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Letters in Drug Design & Discovery, 2019, 16, 994-1005

RESEARCH ARTICLE
ISSN: 1570-1808
eISSN: 1875-628X

Benzo[d]imidazol-5-yl)-5-(substituted)-1,3,4-Oxadiazoles: Synthesis, Anti- Impact

Factor:
0.953

cancer, Antimicrobial and In Silico Studies

BENTHAM
SCIENCE

Naveen Kumara,b, Swamy Sreenivasab,*, Bhuvanesh Sukhlal Kalalc, Vasantha Kumara, Bantwal
Shivarama Hollaa, Vinitha Ramanath Paic, Nadigar Revansiddappa Mohand and Shivaraj
Govindaiahb

a
Department of Chemistry, Sri Dharmasthala Manjunatheshwara College (Autonomous), Ujire, Karnataka, India;
b
Department of Studies and Research in Organic Chemistry, Tumkur University, Tumkur, Karnataka, India; cDepart-
ment of Biochemistry, Yenepoya University, Deralakatte, Mangalore, Karnataka, India; dNational Assessment and Ac-
creditation Council, Nagarbhavi, Bangalore- 560072, Karnataka, India
Letters in Drug Design & Discovery

ARTICLE HISTORY
Received: July 1, 2018
Revised: November 8, 2018
Accepted: December 12, 2018
DOI:
10.2174/1570180816666181220123924

noma cancer cell line with IC50 47.06 µM and 36.76 µM, respectively. In silico studies also revealed
Keywords: Benzimidazole, oxadiazole, antimicrobial, anticancer, docking, 2OH 4 protein, VEGFR-2 tyrosine kinase.
Abstract: Background: Cancer is a fatal disease for mankind; continuous research is still going on
for the invention of potent anticancer drugs. In this view, 1, 3, 4-Oxadiazoles are privileged mole-
cules which attracted medicinal chemists towards their anticancer properties.
Methods: A new series of benzo[d]imidazol-5-yl)-5-(substituted)-1,3,4-oxadiazole derivatives was
synthesized in an efficient ‘one-pot’ nitro reductive cyclization using sodium dithionite as a cyclizing
agent by a conventional method with good yield. All the structures of the synthesized molecules were
characterized by IR, 1H NMR, HRMS and Mass spectral analysis. Anticancer activity screening
against A375 melanoma cancer cell line and MDA-MB-231 breast cancer cell line along with anti-
microbial activity were carried out using agar well diffusion method.
Results: Compounds 8a and 8j of the series emerged as potent anticancer agents against A375 mela-
that compounds 8a and 8j showed highest interaction with 2OH4 protein of VEGFR-2 tyrosine ki-
nase. Substantial antibacterial and antifungal activities against the tested microorganism were ob-
served for compounds 8j and 8g.
Conclusion: Potent anticancer property has been observed with 1,3,4-Oxadiazole linked tetrafluro
substituted benzene ring 8j indicating that future research on these type of molecules can be
continued to improve the anticancer activity.

1. INTRODUCTION cytotoxicity and genotoxicity on normal host cells and stimu-

Cancer diseases are commonly prevailing throughout the
developed and developing countries as a matter of growing
circumstance; studies in the area of finding new anticancer
drugs are well documented in the field of research from
many years. Food and Drug Administration (FDA) had ap-
proved many anticancer drugs with heterocyclic core moie-
ties (Mercaptopurine, Melphalan, Vincristine) for the treat-
ment of different types of cancers. A fundamental goal of
medicinal research and drug discovery is to develop new
anticancer therapeutic drugs with less adverse effects of
*Address correspondence to this author at the Associate Professor and
Chairman, Department Studies and Research in Organic Chemistry, Tumkur
University, P.O. Box: 572-103, Tumkur, India; Tel/Fax: +91 8162270719,
+91 96202 30672; E-mail: drsreenivasa@yahoo.co.in
lating secondary malignancy [1]. Breast cancer is one the
most common types of cancer along with another common
disease skin cancer. Mainly two receptors such as tyrosine
kinases (RTKs), Vascular Endothelial Growth Factor Receptor
1 (VEGFR-1) kinase and Vascular Endothelial Growth
Factor Receptor 2 (VEGFR-2) kinase [2, 3] are responsible
for Vascular Endothelial Growth Factor (VEGF) biological
effects. It has been proven that the reticence of VEGF sig-
nals blocks angiogenesis in tumors as well as alters or termi-
nates tumor vessels [4]. Consequently, VEGFR-2 becomes
an attractive target for in silico biological cancer studies.
Benzimidazole and its derivatives had gained significant
importance in the field of research because of their in vivo
increased stability and bioavailability [5, 6]. From the past
several decades, researchers have been working on various
biological applications of benzimidazole acid derivatives

1875-628X/19 $58.00+.00 ©2019 Bentham Science Publishers

Benzo[d]imidazol-5-yl)-5-(substituted)-1, 3, 4-Oxadiazoles
[7, 8]. 1, 2-disubstituted benzimidazole-5-carboxylic deriva-
tives are mainly known to possess Hepatitis-C virus (HCV)
inhibitory activity and in recent years this moiety has gained
extensive interest in anticancer drug discovery [9]. Moreo-
ver, these benzimidazole derivatives are known to possess
various biological activities such as antimicrobial [10-13],
anti-inflammatory [14-17], anti-allergic [18], antioxidant
[19-22], anti-tubercular [23, 24], anthelmintic [25], antiviral
[26, 27], antimalarial [28] and anticancer [29-33] properties.
Oxadiazole scaffold is found in many anticancer drugs,
particularly Zibotentan, a well-known anti-cancer drug used
in the chemotherapeutic treatment, consists of 1,3,4-
oxadiazole ring system [34]. It acts as a bioesteric replace-
ment for amide, ester group and increases lipophilic prop-
erty of the molecule [35]. 1,3,4-Oxadiazoles found to ex-
hibit different biological properties like anticancer activi-
ty [36-40], histone deacetylase inhibitor [41] and MetAP2
inhibitor [42] along with antimicrobial [43, 44],
anti-inflammatory [45-48], anti-tubercular [49-51], antidi-
abetic [52-54], anti-proliferative [55-57], anti-depressant
[58-60], and anti-anxiety properties [61]. Ability to form
strong hydrogen bonding with receptor molecules in the
human body result in enhanced drug action have made
1,3,4-oxadiazole scaffolds more interesting candidates to
combine with benzimidazole. Based on the importance of
benzimidazole and [1, 3, 4]-oxadiazoles potency, in our
present work, we incorporated [1, 3, 4]-oxadiazole ring at
5th position of 1-methyl-2-substituted benzimidazole ring
system. In order to identify the pharmacological potency
of all the synthesized new series of benzo[d]imidazol-5-
yl)-5-(substituted)-1, 3, 4-oxadiazole derivatives, we
screened the compounds for in vitro antimicrobial activities
like antibacterial against Staphylococcus aureus, Escherichia
coli bacterial strains, antifungal activity against Candida
albicans, Aspergillus flavus fungal strains and anticancer
activity against A375 melanoma cancer, MDA-MB-231
breast cancer cell lines. In silico study was performed to un-
derstand the interaction at the active site of the protein and
benzimidazole derivatives using the protein PDB: 2OH4
VEGFR-2 Tyrosine Kinase Receptors and to Correlate the
activity of the compounds by in silico study.
2. MATERIALS AND METHODS
2.1. General
All the chemicals used were acquired from Merck, Spec-
trochem and Sigma-Aldrich Company and were of analytical
grade. Melting point (mp) of the all the newly synthesized
compounds was determined using open capillary tubes. FT-
IR absorption spectra were obtained in the range 4000-400
cm-1 on Shimadzu IR Affinity-1. 1H NMR spectra were rec-
orded on Bruker Avance III at 400.23 MHz with internal
standard tetramethylsilane and chemical shifts were ex-
pressed in parts per million (ppm). LC-MS and HRMS were
recorded on Water, synapt G2 high detection mass spec-
trometry. Reactions were monitored by Thin Layer Chro-
matography using precoated silica plates 60 F254
aluminium sheets and visualization was done by UV light
at 254 nm. Compounds were synthesized according to re-
ported procedures and purifications were done by recrys-
tallization.
Letters in Drug Design & Discovery, 2019, Vol. 16, No. 9 995
2.2. Chemistry
2.2.1 Synthesis of Ethyl 4-Chloro-3-Nitrobenzoate (2)
Compound ethyl 4-chloro-3-nitrobenzoate was synthe-
sized according to literature procedure. m.p 60-62°C (lit. 59-
61°C) [62, 63].
2.2.2. Synthesis of Ethyl 4-(methylamino)-3-Nitrobenzoate (3)
Compound ethyl 4-(methylamino)-3-nitrobenzoate was
synthesized according literature procedure. m.p 102-103°C
(lit. 101°C) [62, 63].
2.2.3. Synthesis of 2-(3-fluoro-4-methylphenyl)-1-Methyl-
1H-Benzo[d]imidazole-5-Carboxylate (5)
To a clear mixture of ethyl-3-nitro-4-(methylamino)
benzoate (3) (2.24 g, 10 mmol) and 3-fluoro-4-methyl
benzaldehyde (1.21 mL, 10 mmol) (4) in dimethyl sulfoxide
(DMSO) (20 mL), reductive cyclizing agent sodium
dithionite (5.22 g, 30 mmol) was added and stirred at reflux
temperature for 3 hours. Progress and completion of the
reaction were monitored by TLC. The reaction mixture was
then poured on to ice chips. Crude product separated as
white solid was filtered, washed with cold water and dried.
Recrystallization in ethanol, yielded white precipitate of pure
2-(3-fluoro-4-methylphenyl)-1-methyl-1H-benzo[d] imidaz-
ole-5-carboxylate (5) in good yield: 90 %; mp 175-177oC;
C18H17FN2O2; LC-MS m/z (%) 313.21 (M+1) (calculated
312.12); IR (KBr) cm-1 2980 (C-H), 1706 (C=O), 1622
(C=N), 1469 (C=C), 1088 (C-F); 1H NMR (400 MHz,
DMSO) δ 1.36 (t, J = 7.2 Hz, CH3 , 3H), 2.50 (s, 3H, CH3),
3.95 (s, 3H, N-CH3), 4.37 (m, CH2, 2H), 7.54 (t, J = 8.0 Hz,
Ar, 1H), 7.71(dd, Ar, 2H), 7.83 (d, J = 8.8 Hz, Ar, 1H),7.99
(d, J = 10.0Hz, Ar, 1H), 8.29 (s, Ar, 1H).
2.2.4. Synthesis of 2-(3-fluoro-4-methylphenyl)-1-Methyl-
1H-Benzo[d]imidazole-5-Carbo Hydrazide (6)
A mixture of ethyl 2-(3-fluoro-4-methylphenyl)-1-
methyl-1H-benzo[d]imidazole-5-carboxylate (3.12 g, 10
mmol) (5) and hydrazine hydrate (1.6 mL, 50 mmol) in
ethanol (50 mL) was stirred under reflux for 8 hours. The
reaction was monitored by thin layer chromatography and
after the completion of reaction, the mixture was quenched
into ice pieces. White solid crude obtained was filtered,
washed and dried. Pure pre-final compound 2-(3-fluoro-4-
methylphenyl) -1-methyl-1H-benzo[d]imidazole-5-carbohy-
drazide (6) in good yield was obtained further recrystallized
using ethanol. yield: 85 %; C16H15FN4O; mp 198-200ºC; LC-
MS m/z (%) 299.24 (M+1) (calculated 298.12); IR (KBr) cm-
1
3460 (NH2, Asymmetric), 3284 (NH2, Symmetric), 3336
(NH), 2972 (C-H), 1633 (C=O), 1608 (C=N), 1525 (C=C),
1047 (C-F); 1H NMR (400 MHz, DMSO) δ 2.50 (s, 3H, CH3),
3.90 (s, 3H, N-CH3), 4.50 (s, NH2, 2H), 7.50 (t, J = 8.0 Hz,
Ar, 1H), 7.69 (m, Ar, 3H), 7.86 (d, J = 9.6 Hz, Ar, 1H), 8.18
(s, 1H, Ar), 9.77 (s, NH, 1H).
2.2.5. General Procedure for the Synthesis of Ben-
zo[d]imidazol-5-yl)-5-(substituted)-1, 3, 4-Oxadiazole De-
rivatives (8a-j)
Mixture of 2-(3-fluoro-4-methylphenyl)-1-methyl-1H-
benzo[d]imidazole-5-carbohydrazide (2.98g, 10 mmol) (6)
with different aromatic substituted carboxylic acids 7(a-j)
996 Letters in Drug Design & Discovery, 2019, Vol. 16, No. 9
(10mmol) in phosporousoxychloride (30mL) was
refluxed for 4 hours. Completion of the reactions was
monitored by TLC. After the completion of the reaction,
the reaction mixture was poured on to crushed ice. Solid
obtained was filtered, washed with water, dried and
recrystallized from ethanol to yield benzo[d]imidazol-5-yl)-5-
(substituted)-1,3,4-oxadiazole derivatives (8a-j) in good yield.
2.2.6. 2-(2-(3-fluoro-4-methylphenyl)-1-Methyl-1H-Benzo-
[d]imidazol-5-yl)-5-(furan-2-yl)-1, 3, 4-oxadiazole (8a)
Yield: 62%; mp 212 ºC; C21H15FN4O2, HRMS m/z (%)
375.1261 (M+1) (calculated 375.1213 ) ; IR (KBr) cm-1 2956
(C-H), 1608 (C=N), 1548 (C=C), 1091 (C-F); 1H NMR (400
MHz; DMSO): δ 2.32 (s, 3H, CH3), 3.93 (s, 3H, N-CH3),
6.82-6.80 (m, Ar, 1H), 7.51-7.45 (m, Ar, 2H), 7.66-7.61 (m,
Ar, 2H), 7.86 (d, J = 8.4 Hz, Ar, 1H), 8.01 (d, J = 10.0 Hz,
Ar, 1H), 8.07 (s, Ar, IH), 8.32 (s, Ar, 1H).
2.2.7. 2-(2-(3-fluoro-4-methylphenyl)-1-Methyl-1H-Benzo-
[d]imidazole-5-yl)-5-phenyl-1, 3, 4-Oxadiazole (8b)
Yield: 80%; mp 206-208 ºC; C23H17FN4O; HRMS m/z
(%) 385.1466 (M+1) (calculated 385.1420 ); IR (KBr) cm-1
2926 (C-H), 1614 (C=N), 1573 (C=C), 1095 (C-F); 1H NMR
(400 MHz; DMSO): δ 2.37 (s, 3H, CH3), 3.99 (s, 3H, N-
CH3), 8.48 (s, Ar, 1H), 8.20-8.14 (m, ArH, 3H), 7.97 (d, J = 8.4
Hz, Ar, 1H), 7.74-7.66 (m, Ar, 5H), 7.56 (t, J = 8.0 Hz, Ar, 1H).
2.2.8. 2-(3-chlorophenyl)-5-(2-(3-fluoro-4-methylphenyl)-1-
Methyl-1H-Benzo[d]imidazol-5-yl)-1, 3, 4-Oxadiazole (8c)
Yield: 86%; mp 244 ºC; C23H16ClFN4O; HRMS m/z (%)
419.1079(M+) (calculated 418.8507 ); IR (KBr) cm-1 2919
(C-H), 1617 (C=N), 1575(C=C), 1057 (C-F), 884 (C-Cl); 1H
NMR (400 MHz; DMSO): δ 2.32 (s, 3H, CH3), 3.93 (s, 3H,
N-CH3), 8.48 (s, Ar, 1H), 8.21 (s, Ar, 1H), 8.10-8.08 (m, Ar,
2H),7.818 (d, J =7.6 Hz, Ar, 1H), 7.67-7.62 (m, Ar, 4H),
7.49 (t, J = 7.6Hz, Ar, 1H).
2.2.9. 2-(4-chlorophenyl)-5-(2-(3-fluoro-4-methylphenyl)-1-
Methyl-1H-Benzo[d]imidazole-5-yl)-1, 3, 4-Oxadiazole (8d)
Yield: 76%; mp 216-218 ºC; C23H16ClFN4O; HRMS m/z
(%) 419.1081 (M+) (calculated 418.8507 ); IR (KBr) cm-1
2947 (C-H), 1610 (C=N), 1546 (C=C), 1093 (C-F), 891 (C-
Cl); 1H NMR (400 MHz; DMSO) δ 2.36 (s, 3H, CH3), 3.97
(s, 3H, N-CH3), 8.481 (s, Ar, 1H), 8.22-8.10 (m, Ar, 3H),
7.90 (d, J = 7.6 Hz, Ar, 1H), 7.73-7.66 (m, Ar, 4H), 7.55 (t, J
= 8.0 Hz, Ar, 1H).
2.2.10. 2-(2-(3-fluoro-4-methylphenyl)-1-Methyl-1H-Benzo-
[d]imidazole-5-yl)-5-(4-flurophenyl)-1, 3, 4-Oxadiazole (8e)
Yield: 83%; mp 230-232 ºC; C23H16F2N4O; HRMS m/z
(%) 403.1376 (M+1) (calculated 403.1326) ; IR (KBr) cm-1
294 (C-H), 1623 (C=N), 1568 (C=C), 1091 (C-F) ; 1H NMR
(400 MHz; DMSO), δ 2.38 (s, 3H, CH3), 4.02 (s, 3H, N-CH3),
8.505 (s, Ar, 1H), 8.28-8.22 (m, Ar, 3H), 8.08 (d, J = 8.4 Hz,
Ar, 1H), 7.76 (d, J=10.4 Hz, Ar, 1H), 7.71 (d, J = 7.6Hz, Ar,
1H), 7.61 (t, J = 7.6 Hz, Ar, 1H), 7.51 (t, J = 8.4 Hz, Ar, 2H).
2.2.11. 2-(2-(3-fluoro-4-methylphenyl)-1-Methyl-1H-Benzo-
[d]imidazole-5yl)-5-(p-tolyl)-1, 3, 4-Oxadiazole (8f)
Yield: 80%; mp 220ºC; C24H19FN4O; HRMS m/z (%)
399.1625(M+1) (calculated 399.1576) ; IR (KBr) cm-1 2944
Kumar et al.
(C-H), 1614 (C=N), 1542 (C=C), 1093 (C-F), 891; 1H NMR
(400 MHz; DMSO), δ 2.35 (s, 3H, CH3), 2.42 (s, 3H, CH3),
3.96 (s, 3H, N-CH3), 8.44 (s, Ar, 1H), 8.09-8.00 (m, Ar, 3H),
7.88 (d, J = 8.4 Hz, Ar, 1H), 7.73-7.65 (m, Ar, 2H), 7.52 (t, J
= 7.6 Hz, Ar, 1H),7.45 (d, J = 7.6 Hz, Ar, 2H).
2.2.12. 2-(2-(3-fluoro-4-methylphenyl)-1-Methyl-1H-Benzo-
[d]imidazol-5-yl)-5-(4-(trifluoromethoxy) phenyl)-1, 3, 4-
Oxadiazole (8g)
Yield: 84%; mp 252 ºC; HRMS m/z (%) 469.1296 (M+1)
(calculated 469.1243) ; C24H16F4N4O2; IR (KBr) cm-1 2948
(C-H), 1602 (C=N), 1548 (C=C), 1098 (C-F); 1H NMR (400
MHz; DMSO), δ 2.35 (s, 3H, CH3), 3.96 (s, 3H, N-CH3),
8.40 (s, Ar, 1H,), 8.15 - 8.02 (m, ArH, 3H), 7.78 (d, J = 8.4
Hz, ArH, 1H), 7.73 - 7.62 (m, ArH, 2H), 7.67 (t, J = 7.6 Hz,
ArH, 1H), 7.35 (d, J = 7.6, ArH, 2H).
2.2.13. 2-(2, 4-dichlorophenyl)-5-(2-(3-fluro-4-methylphenyl)-
1-Methyl-1H-Benzo[d]imidazole-5-yl)-1, 3, 4-Oxadiazole
(8h)
Yield: 88%; mp 256 ºC; LC-MS m/z (%) 452.79 (M+)
(calculated 453.29) ; C23H15Cl2FN4O; IR (KBr) cm-1 2984
(C-H), 1610 (C=N), 1567 (C=C), 848 (C-Cl), 1067 (C-F); 1H
NMR (400 MHz; DMSO), δ 2.36 (s, 3H, CH3), 3.99 (s, 3H,
N-CH3), 8.50 (s, 1H, ArH), 8.02 (d, J = 8.2 Hz, ArH, 1H),
7.52 (s, 1H, ArH), 8.11 (d, J = 8.8, ArH Hz, 1H), 7.73 (d, J
=7.6 Hz, ArH, 2H), 7.60 (t, J = 7.6 Hz, ArH, 1H,).
2.2.14. 2-(2, 4-difluorophenyl)-5-(2-(3-fluoro-4-methylphenyl)-
1-Methyl-1H-Benzo[d]imidazol-5-yl)-1, 3, 4-Oxadiazole
(8i)
Yield: 81%; mp 220 ºC; HRMS m/z (%) 421.1289 (M+1)
(calculated 421.1232); C23H15F3N4O; IR (KBr) cm-1 2950
(C-H), 1623 (C=N), 1576 (C=C), 1098 (C-F). 1H NMR (400
MHz, DMSO) δ 2.37 (s, 3H, CH3), 3.98 (s, 3H, N-CH3), 8.50
(s, Ar,1H), 8.1 (d, J = 8.2 Hz, Ar, 1H), 7.56 (s, Ar, 1H,), 8.21
(d, J = 8.8 Hz, Ar, 1H),7.73 (d, , J = 7.6 Hz , Ar, 2H), 7.60
(t, J = 7.6 Hz, Ar, 1H).
2.2.15. 2(2-(3-fluoro-4-methylphenyl)-1-Methyl-1H-benzo-
[d]imidazole-5-yl)-5-(2, 3, 4, 5-tetraflurophenyl)-1, 3, 4-
Oxadiazole (8j)
Yield: 82 %; mp 263 ºC; HRMS m/z (%) 457.0697 (M+1)
(calculated 457.1043) ; C23H13F5N4O; IR (KBr) cm-1 2943
(C-H), 1622 (C=N), 1564 (C=C), 1091 (C-F) 1H NMR (400
MHz, DMSO), δ 2.37 (s, 3H, CH3), 3.99 (s, 3H, N-CH3),
8.528 (s, Ar,1H), 8.353 (m, Ar, 1H), 8.17 (d, J = 8.8 Hz,
Ar,1H), 7.98 (d, J = 8.8 Hz, Ar, 1H), 7.67-7.73 (m, Ar, 2H),
7.565 (t, J = 7.6 Hz, Ar, 1H).
2.3. Biological Assay
2.3.1. Cell Culture
The human melanoma cell line (A375) and human breast
cancer cell line (MDA-MB231) were procured from the
National Centre for Cell Science (NCCS Pune, India); and
human dermal fibroblast (HDF) cells isolated from foreskins
discarded in surgical procedures of healthy donors. Cells
cultured in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% heat-inactivated fetal bovine serum,
2 mmol/L glutamine, 1% antibiotics solution (100 U/mL
penicillin G and 100 mg/mL streptomycin). Cells were
Benzo[d]imidazol-5-yl)-5-(substituted)-1, 3, 4-Oxadiazoles
grown in 25 cm2 tissue culture flasks and maintained at 37oC
in a humidified incubator with 5 % CO2. All the consuma-
bles and plastic-wares were procured from Himedia, India
unless stated otherwise.
2.3.1.1. Cell Viability Assays
In vitro cytotoxicity was performed by 3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-di-phenyltetrazoliumbromide (MTT) as-
say [64]. When the density reached approximately 70 % con-
fluency, the cell culture was trypsinized and 100 µL cell sus-
pension (a density of 5000 cells/well) was added in 96-well
plates plate and allowed to adhere overnight. Next day, the
compounds were dissolved in DMSO by heating at 95̊ C for
10 minutes using Digital Heating Cooling Dry bath (Thermo
Scientific); cooled to room temperature and final stock con-
centration of 1mg/mL was prepared in DMEM (final DMSO
concentration was < 1 %). A serial dilution of drugs, ranging
from 2.5 to 320μg/mL, was prepared in DMEM and cells
were treated in triplicates. Untreated cells represented a con-
trol group and Cisplatin treated as a positive control. After
48 h of incubation, the media was aspirated, 100 µL MTT
(1 mg/mL) was added to each well and the plate was incu-
bated at 37oC for 4h. After incubation, the media/MTT solu-
tion was aspirated and 100 μL DMSO was added to all the
wells. Using multi-mode micro plate reader (FLUOstar
Omega; BMG Labtech), a uniform shaking (300 rpm for 5
min) was applied to dissolve Formosan crystals and the ab-
sorbance was recorded at 570 nm. Cell viability was calcu-
lated using the following equation: (test-sample OD-blank
control OD)/ (untreated control OD-blank control OD) × 100
%. A non-linear regression graph was plotted between cell
survival (%) and log10 drug/compound concentration and
the 50 % minimum inhibitory concentration (IC50) was de-
termined using Prism software version 7 (GraphPad Soft-
ware Inc., San Diego, CA, USA).
2.3.2. Microbial Study
Test systems, bacterial strains Staphylococcus aureus
Gram +ve (NCIM-5022) and Escherichia coli Gram –ve
(NCIM-5051) and fungal molds Candida albicans (ATCC-
10231) and Aspergillus flavus (ATCC-9643), were procured
from CSIR National Chemical Laboratory (NCL), Pune.
They were preserved on nutrient agar media slants and pota-
to dextrose agar (PDA) slants respectively at 4oC. Standards
drugs Ciprofloxacin (CIP) and Greseofulvin (GRIS) refer-
ence samples were purchased from Himedia, Mumbai,
INDIA. Dimethyl sulfoxide AR grade was used as control.
Antibacterial activity of compounds (8a-j) was tested
against Escherichia coli and Staphylococcus aureus bacterial
strains by agar well diffusion technique [65, 66]. Test
compounds were dissolved in negative control (CON)
dimethyl sulfoxide (DMSO) to attain different concen-
trations 10, 20, 30, 40, 50 and 60µg 50µL-1 and positive
control Ciprofloxacin (CIPR) (10μg50μL-1) was taken as a
standard. Nutrient agar media plates were prepared by pour-
ing melted agar which was cooled to 45 oC into the sterilized
petri dish and was allowed to solidify. Sterile agar media
Petri dish swabbed using sterile L-Shaped glass rod with
approximately 100μL of day-old matured broth culture of
individual bacterial strains. Five even bores of 6 m Min
diameter were bored in each petri plate using cork borer to
accommodate 50μL of the solution in each well. Sterile
micropipette tips were utilized to load 50μL of sample,
Letters in Drug Design & Discovery, 2019, Vol. 16, No. 9 997
control, and standard into the agar wells with precision.
After completion of inoculation, petri plates were kept under
incubation at 37oC for 36 hours. Zone of inhibition diameters
were measured in mm after the incubation time of 36 hours.
The experiment was performed in triplicates, average values
zone of inhibition are reported in Table 2.
Antifungal activity of all the benzimidazole derivatives.
(8a-j) was carried out against fungi such as Candida albicans
and Aspergillus flavus on potato dextrose agar media know
as poisoned food assay [65,66]. Test samples were dissolved
in negative control (CON) dimethyl sulfoxide (DMSO) to
attain different concentrations 10, 20, 30, 40 and 50µg /50µL-1
were used to assess the dose-dependent activity and positive
control Griseofulvin (GFIR) (10µg 50μL-1) was taken as a
standard. Fungal spores from the stock slants were trans-
ferred into sterile 5mL of 1% saline solution. About 15mL of
sterilized molten PDA medium was poured into each steri-
lized petri plates and was allowed to solidify, small portions
of the mycelium of each fungus were inoculated carefully on
PDA media plates with L-shape sterilized glass rod. After
inoculation, plates were incubated for five days at (25 ± 2)oC
to achieve uniform growth. Sterile cork borer was made use
to bore 5 wells of 6 mM diameter in each sterile petriplate.
Micropipette sterile tips were utilized to load 50 μL of
sample, control and standard into wells with care. Inoculated
petri plates were kept in incubation chamber at 27oC for about
2-3 days or until activity was observed. After completion of the
incubation period, the zone of inhibition diameter was measured
in mM. Experiments were repeated in triplicates and average
values calculated are represented in Table 3.
2.4. In Silico Predictions
A preliminary test of the drug-likeness of the title com-
pounds was calculated in accordance with Lipinski's rule of
five [67, 68]. All the newly synthesized compounds were
subjected to a computational program using QIKPROP [69]
module of Schrodinger software for the in silico determina-
tion of pharmacokinetic properties such as absorption, distri-
bution, metabolism and excretion (ADME). The properties
such as the percentage of human oral absorption, aqueous
solubility, IC50 value for blockage of HERG K+ channels
and blood-brain barrier permeability were also predicted to
know about the basic bioactivity of the compounds.
2.4.1. Docking Preparations
The 3D co-ordinates of crystallographic structure of
VEGFR-2 and TIE-2 kinase protein complex [70] (PDB ID:
2OH4) was downloaded from Brookheaven protein Data
Bank (www.rscb.com). The protein complex was pre-
processed and prepared by Protein Preparation Wizard [71]
in Maestro of Schrödinger software. The minimization of the
complex was continued using OPLS-2005 (Optimized
Potential for Liquid Simulations) force field [72] until the
root mean square deviation (RMSD) reached the value of 0.3
Å. The ligands for docking studies were prepared using Lig-
Prep [73], Schrödinger. The ligands were geometrically re-
fined and assigned appropriate protonation state at pH 7.0 ±
2.0. The preprocessed protein was then used to generate grid
for docking studies. The grid was assigned by picking the
ligand as the center of the grid and then the grid box was
generated by applying default parameters. The docking was
998 Letters in Drug Design & Discovery, 2019, Vol. 16, No. 9
Scheme 1. Reagents and Condition: (i) Ethanol, H2SO4, reflux, 4-6 h; (ii) CH3NH2, THF, TEA, r.t, 12 h; (iii) Na2S2O4, DMSO, 90º C, 3 h;
(iv) NH2NH2.2H2O, EtOH, reflux, 8 h; (v) R-CO2H (7a-j), POCl3, reflux 4 h.
carried out using GLIDE, Schrödinger. GLIDE XP (extra
precision) method was followed for docking calculations
[74]. Conformers were generated using torsional search
method with distance-dependent dielectric solvation
treatment and OPLS–2005 force field. The extra precision
(XP) mode of docking was used to find the best fit molecules
in the active site of VEGFR-2 and TIE-2 kinase protein
using Glide48 application of Schrödinger software suite.
3. RESULTS AND DISCUSSION
3.1. Synthesis
The new series of fluorinated benzo[d]imidazol-5-yl)-5-
(substituted)-1, 3, 4-oxadiazole derivatives (8a-j) were syn-
thesized by a series of reactions as depicted in Scheme 1.
The compound ethyl 4-chloro-3-nitrobenzoate (2) was syn-
thesized by esterification of 4-chloro-3-nitrobenzoic acid (1)
as per literature procedure [75]. The compound ethyl 4-
(methylamino)-3-nitrobenzoate (3) was synthesized by nu-
cleophilic displacement of chlorine atom of the compound
(2) by methylamine in the presence of triethylamine as a
base in tetrahydrofuran at room temperature [76]. The key
intermediate ethyl 2-(3-fluoro-4-methylphenyl)-1-methyl-
1H-benzo[d] imidazole-5-carboxylate (5) was obtained by
‘One pot’ nitro reductive cyclisation of compound (3) with
3-fluoro-4-methylbenzaldehyde (4) using reductive cyclizing
agent, sodium dithionite in dimethyl sulfoxide at 90ºC. This
synthetic protocol as described in the previous studies is
highly efficient in terms of yield, purity and reaction time.
The product was obtained in high purity, which was con-
firmed by spot to spot conversion in TLC & yield of 95%
indicates its efficiency. This reductive cyclisation reaction
was completed within 3hrs, which shows the highly conven-
ient route to access benzimidazole compare to other tradi-
tional methods. Generally in stepwise cyclisation method
initially requires reducing agent for conversion of nitro
group to an amine followed by cyclisation which requires
Kumar et al.
strong acidic or oxidizing conditions. Hence, compared to
stepwise methods, ‘One pot’ reductive cyclisation method is
very simple, appropriate and cost effective. This ‘one-pot’
method of preparation works very effectively with alkyl
substituted amines as well [76].
The compound (5) was refluxed with hydrazine hydrate
in ethanol to obtain 2-(3-fluoro-4-methylphenyl)-1-methyl-
1H-benzo[d]imidazole-5-carbo hydrazide (6) as pre-final
compound. Dehydrative cyclization of compound (6) was
achieved with ten different substituted aromatic carbox-
ylic acids (7a-j) in phosphorous oxychloride to get
benzo[d]imidazol-5-yl)-5-(substituted)-1,3,4-oxadiazole de-
rivatives (8a-j) in good yield. Purification of the compounds
was done by recrystallization in ethanol.
3.2. Biological Activity
3.2.1. Anticancer Activity
All the synthesized target compounds were screened for
their anticancer activity against two cancer cell lines, human
breast cancer MDA-MB-231 and melanoma cancer A375
cell lines using MTT assay. Results of the study are depicted
in Table 1. From the results, it is clear that all the compounds
showed good to moderate activity against both the cancer
cell lines. Among the screened derivatives, Compound 8j
with tetraflourophenyl unit exhibited prominent activity
against A375 cell line with IC50 36.76±0.66µM and shows
poor activity against MDA-MB-231 cell line with
IC50105.77±1µM.Compound 8a with furyl group in the
oxadiazole ring emerged as potential compound against both
cell lines with IC50 47.06±1.1µM against A375 cell line and
IC50 56.12±0.9 µM against MDA-MB-231 cell line. Further,
Compound 8g with trifluoromethoxyphenyl group shows
moderate activity against A375 cell line with IC50 52.88±1.2
µM and MDA-MB-231 cell line with IC50 81.12±1.5 µM.
Compound 8h with 2, 4,-dichloro phenyl ring in the
oxadiazole part shows moderate activity IC 50 62.18±1.3 µM
Benzo[d]imidazol-5-yl)-5-(substituted)-1, 3, 4-Oxadiazoles Letters in Drug Design & Discovery, 2019, Vol. 16, No. 9 999

against A375cell line andIC50 61.38±1.2 µM against MDA- Table 2. Antibacterial activity of compounds (8a-j).
MB-231 cell line. Compound 8d with 4-chloro phenyl shows
moderate activity IC50 75.32±1.4µM against A375cell line
but it fails to show activity against MDA-MB-231 cell line Zone of Inhibition (mm) (Mean±SE)
and 8i with 2, 4-difluoro phenyl group displayed moderate Sl. No. Compound
activity against A375 cell line with IC50 91.41±2.3 µM and S. Aureus E. Coli
IC50 67.53±1.2 µM against MDA-MB-231 cell line. Other
compounds are inactive against both the cell lines. From the 1 8a 19.22±0.32** 21.38±0.56**
anticancer study, it is clear that fluorine substitution has
2 8b 14.80±0.37 17.08±0.33
more effect on increasing its bioefficacy, and gives evidence
for the role of fluorine atom in the increased biological 3 8c 11.92±0.65 16.18±0.42
profile of the synthesized compounds.
4 8d 14.15±0.42 14.32±0.22
Table 1. Anticancer activity of compounds 8a-j (IC50 in μM)a.
5 8e 17.09±0.52* 22.45±0.25*

Sl. 6 8f 13.15±0.18 17.72±0.36

Compound A375b MDA-MB-231c
No.
7 8g 19.95±0.29** 24.38±0.23**
1 8a 47.06±1.1 56.12±0.9
8 8h 15.22±0.23* 22.11±0.41*
2 8b ----------- 235.37±2.3
9 8i 16.28±0.15* 21.78±0.16*
3 8c 119.63±0.69 106.55±1.8
10 8j 20.80±0.16** 25.65±0.17**

4 8d 75.32±1.4 124.98±2 11 CIPRO 21.00±0.12 26.7±0.33

5 8e 99.29±2.1 135.56±1.6 *, ** represents the significance of standard deviation observed for standard
drug and synthesized molecules during biological analysis in triplicates.
6 8f ----------- 181.01±2.5
3.3.2. Antifungal Activity
7 8g 52.88±1.2 81.12±1.5
All the synthesized target compounds were screened for
8 8h 62.18±1.3 61.38±1.2
their antifungal activities against C. albicans and A. flavus
9 8i 91.41±2.3 67.53±1.2 bacterial strains on potato dextrose agar media to known as
poisoned food assay [47, 48]. Results of antifungal activity
10 8j 36.76±0.66 105.77±1 are depicted in Table 3 From the antifungal screening studies.
11 Cisplatind 8.28±0.4 24.57±1.2
Table 3. Antifungal activity of compounds (8a-j).
a
IC50 is the concentration of compound required for 50% inhibition of cell viability
determined using MTT assay. Cells were treated with concentrations ranging from 2.5
to 320 µg/mL for 48 hrs. The resultsrepresent the mean ± standard deviations of three Zone of Inhibition (mm) (Mean±SE)
replicates. bA375 is human metastatic melanoma cancer cell line. cMDA-MB-231 is Sl. No. Compound
human breast cancer cell line. dCisplatin is used as positive control. C. albicans A. flavus

1 8a 21.67±0.21** 22.23±0.24**
3.3. Antimicrobial Activity
2 8b 14.31±0.31 16.26±0.21*

3.3.1. Antibacterial Activity 3 8c 13.45±0.61 17.61±0.19

All the target compounds were screened for their
4 8d 14.19±0.82 17.54±0.69
antibacterial activity against Gram +ve Staphylococcus aure-
us (NCIM-5022) and Gram-ve Escherichia coli (NCIM- 5 8e 19.82±0.28* 21.26±0.58**
5051) bacterial strains Antibacterial activity was carried out
by agar well diffusion technique. Results of antibacterial 6 8f 12.75±0.14 11.21±0.17
activity are depicted in Table 2. From the results of
7 8g 21.25±0.26** 23.87±0.14**
antibacterial activity, it is clear that some of the compounds
showed good activity against bacterial strains. Among the 8 8h 22.24±0.14** 22.55±0.16**
tested compounds, 8j with tetrafluorophenyl group exhibited
as potent activity against E. coli and S. Aureus bacterial 9 8i 12.28±0.14* 14.23±0.21
strains. Compound 8g with electron releasing 4-triflour-
omethoxy phenyl ring displayed remarkable activity against 10 8j 22.92±0.34** 24.78±0.33**
both the bacterial strains. Compound 8a with furyl ring
11 GRV 23.12±0.12** 25.21±0.19**
displayed moderate activity against E. coli and S. aureus
bacterial strains. Remaining compounds did not exhibit *, ** represents the significance of standard deviation observed for standard
promising activity against both the strains. drug and synthesized molecules during biological analysis in triplicates.
1000 Letters in Drug Design & Discovery, 2019, Vol. 16, No. 9 Kumar et al.

studies, compound 8j with tetrafluorophenyl ring exhibited
potent activity against C. albicans and A. flavus fungal
strains.Compound 8g with 4-triflouromethoxy ring and 8h
with 2,4-dichlorophenyl ring showed good activity against
both the strains. Compound 8e with 4-flouro substitution and
compound 8a with furyl ring exhibited moderate activity
against C. albicans and A. flavus molds. Increased
antimicrobial activity of compounds 8j and 8g was due to the
presence of fluorinated groups which has enhanced the
lipophilicity of these molecules. Remaining compounds did
not show remarkable activity against both the strains.
3.4. Molecular Docking Studies
In order to understand the possible mode of action of the
newly synthesized benzimidazole-oxadiazole molecules for
the observed anticancer activity, molecular docking studies
were performed. VEGFR-tyrosine kinase was selected as a
target protein for the docking studies based upon the
reported literature of benzimidazole scaffold.
Molecular docking studies were performed on 2OH4
protein of VEGFR-2 protein using Glide software of
Schrodinger molecular docking tool kit. The Lipinski’s rule of
five values of compounds (8a-j) indicates that the
compounds are endowed with drug-like properties.
Compound 8e showed no violation of Lipinski’s rule of five.
All other compounds showed one violation due to their
partition coefficient value between octanol and water being
more than 5 and the molecular weight greater than 500 amu.
The drug−like prediction results by Qikprop are tabulated in
Tables (4 and 5). The tested compounds have a good
percentage of human oral absorption. Predicted aqueous
solubility (QPlogS) parameter indicated that all the
compounds having poor aqueous solubility. The prediction
of blood-brain barrier permeability (QPlogBB) for the tested
compounds was assessed and all the compounds were
predicted to have acceptable values. The IC50 value of
HERG K+ channel blockage (QPlogHERG) was also
predicted and the results showed that all the tested
compounds have a moderate range of values.
Docking studies were carried out to evaluate the binding
affinity and the interactions between the synthesized
compounds and the VEGFR-2 tyrosine kinase (PDB ID:
2OH4). The docking score of the compounds (8a-j) varies
from -10.205 to -7.029. The evdw and ecoul are the
predicted van der Waals’ interaction and electrostatic
interaction energies between ligand and the protein docking
results in Table 6 clearly indicate that the water molecules
present in the active site of VEGFR-tyrosine kinase protein
plays an important role in binding
All the target compounds were docked at the binding
site of VEGFR-tyrosine kinase protein. The docking pic-
tures are depicted in Figs. (1 and 2). The binding site com-
prises of polar and hydrophobic residues which involve in
the binding with target molecules. Oxadiazole ring of the
molecule surrounded by hydrophobic pocket of the
VEGFR-2 tyrosine kinase. Oxygen of the oxadiazole ring
of 8j interacts with ASP1044 at a distance of 2.34 Å. Tetra-
fluorophenyl ring attached to the oxadiazole involve in the π-
π interaction with HIE 1024. Benzimidazole unit of the tar-
get compound also surrounded by hydrophobic residues.
Benzene ring of the benzimidazole involves in the π-π bond-
ing with LYS866. Similarly, compound 8a with furyl substi-
tution binds in the same pocket as that of 8j. The furyl ring
involves π-π interaction with HIE 1024. Oxygen of the
oxadiazole ring of 8a interacts with ASP1044 at a distance of
2.25 A Protonated N-H of imidazole forms hydrogen bond-
ing with PHE 1045. Also PHE 1045 involves π-π interaction
with 3-fluoro-4-methyl phenyl ring of the benzimidazole.
The benzene ring of the benzimidazole makes strong π-π
bonding with LYS 866.

Table 4. Lipinski’s rule of five factors of the compounds (8a-j).

Factors of Lipinski’s Rule of Five

Compound mol_MW(< 500) Donor HB (< 5) Accpt HB (<10) QPlogPo/w (< 5) Rule of Five

8a 374.373 0 4.5 4.681 0

8b 384.412 0 4 5.481 1

8c 418.857 0 4 5.976 1

8d 418.857 0 4 5.987 1

8e 402.402 0 4 5.721 1

8f 398.439 0 4 5.796 1

8g 468.41 0 4 6.634 1

8h 453.302 0 4 6.449 1

8i 420.393 0 4 5.924 1

8j 456.374 0 4 6.343 1
Benzo[d]imidazol-5-yl)-5-(substituted)-1, 3, 4-Oxadiazoles Letters in Drug Design & Discovery, 2019, Vol. 16, No. 9 1001

Table 5. Lipinski’s rule of five factors of the compounds (8a-j).

Pharmacokinetic Properties

Compound Percent Human Oral Absorptiona QPlogSb QPlogHERGc QPlogBBd

8a 100 -6.609 -6.646 -0.165

8b 100 -7.53 -7.018 -0.092

8c 100 -8.298 -6.932 0.067

8d 100 -8.3 -6.922 0.068

8e 100 -7.906 -6.889 0.017

8f 100 -8.139 -6.927 -0.113

8g 100 -9.067 -7.022 0.111

8h 100 -8.851 -6.797 0.286

8i 100 -8.153 -6.777 0.139

8j 100 -8.791 -6.557 0.322

a
Percentage of human absorption, Predicted aqueous solubility; S in mol/L, Predicted IC50 value for blockage of HERG K+ channels, Predicted blood brain barrier permeability.
b c d

Table 6. Molecular docking results of compounds (8a-j) with 2OH4.

Glide Glide Glide Glide Interacting

Compound
gscore evdw ecoul energy Residues

8a -10.205 -48.014 -4.153 -52.167 LYS 866, ASP 1044, PHE 1045, HIE 1024

8b -9.866 -50.1 -3.13 -53.23 LYS 866, ASP 1044, PHE 1045

8c -9.808 -46.371 -0.852 -47.223 LYS 866, ASP 1044, HIE 1024

8d -9.633 -51.715 -0.325 -52.04 LYS 866, ASP 1044

8e -9.744 -51.171 -0.327 -51.498 LYS 866, ASP 1044

8f -9.722 -51.264 0.115 -51.149 LYS 866, ASP 1044

8g -7.029 -32.551 -4.386 -36.937 LYS 866, PHE1045, H2O

8h -10.127 -49.122 -3.993 -53.116 LYS 866, ASP 1044, PHE 1045

8i -9.769 -52.056 2.354 -49.702 LYS 866, ASP 1044

8j -10.152 -50.226 0.297 -49.929 LYS 866, ASP 1044, HIE 1024

Fig. (1). Docking pose of the compound 8j with protein VEGFR-2 tyrosine kinase.
1002 Letters in Drug Design & Discovery, 2019, Vol. 16, No. 9 Kumar et al.

Fig. (2). Docking pose of the compound 8a with protein VEGFR-2 tyrosine kinase.

CONCLUSION
FUNDING
In the present study we designed and synthesized a series
of benzo[d]imidazol-5-yl)-5-(substituted)-1,3,4-oxadiazole None.
derivatives. The core moiety was prepared in good yield and
purity via ‘one-pot’ reductive cyclization method was found CONFLICT OF INTEREST
to be superior over all other conventional routes of synthesis.
The authors declare no conflict of interest, financial or
All the final compounds were screened for their anticancer l,
otherwise.
antimicrobia activities and in silico study was also performed.
Compound 8j and 8a emerged as a promising anticancer
agent against melanoma cancer cell line. These compounds ACKNOWLEDGEMENTS
are found to be more selective towards melanoma cancer
The authors are indebted to Dr. Senthamaraikannan Ka-
than the breast cancer cell lines. Further, Compound 8j and
bilan, Drug Discovery Lab, Annamalai University, Tamilna-
8g emerged as a prominent antimicrobial agent against all
du for molecular docking studies. We are gratefully ac-
the tested microorganisms. Form the study it was evident knowledging the Manipal University (Maniapl, Karnataka,
that fluorinated substitution plays an important role in
India) for providing spectral data. We are also thankful to
enhancing biological activity. In supplementary material,
Yenepoya Research Centre Deralakatte (Mangalore, Karna-
molecular docking studies suggest that target compounds 8a
taka, India) for their help in carrying out the anticancer
and 8j bind well at the binding pocket of 2OH4 protein of
screening studies. We are grateful to Sri Siddaganga Phar-
VEGFR-2 tyrosine kinase acceptor with remarkable
macy College (Tumkur, Karnataka, India) for providing the
hydrogen bonding and π−π interaction. According to the facility for us to perform the antimicrobial studies.
docking score, we can suggest that 8a and 8j molecules are
active same as in vitro studies. Among all the molecules,
compound 8j displayed as an active molecule in both SUPPLEMENTARY MATERIAL
anticancer and antimicrobial activity. So there is scope for Supplementary material is available on the publisher’s
future studies. web site along with the published article.

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