Synthesis of indole–quinoline–oxadiazoles: their anticancer potential and computational tubulin binding studies

Article abstract of DOI:10.1007/s11164-015-2412-8

Abstract: Small hybrid molecules with two or more structural pharmacores having different biological functions and distinct activity have gained a significant role in cancer drug development to combat various types of malignancies. The present study describes an efficient, clean and strategic synthesis of 12 new substituted quinoline–indole–oxadiazole hybrids from substituted 2-(quinolin-8-yloxy)acetohydrazides and indole-3-carboxylic acids by employing T3P as a green catalyst. Structures of the newly synthesized compounds were established by IR, ¹H NMR, ¹³C NMR, DEPT C-NMR and MS spectroscopic evidence, as well as CHN analysis data. All indole–quinoline–oxadiazoles were tested for their in vitro cytotoxic potential in breast adenocarcinoma (MCF7) and normal kidney (vero) cell lines using MTT assay. 8-((5-(3-(1H-indol-3-yl)propyl)-1,3,4-oxadiazol-2-yl)methoxy)quinoline (3d) exhibited a low IC50 value and a high selectivity index to MCF7 cells and also displayed a mitotic block in flow cytometric cell cycle progression analysis. Microtubule disruption can induce G₂/M phase cell cycle arrest leading to abnormal mitotic spindle formation. Ligand 3d demonstrated its capability of being a probable tubulin inhibitor when docked in the colchicine domain of tubulin. Graphical Abstract: New series of 8-((5-((1H-indol-3-yl)methyl)-1,3,4-oxadiazol-2-yl)methoxy)quinolines were synthesized using T3P as a green catalyst and screened for their cytotoxic and antimitotic potential. The most active 8-((5-(3-(1H-indol-3-yl)propyl)-1,3,4-oxadiazol-2-yl)methoxy)quinoline?3d, displayed good binding interactions with the colchicine binding cavity of microtubule.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s11164-015-2412-8

Res Chem Intermed
DOI 10.1007/s11164-015-2412-8
Synthesis of indole–quinoline–oxadiazoles: their
anticancer potential and computational tubulin binding
studies
Pooja R. Kamath1 Dhanya Sunil1 Abdul A. Ajees2
•
•
Received: 3 October 2015 / Accepted: 28 December 2015
Ó Springer Science+Business Media Dordrecht 2016
Abstract Small hybrid molecules with two or more structural pharmacores having
different biological functions and distinct activity have gained a significant role in cancer
drug development to combat various types of malignancies. The present study describes
an efficient, clean and strategic synthesis of 12 new substituted quinoline–indole–oxa-
diazole hybrids from substituted 2-(quinolin-8-yloxy)acetohydrazides and indole-3-car-
boxylic acids by employing T3PÒ as a green catalyst. Structures of the newly
synthesized compounds were established by IR, 1H NMR, 13C NMR, DEPT C-NMR and
MS spectroscopic evidence, as well as CHN analysis data. All indole–quinoline–oxa-
diazoles were tested for their in vitro cytotoxic potential in breast adenocarcinoma
(MCF7) and normal kidney (vero) cell lines using MTT assay. 8-((5-(3-(1H-indol-3-
yl)propyl)-1,3,4-oxadiazol-2-yl)methoxy)quinoline (3d) exhibited a low IC50 value and
a high selectivity index to MCF7 cells and also displayed a mitotic block in flow
cytometric cell cycle progression analysis. Microtubule disruption can induce G2/M
phase cell cycle arrest leading to abnormal mitotic spindle formation. Ligand 3d
demonstrated its capability of being a probable tubulin inhibitor when docked in the
colchicine domain of tubulin.
Graphical Abstract New series of 8-((5-((1H-indol-3-yl)methyl)-1,3,4-oxadiazol-2-
yl)methoxy)quinolines were synthesized using T3PÒ as a green catalyst and screened for
their cytotoxic and antimitotic potential. The most active 8-((5-(3-(1H-indol-3-yl)pro-
pyl)-1,3,4-oxadiazol-2-yl)methoxy)quinoline 3d, displayed good binding interactions
with the colchicine binding cavity of microtubule.
&
Dhanya Sunil
dhanyadss3@gmail.com
1
2
Department of Chemistry, Manipal Institute of Technology, Manipal University,
Manipal 576104, India
Department of Atomic and Molecular Physics, Manipal Institute of Technology, Manipal
University, Manipal, India
123

P. R. Kamath et al.
Keywords Indole–quinoline–oxadiazole Á Cytotoxicity Á Mitosis Á Tubulin
Introduction
Cancer is becoming a worldwide pandemic and chemotherapy remains the most
widely employed current treatment option. The failure of many successful
contemporary chemotherapeutic drugs is mainly attributed to their side effects
and the mechanism by which cancer cells develop resistance to these drugs,
resulting in increased efflux of chemotherapeutic drugs from cancer cells. Hence,
new advances in the development of anticancer drugs and more effective treatment
approaches are of extreme relevance in drug discovery and chemotherapy. Based on
considerable developments in cancer biology, major research in these areas is
presently focused on developing drugs capable of reversing multidrug resistance
with less toxic side effects that act via more cancer-specific mechanisms aimed at
the corresponding molecular targets [1].
1,3,4-Oxadiazole, a heterocyclic nucleus, has been widely exploited for various
spectra of therapeutic applications like HIV-integrase inhibitor raltagravir,
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Synthesis of indole–quinoline–oxadiazoles: their…
antibacterial furamizole, antihypertensive agents tiodazosin and nesapidil and
anticancer agent zibotentan [2–6]. Past literature also discloses the extensive
therapeutic potential and attractive anticancer activity of 1,3,4-oxadiazole incorpo-
rated with a highly biologically potent indole pharmacore [7–11]. The prominent
role of the active quinoline nucleus has been well known in the search for molecules
of pharmaceutical interest [12–18]. The present study aims at a propyl phosphonic
acid cyclic anhydride (T3PÒ) mediated efficient cyclodehydration synthetic strategy
of 1,3,4-oxadiazole template linking the two bioactive indole and quinoline moieties
under mild reaction conditions with easy work-up procedures yielding pure
products.
Microtubules, a major component of the eukaryotic cytoskeleton, play a crucial
role during mitosis in proper spindle formation and are among the most effective
and promising targets for anticancer therapy. Microtubule targeting drugs interact
with tubulin primarily through three different binding sites: the taxane widely used
in the treatment of lung, breast, ovarian and bladder cancers binds at the inner
surface of the b subunit promoting tubulin stabilization; vinca alkaloids employed in
the treatment of leukaemia, bladder and breast cancer bind with high affinity to one
or few tubulin molecules at the tip of microtubules promoting depolymerization;
and colchicine interacts with b-tubulin at its interface with a-tubulin, resulting in
inhibition of tubulin polymerization [19, 20]. The limitations of current anti-tubulin
drugs like drug resistance, toxicity and bioavailability warrants the identification of
new potent tubulin inhibitors. The current work also focuses on the in vitro
anticancer activity of the synthesized hybrid molecules and their in silico docking
studies with tubulin as the target to explore their potential as microtubule inhibitors.
Experimental section
Chemistry
Chemicals and solvents for the present study were procured commercially from
Sigma Aldrich, Merck and Himedia. Thin layer chromatography was performed
using pre-coated aluminum sheets with Aluchrosep Silica Gel 60/UV254 and the
spots were visualized in a UV chamber. Melting points of synthesized 1,3,4-
oxadiazoles were checked by the open capillary method and are uncorrected. IR
spectra in KBr pellets, NMR spectra with tetramethylsilane as internal standard and
mass spectra were recorded in a Schimadzu FTIR 8400S spectrophotometer, Bruker
400 MHz instrument and Agilent 6510 series mass spectrometer, respectively.
Elemental analysis of the compounds was carried out using a Flash thermo 1112
series CHN analyser.
General procedure for preparation of 8-((5-((1H-indol-3-yl)methyl)-1,3,4-oxadia-
zol-2-yl)methoxy)quinolines 3(a–l) 2-(Quinolin-8-yloxy)acetohydrazides 1(a–c)
(1 mmol) and indole 3-carboxylic acids 2(a–d) (1 mmol) was refluxed in nitrogen
atmosphere with T3PÒ (2 mmol) and TEA (4 mmol) in ethyl acetate medium for
7 h at 80 °C. The reaction mixture was cooled, poured on to ice-cold sodium
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P. R. Kamath et al.
bicarbonate solution and extracted with ethyl acetate. The organic solvent was
evaporated and the crude product was re-crystallized from hexane.
8-((5-(1H-indol-3-yl)-1,3,4-oxadiazol-2-yl)methoxy)quinoline (3a) Dark brown
solid (92.23 %); m.p. 178–180 °C; IR (KBr) [cm-1]: 3207 (indole NH str.), 3062
(Ar. C–H str.), 2983 (methylene C–H asym. str.), 2858 (methylene C–H sym. str.),
1
1620 (C=N str.), 1573 (Ar. C=C str.), 1259 (C–O–C str.); H NMR (400 MHz,
DMSO-d6) [ppm]: d 4.83 (s, 2H, OCH2), d 6.95 (s, 1H, indole 2-H), d 7.06 (d, 2H,
quinoline 5-H and 7-H), d 7.34–7.29 (m, 2H, quinoline 3-H and 6-H), d 7.59–7.53
(m, 4H, indole), d 8.34 (d, 1H, quinoline 4-H), d 8.90 (d, 1H, quinoline 2-H), d 9.90
(s, 1H, indole N–H); 13C NMR (100 MHz, DMSO-d6) d = 174.97, 171.49, 149.75,
140.33, 136.79, 136.59, 129.65, 127.63, 127.29, 122.78, 122.47, 121.56, 121.29,
118.77, 118.71, 118.58, 114.49, 114.33, 112.30, 68.49; MS (m/z): 343 (M ? 1);
Anal. Calcd. for C20H14N4O2: C, 70.17; H, 4.12; N, 16.37; found: C, 70.37; H, 4.14;
N, 16.39.
8-((5-((1H-indol-3-yl)methyl)-1,3,4-oxadiazol-2-yl)methoxy)quinoline (3b) Dark
brown solid (87.78 %); m.p. 128–130 °C; IR (KBr) [cm-1]: 3238 (indole NH
str.), 3060 (Ar. C–H str.), 2983 (methylene C–H asym. str.), 2844 (methylene C–H
1
sym. str.), 1618 (C=N str.), 1571 (Ar. C=C str.), 1259 (C–O–C str.); H NMR
(400 MHz, DMSO-d6) [ppm]: d 2.74 (s, 2H, CH2), d 4.85 (s, 2H, OCH2), d 6.93 (s,
1H, indole 2-H), d 7.07 (d, 2H, quinoline 5-H and 7-H), d 7.35–7.30 (m, 2H,
quinoline 3-H and 6-H), d 7.59–7.53 (m, 4H, indole), d 8.37 (d, 1H, quinoline 4-H),
d 8.83 (d, 1H, quinoline 2-H), d 9.89 (s, 1H, indole N–H); 13C NMR (100 MHz,
DMSO-d6) d = 174.95, 171.48, 149.75, 140.30, 136.77, 136.58, 129.63, 127.62,
127.28, 122.75, 122.45, 121.57, 121.28, 118.78, 118.73, 118.55, 114.48, 114.34,
112.33, 68.49, 31.88; MS (m/z): 357 (M ? 1); Anal. Calcd. for C20H16N4O2: C,
70.77; H, 4.53; N, 15.72; found: C, 70.82; H, 4.54; N, 15.75.
8-((5-(2-(1H-indol-3-yl)ethyl)-1,3,4-oxadiazol-2-yl)methoxy)quinoline (3c) Light
brown solid (90.59 %); m.p. 232–234 °C; IR (KBr) [cm-1]: 3218 (indole NH
str.), 3060 (Ar. C–H str.), 2948 (methylene C–H asym. str.), 2850 (methylene C–H
1
sym. str.), 1618 (C=N str.), 1571 (Ar. C=C str.), 1259 (C–O–C str.); H NMR
(400 MHz, DMSO-d6) [ppm]: d 2.23 (t, 2H, CH2), d 2.72 (t, 2H, CH2), d 4.85 (s,
2H, OCH2), d 6.93 (s, 1H, indole 2-H), d 7.11 (d, 2H, quinoline 5-H and 7-H), d
7.32–7.27 (m, 2H, quinoline 3-H and 6-H), d 7.59–7.52 (m, 4H, indole), d 8.33 (d,
1H, quinoline 4-H), d 8.87 (d, 1H, quinoline 2-H), d 9.93 (s, 1H, indole N–H); 13C
NMR (100 MHz, DMSO-d6) d = 174.98, 171.48, 149.76, 140.32, 136.76, 136.53,
129.61, 127.65, 127.23, 122.77, 122.48, 121.55, 121.29, 118.76, 118.71, 118.57,
114.47, 114.33, 112.35, 68.41, 33.87, 33.42; MS (m/z): 371 (M ? 1); Anal. Calcd.
for C22H18N4O2: C, 71.34; H, 4.90; N, 15.13; found: C, 71.49; H, 4.92; N, 15.16.
8-((5-(3-(1H-indol-3-yl)propyl)-1,3,4-oxadiazol-2-yl)methoxy)quinoline (3d) Light
brown solid (91.13 %); m.p. 242–244 °C; IR (KBr) [cm-1]: 3332 (indole NH
str.), 3049 (Ar. C–H str.), 2929 (methylene C–H asym. str.), 2835 (methylene C–H
1
sym. str.), 1614 (C=N str.), 1564 (Ar. C=C str.), 1259 (C–O–C str.); H NMR
(400 MHz, DMSO-d6) [ppm]: d 1.88 (t, 2H, CH2), d 2.21 (m, 2H, CH2), d 2.70 (t,
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Synthesis of indole–quinoline–oxadiazoles: their…
2H, CH2), d 4.86 (s, 2H, OCH2), d 6.96 (s, 1H, indole 2-H), d 7.08 (d, 2H, quinoline
5-H and 7-H), d 7.33–7.28 (m, 2H, quinoline 3-H and 6-H), d 7.58–7.52 (m, 4H,
indole), d 8.36 (d, 1H, quinoline 4-H), d 8.88 (d, 1H, quinoline 2-H), d 9.91 (s, 1H,
indole N–H); 13C NMR (100 MHz, DMSO-d6) d = 174.99, 171.49, 149.77, 140.30,
136.78, 136.51, 129.60, 127.62, 127.20, 122.79, 122.46, 121.53, 121.28, 118.79,
118.73, 118.56, 114.46, 114.37, 112.36, 68.40, 33.89, 33.44, 26.38; MS (m/z): 385
(M ? 1); Anal. Calcd. for C23H20N4O2: C, 71.86; H, 5.24; N, 14.57; found: C,
72.05; H, 5.26; N, 14.60.
8-((5-(1H-indol-3-yl)-1,3,4-oxadiazol-2-yl)methoxy)-2-methylquinoline (3e) Cream
solid (85.28 %); m.p. 128–130 °C; IR (KBr) [cm-1]: 3211 (indole NH str.), 3062
(Ar. C–H str.), 2929 (methylene C–H asym. str.), 2862 (methylene C–H sym. str.),
1
1627 (C=N str.), 1569 (Ar. C=C str.), 1238 (C–O–C str.); H NMR (400 MHz,
DMSO-d6) [ppm]: d 2.48 (s, 3H, CH3), d 4.80 (s, 2H, OCH2), d 6.91 (s, 1H, indole
2-H), d 7.05 (d, 2H, quinoline 5-H and 7-H), d 7.36–7.30 (m, 1H, quinoline 6-H), d
7.58–7.52 (m, 4H, indole), d 8.35 (d, 2H, quinoline 3-H and 4-H), d 9.92 (s, 1H,
indole N–H); 13C NMR (100 MHz, DMSO-d6) d = 174.96, 171.50, 150.75, 140.31,
136.78, 136.57, 129.64, 127.65, 127.27, 122.79, 122.47, 121.57, 121.28, 112.03
118.76, 118.73, 118.59, 114.48, 114.32, 68.47, 27.32; MS (m/z): 357 (M ? 1);
Anal. Calcd. for C22H16N4O2: C, 70.77; H, 4.53; N, 15.72; found: C, 71.02; H, 4.54;
N, 15.76.
8-((5-((1H-indol-3-yl)methyl)-1,3,4-oxadiazol-2-yl)methoxy)-2-methylquinoline (3f)
Light brown solid (93.21 %); m.p. 208–210 °C; IR (KBr) [cm-1]: 3290 (indole NH
str.), 3050 (Ar. C–H str.), 2921 (methylene C–H sym. str.), 2875 (methylene C–H
1
asym. str.), 1666 (C=N str.), 1566 (Ar. C=C str.), 1234 (C–O–C str.); H NMR
(400 MHz, DMSO-d6) [ppm]: d 2.47 (s, 3H, CH3), d 2.73 (s, 2H, CH2), d 4.83 (s,
2H, OCH2), d 6.93 (s, 1H, indole 2-H), 7.06 (d, 2H, quinoline 5-H and 7-H), d
7.35–7.29 (m, 1H, quinoline 6-H), d 7.59–7.53 (m, 4H, indole), d 8.37 (d, 2H,
quinoline 3-H and 4-H), d 9.94 (s, 1H, indole N–H); 13C NMR (100 MHz, DMSO-
d6) d = 174.95, 171.52, 150.74, 140.30, 136.76, 136.56, 129.63, 127.62, 127.26,
122.77, 122.46, 121.55, 121.28, 112.04, 118.75, 118.72, 118.58, 114.47, 114.30,
68.46, 31.87, 27.33; MS (m/z): 371 (M ? 1); Anal. Calcd. for C22H18N4O2: C,
71.34; H, 4.90; N, 15.13; found: C, 71. 53; H, 4. 91; N, 15.16.
8-((5-(2-(1H-indol-3-yl)ethyl)-1,3,4-oxadiazol-2-yl)methoxy)-2-methylquinoline (3g)
Dark brown solid (92.57 %); m.p. 230–232 °C; IR (KBr) [cm-1]: 3232 (indole
NH str.), 3055 (Ar. C–H str.), 2947 (methylene C–H asym. str.), 2830 (methylene
C–H sym. str.), 1652 (C=N str.), 1566 (Ar. C=C str.), 1234 (C–O–C str.); 1H NMR
(400 MHz, DMSO-d6) [ppm]: d 2.22 (t, 2H, CH2), d 2.45 (s, 3H, CH3), d 2.70 (t,
2H, CH2), d 4.82 (s, 2H, OCH2), d 6.94 (s, 1H, indole 2-H), d 7.08 (d, 2H, quinoline
5-H and 7-H), d 7.33–7.27 (m, 1H, quinoline 6-H), d 7.58–7.52 (m, 4H, indole), d
8.38 (d, 2H, quinoline 3-H and 4-H), d 9.95 (s, 1H, indole N–H); 13C NMR
(100 MHz, DMSO-d6) d = 174.93, 171.50, 150.73, 140.29, 136.75, 136.54, 129.62,
127.61, 127.25, 122.75, 122.44, 121.53, 121.26, 121.05, 118.74, 118.71, 118.55,
114.47, 114.28, 112.08, 68.45, 33.86, 33.40, 27.31; MS (m/z): 385 (M ? 1); Anal.
123

P. R. Kamath et al.
Calcd. for C23H20N4O2: C, 71.86; H, 5.24; N, 14.57; found: C, 72.11; H, 5.25; N,
14.60.
8-((5-(3-(1H-indol-3-yl)propyl)-1,3,4-oxadiazol-2-yl)methoxy)-2-methylquinoline (3h)
Dark brown solid (97.25 %); m.p. 162–164 °C; IR (KBr) [cm-1]: 3265 (indole NH
str.), 3056 (Ar. C–H str.), 2935 (methylene C–H asym. str.), 2890 (methylene C–H
1
sym. str.), 1652 (C=N str.), 1566 (Ar. C=C str.), 1234 (C–O–C str.); H NMR
(400 MHz, DMSO-d6) [ppm]: d 1.87 (t, 2H, CH2), d 2.20 (m, 2H, CH2), d 2.42 (s,
3H, CH3), d 2.72 (t, 2H, CH2), d 4.80 (s, 2H, OCH2), d 6.92 (s, 1H, indole 2-H), d
7.06 (d, 2H, quinoline 5-H and 7-H), d 7.32–7.26 (m, 1H, quinoline 6-H), d
7.56–7.50 (m, 4H, indole), d 8.37 (d, 2H, quinoline 3-H and 4-H), d 9.96 (s, 1H,
indole N–H); 13C NMR (100 MHz, DMSO-d6) d = 174.91, 171.49, 150.70, 140.25,
136.74, 136.51, 129.60, 127.63, 127.22, 122.71, 122.42, 121.50, 121.25, 118.70,
118.73, 118.54, 114.46, 114.27, 112.10, 68.43, 33.85, 33.41, 28.38 27.31; MS (m/z):
399 (M ? 1); Anal. Calcd. for C24H22N4O2: C, 72.34; H, 5.57; N, 14.06; found: C,
72.44; H, 5.58; N, 14.09.
8-((5-(1H-indol-3-yl)-1,3,4-oxadiazol-2-yl)methoxy)-2,4-dichloroquinoline (3i)
Brown solid (89.98 %); m.p. 254–256 °C; IR (KBr) [cm-1]: 3261 (indole NH
str.), 3085 (Ar. C–H str.), 2947 (methylene C–H asym. str.), 2858 (methylene C–H
1
sym. str.), 1677 (C=N str.), 1566 (Ar. C=C str.), 1234 (C–O–C str.); H NMR
(400 MHz, DMSO-d6) [ppm]: d 4.96 (s, 2H, OCH2), d 7.01–6.97 (t, 1H, indole
5-H), d 7.08–7.05 (t, 1H, indole 6-H), d 7.18 (s, 1H, indole 2-H), d 7.36 (d, 1H,
indole 4-H), d 7.58 (d, 1H, indole 7-H), d 7.82–7.81 (m, 1H, quinoline 3-H), d 8.05
(s, 1H, quinoline 6-H), d 8.64 (d, 1H, quinoline 4-H), d 8.97 (d, 1H, quinoline 2-H),
d 10.18 (s, 1H, indole N–H); 13C NMR (100 MHz, DMSO-d6) d = 172.00, 170.59,
155.73, 149.68, 140.31, 136.72, 136.60, 128.19, 126.29, 123.83, 122.78, 121.40,
118.80, 118.65, 118.54, 114.47, 114.36, 112.37, 111.78, 72.73; MS (m/z): 412
(M ? 1), 414 (M ? 3), 416 (M ? 5); Anal. Calcd. for C20H12Cl2N4O2: C, 58.41;
H, 2.94; N, 13.62; found: 58.61; H, 2.95; N, 13.66.
8-((5-((1H-indol-3-yl)methyl)-1,3,4-oxadiazol-2-yl)methoxy)-2,4-dichloroquinoline (3j)
Brown solid (90.28 %); m.p. 258–260 °C; IR (KBr) [cm-1]: 3250 (indole NH str.),
3091 (Ar. C–H str.), 2906 (methylene C–H asym. str.), 2840 (methylene C–H sym.
str.), 1620 (C=N str.), 1564 (Ar. C=C str.), 1260 (C–O–C str.); 1H NMR (400 MHz,
DMSO-d6) [ppm]: d 2.56 (s, 2H, CH2), d 4.93 (s, 2H, OCH2), d 7.03–6.99 (t, 1H,
indole 5-H), d 7.09–7.06 (t, 1H, indole 6-H), d 7.15 (s, 1H, indole 2-H), d 7.35 (d,
1H, indole 4-H), d 7.56 (d, 1H, indole 7-H), d 7.80–7.79 (m, 1H, quinoline 3-H), d
8.04 (s, 1H, quinoline 6-H), d 8.63 (d, 1H, quinoline 4-H), d 8.99 (d, 1H, quinoline
2-H), d 10.14 (s, 1H, indole N–H); 13C NMR (100 MHz, DMSO-d6) d = 172.01,
170.58, 155.75, 149.66, 140.32, 136.71, 136.61, 128.18, 126.28, 123.85, 122.77,
121.41, 118.82, 118.67, 118.53, 114.46, 114.35, 112.38, 111.79, 72.70, 31.20; MS
(m/z): 426 (M ? 1), 428 (M ? 3), 430 (M ? 5); Anal. Calcd. for C21H14Cl2N4O2:
C, 59.31; H, 3.32; N, 13.17; found: 59.53; H, 3.34; N, 13.20.
8-((5-(2-(1H-indol-3-yl)ethyl)-1,3,4-oxadiazol-2-yl)methoxy)-2,4-dichloroquinoline
(3k) Brown solid (90.28 %); m.p. 240–242 °C; IR (KBr) [cm-1]: 3205 (indole NH
123

Synthesis of indole–quinoline–oxadiazoles: their…
str.), 3058 (Ar. C–H str.), 2933 (methylene C–H asym. str.), 2869 (methylene C–H
1
sym. str.), 1685 (C=N str.), 1591 (Ar. C=C str.), 1230 (C–O–C str.); H NMR
(400 MHz, DMSO-d6) [ppm]: d 2.57 (t, 2H, CH2), d 2.96 (t, 2H, CH2), d 4.96 (s,
2H, OCH2), d 7.00–6.98 (t, 1H, indole 5-H), d 7.08–7.05 (t, 1H, indole 6-H), d 7.16
(s, 1H, indole 2-H), d 7.33 (d, 1H, indole 4-H), d 7.55 (d, 1H, indole 7-H), d
7.79–7.78 (m, 1H, quinoline 3-H), d 8.02 (s, 1H, quinoline 6-H), d 8.61 (d, 1H,
quinoline 4-H), d 8.98 (d, 1H, quinoline 2-H), d 10.15 (s, 1H, indole N–H); 13C
NMR (100 MHz, DMSO-d6) d = 171.99, 170.57, 155.71 149.67, 140.32, 136.71,
136.58, 128.17, 126.28, 123.84, 122.77, 121.39, 118.79, 118.67, 118.56, 114.46,
114.37, 112.39, 111.79, 72.74, 34.46, 33.49; MS (m/z): 440 (M ? 1), 442 (M ? 3),
444 (M ? 5); Anal. Calcd. for C22H16Cl2N4O2: C, 60.15; H, 3.67; N, 12.75; found:
60.34; H, 3.69; N, 12.78.
8-((5-(3-(1H-indol-3-yl)propyl)-1,3,4-oxadiazol-2-yl)methoxy)-2,4-dichloroquinoline
(3l) Dark red solid (94.07 %); m.p. 184–186 °C; IR (KBr) [cm-1]: 3267 (indole
NH str.), 3058 (Ar. C–H str.), 2933 (methylene C–H asym. str.), 2839 (methylene
C–H sym. str.), 1608 (C=N str.), 1577 (Ar. C=C str.), 1240 (C–O–C str.); 1H NMR
(400 MHz, DMSO-d6) [ppm]: d 1.86 (t, 2H, CH2), d 2.56–2.54 (m, 2H, CH2), d 2.95
(t, 2H, CH2), d 4.97 (s, 2H, OCH2), d 7.01–6.97 (t, 1H, indole 5-H), d 7.06–7.03 (t,
1H, indole 6-H), d 7.18 (s, 1H, indole 2-H), d 7.32 (d, 1H, indole 4-H), d 7.56 (d,
1H, indole 7-H), d 7.77–7.75 (m, 1H, quinoline 3-H), d 8.06 (s, 1H, quinoline 6-H),
d 8.63 (d, 1H, quinoline 4-H), d 8.96 (d, 1H, quinoline 2-H), d 10.11 (s, 1H, indole
N–H); 13C NMR (100 MHz, DMSO-d6) d = 172.07, 170.60, 155.73, 149.68,
140.33, 136.75, 136.62, 128.19, 126.27, 123.86, 122.78, 121.42, 118.83, 118.69,
118.51, 114.48, 114.33, 112.39, 111.77, 72.68, 34.45, 33.50, 27.39; MS (m/z): 454
(M ? 1), 456 (M ? 3), 458 (M ? 5); Anal. Calcd. for C23H18Cl2N4O2: C, 60.94;
H, 4.00; N, 12.36; found: 61.14; H, 4.02; N, 12.39.
Biological studies
Anticancer studies
MCF-7 and non-cancerous kidney vero cell lines, procured from the National Centre
for Cancer Studies, Pune, India, were cultured at 37 °C in 5 % CO2 atmosphere in
Dulbecco’s modified Eagles medium containing 10 % fetal bovine serum.
MTT assay
MTT is a standard in vitro colorimetric assay used to monitor the extent of cell
proliferation and viability as well as cytotoxicity. The MCF7 and normal vero cells
were trypsinized and counted using standard procedures. Cells were seeded at a
density of 5 9 103 cells in 96-well plates and were incubated at 37 °C for 24 h in
CO2 atmosphere. Subsequently, cells were treated with different concentrations of
test compounds (50–400 lM) in triplicates and incubated for 48 h at 37 °C.
Vehicles and their dilutions used to dissolve the test compounds were used as
controls. After 24 h, 30 lL of MTT reagent (4 mg/mL) was added to each well and
123

P. R. Kamath et al.
further incubated for an additional 4 h at 37 °C. Plates were centrifuged, media was
aspirated, 100 lL of DMSO was used to dissolve the formazan crystals and finally
the optical density was noted at 540 nm in an ELISA plate reader (ELx800; BioTek,
VT, USA) [21, 22].
Flow cytometry
Flow cytometric cell sorter analysis was performed to study the effect of test
compounds on cell cycle progression. Around 2 9 106 cells/mL were cultured and
incubated for 48 h with 0.5 lM vincristine and 5 lM test compound. Later, the cells
were washed with ice-cold PBS and fixed with 2 mL of 70 % ice-cold alcohol for
30 min at 4 °C. Then, the cells were centrifuged, and the alcohol was discarded and
washed with 2 mL phosphate buffer saline (PBS). About 100 mg/L of RNase was
added to the pellets and incubated for 1 h at room temperature. Then, 1 mL of PBS
and 5 g/L of propidium iodide was added and incubated for 30 min in the dark.
Finally, the cells were acquired using a BD AccuriTM C6 flow cytometer and cell
cycle distribution was examined [21].
In-silico molecular modelling and docking studies
Microtubules are heterodimers consisting of a- and b-tubulin with 453 and 455
amino acids, respectively, and are critical for cell growth and proliferation. They
undergo conformational changes and switch from straight to curved conformation or
vice versa based on the inhibitors. Tubulin inhibitors are classified based on their
microtubule stabilizing and destabilizing mechanism. Tubulin has three major
binding sites, one each for taxol and vinca-alkaloids which cause microtubule
polymerization and one for colchicine that acts via a microtubule depolymerisation
mechanism. Hence, microtubule dynamics is an important target for anticancer drug
discovery and docking studies has been carried out with 3d in all the three major
inhibitor sites. The structure of the ligand molecules—colchicine (CN2), docetaxel
(TXL), vinblastine (VLB) and 3d in MDL format—were drawn using MarvinSketch
14.11.17.0 (http://www.chemaxon.com). The MOL2 format of these structures was
generated using Open Babel software [23]. As of now, a and b tubulin protein date
bank (PDB) has eight tubulin structures complexed with CN2 (PDB IDs: 1SA0,
1Z2B, 3DU7, and 4O2B), TXL (PDB ids: 1IA0, and 1TUB) and VLB (PDB ids:
1Z2B, and 4EB6). High-resolution tubulin crystal structures complexed with CN2
(PDB ID: 1SA0), TXL (PDB ID: 1TUB) and VLB (PDB ID: 1Z2B) were selected
from a and b tubulin PDB for further studies. Input files necessary for docking
studies were prepared by removing the bound ligands, ions and water, and later
polar hydrogen atoms were added to the protein molecule using AutoDockTools
(v.1.5.6) [24]. The grid maps were created for both receptor and ligands based on
the corresponding ligand binding sites. The docking studies were performed using
AutoDock Vina (v.1.1.2) [25] and the docking parameters were kept as default
values. Molecular interactions were calculated using CONTACT program accessi-
ble in CCP4 suite [26] and surface area specifications were calculated by the
PDBePISA server (http://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver). The
123

Synthesis of indole–quinoline–oxadiazoles: their…
structural models were generated by Pymol (www.pymol.org, PyMol-Version 1.6.
¨
Schrodinger, LLC.).
Results and discussion
Chemistry
Indole-3-carboxylic acids 1(a–c) were appropriately selected to fix four different
alkyl chain spacers at a and b tubulin C-5 position of a and b tubulin oxadiazole
ring for the present study. Substituted 2-(quinolin-8-yloxy)acetohydrazides 2(a–d)
were prepared from substituted quinolin-8-ols via two-step reaction methods as
provided by a and b tubulin literature [27]. The cyclization reaction of equimolar
quantities of indole-3-carboxylic acids 2(a–d) and 2-(quinolin-8-yloxy)acetohy-
drazides 1(a–c) were carried out in nitrogen atmosphere using T3PÒ to synthesize
the respective indole–quinoline–oxadiazoles 3(a–l) [28]. The advantages of T3PÒ,
like less tedious work-up processes and pure products with better yield, wwereas
exploited in the current cyclization reaction. The reaction conditions were optimized
by changing the molar ratios of T3PÒ and TEA (triethyl amine) under various
reaction temperatures and reaction times. Equimolar amounts of reactants 1(a–c)
and 2(a–d) with 2 equivalent of T3PÒ and 4 equivalent of TEA on refluxing in ethyl
acetate medium at 80 °C for 7 h indicated pure product formation. The synthetic
pathway of 8-((5-((1H-indol-3-yl)methyl)-1,3,4-oxadiazol-2-yl)methoxy)quinoline
derivatives 3(a–l) is depicted in Scheme 1.
The T3PÒ-mediated dehydration mechanism of acid and hydrazide involves the
well-established conversion of the oxygen of indole-3-carboxylic acid into an ionic
leaving group that will lead to the formation of 1,2-diacyl hydrazines I (Fig. 1). The
phosphorous atom of T3PÒ will then attack the oxygen of the carbonyl group of 1,2-
diacyl hydrazine I to produce an intermediate II that would further cyclize to
intermediate III, followed by nitrogen-aided removal of the anion to give the final
oxadiazole derivative IV. The ionic byproduct can be easily extracted by aqueous
work-up with minor product loss. The proposed cyclodehydration mechanism
supported by T3PÒ is outlined in Fig. 1.
The carbonyl absorption peaks of the two reactants, 2-(quinolin-8-yloxy)aceto-
hydrazides and indole-3-carboxylic acids, were observed at 1670–1680 and
1715–1725 cm-1 in their respective IR spectra. The absence of these carbonyl
absorptions in the IR spectra of oxadiazole confirms the formation of the cyclized
product. The 1H NMR of the reactants showed signals at 2 ppm that corresponds to
NH2 protons and 11 ppm for the acid proton. These characteristic peaks were also
1
not observed in the HNMR spectra of oxadiazoles which further substantiates the
complete cyclization of the reactants. The OCH2 protons of oxadiazoles were found
to resonate in the deshielded region at 4.80–4.97 ppm, while the methylene protons
were observed upfield in the range of 1.8–2.7 ppm, which was further confirmed by
the DEPT C NMR-spectra. The mass spectra of oxadiazoles displayed the molecular
ion peaks as per their respective molecular masses.
123

P. R. Kamath et al.
Scheme 1 Synthetic pathway for 8-((5-((1H-indol-3-yl)methyl)-1,3,4-oxadiazol-2-yl) methoxy)quinolines 3(a–i)
Biological activity
All the synthesized oxadiazoles (3a–l) were subjected to MTT assay in order to
analyze their in vitro cytotoxicity profile and the results are represented in Table 1.
The assay was performed in triplicate and the results were statistically analyzed
using Graph Pad Prism, 5. Compound 3d was found to significantly reduce the
viability of MCF7 cells with an IC50 value of 8.62 lM. It also displayed higher
selectivity (fourfold) to breast adenocarcinoma cells than to vero cells, whereas the
selectivity index of vincristine was three. The active compound 3d is derived from
indole-3-butyric acid and 2-(quinolin-8-yloxy)acetohydrazide.
Apoptosis induced by tubulin-interfering agents is widely testified to follow G2/
M cell cycle arrest. To study the effect of compound 3d on different phases of the
MCF7 cell cycle, flow cytometric studies were carried out. A normal cell cycle
pattern was observed in the histogram of DMSO-treated control cells is presented in
Fig. 2. The standard antimitotic drug vincristine (0.5 lM), exhibited G2/M phase
cell cycle arrest in breast adenocarcinoma cells at the end of 48 h, as shown in
Fig. 2. A cell cycle arrest in the mitotic phase was seen in the histogram of MCF7
cells when subjected to a 48-h treatment with oxadiazole 3d. A lesser number of
cells were observed in the S phase (7.2 %) when incubated with 3d compared with
123

Synthesis of indole–quinoline–oxadiazoles: their…
Fig. 1 Proposed mechanism for the formation of 1,3,4-oxadiazole mediated by T3PÒ and TEA
Table 1 In vitro cell growth
Comp. no.
IC50 (lM)
MCF-7
IC50 (lM)
VERO
inhibition by indole–quinoline–
oxadiazole 3(a–l) using MTT
assay
Vincristine
0.3 0.02
205.5 5.89
[200
0.9 0.04
[200
3a
3b
3c
3d
3e
3f
[200
[200
[200
8.62 – 1.23
[200
38.09 – 1.87
[200
[200
[200
3g
3h
3i
[200
[200
136.6 3.57
[200
[200
[200
3j
[200
[200
3k
3l
[200
[200
Bold distinguishes the most
active molecule
[200
[200
control, with 11.8 % of MCF-7 cells permitting the cells to move to mitotic phase.
Moreover, a rise in the number of mitotic cells from 19.5 % (as seen in the control)
to 23.7 % in the 3d-treated cells was visible in the cell cycle histogram, which is
indicative of mitotic cell cycle arrest.
123

P. R. Kamath et al.
Fig. 2 Detection of cell cycle arrest using flow cytometry. A reduction in the percentage of cells in the S
phase allowing cells to move into the mitotic phase and accumulation of cells in mitotic phase is seen in
3d-treated cells when compared to control
Molecular modeling studies
The interaction of tubulin with a large number of drugs that inhibit or promote
microtubule assembly has led to the identification of three drug binding sites,
namely colchicine, taxol and vinca sites on the protein. In order to investigate the
extent of interactions involved between the most active compound 3d with the three
known binding sites of microtubules, molecular docking studies were carried out.
Initial docking was performed with the control molecules—colchicine, pacilitaxel,
and vinblastine—to check the accuracy of molecular docking studies. Later, 3d was
docked into these binding sites with three separate seeds. Autodock Vina yielded 20
different conformations for each binding site. Selection of the final models was done
based on the binding affinity values. The respective binding energies and
computational molecular interaction details for each of the ligands are presented
in Table 2. The binding affinities for all the controls were on par with the reported
values. The superimposed models of 3d and controls in their respective binding
pockets are shown in Fig. 3. The binding models of 3d to three tubulin binding sites
are presented in Fig. 4. Oxadiazole 3d was found to make hydrogen bonds with
Asn101, Ala317 and Lys 352 amino acids when docked at the colchicine site.
Interestingly, 3d also covered similar orientation of colchicine (Fig. 3a) and was
found to make hydrophobic contacts with seven amino acids (Table 2) identical to
that observed with colchicine. This orientation played a key role in allowing the two
nitrogen atoms of the oxadiazole ring of 3d to make hydrogen bonds with the
˚
˚
hydrogen attached to the hydroxyl groups of Lys353 (3.09 A), Ala317 (2.36 A) and
˚
amine group of Asn101 (3.24 A) of the tubulin amino acids. The oxygen atom
present between quinoline and oxadiazole moieties were involved in the interaction
with tubulin amino acids apart from the nitrogen atoms in the rings. Surprisingly,
the indole moiety of 3d did not show any appreciable interaction with the tubulin
amino acids. In the taxol binding site, 3d was found to interact through two
hydrogen bonds (His229 and Thr226) and eight hydrophobic contacts within the 4.0
˚
A cut-off from tubulin amino acids. In the crystal structure with tubulin, TXL also
displayed similar hydrogen bonds with His229. Similar results were obtained when
3d was docked at the VLB site. The hydrogen bond details and hydrophobic
123

Synthesis of indole–quinoline–oxadiazoles: their…
Table 2 Docking results of ligand 3d at colchicine, paclitaxel and vinblastine binding site of
microtubule
At colchicine binding site
CN2a
At paclitaxel biding site
TXLa
At vinblastine biding site
VLBa
3d
3d
3d
Molecular
formula
C22H25NO6S C23H20N4O2 C43H53NO14 C23H20N4O2 C46H58N4O9 C23H20N4O2
Molecular
weight
(g/mol)
431.50
630
384.439
227
807.88
865
384.439
227
810.97
927
384.439
227
Over all
surface
2
˚
area (A )
Binding
affinity
(kcal/mol)
-7.4
-9.3
-8.6
-8.7
-9.6
-7.2
Tubulin
amino
acids
Ser178
Val181
Cys241
Leu242
Leu248
Ala250
Lys254
Leu255
Asn258
Met259
Ala316
Lys352
Ile378
Asn101
Cys241
Leu248
Ala250
Lys254
Leu255
Ala316
Ala317
Val318
Lys352
Thr353
Ala354
Val23
Leu217
Leu219
Asp226
His229
Leu230
Phe272
Leu275
Thr276
Ser277
Leu371
Lys176
Val177
Tyr210
Phe214
Thr221
Pro222
Thr223
Tyr224
Leu227
Pro325
Val328
Val353
Ile355
Val177
Tyr224
Asn249
Pro325
Val328
Asn329
Ile332
Asp226
His229
Ser236
Phe272
Leu275
Thr276
Pro360
Arg369
Gly370
Leu371
Val353
Ile355
Asn329
335.8
Maximum
interface
407.2
124.7
557.7
139.8
129.3
2
˚
area (A )
Tubulin amino acids involved in possible hydrogen bonds are shown in bold
a
Controls
contacts compared to VLB are summarized in Table 2. In general, the docking
scores, molecular interactions and interaction surface obtained for 3d well fitted
with the colchicine site. The binding affinity and molecular interactions suggest
better compatibility for 3d with the colchicine site, in comparison with the TXL and
VLB sites.
The introduction of methylene linkers between C-3 of the indole ring and C-5 of
the oxadiazole moiety was reported to show promising activity [7, 21]. This study
aims to incorporate diversity in methylene linker substitution at C-5 positions of the
oxadiazole ring connected to C-3 of the highly bioactive indole group. The effect of
123

P. R. Kamath et al.
Fig. 3 Superimposed models of tubulin bound ligand 3d on a colchicine, b pacilitaxel, and c vinblastine
in their corresponding binding sites extracted from the crystal structures. Oxadiazole 3d: carbon (yellow),
oxygen (red), nitrogen (dark blue); CN2, TXL, VLB: carbon (green), nitrogen (blue) and oxygen (red).
(Color figure online)
Fig. 4 Computational docking models: 3d docked at a colchicine, b pacilitaxel, and c vinblastine sites of
tubulin
the electron-donating methyl group and the electro-withdrawing chlorine atom
attached to the quinoline moiety on the cytotoxic and antimitotic profile of indole–
quinoline–1,3,4-oxadiazole derivatives was explored. The SAR study reveals that a
longer three-methylene side chain linker (n = 3) at C-5 position of oxadiazole
without any electron-donating or -withdrawing substituents on the quinoline ring
has a relatively enhanced activity. The molecular docking results show that the
nitrogens of the oxadiazole and quinoline rings are essential as recognition features
with the key amino acid residues at the enzyme binding site for tubulin inhibition. A
flexible oxo-methylene linker is essential to permit hydrophobic recognition of
phenyl rings with the hydrophobic area of amino acids at the enzyme pocket [29].
The introduction of a methyl group at the C-2 position and two chloro substituents at
the C-5 and C-7 positions of the quinoline ring did not increase the anticancer
activity of the oxadiazole derivatives.
Conclusions
The present study discusses a convenient, efficient and green synthetic route to
synthesize 12 new 8-((5-((1H-indol-3-yl)methyl)-1,3,4-oxadiazol-2-yl)methoxy)quino-
lines from substituted 2-(quinolin-8-yloxy) acetohydrazides and indole-3-carboxylic
acids with T3PÒ as catalytic dehydrocyclization agent. Oxadiazole 3d inhibited breast
adenocarcinoma cell proliferation with a low IC50 value, high selectivity index and G2/
123

Synthesis of indole–quinoline–oxadiazoles: their…
M cell cycle arrest advocating its potential to act as a lead anticancer and antimitotic
molecule. Good binding interactions of oxadiazole 3d with the colchicine binding
cavity of microtubules correlates well with the antiproliferative activity and holds great
promise as a potential tubulin inhibitor causing mitotic cell cycle arrest leading to
apoptosis. Although colchicine is used in the treatment of gouty arthritis and familial
Mediterranean fever, neither colchicine nor any related compounds bind to colchicine
binding sites due to their toxicity. Based on these facts, 3d and its derivatives may serve
as a potent alternative.
Acknowledgment The authors are grateful to the Department of Science and Technology, Science and
Engineering Research Board, India, for supporting the computational facilities through Grant SB/FT/LS-
273/2012.
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