Design and synthesis of newer 1,3,4-oxadiazole and 1,2,4-triazole based Topsentin analogues as anti-proliferative agent targeting tubulin

B i o o r g an i c C h e m i s tr y 9 5 (2 020 )1 03519

Contents lists available at ScienceDirect

Bioorganic Chemistry

journal homepage: www.elsevier.com/locate/bioorg

Design and synthesis of newer 1,3,4-oxadiazole and 1,2,4-triazole based

Topsentin analogues as anti-proliferative agent targeting tubulin

Fatima Naaz^a, Faiz Ahmad^b, Bilal Ahmad Lone^b, Yuba Raj Pokharel^b, Neeraj Kumar Fuloria^c,

⁎

Shivkanya Fuloria^c, Manickam Ravichandran^d, Lalitha Pattabhiraman^e, Syed Shaﬁ^f,,

⁎

M. Shahar Yar^a,

^aDepartment of Pharmaceutical Chemistry, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India

^bFaculty of Life Sciences and Biology, South Asian University, New Delhi 110021, India

^cFaculty of Pharmacy, AIMST University, Semeling Campus, Jalan Bedong-Semeling, Bedong, Kedah Darul Aman 08100, Malaysia

^dFaculty of Applied Science, AIMST University, Semeling Campus, Jalan Bedong-Semeling, Bedong, Kedah Darul Aman 08100, Malaysia

^eFaculty of Medical Sciences, AIMST University, Semeling Campus, Jalan Bedong-Semeling, Bedong, Kedah Darul Aman 08100, Malaysia

^fDepartment of Chemistry, School of Chemical and Life Science, Jamia Hamdard, New Delhi, India

A R T I C L E I N F O

A B S T R A C T

Keywords:

A set of two series of 1,3,4-oxadiazole (11a-n) and 1,2,4-Triazole (12a, c, e, g, h, j-n) based topsentin analogues

were prepared by replacing imizadole moiety of topsentin through a multistep synthesis starting from indole. All

the compounds synthesized were submitted for single dose (10 µM) screening against a NCI panel of 60-human

cancer cell lines. Among all cancer cell lines, colon (HCC-2998) and Breast (MCF-7, T-47D) cancer cell lines were

found to be more susceptible for this class of compounds. Among the compounds tested, compounds 11a, 11d,

11f, 12e and 12h, were exhibited good anti-proliferative activity against various cancer cell lines. Compounds

11d, 12e and 12h demonstrated better activity with IC₅₀2.42 µM, 3.06 µM, and 3.30 µM respectively against

MCF-7 human cancer cell line than that of the standard drug doxorubicin IC₅₀6.31 µM. Furthermore, 11d

induced cell cycle arrest at G0/G1 phase and also disrupted mitochondrial membrane potential with reducing

cell migration potential of MCF-7 cells in dose dependent manner. In vitro microtubule polymerization assays

found that compound 11d disrupt tubulin dynamics by inhibiting tubulin polymerization with IC₅₀3.89 μM

compared with standard nocodazole (IC₅₀2.49 μM). In silico docking studies represented that 11d was binding at

colchicine binding site of β-tubulin. Compound 11d emerged as lead molecule from the library of compounds

tested and this may serve as a template for further drug discovery.

1,3,4-oxadiazole

1,2,4-Triazole

Topsentin

Anti-proliferation

Colchicine

Tubulin

Cancer cell line

1. Introduction

taxol and Vinca binding sites of tubulin respectively (due to the over

expression of eﬄux pumps on cancer cells) [5,6], colchicine binding

Cancer comprises of more than 200 diseases which are illustrated by

the uncontrolled proliferation of cells with poor clinical prognosis [1].

There are various ways to control the cell proliferation. The new era of

research is paying attention towards the development of anti-cancer

agents that blocks the mitotic spindle during cell division [2]. These

mitotic spindles are the rays which arise from microtubules and these

microtubules are formed by polymerization of α- and β- tubulin het-

erodimer [3]. Compounds like colchicines, paclitaxel, vincristine are

well known for tubulin inhibitors which interacts at diﬀerent sites of β-

tubulin and inhibit cell division by interfering with microtubule dy-

namics [4]. Due to the escalating multidrug resistance (MDR) of human

cancer cells to the drugs i.e. paclitaxel and vincristine which interacts at

site inhibitors (CBSIs) have attracted great attention in search of new

chemical entities that interact diﬀerently with tubulin [7]. Several

natural as well as synthetic compounds with cis restricted geometries

were developed which interact the tubulin at colchicine binding site

[8]. Among the scaﬀolds, indole moiety has found to be versatile and

several indole containing compounds were developed with excellent

anti-proliferative activity [9]. Several indole based alkaloids have been

explored to develop new anti-cancer agents from the natural as well as

synthetic sources.

Topsentin is a bis-indole alkaloids found in several Mediterranean

shallow-water Sponges such as Topsentia genitrix, Spongosporites ruetzleri,

Spongosorites genitrix and sponge Hexadella sp [10]. It has received major

^⁎Corresponding authors.

E-mail addresses: syedshaﬁ@jamiahamdard.ac.in (S. Shaﬁ), yarmsy@rediﬀmail.com (M. Shahar Yar).

https://doi.org/10.1016/j.bioorg.2019.103519

Received 19 September 2019; Received in revised form 1 November 2019; Accepted 12 December 2019

A v a i l a b l e o n l i n e 2 3 D e ce m b e r 2 019

F. Naaz, et al.

BioorganicChemistry95(2020)103519

Fig. 1. Topsentin and other secondary metabolite of marine origin.

interest because of its broad spectrum of biological activities including

anti-viral [11], anti-inﬂammatory [12], Anti-Alziemers [10], and anti-

proliferative [13] (IC₅₀between 6 and 20 µg/mL) activities [11]. Var-

ious other bis-indole containing secondary metabolites (1–5) [14] have

also isolated from the marine invertebrate which have similar structural

framework as that of topsentin as shown in Fig. 1.

2. Materials and methods

2.1. General

All the chemicals and reagents used in the study were purchased

from Merck (India), Spectrochem, and Sigma Aldrich which were of

reagent grade. TLC was performed for indication of reaction completion

on 0.25 mm silica gel 60-F254 plates. UV light was used to visualize

spots. Melting points of all the synthesized compounds were measured

using Buchi labortechnik AG 9230 automated melting point apparatus

(Switzerland); IR spectra were recorded on Bruker ALPHA FT-IR

Inspired by reported anti-proliferative activity of topsentin and its

similarities with the structural template designed to develop the mo-

lecules that occupies the colchicines binding site, we aimed at the

synthesis of 1,3,4-oxadiazole and 1,2,4-triazole based topsentin analo-

gues that occupies colchicines binding site as demonstrated in Fig. 2.

Fig. 2. Design strategy of 1,3,4-oxadiazole (11a-n) and 1,2,4-triazole (12a, c, e, g, h, j-n) based Topsentin analogues.

2

F. Naaz, et al.

BioorganicChemistry95(2020)103519

spectrometer (Germany), ¹H and ¹³C NMR spectra were determined on

a Bruker (300 MHz and 75 MHz) spectrometer and chemical shifts were

expressed as ppm against TMS as internal reference. Mass spectras were

recorded on ESI-MS. All compounds prepared in this paper are novel

and conﬁrmed with spectral data. All the synthesized compounds were

recrystallized in methanol and were puriﬁed by column chromato-

graphy.

2.2.5.1. (1H-indol-3-yl)(5-((4-nitrophenyl)amino)-1,3,4-oxadiazol-2-yl)

methanone (11a). Orange solid; 96% yield; mp 299.7 °C; IR (cm⁻¹):

3449, 3363, 3202, 3119, 3057, 2988, 2935, 1644, 1593, 1596, 1551,

1390, 1325, 1224, 1156, 1087, 843, 792, 735, 665, 609, 530; ¹H NMR

(300 MHz, DMSO‑d₆) δ (ppm): 12.40 (s, 1H), 11.81 (s, 1H), 8.89 (s,

1H), 8.32 (d, 3H, J = 9.3 Hz), 7.84 (d, 2H, J = 9 Hz), 7.58 (d, 1H,

J = 6.9 Hz), 7.30 (t, 2H, J = 3 Hz); ¹³C NMR (75 MHz, DMSO‑d₆):

170.7, 160.3, 157.1, 154.9, 151.9, 138, 137, 126.4, 125.9, 124.1,

123.2, 121.7, 117.6, 113.8, 113.1; MS (ESI) m/z: [M+1]⁺350.1.

2.2. Chemistry

2.2.1. Procedure for synthesis of 2-(1H-indol-3-yl)-2-oxoacetyl chloride

(7)

2.2.5.2. (1H-indol-3-yl)(5-((2-methoxyphenyl)amino)-1,3,4-oxadiazol-2-

yl)methanone (11b). Yellow solid; 94% yield; mp 253 °C; 3456, 3211,

3040, 2965, 2892, 2847, 1637, 1610, 1589, 1545, 1505, 1554, 1222,

1153, 1126, 1069, 1030, 884, 855, 729, 630, 568, 520; ¹H NMR

(300 MHz, DMSO‑d₆) δ (ppm): 12.37 (s, 1H), 10.18 (s, 1H), 8.89 (s,

1H), 8.30 (d, 1H, J = 4.80 Hz), 8.01 (d, 1H, J = 7.5 Hz), 7.58 (d, 1H,

J = 5.1 Hz), 7.30 (d, 2H, J = 3.3 Hz), 7.11 (s, 2H), 7.03 (d, 1H,

J = 6.0 Hz), 3.89 (d, 3H, J = 11.4); ¹³C NMR (75 MHz, DMSO‑d₆):

170.9, 161.8, 157, 159.9, 137.9, 136.9, 127.3, 126.5, 124.6, 124,

123.1, 121.7, 121, 120.4, 113.8, 113.1, 112, 56.3; MS (ESI) m/z: [M

+1]⁺335.2.

10 g of indole (6) was dissolved in 100 mL of diethyl ether. After

complete dissolution, 10 mL of oxylyl chloride was added slowly to

reaction mixture and stirred for 15 min. Orange colour precipitate 2-

(1H-indol-3-yl)-2-oxoacetyl chloride (7) occurred in the reaction was

ﬁltered oﬀ and dried under high vaccum pump and immediately pur-

sued for the further reaction without any delay.

2.2.2. Procedure for synthesis of 2-(1H-indol-3-yl)-2-oxoacetohydrazide

(8)

Immediately, dried precipitate of compound 7 (10 g) was trans-

ferred slowly into a round bottomed ﬂask with 100 mL of hydrazine

hydrate for 15 min while stirring at ambient temperature. The reaction

mixture was further allowed to stir at ambient temperature for further

30 min. After the completion of the reaction monitored by TLC (1:1

ratio of Ethyl acetate: Hexane), the reaction mixture was poured onto

crushed ice. Creamish oﬀ white precipitates thus formed was ﬁltered oﬀ

and dried under vaccum. Creamish oﬀ white solid; 98% yield; ¹H NMR

(400 MHz, DMSO‑d₆) δ (ppm): 12.23 (s, 1H), 10.01 (s, 1H), 8.70 (s,

1H), 8.22–8.20 (m, 1H), 7.55–7.52 (m, 1H), 7.29–7.23 (m, 2H), 4.58 (s,

2H);MS (ESI) m/z: [M+1]⁺204.1.

2.2.5.3. (1H-indol-3-yl)(5-((4-methoxyphenyl)amino)-1,3,4-oxadiazol-2-

yl)methanone (11c). Yellow solid; 97% yield; mp 315.9 °C; 3432, 3347,

3188, 3055, 2994, 2937, 2899, 2811, 2757, 1650, 1599, 1509, 1554,

1223, 1159, 1081, 1041, 857, 827, 799, 772, 736, 602, 572, 530; ¹H

NMR (300 MHz, DMSO‑d₆) δ (ppm): 12.35 (s, 1H), 10.82 (s, 1H), 8.87

(s, 1H), 8.29–8.27(m, 1H), 7.59–7.53 (m, 3H), 7.32–7.27(m, 2H),

7.00–6.97 (m, 2H), 3.75 (s, 3H); ¹³C NMR (75 MHz, DMSO‑d₆):

170.9, 161.3, 156.7, 155.3, 137.8, 136.9, 131.8, 126.4, 124, 123.1,

121.7, 119.5, 115.8, 113.7, 113.1, 55.7; MS (ESI) m/z: [M+1]⁺335.1.

2.2.5.4. (5-((2-Fluorophenyl)amino)-1,3,4-oxadiazol-2-yl)(1H-indol-3-

yl)methanone (11d). Yellow solid; 95% yield; mp 282 °C; IR (cm⁻¹):

3506, 3428, 3188, 3053, 2992, 2938, 2899, 2816, 1649, 1598, 1508,

2.2.3. General procedure for the synthesis of 2-(2-(1H-indol-3-yl)-2-

oxoacetyl)-N-phenylhydrazine carbothioamide (10a-n)

0.5 g of compound 8 2-(1H-indol-3-yl)-2-oxoacetohydrazide and

0.53 g (1.2 equivalents) of diﬀerent substituted aryl and alkyl iso-

thiocyanates (9a-n) were reﬂuxed in absolute ethanol (5 mL) for 6 hr.

Solid precipitate was formed in the reaction mixture was ﬁltered of and

washed with cold alcohol to yield compounds (10a-n). TLC was run in

TEF 4:5:1 which indicate the completion of the reaction.

1554, 1395, 1228, 1159, 1080, 1040, 857, 798, 736, 605, 566, 535; ¹

H

NMR (300 MHz, DMSO‑d₆) δ (ppm): 12.38 (s, 1H), 10.85 (s, 1H), 8.85

(s, 1H), 8.31–8.28 (m, 1H), 8.11 (t, 1H, J = 9 Hz), 7.60–7.57 (m, 1H),

7.35–7.25 (m, 4H), 7.18 (t, 1H, J = 6 Hz); ¹³C NMR (75 MHz,

DMSO‑d₆): 170.8, 161.4, 157.2, 154.8, 151.6, 137.9, 136.9, 126.4,

126.3, 125.2, 125, 124.9, 124.1, 123.1, 121.9, 121.7, 116.3, 116,

113.8, 113.1; MS (ESI) m/z: [M+1]⁺323.1.

2.2.4. Typical procedure for synthesis of 2-(2-(1H-indol-3-yl)-2-

oxoacetyl)-N-phenylhydrazinecarbothioamide (10a)

2.2.5.5. (5-((4-Fluorophenyl)amino)-1,3,4-oxadiazol-2-yl)(1H-indol-3-

yl)methanone (11e). Yellow solid; 94% yield; mp 332.7 °C; IR (cm⁻¹):

3506, 3428, 3188, 3053, 2992, 2938, 2899, 2816, 1649, 1598, 1508,

1555, 1395, 1290,1228, 1159, 1080, 1040, 857, 798, 736, 605, 565,

535; ¹H NMR (300 MHz, DMSO‑d₆) δ (ppm): 12.37 (s, 1H), 11.09 (s,

1H), 8.88 (s, 1H), 8.31–8.28 (m, 1H), 7.68–7.64 (m, 2H), 7.60–7.57 (m,

1H), 7.31–7.23 (m, 4H); ¹³C NMR (75 MHz, DMSO‑d₆): 170.9, 161,

159.7, 156.7, 137.9, 136.9, 135, 126.4, 124.1, 123.1, 121.7, 119.5,

119.4, 116.4, 116.1, 113.7, 113.1; MS (ESI) m/z: [M+1]⁺323.1.

To 10 mL of absolute ethanol, 2 g of compound 8 and 2.16 g (1.2

equivalents) of 1-isothiocyanato-4-nitrobenzene 9a was added and re-

ﬂuxed for 6 hr. after completion of reaction; solid precipitate was

formed in reaction mixture. These Solid precipitate was ﬁltered oﬀ and

dried to aﬀord compound 10a. Yellow solid; 86% yield; ¹H NMR

(400 MHz, DMSO‑d₆)

δ (ppm): 12.29 (s, 1H), 10.85 (s, 1H),

10.19–10.13 (m, 2H), 8.79 (d, J = 2.8 Hz, 1H), 8.21–8.21 (m, 3H), 7.91

(d, J = 8.8 Hz, 2H), 7.57–7.55 (s, 1H), 7.30–7.27 (m, 2H); MS (ESI) m/

z: [M−1]⁺382.3.

2.2.5.6. (5-((3-Chlorophenyl)amino)-1,3,4-oxadiazol-2-yl)(1H-indol-3-

yl)methanone (11f). Yellow solid; 97% yield; mp 321.3 °C; IR (cm⁻¹):

3448, 3361, 3202, 3118, 3058, 2988, 2937, 1643, 1594, 1595, 1551,

1224, 1156, 1088, 1012, 887, 850, 792, 735, 664, 609, 529; ¹H NMR

(300 MHz, DMSO‑d₆) δ (ppm): 12.38 (s, 1H), 11.29 (s, 1H), 8.88 (s,

1H), 8.31–8.28 (m, 1H), 7.81 (s, 1H), 7.60–7.52 (m, 2H), 7.45–7.39 (m,

1H), 7.31–7.26 (m, 2H), 7.12 (d, 1H, J = 7.2 Hz); ¹³C NMR (75 MHz,

DMSO‑d₆): 170.8, 164.2, 160.6, 156.8, 150.1, 138, 136.9, 134, 131.3,

126.4, 124.1, 123.1, 122.5, 121.7, 117.2, 116.4, 113.7, 113.1; MS (ESI)

m/z: [M+1]⁺339.

2.2.5. General procedure for the synthesis of (1H-indol-3-yl)(5-

(phenylamino)-1,3,4-oxadiazol-2-yl)methanone (11a-n)

0.5

g

of

2-(2-(1H-indol-3-yl)-2-oxoacetyl)-N-phenylhy-

drazinecarbothioamide (10a-n) and 0.832 g (3 equivalents) of N-(3-

Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC.HCl)

with catalytic amount of Hydroxybenzotriazole (HOBt) were added to

5 mL of dry dimethylformamide (DMF). The reaction mixture was

stirred for 3–5 h at room temperature under inert atmosphere. After the

completion of reaction monitored by TLC (TEF, 4:5:1), the reaction

mixture was poured onto ice. The precipitate formed was ﬁltered oﬀ,

washed with excess of water, dried under high vaccum and re-

crystallized in ethanol to aﬀord the title compound 11a-n in pure form.

2.2.5.7. (5-((4-Chlorophenyl)amino)-1,3,4-oxadiazol-2-yl)(1H-indol-3-

yl)methanone (11g). Yellow solid; 98% yield; mp 334.4 °C; IR (cm⁻¹):

3

F. Naaz, et al.

BioorganicChemistry95(2020)103519

3445, 3358, 3325, 3286, 3209, 3118, 3058, 3028, 2991, 2940, 1644,

1597, 1593, 1552, 1390, 1222, 1157, 1092, 1012, 854, 829, 794, 736,

667, 605, 527; ¹H NMR (300 MHz, DMSO‑d₆) δ (ppm): 12.40 (s, 1H),

11.24 (s, 1H), 8.88 (s, 1H), 8.30 (d, 1H, J = 4.5 Hz), 7.70–7.65 (m, 2H),

7.60–7.57 (s, 1H), 7.49–7.45 (m, 2H), 7.31–7.28 (m, 2H); ¹³C NMR

(75 MHz, DMSO‑d₆): 170.9, 160.8, 156.8, 137.9, 137.6, 136.9, 129.5,

126.5, 126.4, 124.1, 123.1, 121.7, 119.4, 113.7, 113.1 MS (ESI) m/z:

[M+1]⁺339.1.

methanone (11m). Creamish white solid; 97% yield; mp 306.3 °C; IR

(cm⁻¹): 3448, 3362, 3202, 3119, 3057, 2988, 2935, 1644, 1593, 1596,

1551, 1224, 1156, 1087, 843, 792, 735, 665, 609, 530; ¹H NMR

(300 MHz, DMSO‑d₆) δ (ppm): 12.28 (s, 1H), 8.81 (s, 1H), 8.26 (t, 2H,

J = 7.8 Hz), 7.57–7.54 (m, 1H), 7.30–7.23 (m, 2H), 1.97 (s, 2H), 1.73

(s, 2H), 1.58 (d, 1H, J = 10.8 Hz), 1.36–1.26 (m, 5H); ¹³C NMR

(75 MHz, DMSO‑d₆): 171.1, 163.9, 156.9, 137.4, 136.9, 126.5, 123.9,

122.9, 121.7, 113.6, 113, 52.3, 32.6, 25.5, 24.7; MS (ESI) m/z: [M

+1]⁺311.1.

2.2.5.8. (5-((3-Bromophenyl)amino)-1,3,4-oxadiazol-2-yl)(1H-indol-3-

yl)methanone (11h). Yellow solid; 92% yield; mp 277.3 °C; IR (cm⁻¹):

3446, 3358, 3326, 3286, 3209, 3118, 3058, 3028, 2990, 2940, 1643,

1596, 1541, 1593, 1552, 1390, 1222, 1157, 1091, 854, 829, 793, 736,

661, 607, 528; ¹H NMR (300 MHz, DMSO‑d₆) δ (ppm): 12.39 (s, 1H),

11.29 (s, 1H), 8.89 (s, 1H), 8.31–8.28 (m, 1H), 7.96 (s, 1H), 7.58 (d, 2H,

J = 6 Hz), 7.39–7.20 (m, 4H); ¹³C NMR (75 MHz, DMSO‑d₆): 170.8,

162.1, 157.3, 137.9, 136.6, 133.6, 129, 127.2, 126.4, 123.1, 121.7,

117, 113.7, 113.1; MS (ESI) m/z: [M]⁺382, [M+1]⁺383, [M+2]⁺

384.

2.2.5.14. (5-(Ethylamino)-1,3,4-oxadiazol-2-yl)(1H-indol-3-yl)

methanone (11n). Creamish white solid; 80% yield; mp 232.9 °C; IR

(cm⁻¹): 3496, 3440, 3348, 3268, 3223, 3161, 3117, 3016, 2982, 2936,

2887, 2859, 2813, 1607, 1598, 1546, 1229, 1127, 1053, 854, 795, 769,

737, 693, 647, 594, 542, 530; ¹H NMR (300 MHz, DMSO‑d₆) δ (ppm):

12.29 (s, 1H), 8.81 (s, 1H), 8.26 (d, 2H, J = 7.5 Hz), 7.56 (d, 1H,

J = 8.1 Hz), 7.27 (t, 2H, J = 3.6), 3.91–3.83 (m, 2H), 1.23–1.18 (m,

3H); ¹³C NMR (75 MHz, DMSO‑d₆): 171.1, 164.4, 157, 137.4, 136.8,

126.4, 123.9, 123, 121.7, 113.6, 113, 37.9, 15.9; MS (ESI) m/z: [M

+1]⁺255.4.

2.2.5.9. (5-((2-Bromophenyl)amino)-1,3,4-oxadiazol-2-yl)(1H-indol-3-

yl)methanone (11i). Yellow solid; 94% yield; mp 256.4 °C; IR (cm⁻¹):

3447, 3359, 3206, 3118, 3058, 2989, 2939, 1643, 1595, 1594, 1551,

1390, 1223, 1156, 1090, 853, 831, 793, 736, 663, 609, 528; ¹H NMR

(300 MHz, DMSO‑d₆) δ (ppm): 12.35 (s, 1H), 10.38 (s, 1H), 8.86 (s,

1H), 8.29–8.26 (m, 1H), 7.89 (d, 1H, J = 8.1 Hz), 7.73 (d, 1H,

J = 7.8 Hz), 7.59–7.56 (m, 1H), 7.47 (t, 1H, J = 7.5), 7.32–7.25 (m,

2H), 7.18 (t, 1H, J = 7.5 Hz); ¹³C NMR (75 MHz, DMSO‑d₆): 170.8,

162.1, 157.3, 137.9, 136.9, 136.6, 133.6, 129, 127.2, 126.4, 125.2,

124.1, 123.1, 121.7, 117, 113.7, 113.1; MS (ESI) m/z: [M]⁺382, [M

+1]⁺383, [M+2]⁺384.

2.2.6. General procedure for the synthesis of (1H-indol-3-yl)(5-mercapto-

4-phenyl-4H-1,2,4-triazol-3-yl)methanone (12a, c, e, g, h, j-n)

To the methanolic solution of 2-(2-(1H-indol-3-yl)-2-oxoacetyl)-N-

phenylhydrazinecarbothioamide (0.1 g) (10a-n) 2 N solution of NaOH

(10 mL) was added to it and reﬂuxed for 12 h. After completion, re-

action was monitored by TLC (run in TEF 4:5:1), reaction mixture was

poured onto crushed ice and was acidiﬁed with 2 N HCl to give the solid

precipitate. The solid precipitate was ﬁltered oﬀ, washed with excess of

water, dried under high vaccum and recrystallized in ethanol to yield

pure compounds (12a, c, e, g, h, j-n).

2.2.5.10. (5-((4-Ethoxyphenyl)amino)-1,3,4-oxadiazol-2-yl)(1H-indol-3-

yl)methanone (11j). Yellow solid; 96% yield; mp 358.7 °C; 3505, 3427,

3188, 3053, 2992, 2939, 2816, 1650, 1598, 1508, 1555, 1395, 1229,

1159, 1080, 1040, 798, 736, 605; 1H NMR (300 MHz, DMSO‑d₆) δ

(ppm): 12.36 (s, 1H), 10.83 (s, 1H), 8.88 (s, 1H), 8.31–8.28 (m, 1H),

7.59–7.53 (m, 3H), 7.38–7.26 (m, 2H), 6.97 (d, 2H J = 9 Hz); 4.00 (q,

2H, J = 6.6 Hz), 1.32 (t, 3H, J = 6.9); ¹³C NMR (75 MHz, DMSO‑d₆):

170.9, 161.3, 156.6, 154.6, 137.8, 136.9, 131.6, 126.4, 124, 123.1,

121.7, 119.4, 115.4, 113.7, 113.1, 63.6, 15.1; MS (ESI) m/z: [M+1]⁺

349.1.

2.2.6.1. (1H-indol-3-yl)(5-mercapto-4-(4-nitrophenyl)-4H-1,2,4-triazol-3-

yl)methanone (12a). Orange solid; 96% yield; mp 290.5 °C; IR (cm⁻¹):

3439, 3398, 3289, 3228, 3107, 2575, 1709, 1591, 1509, 1448, 1342,

1087, 850, 792, 742, 676, 614; ¹H NMR (300 MHz, DMSO‑d₆) δ (ppm):

12.42 (s, 1H), 11.87 (s, 1H), 8.90 (d, 1H, J = 2.1 Hz), 8.32 (d, 3H

J = 8.7 Hz), 7.84 (d, 2H, J = 8.7 Hz), 7.58 (d, 1H, J = 5.7 Hz),

7.31–7.29 (m, 2H);¹³C NMR (75 MHz, DMSO‑d₆): 170.7, 160.2, 157.1,

144.7, 142, 138.1, 136.9, 126.3, 125.9, 124.1, 123.2, 121.7, 117.6,

113.7, 1133.2; MS (ESI) m/z: [M]⁺365.

2.2.6.2. (1H-indol-3-yl)(5-mercapto-4-(4-methoxyphenyl)-4H-1,2,4-

triazol-3-yl)methanone (12c). Yellow solid; 87% yield; mp 253.9 °C; IR

(cm⁻¹): 3484, 3336, 3275, 3120, 3043, 2911, 2535, 1639, 1597, 1509,

1433, 1300, 1235, 1141, 1009, 750, 609, 570; ¹H NMR (300 MHz,

DMSO‑d₆) δ (ppm): 14.33 (s, 1H), 12.36 (s, 1H), 10.83 (s, 1H), 8.88 (d,

1H, J = 3.3 Hz), 8.53 (d, 1H, J = 3 Hz), 8.04 (d, 1H, J = 7.5 Hz),

7.56–7.52 (m, 2H), 7.37–7.13 (m, 3H), 7.02 (d, 2H, J = 8.7 Hz), 3.80

(s, 3H); ¹³C NMR (75 MHz, DMSO‑d₆):169.8, 159.7, 139.2, 137.8,

137.1, 136.9, 129.4, 127.7, 126, 124.1, 123.1, 121.7, 121.5, 119.4,

114.8, 114.4, 113, 55.8; MS (ESI) m/z: [M+1]⁺351.2.

2.2.5.11. (5-(Benzylamino)-1,3,4-oxadiazol-2-yl)(1H-indol-3-yl)

methanonev (11k). Creamish white solid; 95% yield; mp 301.6 °C; IR

(cm⁻¹): 3448, 3360, 3203, 3118, 3058, 2988, 2937, 1643, 1594, 1595,

1551, 1390, 1223, 1156, 1089, 1012, 977, 887, 851, 792, 735, 664,

609, 529; ¹H NMR (300 MHz, DMSO‑d₆) δ (ppm): 12.29 (s, 1H), 8.81

(d, 2H, J = 4.2 Hz), 8.27 (d, 1H, J = 6 Hz), 7.55 (d, 1H, J = 7.5 Hz),

7.39–7.34 (m, 4H), 7.31–7.26 (m, 3H), 4.52 (d, 2H, J = 5.7 Hz); ¹³C

NMR (75 MHz, DMSO‑d₆): 171, 164.6, 157.2, 138.7, 137.5, 136.9,

128.9, 127.8, 127.7, 126.4, 123.9, 123, 121.7, 113.6, 113, 46.4; MS

+

(ESI) m/z: [M+1] 319.1.

2.2.6.3. (4-(4-Fluorophenyl)-5-mercapto-4H-1,2,4-triazol-3-yl)(1H-indol-

3-yl)methanone (12d). Yellow solid; 87% yield; mp 278.3 °C; IR

(cm⁻¹): 3387, 3356, 3217, 3049, 2922, 2581, 1611, 1578, 1502,

1447, 1388, 1223, 1201, 1136, 1057, 881, 851, 779, 730, 658, 628,

572; ¹H NMR (300 MHz, DMSO‑d₆) δ (ppm): 14.41 (s, 1H), 12.38 (s,

1H), 11.11 (s, 1H), 8.88 (s, 1H), 8.56 (s, 1H), 8.03 (d, 1H, J = 7.8 Hz),

7.67–7.63 (m, 1H), 7.58–7.52 (m, 1H), 7.47–7.43 (m, 1H), 7.36–7.17

(m, 3H); MS (ESI) m/z: [M−2]⁺336.3.

2.2.5.12. (5-(Butylamino)-1,3,4-oxadiazol-2-yl)(1H-indol-3-yl)

methanone (11l). Creamish white solid; 89% yield; mp 256 °C; IR

(cm⁻¹): 3517, 3448, 3362, 3202, 3118, 3058, 2988, 2936, 1644, 1594,

1596, 1551, 1390, 1224, 1156, 1088, 846, 792, 735, 664, 609, 530; ¹

H

NMR (300 MHz, DMSO‑d₆) δ (ppm): 12.28 (s, 1H), 8.82 (s, 1H),

8.29–8.23 (m, 2H), 7.58–7.55 (m, 1H), 7.31–7.24 (m, 2H), 3.30 (q, 2H,

J = 6.6 Hz), 1.63–1.53 (m, 2H), 1.43–1.31 (m, 2H), 0.92 (t, 3H,

J = 7.2 Hz); ¹³C NMR (75 MHz, DMSO‑d₆): 171.1, 164.6, 157, 137.4,

136.9, 126.5, 123.9, 122.9, 121.7, 113.6, 113, 42.6, 31.2, 19.8, 15; MS

(ESI) m/z: [M+1]⁺285.1.

2.2.6.4. (4-(4-Chlorophenyl)-5-mercapto-4H-1,2,4-triazol-3-yl)(1H-indol-

3-yl)methanone (12g). Yellow solid; 65% yield; mp 291.5 °C; IR

(cm⁻¹): 3444, 3230, 3102, 3046, 2586, 1633, 1598, 1482, 1419,

1304, 1237, 1090, 1003, 938, 856, 750, 676, 606, 540; ¹H NMR

2.2.5.13. (5-(Cyclohexylamino)-1,3,4-oxadiazol-2-yl)(1H-indol-3-yl)

4

F. Naaz, et al.

BioorganicChemistry95(2020)103519

(300 MHz, DMSO‑d₆) δ (ppm): 14.38 (s, 1H), 12.37 (s, 1H), 11.22 (s,

1H), 8.88 (s, 1H), 8.04 (d, 1H, J = 7.5 Hz), 7.68–7.53 (m, 3H),

7.47–7.38 (m, 2H), 7.29–7.18 (m, 2H); ¹³C NMR (75 MHz, DMSO‑d₆):

174, 169.7, 148.3, 139.1, 137, 134.3, 134, 130.2, 129.4, 126.1, 124.2,

123.2, 121.5, 119.4, 114.6, 112.7; MS (ESI) m/z: [M−1]⁺353.2.

¹³C NMR (75 MHz, DMSO‑d₆): 175.2, 168.2, 147.8, 138.7, 136.9,

126.2, 124.0, 123.0, 121.7, 114.5, 113.0, 42.8, 14.2; MS (ESI) m/z:

[M−1]⁺271.2.

2.3. Pharmacology

2.2.6.5. (4-(3-Bromophenyl)-5-mercapto-4H-1,2,4-triazol-3-yl)(1H-indol-

3-yl)methanone (12h). Yellow solid; 65% yield; mp 193.8 °C; IR

(cm⁻¹): 3460, 3416, 3264, 3144, 3003, 2914, 2845, 2555, 1701,

1598, 1542, 1505, 1438, 1284, 1210, 1115, 1066, 1015, 878, 844, 735,

622; ¹H NMR (300 MHz, DMSO‑d₆) δ (ppm): 14.49 (s, 1H), 12.40 (s,

1H), 11.31 (s, 1H), 8.89 (d, 1H, J = 3 Hz), 8.57 (d, 1H, J = 3.3 Hz),

7.96 (s, 1H), 7.58–7.50 (m, 2H), 7.48–7.20 (m, 4H); ¹³C NMR (75 MHz,

DMSO‑d₆): 177.1, 174, 163.3, 139.3, 136.7, 130.9, 130.3, 128.7, 127.6,

125.9, 125.4, 122.6, 123.4, 120.9, 112; MS (ESI) m/z: [M−1]⁺400.2.

2.3.1. Sixty cancer cell line screening at the NCI

All the synthesized compounds were screened for their anti-pro-

liferative activity against a panel of 60 cancer cell line at National

Cancer Institute, Bethesda, MD, USA as per the standard procedure

given at http://www.dtp.nci.nih.gov [15]. RPMI 1640 medium (5%

fetal bovine serum and 2 mM ^L-glutamine) was used to grow the human

tumor cell lines. All the tumor cells were incubated into 96-well mi-

crotiter plate. Then this plated was placed for incubation at 37 °C for

24 h. After that two plates of each cell line were ﬁxed with TCA in situ

and optical density was measured at this point which represented the

cell population of each cell line at the time of compound addition

(ODtzero). On the other hand, all the tested compounds were dissolved

in DMSO to yield 400-fold desired ﬁnal concentration and stored at

−80 °C. These frozen compounds were thawed and their aliquot part

was diluted to 10⁻⁴M concentration with the medium containing

50 µg/mL of gentamicin at the time of compound addition. Control

sample was made with DMSO only. 100 µl of the tested compounds

from the aliquot parts were added to appropriate 96-well microtiter

plate containing 100 µl of medium ensuing in the required ﬁnal drug

concentrations of 10⁻⁵M and 0 M (control). After addition of tested

compounds, 96-well microtiter plate was incubated for 48 h at 100%,

5% CO₂, 95% air, 100% relative humidity. Cold TCA was used to stop

the assay for adherent cells. Further on 50 mL of 50% (w/v) TCA was

used to ﬁx the cell and incubated for 1 hr at 4 °C. The supernatant was

removed, and the 96-well microtiter plates were rinsed ﬁve times with

water and air dried. 100 mL solution of protein binding dye, Sulfor-

hodamine B (SRB) was made at 0.4% (w/v) in 1% acetic acid and was

added to each well of the plates. These Plates were placed at room

temperature for incubation for 10 min then were washed with 1%

acetic acid ﬁve times to remove unbound dye. Then the plates were

treated with 10 Mm trizma base, so that unbound dye was solubilized

with trizma base. The absorbance was measured at a wavelength of

515 nm on an automated plate reader and results for each tested

compounds were calculated as the percent of tumor growth of the

treated cells in comparison with the untreated control cells. Optical

density (OD) was recored for SRB-derived color just before exposing the

cells to the test compound (ODtzero) and after 48hrs exposure to the

test compound (ODtest) or the control vehicle (ODctrl).

2.2.6.6. (4-(4-Ethoxyphenyl)-5-mercapto-4H-1,2,4-triazol-3-yl)(1H-indol-

3-yl)methanone (12j). Orange solid; 60% yield; mp 262 °C; IR (cm⁻¹):

3455, 3304, 3231, 3099, 3041, 2973, 2594, 1639, 1604, 1514, 1480,

1432, 1304, 1268, 1229, 1146, 1119, 1033, 857, 825, 747, 647; ¹H

NMR (300 MHz, DMSO‑d₆) δ (ppm): 14.32 (s, 1H), 12.37 (s, 1H), 10.83

(s, 1H), 8.88 (d, 1H, J = 2.1 Hz), 8.54 (d, 1H, J = 2.4 Hz), 8.04 (d, 1H,

J = 7.8 Hz), 7.54 (d, 1H, J = 8.1 Hz), 7.29–7.18 (m, 3H), 7.00 (d, 2H,

J = 7.8 Hz), 4.06 (q, 2H, J = 6.9 Hz), 1.35 (t, 2H, J = 6.9 Hz); ¹³C

NMR (75 MHz, DMSO‑d₆): 174.4, 169.8, 159.0, 148.8, 139.2, 137.1,

129.4, 127.5, 126.0, 124.1, 123.1, 121.5, 115.4, 114.9, 114.8, 113.1,

63.7, 15.11; MS (ESI) m/z: [M+1]⁺365.2.

2.2.6.7. (4-Benzyl-5-mercapto-4H-1,2,4-triazol-3-yl)(1H-indol-3-yl)

methanone (12k). Creamish white solid; 98% yield; mp 258.8 °C; IR

(cm-1): 3474, 3396, 3289, 3121, 3093, 2975, 2925, 2643, 1673, 1633,

1524, 1478, 1436, 1417, 1360, 1268, 1120, 1011, 884, 855, 774, 727,

629, 551; ¹H NMR (300 MHz, DMSO‑d₆) δ (ppm): 14.53 (s, 1H), 12.34

(s, 1H), 8.54 (s, 1H), 7.55 (d, 1H, J = 1.5 Hz), 7.38–7.21 (m, 7H), 5.62

(s, 2H); ¹³C NMR (75 MHz, DMSO‑d₆): 174.9, 169.3, 147.5, 138.6,

136.9, 128.8, 127.7, 127.5, 126.3, 124.1, 123.2, 121.7, 114.2, 113.1,

47.4; MS (ESI) m/z: [M+1]⁺335.2.

2.2.6.8. (4-Butyl-5-mercapto-4H-1,2,4-triazol-3-yl)(1H-indol-3-yl)

methanone (12i). Pale Yellow solid; 70% yield; mp 225.9 °C; IR (cm⁻¹):

3367, 3100, 3043, 2960, 2925, 2560, 1613, 1581, 1512, 1474, 1418,

1361, 1270, 1196, 1118, 998, 884, 854, 746, 643, 560, 529; ¹H NMR

(300 MHz, DMSO‑d₆) δ (ppm): 14.29 (s, 1H), 12.35 (s, 1H), 10.83 (s,

1H), 8.52 (s, 1H), 8.27 (d, 1H, J = 5.1 Hz), 7.56 (d, 1H, J = 7.2 Hz),

7.29 (m, 2H), 4.32–4.27 (m, 2H), 1.71–1.66 (m, 2H), 1.34–1.27 (m,

2H), 0.88 (t, 2H, J = 7.2 Hz); ¹³C NMR (75 MHz, DMSO‑d₆): 175.3,

168.5, 147.8, 138.7, 137, 126.3, 124.18, 123.2, 121.77, 114.5, 113.1,

44.4, 30.7, 19.7, 14; MS (ESI) m/z: [M+1]⁺301.2.

2.3.2. Anti-proliferative assay against MCF-7 cancer cell line

Compounds 11a, 11d, 11f, 12e, and 12h, showed consistent ac-

tivity against MCF-7 Breast cancer cell line with lower % growth and

was selected to evaluate its anti-proliferative activity against MCF-7

cancer cell line using MTT assay [16]. Doxorubicin and combretastatin

A-4 (CBSIs) were used as a standard. Cancer cells were seeded on 96-

well plate (3–5 × 10³cells per well) and incubated for 48 h at 37 °C

then treated with diﬀerent concentration of the test compound with

respective to vehicle control. After 48 h of incubation, it was exposed to

20 µl of freshly prepared MTT reagent (3-(4, 5-dimethylthiazol-2-yl)-

2,5-diphenyl tetrazolium bromide) incubated for 2 h. The supernatant

from each well was removed and replaced with DMSO (100 µl) to

dissolve formazan crystal. The optical density (absorbance) was re-

corded at 570 nm with reference wavelength of 620 nm and its half

2.2.6.9. (4-Cyclohexyl-5-mercapto-4H-1,2,4-triazol-3-yl)(1H-indol-3-yl)

methanone (12m). Creamish white solid; 92% yield; mp 251.3 °C; IR

(cm⁻¹): 3443, 3334, 3206, 3161, 2927, 2569, 1713, 1626, 1518, 1430,

1341, 1204, 1149, 1119, 1021, 856, 744, 650, 606; ¹H NMR (400 MHz,

DMSO‑d₆) δ (ppm): 14.19 (s, 1H), 12.44 (s, 1H), 8.33 (s, 1H), 7.81 (s,

1H), 7.56–7.50 (m, 3H), 7.32–7.28 (m, 2H), 4.66 (t, J = 11.6 Hz, 1H),

2.24–2.15 (m, 2H), 1.84–1.52 (m, 4H), 1.32–1.08 (m, 4H); ¹³C NMR

(75 MHz, DMSO‑d₆): 174.6, 162.7, 157, 136.7, 130.9, 125.4, 120.9,

112.0, 52.9, 25.9; MS (ESI) m/z: [M−1]⁺325.3.

2.2.6.10. (4-Ethyl-5-mercapto-4H-1,2,4-triazol-3-yl)(1H-indol-3-yl)

methanone (12n). Pale yellow solid; 81% yield; mp 276.3 °C; IR

(cm⁻¹): 3408, 3278, 3102, 3042, 2878, 2582, 1649, 1607, 1512,

1478, 1445, 1341, 1277, 1203, 1146, 1118, 1086, 987, 860, 792, 777,

743, 652, 604; ¹H NMR (300 MHz, DMSO‑d₆) δ (ppm): 14.30 (s, 1H),

12.34 (s, 1H), 8.52 (s, 1H), 8.29–8.26 (m, 1H), 7.57–7.55 (m, 1H),

7.30–7.27 (m, 2H), 4.32 (q, 2H, J = 6.6 Hz), 1.30 (t, 3H, J = 6.9 Hz);

maximal inhibitory concentration (IC₅₀

) was calculated from the

equation of the dose response curve.

2.3.3. Cell cycle analysis

Eﬀect of 11d on the cell cycle of MCF-7 cell line was evaluated by

using ﬂow cytometry at 0.5 µM, 1 µM, 2 µM, 5 µM and 10 µM con-

centration. These cells were seeded into 6 well plates and placed for

5

F. Naaz, et al.

BioorganicChemistry95(2020)103519

Scheme 1. Protocol for the synthesis of indole linked 1,3,4-oxadiazole conjugates (11a-n) and 1,2,4-triazole conjugates (12a, c, e, g, h, j-n).

incubation at 37 °C for 24 h which lead to attachment of cells with

50–60% conﬂuency. Further on it was treated with vehicle control 0.1%

DMSO and 11d (0.5, 1, 2, 5 and 10 µM) and incubated for 72 h. The

collected cells were harvested and ﬁxed with 75% ice cold ethanol at

4 °C. Excess of ethanol was removed and cells were washed with PBS.

Cells were treated and stained with RNase (50 U/mL) and Propidium

Iodide (20 μg/mL) staining solution for 20 min at room temperature in

dark and was further subjected for ﬂow cytometry (Bectone Dickinson,

FACS Calibur system) [17].

2.3.7. Molecular docking

To view binding pattern of all the synthesized compounds for anti-

proliferative activity, molecular docking was done at colchicine binding

site (chain B) of β-tubulin (PDB code: 1SA0) [21]. The docking simu-

lations of all the compounds were performed using Maestro, version 9.6

implemented from Schrodinger software suite. Synthesized compounds

were sketched in 3D format using the build panel and were also pre-

pared for docking using lig prep application. The proteins were taken

from Protein data bank and were prepared for docking by solvent re-

moving, hydrogen adding and further minimization in the presence of

bound ligand (COL) using protein preparation wizard. Grids for mole-

cular docking were generated with bound co-crystallized ligand. For

validation of docking parameters co-crystal ligand (COL) was re-docked

at catalytic site of protein. RMSD between co-crystal and re-docked

pose was found to be 0.235 Å [22]. Compounds were docked using

Glide extra-precision (XP) mode, with up to three poses saved per

molecule.

2.3.4. Wound healing assay

To evaluate the eﬀect of 11d on cell migration, wound healing assay

was performed [18]. Firstly, MCF-7 cells (3 x10⁵) were seeded and

grown to each well of 6 well plates until mono-layer was formed. Then

by using 200 µl pipette tip,

a Scratches were made in con-

ﬂuentmonolayers. After this, the wounds were washed with PBS to

remove non-adherent cell debris. Cells were further treated with 5 µM

and 10 µM of 11d. Cells which migrated across the wound area were

captured with an inverted microscope and photographed for indicated

time (0, 8, 16 and 24 h).

3. Result and discussion

3.1. Chemistry

2.3.5. Mitochondrial membrane potential analysis

JC-1 staining method was opted to study the eﬀect of 11d on mi-

tochondrial membrane potential of MCF-7 cells [19]. Brieﬂy, MCF-7

cells treated with 11d at 5 µM and 10 µM for 72 h and were incubated

with 10 µM JC-1 dye diluted in PBS for 20 min at 37 °C, 5% CO₂in-

cubator. Monomer was treated with excited wavelength of 488 nm. A

green ﬂuorescence is emitted by JC-1 monomers with a maximum

emission at 530 nm, where as J-aggregates emitted orange-red ﬂuor-

escence maximum at 590 nm.

Above designed Indole linked 1,3,4-oxadiazole conjugates (11a-n)

and 1,2,4-triazole (12a, c, e, g, h, j-n) and were synthesized as outline

in Scheme 1. Indole 6 was reacted with the oxalyl chloride to give 2-

(1H-indol-3-yl)-2-oxoacetyl chloride 7 [23] which was then reacted

with the hydrazine hydrate to form 2-(1H-indol-3-yl)-2-oxoacetohy-

drazide 8. Compound 8 was further reﬂuxed with diﬀerent aliphatic as

well as aryl isothiocyanates (9a-n) to aﬀord diﬀerent substituted thio-

semicarbazides (10a-n). These thiosemicarbazides (10a-n) were cy-

clized under coupling conditions with N-Ethyl-N′-(3-dimethylamino-

propyl)carbodiimide hydrochloride (EDC.HCl) & Hydroxybenzotriazole

(HOBt) to aﬀord substituted 1,3,4-oxadiazoles (11a-n) in very good

yields (90–95%). On the another side, thiosemicarbazides (10a-n) cy-

clized with 2 N NaOH to aﬀord 5-mercapto-1,2,4-triazoles (12a, c, e, g,

h, j-n) in 60–70% yields as illustrated in Table 1.

2.3.6. In vitro tubulin polymerization inhibitory assay

The compounds 11d and 12e were monitored for their anti-tubulin

activity. Study involved in vitro assay to monitor the time dependent

polymerization of tubulin into microtubules using ﬂuorescence-based

tubulin polymerization assay kit (BK011, Cytoskeleton, Inc.) as per

manufacturer’s protocol [20]. Experiment involved measurement of

ﬂuorescence emission at 420 nm (excitation wavelength at 360 nm) for

1 h in a multimode plate reader (Tecan M200 inﬁnite).For the two test

compounds (11d and 12e), standard (Nocodazole) and control (zero

drug), the reaction mixture was prepared separately as per speciﬁcation

of cytoskeleton protocol for tubulin inhibition. In brief, 2 mg/ml tu-

bulin in 80 mM PIPES with pH 6.9, 2 nM MgCl₂, 0.5 mM EGTA, 1.0 mM

GTP and 15% tubulin glycerol were taken in a ﬁnal volume of 50 µl and

transferred into each well of 96 well microplate (Prior to transfer of

reaction mixture the plate was warmed up to 37 °C for 10 min. For

determination of IC₅₀values of two compounds against tubulin poly-

merization, the compounds were pre-incubated with tubulin at varying

concentrations (1, 5 and 10 µM).

Formation of N-substituted 2-amino-1,3,4-oxadiazole (11a-n) from

thiosemicarbazides (10a-n) was conﬁrmed by the appearance of a

singlet corresponds to eNH proton at in the rang of δ 10.18–11.81 and

disappearance of two NH peak at 10.85 and 10.13 corresponds to

amides in ¹H NMR (supplementary data). The presence of two siglets in

the range at δ 12.29–12.39 and δ 10.18–11.81 corresponds to indolyl

eNH and eNH of 2-amino-1,3,4-oxadiazole (11a-n) respectively was

evidenced by D₂O exchange ¹H NMR spectra of 11c (supplementary

data). While ¹³C NMR gave two characteristic peaks for these derivative

i.e C]O at

δ 171.11–170.75 ppm and eOeC]N)eNH at δ

160.37–164.62 ppm. All the intermediates and target compounds were

ﬁnally conﬁrmed by their mass spectras.

Formation of thiosemicarbazide (10a-n) from hydrazide (8) was

6

F. Naaz, et al.

BioorganicChemistry95(2020)103519

conﬁrmed by the appearance of four singlets corresponds to eNH

groups at δ 12.29, 10.85, 10.19, and 10.13 ppm in ¹H NMR (10a). Base

catalised cyclization was conﬁrmed by the appearance of a singlet

corresponds to eSH proton at δ 14.30–14.66 ppm and the dis-

appearance of three singlets corresponding to eNH groups of thiose-

micarbazide. 5-mercapto-1,2,4-triazoles with aromatic substitution on

nitrogen (12a, c, e, g, h, j) were observed as tautomers in diﬀerent

ratios as observed from ¹H NMR (supplementary data). Appearance of a

singlet corresponds to eNH proton at δ 10.43–11.87 ppm conﬁrmed the

existance of it’s tautomer. It was further conﬁrmed by IR which ob-

tained absorption bands for SeH at 2535–2643 cm⁻¹, C]N (triazole

ring) stretching bands at 1578–1633 cm⁻¹and C]S at

1210–1237 cm⁻¹. Whereas, aliphatic substituted compounds (12k-n)

were not tautomerized as only singlet corresponds to eSH proton was

observed in the region of δ 14.16–14.53 ppm in 1H NMR (supple-

mentary data).

3.2. Biological evaluation

3.2.1. Anti-proliferative investigation against 60 cell lines

All the synthesized compounds were submitted to National Cancer

Institute (NCI), Germantown, MD, USA for their anti-proliferative ac-

tivity against 60 cancer cell lines under nine diﬀerent cancer cell types

(leukemia, lung, colon, CNS, melanoma, ovarian, renal, prostate and

breast cancers) with their sub-panels. All these compounds were se-

lected for single concentration of 10 µM and their growth percent data

Table 1

Synthesized indole linked 1,3,4-oxadiazole analogues (11a-n) and 1,2,4-triazole (12a, c, e, g, h, j-n).

Thiosemicarbazide

(10)

Indole linked 1,3,4-

Oxadiazole (11)

Indole linked 1,2,4-

triazole (12)

Code

a

-

b

c

-

d

e

f

-

g

(continued on next page)

7

F. Naaz, et al.

BioorganicChemistry95(2020)103519

Table 1 (continued)

h

i

-

O

S

O

N

H

N

NH

H

SH

O

HN

j

N

H

OC₂H₅

O

N

O

N

NH

k

l

N

H

m

n

was demonstrated in Tables 2 and 3. Among the 1,3,4-oxadiazole based

compounds (11a-n), compounds 11a, 11d, 11e, 11f, 11g and 11k

demonstrated considerable anti-proliferative activity against most of

the cancer cell lines. Among all, compound 11d showed potent activity

against non small lung NCI-H460, colon HCC-2998, Melanoma SK-MEL-

5, UACC-62 and breast MCF-7 cancer cell line with % growth of only

19.72–28.16 as shown in Table 2. It also exhibited moderate activity

against colon HCT-116 and renal ACHN cancer cell line with 46.71%

growth and 37.98% growth respectively (in Table 2). Compound 11a,

11f, and 11g displayed moderate activity against colon HCC-2998 with

37.57–48.72% growth whereas compound 11a, 11d and 11f exhibited

good to moderate activity of growth % of 31.16–41.13 of colon KM12

cancer cell line. Compound 11a, 11f, 11g and 11k showed good to

moderate activity against breast MCF-7 cancer cell line with % growth

of 31.64–47.61 whereas compound 11a, 11d, 11e, 11f, 11g and 11k

demonstrated moderate anti-cancer activity against breast T-47D

cancer cell line. Compound 11a, 11g and 11f also exhibited moderate

activity with 50.60% growth against leukemia HL-60 (TB), 45.01%

growth of colon HCC-299 and 45.54% growth of melanoma UACC-62

respectively.

Among the 1,2,4-triazole based compounds (12a, c, e, g, h, j-n),

compounds 12e and 12h were found to be best when compared to other

compounds. These compounds exhibited good to moderate activity

with 33–50% growth of colon HCC-2998, colon HCT-15, colon KM 12,

breast T-47D and breast MCF-7 cancer cell lines as illustrated in

Table 3. Compound 12e and 12h was found to be potent against breast

T-47D cancer cell line with % growth of 25.26% and 26.08% respec-

tively. Herein, Compound 12e also demonstrated good anti-pro-

liferative activity with % growth of 34.23% against Melanoma SK-MEL-

5 cancer cell line. While compound 12h also illustrated moderate ac-

tivity with % growth of 51.87% against Melanoma UACC-62 cancer cell

line. Compound 12a and 12g showed moderate activity against breast

T-47D cancer cell line with % growth of 40.36 and 43.78% respectively.

After over viewing the obtained NCI anti-proliferative data from

both the series, it has observed that most of the synthesized compounds

displayed anti-proliferative activity against melanoma, colon and breast

cancer. Compounds 11a, 11d, 11f 12e, and 12h, showing better ac-

tivity were taken for further studies.

3.2.2. IC₅₀value against MCF-7 cancer cell line

Among all the 60 cancer cell lines, MCF-7 & T-47D cell lines of

breast cancer and HCC-2998, HCT-15 & KM 12 cell lines of colon cancer

were found to be more susceptible for these classes of compounds.

From all the synthesized compounds 11a-n and (12a, c, e, g, h, j-n),

compounds 11a, 11d 11f, 12e, and 12h, exhibited 21.88–37.13% of

growth of MCF-7 breast cancer cell line according to obtained NCI anti-

8

F. Naaz, et al.

BioorganicChemistry95(2020)103519

Table 2

Anti-proliferative activity of synthesized indole linked 1,3,4-oxadiazole analogues (11a-n) against NCI panel of 60 human cancer cell lines.

Cell line/panel

Growth percent

Subpanel tumor cell lines

11a

11b

11c

11d

11e

11f

11g

11h

11i

11j

11k

11l

11m

11n

Leukemia

CCRF-CEM

HL-60(TB)

K-562

95.07

50.60

101.20

92.72

90.01

86.20

102.86

91.79

98.67

102.85

97.62

98.78

87.44

87.08

94.31

99.27

91.15

90.28

100.34

72.56

104.02

101

94.63

91.15

81.34

98.48

107.01

92.01

95.03

97.43

97.53

98.20

95.58

107.32

104.27

104.15

107.78

103.65

106.18

97.61

87.40

100.36

104.24

100.40

90.11

102.13

104.74

98.19

102.71

112.01

106.08

93.62

94.02

101.20

112.81

104.74

96.18

107.02

103.55

80.01

111.70

107.93

102.47

116.09

109.48

103.79

97.89

91.12

87.04

105.13

70.12

91.12

102.07

94.65

82.55

MOLT-4

RPMI-8226

SR

101.90

99.55

79.47

91.69

102.62

106.39

95.46

Non-small cell lung cancer

A549/ATCC

EKVX

94.54

74.19

76.32

78.43

77.97

87.60

69.71

91.13

95.24

101.08

93.49

90.52

82.08

81.91

84.73

83.85

95.11

103.93

98.10

97.45

90.56

88.96

97.99

97.72

92.95

104.52

100.95

62.78

86.34

67.15

97.02

84.15

65.08

85.42

28.16

70.60

95.94

98.11

77.20

96.76

89.72

90.27

100.96

101.27

102.91

80.31

85.73

70.63

99.93

85.58

82.90

86.74

56.77

79.24

88.62

98

92.71

98.38

79.60

100.55

85.20

91.30

86.55

100.73

101.13

88.21

104.45

79.18

97.89

85.56

88.13

94.58

99.17

98.15

88.96

109.3

97.73

97.46

88.09

89.99

107.20

99.77

101.61

84.81

97.15

68.64

105.11

93.40

88.58

90.57

70.11

87.57

99.87

113.25

84.29

104.66

93.86

89.73

104.19

107.50

91.86

99.40

103.96

106.46

106.72

93.44

96.15

93.92

106.73

81.25

106

79.38

88.69

92.66

90.06

88.59

87.21

104.13

91.43

HOP-62

84.68

84.60

84.05

88.36

98.81

100.21

99.53

HOP-92

NCI-H226

NCI-H23

NCI-H322M

NCI-H460

NCI-H522

Colon cancer

COLO 205

HCC-2998

HCT-116

HCT-15

97.62

48.72

81.76

49.77

104.20

41.13

103.80

108.09

85.01

78.31

86.10

100.78

81.84

95.33

103.62

91.48

89.80

98.02

102.93

91.40

103.79

105.32

26.21

46.71

56.70

91.30

31.16

99.24

107.64

54

94.72

37.57

76.68

64.94

109.73

37.98

101.59

94.09

45.01

91.39

64.06

92.98

53.76

100.76

102.28

74.74

96.66

66.96

105.77

72.73

100.84

98.86

88.64

88.38

80.09

100.39

81.68

98.73

102.94

98.17

102.25

72.63

87.46

65.63

108.04

55.42

101.35

112.99

92.82

110.76

98.45

107.06

96.83

91.38

97.49

105.56

91.39

101.55

93.03

79.24

101.41

57.35

99.07

88.45

90.82

107.62

89.80

105.99

103.81

97.33

101.41

103.41

81.82

HT29

101.37

67.75

KM12

SW-620

103.75

100.08

107.45

CNS cancer

SF-268

SF-295

SF-539

SNB-19

SNB-75

U251

92.97

89.87

105.88

103.58

105.93

97.62

98.33

102.66

107.13

112.88

105.30

87.10

104.22

107.41

110.54

98.02

97.71

102.23

106.60

106.27

95.98

104.79

107.59

103.53

96.82

102.84

108.17

109.6

105.93

107.98

112.85

100.48

98.94

94.49

101.07

111.44

110.52

103.57

112.79

104.52

100.83

98.03

101.37

102.34

94.29

112.75

100.46

101.80

98.51

103.36

101.41

103.48

89.58

106.04

107.33

103.78

100.86

100.35

108.54

109.35

104.95

95.39

92.31

103.52

97.69

108.02

91.00

85.85

92.45

94.16

89.53

89.25

101.86

101.37

96.73

100.33

94.73

97.04

94.67

96.91

96.59

98.62

99.43

103.81

Melanoma

LOX IMVI

MALME-3M

M15

90.27

101.45

93.20

104.86

91.16

107.31

77.37

97.91

65.61

94.32

92.60

99.79

111.34

87.86

115.64

88.21

103.85

90.68

99.23

109.56

100.59

108.10

99.71

115.69

98.66

96.34

89.77

99.50

111.73

98.15

101.91

88.29

104.02

21.32

79.05

19.72

101.21

104.91

104.84

103.27

95.45

96.23

100.04

100

97.40

102.01

95.87

111.67

105.33

107.67

79.78

87.73

82.48

96.75

96.85

98.57

103.43

102.93

106.22

78.96

89.58

64.16

96.02

105.17

92.49

100.50

110.16

104.26

89.45

92.61

83

102.62

109.94

96.64

110.02

98.08

110.30

95.44

83.16

92.15

99.68

102.90

96.69

107.73

86.59

98.49

71.45

91.73

76

98.78

100.13

95.38

100.04

96.27

102.86

104.95

111.04

90.37

121.64

105.81

99.57

92.92

MDA-MB-435

SK-MEL-2

SK-MEL-28

SK-MEL-5

UACC-257

UACC-62

103.77

92.09

106.26

54.80

89.06

45.54

105.97

100.28

111.33

100.53

112.24

94.05

110.67

88.61

113.45

97.96

111.07

103.76

104.03

95.98

100.93

90.54

107.77

93.26

Ovarian cancer

IGROV1

60.15

89.66

76.63

87.93

97.59

97.96

87.63

62.59

95.11

100.03

119.53

94.72

95.51

93.06

96.31

110.08

92.55

109.43

99.99

101.92

90.71

66.60

83.57

89.42

77.39

95.42

98.68

82.38

79.93

102.70

99.50

85.82

102.07

104.72

77.33

65.58

94.25

99.56

91.64

93.85

105.02

68.99

74.07

95.15

111.74

82.31

96.41

101.11

79.42

70.05

92.33

94.68

86

72.62

97.17

105.99

99.55

94.91

101.70

83.04

89.71

105.80

110.47

111.09

96.51

99.37

89.83

80.37

104.34

98.30

87.50

99.59

115.13

75.30

93.22

100.68

108.71

112.17

105.21

98.39

88.4

103.08

103.65

99.29

85.62

95.38

90.87

93.75

101.65

101.18

87.35

OVCAR-3

OVCAR-4

OVCAR-5

105.76

102.63

95.40

OVCAR-8

97.60

101.45

71.52

NCI/ADR-RES

SK-OV-3

105.64

Renal cancer

786-0

106.97

85.72

83.89

115.68

98.74

101.48

63.60

98.22

96.43

105.10

37.98

68.86

106.37

94.15

72.87

90.49

104.82

106.60

93.17

109.71

77.05

92.52

109.85

97.78

96.61

89.70

108.34

97.03

100.78

108.75

96.89

94.98

88.82

107.59

95.74

93.12

116.10

99.92

113.56

92.83

103.07

90.80

87.43

124.81

93.45

112.38

86.97

105.99

102.66

103.08

105.32

97.58

103.50

99.59

98.02

111.24

101.54

94.29

93.20

104.04

105.40

96.48

108.77

108.59

100.61

107.60

98.56

97.88

89.71

87.90

127.03

108.57

117.18

71.89

ACHN

100.98

96.71

100.82

97.25

CAK-1

RXF 393

SN 12C

TK-10

105.01

100.66

135.42

78.69

110.92

101.66

117.43

93.77

111.59

100.08

106.99

98.15

111.61

96.47

113.25

96.07

118.77

91.26

98.56

UO-31

85.61

Prostate cancer

PeCe3

77.22

85.07

96.49

87.91

80.01

97.66

97.80

89.55

86.45

85.87

87.59

94.10

89.38

94.46

99.62

85.08

109

DU-155

108.71

116.37

112.68

110.88

109.50

106.88

107.04

106.84

112.13

106.84

106.67

Breast cancer

MCF7

31.64

79.40

98.17

104.07

42.37

103.49

86.68

74.86

88.31

98.62

115.77

71.96

102.17

95.88

73.33

21.88

66.74

89.94

88.70

39.84

87.94

59.10

89.23

97.11

106.41

43.24

86.68

96.00

33.69

75.34

101.28

101.10

39.87

91.88

87.58

47.61

93.78

105.01

106.58

41.97

86.34

92.08

84.53

79.94

98.60

101.68

67.87

103.26

94.13

77.21

77.70

31.92

83.55

98.24

98.57

38.45

85.07

92.20

90.93

67.70

79.72

90.07

98.97

93.18

64.70

111.35

96.18

MDA-MB-231/ATCC

HS 578T

100.28

108.83

108.06

68.41

88.94

105.63

101.44

113.07

82.42

92.81

111.46

97.28

104.32

103.40

79.56

98.49

BT-549

107.55

63.30

116.56

67.60

T-47D

MDA-MB-468

Mean GP

98.82

103.33

96.01

93.55

103.31

100.46

107.45

99.84

98.38

100.14

9

F. Naaz, et al.

BioorganicChemistry95(2020)103519

Table 3

Anti-proliferative activity of synthesized indole linked 1,2,4-triazole analogues (12a, c, e, g, h, j-n) against NCI panel of 60 human cancer cell lines.

Growth percent

Cell line/panel

12a

12c

12e

12g

12h

12j

12k

12l

12m

12n

Leukemia

CCRF-CEM

HL-60(TB)

K-562

85.06

61.41

83.33

85.96

68.41

92.01

96.90

83.83

84.88

87.73

80.97

97.17

88.63

78.12

86.45

88.94

79.06

86.32

92.02

68.91

94.74

83.15

91.02

97.61

90.63

88.79

84.57

90.19

84.09

76.77

83.11

97.20

88.45

103.06

80.08

96.19

92.52

84.40

75.07

96.86

81.06

84.30

91.33

90.85

76.12

102.71

78.67

91.67

94.49

89.29

87.54

92.82

84.23

86.51

93.26

88.31

88.77

93.43

69.25

97.11

MOLT-4

RPMI-8226

SR

Non-small cell lung cancer

A549/ATCC

EKVX

80.99

94.60

87.40

80.33

77.83

91.84

94.66

95.67

84.66

98.61

94.77

86.59

76.55

84.15

100.19

102.35

98.66

83.89

88.03

78.20

84.54

89.51

84.79

96.05

88.90

100.47

84.74

93.95

90.60

90.22

91.97

90.01

94.16

100.26

102.63

75.02

86.47

70.94

81.73

84.25

83.08

87.20

77.68

75

91.46

98.45

94.95

79.64

92.34

100.30

98.41

101.50

90.31

88.82

98.50

98.70

99.50

96.46

99.46

99.42

98.18

94.45

92.25

90.25

85.99

89.43

92.51

92.63

96.71

98.69

83.27

89.15

100.06

87.27

86.04

96.60

97.40

93.98

99.18

87.60

82.02

75.32

71.88

75.10

70.59

80.03

94.36

96.37

81.63

HOP-62

HOP-92

NCI-H226

NCI-H23

NCI-H322M

NCI-H460

NCI-H522

79.98

Colon cancer

COLO 205

HCC-2998

HCT-116

HCT-15

96.82

58.50

90.26

64.75

98.62

67.38

99.95

101.49

99.84

89.73

73.65

94.57

78.16

96.59

109.94

39.45

89.42

41.72

100.98

49.96

93.71

104.24

81.79

100

98.99

38.77

89.72

40.88

96.22

50.69

94.56

107.97

110.86

98.88

100.79

98.31

88.55

96.50

107.63

100.96

91.06

106.24

101.48

92.39

99.04

99.76

92.25

101.25

73.84

104.70

92.54

98.80

89.88

83.93

96.44

101.91

98.91

97.99

104.94

94.68

91.43

101.20

63.68

89.47

65.95

107.41

95.68

HT29

104.79

91.11

KM12

SW-620

101.03

CNS cancer

SF-268

SF-295

SF-539

SNB-19

SNB-75

U251

95.37

97.87

97.52

95.47

96.95

109.16

94.10

88.48

106.67

95.41

101.55

105.99

93.78

83.19

97.31

92.60

97.30

90.50

98.06

90.43

89.05

93.05

96.92

89.69

99.41

108.56

103.46

83.03

96.44

93.75

103.52

94.10

102.12

107.67

89.03

101.25

107.23

100.98

95.76

103.80

107.34

92.99

102.55

101.70

100.40

81.17

102.32

96.20

116.18

95.37

115.40

87.05

88.12

86.07

105.43

104.30

99.06

103.70

100.04

106.27

Melanoma

LOX IMVI

MALME-3M

M15

100.43

105.55

95.88

104.39

86.37

107.41

88.31

99

100.44

104.62

104.54

102.30

100.37

117.24

89.97

98.63

98.67

99.86

107.33

86.70

119.74

34.23

90.91

78.28

96.83

93.21

95.39

99.29

102.15

85.88

105.40

27.98

97.41

51.87

98.13

94.91

108.85

88.54

99.56

99.33

111.40

105.02

123.47

92.43

96.36

111.45

92.42

98.70

91.93

116.82

99.91

94.83

81.70

89.48

106.92

88.33

97.78

97.06

115.40

100.30

89.22

82.83

96.29

100.62

100.92

106.17

86.32

116.24

87.25

93.72

96.56

110.70

103.98

108.53

89.18

107.35

101.60

101.16

105.94

118.77

98.41

MDA-MB-435

SK-MEL-2

SK-MEL-28

SK-MEL-5

UACC-257

UACC-62

120.96

84.66

101.79

81.94

102.96

87.29

98.71

93.09

91.81

Ovarian cancer

IGROV1

73.10

95.25

85.01

100.67

97.13

106.05

95.44

77.31

92.68

84.10

106.57

97.17

104.36

84.07

71.97

91.23

92.75

96.29

97.06

101.18

83.27

75.74

99.38

103.02

12.83

97.79

102.95

89.01

60.57

86.41

87.12

89.71

93.46

105.52

91.78

90.68

97.82

84.37

118.21

98.41

98.71

91.81

100.05

108.13

100.62

115.65

95.47

87.70

106.47

92.43

133.13

93.13

106.60

88.42

93.72

96.44

92.89

115.32

94.55

101.29

89.90

63.45

92.39

94.20

124.07

96.10

103.89

90.38

OVCAR-3

OVCAR-4

OVCAR-5

OVCAR-8

NCI/ADR-RES

SK-OV-3

108.56

96.67

Renal cancer

786-0

115.27

87.87

96.08

85.39

101.52

92.79

116.96

75.19

103.85

107.21

102.97

84.07

105.22

92.82

107.71

108.10

113.77

89.33

106.52

113

103.37

117.92

100.30

77.25

100.24

97.53

97.81

100.82

100.27

80.51

99.82

93.97

97.61

86.46

101.95

92.05

102.83

107.25

99.62

102.89

97.06

97.58

80.81

105.88

94.55

118.22

75.98

A498

ACHN

105.66

80.03

112.05

87.52

101.51

79.33

CAK-1

73.24

RXF 393

SN 12C

TK-10

102.54

110.65

125.22

89.47

101.64

108.03

115.18

86.62

104.42

96.25

115.97

98.40

107.40

90.05

120.96

100.80

122.78

88.71

115.58

81.60

106.12

86.64

134.46

86.29

132.04

90.12

UO-31

Prostate cancer

PC-3

83.87

115.44

66.13

86.77

95.40

99.73

87.84

89.15

96.56

93.83

94

85.64

DU-155

108.91

106.09

103.45

101.88

101.33

103.74

104.99

102.26

Breast cancer

MCF7

63.32

91.33

92.83

116.68

40.36

66.13

89.01

95.68

112.50

50.91

37.13

86.35

97.42

120.43

25.26

54.78

97.70

100.02

115.16

43.78

33.39

76.89

93.76

119.9

26.08

85.38

96.10

99.0

85.91

105.74

92.45

113.71

76.83

73.18

96.41

90.97

115.19

77.54

84.77

89.31

86.18

122.84

82.22

76.35

87.94

97.87

122.95

70.04

MDA-MB-231/ATCC

HS 578T

BT-549

111.78

83.01

T-47D

(continued on next page)

10

F. Naaz, et al.

BioorganicChemistry95(2020)103519

Table 3 (continued)

Growth percent

Cell line/panel

12a

12c

12e

12g

12h

12j

12k

12l

12m

12n

MDA-MB-468

105.90

97.43

92.23

96.95

98.67

102.22

100.30

109.29

106.81

99.89

Mean GP

90.96

94.56

88.54

95.05

85.56

98.08

96.97

96.23

95.52

93.57

Table 4

IC₅₀proﬁle for compound 11a, 11d, 11f, 12e, 12h, with doxorubicin (standard) and Combretastatin A-4 (standard) against MCF-7 cancer cell line.

11a (μΜ)

11d (μΜ)

11f (μΜ)

12e (μΜ)

12h (μΜ)

Doxorubicin (μΜ)

CA-4 (nΜ)

IC₅₀

5.98

2.42

8.11

3.06

3.30

6.31

2.16

Fig. 3. Structure Activity Relationship for synthesized analogues.

proliferative data. These compounds were screened to evaluate their

IC₅₀values against MCF-7 by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide) cytotoxicity assay. Doxorubicin (con-

sidered in many chemotherapy regimens, particularly found eﬀective in

treatment of breast cancer) and combretastatin A-4 (CBSIs) were used

as standard. IC₅₀values for compound 11a, 11d, 11f, 12e, and 12h,

were calculated by dose response curve (supplementary data) and

shown in Table 4. Among all the selected compounds, compound 11d

demonstrated best cytotoxicity with IC₅₀2.42 µM when compared with

standard doxorubicin (IC₅₀6.31 µM). While, compounds 11a, 12e and

12h, also exhibited good cytotoxicity against MCF-7 breast cancer cell

line with IC₅₀5.98 µM, 3.06 µM, and 3.30 µM and respectively in

comparison to doxorubicin. Compound 11f displayed moderate anti-

cancer activity with IC₅₀8.11 µM than doxorubicin.

attached to indolyl ring; (ii) Nature of the group attached to eNH/

eNe; (iii) Nature of the substituents on the aromatic ring.

Analogues with 1,3,4-oxadiazole moiety showed good activity when

compared to analogues with 1,2,4-triazole moiety. So, the preferences

for anti-proliferative activity of the ﬁve membered heterocyclic rings is

oxadiazole > triazole. With respect to nature of the substituent’s at-

tached to 1,3,4-oxadiazole and 1,2,4-triazole ring, it has seen that aryl

substitution on both the rings (11a, 11d, 11e, 11f, 11g, 12a, 12d, 12e,

12g) showed superior anti-proliferative activity than the aliphatic

substitution (11l, 11m, 11n, 12l, 12m, 12n). When both the hetero-

cyclic moieties substituted with aliphatic groups they result in the loss

of activity. However when 1,3,4-oxadiazole moiety substituted with

benzyl group (11k), it retained the activity and exhibited good anti-

proliferative activity against breast cancer cells (MCF-7 with 31.92%

growth and T-47D with 38.45%) whereas compound 12k (analogues

with 1,2,4-triazole ring attached to benzyl substitution) had no anti-

proliferative property.

3.2.3. Structure activity relationship

Structure activity relationship has been drawn on the basis oﬀ-

ollowing three parameters (i) Nature of the heterocyclic moiety

Nature and positioning of the functional group on aromatic group

11

F. Naaz, et al.

BioorganicChemistry95(2020)103519

Fig. 4. Compound 11d induced cell arrest at G0/G1 phase in MCF-7 Cells. (A) 5 and 10 µM concentration of 11d was incubated with MCF-7 cells for 48 h. Population

of cells in diﬀerent phases of cell cycle were analyzed by Modit LT software and obtained result was shown in histogram. (B) Bar graph showing percentage of cells in

diﬀerent phases of cell cycle. Data are represented as mean

SD of separate independent experiments, where (*) indicated P < 0.05 and (**) indicated P < 0.01

compared to vehicle control as determined by t-test.

greatly inﬂuence the activity. Electron withdrawing groups on aromatic

ring displayed better activity compared to electron donating groups.

Among the electron withdrawing groups halogens have enhanced the

activity remarkably. The activity proﬁles of the substituent’s was ob-

served as F > Br > NO₂> Cl. whereas aromatic substitution of the

ﬂuorine atom displayed promising anti-proliferative activity among all

the synthesized compounds. On the basis of the positioning of the

substituent on aromatic ring, ortho- and para- substitution was found to

be preferable for the activity as shown in Fig. 3.

3.2.4. Cell cycle analysis

Among all the potent anti-proliferative compounds, compound 11d

exhibited best IC₅₀value (2.42 µM) against MCF-7 cell line than that of

standard doxorubicin. Thus compound 11d was selected to examine its

eﬀect on progression of cell cycle on MCF-7 cell line.

Distribution proﬁle in diﬀerent phases of cell cycle was determined

by ﬂow cytometry. The results depicted in Fig. 4(A) showed that there

was increase of cells in G0/G1 phase from 52.38

0.42 to

65.43

1.37 in dose depended manner (at concentration of 0.5 µM,

Among the compounds tested, it has seen compound 11d (o-ﬂuoro)

illustrated the best anti-proliferative activity. Compound 11d emerged

as a lead out of the small library of compound screened and was se-

lected for further biological studies.

1 µM, 2 µM, 5 µM and 10 µM). The percentages of cells in diﬀerent

phases of cell cycle were represented in Fig. 4(B). Compound 11d

provoked cell cycle arrest in G0/G1 phase of cell cycle in MCF-7 cell

line at concentration of 10 µM (p < 0.05).

12

F. Naaz, et al.

BioorganicChemistry95(2020)103519

Fig. 5. Eﬀects of 11d on the migrating ability of MCF-7 cells. (A) MCF-7 cells were grown on 6 wells plate until monolayer was formed, following which a scratch was

made and cells were treated with 5 and 10 µM of 11d and photographs were taken at indicated time points (0,8,16 and 24 h). (B) A signiﬁcant diﬀerence in arbitrary

unit analyzed by J software as shown in graph. Data are represented as mean

SD, where (**) indicated P < 0.01 and (***) indicated P < 0.001 compared to

vehicle control as determined by t-test.

Fig. 6. Eﬀect of 11d on the mitochondrial membrane potential: MCF-7 cells were treated with 5 and 10 µM and changes in mitochondrial membrane potential were

determined by staining with JC-1 dye. Cells undergoing apoptosis and with compromised mitochondrial membrane potential emits green ﬂuorescence whereas cells

with polarized potential emit red ﬂuorescence as evident from the photographs.

cyclin-dependent kinases (cdk) inhibitor by monitoring p21waf1-Chk1-

cdc25C-cdc2-checkpoint-pathway. Oncogene induced over expression

of p21^waf1which produced opposite eﬀects on that of Chk1-cdc25C-

cdc2 pathway and enhanced cell arrest at G1 phase [25]. Along with

this, Marc et al in 1996 also reported that taxol induces cell arrest at

late-G1 in transformed and non-transformed REF-52 and WI-38 mam-

malian ﬁbroblast cells which is characterized by partially depho-

sphorylated pRb, and inactive cdk2 kinase (cyclin-dependent kinases)

[26]. Here in, Zoe et al. in 1999, determined eﬀect of the MTIs (Mi-

crotubule inhibitors), Taxol and vincristine, on the cell cycle kinetics

using two isogenic cell lines, HCT116 p21+/+ and HCT116 p21−/−.

It was examined that p21-deﬁcient cells illustrated dose-dependent,

enhanced chemosensitivity to MTIs and these MTIs induced arrest at G1

[27].

Table 5

IC₅₀values for the tested compounds against tubulin polymerization.

Standard (NOC)

11d

12e

IC₅₀(μ M)

2.49

3.89

8.32

Generally it has observed that microtubule disrupting agents arrest

cell cycle at G2/M phase but various other studies are reported that

microtubules disrupting agents can also induced cell arrest at G1 phase

by altering various gene products i.e p53, pRb, p16^INK4A, or p21^Waf1/

^Cip1, which are involved in cell division at G1-S phase [24]. Charlie

et al. in 2002 reported that disrupted microtubules can also cause cell

arrest during interphase at G₁via over expression of p21^waf1(p21), a

13

F. Naaz, et al.

BioorganicChemistry95(2020)103519

Table 6

tubulin with IC₅₀3.89 µM and 8.32 µM, whereas the standard noco-

dazole exhibited IC₅₀2.49 µM against tubulin polymerization as illu-

strated in Table 5. In vitro assay results revealed that 12d exhibited

good tubulin polymerization inhibitory activity than that of 11e but

showed comparative activity to that standard.

Docking score of synthesized compounds (11a-n) and (12a, c, e, g, h, j-n) for

tubulin binding site.

S. No. Code

Docking score

(kcal/mol)

Code

Docking score (kcal/

mol)

1

11a

11b

11c

11d

11e

11f

−5.08

−5.90

−6.48

−6.28

−5.77

−6.44

−6.23

−6.38

−5.63

−3.59

−6.10

−5.40

−5.71

−4.89

12a

12b

12c

12d

12e

12f

−6.16

–

3.2.8. Molecular docking study

2

In silico binding aﬃnities of all the synthesized compounds were

determined by using schrodinger software at colchicines binding site of

β-tubulin (PDB code 1SA0). Docking score of all the compounds were

demonstrated in Table 6. Binding pose of most potent compounds 11d

and 12e was investigated and compared with standard nocodazole as

shown in Fig. 7. Compound 11d was interacted with VAL 238 amino

acid residue through hydrogen bonding with bond length of 2.1 Å. It

exhibited hydrophobic interaction with LEU 242, CYS 241, ILE 378,

VAL 318, ALA 317, ALA 316, VAL 315, ALA 354, VAL 351, MET 259,

LEU 255, LEU 252, ALA 250, LEU 248 amino acid residue and also

exhibited other interaction with THR 239, THR 314, LYS 352, ASN 350,

ASN 349, ASN 258 amino acids residue of β-tubulin complex. Com-

pound 12e displayed no hydrogen bond but showed hydrophobic in-

teraction with MET 259, VAL 257, LEU 255, LEU 248, ALA 250, VAL

238, ILE 378, CYS 241, LEU 242, VAL 318, ALA 317, ALA 316, VAL

315, ALA 354 amino acid residues. It also exhibited other kinds of in-

teractions with various residues of β-tubulin complex (ASN 258, LYS

254, THR 239, THR 314, LYS 352, ASN 350). Nocodazole (standard)

illustrated hydrogen bonding with ASN 258 and ASN 349 with bond

length 2.3 Å and 1.8 Å respectively. It also exhibited hydrophobic in-

teraction with ILE 347, PRO 261, MET 259, LEU 255, LEU 248, CYS

241, ALA 354, VAL 351, ILE 347, VAL 315, ALA 316, ALA 317, VAL 318

amino acid. Nocodazole also demonstrated other interaction with ASN

258, ASN 349, ASN 350, LYS 352, THR 353 and THR 314 amino acid

residues as shown in Fig. 7.

3

−6.43

–

4

5

−6.66

–

6

7

11g

11h

11i

12g

12h

12i

−6.60

−6.28

–

8

9

10

11

12

13

15

16

11k

11j

12j

−6.44

−6.73

−5.77

−5.17

−3.79

12k

12l

11l

11m

11n

12m

12n

Nocodazole −5.18

Above studies revealed that MTIs in tumor cells may also caused cell

arrest at G₁-S via involvement of various gene products.

3.2.5. Cell migration analysis

Among various others anti-cancer eﬀects exhibited by microtubules

interacting agents. Interference with cell migration was one of the

parameter which has been also reported. So, compound 11d was

evaluated for cell migration by wound healing assay on MCF-7 cell line.

The migration potential of compound 11d was evaluated at 0 h, 8 h,

16 h and 24 h.

As depicted in Fig. 5(A), Cell free area was signiﬁcantly higher in

treated cell as compared to control with passage of time which in-

dicated potential anti-migratory eﬀect of compound 11d. Thus com-

pound 11d inhibited the migration of MCF-7 cells in dose dependent

manner. The graphical representation of signiﬁcant diﬀerence in the

wound area shown by 11d with respect to control was demonstrated in

Fig. 5(B).

Compounds 11d and 12e were superimposed with standard drugs

nocodazole at colchicine binding site of β-tubulin complex.

Benzimidazole ring of nocodazole was superimposed with 2-ﬂuoro

phenyl ring of compound 11d and 4-ﬂuoro phenyl ring of compound

12e whereas thiophene ring of nocodazole was superimposed with

1,3,4-oxadiazole ring of compound 11d and carbonyl group of com-

pound 12e as illustrated in Fig. 7(E,F). Docking score of 11d

(−6.28 kcal/mol) and 12e (−6.66 kcal/mol) was also better than that

of nocodazole (−5.18 kcal/mol) as illustrated in Table 6.

3.2.6. Mitochondrial membrane potential (MMP) analysis

Change in the mitochondrial membrane potential is one of the early

and prominent features of cells undergoing apoptosis. So, to evaluate

whether 11d induced apoptosis was involved in damaging the integrity

of mitochondrial membrane. MMP of 11d was conducted using a ca-

tionic dye JC-1by ﬂuorescence microscopy on MCF-7 cells. When these

cells were treated with 5 µM and 10 µM of 11d, there was increase in

green ﬂuorescence in the cytoplasm as dose dependent manner. This

characterized that JC-1 monomers when treated with 10 µM of 11d,

there was more accumulated of the dye in the cytoplasm of cells than

the monomer treated with 5 µM due to compromised MMP. On the

other hand, control cells having negative and intact MMP allowed ca-

tionic JC-1 dye to enter mitochondria and formed aggregates which

were illustrated by red ﬂuorescence in the cytoplasm as shown in Fig. 6.

Hence microscopic images were suggesting that 11d perturbed mi-

tochondrial membrane potential (MMP) of MCF-7 cells in the process of

apoptosis.

4. Conclusion

A series of twenty four 1,3,4-oxadiazole and 1,2,4-triazole based

topsentin analogues were synthesized by simple synthetic methodology

and evaluated for their anti-proliferative activity. Anti-proliferative

screening suggested that compounds 11a, 11d, 11f, 12e, 12h, showed

good to moderate activity against a panel of various NCI cancer cell

lines. Compounds 11d and 12e were found to be best compounds from

both the series. The most potent Compound 11d showed good speciﬁ-

city against non-small cell lung cancer (NCI-H460), Colon cancer (HCC-

2998, HCT-116, KM12), Melanoma (SK-MEL-5, UAAC-62), Renal

(ACHN), and Breast (MCF-7, T-47D) Compound 11d also demonstrated

good anti-proliferative activity against MCF-7 cancer cell line cancer

cell line with best IC₅₀2.42 μΜ. Whereas, compound 12e and 12h

displayed good IC₅₀3.06 μM and 3.30 μM against MCF-7 cancer cell

line. Further mechanistic studies illustrated that 11d induced cell arrest

at G0/G1 phase and also depolarized mitochondria membrane potential

of MCF-7 cell line. Wound healing assay revealed that 11d reduced cell

growth by lowering cell migration potential in dose dependent manner.

3.2.7. In vitro tubulin polymerization inhibitory activity

Compounds 11d and 12e with best IC₅₀against MCF-7 cancer cell

line were selected for their in vitro tubulin polymerization assay and

were compared with standard nocodazole. Compounds 11d and 12e

disturbed the microtubule dynamic by inhibiting polymerization of

14

F. Naaz, et al.

BioorganicChemistry95(2020)103519

Fig. 7. (A) (i) Binding pose of 11d (cyan) at colchicine binding site of β-tubulin are shown in stick representation while protein is render in cartoon representation

and black colour dotted line is depicted hydrogen bonding with VAL 238 residues as ball and stick. (ii) Lig plot of 11d in the binding pocket of β-tubulin; (B) (i)

Binding pose of 12e (cyan) at colchicine binding site of β-tubulin (PDB code: 1SA0) are shown in stick representation while protein is rendered in cartoon re-

presentation (ii) Lig plot of 12e in the binding pocket of β-tubulin; (C) (i) Binding pose of nocodazole (cyan) at colchicine binding site of β-tubulin are shown in stick

representation while protein is render in cartoon representation and black colour dotted line is depicted hydrogen bonding with ASN 258 and ASN 349 residues as

ball and stick. (ii) Lig plot of nocodazole in the binding pocket of β-tubulin; D) Superimposition of docking pose of 11d (pink) and 12e (cyan) with nocodazole

(yellow) at colchicine binding site of β-tubulin.

To ﬁnd the mode of action for compounds 12e and 11d for exhibiting

anti-proliferative activity, In-vitro polymerization assay was done and

indicated that 11d potently inhibited tubulin polymerization with IC₅₀

3.89 than 12e. In silico molecular docking results showed that com-

pounds 11d and 12e were occupying colchicine binding site of β-tu-

bulin with good docking score −6.66 kcal/mol and −6.28 kcal/mol

respectively when compared to nocodazole (−5.18 kcal/mol). Further

on 11d will be selected as the lead molecule for further investigation.

Acknowledgments

FN would like to express their deep sense of gratitude to Jamia

Hamdard for ﬁnancial support under Jamia Hamdard-Silver Jubilee