]-5-[2-(phenylamino)-
NH, C=C), 1292 (C=S). MS m/z: 392 (M+). Anal. Calcd for phenyl]-1,3,4-oxadiazole-2(3H)-thione (4m): The crude prod-
C21H17FN4OS (392.45): C, 64.27; H, 4.37; N, 14.28. Found: C, uct was crystallized from ethanol. Yield 70%, mp 226–228°C.
64.10; H, 4.40; N, 14.00.
1H-NMR (DMSO-d6) δ: 5.76 (2H, s, CH2), 6.92 (2H, d,
3-[(4-Chlorophenyl)aminomethyl]-5-[2-(phenylamino)- J=7.8Hz, H-2′,6′ Ar), 7.03 (1H, d, J=7.8Hz, H-3 Ar),
phenyl]-1,3,4-oxadiazole-2(3H)-thione (4i): The crude product 7.07–7.22 (2H, m, H-5,4′ Ar), 7.24–7.33 (3H, m, H-4,3′,5′
was crystallized from ethanol. Yield 62%, mp 110–112°C. Ar), 7.37–7.41 (2H, m, H-5″,6″ Ar), 7.69 (1H, d, J=7.8Hz, H-6
1H-NMR (DMSO-d6) δ: 5.36 (2H, s, CH2), 6.89 (2H, d, Ar), 7.74 (2H, d, J=7.8Hz, H-4″,7″ Ar), 8.30 (1H, s, NH exch.
J=7.8Hz, H-2″,6″ Ar), 6.94 (2H, d, J=7.8Hz, H-2′,6′ Ar), 7.08 D2O), 9.18 (1H, s, NH exch. D2O). IR (KBr) cm−1: 3444, 3336,
(1H, d, J=9Hz, H-3 Ar), 7.26 (2H, t, H-5,4′ Ar), 7.33–7.53 (2 NH), 3051 (CH Ar), 2924, 2854 (CH aleph), 1650, 1610,
(3H, m, H-4,3′,5′ Ar), 7.71 (2H, d, J=6.3Hz, H-3″,5″ Ar), 7.91 1597, 1570 (C=N, NH, C=C), 1280 (C=S). MS m/z: 431 (M+).
(1H, d, J=6.3Hz, H-6 Ar), 8.22 (1H, s, NH exch. D2O), 9.94 Anal. Calcd for C22H17N5OS2 (431.53): C, 61.23; H, 3.97; N,
(1H, s, NH exch. D2O). IR (KBr) cm−1: 3344, 3275 (2 NH), 16.23. Found: C, 61.71; H, 4.12; N, 16.42.
3035 (CH Ar), 2951, 2924 (CH aleph), 1656, 1597, 1573 (C=N,
General Procedure for Synthesis of 5-[2-(Phenylamino)-
NH, C=C), 1276 (C=S). MS m/z: 408 (M+). Anal. Calcd for phenyl]-2-substituted Thio-1,3,4-oxadiazole (5a,b) 5-Sub-

February 2013
157
stituted-1,3,4-oxadiazole-2-thiol
3 (2.69g, 10mmol), ethyl Emax model.
iodide or benzyl chloride (10mmol) and anhydrous K2CO3
(2.8g, 20mmol) in dry acetone (30mL) were refluxed with
stirring for 14h. After completion of the reaction, the reaction
mixture was cooled and poured onto ice-water. The separated
[D]m
Kdm +[D]m




% Cell viability=(100 − R)× 1−
+R






solid was filtered, washed with water and dried to afford the Where R is the residual unaffected fraction (the resistance
title compounds in 65–70% yield. fraction), [D] is the drug concentration used, Kd is the drug
2-Ethylthio-5-[2-(phenylamino)phenyl]-1,3,4-oxadiazole concentration that produces a 50% reduction of the maxi-
(5a): The crude product was crystallized from ethanol. Yield mum inhibition rate and m is a Hill-type coefficient. IC50 was
1
65%, mp 89–92°C. H-NMR (CDCl3) δ: 1.54 (3H, t, CH2CH3), defined as the drug concentration required to reduce fluores-
3.33 (2H, q, CH2CH3), 6.81 (2H, d, J=6.9Hz, H-2′,6′ Ar), 6.86 cence to 50% of that of the control (i.e., Kd=IC50 when R=0
(1H, d, J=6.6Hz, H-3 Ar), 7.09 (2H, t, H-5,4′ Ar), 7.13–7.39 and Emax=100−R).41)
(3H, m, H-4,3′,5′ Ar), 7.81 (1H, d, J=8.4Hz, H-6 Ar), 9.23
QSAR. Computational Method All the computational
(1H, s, NH exch. D2O). IR (KBr) cm−1: 3290 (NH), 3035 (CH works were performed on Molecular Operating Environment
Ar), 2962, 2924, 2850 (CH aleph), 1610, 1597, 1577, 1550 (C= software (MOE version 2008.10.2).42) The structures of 18
N, NH, C=C). MS m/z: 297 (M+). Anal. Calcd for C16H15N3OS compounds used as training set were sketched using molecular
(297.37): C, 64.62; H, 5.08; N, 14.13. Found: C, 64.79; H, 5.18; builder of MOE and each structure was subjected to energy
N, 13.97.
minimization up to 0.01kcal/molÅ using the MMFF94x force
2-Benzylthio-5-[2-(phenylamino)phenyl]-1,3,4-oxadiazole field. Optimization methods were used followed by conforma-
(5b): The crude product was crystallized from ethanol. Yield tional search of each energy-minimized structure. The most
1
70%, mp 113–115°C. H-NMR (CDCl3) δ: 4.54 (2H, s, CH2), stable conformer of each structure was selected and saved
6.83 (2H, d, J=7.8Hz, H-2′,6′ Ar), 6.86 (1H, d, J=6.6Hz, H-3 into database to generate the common descriptors. QuaSAR
Ar), 7.15 (2H, t, H-5,4′ Ar), 7.27–7.38 (3H, m, H-4,3′,5′ Ar), descriptor module of MOE was used to calculate descriptors
7.40–7.51 (5H, m, H-2″,3″,4″,5″,6″ Ar), 7.78 (1H, d, J=8.7Hz, for each molecule. The probability density functions used are
H-6 Ar), 9.21 (1H, s, NH exch. D2O). 13C-NMR (CDCl3) δ: Gaussian. The root mean square deviation (RMSD) tolerance
36.73 (SCH2), 106.90 (C-4′ NHC6H5), 113.85 (C-5), 117.72 was set to 0.5Å. Regression analysis was performed using
(C-3), 122.44 (C-2′,6′ NHC6H5), 123.61 (C-1), 128.04 (C-4″ HT29 IC50 as dependent factor and the calculated descriptors
CH2C6H5), 128.13 (C-6), 128.75 (C-2″,6″ CH2C6H5), 129.01 as predictable variables.
(C-3″,5″ CH2C6H5), 129.29 (C-3′,5′ NHC6H5), 132.26 (C-4),
In this study, the pool of descriptors was optimized using
135.48 (C-1′ NHC6H5), 140.52 (C-1″ CH2C6H5), 144.13 (C-2), principal components analysis (PCA). The optimization start-
162.00 (C-5 oxadiazole), 165.46 (C-2 oxadiazole). IR (KBr) ed with the reduction in the number of molecular descriptors
cm−1: 3302 (NH), 3039 (CH Ar), 2920, 2840 (CH aleph), 1640, by the determination of the highly inter-correlated descriptor
1600, 1581, 1546 (C=N, NH, C=C). MS m/z: 359 (M+). Anal. pairs and only one from each pair was selected; then the de-
Calcd for C21H17N3OS (359.44): C, 70.17; H, 4.77; N, 11.69. scriptors with insignificant variance through the data set were
Found: C, 70.30; H, 4.60; N, 11.78.
also rejected. QSAR model was then constructed after ensur-
Antitumor Screening All newly synthesized compounds ing reasonable correlation of antiproliferative activity with the
were tested against the tumor cell line HT29 (Colon adeno- individual descriptors and minimum inter-correlation among
carcinoma cell line) and MCF7 (Breast cancer cell line) at the descriptors used in the derived model. The quality of the
Department of Pharmacology and Toxicology, Faculty of model was assessed using the statistical parameter r2 and q2.
Pharmacy, Ain Shams University using the Sulfo-Rhodamine
Molecular Descriptors mr; molar refractivity (Physical
Properties): including implicit hydrogens and calculated by
B stain (SRB) assay by the method of Skehan et al.40)
Procedure Cells were plated in 96-multiwell plate using the Lorentz–Lorenz equation,46) where n is the refrac-
(104 cells/well) for 24h before treatment with the compounds tive index, MW is the molecular weight, and d is the density
to allow attachment of cell to the wall of the plate. Test com- of the substance.
pounds were dissolved in dimethylsulfoxide (DMSO) and
diluted to the appropriate volume. Different concentrations of
the compound under test (0.001, 0.01, 0.1, 1, 10, 100µm) were
n2 1×MW
mr =
n2 +2×d
added to the cell monolayer. Triplicate wells were prepared for mr is primarily a measure of the bulk and polarizability of
each individual dose. Monolayer cells were incubated with the substituent with no dependence on conformation.
compounds for 48h at 37°C and in atmosphere of 5% CO2.
After 48h, cells were fixed, washed and stained for 30min tance Matrix Descriptors): half the sum of all the distance
weinerPol; Wiener polarity number (Adjacency and Dis-
with 0.4% (w/v) SRB dissolved in 1% acetic acid. Excess stain matrix entries).
was washed with acetic acid and attached stain was recovered
vsurf_S; Interaction field surface area (Surface Area, Vol-
with Tris ethylenediaminetetraacetic acid (Tris EDTA) buffer. ume and Shape Descriptors): depend on the structure connec-
Color intensity was measured in an enzyme-linked immu- tivity and conformation (dimensions are measured in Å). The
nosorbent assay (ELISA) reader. The relation between the vsurf_ descriptors are similar to the VolSurf descriptors and
surviving fraction and drug concentration was plotted to get have been shown to be useful in pharmacokinetic property
the survival curve of each tumor cell line after the specified prediction, Table 2.
compound. IC50 and R fraction of the tested compounds were
illustrated in Table 1.
Validation and Cross-Validation of the Model Cross-
validation statistical technique has been applied to estimate
The dose response curve of compounds was analyzed using the quality with regard to predictive ability of the generated

158
Vol. 61, No. 2
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The observed activities (Obs. IC50) together with the pre-
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lated using multi-linear regression (MLR) are listed in 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.
The observed IC50 is plotted against their predicted values
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Outliers Compounds 4j, 4l, 5a were considered as outli-
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these outliers from the main data set and formulating another
QSAR can resolve the problem. Outliers may be acting by a
different mechanism.
Model Evaluation By evaluation of the model and its trial
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QSAR study.
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