Synthesis, anticancer evaluation and molecular docking studies of 2,5-bis(indolyl)-1,3,4-oxadiazoles, Nortopsentin analogues

Article abstract of DOI:10.1016/j.molstruc.2020.127875

A series of ten novel 2,5-bis(indolyl)-1,3,4-oxadiazoles were designed and synthesized. All these compounds were evaluated for their cytotoxicity against four cancer cell lines namely A549, MDA-MB-231, MCF-7 and HeLa using MTT reduced assay. Among them, compound 12e exhibited good cytotoxicity on MCF-7 cell line with IC₅₀ value of 1.8 μM and it was identified as a promising drug lead when compared to the standard drug doxorubicin (IC₅₀ value 0.98 μM). Compound, 12h exhibited better antitumor activity against three cancer cell lines i.e., lung (A549), breast (MCF-7) and cervical (HeLa) with IC₅₀ values of 3.3 μM, 2.6 μM and 6.34 μM respectively. The impact of these compounds on colchicine binding site of tubulin polymer was carefully investigated using molecular docking studies and interpretations between actives and inactive were explained.

Full text of DOI:10.1016/j.molstruc.2020.127875

Journal Pre-proof
Synthesis, anticancer evaluation and molecular docking studies of 2,5-
bis(indolyl)-1,3,4-oxadiazoles, Nortopsentin analogues
Reddymasu Sreenivasulu, Mandava Bhuvan Tej, Surender Singh Jadav, Pombala
Sujitha, C. Ganesh Kumar, Rudraraju Ramesh Raju
PII:
S0022-2860(20)30199-X
DOI:
https://doi.org/10.1016/j.molstruc.2020.127875
MOLSTR 127875
Reference:
To appear in: Journal of Molecular Structure
Received Date: 29 December 2019
Revised Date: 29 January 2020
Accepted Date: 7 February 2020
Please cite this article as: R. Sreenivasulu, M.B. Tej, S.S. Jadav, P. Sujitha, C.G. Kumar, R.R.
Raju, Synthesis, anticancer evaluation and molecular docking studies of 2,5-bis(indolyl)-1,3,4-
oxadiazoles, Nortopsentin analogues, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/
j.molstruc.2020.127875.
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Guntur, India.
Date: 29-01-2020.
To
The Editor
J. Mol. Struct.
Sub: Credit Author Statement - reg.
Respected Sir,
It is informed to your kind notice that The corresponding author is responsible for ensuring
that the descriptions are accurate and agreed by all authors.
Thanking you sir,
Yours Sincerely
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Corresponding author

Synthesis, anticancer evaluation and molecular docking studies
of 2,5-Bis(indolyl)-1,3,4-oxadiazoles, Nortopsentin analogues
Reddymasu Sreenivasulu1, Mandava Bhuvan Tej2, Surender Singh Jadav3, Pombala
Sujitha4, C. Ganesh Kumar4, Rudraraju Ramesh Raju1*
1Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar – 522 510,
Andhra Pradesh, India.
2Department of Pharmacy, Sri Ramachandra Institute of higher education and Research,
Sri Ramachandra Nagar, Porur, Chennai – 600116, Tamil Nadu, India.
3 Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology,
Mesra, Ranchi, Jharkhand – 835 215, India
4Medicinal Chemistry and Pharmacology division, CSIR – Indian Institute of Chemical
Technology, Uppal Road, Tarnaka, Hyderabad – 500 007, Telangana, India.
*Corresponding author Email: rrraju1@gmail.com
Graphicalabstract:
Abstract: A series of ten novel 2,5-bis(indolyl)-1,3,4-oxadiazoles were designed and
synthesized. All these compounds were evaluated for their cytotoxicity against four
cancer cell lines namely A549, MDA-MB-231, MCF-7 and HeLa using MTT reduced
assay. Among them, compound 12e exhibited good cytotoxicity on MCF-7 cell line with
IC50 value of 1.8 µM and it was identified as a promising drug lead when compared to
the standard drug doxorubicin (IC50 value 0.98ꢀM). Compound, 12h exhibited better
antitumor activity against three cancer cell lines i.e., lung (A549), breast (MCF-7) and

cervical (HeLa) with IC50 values of 3.3 µM, 2.6 µM and 6.34 µM respectively. The
impact of these compounds on colchicine binding site of tubulin polymer was carefully
investigated using molecular docking studies and interpretations between actives and
inactive were explained.
Key Words: bis - indolyl – oxadiazoles; nortopsentin analogues; marine alkaloids,
Cytotoxicity and MTT reduced assay.
1. Introduction: Over the past decade, a large number of marine alkaloids with unique
chemical structures such as Topsentins, Dragmacidins, Coscinamides and Nortopsentins
were isolated from different marine sponge metabolites, which exhibited potent
biological activities such as antitumor, antiviral, antimicrobial, anti-inflammatory and
HIV-inhibitory activities [1-7]. Nortopsentins A-C and the analogue D having 2,4-bis (3’-
indolyl)imidazole skeleton, exhibited invitro cytotoxicity against P338cells (IC50,4.5-
20.7µM), antifungal activity against Candida albicans, and antimicrobial activity against
Bacillus subtillis. Further, N-methyl derivatives of Nortopsentins were found to show
significant improvement in P338 activity when compared to the activity of parent
compounds (IC50, 0.8-2.1 µM) [8-10].
Analogues of Nortopsentins, where the imidazole moiety was replaced by
thiazole [11], thiophene [12], pyrazole [13], furan – isoxazole [14], pyrrole [15],
triazinones [16], and Pyridine [17] etc. were reported previously by other researchers.
These analogues were reported to have exhibited strong inhibitory activity against tumor
cell lines (GI50<0.01 – 89.4 µM). The structures of Nortopsentins and its analogues are
shown in Figure 1.
<Figure 1>
Oxadiazoles exhibit a variety of chemical and biological properties. More
importantly, heterocyclic compounds containing 1,3,4-oxadiazole moiety exhibit wide

range of biological activites like antimicrobial [18], antiviral [19], fungicidal [20] and
anticonvulsant, as well as inhibitory action on tyrosinase [21] and HDACs [22]. Apart
from these biological activities, 1,3,4-oxadiazoles were also identified for their electron
conducting properties and applications in light – emitting devices (LEDs) [23]. 1, 3, 4 –
oxadiazole scaffold is present in many anticancer drugs and provides tunable cytotoxic
properties. Some of the heterocyclic compounds containing 1,3,4-oxadiazole moiety were
identified to act as tubulin binding agents and DNA intercalators [24] and are highlighted
in various drug discovery programs [25].
Previously, we reported the design and synthesis of different heterocyclic systems
with potent antitumor activities against a panel of various human cancer cell lines [26-
39]. We also designed and synthesized new series of bis indolyl tri keto diazo compounds
and 3,5-bis(3-indolyl) triazinones. These derivatives were screened for their anticancer
activities in vitro towards four different human cancer cell lines viz., HeLa, MCF-7,
MDA-MB-231 and A549 cell lines [40]. In continuation of our research work, we
prompted the synthesis of Bis (indolyl) – oxadiazoles as new analogs of Nortopsentins.
We report a simple method for preparation of N-methyl and N, N – dimethyl Bis
(indolyl) – oxadiazoles via the cyclo condensation of carbohydrazide with carboxylic
acid in the presence of polyphosphoric acid as the dehydrating agent. All the compounds
synthesized were screened for their antitumor activity against lung (A549), breast (MDA-
MB-231, MCF-7) and Cervical (HeLa) cancer cell lines. The anti-proliferative properties
of bis-indole derived hydrazide complexes and 1, 3, 4-oxadiazoles are already
established. They play an effective role in inhibition of mitotic spindle formation thereby
altering tubulin polymerization. The colchicine binding site of tubulin is targeted for the
present class of analogs using molecular docking simulations. The binding mode of
nortopsentin analogs was studied using Schrodinger’s LLC.
2. Experimental section:
2.1. General
All chemicals and reagents were obtained from Aldrich (Sigma–Aldrich, St. Louis, MO,
USA), Lancaster (Alfa Aesar, Johnson Matthey Company, Ward Hill, MA, USA) and
were used without further purification. Reactions were monitored by TLC, performed on

silica gel glass plates containing 60 F-254, and visualization on TLC was achieved by
UV light or iodine indicator. Proton spectra were recorded on a Gemini Varian-VXR-
unity (400 MHz) instrument. Chemical shifts (δ) are reported in ppm downfield from
internal TMS standard. Mass ESI spectra were recorded on Micro mass, Quattro LC
using ESI+ software with capillary voltage 3.98 kV and ESI mode positive ion trap
detector. Melting points were determined with an electro thermal melting point apparatus.
General procedure for the preparation of 5 – substituted indole – 3 – carboxylic
acids (7a-7b): 6g of substituted indole – 3 – aldehydes (6a-6b) were respectively
dissolved in 600 mL of acetone. KMnO4 solution (9g KMnO4 soluble in 180 mL water)
was added dropwise for 30 minutes to this reaction mixture and the reaction mass was
stirred for 6 hours at room temperature. The reaction mixture was then quenched by
adding 20 mL of 30% H2O2, filtered and concentrated on rotavapor. Later, it was
acidified by conc. HCl, filtered, dried and recrystallized from methanol. 5 – substituted
indole – 3 – carboxylic acids 7a-7b was obtained as the product.
1H-indole-3-carboxylic acid (7a) [42]: yellow colored solid. Yield: 63%. m.p:230-
232oc.
5-Bromo-1H-indole-3-carboxylic acid (7b): yellow colored solid. yield: 65%. m.p:230-
1
232oc; IR (KBr): 3349, 2914, 2574, 1643 cm-1; H NMR (400 MHz, DMSO-d6): δ 7.30
(dd, 1H, J = 2Hz, 2Hz), 7.435 (d, 1H, J = 8.8Hz), 8.05 (d, 1H, J = 2.8Hz), 8.12 (d, 1H,
13
J = 2Hz), 12.01 (s, 1H), 12.13 (s, 1H) ppm; C NMR (400 MHz, DMSO-d6): δ 107.06,
113.79, 114.28, 122.68, 124.70, 127.79, 133.45, 135.16, 165.45 ppm; HRMS calculated
for C9H6NO2BrNa: 261.94741; found: 261.94711.
General procedure for the preparation of 5 – substituted – 3 – carbomethoxy
Indoles (8a-8b):
4g of 5-substituted indole – 3 – carboxylic acids were dissolved in 10 volumes (400ml) of
methanol and a catalytic amount of conc. H2SO4 was added slowly. The reaction mixture
was refluxed for 5 hours and then cooled to room temperature. Ice cold water was added

and the product was filtered when yellow coloured solid was obtained. This solid on
recrystallization from ethanol yielded 5 – substituted -3- carbomethoxy indoles 21a-21b.
Methyl 1H-indole-3-carboxylate (8a) [43]: yellow colored solid. Yield: 66%. m.p:149-
151oc.
Methyl -5-bromo-1H-indole-3-carboxylate (8b): yellow colored solid. Yield. 62%.
m.p: 210-212oc; IR (KBr): 3213, 3114, 2929, 1745 cm-1; 1H NMR (400 MHz, CDCl3): δ
3.93 (s, 3H), 7.28 (d, 1H, J = 8.8Hz), 7.35 (dd, 1H, J = 1.6Hz, 1.6Hz), 7.91 (d, 1H, J =
2.8Hz), 8.32 (s, 1H), 8.67 (s, 1H) ppm; 13C NMR (400 MHz, CDCl3+DMSO-d6): δ
49.93, 105.91, 112.88, 113.75, 122.40, 124.26, 126.65, 131.89, 134.33, 164.13 ppm;
HRMS calculated for C10H9NO2Br: 253.98112; found: 253.98102.
General procedure for the preparation of 5 – substituted – 1 – methyl – 3 –
Carbomethoxy indoles (9a-9b): A mixture of 5 – substituted – 3 – carbomethoxy
indoles (3g, 17 mmol), Potassium carbonate (1.5g), N,N-dimethyl formamide (21 mL, 7
vol.) and dimethyl carbonate (4.286 mL, 50 mmol) were stirred and refluxed at 1300C for
3.5 hours. The reaction mixture was allowed to cool to room temperature followed by
addition of ice. The yellow color solid obtained was filtered and recrystallized from
ethanol which afforded 5 – substituted – 1 – methyl – 3 – carbomethoxy indoles 9a-9b.
Methyl 1-methyl-1H-indole-3-carboxylate (9a) [44]: yellow color solid. Yield: 60%.
m.p: 81-83oc;
Methyl 5-bromo-1-methyl-1H-indole-3-carboxylate (9b): yellow color solid. Yield:
61%. m.p: 65-67oc; IR (KBr): 3246, 3100, 2940, 1745 cm-1; 1H NMR (400 MHz, CDCl3):
δ 3.81 (s, 3H), 3.90 (s, 3H), 7.19 (d, 1H, J = 8.8Hz), 7.36 (dd, 1H, J = 1.6Hz,1.6Hz), 7.75
(s, 1H), 8.29 (d, 1H, J = 1.6Hz) ppm; 13C NMR (400 MHz, CDCl3): δ 33.52, 51.03,
106.53, 111.17, 115.52, 124.13, 125.64, 127.97, 135.75, 135.86, 164.86 ppm; HRMS
calculated for C11H11NO2Br: 267.99677; found: 267.99707.

General procedure for the preparation of 5 – substituted – 1 – methyl indole - 3 –
Carbohydrazides (10a-10b):
5-substituted – 1 – methyl – 3 - carbomethoxy indoles (2g, 10mmol) were dissolved in
ethanol (10ml, 5vol) and hydrazine hydrate (1.55ml, 32mmol) was slowly added. The
reaction mixture was refluxed for 8 hours and allowed to cool down to room temperature.
The reaction mixture was then poured into crushed ice, filtered and recrystallized from
ethanol which yielded 5-substituted-1-methyl indole-3-carbohydrazides 10a-10b.
1-methyl-1H-indole-3-carbohydrazide (10a): yellow colored solid. Yield: 61%. m.p:
150-152oc; 1H NMR (400 MHz, CDCl3): δ 3.80 (s, 3H), 4.14 (s, 2H), 7.24-7.32 (m, 3H),
7.34 (d, 1H, J = 7.6Hz), 7.66 (s, 1H), 7.93(d, 1H, J = 7.6Hz) ppm.
5-bromo-1-methyl-1H-indole-3-carbohydrazide (10b): yellow colored solid. Yield:
74%. m.p: 250-252oc; 1H NMR (400 MHz, CDCl3): δ 3.81 (s, 3H), 4.11 (s, 2H), 7.04 (s,
1H), 7.21 (d, 1H, J = 8.8Hz), 7.38 (dd, 1H, J = 1.2Hz, 1.6Hz), 7.60 (s, 1H), 8.13 (s, 1H)
ppm; HRMS calculated for C10H11N3OBr: 268.00800; found: 268.00775.
General procedure for the synthesis of Bis (indolyl) oxadiazoles:
5-Bromo-1-methyl-indolyl-3-carbohydrazide or 1-methyl-indolyl-3-carbohydrazide
(500mg, 1.0 eq.) was added to different indole-3-carboxylic acids (1.87 mmol, 1.0 eq.).
To this reaction mixture, 12 volumes of polyphosphoric acid was added and heated at
90oc over a period of 3.5 h to 4 h. The reaction mixture was cooled to room temperature
and followed by addition of ice. The PH of the above reaction mixture was brought to
neutral by using sodium bicarbonate solution. The solid obtained was separated and
dried. The crude compound was purified by column chromatography with DCM solvent.
The solvent was concentrated with rotary under cooling conditions when yellow colored
solid was formed. It was washed with n-hexane and ether solvent to remove non-polar
and semi-polar impurities, which yielded the pure compound in yellow colour .
Synthesis of 5-bromo-1-methyl-3-[5-(1-methyl-1H-indol-3-yl)-1, 3, 4-oxadiazol-2-yl]-
1H-indole (12a): This compound 12a was prepared following the above general

procedure employing 500 mg 10b (1.0 eq.) with 1.86 mmol 1-methyl indole -3-
carboxylic acid 11a (1.0 eq.) afforded 12a in yellow color solid. Yield: 74% (563 mg).
m.p.: 168-170oc; 1H NMR (DMSO-d6, 400 MHz): δ 3.95 (s, 6H), 7.48-7.50 (d, 3H, J=9.6
MHz), 7.62-7.64 (d, 2H, J=8.8 MHz), 8.3 (s, 4H) ppm; 13C NMR (DMSO-d6, 400 MHz):
δ 159, 136, 133, 126, 125, 123, 114, 113, 98, 33 ppm; MS (ESI): m/z = 407 [M+H]+, 408
[M+2H]+.
3, 3'-(1,3,4-oxadiazole-2,5-diyl)bis(5-bromo-methyl 1H indole) (12b): This compound
12b was prepared following the above general procedure employing 500 mg of 10b (1.0
eq.) with 1.86 mmol 5-bromo-1-methyl-1H-indole-3-carboxylic acid 11b (1.0 eq.) which
1
afforded 12b in yellow color solid. Yield: 75% (683 mg). m.p.: 197-199oc; H NMR
(DMSO-d6, 400 MHz): δ 3.95 (s, 6H), 7.48-7.50 (d, 2H, J=8.4 MHz), 7.62-7.64 (d, 2H,
13
J=8.4 MHz), 8.30 (m, 4H) ppm; C NMR (DMSO-d6, 400 MHz): δ 146, 140, 131, 130,
127, 116, 114, 113, 111, 33 ppm;MS (ESI): m/z = 485 [M+H]+, 486 [M+2H]+.
5-methoxy-1-methyl-3-[5-(1-methyl-1H-indol-3-yl)-1,3,4-oxadiazol-2-yl]-1H-indole
(12c): The compound 12c was prepared following the above general procedure
employing 500 mg of 10a (1.0 eq.) with 2.64 mmol 5-methoxy-1-methyl indole -3-
carboxylic acid 11c (1.0 eq.) which afforded 12c as a yellow colored solid. yield: 75%
1
(710 mg); m.p.: 161-163oc; H NMR (DMSO-d6, 400 MHz): δ 3.73 (s, 6H), 6.82-6.97
(m, 4H), 7.31-7.43 (m, 4H) ppm; 13C NMR (DMSO-d6, 400 MHz): δ 152, 146, 140, 130,
128, 125, 121, 116, 114, 113, 112, 111, 110, 109, 56, 33 ppm; MS (ESI): m/z = 359
[M+H]+.
1-methyl-3-[5-(1-methyl-1H-indol-3-yl)-1,3,4-oxadiazol-2-yl]-5-nitro-1H-indole
(12d): The compound 12d was prepared following the above general procedure
employing 500 mg of 10a (1.0 eq.) with 2.64 mmol 5-Nitro-1-methyl indole -3-
carboxylic acid 11d (1.0 eq.) which afforded 12d as a yellow colored solid. yield: 79%
1
(780 mg); m.p.: 111-113oc; H NMR (DMSO-d6, 400 MHz): δ 2.379 (s, 6H), 7.279 (s,
3H), 7.363-7.383 (d, 3H, J=8.0 MHz), 7.707-7.727 (d, 3H, J=8.0 MHz) ppm; 13C NMR

(DMSO-d6, 400 MHz): δ 146, 144, 140, 137, 129, 128, 124, 122, 121, 116, 114, 112,
111, 110, 108, 32.8 ppm; MS (ESI): m/z = 374 [M+H]+.
3-[5-(5-bromo-1-methyl-1H-indol-3-yl)-1,3,4-oxadiazol-2-yl]-5-methoxy-1-methyl-
1H-indole (12e): The compound 12e was prepared following the above general
procedure employing 500 mg of 10b (1.0 eq.) with 1.86 mmol 5-methoxy-1-methyl-1H-
indole-3-carboxylic acid 11c (1.0 eq.) which afforded 12e as a yellow colored solid.
yield: 68% (556 mg). m.p.: > 250oc; 1H NMR (DMSO-d6, 400 MHz): δ 3.67 (s, 6H),
3.78 (s, 3H), 6.22 (s, 1H), 6.69-6.81 (m, 2H), 6.87 (s, 1H), 7.01 (s, 1H), 7.09-7.17 (m,
2H), 7.47 (s, 1H) ppm; 13C NMR (DMSO-d6, 400 MHz): δ 155, 145, 140, 135, 131,
129.3, 129.0, 115, 114.6, 114.1, 113.8, 112.2, 111.9, 108.8, 56.1, 32.5 ppm; (ESI): m/z =
437 [M+H]+, 438 [M+2H]+.
3-[5-(1H-indol-3-yl)-1,3,4-oxadiazol-2-yl]-1-methyl-1H-indole (12f): This compound
12f was prepared following the above general procedure employing 500 mg 10a (1.0 eq.)
with 2.64 mmol indole -3- carboxylic acid 11e (1.0 eq.) which afforded 12f as a yellow
colored solid. yield 66% (548 mg). m.p.: 108-110oc; IR (KBr): 3363, 2977, 1541, 1457,
1
1377 cm-1; H NMR (DMSO-d6, 400 MHz): δ 3.64 (s, 3H), 6.83 (s, 1H), 6.88 (s, 1H),
7.09-7.23 (m, 3H), 7.27-7.41 (m, 3H), 7.62-7.74 (m, 2H), 8.23 (brs, 1H) ppm; (ESI): m/z
= 315 [M+H]+.
3-[5-(5-bromo-1H-indol-3-yl)-1,3,4-oxadiazol-2-yl]-1-methyl-1H-indole (12g): The
compound 12g was prepared following the above general procedure employing 500 mg
10a (1.0 eq.) with 2.64 mmol 5-bromo-indole -3- carboxylic acid 11f (1.0 eq.) which
afforded 12g in yellow color solid. yield 64% (666 mg). m.p.: 119-121oc; IR (KBr):
1
3367, 2979, 1540, 1383 cm-1; H NMR (DMSO-d6, 400 MHz): δ 3.64 (s, 3H), 6.43 (s,
1H), 7.17-7.31 (m, 4H), 7.31 (d, 1H), 7.51-7.64 (m, 3H), 8.31 (brs, 1H) ppm; (ESI): m/z
= 393 [M+H]+, 394 [M+2H]+.
1-methyl-3-[5-(5-nitro-1H-indol-3-yl)-1,3,4-oxadiazol-2-yl]-1H-indole (12h): The
compound 12h was prepared following the above general procedure employing 500 mg

of 10a (1.0 eq.) with 2.64 mmol 5-nitro indole -3- carboxylic acid 11g (1.0 eq.) which
afforded 12h as a yellow colored solid. yield: 71% (674 mg). m.p.: 117-119oc; IR (KBr):
1
3369, 2978, 1630, 1533, 1383 cm-1; H NMR (DMSO-d6, 400 MHz): δ 3.67 (s, 3H),
6.79-6.88 (m, 2H), 7.14-7.28 (m, 4H), 7.45 (m, 1H), 8.22 (m, 2H), 8.46 (s, 1H) ppm;
(ESI): m/z = 360 [M+H]+.
5-bromo-3-[5-(1H-indol-3-yl)-1,3,4-oxadiazol-2-yl]-1-methyl-1H-indole (12i): The
compound 12i was prepared following the above general procedure employing 500 mg
10b (1.0 eq.) with 1.86 mmol 1H-indole-3-carboxylic acid 11e (1.0 eq.) which afforded
12i as a yellow colored solid. Yield: 71% (774 mg). m.p.: 263-265oc; IR (KBr): 3368,
1
2991, 1498, 1417, 1298 cm-1; H NMR (DMSO-d6, 400 MHz): δ 3.64 (s, 3H), 6.55-6.63
(m, 2H), 7.17 (d, 1H), 7.21-7.39 (m, 3H), 7.46-7.61 (m, 3H), 8.33 (s, 1H) ppm; (ESI):
m/z = 393 [M+H]+, 394 [M+2H]+.
5-bromo-3-[5-(5-bromo-1H-indol-3-yl)-1,3,4-oxadiazol-2-yl]-1-methyl-1H-indole
(12j): The compound 12j was prepared following the above general procedure employing
500 mg 10b (1.0 eq.) with 1.86 mmol 5-bromo-1H-indole-3-carboxylic acid 11f (1.0 eq.)
which afforded 12j as a yellow colored solid. yield: 67% (592 mg). m.p.: 198-200oc; IR
1
(KBr): 3372, 2977, 1638, 1540, 1375 cm-1; H NMR (DMSO-d6, 400 MHz): δ 3.66 (s,
3H), 6.88-6.94 (m, 2H), 7.22-7.32 (m, 2H), 7.37 (d, 1H), 7.44 (d, 1H), 7.54 (s, 1H), 7.61
(s, 1H), 8.29 (brs, 1H) ppm; (ESI): m/z = 472 [M+H]+, 473 [M+2H]+.
2.2. In vitro cytotoxicity assay
Cytotoxicity of the synthesized compounds was assessed on the basis of
measurement of the in vitro growth in the 96 well microtiter plates by cell-mediated
reduction of a tetrazolium salt to water insoluble formazan crystals by a previously
described method (Mosmann 1983). Cell lines for testing in vitro cytotoxicity included
HeLa-derived from human cervical cancer cells (ATCC No. CCL-2), A549 derived from
human alveolar adenocarcinoma epithelial cells (ATCC No. CCL-185), MDA-MB-231
derived from human breast adenocarcinoma cells (ATCC No. HTB-26), MCF7 derived

from human breast adenocarcinoma cells (ATCC No. HTB-22) and HEK-293 derived
from normal human embryonic kidney cells (ATCC No. CRL-1573) were obtained from
American Type Culture Collection, Manassas, VA, USA. All tumor cell lines were
maintained in a modified DMEM medium supplemented with 10% fetal bovine serum,
along with 1% non-essential amino acids except L-glutamine, 0.2% sodium hydrogen
carbonate, 1% sodium pyruvate and 1% antibiotic mixture (10 000 units penicillin and 10
mg of streptomycin per mL). The cells were washed and re-suspended in the above
medium and then 100 mL of this suspension was seeded into a 96-well bottom plate. The
cells were kept at 37 °C in a humidified incubator (Model 2406 Shellab CO2 incubator,
Sheldon, Cornelius, OR) under 5% CO2 atmosphere. After incubation for 24 h, the cells
were treated for 2 days with the test compounds at concentrations ranging from 0.1–100
mM in DMSO and were assayed at the end of the second day. After incubation for 48 h,
the cells were subjected to MTT colorimetric assay (5 mg mL-1). The effects of different
test compounds on the viability of the tumor cell lines were measured at 540 nm using a
multimode reader (Infinite® M200Pro, Tecan, Switzerland). Doxorubicin was used as
positive control for comparison purpose and 1% DMSO as a vehicle control. In order to
account for the toxicity of DMSO, the values obtained for the DMSO control were
subtracted from those of the test compounds. Dose-response curves were plotted for the
test compounds and controls after correction by subtracting the background absorbance
from that of the blanks. The antitumor potency of the compounds indicated by IC50
values (50% inhibitory concentration) was calculated from the plotted absorbance data
for the dose-response curves. Statistical analysis was performed using GraphPad PRISM
software version 3.0 (GraphPad Software, Inc, La Jolla, CA, USA). IC50 values (in µM)
are expressed as the mean ± S.D of four independent experiments. All experimental data
were compared using Student’s t-test. In all comparisons, p < 0.05 was considered
statistically significant.
2.3. Molecular docking protocol
The present molecular studies were carried out using Maestro 8.5 (Schrodingers LLC)
installed in RHEL 5.0 platform. Maestro 10.1 (Academic version) was utilized for
docking interpretation. 3-D X-ray crystallographic tubulin complexed with colchicine

as reference ligand at resolution 3.58 Å with r value 0.233 (obs.) was obtained from the
protein data bank (PDB: 1SA0). Among all homo and heterodimeric chains, chain B was
picked up for protein preparation and colchicine binding site was employed for the
simulation studies. All the ligands were sketched using Chemdraw, saved in .mol format,
imported in maestro panel and minimized by macro model multiple minimizations. Glide
5.0 was employed for molecular docking simulation.
3. Results and Discussion
3.1. Chemistry
Indole and 5 – bromo indole were converted into indole -3- carboxaldehydes 6a-
6b by Vilsmeir – Haack reaction [41]. Oxidation of aldehyde derivatives with potassium
permanganate and acetone yielded corresponding carboxylic acid 7a-7b derivatives. The
derivatives were subjected to simple esterification with conc. H2SO4 as a dehydrating
agent. Further, the ring nitrogen was protected via methylation using dimethyl carbonate
and K2CO3 followed by reflux in DMF for 4 hours. Finally, the N-protected ester formed
was refluxed with ethanolic hydrazine hydrate which resulted in the formation of the
carbohydrazide derivatives 10a-10b. These carbohydrazide derivatives 10a-10b so
formed were further reacted with different indole -3-carboxylic acids 11a-11g in the
presence of solid phase catalyst polyphosphoric acid at 90oC for over 3-4 hours which
yielded bis(indolyl)oxadiazole derivatives. (Scheme 1)
<Scheme 1>
3.2. In vitro cytotoxicity
Ten compounds were evaluated for their anticancer activity in select five human
cancer cell lines viz. HeLa (Cervical cancer cells), MDA-MB-231 & MCF (Human breast
adenocarcinoma cells), A549 (Human alveolar adenocarcinoma cells), and HEK-293
(normal human embryonic kidney cells) as per the reported standard protocol [Mosmann
1983]. IC50 value is defined as the drug concentration that produces 50% inhibition of the
tumor cells and are represented in µM. The IC50 values of the compounds evaluated

ranged between 1.8 - 42.3 ꢀM as illustrated in table 1. The compounds 12e and 12h
showed good cytotoxicity with IC50 values of 1.8 ꢀM and 2.6ꢀM on breast cancer cell
line MCF-7. Three compounds 12e, 12f and 12h showed better cytotoxicity on cervical
cancer cell line (HeLa) with IC50 values of 9.23 ꢀM, 9.4 ꢀM and 6.34 ꢀM. The
compound 12h showed good cytotoxicity with IC50 values of 3.3 ꢀM on lung cancer cell
line A549. Further, compound 12e was identified as a promising drug lead which
exhibited promising cytotoxicity with an IC50 value of 1.8 ꢀM towards MCF-7 when
compared to the standard drug doxorubicin (IC50 value 0.98ꢀM). The compounds 12a,
12b, 12g and 12i showed moderate cytotoxicity on cervical cancer cell line HeLa. The
compound 12d exhibited moderate cytotoxicity on breast cancer cell line MCF-7. The
compounds 12e and 12h showed moderate cytotoxicity with an IC50 value of 12.17 ꢀM
and 10.23ꢀM, particularly on breast cancer cell line MDA-MB-231. The compound 12j
showed no cytotoxicity among the four cancer cell lines. Interestingly, all the compounds
exhibited no cytotoxicity on the normal human embryonic kidney cells, HEK-293. The
anti-tumor activities of ten compounds among the five cancer cell lines are shown in table
1.
<Table 1>
3.3. Molecular docking simulation
The bisindole bearing 1, 3, 4-oxadiazoles and nortopsentin derived analogs were screened
invitro for their anti-proliferative activity using different cell lines. The colchicine
binding site of tubulin polymer was employed for these comparative studies through
insilico simulations using Glide 5.0. The hydrophobic and hydrophilic active site of
colchicine consists of a few noticeable crucial residues like Cys241, Val238, Ala316,
Ala317, Leu242, Val318, Leu252, Thr314, Val315, Asn349 and Met259. All the ligands
consist of indole rings on either side of the 1,3,4-oxadiazole ring. Therefore, a careful
investigation of the binding pattern of ligands provided a few specific points which are
helpful for correlating in-vitro data. The first substituted indole ring was observed to be
similarly bound at colchicine active site. The compound 12c can be compared with active
compound 12e, the presence of bromo group on indole ring of compound 12e and h-bond

(2.96Å) of methoxy group with Lys254 might be a route cause for the anti-proliferative
activity and this was absent in 12c, 12a, 12b, and 12d. Compound 12h can be compared
with compound 12d, the absence of N-methyl ring made both differ in their binding
pattern and might be a valid reason behind potency of compound 12h. Binding of indole
with the nitro group was observed to be present near to catalytic Cys241. The NH of free
indole ring of compound 12f was observed to be involved in H-bond with Cy241 (3.15Å)
and Val238 (3.25Å) which may be the reason for its exhibiting least activity. In the case
of compound 12i h-bond with Val238 (2.92 Å) was observed. The binding mode of
compound 12j was not studied. The conclusive pictures of docking studies were
presented in following figure 2.
<Figure 2>
4. Conclusion: We have designed and synthesized a novel series of ten novel 2,5-
bis(indolyl)-1,3,4-oxadiazoles and evaluated for their cytotoxicity against four cancer cell
lines namely A549, MDA-MB-231, MCF-7 and HeLa using MTT reduced assay.
Among them, the compound 12e exhibited good cytotoxicity on MCF-7 cell line with
IC50 value of 1.8 µM and it was identified as a promising drug lead when compared to
the standard drug doxorubicin (IC50 value 0.98ꢀM). Compound, 12h exhibited better
antitumor activity against three cancer cell lines i.e., lung (A549), breast (MCF-7) and
cervical (HeLa) with IC50 values of 3.3 µM, 2.6 µM and 6.34 µM respectively. The
impact of these compounds on colchicine binding site of tubulin polymer was carefully
investigated using molecular docking studies and interpretations between actives and
inactive were explained.
Acknowledgements
The authors are thankful to University Grants Commission, New Delhi, India for
financial assistance in the form of Major Research Project F.No. 42-285/2013 (SR).

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Figure 1: Structures of Nortopsentin analogues

CHO
COOH
CH3OH, Conc.H2SO4
R
R
R
i) POCl3,DMF
KMnO4/Acetone
4 - 5 hours,rt
6 hours, Reflux
ii) Aqu.NaOH, reflux
N
N
N
H
H
H
6a - 6b
7a - 7b
5a - 5b
6a, R=H
6b, R=Br
O
7a, R=H
7b, R=Br
5a, R=H
5b, R=Br
O
H
N
O
OCH3
OCH3
NH2
R
R
R
NH2NH2H2O/Ethanol
Reflux, 8hours
DMC, DMF
K2CO3,Reflux
N
N
N
H
10a - 10b
9a - 9b
CH3
CH3
8a - 8b
10a, R=H
10b, R=Br
R2
9a, R=H
9b, R=Br
8a, R=H
8b, R=Br
R
O
OH
N
N
R2
PPA
90oC, 3 - 4 h
+
O
N
N
N
12a - 12j
R1
R1
11a - 11g
H3C
Where, 11a, R1=CH3, R2=H
11b, R1=CH3, R2=Br
11c, R1=CH3, R2=OCH3
11d, R1=CH3, R2=NO2
11e, R1= H, R2=H
12a, R=Br, R1=CH3, R2=H
12b, R=Br, R1=CH3, R2=Br
12c, R=H, R1=CH3, R2=OCH3
12d, R=H, R1=CH3, R2=NO2
12e, R=Br, R1=CH3, R2=OCH3
12f, R=H, R1= H, R2=H
12g, R=H, R1= H, R2=Br
11f, R1= H, R2=Br
11g, R1=H, R2= NO2
12h, R=H, R1=H,
R2= NO2
12i, R=Br, R1= H, R2=H
12j, R=Br, R1= H, R2=Br
Scheme 1: Synthesis of 2, 5-Bis (indolyl)-1, 3, 4-oxadiazoles
Table 1. Anti-tumor activity of the synthesized compounds
IC50 (µM)
Test
compound
MDA-MB-
A549
MCF-7
30.9 ± 0.48
24 ± 0.5
HeLa
HEK 293
231
-
-
-
-
14.8 ± 0.39
19.5 ± 0.43
42.3 ± 0.96
-
-
-
12a
38.9 ± 0.85
-
12b
32.2 ± 0.9
12c

-
-
19.2 ± 0.8
33.2 ± 0.45
9.23 ± 0.58
9.4 ± 0.37
16.3 ± 0.33
6.34± 0.56
19.8 ± 0.45
-
-
-
-
-
-
-
-
12d
12e
24.2 ± 0.89
12.17±1.1
1.8± 0.9
-
-
24.5 ± 0.9
12f
-
-
-
12g
10.23 ± 1.3
12h
3.3 ±0.85
2.6 ± 0.89
-
-
-
12i
-
-
-
-
12j
0.89± 0.26
0.36 ± 0.14
0.47 ± 0.4
0.98 ± 0.14
Doxorubicin
- No activity
A549
- Human alveolar adenocarcinoma cell line
- Human Cervical cancer cell line
HeLa
MDA-MB-231
MCF-7
- Human breast adenocarcinoma cell line
- Human breast adenocarcinoma cell line
- Human embryonic kidney cell line
HEK 293

A
B
D
C
Figure 2: A) Surface diagram of colchicine and GDP binding domain; B) Superpose of
12a-12j at tubulin active site; C) Interactions of compound 12e; D) 2D-interaction
diagram of 12e. Green thick stick indicates compound 12a-12j (Figure B & D), yellow
dotted line indicates h-bond. Incase of Figure D, green dotted line indicates h-bond.

Highlights
•
We have design and synthesized a new series of N-methyl and N,N-dimethyl 2,5-
Bis(indolyl)-1,3,4-oxadiazoles, Nortopsentin analogues.
•
All these analogs screened for their anticancer activities against A549, MDA-MB-
231, MCF-7 and HeLa cancer cell lines and Normal human embryonic kidney cell
line (HEK 293).
•
•
•
The compounds 12e and 12h showed potent anticancer activities against MCF-7
cancer cell line with IC50 values of 1.8± 0.9 µM and 2.6 ± 0.89 µM respectively.
It is important to note that all the compounds exhibited no cytotoxicity on the normal
human embryonic kidney cells, HEK-293.
The impact of these compounds on colchicine binding site of tubulin polymer was
carefully investigated using molecular docking studies

Guntur, India.
Date: 29-12-2019.
To
The Editor
Journal of Molecular Structure
Respected Sir,
It is informed to your kind notice that We submit our original research article entitled
“Synthesis, anticancer evaluation and molecular docking studies of 2,5-Bis(indolyl)-1,3,4-
oxadiazoles, Nortopsentin analogues” for your Journal. We have no any conflict of interest
statement. .
Thanking you sir,
Yours Sincerely
Reddymasu Sreenivasulu
First author