Design and synthesis of new 2,5-disubstituted-1,3,4-oxadiazole analogues as anticancer agents

Article abstract of DOI:10.1007/s00044-016-1672-1

In continuance of our search for new anticancer agents, we report herein the design, synthesis, and anticancer evaluation of oxadiazole analogues. Two series (4a-h and 4i-q) of new oxadiazole analogues were designed based on heterocyclic (1,3,4-oxadiazole

Full text of DOI:10.1007/s00044-016-1672-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1672-1
ORIGINAL RESEARCH
Design and synthesis of new 2,5-disubstituted-1,3,4-oxadiazole analogues
as anticancer agents
Mohit Agarwal1 Vikram Singh1 Sachin Kumar Sharma1 Piush Sharma1
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Md. Yousuf Ansari2 Surender Singh Jadav3 Sabina Yasmin3
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Reddymasu Sreenivasulu4 Mohd. Zaheen Hassan5 Vipin Saini6
Mohamed Jawed Ahsan1
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Received: 19 December 2015 / Accepted: 4 July 2016
© Springer Science+Business Media New York 2016
Abstract In continuance of our search for new anticancer
agents, we report herein the design, synthesis, and antic-
ancer evaluation of oxadiazole analogues. Two series (4a-h
and 4i-q) of new oxadiazole analogues were designed based
on heterocyclic (1,3,4-oxadiazole)-linked aryl core of IMC-
038525 (tubulin polymerization inhibitor), NSC 776715,
and NSC 776715 and synthesized. All the compounds were
fully characterized by infrared, nuclear magnetic resonance
spectroscopy, and mass spectral data and the purity of
compounds was checked by elemental analysis (C, H, and
N analysis). Further seven compounds were evaluated for
anticancer activity on nine different panels of 60 cell lines
(60 NCI cancer cell lines) according to the National
Cancer Institute screening protocol and percent growth and
percent growth inhibition was calculated at 10 µM drug
concentration. Ten compounds were evaluated for antic-
ancer activity on two cancer cell lines (HeLa and MDA-
MB-435) as per the standard protocol reported at four dif-
ferent drug concentrations (10−7, 10−6, 10−5, and 10−4 µM)
and GI50, LC50, and TGI dose-related parameters were
calculated. The compound 4j showed maximum anticancer
activity at 10 µM, and was found to have higher sensitivity
against MOLT-4, IGROV1, HCT-116, and K-562 with
percent growth inhibitions of 50.38, 48.45, 46.26, and 46.26
respectively. The compound 4j showed superior anticancer
activity than imatinib on 41 human cancer cell lines. The
compound 4p showed anticancer activity with GI50 of 36.7
and 46.5 µM against HeLa and MDA-MB-435 cell lines,
respectively.
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Keywords Anticancer Adriamycin IMC-038525
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Oxadiazole One dose assay Cancer cell lines
Mohit Agarwal and Vikram Singh contributed to this work equally.
* Mohamed Jawed Ahsan
jawedpharma@gmail.com
Introduction
1
Department of Pharmaceutical Chemistry, Maharishi Arvind
Cancer is a disease associated with uncontrolled cell growth
and proliferation and is responsible for nearly 13 % of total
death tolls worldwide. A total of 1,658,370 new cancer
cases and 589,430 cancer deaths are projected to occur in
the United States in 2015 (Siegel et al., 2015). In India the
total number of cancer cases are likely to go up from
979,786 cases in the year 2010 to 1,148,757 cases in the
year 2020 (Takiar et al., 2010). In spite of considerable
advancement in the understanding of molecular mechan-
isms of cancer pathogenesis, yet no specific treatment is
available to alleviate the disease completely. Despite the
availability of improved drugs and targeted cancer thera-
pies, it is expected that the new cases of cancer will jump to
College of Pharmacy Ambabri Circle, Jaipur, Rajasthan 302 039,
India
2
Pharmacoinformatics Department, National Institute of
Pharmaceutical Education and Research (NIPER), Hajipur 844
102 Bihar, India
3
Department of Pharmaceutical Chemistry, Birla Institute of
Technology, Mesra, Ranchi, Jharkhand 835 215, India
4
Department of Chemistry, , Acharya Nagarjuna University,
Nagarjuna Nagar 522 510 Andhra Pradesh, India
5
School of Chemical Science, University Sains Malaysia, Penang
118 00, Malaysia
6
M. M. College of Pharmacy, Maharishi Markandeshwar
University, Mullana, Ambala, Haryana 133207, India

Med Chem Res
19.3 million worldwide by 2025 (WHO World Cancer
Report, 2014). The therapeutic approach of cancer includes
chemotherapy, radiotherapy, surgery, immunotherapy,
monoclonal antibody therapy, hormonal therapy, targeted
therapy, and angiogenesis inhibition. In chemotherapy the
chemo-preventive molecules act by various molecular
mechanisms and may involve inhibition of initiation, pro-
motion, progression, and metastasis of cancerous cells, but
this process can also kill the normal cells. The use of
anticancer drugs not only affects the normal human cells but
are also restricted to their toxic potentials, resistance, and
genotoxicity. Due to these complications the demand for
relatively more effective and safer agents for cancer therapy
is today’s need to combat against cancer (Aydemir and
Bilaloglu, 2003).
The heterocyclic oxadiazoles being rich in biological
potential as antitubercular (Karabanovich et al., 2016),
anticancer (Abdel-Aziz et al., 2016), anticonvulsant (Rajak
et al., 2013), antimicrobial (Bakht et al., 2010), anti-HIV
(Khan et al., 2012), anti-inflammatory (Ramaprasad et al.,
2013) inspired us to go further and explore oxadiazoles .
Moreover, 1,3,4-oxadiazole heterocycles are very good
bioisosteres of amides and esters, and to a large extent
enhance the pharmacological activity by participating in
hydrogen bonding interactions with receptors (Zhang et al.,
2014). In the present investigation the heterocyclic (oxa-
diazole) linked aryl core of IMC-038525 (tubulin poly-
merization inhibitor), NSC 776715, and NSC 776716 was
taken and two newer series of oxadiazoles (4a-g and 4i-o)
were synthesized (Tuma et al., 2010; Ahsan et al., 2014).
The design of the title compounds (4a-g and 4i-o) is shown
in Fig. 1. Furthermore, the aryl group of the title compounds
(4a-g) was replaced with five-member heteroaryl (2-furyl)
group (4h) in the first series of oxadiazoles. The second
series of oxadiazoles (4i-q) was designed with the incor-
poration of methylene linker with a hope to increase the
pharmacological profile. The methylene linker (-CH2-) is
suggested to offer flexibility to the molecule due to sp3
hybridization and would result in increased pharmacologi-
cal activity. The terminal aryl group (4i-o) was further
replaced with five-member heteroaryl (2-furyl) group (4p)
and aliphatic alkyl (ethyl) group (4q) (Fig.2).
Experimental
General
All the chemicals were procured from Merck (Mumbai) and
S.D. Fine Chemicals (Mumbai). Melting points were
determined by open tube capillary method and are uncor-
rected. Purity of the compounds was checked on thin layer
chromatography (TLC) plates (silica gel G) using mobile
phase chloroform/methanol (8:2) and toluene/ethylacetate/
formic acid (5:4:1); the spots were located under iodine
vapor or UV light. Infra spectra were obtained on a Schi-
madzu 8201 PC, FT-IR spectrometer (KBr pellets). 1H
NMR and 13C NMR spectra were recorded on a Bruker AC
400 MHz spectrometer using Tetramethylsilane as internal
standard in dimethylsulphoxide (DMSO)d6. Mass spectra
were recorded on a Bruker Esquire LCMS using ESI and
elemental analyses were performed on Perkin-Elmer 2400
Elemental Analyzer.
Fig. 1 Rationally design
Core skeleton of IMC-038525
methylene linker provide flexibility to the
molecule due to sp3 hybridization
template for target molecules
obtained from anticancer drugs
IMC-038525, and reported
compounds (Ahsan et al., 2014)
Cl
H
N N
O
R2 = 4-F (NSC: 776716)
R2 = 4-OCH3 (NSC: 776715)
N
N N
N
O
R2
F
H
4i-o
R2
O
F
N N
O
N N
O
O
N
N
R2
H
H
NH
4a-g
N
IMC-038525

Med Chem Res
Fig. 2 Rationale template and
modification for target
molecules and introduction of
methylene linkage (-CH2-) to
provide flexibility
heteroaryl ring
(2-furyl)
heteroaryl ring
(2-furyl)
F
N
N
H
N
N N
N
O
O
H
F
methylene linker provide
flexibility to the molecole
due to sp3 hybridization
4h
4p
F
H
N
N
N
N
N
O
F
N
O
H
R
4i-o
R
4a-g
H
N N
O
N
F
4q
alkyl (ethyl)
Method for the synthesis of 4-fluorophenyl urea (2a)
throughout by TLC using chloroform/ methanol (8:2) and
acetone/n-hexane (8:2) as mobile phase.
4-Fluoro aniline (1) (0.1 mol; 11.1 g) was dissolved in 20 ml
glacial acetic acid and 10 ml hot water, and sodium cyanate
(0.1 mol; 6.5 g) in 80 ml of hot water was added with stir-
ring. It was then allowed to stand for 30 min, then cooled in
ice bath, filtered with suction, dried, and recrystallized from
boiling water to obtain 4-fluorophenyl urea (2a) (Azam et
al., 2010).
N,5-Bis(4-fluorophenyl)-1,3,4-oxadiazol-2-amine (4a)
IR (KBr) vmax: 3352 (NH), 1511 (C=N), 1252 (C–O–C,
1
oxadiazole stretch), 786 (C–F) cm−1; H NMR (400 MHz,
DMSO-d6): δ 7.21–7.24 (2H, d, J = 8.8 Hz, ArH), 7.27–
7.29 (2H, d, J = 8.8 Hz, ArH), 7.47–7.49 (2H, d, J = 4.9 Hz,
ArH), 7.62–7.63 (2H, d, J = 4.9 Hz, ArH), 8.81 (1H, s,
ArNH); 13C NMR (100 MHz, DMSO-d6): δ 162.2, 164.9,
152.1, 149.3, 138.9, 129.6, 121.2, 117.4, 116.8, 116.2;
EIMS m/z = 274 [M]+, 276 [M+2]+. Cal/Ana: [C (61.54)
61.58 H (3.32) 3.23 N (15.38) 15.32].
Method for the synthesis of 4-fluorophenyl
semicarbazide (3a)
Equimolar quantities of 4-fluorophenyl urea (2a) (0.05 mol;
7.70 g) and hydrazine hydrate (AR 99–100 %) (0.05 mol;
2.5 ml) in 50 ml ethanol were refluxed for 24 h with stirring.
The two-thirds volume of alcohol was distilled by vacuum
distillation and then the reaction mixture was poured into
the crushed ice. The resultant precipitate was filtered,
washed with water, and dried. The solid mass was recrys-
tallized from 25 ml absolute ethanol to obtain 4-fluor-
ophenyl semicarbazide (3) (Azam et al., 2010).
5-(4-Chlorophenyl)-N-(4-fluorophenyl)-1,3,4-oxadiazol-2-
amine (4b)
IR (KBr) vmax: 3334 (NH), 1507 (C=N), 1249 (C–O–C,
1
oxadiazole stretch), 806 (C–F), 694 (C–Cl) cm−1; H NMR
(400 MHz, DMSO-d6): δ 7.11–7.13 (2H, d, J = 8.8 Hz,
ArH), 7.45–7.47 (2H, d, J = 8.3 Hz, ArH), 7.61–7.62 (2H, d,
J = 4.9 Hz, ArH), 7.85–7.88 (2H, d, J = 8.4 Hz, ArH), 8.98
(1H, s, ArNH); 13C NMR (100 MHz, DMSO-d6): δ 164.2,
152.9, 149.3, 138.9, 134.7, 129.9, 128.7, 124.4, 117.8,
116.1; EIMS m/z = 290 [M]+, 292 [M+2]+, 294 [M+4]+. Cal/
Ana: [C (58.02) 58.06 H (3.13) 3.16 N (14.51) 14.42].
General method for the synthesis of N-(4-fluorophenyl)-
5-aryl-1,3,4-oxadiazol-2-amine analogues (4a-h)
4-Fluorophenyl semicarbazide (0.005 mol; 0.85 g) (3a) and
aromatic aldehydes (0.005 mol) were refluxed for 10–12 h
using 20 mol% NaHSO3 and ethanol-water system (1:2, v/
v) solvent (Sangshetti et al., 2011). After completion of
reaction, the excess solvent was removed and the con-
centrate was poured into crushed ice filter, washed with
water, dried, and recrystallized with absolute ethanol to
obtain the final product (4a–x). The reaction was monitored
4-{5-[(4-Fluorophenyl)amino]-1,3,4-oxadiazol-2-yl}phenol
(4c)
IR (KBr) vmax: 3412 (OH), 3334 (NH), 1508 (C=N), 1243
1
(C–O–C, oxadiazole stretch), 794 (C–F) cm−1; H NMR
(400 MHz, DMSO-d6): δ 7.02–7.04 (2H, d, J = 8.1 Hz,
ArH), 7.35–7.37 (2H, d, J = 8.1 Hz, ArH), 7.60–7.62 (2H,

Med Chem Res
d, J = 8.2 Hz, ArH), 7.85–7.87 (2H, d, J = 8.2 Hz, ArH),
8.91 (1H, s, ArNH), 10.52 (1H, s, OH); 13C NMR (100
MHz, DMSO-d6): δ 164.3, 158.7, 152.4, 149.3, 138.6,
128.9, 118.4, 117.8, 116.9, 116.1; EIMS m/z = 272 [M]+,
274 [M+2]+. Cal/Ana: [C (61.99) 62.00 H (3.72) 3.75 N
(15.49) 15.52].
cm−1; 1H NMR (400 MHz, DMSO-d6): δ 3.77 (3H, s,
OCH3), 6.94–6.96 (2H, d, J = 8.0 Hz, ArH), 7.02–7.04 (2H,
d, J = 8.0 Hz, ArH), 7.38 (1H, s, ArH), 7.61–7.64 (2H, m,
ArH), 8.72 (1H, s, ArNH), 10.56 (1H, s, OCH3); 13C NMR
(100 MHz, DMSO-d6): δ 164.3, 152.1, 151.2, 149.9, 145.1,
138.1, 121.2, 119.9, 117.9, 117.3, 116.3, 112.3, 56.6; EIMS
m/z = 301 [M]+, 302 [M+1]+. Cal/Ana: [C (59.80) 59.90 H
(4.01) 3.98 N (13.95) 13.96].
2-{5-[(4-Fluorophenyl)amino]-1,3,4-oxadiazol-2-yl}phenol
(4d)
IR (KBr) vmax: 3424 (OH), 3324 (NH), 1507 (C=N), 1263
N-(4-Fluorophenyl)-5-(furan-2-yl)-1,3,4-oxadiazol-2-amine
(4h)
1
(C–O–C, oxadiazole stretch), 796 (C–F) cm−1; H NMR
(400 MHz, DMSO–d6): δ 7.05–7.07 (2H, d, J = 8.0 Hz,
ArH), 7.23–7.25 (2H, d, J = 8.0 Hz, ArH), 7.63–7.82 (4H,
m, ArH), 8.87 (1H, s, ArNH), 10.49 (1H, s, OH); 13C NMR
(100 MHz, DMSO-d6): δ 164.3, 155.2, 152.4, 149.1, 138.2,
130.1, 128.1, 121.8, 117.9, 116.9, 116.3, 112.5; EIMS m/z
= 272 [M]+, 274 [M+2]+. Cal/Ana: [C (61.99) 67.00 H
(3.72) 3.75 N (15.49) 15.52].
IR (KBr) vmax: 3333 (NH), 1509 (C=N), 1254 (C–O–C,
1
oxadiazole stretch), 802 (C–F) cm−1; H NMR (400 MHz,
DMSO-d6): δ 6.92–6.94 (2H, d, J = 8.1 Hz, ArH), 6.97–
6.99 (2H, d, J = 8.1 Hz, ArH), 7.06–7.12 (3H, m, ArH),
8.48 (1H, s, ArNH); 13C NMR (100 MHz, DMSO-d6): δ
157.3, 152.1, 149.9, 147.4, 142.9, 117.9, 116.3, 105.3,
107.7; EIMS m/z = 245 [M]+, 246 [M+1]+. Cal/Ana: [C
(58.78) 58.80 H (3.29) 3.27 N (17.14) 17.16].
N-(4-Fluorophenyl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-
2-amine (4e)
IR (KBr) vmax: 3380 (NH), 1510 (C=N), 1253 (C–O–C,
Method for the synthesis of ethyl [(4-fluorophenyl)
amino]acetate (2b)
1
oxadiazole stretch), 1168 (O–CH3), 806 (C–F) cm−1; H
NMR (400 MHz, DMSO-d6): δ 3.77 (3H, s, OCH3), 6.94–
6.96 (2H, d, J = 8.2 Hz, ArH), 7.02–7.04 (2H, d, J = 8.2 Hz,
ArH), 7.62–7.64 (2H, d, J = 8.0 Hz, ArH), 7.75–7.77 (2H,
d, J = 8.0 Hz, ArH), 8.67 (1H, s, ArNH); 13C NMR (100
MHz, DMSO-d6): δ 164.0, 160.1, 152.0, 149.9, 138.7,
128.5, 118.5, 117.9, 116.3, 114.9, 56.2; EIMS m/z = 285
[M]+, 286 [M+1]+. Cal/Ana: [C (63.15) 63.19 H (4.24) 4.25
N (14.73) 14.76].
4-Fluoroaniline (1) (0.1 mol; 11.1 g) and ethylchoroacetate
(0.2 mol; ~24 ml) were taken in a round bottom flask and
suspended in 100 ml ethanol and (0.15 mol; ~20.1 g)
anhydrous sodium acetate trihydrate was added to the
mixture. The mixture was refluxed for 48 h on sand bath
with vigorous stirring, then cooled and the excess
solvent removed under reduced pressure. The residual
mass was triturated with ice water to remove sodium acetate
trihydrate and extracted with ethylacetate (3 × 50 ml)
and the ethylacetate layer was washed with 10 %
sodium hydroxide solution (3 × 30 ml) followed by
water (3 × 30 ml) and then dried over anhydrous sodium
sulphate and evaporated to dryness to obtain ethyl[(4-
fluorophenyl)amino]acetate (2b) as light brown solid (Fin-
ger et al., 1965).
5-(3,4-Dimethoxyphenyl)-N-(4-fluorophenyl)-1,3,4-
oxadiazol-2-amine (4f)
IR (KBr) vmax: 3368 (NH), 1512 (C=N), 1250 (C–O–C,
1
oxadiazole stretch), 1167 (O–CH3), 806 (C–F) cm−1; H
NMR (400 MHz, DMSO-d6): δ 3.80 (6H, s, OCH3), 7.04–
7.06 (2H, d, J = 8.1 Hz, ArH), 7.11–7.13 (2H, d, J = 8.2 Hz,
ArH), 7.62–7.66 (2H, m, ArH), 7.85 (1H, s, ArH), 8.61
(1H, s, ArNH); 13C NMR (100 MHz, DMSO-d6): δ 164.5,
152.1, 150.2, 149.9, 149.1, 138.7, 120.5, 119.5, 117.1,
116.6, 115.8, 112.7, 56.2; EIMS m/z = 315 [M]+, 316 [M
+1]+. Cal/Ana: [C (60.95) 60.91 H (4.48) 4.45 N (13.33)
13.36].
Method for the synthesis of 2-[(4-fluorophenyl)amino]
acetohydrazide (3b)
Ethyl[(4-fluorophenyl)amino]acetate
(2b)
(0.075 mol;
14.79 g) and hydrazine hydrate (0.15 mol; ~7.5 ml) was
refluxed in ethanol for 22 h on water bath. The two-third
volume of reaction mixture was removed under reduced
pressure and then poured into crushed ice to obtain 2-[(4-
chlorophenyl)amino]acetohydrazide (4) as light brown solid
(Finger et al., 1965).
4-{5-[(4-Fluorophenyl)amino]-1,3,4-oxadiazol-2-yl}-2-
methoxyphenol (4g)
IR (KBr) vmax: 3412 (OH), 3328 (NH), 1507 (C=N), 1252
(C–O–C, oxadiazole stretch), 1167 (O–CH3), 802 (C–F)

Med Chem Res
General method for the synthesis of N-(4-fluorophenyl)
[5-aryl/alkyl-1,3,4-oxadiazol-2-yl]methyl amine
analogues (4i-q)
115.1, 51.4; EIMS m/z = 285 [M]+, 286 [M+1]+. Cal/Ana:
[C (63.15) 63.10 H (4.24) 4.26 N (14.73) 14.76].
2-(5-{[(4-Fluorophenyl)amino]methyl}-1,3,4-oxadiazol-2-
2-[(4-Fluorophenyl)amino]acetohydrazide (3b) (0.001 mol;
0.91 g) and aromatic/aliphatic aldehydes was refluxed in
ethanol-water system (1:2, v/v) solvent and 20 mol%
NaHSO3 for 10–12 h (Sangshetti et al., 2011). After com-
pletion of reaction the excess solvent was removed and the
concentrate was poured into crushed ice filter, washed with
water, dried and recrystallized with absolute ethanol to
obtain the final product (4i-q). The completion of reaction
was monitored throughout by TLC using chloroform/
methanol (8:1) and acetone/n-hexane (8:2) as mobile phase.
yl)phenol (4l)
IR (KBr) vmax: 3429 (OH), 3349 (NH), 1507 (C=N), 1252
(C–O–C, oxadiazole stretch), 804 (C–F) cm−1; H NMR
1
(400 MHz, DMSO-d6): δ 4.32 (2H, s, CH2), 6.81–6.83 (2H,
d, J = 8.3 Hz, ArH), 6.93–6.95 (2H, d, J = 8.3 Hz, ArH),
7.08–7.10 (2H, d, J = 8.1 Hz, ArH), 7.67–7.69 (2H, d, J =
8.1 Hz, ArH), 8.52 (1H, s, ArNH), 10.42 (1H, s, OH); 13C
NMR (100 MHz, DMSO-d6): δ 164.5, 158.5, 151.3, 149.7,
143.2, 128.8, 118.2, 116.8, 116.1, 115.3, 51.6; EIMS m/z =
285 [M]+, 286 [M+1]+. Cal/Ana: [C (63.15) 63.12 H (4.24)
4.26 N (14.73) 14.72].
4-Fluoro-N-{[5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl]
methyl}aniline (4i)
4-Fluoro-N-{[5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl]
IR (KBr) vmax: 3349 (NH), 1509 (C=N), 1256 (C–O–C,
methyl}aniline (4m)
1
oxadiazole stretch), 802 (C–F) cm−1; H NMR (400 MHz,
DMSO-d6): δ 4.32 (2H, s, CH2), 6.92–6.94 (2H, d, J = 8.1
Hz, ArH), 6.98–7.00 (2H, d, J = 8.1 Hz, ArH), 7.07–7.08
(2H, d, J = 4.9 Hz, ArH), 7.85–7.86 (2H, d, J = 4.9 Hz,
ArH), 8.53 (1H, s, ArNH); 13C NMR (100 MHz, DMSO-
d6): δ 164.2, 162.9, 151.1, 149.3, 143.9, 129.6, 121.2,
116.8, 116.2, 115.9, 51.6; EIMS m/z = 287 [M]+, 288
[M+1]+. Cal/Ana: [C (62.72) 62.78 H (3.86) 3.85 N (14.63)
14.59].
IR (KBr) vmax: 3270 (NH), 1508 (C=N), 1249 (C–O–C,
1
oxadiazole stretch), 1166 (O–CH3), 813 (C–F) cm−1; H
NMR (400 MHz, DMSO-d6): δ 3.80 (3H, s, OCH3), 4.30
(2H, s, CH2), 7.02–7.04 (2H, d, J = 8.1 Hz, ArH), 7.23–
7.25 (2H, d, J = 8.1 Hz, ArH), 7.63–7.65 (2H, d, J = 8.0 Hz,
ArH), 7.85–7.87 (2H, d, J = 8.0 Hz, ArH), 8.61 (1H, s,
ArNH); 13C NMR (100 MHz, DMSO-d6): δ 164.3, 160.9,
151.9, 149.3, 138.7, 128.7, 118.4, 117.7, 116.4, 114.2,
56.2, 51.6; EIMS m/z = 299 [M]+, 300 [M+1]+. Cal/Ana: [C
(64.21) 64.19 H (4.71) 4.72 N (14.04) 14.06].
N-{[5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl]methyl}-4-
fluoroaniline (4j)
N-{[5-(3,4-Dimethoxyphenyl)-1,3,4-oxadiazol-2-yl]methyl}-
IR (KBr) vmax: 3343 (NH), 1512 (C=N), 1251 (C–O–C,
oxadiazole stretch), 806 (C–F), 696 (C–Cl) cm−1; 1H NMR
(400 MHz, DMSO-d6): δ 4.35 (2H, s, CH2), 6.91–6.93 (2H,
d, J = 8.0 Hz, ArH), 6.99–7.01 (2H, d, J = 8.0 Hz, ArH),
7.37–7.38 (2H, d, J = 4.9 Hz, ArH), 7.65–7.66 (2H, d, J =
4.9 Hz, ArH), 8.69 (1H, s, ArNH); 13C NMR (100 MHz,
DMSO-d6): δ 164.3, 151.1, 149.7, 143.8, 129.6, 128.2,
124.4, 116.8, 115.9, 51.6; EIMS m/z = 303 [M]+, 305
[M+2]+. Cal/Ana: [C (59.32) 59.28 H (3.65) 3.66 N (13.84)
13.89].
4-fluoroaniline (4n)
3318 (NH), 1512 (C=N), 1253 (C–O–C, oxadiazole
stretch), 1167 (O–CH3), 804 (C–F) cm−1; H NMR (400
1
MHz, DMSO-d6): δ 3.81 (3H, s, OCH3), 3.85 (3H, s,
OCH3), 4.31 (2H, s, CH2), 7.11–7.13 (2H, d, J = 8.1 Hz,
ArH), 7.22–7.24 (2H, d, J = 8.1 Hz, ArH), 7.66 (1H, s,
ArH), 7.72–7.74 (2H, d, J = 8.3 Hz, ArH), 8.61 (1H, s,
ArNH); 13C NMR (100 MHz, DMSO-d6): δ 164.3, 160.6,
152.1, 149.8, 138.6, 128.5, 118.6, 117.9, 116.3, 114.9,
56.2, 51.6; EIMS m/z = 329 [M]+, 330 [M+1]+. Cal/Ana: [C
(62.00) 62.05 H (4.90) 4.88 N (12.76) 12.74].
4-(5-{[(4-Fluorophenyl)amino]methyl}-1,3,4-oxadiazol-2-
yl)phenol (4k)
4-(5-{[(4-Fluorophenyl)amino]methyl}-1,3,4-oxadiazol-2-
IR (KBr) vmax: 3421 (OH), 3312 (NH), 1509 (C=N), 1253
yl)-2-methoxyphenol (4o)
1
(C–O–C, oxadiazole stretch), 802 (C–F) cm−1; H NMR
(400 MHz, DMSO-d6): δ 4.35 (2H, s, CH2), 6.71–6.73 (2H,
d, J = 8.0 Hz, ArH), 6.82–6.84 (2H, d, J = 8.1 Hz, ArH),
6.94–7.38 (4H, m, ArH), 8.61 (1H, s, ArNH), 10.51 (1H, s,
OH); 13C NMR (100 MHz, DMSO-d6): δ 165.4, 155.5,
151.9, 149.5, 143.8, 130.6, 128.5, 121.9, 116.9, 116.3,
IR (KBr) vmax: 3412 (OH), 3328 (NH), 1507 (C=N), 1252
(C–O–C, oxadiazole stretch), 1167 (O–CH3), 802 (C–F)
cm−1; 1H NMR (400 MHz, DMSO-d6): δ 3.81 (3H, s,
OCH3), 4.32 (2H, s, CH2), 6.91–6.93 (2H, d, J = 8.1 Hz,
ArH), 7.05–7.06 (2H, d, J = 8.1 Hz, ArH), 7.41 (1H, s,

Med Chem Res
ArH), 7.61–7.63 (2H, d, J = 8.2 Hz, ArH), 8.67 (1H, s,
ArNH), 10.52 (1H, s, OH); 13C NMR (100 MHz, DMSO-
d6): δ 164.3, 152.5, 151.9, 149.2, 145.8, 138.7, 121.1,
119.9, 117.3, 117.1, 116.4, 112.2, 56.6, 52.6; EIMS m/z =
315 [M]+, 316 [M+1]+. Cal/Ana: [C (60.95) 60.91 H (4.48)
4.50 N (13.33) 13.36].
calculated at each of the drug concentrations levels as: [(Tf
− Ti)/(C − Ti)] × 100 for concentrations for which Tf ≥ Ti and
[(Tf − Ti)/Ti] × 100 for concentrations for which Tf o Ti.
Anticancer activity of the 10 compounds (4a, 4c, 4d, 4g,
4h, 4k, 4l, 4o, 4p, and 4q) was estimated in two different
human cell lines (HeLa and MDA-MB-435) using the sul-
forhodamine B (SRB) protocol (Vichai and Kirtikara, 2006;
Prabhakaran et al., 2014). The cells were inoculated into 96-
well microtiter plates (90 μl/well) at appropriate plating
densities depending on the doubling time of individual cell
lines. The microtiter plates were incubated at 37 °C in a
carbon dioxide (CO2) incubator for 24 h prior to addition of
experimental drugs. The cells were then incubated for 48 h
with the test compounds and the experiment was concluded
by adding 30 % chilled trichloro acetic acid to the wells.
Cells were stained using 0.4 % SRB in 1 % acetic acid. The
SRB dye bound to the fixed cells was eluted using 10 mM
unbuffered Tris solution and the optical density was mea-
sured using enzyme-linked immunosorbent assay micro
plate reader. Percent growth (GP) was calculated on a plate-
by-plate basis for test wells relative to control wells and was
expressed as the ratio of average absorbance of the test well
to the average absorbance of the control wells multiplied by
100. Using the six absorbance measurements (time zero
(Tz), control growth (C), and test growth in the presence of
drug at the four concentration levels (Ti)], the percentage
growth was calculated at each of the drug concentration
levels. Percentage growth inhibition was calculated using
the formulae
4-Fluoro-N-{[5-(furan-2-yl)-1,3,4-oxadiazol-2-yl]methyl}
aniline (4p)
IR (KBr) vmax: 3331 (NH), 1507 (C=N), 1249 (C–O–C,
1
oxadiazole stretch), 804 (C–F) cm−1; H NMR (400 MHz,
DMSO-d6): δ 4.32 (2H, s, CH2), 6.91–6.93 (2H, d, J = 8.2
Hz, ArH), 6.98–7.00 (2H, d, J = 8.2 Hz, ArH), 7.09–7.18
(3H, m, ArH), 8.68 (1H, s, ArNH); 13C NMR (100 MHz,
DMSO-d6): δ 157.6, 152.3, 149.5, 147.3, 142.7, 117.7,
116.4, 105.8, 107.7, 52.6; EIMS m/z = 259 [M]+, 260
[M+1]+. Cal/Ana: [C (60.23) 60.28 H (3.89) 3.87 N (16.21)
16.19].
N-[(5-Ethyl-1,3,4-oxadiazol-2-yl)methyl]-4-fluoroaniline
(4q)
IR (KBr) vmax: 3321 (NH), 1507 (C=N), 1250 (C–O–C,
1
oxadiazole stretch), 806 (C–F) cm−1; H NMR (400 MHz,
DMSO-d6): δ 1.34–1.39 (3H, m, CH3), 2.55–2.59 (2H, m,
CH2), 4.31 (2H, s, CH2), 6.93–6.95 (2H, d, J = 8.1 Hz,
ArH), 6.97–6.99 (2H, d, J = 8.1 Hz, ArH), 8.61 (1H, s,
ArNH); 13C NMR (100 MHz, DMSO-d6): δ 156.6, 152.3,
149.5, 143.3, 116.4, 115.8, 52.6, 23.1, 14.9; EIMS m/z =
221 [M]+, 222 [M+1]+. Cal/Ana: [C (59.72) 59.76 H (5.47)
5.50 N (18.99) 18.93].
ꢀ
ꢁ
TiÀTz
CÀTz
´ 100 for concentrations Ti ꢀ Tz i:e:
TiÀTz is positive or zero
In vitro anticancer activity
ꢀ
ꢁ
Ti À Tz
´ 100 for concentrations Ti < Tz i:e:
Ti À Tz is negative
The anticancer screening was carried out on nine different
panels of cancer cell lines viz. leukemia, melanoma, lung,
colon, CNS, ovarian, renal, prostate, and breast cancers cell
lines, nearly 60 in number according to the reported NCI
US protocol (http://dtp.nci.nih.gov; Boyd and Paull, 1995;
Monks et al., 1991; Shoemaker 2006). Seven compounds
(4b, 4e, 4f, 4i, 4j, 4m, and 4n) were selected for anticancer
screening at National Cancer Institute (NCI), USA. NCI
selected the compounds for anticancer screening based on
the novelty of heterocyclic ring system, drug-like properties
utilizing the concept of privileged scaffolds, structure based
on computer-aided drug design, etc., while the structures
containing problematic linkage or functional groups (e.g.
nitro, nitroso, -N-N-, -N=N-, imine, semicarbazone, thia-
mides, thioureas) are avoided (http://dtp.nci.nih.gov). Using
the seven absorbance measurements (time zero (Ti), control
growth (C), and test growth in the presence of drug at the
five concentration levels (Tf)), the percentage growth was
Tz
Three-dose response parameters (GI50, TGI, and LC50)
were calculated for each of the experimental agents. Growth
inhibition of 50 % (GI50) was calculated from 100 × [(Ti −
Tz)/(C − Tz)] = 50, which was the drug concentration
resulting in a 50 % reduction in the net protein increase (as
measured by sulforhodamine B, SRB staining) in control
cells during the drug incubation. The total growth inhibition
(TGI) was calculated from Ti=Tz, which was the drug
concentration resulting in TGI and signified the cytostatic
effect. The LC50 was calculated from 100 × [(Ti − Tz)/(C −
Tz)] = −50, which was the drug concentration resulting in a
net loss of cells following treatment which indicated the
concentration of drug resulting in a 50 % reduction in the
measured protein at the end of the drug treatment as com-
pared to that at the beginning.

Med Chem Res
Results and discussion
Chemistry
2-[(4-fluorophenyl)amino]acetohydrazide (3b) as light
brown solid (Finger et al., 1965). In the final step equimolar
quantity of 2-[(4-fluorophenyl)amino]acetohydrazide (3a)
and aromatic/aliphatic aldehyde was refluxed for 10–12 h in
ethanol-water system (1:2, v/v) solvent using 20 mol%
NaHSO3 to obtain N-(4-fluorophenyl)[5-aryl/alkyl-1,3,4-
oxadiazol-2-yl]methyl amine analogues (4i-q). The com-
pletion of reaction was monitored throughout by TLC using
mobile phase chloroform/methanol (8:2), acetone/n-hexane
(8:2), and toluene/ethylacetate/formic acid (5:4:1) and the
spots were identified either in iodine vapor or UV chamber.
The yields of the title compounds were ranging between 62
and 85 % after recrystallization with absolute ethanol. The
oxadiazole analogues (4a-q) were synthesized as per the
reported method (Sangshetti et al., 2011).
The structures of the newly synthesized compounds were
confirmed by using modern analytical techniques viz. FT-
IR, NMR (1H and 13C), and mass spectral data. The purity
of the synthesized compounds was checked by elemental
analysis (C, H, and N analysis). Both the analytical and
spectral data of the compounds were in full agreement with
the proposed structure. In general, the IR spectra of the
compounds afforded oxadiazole stretching (C-O-C) at
1243–1263 cm−1, C=N stretching at 1507–1512 cm−1 and
NH stretching at 3270–3349 cm−1 while OH stretching was
observed at 3412–3429 cm−1 bands. In the Nuclear Mag-
netic Resonance spectra (1H NMR) the signals of the
respective protons of the oxadiazole analogues (4a-q) were
verified on the basis of their chemical shifts, multiplicities,
and coupling constants in DMSO-d6. The spectra of the
The title compounds (4a-q) were synthesized as per the
synthetic protocol outlined in Schemes 1 and 2 and thier
physical constants are given in Table 1. The oxadiazole
analogues of the series (4a-h) was synthesized starting from
4-fluroaniline (1). In the initial step 4-fluorophenyl urea (2a)
was synthesized from 4-fluroaniline (1) and sodium cyanate
in the glacial acetic acid as per the reported method (Azam
et al., 2010). In the subsequent step 4-fluorophenyl urea
(2a) and hydrazine hydrate was refluxed in ethanol for 24 h
to obtain 4-fluorophenyl semicarbazide (3a). In the final
step equimolar quantity of 4-fluorophenyl semicarbazide
(3a) and aromatic aldehydes was refluxed for 10–12 h using
20 mol% NaHSO3 and ethanol-water system (1:2, v/v)
solvent to obtain N-(4-fluorophenyl)-5-aryl-1,3,4-oxadia-
zol-2-amine analogues (4a-h). The oxadiazoles of the series
(4i-q) were synthesized starting from 4-fluoroaniline. In the
initial step equimolar quantity of 4-fluoroaniline (1) (0.1
mol; 11.1) and ethylchoroacetate (0.2 mol; ~24 ml) were
taken in a round bottom flask and suspended in 80–100 ml
ethanol and anhydrous sodium acetate trihydrate (0.15 mol;
~20.1 g) was added to the mixture. The mixture was
refluxed for 48 h on sand bath with vigorous stirring to
obtain intermediate semisolid ethyl[(4-fluorophenyl)amino]
acetate (2b). In the subsequent step compound 3b (0.075
mol; 14.79 g) and hydrazine hydrate (0.15 mol; ~7.5 ml)
was refluxed in ethanol for 22 h on water bath to obtain
Scheme 1 Protocol for the
synthesis of N-(4-fluorophenyl)-
5-aryl-1,3,4-oxadiazol-2-amine
analogues (4a-h): (i) Reagents:
NaCNO/AcOH; (ii)
H
N
O
O
N
N
F
4h
NH2NH2H2O/EtOH; (iii) and
(iv) ArCHO/ 20 mol% NaHSO3/
EtOH-H2O system (1:2, v/v)
iv
H
N
H
N
H
N
NH2
NH2
NH2
O
O
ii
i
F
F
F
3a
2a
1
iii
H
N
O
N
N
R
F
4a-g
R = 4-F, 4-Cl, 4-OH, 2-OH, 4-OCH3
R = 3,4-(OCH3)2, 4-OH-3-OCH3

Med Chem Res
F
O
N
H
O
N
N
4p
iv
F
H
N
O
H
N
O
NH2
NH2
O
N
H
O
N
H
N
v
ii
N
i
F
F
F
3b
4q
2b
1
iii
F
O
N
H
N
N
R
4i-o
R = 4-F, 4-Cl, 4-OH, 2-OH, 4-OCH3
R = 3,4-(OCH3)2, 4-OH-3-OCH3
Scheme 2 Protocol for the synthesis of N-(4-fluorophenyl)[5-aryl/
alkyl-1,3,4-oxadiazol-2-yl]methyl amine analogues (4i-q): (i)
Reagents: ClCH2COOEt; sodium acetate trihydrate/ EtOH; (ii)
NH2NH2H2O/EtOH; (iii), (iv) and (v) ArCHO/RCHO/ 20 mol%
NaHSO3/EtOH-H2O system (1:2, v/v)
compounds showed a singlet at δ 3.77–3.85 ppm corre-
sponding to OCH3 group; a singlet at δ 4.30–4.35 ppm
corresponding to methylene (-CH2-) linkage; singlet/doub-
let/multiplet at δ 6.92–7.88 ppm corresponding to aromatic
protons (ArH); a singlet at 8.52–8.98 ppm corresponding to
aromatic NH (ArNH) and a singlet at 10.42–10.56 ppm
corresponding to phenolic (OH) group. The nature of car-
bon atoms was characterized and verified by 13C NMR,
while the mass spectra of the compounds showed molecular
ion peak M+, (M+1)+,and (M+2)+.
respectively. The compound 4e showed maximum sensi-
tivity towards T-47D, K-562, A498, NCI-H522, PC-3, and
HCT-116 with percent GIs of 44.70, 43.63, 40.94, 39.54,
38.84, and 32.58, respectively. The compound 4i showed
maximum sensitivity towards MDA-MB-231/ATCC,
NCI-H522, and A549/ATCC with percent GIs of 41.48,
41.39, and 33.28, respectively. The compound 4m showed
maximum sensitivity towards T-47D, UO-31, and HCT-116
with percent GIs of 43.68, 38.24, and 32.77, respectively.
The compound 4n showed maximum sensitivity towards
T-47D, PC-3, UO-31, K562, RPMI-8226, and HOP-92
with percent GIs of 46.11, 44.06, 39.88, 38.55, 33.86, and
32.96, respectively. The compounds 4b, 4e, 4f, 4i, and 4n
showed moderate activity; however, the compound 4m
showed mean growth percent (GP) of 90.29 but found to be
inactive towards cancer cell lines. The compound 4j
showed maximum sensitivity towards MOLT-4, IGROV1,
HCT-116, K-562, RPMI-8226, PC-3, UO-31, MCF-7,
MDA-MB-231/ATCC, NCI-H522, MDA-MB-468, T-47D,
A549/ATCC, and HOP-92 with percent GIs of 50.38,
48.45, 46.26, 46.25, 45.94, 45.73, 45.67, 44.86, 44.43,
43.50, 43.12, 43.01, 33.87, and 32.95, respectively.
The compound 4j showed comparatively higher activity
among the series (4a-q) (Table 2). The compound that
showed growth inhibitions of ≥32 % was considered
to be active towards that particular cell lines and marked
with bold figure in Table 2 (Corona et al., 2009). When
Anticancer activity
Seven compounds (4b, 4e, 4f, 4i, 4j, 4m, and 4n) were
evaluated for their anticancer activity as per the NCI US
standard protocol at single dose assay (10 µM concentra-
tion) on leukemia, melanoma, lung, colon, CNS, ovarian,
renal, prostate, and breast cancers cell lines (nearly 60 in
numbers) and growth percent (GP) and percent growth
inhibition (%GI) were calculated. Ten compounds (4a, 4c,
4d, 4g, 4h, 4i, 4k, 4l, 4o, and 4p) were evaluated for
anticancer activity on HeLa (Cervix) and MDA-MB-435
(Breast) cancer cell lines at four different drug concentration
(10−7, 10−6, 10−5, and 10−4 µM) and three-dose response
parameters (GI50, TGI, and LC50) were calculated. The
compound 4b showed maximum sensitivity towards CCRF-
CEM and T-47D with %GIs of 41.55 and 37.80,

Med Chem Res

Med Chem Res
Table 2 In vitro anticancer activity of 2,5-disubstituted-1,3,4-oxadiazole analogues (4a-q)
Compound
Cancer cell lines assay in single dose assay 10 µM concentration
Mean growth
percent (GP)
Range of
GP
The most sensitive cell lines
GP of the most
sensitive cell line
% growth
inhibition (GI)
4a
4b
117.6
95.15
116.9-118.3
HeLa (Cervix Cancer)
118.3
116.9
58.45
66.20
70.64
74.03
75.43
77.42
97.7
−18.3
−16.9
41.55
37.80
29.36
25.97
24.57
22.58
2.3
MDA-MB-435 (Breast Cancer)
CCRF-CEM (Leukemia)
58.45-137.03
T-47D (Breast Cancer)
K-562 (Leukemia)
NCI-H522 (Non-Small Cell Lung Cancer)
MCF-7 (Breast Cancer)
SK-OV-3 (Renal Cancer)
4c
4d
4e
109.2
110.1
85.81
97.7-120.7
HeLa (Cervix Cancer)
MDA-MB-435 (Breast Cancer)
HeLa (Cervix Cancer)
120.7
105.4
114.8
55.30
56.37
59.06
60.42
61.16
67.42
68.60
69.74
80.63
82.37
85.19
87.39
87.70
127.4
117.8
116.0
123.7
58.52
58.61
66.72
68.64
70.34
70.86
49.62
51.55
53.74
53.75
54.06
54.27
54.33
55.16
55.57
56.50
56.88
56.99
66.13
67.05
−20.7
−5.4
105.4-114.8
55.30-131.98
MDA-MB-435 (Breast Cancer)
T-47D (Breast Cancer)
−14.8
44.70
43.63
40.94
39.58
38.84
32.58
31.40
30.26
19.37
17.63
14.81
12.61
12.30
−27.4
−17.8
−16.0
−23.7
41.48
41.39
33.28
31.36
29.66
29.14
50.38
48.45
46.26
46.25
45.94
45.73
45.67
44.86
44.43
43.50
43.12
43.01
33.87
32.95
K-562 (Leukemia)
A498 (Renal Cancer)
NCI-H522 (Non-Small Cell Lung Cancer)
PC-3 (Prostate cancer)
HCT-116 (Colon Cancer)
HT29 (Colon Cancer)
A549/ATCC (Non-Small Cell Lung Cancer)
HOP-92 (Non-Small Cell Lung Cancer)
PC-3 (Prostate cancer)
4f
99.13
80.63-114.27
NCI-H226 (Non-Small Cell Lung Cancer)
RPMI-8226 (Leukemia)
SNB-75 (CNS cancer)
4g
4h
4i
122.6
119.8
89.47
117.8-127.4
116-123.7
HeLa (Cervix Cancer)
MDA-MB-435 (Breast Cancer)
HeLa (Cervix Cancer)
MDA-MB-435 (Breast Cancer)
MDA-MB-231/ATCC (Breast Cancer)
NCI-H522 (Non-Small Cell Lung Cancer)
A549/ATCC (Non-Small Cell Lung Cancer)
U251 (CNS cancer)
58.52-118.31
HS 578T (Breast Cancer)
UACC-257 (Melanoma)
4j
76.12
49.62-126.50
MOLT-4 (Leukemia)
IGROV1 (Ovarian Cancer)
HCT-116 (Colon Cancer)
K-562 (Leukemia)
RPMI-8226 (Leukemia)
PC-3 (Prostate cancer)
UO-31 (Renal Cancer)
MCF-7 (Breast Cancer)
MDA-MB-231/ATCC (Breast Cancer)
NCI-H522 (Non-Small Cell Lung Cancer)
MDA-MB-468 (Breast Cancer)
T-47D (Breast Cancer)
A549/ATCC (Non-Small Cell Lung Cancer)
HOP-92 (Non-Small Cell Lung Cancer)

Med Chem Res
Table 2 continued
Compound
Cancer cell lines assay in single dose assay 10 µM concentration
Mean growth
percent (GP)
Range of
GP
The most sensitive cell lines
GP of the most
sensitive cell line
% growth
inhibition (GI)
4k
4l
119.1
110.2-127.9
114-119.4
HeLa (Cervix Cancer)
110.2
127.9
119.4
114.0
56.32
61.76
67.23
70.58
71.23
53.89
55.94
60.12
61.65
66.14
67.04
68.79
69.80
106.7
119.5
119.3
122.0
114.1
122.5
110.2
−27.9
−19.4
−14.0
43.68
38.24
32.77
29.42
28.77
46.11
44.06
39.88
38.35
33.86
32.96
31.21
30.20
−6.7
MDA-MB-435 (Breast Cancer)
HeLa (Cervix Cancer)
116.7
90.29
MDA-MB-435 (Breast Cancer)
T-47D (Breast Cancer)
4m
56.32-118.03
UO-31 (Renal Cancer)
HCT-116 (Colon Cancer)
SK-OV-3 (Ovarian Cancer)
HOP-62 (Non-Small Cell Lung Cancer)
T-47D (Breast Cancer)
4n
87.65
53.89-137.74
PC-3 (Prostate cancer)
UO-31 (Renal Cancer)
K-562 (Leukemia)
RPMI-8226 (Leukemia)
HOP-92 (Non-Small Cell Lung Cancer)
IGROV1 (Ovarian Cancer)
MDA-MB-231/ATCC (Breast Cancer)
HeLa (Cervix Cancer)
4o
4p
4q
113.1
120.6
118.3
106.7-119.5
119.3-122
MDA-MB-435 (Breast Cancer)
HeLa (Cervix Cancer)
−19.5
119.3
−22.0
−14.1
−22.5
MDA-MB-435 (Breast Cancer)
HeLa (Cervix Cancer)
114.1-122.5
MDA-MB-435 (Breast Cancer)
60
50
40
30
20
10
0
% GI of 4j
% GI of Imatinib
-10
-20
-30
Fig. 3 The comparison of percent growth inhibition (% GI) expressed by compound 4j and Imatinib against the NCI human cancer cell lines at 10 µM
compared with the standard drug imatinib, the compound 4j
showed superior anticancer activity over nearly 41 cancer
cell lines. The comparison of %GIs expressed by com-
pound 4j (blue) and imatinib (red) is shown in Fig. 3.
The compounds 4a, 4c, 4d, 4g, 4h, 4k, 4l 4o, 4p, and 4q
showed less anticancer activity with mean growth percent

Med Chem Res
Table 3 The percent control
growth of selected compounds at
different molar concentrations
Compound % control growth
Molar drug concentrations
HeLa
MDA-MB-435
10−7 10−6
10−7 M
10−6
M
10−5
M
10−4
62.2
M
M
M
10−5
M
10−4
M
4a
4c
107.6
109.7
94.1
118.3
97.7
116.9
107.1
112.3
120.5
115.1
104.7
89.3
118.9
116.8
117.0
118.0
122.5
127.7
114.3
123.9
120.9
121.2
10.7
116.9
120.7
114.8
117.8
123.7
127.9
114.0
119.5
122.0
122.5
49.8
110.9
108.8
114.7
113.7
118.1
126.0
112.6
113.2
100.4
120.4
−70.7
86.1
33.9
87.2
59.9
86.7
85.6
74.9
83.9
−13.1
83.5
4d
4g
4h
4k
4l
105.4
127.4
116.0
110.2
119.4
106.7
119.3
114.1
−72.7
113.7
110.2
105.1
121.2
104.5
107.2
95.7
−29.6
64.7
30.2
−40.3
21.0
4o
4p
4q
ADR
94.1
45.0
−26.3
37.7
−30.1
83.8
110.1
−71.0
−74.8
−29.4
−65.3
ADR Adriamycin
Fig. 4 a Growth curve of
selected oxadiazole analogues
over MDA-MB-435 (Human
Breast Cancer cell line) at
different molar concentrations
(µM). b Growth curve of
selected oxadiazole analogues
over HeLa (Human Cervix
Cancer cell line) at different
molar concentrations (µM)
a
Growth Curve: Human Breast Cancer Cell line (MDA-MB-435)
4a
4c
4d
4g
4h
4k
4l
150
100
50
0
10-7M
10-6M
10-5M
10-4M
4o
4p
4q
-50
-100
Molar Drug Concentrations
ADR
4a
b
Growth Curve: Human Cervix Cancer Cell line (HeLa)
4c
150
100
50
4d
4g
4h
4k
4l
0
4o
10-7M
10-6M
10-5M
10-4M
4p
4q
ADR
Molar Drug Concentrations
-50
-100
of 109.1–20.6 at 10 µM and the anticancer activity of
compounds was found to be increased with increase in
concentration (100 µM). The compound 4l showed
higher anticancer activity than standard drug adriamycin at
100 µM against HeLa cell lines while compound 4p showed
maximum sensitivity against MDA-MB-435 cell lines at
100 µM but comparatively less active than standard drug
adriamycin. The anticancer activity of 10 compounds

Med Chem Res
a
Compound 4a (GI50 = 49.1 µM)
Compound 4g (GI50 = 49.1 µM)
Compound 4l (GI50 = 42.0 µM)
Compound 4p (GI50 = 36.7 µM)
Control
Compound 4c (GI50 = 48.69 µM)
Compound 4k (GI50 = 75.31 µM)
Compound 4o (GI50 = 65.3 µM)
Fig. 5 a Images of growth control of MDA-MB-435 cancer cell line by oxadiazole analogues. b Images of growth control of HeLa cancer cell line
by oxadiazole analogues

Med Chem Res
b
Compound 4c (GI50 = 80.9 µM)
Control
Compound 4k (GI50 = 75.31 µM)
Compound 4p (GI50 = 46.5 µM)
Fig. 5 Continued
Table 4 Calculated values of LC50, TGI, and GI50 of the selected
oxadiazole analogues
Ten compounds (4a, 4c, 4d, 4g, 4h, 4i, 4k, 4l, 4o, and
4p) were evaluated for anticancer activity on HeLa (Cervix)
and MDA-MB-435 (Breast) cancer cell lines at four dif-
ferent drug concentration (10−7, 10−6, 10−5, and 10−4 µM)
and three-dose response parameters (GI50, TGI, and LC50)
were calculated (Table 4). The compound 4p showed
anticancer activity having GI50 of 36.7 µM against HeLa
and 46.5 µM against MDA-MB-435 cell lines. The LC50
was found to be 4100 µM for both the cell lines; however,
TGI value were ranging between 81.5 and 4100 µM over
HeLa and 76.3 and 4100 µM over MDA-MB-435 cell
lines. The images of growth control over cancer cell lines by
10 oxadiazoles are shown in Fig. 5a (MDA-MB-435) and
Fig 5b (HeLa).
The structure activity relationship was established with
the anticancer data. The oxadiazole-linked aryl nucleus
with 4-chloro substitution showed maximum anticancer
activity and followed by 4-fluoro, 4-methoxy and 3,4-
dimethoxy substitution. Substitution with 4-hydroxy and 2-
hydroxy was not very significant. The order of pharmaco-
logical activity followed as 4-Cl 4 4-F 4 4-OCH3 4 3,4-
(OCH3)2. The replacement of aryl ring with either
five heteroaryl (2-furyl) or alkyl group reduced the
anticancer activity. Furthermore, introduction of the
methylene (-CH2-) linkage was found to increase the
Compound
Micro molar drug concentrations calculated from graph
HeLa
LC50
MDA-MB-435
TGI
GI50
LC50
TGI
GI50
4a
4c
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
54.42
81.5
49.1
48.69
>100
49.1
>100
75.31
42
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
70.6
>100
>100
>100
>100
>100
>100
>100
>100
76.3
>100
80.9
89.95
>100
81.5
4d
4g
4h
4k
4l
>100
>100
>100
75.31
>100
>100
46.5
>100
>100
73.75
>100
76.3
4o
4p
4q
ADR
65.3
36.7
83.8
<0.1
>100
<0.1
>100
1.7
>100
<0.1
ADR Adriamycin
against HeLa and MDA-MB-435 at four different con-
centrations (10−7, 10−6, 10−5, and 10−4 µM) is given in
Table 3. The graphical presentation of growth curve of 10
oxadiazole analogues at different molar concentrations (µM)
is shown in Fig. 4a (MDA-MB-435) and Fig. 4b (HeLa).

Med Chem Res
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applications of the National Cancer Institute in vitro anticancer
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activity might be due to flexibility offered to the molecule
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Conclusion
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Two series of oxadiazoles were synthesized with and without
methylene linkage (-CH2-) in satisfactory yield. All the oxa-
diazole analogues were evaluated for their anticancer
screening as per the NCI US standard protocol. Some of the
oxadiazoles showed significant anticancer activity in one dose
assay at 10 µM. Furthermore, introduction of methylene
(-CH2-) was found to be promising in improving biological
profile. Among the series of oxadiazoles, the compound 4j
showed anticancer activity superior to that of the standard
drug Imatinib over 41 cancer cell lines could be considered as
lead for further optimization and drug discovery.
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Acknowledgments Antiproliferative data were provided by National
Cancer Institute (NCI US), Bethesda, MD, USA and Anticancer Drug
Screening Facility (ACTREC), Navi Mumbai, India. We are grateful
for all help provided by Prof. Doug Smallwood and Mr. Mohammed
Nayel NCI US), Bethesda, MD, USA. We are also grateful for all help
provided by Dr. Jyoti Kode, ACTREC, Navi Mumbai, India. The
people holding the management of Maharishi Arvind College of
Pharmacy, Jaipur, Rajasthan, India is acknowledged for providing
research facilities. We are also grateful to Dr. Reddy Institute of Life
Science, Hyderabad, Andhra Pradesh, India for providing spectral data
of synthesized compounds. The author VS is thankful to DST, Jaipur
for partial financial support (1156/2015).
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R, Sharma PC, Pawar RS, Kharya MD (2013) Novel limonene
and citral based 2,5-disubstituted-1,3,4-oxadiazoles: a natural
product coupled approach to semicarbazones for antiepileptic
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Compliance with ethical standards
Ramaprasad GC, Kalluraya B, Kumar BS, Mallaya S (2013)
Synthesis of new oxadiazole derivatives as anti-inflammatory,
analgesic, and antimicrobial agents. Med Chem Res 22:5381–
5389
Conflict of interest The authors declare that they have no conflict of
interest.
Sangshetti JN, Chabukswar AR, Shinde DB (2011) Microwave
assisted one pot synthesis of some novel 2,5-disubstituted 1,3,4-
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