Design, synthesis, modelling studies and biological evaluation of 1,3,4-oxadiazole derivatives as potent anticancer agents targeting thymidine phosphorylase enzyme

Article abstract of DOI:10.1016/j.bioorg.2021.104873

A series of novel 1,3,4-oxadiazole derivatives with substituted phenyl ring were designed and synthesized with an objective of discovering newer anti-cancer agents targeting thymidine phosphorylase enzyme (TP). The 1,3,4-oxadiazole derivatives were synthesized by simple and convenient methods in the lab. Chemical structure of the all the synthesized compounds were characterized by IR, ¹H NMR and mass spectral methods and evaluated for cytotoxicity by MTT method against two breast cancer cell lines (MCF-7 and MDA-MB-231). Further, results of TP assay identified that 1,3,4-oxadiazole molecules displayed anti-cancer activity partially by inhibition of phosphorylation of thymidine. The TP assay identified SB8 and SB9 as potential inhibitors with anti-cancer activity against both the cell lines. The molecular docking studies recognized the orientation and binding interaction of molecule at the active site amino acid residues of TP (PDB: 1UOU). Acute toxicity studies of compound SB8 at the dose of 5000 mg/kg has identified no signs of clinical toxicity was observed. The SARs study of synthesized derivatives revealed that the substitution of phenyl ring with electron withdrawing group at ortho position showed significant TP inhibitory activity compared to para substitution. The experimental data suggests that 1,3,4-oxadiazole with substituted phenyl can be taken as a lead for the design of efficient TP inhibitors and active compounds which can be taken up for further studies.

Full text of DOI:10.1016/j.bioorg.2021.104873

Bioorganic Chemistry 111 (2021) 104873
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis, modelling studies and biological evaluation of
1,3,4-oxadiazole derivatives as potent anticancer agents targeting
thymidine phosphorylase enzyme
Shalini Bajaj1, Maushmi S. Kumar1, Hussain Tinwala, Mayur YC*
Shobhaben Pratapbhai Patel School of Pharmacy Technology Management, SVKM’s NMIMS, Mumbai, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of novel 1,3,4-oxadiazole derivatives with substituted phenyl ring were designed and synthesized with
an objective of discovering newer anti-cancer agents targeting thymidine phosphorylase enzyme (TP). The 1,3,4-
oxadiazole derivatives were synthesized by simple and convenient methods in the lab. Chemical structure of the
all the synthesized compounds were characterized by IR, 1H NMR and mass spectral methods and evaluated for
cytotoxicity by MTT method against two breast cancer cell lines (MCF-7 and MDA-MB-231). Further, results of
TP assay identified that 1,3,4-oxadiazole molecules displayed anti-cancer activity partially by inhibition of
phosphorylation of thymidine. The TP assay identified SB8 and SB9 as potential inhibitors with anti-cancer
activity against both the cell lines. The molecular docking studies recognized the orientation and binding
interaction of molecule at the active site amino acid residues of TP (PDB: 1UOU). Acute toxicity studies of
compound SB8 at the dose of 5000 mg/kg has identified no signs of clinical toxicity was observed. The SARs
study of synthesized derivatives revealed that the substitution of phenyl ring with electron withdrawing group at
ortho position showed significant TP inhibitory activity compared to para substitution. The experimental data
suggests that 1,3,4-oxadiazole with substituted phenyl can be taken as a lead for the design of efficient TP in-
hibitors and active compounds which can be taken up for further studies.
1,3,4-oxadiazole
Anti-cancer
Thymidine phosphorylase
Breast cancer
Docking
1. Introduction
heterocyclic compounds have proved to be a boon for the development
of newer chemotherapeutic molecules.
Cancer still remains the second leading cause of mortality all over the
globe. With increasing number of cancer incidences in developed as well
developing countries, cancer is one of the major health research area
being explored by researchers all over the globe. According to the
American Cancer Society in 2019, around 1,762,450 new cancer cases
and 606,880 cancer deaths were projected to occur in the United States
alone [1]. At present, chemotherapy is one of the most important
treatment available for treatment of cancer. The ultimate goal of
chemotherapy is to kill the cancerous cells while safeguarding the sur-
rounding healthy cells. The success of chemotherapy is hindered by
obstacles such as toxicity of chemotherapeutics on the healthy cells,
higher dose regime and developing resistance over existing chemo-
therapeutic drugs. The only feasible solution to all the above challenges
is developing newer chemotherapeutic molecules with toxicity specific
to cancerous cell at a lower dose. In this endeavour, nitrogen containing
In recent years, studies pertaining to structural activity relationships
(SARs) with target structures along with the underlying mechanisms
involving 1,3,4-oxadiazole moiety have carved their niche owing to the
versatility of the core ring which enables various substitutions at the
carbon and nitrogen atoms of the ring thereby generating various mono-
and di- substituted 1,3,4-oxadiazole derivatives. These derivatives have
exhibited a wide array of pharmacological activities including antibiotic
[2] anti-malarial [3,4], anti-viral [5,6], analgesic [7] anti-inflammatory
[8] vasodilation [9] anti-cancer [10–12] and many others [13–15]. In
the last decade, 1,3,4-oxadiazole has been used as a motif to develop
newer chemotherapeutic molecules for improved and efficient chemo-
therapeutic application. Over recent years, newer hallmarks have been
explored in search for a potential molecular target for treating cancer
[16].
The 1,3,4-oxadiazole ring containing compound showed potent anti-
* Corresponding author at: Department of Pharmaceutical Chemistry, SPPSPTM, SVKM’s NMIMS University, Vile Parle (West), Mumbai 400056, India.
E-mail address: mayuryc@hotmail.com (M. YC).
1
Both the authors have contributed equally.
https://doi.org/10.1016/j.bioorg.2021.104873
Received 24 April 2020; Received in revised form 24 March 2021; Accepted 25 March 2021
Available online 29 March 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

S. Bajaj et al.
Bioorganic Chemistry 111 (2021) 104873
Fig. 1. Design approach of 1,3,4-oxadiazole derivatives.
Fig. 2. Thymidine phosphorylase as a potential target for cancer treatment.
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Bioorganic Chemistry 111 (2021) 104873
Scheme 1. Synthesis of 1,3,4-oxadiazole derivatives from different acids.
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Bioorganic Chemistry 111 (2021) 104873
cancer activity. Zibotentan (ZD4054) is under clinical trial for its anti-
cancer activity that contain 1,3,4-oxadiazole ring [17] (Fig. 1). Many
researchers have reported different derivatives of 1,3,4-oxadiazole
which showed potent anti-cancer activity against breast cancer. Target
specific anti-cancer activity is one of the most encouraging strategies. In
this research work, we mainly focus on 1,3,4-oxadiazole derivatives
induced anti-cancer activity against breast cancer with inhibition of
thymidine phosphorylase (TP) enzyme [18–21]. TP is overexpressed in
many types of cancer specially in breast cancer [22,23]. The TP converts
Table 1
Physiochemical properties of 1,3,4-oxadiazole molecules.
Comp. No.
R1
R2
Mol. Formula
Mol Wt.
λmax (nm)
SB1.
4-chloro
4-chloro
4-chloro
4-chloro
4-chloro
4-chloro
2-chloro
2-chloro
2-chloro
2-chloro
2-chloro
2-floro
styryl
4-chloro
2-chloro
2-floro
C14H8Cl2N2O
291
291
275
287
271
302
291
275
287
271
302
258
274
283
283
256
256
248
407.0, 262.0
407.0, 259.0
407.0, 257.0
408.0, 263.0
409.0, 262.0
406.0, 259.0
407.0, 258.0
408.0, 264.0
408.0, 262.0
407.0, 258.0
407.0, 261.0
407.0, 259.0
406.0, 257.0
407.0, 259.0
407.0, 261.0
408.0, 262.0
406.0, 261.0
407.0, 262.0
thymidine into thymine and 2-deoxy-α-D-ribose-1-phosphate (dRib-1-P)
SB2.
C
C
14H8Cl2N2O
14H8ClFN2O
SB3.
[24]. This metabolite of TP is involved in promoting tumor growth by
enhancing angiogenesis induced metastasis and resistance to apoptosis
which establishes TP as a potential target for cancer treatment (Fig. 2).
Due to this, inhibition of TP activity may assist as probable therapeutic
approach for preventing the growth of tumor cells and give insights in
development of potent derivatives against cancer.
SB4.
2-methoxy
4-amino
4-nitro
C15H11ClN2O2
SB5.
C14H10ClN3O
SB6.
C14H8ClN3O3
SB7.
2-chloro
2-floro
C
C
14H8Cl2N2O
14H8ClN2FO
SB8.
SB9.
2-methoxy
4-amino
4-nitro
C15H11ClN2O2
SB10.
SB11.
SB12.
SB13.
SB14.
SB15.
SB16.
SB17.
SB18.
C
14H10ClN3O
C14H8ClN3O3
C14H8F2N2O
In view of the significant studies on anti-cancer activity of 1,3,4-oxa-
diazole derivatives, and in continuation of our previous work on its TP
inhibitory activity we aim for newer 1,3,4-oxadiazole heterocyclic
pharmacophores targeting cancer via inhibition of TP activity [25].
In this research work, we have attempted to synthesize a novel series
of 1,3,4-oxadiazole derivatives, characterized and tested them for in
vitro activity in human MCF-7 and MDA-MB-231 breast cancer cell lines
for inhibition of TP activity. This study also highlights the molecular
docking on TP receptor and active compounds were evaluated for in vivo
acute toxicity studies and were correlated with predicated LD50 values
[26,27]. We also compared the 1,3,4-oxadiazole structural modifica-
tions and have proven to give more cytotoxic drugs with TP inhibitory
activity.
2-floro
styryl
C18H14N2O
styryl
4-chloro
2-chloro
4-chloro
2-chloro
styryl
C
C
16H11ClN2O
16H11ClN2O
styryl
H
C14H8ClN2O
H
C
C
14H8ClN2O
16H12N2O
H
2.2. Biological activity
2.2.1. Cell proliferation assay (MTT)
All the synthesized derivatives of 1,3,4-oxadiazole (SB1-SB18) were
screened for in vitro cytotoxicity studies against human breast cancer cell
lines MCF-7, MDA-MB-231 and VERO cell lines using MTT assay
(Table 2). The cytotoxicity of all the synthesized derivatives against
MCF-7 and MDA-MB-231 cell lines are shown in Fig. 3. The data from
Table 2 and Fig. 3, some of the compounds have showed good cyto-
toxicity against MCF-7 and MDA-MB-231 cell lines. The results of anti-
cancer activity are expressed in terms of compound concentration
required to reduce the viability of the cells by 50% with respect to the
2. Results and discussion
2.1. Chemistry
In the present study, 18 derivatives of 1,3,4-oxadiazole have been
synthesized as highlighted in Scheme 1. The intermediate (3a-3r) was
prepared by the esterification of 4-chlorobenzoic acid, 2-chlorobenzoic
acid, 2-fluorbenzoic acid, styryl and benzoic acid followed by reaction
with hydrazine hydrate. The final compounds (SB1-SB18) were pre-
pared by the reaction of 3a-3r with different benzoic acid derivatives
such as 4-chloro, 2-chloro, 2-fluro, 2-methoxy, 2-amino, 4-nitro and
styryl in presence of phosphoryl chloride (POCl3) as cyclizing agent
(Scheme 1).
control (IC50
μM). Compounds also found to be active against TP
Table 2
In vitro IC50, CC50 and SI index of synthesized compounds (IC50 is the concen-
tration at which 50% cell death occur and CC50 is the concentration at which
50% cells survive).
The depicted structures of all the synthesized derivatives were
strengthened by the different spectroscopy analysis such as IR, 1H NMR,
13C NMR and mass spectral data. The physicochemical property of all
the synthesized derivatives is depicted in Table 1.
Comp. IC50 (µM)
CC50 (µM)Vero SI of
SI of
Code
cell lines
MCF-7 MDA-MD-231
MCF-7
MDA-MB-231
SB1.
18.89 ± 0.69 19.02 ± 0.27
55.01
35.18
33.52
71.08
27.12
56.09
59.71
39.03
26.69
29.45
45.02
23.15
48.08
29.01
77.45
65.28
72.16
54.11
ND
2.90
3.52
7.44
1.1
2.89
5.98
4.36
1.45
8.86
1.35
1.16
7.99
11.75
2.15
1.36
1.89
0.77
1.44
2.57
2.96
3.74
5.07
ND
IR spectral data of synthesized compounds 3a-3r showed charac-
SB2.
9.98 ± 0.54
4.50 ± 0.2
5.98 ± 0.13
7.69 ± 1.92
49.01 ± 0.3
6.57 ± 1.49
41.40 ± 0.92
51.39 ± 0.2
4.88 ± 1.74
2.27 ± 0.73
teristic peaks at
υ
cmꢀ 1 794, 1434 and 710 due to presence of C Cl,
SB3.
–
ꢀ 1
SB4.
61.49 ± 1.2
8.86 ± 0.45
57.01 ± 1.3
67.91 ± 0.4
2.50 ± 0.35
1.85 ± 0.28
–
C
F and styryl respectively. Compound SB1 showed peak at 794 cm
SB5.
3.06
0.98
0.98
15.60
14.42
1.85
3.66
2.05
0.82
1.20
1.01
3.67
3.83
4.79
ND
–
due to C Cl stretching of chlorine group. Compound SB3 showed
SB6.
characteristic IR peaks at
υ
cmꢀ 1 1449 and 825 due to stretching of
SB7.
–
–
SB8.
aromatic C F and C Cl group. Compound SB6 showed peak at 1366
υ
cmꢀ 1 due to presence of nitro group.
SB9.
SB10.
SB11.
SB12.
SB13.
SB14.
SB15.
SB16.
SB17.
SB18.
aDOX
15.86 ± 1.27 13.68 ± 1.72
12.28 ± 0.98 32.88 ± 1.21
11.28 ± 0.38 12.24 ± 0.75
58.01 ± 0.88 62.05 ± 0.24
28.57 ± 1.29 20.07 ± 1.91
25.54 ± 1.17 30.14 ± 1.44
17.74 ± 0.68 21.98 ± 1.22
18.82 ± 0.74 19.25 ± 0.92
11.28 ± 1.02 10.67 ± 1.86
The 1H NMR spectra of compound SB13 showed signals at δ 7.2–7.8
indicating the presence of phenyl ring aromatic protons. The mass
spectrum of compound SB1 displayed a molecular ion peak at m/z 443
and base peak at m/z 230, compound SB3 showed molecular ion peak at
m/z 411 and base peak at m/z 275 and compound SB12 showed mo-
lecular ion peak at m/z 539.1 and base peak at m/z 259. The possible
fragment of base peak of compound SB1 and SB12 are 1-chloro-4-meth-
ylbenzene, 1-fluoro-2-methylbenzene respectively. Similarly, all the
synthesized derivatives showed their respective m/z value peaks ac-
cording to the molecular mass of the compounds.
0.21 ± 0.03
0.28 ± 0.04
IC50 = The required concentration of compound for inhibition of cell prolifer-
ation by 50%.
ND = Not Determined.
a
DOX = Doxorubicin, positive control compound.
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Bioorganic Chemistry 111 (2021) 104873
Fig. 3. Cytotoxicity and TP inhibitory profile of 1,3,4-oxadiazole derivatives.
the compounds is shown in Table 3. The structure activity relationship
for TP inhibitory activity revealed that substitution of 4-chloro phenyl at
2nd and 5th position of 1,3,4-oxadiazole ring have more potent activity.
Compounds SB9 and SB8 showed highest IC50 value of 24.03 ± 3.78 and
Table 3
In vitro thymidine phosphorylase (TP) inhibitory assay
(IC50) results for synthesized compounds.
Comp. Code
IC50 (µM)
25.27 ± 1.8
4.42 M.
μM compared to IC50 value of standard drug (7-DX) 38.68 ±
SB1.
52.81 ± 2.8
28.91 ± 0.98
32.71 ± 2.6
52.49 ± 1.2
25.96 ± 2.54
50.47 ± 4.2
48.01 ± 0.36
25.27 ± 1.8
24.03 ± 3.78
26.28 ± 4.5
34.49 ± 3.5
51.66 ± 1.4
52.90 ± 3.7
58.22 ± 1.4
36.48 ± 1.3
44.34 ± 1.9
49.5 ± 0.68
40.45 ± 3.3
38.68 ± 4.42
μ
SB2.
SB3.
2.2.2.1. Determination of the significance of correlation coefficient.
Moreover, to determine the significance of IC50 in both cell lines and TP
inhibitory activity for two most active compounds (SB8 and SB9),
correlations for both cell lines and TP inhibitory IC50 activity were
generated and presented in Fig. 4. Correlation between IC50 value of
both cell lines and TP inhibitory activity for compound SB8 showed
regression coefficient R2 = 0.8147 and for compound SB9 regression
coefficient R2 = 0.7642 (Fig. 4). This indicates that 1,3,4-oxadiazole
molecules with cytotoxicity also have potent TP inhibitory activity.
SB4.
SB5.
SB6.
SB7.
SB8.
SB9.
SB10.
SB11.
SB12.
SB13.
SB14.
SB15.
SB16.
SB17.
SB18.
b7-DX
2.3. Structure-activity correlation of synthesized compounds with TP
inhibitory activity
b
The anti-cancer activity of all the synthesized derivatives of 1,3,4-
oxadiazole against MCF7, MDA-MD-231 breast cancer cell lines and
TP inhibitory activity explores the following assumptions about the
structure–activity relationships (SARs) (Fig. 5):
7-DX = 7-Deazaxanthine, standard compound
enzyme. Compound SB9, SB8 and SB3 showed most potent anti-cancer
activity against MCF-7 with IC50 value of 1.85 ± 0.28 μM, 2.50 ± 0.35
μ
M and 4.50 ± 0.2
μ
M respectively. Substitution of 2-chloro group at R1
position and 2-methoxy at R2 position of 1,3,4-oxadiazole ring showed
potent anti-cancer activity. Compound SB4, SB6, SB7 and SB14 showed
better activity against MDA-MB-231 cell lines comparatively in MCF-7
cell line. Un-substituted phenyl ring attached to 1,3,4-oxadiazole ring
through styryl showed less activity against MCF-7 cancer cell line.
Compound SB9, SB8 and SB2 exhibited good activity against MDA-MB-
(i) Phenyl ring at 2nd and 5th position of 1,3,4-oxadiazole ring is
very important for anti-cancer activity.
(ii) Compound with substituted ortho chloro group at both 2nd and
5th position phenyl ring of 1,3,4-oxadiazole ring showed more
potent anti-cancer activity in MCF-7 cell line.
(iii) Compound with substituted ortho chloro group at 2nd and ortho
methoxy group at 5th position phenyl ring of 1,3,4-oxadiazole
ring highest activity in MDA-MB-231 cell line.
231 cell line with IC50 value 2.27 ± 0.73 μM, 4.88 ± 1.74 μM and 5.98 ±
0.13 μM respectively with IC50 less than 8 μM. The selectivity index (SI)
(iv) Compound with substitution of 2-chloro and 2-floro phenyl at
2nd position and 2-floro, 3-chloro and 2-methoxy group at 5th
position showed more potent anti-cancer along with TP inhibi-
tory activity.
was calculated by dividing CC50 of VERO cell line by IC50 of both MCF-7
and MDA-MB0231 cell line SI= (CG50) / (IC50).
2.2.2. Thymidine phosphorylase inhibitory assay
(v) The replacement of the 2-chlorophenyl with 2-florophenyl at R1
position showed a remarkable reduction in the inhibitory
activity.
Eighteen compounds were evaluated for TP inhibitory activity by
taking 7-deazaxanthine (7-DX) as standard. TP inhibitory activity of all
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Bioorganic Chemistry 111 (2021) 104873
Fig. 4. Correlation between IC50 of MCF-7, MDA-MB-231 cell line and TP inhibitory activity of compound SB8 and SB9.
Fig. 5. Structure-activity relationship of the newly synthesized 1,3,4-oxadiazole derivatives as TP inhibitors and anti-cancer agents.
(viii) Among the different ortho substituents the order of activity was
found to be F > Cl > OCH3.
Table 4
Effect of SB8 compound on body weight of rat.
(ix) While analyzing the derivatives with substituents at para position
of benzene ring, it was evident that the order of activity of
different substituents was Cl > NH2 > NO2.
Group
Dose (mg/kg)
Weekly Body Weight (g) (Mean ± SD)
Day 0
Day 7
Day 14
(x) Among the compounds containing 2-fluoro substituents at R1, 4-
chloro substituent at R2 position showed better activity as
compared to 2-chloro substituent at R2 position compounds.
(xi) Among the compounds containing 4-chloro substituent at R1 and
different substituents at R2 position exhibited cytotoxic potential
in following position 2F > 4NH2 > 2Cl > 2OCH3 > 4Cl > 4NO2.
A
B
C
D
Control
300
155 ± 7
170 ± 9
180 ± 10
183 ± 11
177 ± 12
193 ± 16
197 ± 18
208 ± 19
197 ± 20
213 ± 22
222 ± 12
228 ± 16
2000
5000
(vi) Styryl group attached between phenyl and 1,3,4-oxadiazole
showed minute activity against both MCF-7 and MDA-MB-231
cell lines and TP enzyme.
2.4. Acute oral toxicity
(vii) The derivative of 2-methoxy phenyl have less activity compared
to 2-chlorophenyl and 2-florophenyl derivatives.
Compound SB8 was selected for acute oral toxicity study as the
toxicity prediction tool- Protox server identified its LD50 as 5000 mg/kg.
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S. Bajaj et al.
Bioorganic Chemistry 111 (2021) 104873
Fig. 6. Effect of SB8 compound on body weight. All values are expressed as Mean ± SD.
Fig. 7. Predicted binding mode of Compound SB8.
Rats were kept for overnight fasting before the day of dosing and for 3 h
after dosing. On the first day of dosing, rats were monitored every hour
up to 12 h. for mortality, clinical signs and behavioural changes. And
thereafter, they were observed twice a day for 14 days and body weight
was recorded daily (Table 4). No clinical sign of toxicity was observed
during the period of 14 days under observation among the groups of
control and tested compound. No statistically significant reduction in
body weight in Fig. 6 were observed. Body weight changes if any are an
indicator of adverse side effects, as the animals that survive cannot lose
more than 10% of the initial body weight. There was no mortality
recorded in all the treated groups, and all the treated experimental rats
survived. Present study suggests that compound SB8 is safe for oral
administration of up to 5000 mg/kg in single dose to albino female
Wistar rats and also in correlation with predicted LD50 values by Protox
server.
2.5. Statistical analysis
Data obtained from acute oral studies was analysed by two-way
analysis of variance (ANOVA) using GraphPad Prism 8 statistical soft-
ware package to analyse the degree of variance among groups, and “p”
values less than, or equal to 0.05 (Bonferroni’s test) was considered
significant.
7

S. Bajaj et al.
Bioorganic Chemistry 111 (2021) 104873
Fig. 8. Predicted binding mode of compound SB9.
2.6. Molecular docking
HIS166, GLY145, SER144 and form hydrogen bonds through the py-
rimidine type of nitrogen of the oxadiazole ring. Of the results one of the
best scoring pose of compounds (SB8 and SB9) from the docking studies
is shown in Fig. 7 and Fig. 8 with the residues interacting with ligand
within the binding site. From the results, we found that compounds (SB3
and SB8) exhibited highest docking score (ꢀ 7.29). Most of the com-
pounds showed binding affinity to human thymidine phosphorylase to
lesser or greater extent depicted in Table 5.
Molecular docking studies paves way for the designing of 1,3,4-oxa-
¨
diazole derivatives by using the Schrodinger (Maestro 11.2) software.
The crystal structure of human thymidine phosphorylase is a complex
with a small molecule inhibitor (PDB ID: 1UOU, Resolution: 2.11 Å) was
derived from protein data bank and was used for the study after
refinement. Results suggested that compound (SB1-SB18) depicted
specific Vander Waals (vdW) interactions with surrounding hydropho-
bic residues LEU148, VAL241, VAL208, TYR199, VAL125, ILE218,
3. Conclusion
In summary, a series of newer 1,3,4-oxadiazole derivatives were
designed and synthesized in good yields by convenient method and
characterized by using different spectroscopy methods such as IR, NMR
and MASS. Evaluation of all the derivatives were done against MCF-7
and MDA-MB-231 breast cancer cell lines and all the derivatives
showed significant activity. Among them compound SB9 with 2-chloro
at R1 and 4-methoxy group at R2 position of 1,3,4-oxadiazole scaffold
Table 5
Docking study of 1,3,4-oxadiazole derivatives.
Comp. code
Docking score
Glide score
SB1.
ꢀ 7.29
ꢀ 59.466
ꢀ 59.466
ꢀ 61.224
ꢀ 63.064
ꢀ 48.244
ꢀ 57.918
ꢀ 57.198
ꢀ 55.353
ꢀ 52.92
SB2.
ꢀ 7.29
SB3.
ꢀ 6.719
ꢀ 6.599
ꢀ 6.496
ꢀ 6.32
SB4.
have shown potent cytotoxicity (IC50 = 1.85 ± 0.28
μM) against MCF-7
SB5.
cancer cell line and compound SB8 have shown potent cytotoxicity
SB6.
against MDA-MB-231 cell lines with IC50 = 2.27 ± 0.73
μM. All the
SB7.
ꢀ 6.251
ꢀ 6.111
ꢀ 6.04
synthesized compounds were screened for target specific activity against
TP enzyme, among them compounds SB8, SB9 and SB3 have shown
potent TP inhibitory activity.
SB8.
SB9.
SB10.
SB11.
SB12.
SB13.
SB14.
SB15.
SB16.
SB17.
SB18.
ꢀ 5.941
ꢀ 5.93
ꢀ 53.836
ꢀ 51.296
ꢀ 35.58
The experimental data and SAR study revealed that electron with-
drawing groups at R1 and R2 position of phenyl ring have better cyto-
toxicity and TP inhibitory activity. These studies indicate that the 1,3,4-
oxadiazole scaffold substituted with phenyl ring can be considered as a
lead molecule for designing of potent cytotoxic agents with TP inhibi-
tory activity. Additionally, molecular-docking studies revealed that
ꢀ 5.748
ꢀ 5.665
ꢀ 5.627
ꢀ 5.567
ꢀ 5.437
ꢀ 5.095
ꢀ 5.035
ꢀ 56.042
ꢀ 41.785
ꢀ 47.148
ꢀ 41.845
ꢀ 50.125
ꢀ 17.623
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S. Bajaj et al.
Bioorganic Chemistry 111 (2021) 104873
nitrogen of 1,3,4-oxadiazole ring and both of phenyl ring played an
important role in binding to the active site residues of TP. Compounds
SB8 and SB9 showed the best binding interaction with amino acid res-
idues HIS116, GLY145, TYR199 and SER144 of TP.
mixture was allowed to cool to RT and poured over crushed ice. The
crude solid product was obtained by filtration and dried under vacuum.
The crude solid product so obtained was purified either by recrystalli-
zation in ethanol or by column chromatography as suited [29].
The in vivo acute rat toxicity study of most active compound clearly
suggested that 1,3,4-oxadiazole derivatives with substituted phenyl ring
could be safe and effective heterocycle for discovering future potent
anti-cancer agents targeting thymidine phosphorylase enzyme.
4.3. Structural analysis of the compounds
4.3.1. 2,5-bis(4-chlorophenyl)-1,3,4-oxadiazole (SB1)
◦
Yield: 72%, M.P 252–255 C, Rf = 0.7 (ethyl acetate: chloroform,
8:2), FTIR (CHCl3, ʋ/(cmꢀ 1): 3249 (N H), 1640 (Ar C C), 1266
–
–
4. Experimental section
–
1
–
–
–
–
–
(C N), 1080 (C O), 2342 (Ar C H), 794 (C Cl). HNMR (500 MHz,
4.1. Materials and methods
CHCl3): δ (ppm), 7.55 (d, 4H, J = 7.54 Hz, Ar H), 8.1 (d, 4H, J = 8.09 Hz,
Ar H). 13CNMR (500 MHz, CHCl3): δ (ppm), 122.8 (ArC), 128.8 (ArC),
–
–
–
O), 129.2 (ArC). ESI Mass (m/z): 291
All the reagents and solvents used in this research work were of
analytical grade and obtained from Sigma-Aldrich, S.D. Fine chemicals
and E. Merck India Ltd. All the reaction was carried out in anhydrous
condition. Progress of the reactions was monitored with the help of thin
layer chromatography (silica gel G coated), using ethyl acetate: chlo-
roform as mobile phase. The results were visualized under UV light
cabinet. Open glass capillaries melting point (M.P) apparatus was used
for the measurement of M.P of all synthesized compounds (˚C) and all the
values were used uncorrected. Synthesized compounds were purified
either by recrystallization in ethanol or by column chromatography,
column packed with silica gel of 230–400 mesh. To further confirm the
purity of the compounds, high pressure liquid chromatography (HPLC)
and CHN analysis studies were carried out on Shimadzu. LC 2010 CHT
HPLC High-Performance Liquid Chromatography System using C18
column and water: acetonitrile as mobile phase while maintaining the
flow rate at 1.0 mL/min and 20 µl injection volume. FTIR spectra was
recorded in the range of 4000–400 cmꢀ 1 on Perkin Elmer RX1 Fourier
transform spectrophotometer using KBr pellets. NMR (1H NMR, 13C
NMR) spectra was obtained using Bruker 500 MHz system using CDCl3
as solvent. The spectra were recorded in δ (ppm) employing tetramethyl
silane (TMS) as internal standard. The 1H NMR spectrum (splitting) was
abbreviated are as follows: s (singlet), d (doublet), t (triplet), q (quartet),
q (pentet), m (multiplet). Chemical information such as molecular
weight of synthesized derivatives was determined by mass spectroscopy
(MS) using methanol as solvent. MS/MS spectra of selected fragment
were obtained for confirmation of drug structure (Supplementary In-
formation provided).
135.7 (C Cl), 164.6 (N
C
–
(M+H) +. Anal. Calc. for C14H8Cl2N2O: C, 57.76; H, 2.77; Cl, 24.36; N,
9.62; O, 5.50. Found: C, 58.21; H, 2.99; Cl, 25.13; N, 10.25; O, 4.77.
4.3.2. 2-(2-chlorophenyl)-5-(4-chlorophenyl)-1,3,4-oxadiazole (SB2)
◦
Yield: 69%. M.P 248–250 C, Rf = 0.8 (ethyl acetate: chloroform,
9:1), FTIR (CHCl3, ʋ/(cmꢀ 1): 1639 (Ar C C), 1265 (C N), 1087
–
–
–
–
(C O), 2342 (Ar C H), 794 (C Cl). 1HNMR (500 MHz, CHCl3): δ
(ppm), 7.3 (d, 1H, J = 7.34 Hz), 7.5 (d, 1H, J = 7.46 Hz), 7.6 (d, 3H, J =
7.51 Hz), 8.0 (d, 3H, J = 8.10 Hz). 13CNMR (500 MHz, CHCl3): δ (ppm),
–
–
–
–
–
126.4 (ArC), 128.8 (ArC), 133.2 (C Cl), 131.2 (C Cl), 164.6
+
–
–
C
(N
O), 129.2 (ArC), 122.8 (ArC). ESI Mass (m/z): 290.9 (M+H)
.
–
Anal. Calc. for C14H8Cl2N2O: C, 57.76; H, 2.77; Cl, 24.36; N, 9.62; O,
5.50. Found: C, 56.43; H, 3.29; Cl, 24.13; N, 9.95; O, 5.99.
4.3.3. 2-(4-chlorophenyl)-5-(2-fluorophenyl)-1,3,4-oxadiazole (SB3)
◦
Yield: 68%, M.P 263–265 C, Rf = 0.6 (ethyl acetate: chloroform,
9:1), FTIR (CHCl3, ʋ/(cmꢀ 1): 1544 (Ar C C), 1280 (C N), 1087
–
–
–
–
1
–
–
–
(C O), 2361 (Ar C H), 825 (C Cl), 1449 (C-F). HNMR (500 MHz,
CHCl3): δ (ppm), 7.3 (d, 1H, J = 7.32 Hz), 7.4 (d, 1H, J = 7.56 Hz), 7.5
(d, 1H, J = 8.13 Hz), 7.6 (d, 1H, J = 7.36 Hz), 8.2 (d, 2H, J = 7.58 Hz).
13CNMR (500 MHz, CHCl3): δ (ppm), 114.3 (ArC), 131.9 (ArC), 160.4
–
–
–
O), 127.6 (ArC), 130.7 (ArC). ESI
(C-F), 135.7 (C Cl), 164.6 (N
C
–
Mass (m/z): 275 (M+H) +. Anal. Calc. for C14H8ClFN2O: C, 61.22; H,
2.94; Cl, 12.91; F, 6.92; N, 10.20; O, 5.82. Found: C, 62.47; H, 2.11; Cl,
13.14; F, 5.99; N, 11.19; O, 5.11.
4.3.4. 2-(4-chlorophenyl)-5-(2-methoxyphenyl)-1,3,4-oxadiazole (SB4)
◦
4.2. Chemistry
Yield: 70%, M.P 274–276 C, Rf = 0.6 (ethyl acetate: chloroform,
8:2), FTIR (CHCl3, ʋ/(cmꢀ 1): 1588 (Ar C C), 1466 (C N), 1232
–
–
–
–
1
– – – –
4.2.1. General procedure for the synthesis of esters of substituted benzoic
acids (2a-2r)
(C O C), 2978 (Ar C H), 794 (C Cl). HNMR (500 MHz, CHCl3): δ
(ppm), 7.2 (d, 1H, J = 8.10 Hz), 7.3 (d, 1H, J = 7.6 Hz), 7.8 (d, 2H, J =
8.13 Hz), 7.9 (d, 2H, J = 8.13 Hz), 8.0 (s, 3H). 13CNMR (500 MHz,
To a solution of substituted aromatic benzoic acid (0.04 mol) in
anhydrous ethanol (1.2 mol), conc. sulphuric acid (2.0 mol) was added
drop wise with continuous stirring and refluxed at 70–80 ◦C for 9–10 h.
After cooling, the reaction mixture was poured on crushed ice. Ester in
liquid/solid form was obtained after neutralizing the mixture with
saturated NaHCO3 solution and extracted in ethyl acetate and pass-
through sodium sulphate [28].
–
CHCl3): δ (ppm), 119.9 (ArC), 129.2 (ArC), 135.7 (C Cl), 164.6
–
(N
– –
C O), 127.3 (ArC), 112.6 (ArC), 131.3 (ArC), 159 (C O), 122.8
–
(C C). ESI Mass (m/z): 287 (M+H) +. Anal. Calc. for C15H11ClN2O2: C,
62.84; H, 3.87; Cl, 12.37; N, 9.77; O, 11.16. Found: C, 61.87; H, 4.55; Cl,
12.89; N, 9.79; O, 12.17.
–
4.3.5. 4-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl) aniline (SB5)
4.2.2. General procedure for synthesis of aromatic hydrazide (3a-3r)
To a solution of substituted aromatic ester (1 mol) in ethanol,
mixture of hydrazine hydrate (1.2 mol) in anhydrous ethanol was added
dropwise and refluxed at 60–70 ◦C for 12–18 h. Upon completion of the
reaction, excess of hydrazine hydrate was evaporated under vacuum and
the resulting mixture was allowed to cool, to yield the hydrazide de-
rivatives [28].
Yield: 64%, M.P 282–284 ◦C, Rf = 0.7 (ethyl acetate: chloroform
8:2), FTIR (CHCl3, ʋ/(cmꢀ 1): 1534 (Ar C C), 1467 (C N), 1255
–
–
–
–
1
– – – –
(C
O C), 3022 (Ar C H), 781 (C Cl). HNMR (500 MHz, CHCl3): δ
(ppm), 6.7 (d, 2H, J = 8.2 Hz), 7.7 (d, 2H, J = 8.26 Hz), 7.8 (d, 2H, J =
8.17 Hz). 13CNMR (500 MHz, CHCl3): δ (ppm), 131.3 (ArC), 128.8
–
(ArC), 129.2 (ArC), 114.1 (ArC), 131.3 (ArC), 149.1 (C N), 135.7
+
–
–
–
– –
C O), 122.8 (C C). ESI Mass (m/z): 272 (M+H) .
(C Cl), 164.6 (N
Anal. Calc. for C14H10ClN3O: C, 61.89; H, 3.71; Cl, 13.05; N, 15.47; O,
4.2.3. General procedure for synthesis of 2,5-disubstituted 1,3,4-
oxadiazole derivatives (SB1-SB18).
5.89. Found: C, 62.79; H, 2.59; Cl, 13.89; N, 16.66; O, 6.17.
Substituted aromatic hydrazide (1 mol) was heated to reflux along
with substituted aromatic benzoic acids (0.9 mol) and phosphorous
oxychloride (1.2 mol) for 8 h. After completion of reaction, the reaction
4.3.6. 2-(4-chlorophenyl)-5-(4-nitrophenyl)-1,3,4-oxadiazole (SB6)
◦
Yield: 67%, M.P 278–280 C, Rf = 0.8 (ethyl acetate: chloroform,
9:1), FTIR (CHCl3, ʋ/(cmꢀ 1): 1566 (Ar C C), 1325 (C N), 1189
–
–
–
–
9

S. Bajaj et al.
Bioorganic Chemistry 111 (2021) 104873
– –
O
–
–
–
(C
C), 3053 (Ar C H), 1366 (N O), 742 (C Cl). 1HNMR (500
4.3.12. 2,5-bis(2-fluorophenyl)-1,3,4-oxadiazole (SB12)
◦
MHz, CHCl3): δ (ppm), 7.9 (m, 4H), 8.0 (d, 2H, J = 8.41 Hz), 8.2 (d, 2H,
Yield: 68%, M.P 264–266 C, Rf = 0.8 (ethyl acetate: chloroform,
J = 8.78 Hz). 13C NMR (500 MHz, CHCl3): δ (ppm), 124.1 (ArC), 129.2
8:2), FTIR (CHCl3, ʋ/(cmꢀ 1): 2954(C H), 1587 (Ar C C), 1434 (C-F),
–
–
–
–
–
–
–
–
(ArC), 128.8 (ArC), 130.1 (ArC), 148.0 (C N), 135.7 (C Cl), 164.6
1115 (C N), 1092 (C O), 1HNMR (500 MHz, CHCl3): δ (ppm), 7.3
(N
O), 122.8 (C C). ESI Mass (m/z): 302 (M+H) +. Anal. Calc. for
(2H, J = 7.3 Hz), 7.4 (2H, J = 7.6 Hz), 7.6 (2H, J = 7.9 Hz), 8.2 (2H, J =
–
–
–
C
–
C
14H8ClN3O3: C, 55.74; H, 2.67; Cl, 11.75; N, 13.93; O, 15.91. Found: C,
7.1 Hz). 13CNMR (500 MHz, CHCl3): δ (ppm), 127.4 (ArC), 114.2 (ArC),
–
– –
131.9 (ArC), 160.4 (C-F), 164.6 (N C O), 130.7 (C C). ESI Mass (m/
–
54.47; H, 3.81; Cl, 12.11; N, 12.79; O, 16.17.
z): 259 (M+H) +. Anal. Calc. for C14H8F2N2O: C, 65.12; H, 3.12; F,
14.71; N, 10.85; O, 6.20. Found: C, 66.28, H, 4.20; F, 15.06; N, 13.89; O,
7.80.
4.3.7. 2,5-bis(2-chlorophenyl)-1,3,4-oxadiazole (SB7)
◦
Yield: 76%, M.P 254–256 C, Rf = 0.7 (ethyl acetate: chloroform,
9:1), FTIR (CHCl3, ʋ/(cmꢀ 1): 1665 (Ar C C), 1139 (C N), 933 (C O),
–
–
–
–
–
1
–
–
2341 (Ar C H), 725 (C Cl), 1449 (C-F). HNMR (500 MHz, CHCl3): δ
(ppm), 7.4 (d, 2H, J = 7.45 Hz), 7.5 (d, 2H, J = 7.49 Hz), 7.6 (d, 2H, J =
7.58 Hz), 8.2 (d, 2H, J = 8.12 Hz). 13CNMR (500 MHz, CHCl3): δ (ppm),
4.3.13. 2,5-di((E)-styryl)-1,3,4-oxadiazole (SB13)
◦
Yield: 78%, M.P 263–265 C, Rf = 0.6 (ethyl acetate: chloroform,
8:1), FTIR (CHCl3, ʋ/(cmꢀ 1): 1548 (Ar C C), 1678, 1654 (Al C C),
–
–
–
–
–
–
– –
–
131.2 (ArC), 130.6 (ArC), 130.4 (ArC), 126.4 (ArC), 133.2 (C Cl),
1682 (C N), 1098 (C
O
C), 3892 (Ar C H). 1HNMR (500 MHz,
–
164.6 (N
O), 128.8 (C C). ESI Mass (m/z): 291 (M+H) +. Anal.
CHCl3): δ (ppm), 7.2 (d, 4H, J = 8.11 Hz), 7.3 (d, 2H, J = 15.45 Hz), 7.4
–
–
–
C
–
Calc. for C14H8Cl2N2O: C, 55.76; H, 2.77; Cl, 24.36; N, 9.62; O, 5.50.
(m, 6H), 7.6 (d, 2H, J = 15.45 Hz). 13CNMR (500 MHz, CHCl3): δ (ppm),
–
– –
C O), 118.0 (C C), 133.4 (AlC).
Found: C, 56.80; H, 1.98; Cl, 24.89; N, 8.74; O, 6.10.
128.7 (ArC), 127.2 (ArC), 161.9 (N
–
ESI Mass (m/z): 275.1 (M+H) +. Anal. Calc. for C18H14N2O: C, 78.81; H,
4.3.8. 2-(2-chlorophenyl)-5-(2-fluorophenyl)-1,3,4-oxadiazole (SB8)
5.14; N, 10.21; O, 5.83. Found: C, 79.91, H, 4.92; N, 11.25; O, 6.16.
◦
Yield: 64%, M.P 263–265 C, Rf = 0.6 (ethyl acetate: chloroform,
9:1), FTIR (CHCl3, ʋ/(cmꢀ 1): 1583 (Ar C C), 1632 (C N), 1028
4.3.14. (E)-2-(4-chlorophenyl)-5-styryl-1,3,4-oxadiazole (SB14)
–
–
–
–
1
◦
– –
–
–
(C
O
C), 2987 (Ar C H), 704 (C Cl), 1058 (C-F). HNMR (500 MHz,
Yield: 72%, M.P 278–280 C, Rf = 0.6 (ethyl acetate: chloroform,
9:1), FTIR (CHCl3, ʋ/(cmꢀ 1): 1501 (Ar C C), 1672, 1622 (Al C C),
–
–
–
CHCl3): δ (ppm), 7.3 (d, 1H, J = 7.88 Hz), 7.4 (m, 1H, J = 7.63 Hz), 7.5
(d, 1H, J = 7.61 Hz), 7.8 (d, 1H, J = 7.61 Hz), 7.9 (d, 1H, J = 7.88 Hz).
13CNMR (500 MHz, CHCl3): δ (ppm), 126.4 (ArC), 131.2 (ArC), 130.4
–
–
– – – –
1600 (C N), 1121 (C
O C), 3873 (Ar C H), 744 (C Cl), 1648
–
1
–
–
(C C). HNMR (500 MHz, CHCl ): δ (ppm), 7.2 (d, 2H, J = 8.00 Hz), 7.3
3
–
–
–
–
C O), 128.0
(ArC), 127.6 (ArC), 133.2 (C Cl), 160.4 (C-F), 164.6 (N
(d, 1H, J = 15.46 Hz), 7.4 (m, 3H), 7.6 (d, 1H, J = 15.46 Hz), 7.9 (m,
4H). ESI Mass (m/z): 283 (M+H) +. Anal. Calc. for C16H11ClN2O: C,
76.97; H, 3.92; Cl, 12.54; N, 9.91; O, 5.66. Found: C, 75.83, H, 4.81; Cl,
13.06; N, 10.22; O, 6.23.
(C C). ESI Mass (m/z): 275 (M+H) +. Anal. Calc. for C14H8ClFN2O: C,
61.22; H, 2.94; Cl, 12.91; F, 6.92; N, 10.20; O, 5.82. Found: C, 60.89; H,
3.00; Cl, 12.93; F, 6.94; N, 12.44; O, 5.87.
–
4.3.9. 2-(2-chlorophenyl)-5-(2-methoxyphenyl)-1,3,4-oxadiazole (SB9)
4.3.15. (E)-2-(2-chlorophenyl)-5-styryl-1,3,4-oxadiazole (SB15)
◦
◦
Yield: 57%, M.P 270–272 C, Rf = 0.7 (ethyl acetate: chloroform,
Yield: 69%, M.P 276–278 C, Rf = 0.7 (ethyl acetate: chloroform,
8:2), FTIR (CHCl3, ʋ/(cmꢀ 1): 1499 (Ar C C), 1652 (C N), 1014
9:1), FTIR (CHCl3, ʋ/(cmꢀ 1): 1642 (Ar C C), 1632 (C N), 1087
–
–
–
–
–
–
–
–
1
(C
O
C), 2898 (Ar C H), 711 (C Cl). HNMR (500 MHz, CHCl3): δ
C), 3901 (Ar C H), 765 (C Cl), 1648 (C C). 1HNMR (500
– – – –
(C O
–
–
– –
–
–
(ppm), 3.8 (s, 3H), 7.2 (m, 2H), 7.4 (d, 1H, J = 7.89 Hz), 7.5 (d, 1H, J =
7.59 Hz), 7.9 (m), 8.0 (d, 1H, J = 7.59 Hz). 13CNMR (500 MHz, CHCl3): δ
(ppm), 127.3 (ArC), 126.4 (ArC), 130.6 (ArC), 131.3 (ArC), 119.9 (ArC),
MHz, CHCl3): δ (ppm), 7.2 (d, 2H, J = 8.0 Hz), 7.3 (m, 2H), 7.4 (d, 1H, J
= 7.65 Hz), 7.6 (m, 2H), 7.7 (d, 1H, J = 7.62 Hz), 7.9 (d, 1H, J = 7.89
Hz). ESI Mass (m/z): 283 (M+H) +. Anal. Calc. for C16H11ClN2O: C,
67.97; H, 3.92; Cl, 12.54; N, 9.91; O, 5.56. Found: C, 68.72, H, 2.85; Cl,
12.80; N, 10.52; O, 6.42.
–
–
–
– – –
C O), 128.0 (C C), 112.6 (C C), 159.7
133.2 (C Cl),164.6 (N
(C O), 55.9 (Al C). ESI Mass (m/z): 287 (M+H) +. Anal. Calc. for
–
–
C
15H11ClN2O2: C, 62.84; H, 3.87; Cl, 12.37; N, 9.77; O, 11.16. Found: C,
62.99; H, 3.81, Cl, 12.37; N, 9.82; O, 11.21.
4.3.16. 2-(4-chlorophenyl)-5-phenyl-1,3,4-oxadiazole (SB16)
◦
Yield: 73%, M.P 239–241 C, Rf = 0.8 (ethyl acetate: chloroform,
9:1), FTIR (CHCl3, ʋ/(cmꢀ 1): 1552 (Ar C C), 1642 (C N), 1063
–
–
–
4.3.10. 4-(5-(2-chlorophenyl)-1,3,4-oxadiazol-2-yl) aniline (SB10)
–
1
◦
– – – –
Yield: 59%, M.P 284–286 C, Rf = 0.7 (ethyl acetate: chloroform,
(C O C), 3822 (Ar C H), 723 (C Cl). HNMR (500 MHz, CHCl3): δ
9:1), FTIR (CHCl3, ʋ/(cmꢀ 1): 1522 (Ar C C), 1616 (C N), 1012
(ppm), 7.4 (d, 2H, J = 7.80 Hz), 7.5 (d, 1H, J = 7.45 Hz), 7.9 (m, 4H). ESI
Mass (m/z): 257 (M+H) +. Calc. for C14H9ClN2O: C, 65.51; H, 3.53; Cl,
13.81; N, 10.91; O, 6.23. Found: C, 66.30, H, 3.89; Cl, 12.87; N, 11.22;
O, 7.10.
–
–
–
–
1
– – – –
(C
O C), 2833 (Ar C H), 752 (C Cl). HNMR (500 MHz, CHCl3): δ
(ppm), 6.7 (d, 2H, J = 8.26 Hz), 7.3 (d, 1H, J = 7.93 Hz), 7.4 (d, 1H, J =
7.58 Hz), 7.7 (d, 2H, J = 8.26 Hz), 7.8 (d, 1H, J = 7.93 Hz), 8.0 (d, 1H, J
= 7.58 Hz). 13CNMR (500 MHz, CHCl3): δ (ppm), 114.1 (ArC), 130.6
–
–
(ArC), 126.4 (ArC), 131.3 (ArC), 133.2 (C Cl), 149. 1 (C N), 164.6
4.3.17. 2-(2-chlorophenyl)-5-phenyl-1,3,4-oxadiazole (SB17)
+
◦
–
–
C
–
–
(N
O), 128.0 (C C), 128.8 (C C). ESI Mass (m/z): 272 (M+H)
.
Yield: 61%, M.P 246–248 C, Rf = 0.6 (ethyl acetate: chloroform,
–
9:1), FTIR (CHCl3, ʋ/(cmꢀ 1): 1541 (Ar C C), 1648 (C N), 1043
–
–
–
Anal. Calc. for C14H10ClN3O: C, 61.89; H, 3.71; Cl, 13.05; N, 15.47; O,
5.89. Found: C, 62.01, H, 3.69; Cl, 12.06; N, 15.48; O, 5.78.
–
1
– – – –
(C
O C), 3899 (Ar C H), 742 (C Cl). HNMR (500 MHz, CHCl3): δ
(ppm), 7.3 (d, 1H, J = 7.85 Hz), 7.4 (d, 2H, J = 7. 08 Hz), 7.5 (d, 1H, J =
7.45 Hz), 7.6 (d, 1H, J = 7.65 Hz), 7.8 (d, 1H, J = 7.65 Hz), 7.9 (m, 3H).
ESI Mass (m/z): 257 (M+H) +. Calc. for C14H9ClN2O: C, 65.51; H, 3.53;
Cl, 13.81; N, 10.91; O, 6.23. Found: C, 65.90, H, 3.72; Cl, 13.10; N,
11.61; O, 5.74.
4.3.11. 2-(2-chlorophenyl)-5-(4-nitrophenyl)-1,3,4-oxadiazole (SB11)
◦
Yield: 64%, M.P 274–276 C, Rf = 0.6 (ethyl acetate: chloroform,
9:1), FTIR (CHCl3, ʋ/(cmꢀ 1): 1583 (Ar C C), 1631 (C N), 1101
–
–
–
–
1
– – – –
(C
O C), 3883 (Ar C H), 794 (C Cl). HNMR (500 MHz, CHCl3): δ
(ppm), 7.4 (d, 1H, J = 7.63 Hz), 7.5 (d, 1H, J = 8.09 Hz), 7.8 (d, 1H, J =
7.59 Hz), 7.9 (d, 2H, J = 8.72 Hz), 8.0 (d, 1H, J = 8.09 Hz), 8.2 (d, 2H, J
= 8.72 Hz). 13CNMR (500 MHz, CHCl3): δ (ppm), 126.4 (ArC), 131.2
4.3.18. (E)-2-phenyl-5-styryl-1,3,4-oxadiazole (SB18)
◦
Yield: 75%, M.P 278–280 C, Rf = 0.6 (ethyl acetate: chloroform,
–
–
–
–
(ArC), 130.4 (ArC), 124.1 (ArC), 133.2 (C Cl), 148.0 (C N), 164.6
9:1), FTIR (CHCl3, ʋ/(cmꢀ 1): 1616 (Ar C C), 1698 (C N), 1074
–
–
+
1
–
–
–
–
C
–
–
– – –
(N
O), 128.0 (C C), 122.8 (C C). ESI Mass (m/z): 302 (M+H)
.
(C O C), 3942 (Ar C H), 1644 (C C). HNMR (500 MHz, CHCl ): δ
–
Anal. Calc. for C14H8ClN3O3: C, 55.74; H, 2.67; Cl, 11.75; N, 13.93; O,
(ppm), 7.2 (d, 2H, J = 8.00 Hz), 7.3 (d, 1H, J = 15.45 Hz), 7.4 (d, 23H, J
15.91. Found: C, 56.16, H, 1.89; Cl, 13.06; N, 14.80; O, 16.71.
= 7.33 Hz), 7.5 (m, 2H), 7.6 (d, 2H, J = 7.82 Hz), 7.9 (d, 2H, J = 7.82
10

S. Bajaj et al.
Bioorganic Chemistry 111 (2021) 104873
Hz). ESI Mass (m/z): 249 (M+H) +. Calc. for C16H12N2O: C, 77.40; H,
3–4 h. All the rats were monitored once in a while for every one hour up
to 12 h on first day and subsequently two times a day for checking
mortality, behavioural changes, signs and symptoms of toxicity for 14
days. Weight of all the twenty rats were taken at the interval 7 days from
the day zero to fourteen days and study was conducted two times for
each dose.
4.87; N, 11.28; O, 6.64. Found: C, 78.30, H, 5.82; N, 12.61; O, 6.72.
4.4. Biological evaluation
4.4.1. In vitro cell proliferation assay
Cancer cell lines such as MCF-7, MDA-MB-231 and VERO cell line
(African Green monkey kidney) were purchased from National Centre
for Cell Sciences (NCCS) (Pune, India). In vitro cytotoxic activity of all
the synthesized 2,5-disubstituted 1,3,4-oxadiazole derivatives was
evaluated using colorimetric MTT assay also used for calculating cell
viability. Doxorubicin was used as a standard inhibitor. Briefly, cells
were grown in DMEM media supplemented with foetal bovine serum
(FBS) 10% and penicillin–streptomycin (50 U/ml, 50 mg/ml) at 37 ◦C,
CO2 (5%) and air (95%). Logarithmically, growing cells were seeded
using 96-well plate at different concentrations of the test compounds
ranging from 0.01 to 100 µM. After 24 h of seeding, the cells were
observed under microscope and treated with varying drug concentration
along with DMSO (vehicle control). Each dilution of test compound was
added in triplicate. Following 48-h incubation with the compounds, cells
were incubated with MTT reagent (5 mg/ml) for 4 h, and then 100 µl of
DMSO was added to dissolve formazan crystals. The absorbance was
then measured at 540 nm and 630 nm (background scan) using EPOCH 2
BioTek microplate reader. The results of anti-cancer activity were
expressed as IC50 values in µM range and are presented in Table 2. IC50 is
defined as the compound concentration required to reduce the viability
of the cells by 50% with respect to the control [30]. CC50 value of VERO
cell line and selectivity index also presented in Table 2.
4.5. Molecular docking
All the synthesized molecules were subjected to molecular docking
studies to recognise the binding interactions within the active binding
site residues of TP receptor (PDB ID: 1UOU). Molecular docking was
done by using Glide module of Schrodinger software. Active site binding
residues of TP receptor such as SER117, SER217, HIS116, LYS221,
ARG202, TYR199, THR118, ARG146, LEU148, THR151, GLY152 and
ILE214 are responsible for binding [26]. The ligands were prepared by
computation of charges and setting number of torsions. The refinement
of receptor was proceeded for addition of polar hydrogen, removal of
water molecules and bind ligand present in the crystal structure. Re-
ceptor grid was generated around the binding site of previously attached
ligand. The result of docking is expressed in terms of Glide score and
binding energy (kcal/mol) and a lower scoring conformation is ranked
higher [34].
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
4.4.2. Thymidine phosphorylase inhibitory assay
Synthesized compounds were evaluated for their thymidine phos-
phorylase inhibition activity by employing 7-deazaxanthine as standard.
TP inhibitory assay results are shown in Table 3. The procedure for TP
inhibitory assay was followed from Krenitsky et al., (1979) with modi-
fications and the absorbance was measured at 290 nm spectrophoto-
Acknowledgement
The authors are grateful to the Department of Science and Tech-
nology (DST), New Delhi, for providing Woman Scientist A (WOS-A)
Project to Dr Shalini Bajaj (SR/WOS-A/CS-98/2016).
metrically. Total reaction mixture of volume 200
which consisted of 145 l potassium phosphate buffer (pH 7.4), 30
E. coli TP enzyme (Sigma T2807 500u) at concentration 0.002 U and 20
l thymidine 5ꞌ mono phosphate solution (0.05 U concentration) as
μ
l was placed in wells,
μ
μl of
Appendix A. Supplementary material
μ
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104873.
substrate. Test material 5 μl was added to this mixture and incubated for
20 min at 25 ◦C and taking reading in microplate reader at 290 nm. The
wells containing reaction mixture devoid of substrate was considered
blank and the absorbance of these blank wells was subtracted from wells
containing reaction mixture with substrate to avoid the background
absorbance. Microplate reader took the readings continuously after 10,
20, 30, 40, 50, and 60 mins after incubation. All assays were performed
in triplicate [31,32].
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