Synthesis of Novel Indolyl-1,2,4-triazoles as Potent and Selective Anticancer Agents

Article abstract of DOI:10.1111/j.1747-0285.2010.01051.x

A diverse series of 22 indolyl-1,2,4-triazole congeners (6 and 7) have been synthesized from the reaction of indole-3-carbonitrile (4) or (5) with appropriate acid hydrazides in the presence of potassium carbonate. Synthesized compounds were evaluated for their cytotoxicity against six human cancer cell lines, and some of the compounds displayed promising activity. In particular, 3-(3',4',5'-trimethoxyphenyl)-5-(N-methyl-3'-indolyl)-1,2,4-triazole (7i) and 3-(4'-piperidinyl)-5-(N-methyl-3'-indolyl)-1,2,4-triazole (7n) were the most promising and broadly active compounds against the tested cell lines. It was interesting to note that the trimethoxyphenyl analog 7i showed twofold selective cytotoxicity against PaCa2 cell line (IC₅₀ 0.8μm), whereas piperidinyl analog 7n was found to be selectively cytotoxic against MCF7 cell line (IC₅₀ 1.6μm). Notably, the 4-fluorophenyl derivative 7c exhibited selective cytotoxicity against PC3 cell line (IC₅₀ 4μm). The structure-activity relationship study revealed that substituents including 3,4,5-trimethoxyphenyl, 3,4-dimethoxyphenyl, 4-benzyloxy-3-methoxyphenyl, 4-piperidinyl, 4-fluorophenyl and N-methylindole are beneficial for the activity of indolyl-1,2,4-triazoles (6 and 7).

Full text of DOI:10.1111/j.1747-0285.2010.01051.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2010.01051.x
Chem Biol Drug Des 2011; 77: 182–188
Research Article
Synthesis of Novel Indolyl-1,2,4-triazoles as
Potent and Selective Anticancer Agents
Dalip Kumar1,*, Maruthi Kumar
Narayanam1, Kuei-Hua Chang2 and
Kavita Shah2,*
broad spectrum of biological properties, including antifungal (5), an-
tiviral (6), antimicrobial (7), A2A receptor antagonists (8), and COX-2
inhibitors (9). Further, the five-membered heterocycles linked with
indole moiety are reported for their anticancer activities (Figure 1).
For example, indolyloxazoles such as Labradorin 1 and Labradorin 2
are found to be potential inhibitors of NCI-H460 human lung cancer
cell line with GI50 values 9.8 and 9.6 lg ⁄ mL, respectively (10,11).
Camalexin, an indolylthiazole and its analogs have shown antitumor
activity against the breast cancer cell lines (12,13). The
bis(indole)alkaloids having bis(indolyl)imidazole skeleton and isolated
from the deep sea sponge Spongosorites ruetzler are reported to
display significant inhibitory effects on the growth of a range of
cancer cells. For example, nortopsentin A-C and its N-methyl ana-
logs have demonstrated impressive cytotoxicity against P388 cancer
cell line (14,15). The anticancer activities of these parent natural
products were further improved by introducing a variety of hetero-
cycles at position 3 of indole moiety (16,17). Recently, 1,2,4-triaz-
oles have been reported as combretastatin A-4 analogs to inhibit
tubulin polymerization (18), and some of them also act as ghrelin
receptor antagonists (19).
1Chemistry Group, Birla Institute of Technology and Science, Pilani,
Rajasthan 333 031, India
2Department of Chemistry and Purdue Cancer Center, Purdue
University, 560 Oval Drive, West Lafayette, IN 47907, USA
*Corresponding authors: Dalip Kumar, dalipk@bits-pilani.ac.in;
Kavita Shah, shah23@purdue.edu
A diverse series of 22 indolyl-1,2,4-triazole cong-
eners (6 and 7) have been synthesized from the
reaction of indole-3-carbonitrile (4) or (5) with
appropriate acid hydrazides in the presence of
potassium carbonate. Synthesized compounds
were evaluated for their cytotoxicity against
six human cancer cell lines, and some of the com-
pounds displayed promising activity. In particular,
3-(3¢,4¢,5¢-trimethoxyphenyl)-5-(N-methyl-3¢-indolyl)-
1,2,4-triazole (7i) and 3-(4¢-piperidinyl)-5-(N-methyl-
3¢-indolyl)-1,2,4-triazole (7n) were the most
promising and broadly active compounds against
the tested cell lines. It was interesting to note that
the trimethoxyphenyl analog 7i showed twofold
selective cytotoxicity against PaCa2 cell line (IC50
0.8 lM), whereas piperidinyl analog 7n was found
to be selectively cytotoxic against MCF7 cell line
(IC50 1.6 lM). Notably, the 4-fluorophenyl deriva-
tive 7c exhibited selective cytotoxicity against
PC3 cell line (IC50 4 lM). The structure–activity
relationship study revealed that substituents
including 3,4,5-trimethoxyphenyl, 3,4-dimethoxy-
phenyl, 4-benzyloxy-3-methoxyphenyl, 4-piperidi-
nyl, 4-fluorophenyl and N-methylindole are
beneficial for the activity of indolyl-1,2,4-triazoles
(6 and 7).
The most common method for the preparation of 1,2,4-triazoles
involves condensation of hydrazides with nitriles ⁄ thioamides at
elevated temperatures (20). Recently, N-substituted triazoles have
been prepared in good yields using one-pot reaction of arylam-
ines, N,N-dimethylformamide dimethyl acetal and acylhydrazide in
acetonitrile (21). A solid-phase synthesis of 3-alkylamino-1,2,4-triaz-
oles involves the initial preparation of immobilized N-acyl-benzo-
triazoles followed by reaction with hydrazine (22). Another
efficient route uses the reaction of nitriles with sodium methoxide
to generate methyl imidate ester, which upon treatment with aryl
hydrazides at high temperature produced 1,2,4-triazoles in good
yields (23–27). Recently, Al-masoudi et al. (28) have reviewed
most of the synthetic routes adopted for the efficient preparation
of 1,2,4-triazoles. In our efforts to develop indole-based hetero-
Key words: 1,2,4-Triazoles, anticancer agents, hydrazides, indolyl-
1,2,4-triazoles, indolylazoles
N
N
Received 17 April 2010, revised 18 August 2010 and accepted for publi-
cation 26 September 2010
N
R
X
R
X
N
H
N
H
Nitrogen-containing five-membered heterocycles plays a vital role in
drug discovery to identify novel chemical entities of immense thera-
peutic potential. Triazoles are the most privileged structures that
are widely explored for their range of pharmacological properties
(1–4). In literature, 1,2,4-triazoles are well documented for their
X = O, S, NH
R = alkyl, aryl, heteroaryl
Figure 1: Structures of some 5-(3¢-indolyl)azoles.
182

Potent and Selective Anticancer Agents
cycles as novel and potential anticancer agents, we have reported
indolyloxazoles, indolyl-1,3,4-oxadiazoles, and indolyl-1,3,4-thiadia-
zole (29–31). In view of the interesting activities of various indoly-
lazoles and 1,2,4-triazoles, we report the synthesis of novel
products (4 and 5) were in agreement with the reported in litera-
ture (34,35).
indolyl-1,2,4-triazoles and their anticancer activities against
panel of six human cancer cell lines.
a
General procedure for the synthesis of indolyl-
1,2,4-triazoles 6 and 7
To a mixture of indole-3-carbonitrile 4 or 5 (2 mmol) and potassium
carbonate (0.5 mmol) in n-BuOH (3 mL) was added acid hydrazide
(1 mmol), and the reaction mixture was stirred at 160 ꢀC for 8 h.
The progress of the reaction was monitored by TLC (23). After com-
pletion of reaction, the solvent was removed under reduced pres-
sure, and the residue so obtained was purified by column
chromatography on silica gel with hexane and ethyl acetate as elu-
ent to obtain pure indolyl-1,2,4-triazoles 6 and 7.
Material and Methods
General
The reactions were monitored by thin-layer chromatography (TLC)
using Merck silica gel plate (60 F254, 0.25 mm), and TLC plates
were visualized by fluorescence quenching under UV light (254 nm).
1H NMR spectra were recorded using Bruker Avance (400 MHz)
spectrometer [Bruker Optik GmbH (For FT-IR, NIR, Raman & minispec
TD-NMR Products), Mumbai, India]. Compounds were taken in
DMSO-d6 and CDCl3 for recording the spectra using tetramethylsi-
lane (TMS) as an internal standard, chemical shifts (d) were
reported in ppm and coupling constant J values were expressed in
Hz. Mass spectra were recorded using 'Hewlett-Packard' HP GS ⁄ MS
5890 ⁄ 5972. Melting points were determined using electrothermal
capillary melting point apparatus and are uncorrected. Buchi rotava-
por (BꢀCHI Labortechnik AG, Flawil 1, Switzerland) was used to
distill off the solvents. All commercially available reagents and sol-
vents were purchased from Merck (Merck Limited, Mumbai, India)
and Aldrich (Sigma-Aldrich Corporation, Bangalore, India) and used
as such without further purification. Compounds were purified by
column chromatography using 100–200 mesh silica gel and hexane
and ethyl acetate as eluent.
3-(4¢-Chlorophenyl)-5-(3¢-indolyl)-1,2,4-triazole
(6a)
White solid. Yield 65%, m.p. 263–265 ꢀC. 1H NMR (400 MHz,
DMSO-d6): 11.80 (s, 1H, NH), 9.32 (s, 1H, NH), 8.03 (d, 2H,
J = 8.12 Hz, Ar-H), 7.82 (s, 1H, Ar-H), 7.41 (d, 2H, J = 8.20 Hz, Ar-
H), 7.32-7.20 (m, 3H, Ar-H), 6.92-6.90 (m, 1H, Ar-H). MS (Electro-
spray Ionization (ESI), m ⁄ z): 295.16 (M + H)+.
3-(4¢-Benzyl)-5-(3¢-indolyl)-1,2,4-triazole (6b)
White solid. Yield 72%, m.p. 170–172 ꢀC. 1H NMR (400 MHz,
DMSO-d6): 11.75 (s, 1H, NH), 9.89 (s, 1H, NH), 8.18-8.15 (m, 1H, Ar-
H), 7.86-7.87 (m, 2H, Ar-H), 7.49-7.46 (m, 1H, Ar-H), 7.39-7.33 (m,
3H, Ar-H), 7.31-7.27 (m, 1H, Ar-H), 7.25-7.17 (m, 2H, Ar-H). 4.76 (s,
2H, CH2). MS (ESI, m ⁄ z): 274.13 (M)+.
Experimental
3-(3¢-Methoxyphenyl)-5-(3¢-indolyl)-1,2,4-triazole
(6c)
White solid. Yield 68%, m.p. 165–167 ꢀC. 1H NMR (400 MHz,
DMSO-d6): 11.08 (s, 1H, NH), 9.88 (s, 1H, NH), 8.32-8.17 (m, 2H, Ar-
H), 7.72 (s, 1H, Ar-H), 7.27-7.30 (m, 6H, Ar-H), 3.90 (s, 3H, OCH3).
MS (ESI, m ⁄ z) 291.14 (M + H)+.
General procedure for the preparation of
indole-3-carboxaldehyde (2) and N-methyl
indole-3-carboxaldehyde (3)
Indole-3-carboxaldehyde (2) was synthesized from commercially
available indole as described in the literature (32,33). The N-methyl-
ation of 2 was carried out using dimethylcarbonate in the presence
of potassium carbonate (32,33).
3-(4¢-Hydroxy-3¢-methoxyphenyl)-5-(3¢-indolyl)-
1,2,4-triazole (6d)
General procedure for the synthesis of indole-3-
carbonitriles 4 and 5
White solid. Yield 65%, m.p. 180–182 ꢀC. 1H NMR (400 MHz,
DMSO-d6): 11.80 (s, 1H, NH), 9.79 (s, 1H, NH), 8.30 (d, 1H,
J = 2.8 Hz, Ar-H), 8.22-8.19 (m, 2H, Ar-H), 7.50-7.46 (m, 2H, Ar-H),
7.29-7.20 (m, 3H, Ar-H), 4.90 (s, 1H, CH2), 3.79 (s, 3H, OCH3). MS
(ESI, m ⁄ z): 306.12 (M)+.
To a stirred solution of indole-3-carboxaldehyde 2 or 3 (0.01 mol)
in formic acid (10 mL) was added sodium formate (0.02 mol) and
hydroxylamine hydrochloride (0.01 mol). The reaction mixture was
refluxed for 3 h at 130 ꢀC. After completion of the reaction as
monitored by TLC, the reaction mixture was cooled to room tem-
perature and poured into ice-cold water (100 mL) and extracted
with dichloromethane (2 · 30 mL). The combined extracts were
washed with saturated sodium bicarbonate solution (30 mL) and
then with brine solution (30 mL). Organic phase was separated,
dried over sodium sulfate, and excess of solvent was distilled off.
The residue so obtained was subjected to a silica gel column
chromatography (hexane and ethyl acetate as eluent) to afford
pure indole-3-carbonitriles (4) or (5). The melting points of the
3-(4¢-Benzyloxy-3¢-methoxyphenyl)-5-(3¢-indolyl)-
1,2,4-triazole (6e)
Off-white solid. Yield 65%, m.p. 225 (dec). 1H NMR (400 MHz,
DMSO-d6): 11.30 (s, 1H, NH), 9.89 (s, 1H, NH), 8.26 (d, 1H,
J = 7.84 Hz, Ar-H), 7.69-7.64 (m, 1H, Ar-H), 7.41-7.39 (m, 2H, Ar-H),
7.35-7.23 (m, 5H, Ar-H), 7.28-7.21 (m, 2H, Ar-H), 7.03 (d, 2H,
J = 1.9 Hz, Ar-H), 5.21 (s, 2H, CH2), 3.78 (s, 3H, OCH3). MS (ESI,
m ⁄ z) 398.11 (M + 2)+.
Chem Biol Drug Des 2011; 77: 182–188
183

Kumar et al.
3-(3¢,4¢,5¢-Trimethoxyphenyl)-5-(3¢-indolyl)-1,2,4-
triazole (6f)
3-(3¢,5¢-Dimethoxyphenyl)-5-(N-methyl-3¢-
indolyl)-1,2,4-triazole (7f)
White solid. Yield 74%, m.p. 245–247 ꢀC. 1H NMR (400 MHz,
DMSO-d6): 10.57 (s, 1H, NH), 9.87 (s, 1H, NH), 8.37-8.17 (m, 1H, Ar-H),
7.97 (s, 1H, Ar-H), 7,55-7.24 (m, 3H, Ar-H), 6.90 (d, 2H, J = 2.16 Hz,
Ar-H), 3.93 (s, 6H, OCH3), 3.77 (s, 3H, OCH3). MS (ESI, m ⁄ z)
374.24(M + Na)+.
Off-white solid. Yield 65%, m.p. 183–185 ꢀC. 1H NMR (400 MHz,
DMSO-d6): 11.48 (s, 1H, NH), 8.36 (s, 1H, Ar-H), 7.54 (s, 1H, Ar-H),
7.48-7.39 (m, 1H, Ar-H), 7.10 (d, 2H, J = 8.2 Hz, Ar-H), 6.90 (d, 1H,
J = 7.56 Hz, Ar-H), 6.61-6.48 (m, 2H, Ar-H), 3.90 (s, 6H, OCH3). 3.83
(s, 3H, NCH3). MS (ESI, m ⁄ z) 335.15 (M + H)+.
3-(4¢-Pyridyl)-5-(3¢-indolyl)-1,2,4-triazole (6g)
Pale yellow solid. Yield 62%, m.p. 190–193 ꢀC. H NMR (400 MHz,
DMSO-d6). 12.08 (s, 1H, NH), 9.87 (s, 1H, NH), 8.77 (d, 2H, J = 5.6 Hz,
Ar-H), 8.32 (d, 1H, J = 1.2 Hz, Ar-H), 8.23 (d, 1H, J = 8.8 Hz, Ar-H),
7.94 (d, 2H, J = 7.6 Hz, Ar-H), 7.54 (d, 1H, J = 8.8 Hz, Ar-H), 7.28-7.26
(m, 2H, Ar-H). MS (ESI, m ⁄ z) 263.20 (M + 2)+.
3-(3¢,4¢-Methylenedioxyphenyl)-5-(N-methyl-3¢-
indolyl)-1,2,4-triazole (7g)
1
Brown solid. Yield 68%, m.p. 252–254 ꢀC. 1H NMR (400 MHz,
DMSO-d6): 13.89 (s, 1H, NH), 8.29 (d, 1H, J = 7.56 Hz, Ar-H), 7.97
(s, 1H, Ar-H), 7.67 (d, 1H, J = 7.48 Hz, Ar-H), 7.58-7.54 (m, 2H, Ar-
H), 7.32-7.24 (m, 2H, Ar-H), 7.06 (t, 1H, J = 7.92 Hz, Ar-H), 6.11 (s,
2H, CH2), 3.88 (s, 3H, NCH3). MS (ESI, m ⁄ z) 319.13 (M + H)+.
3-Phenyl-5-(N-methyl-3¢-indolyl)-1,2,4-triazole (7a)
Off-white solid. Yield 65%, m.p. 180–182 ꢀC. 1H NMR (400 MHz,
DMSO-d6): 12.20 (s, 1H, NH), 8.13 (d, 2H, J = 7.80 Hz, Ar-H), 7.97
(s, 1H, Ar-H), 7.48 (d, 1H, J = 7.48 Hz, Ar-H), 7.24-7.14 (m, 5H, Ar-H),
7.12-7.10 (m, 1H, Ar-H), 3.81 (s, 3H, NCH3). MS (ESI, m ⁄ z) 275.20
(M + H)+.
3-(4¢-Benzyloxy-3¢-methoxyphenyl)-5-(N-methyl-
3¢-indolyl)-1,2,4-triazole (7h)
Brown solid. Yield 58%, m.p. 182–184 ꢀC. 1H NMR (400 MHz,
DMSO-d6): 11.70 (s, 1H, NH), 8.26 (d, 1H, J = 7.84 Hz, Ar-H), 7.63-
7.54 (m, 3H, Ar-H), 7.41-7.39 (m, 2H, Ar-H), 7.35-7.23 (m, 5H, Ar-H),
7.15-6.77 (m, 2H, Ar-H), 5.08 (s, 2H, CH2), 3.78 (s, 3H, OCH3), 3.58
(s, 3H, NCH3). MS (ESI, m ⁄ z) 411.16 (M + H)+.
3-(4¢-Chlorophenyl)-5-(N-methyl-3¢-indolyl)-1,2,4-
triazole (7b)
Pale yellow. Yield 62%, m.p. 215–217 ꢀC. 1H NMR (400 MHz,
DMSO-d6,): 13.72 (s, 1H, NH), 8.42 (d, 1H, J = 7.72 Hz, Ar-H), 8.16
(d, 2H, J = 7.72 Hz, Ar-H), 7.98-7.88 (m, 3H, Ar-H), 7.67 (d, 1H,
J = 7.12 Hz, Ar-H), 7.30-7.25 (m, 2H, Ar-H), 3.91 (s, 3H, NCH3). MS
(ESI, m ⁄ z) 308.16 (M)+.
3-(3¢,4¢,5¢-Trimethoxyphenyl)-5-(N-methyl-3¢-
indolyl)-1,2,4-triazole (7i)
Yellow solid. Yield 70%, m.p. 146–148 ꢀC. 1H NMR (400 MHz,
DMSO-d6): 11.90 (s, 1H, NH), 8.57 (d, 1H, J = 2.32 Hz, Ar-H), 8.23
(d, 1H, J = 1.8 Hz, Ar-H), 8.00 (s, 1H, Ar-H), 7.41-7.23 (m, 4H, Ar-H),
3.90 (s, 9H, OCH3), 3.73 (s, 3H, NCH3). MS (ESI, m ⁄ z) 365.16
(M + H)+.
3-(4¢-Fluorophenyl)-5-(N-methyl-3¢-indolyl)-1,2,4-
triazole (7c)
1
Off-white. Yield 65%, m.p. 202–204 ꢀC. H NMR (400 MHz, DMSO-
3-(4¢-N,N-Dimethylaminophenyl)-5-(5¢-bromo-N-
methyl-3¢-indolyl)-1,2,4-triazole (7j)
d6): 12.04 (s, 1H, NH), 9.80 (s, 1H, Ar-H), 8.19 (d, 2H, J = 7.82 Hz,
Ar-H), 7.92-7.89 (m, 1H, Ar-H), 7.35-7.25 (m, 2H, Ar-H), 7.23-7.05 (m,
3H, Ar-H), 3.77 (s, 3H, NCH3). MS (ESI, m ⁄ z) 293.23 (M + H)+.
Off-white solid. Yield 70%, m.p. 226–228 ꢀC. 1H NMR (400 MHz,
DMSO-d6): 11.50 (s, 1H, NH), 8.40 (d, 1H, J = 1.80 Hz, Ar-H), 7.91
(s, 1H, Ar-H), 7.68 (s, 1H, Ar-H), 7.33-7.25 (m, 3H, Ar-H), 6.73-6.68
(m, 2H, Ar-H), 3.83 (s, 3H, NCH3). 3.13 (s, 6H, NCH3). MS (ESI, m ⁄ z)
396.12 (M + H)+.
3-(4¢-Trifluoromethylphenyl)-5-(N-methyl-3¢-
indolyl)-1,2,4-triazole (7d)
Yellow solid. Yield 62, m.p. 197–199 ꢀC. 1H NMR (400 MHz,
DMSO-d6): 13.86 (s, 1H, NH), 8.43 (d, 1H, J = 7.56 Hz, Ar-H), 8.38
(d, 2H, J = 6.84 Hz, Ar-H), 7.95 (s, 1H, Ar-H), 7.72 (d, 2H,
J = 6.64 Hz, Ar-H), 7.41 (d, 1H, J = 7.36 Hz, Ar-H), 7.34-7.28 (m, 2H,
Ar-H), 3.90 (s, 3H, NCH3). MS (ESI, m ⁄ z) 343.12 (M + H)+.
3-(3¢,4¢,5¢-Trimethoxyphenyl)-5-(6¢-bromo-N-
methyl-3¢-indolyl)-1,2,4-triazole (7k)
White solid. Yield 72%, m.p. 160–163 ꢀC. 1H NMR (400 MHz,
DMSO-d6): 11.57 (s, 1H, NH), 8.37 (s, 1H, Ar-H), 7.66 (s, 1H, Ar-H),
7.10 (d, 1H, J = 2.20 Hz, Ar-H), 6.90 (d, 1H, J = 2.16 Hz, Ar-H),
6.62-6.47 (m, 2H, Ar-H), 3.94 (s, 9H, OCH3). 3.83 (s, 3H, NCH3). MS
(ESI, m ⁄ z) 443.11 (M + H)+.
3-(3¢,4¢-Dimethoxyphenyl)-5-(N-methyl-3¢-
indolyl)-1,2,4-triazole (7e)
Off-white solid. Yield 63%; m.p. 200–203 ꢀC. 1H NMR (400 MHz,
DMSO-d6): 13.10 (s, 1H, NH), 8.27 (d, 1H, J = 7.87 Hz, Ar-H), 7.71-
7.64 (m, 3H, Ar-H), 7.27-7.23 (m, 2H, Ar-H), 7.18-7.14 (m, 1H, Ar-H),
7.06 (d, 1H, J = 8.00 Hz, Ar-H), 3.86 (s, 6H, OCH3). 3.68 (s, 3H,
NCH3). MS (ESI, m ⁄ z) 335.20 (M + H)+.
3-(3¢,4¢,5¢-Trimethoxyphenyl)-5-(5¢-bromo-N-
methyl-3¢-indolyl)-1,2,4-triazole (7l)
White solid. Yield 68%, m.p. 209–211 ꢀC. 1H NMR (400 MHz,
DMSO-d6): 13.6 (s, 1H, NH), 8.37 (d, 1H, J = 1.76 Hz, Ar-H), 7.86 (s,
184
Chem Biol Drug Des 2011; 77: 182–188

Potent and Selective Anticancer Agents
1H, Ar-H), 7.47-7.34 (m, 2H, Ar-H), 7.29-7.14 (m, 2H, Ar-H), 3.94 (s,
1H, CH), 1.15 (d, J = 6.6 Hz, 6H, CH3). MS (ESI, m ⁄ z) 255.12
9H, OCH3). 3.83 (s, 3H, NCH3). MS (ESI, m ⁄ z) 443.08 (M + H)+.
(M + H)+.
5-(N-Methyl-3¢-indolyl)-3-(4¢-pyridyl)-1,2,4-
triazole (7m)
Anticancer activity
Pale yellow. Yield 65%, m.p. 214–215 ꢀC. 1H NMR (400 MHz,
DMSO-d6): 13.08 (s, 1H, NH), 8.70-8.69 (m, 1H, Ar-H), 8.44 (d, 1H,
J = 7.44 Hz, Ar-H), 8.10 (s, 1H, Ar-H), 7.93-7.88 (m, 2H, Ar-H), 7.70
(d, 1H, J = 7.40 Hz, Ar-H), 7.42 (d, 1H, J = 7.12 Hz, Ar-H), 7.33-
7.25 (m, 2H, Ar-H), 3.89 (s, 3H, NCH3). MS (ESI, m ⁄ z) 276.21
(M + H)+.
Cell lines and cell culture
The human cancer cell lines, prostate (PC3, DU145 and LnCaP),
breast (MCF7 and MDA-MB-231), and pancreatic (PaCa2), were
obtained from ATCC.
Cytotoxicity assay
Cytotoxic effects were examined in six human cancer cell lines
(LnCaP, DU145, PC3, MCF7, MDA-MB-231, and PaCa2) using 3-(4,5-
dimethyldiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay.
Cells were cultured in RPMI-1640 media supplemented with 10%
heat-inactivated fetal bovine serum and 1% penicillin ⁄ streptomycin.
They were seeded in 96-well plates at a density of 4 · 103 cells
per well for 12 h. Cells were incubated with varying concentrations
(10 nM–1 mM) of the compounds for 48 h at 37 ꢀC. MTT was added
to the final concentration of 0.2 mg ⁄ mL and incubated for 30 min.
The cells were washed twice with PBS and lysed in 100 lL dimeth-
ylsulfoxide, and the absorbance was measured at 570 nm using
Tecan Spectrafluor Plus (Tecan US, Inc. Durham, NC, USA). IC50 val-
ues were determined by a nonlinear regression analysis with a
curve fitting program, GraphPad Prism 5.0 (GraphPad Software, La
Jolla, CA, USA). Activity results obtained are mentioned in the
Table 1.
3-(4¢-Piperidinyl-5-(N-methyl-3¢-indolyl)-1,2,4-
triazole (7n)
Brown solid. Yield 65%, m.p. 147–150 ꢀC. 1H NMR (400 MHz,
DMSO-d6): 11.42 (s, 1H, NH), 7.74-7.71 (m, 2H, Ar-H), 7.50 (d, 1H,
J = 7.64 Hz, Ar-H), 7.30-7.20 (m, 2H, Ar-H), 3.94 (s, 3H, NCH3). 3.41-
3.39 (m, 1H CH), 3.19-3.12 (m, 4H, CH2), 2.59 (s, 1H, NH), 1.15-1.12
(m, 4H, CH2). MS (ESI, m ⁄ z) 281.19 (M)+.
3-(Isobutyl)-5-(N-methyl-3¢-indoly)-1,2,4-
triazole(7o)
Brown solid. Yield 58%, m.p. 130–133 ꢀC. 1H NMR (400 MHz,
DMSO-d6): 11.97 (s, 1H, NH), 8.16 (d, 1H, J = 8.88 Hz, Ar-H), 7.83
(s, 1H, Ar-H), 7.28-7.24 (m, 1H, Ar-H), 7.21-7.19 (m, 2H, Ar-H),
3.86 (s, 3H, NCH3). 2.68 (d, 2H, J = 7.2 Hz, CH2), 1.27-1.23 (m,
Table 1: Anticancer activities of indolyl-1,2,4-triazoles 6a-g and 7a-o against six human cancer cell linesa
Compound
R
R1
R2
R3
LnCaP
DU145
PC3
MCF7
MDA-MB-231
PaCa2
6a
6b
6c
6d
6e
6f
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
H
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
H
H
H
H
4-ClC6H4
CH2C6H5
>103
354.8
154.2
27.7
8.5
670.6
57
231.8
30.6
8.9
>103
>103
58
252.5
41.8
901.4
389.9
331.3
145.8
4
>103
46.4
95.5
43.5
4.2
903.2
182.9
181.8
32.6
7.1
>103
789.3
169.8
135.8
22.5
484.2
>103
282.3
178.6
25.7
631.8
16.4
28.2
16.1
19.6
4.7
581.7
282.6
183.3
39.6
13.3
83.3
>103
231.5
405.5
90.5
>103
14.1
713.5
22.1
41.5
0.8
3-OCH3C6H4
4-OH-3-OCH3C6H3
4-BnO-3-OCH3C6H3
3,4,5-(CH3O)3C6H2
4-pyridyl
C6H5
4-ClC6H4
4-FC6H4
4-CF3C6H4
3,4-(CH3O)2C6H3
3,5-(CH3O)2C6H3
3,4-(OCH2O)C6H3
4-BnO-3-OCH3C6H3
3,4,5-(CH3O)3C6H2
4-N(CH3)2C6H4
3,4,5-(CH3O)3C6H2
3,4,5-(CH3O)3C6H2
4-pyridyl
558.8
>103
150.4
231
72
188
12.2
133.8
22.7
30.4
6.9
145
319.7
142.4
64.3
204.3
41.7
539
6g
7a
7b
7c
7d
7e
7f
449.1
191.1
155.8
128
>103
5.8
8.1
163.8
23.8
23.5
10.2
55.2
16
51.8
725.6
6
64
4.8
7g
7h
7i
20.1
1.4
2.3
7j
75.6
6.3
91
8.6
125.8
10
575.5
13.7
20.5
226
2.6
76.5
286
7k
7l
20.5
153
101.4
204.2
6.9
14.1
91.5
3.2
16
350
1.6
>103
7m
7n
7o
>103
3.2
4-piperidinyl
Isobutyl
188.2
198.5
149
>103
aValues are reported as IC50 in micromolar concentration of the compound required to effect 50% inhibition of the net tumor cell growth. GraphPad Prism 5.0,
a curve-fitting program was used to plot dose response curve by nonlinear regression analysis to calculate IC50 values. The IC50 values <10 lM are indicated in
bold font.
Chem Biol Drug Des 2011; 77: 182–188
185

Kumar et al.
N
N
R1
R2
R1
R2
R1
R2
R1
R2
CHO
CN
(i)
R3
(iii)
(iv)
N
H
N
R
1
N
R
N
R
N
R
6a-g
7a-o
2
3
R = H
R = CH3
4
5
R = H
R = CH3
R = H
R = CH3
(ii)
R = H
R1, R2 = H or Br
3 = alkyl, aryl
R
Scheme 1: Reagents and conditions: (i) POCl3, DMF; (ii) (CH3O)2CO, K2CO3, DMF, 125 ꢀC; (iii) NH2OH. HCl, HCOONa, HCOOH, 130 ꢀC; (iv)
R3CONHNH2, K2CO3, n-BuOH, 160 ꢀC.
erature reports that N-methylated indole moiety is beneficial for the
cytotoxicity. Therefore, we have synthesized a series of compounds
7 bearing N-methylindole moiety. Compounds 7a and 7b with phe-
nyl and 4-chlorophenyl substituents were moderately active. The 4-
fluorophenyl derivative 7c displayed selective cytotoxicity against
PC3 cell line with IC50 4.0 lM. Compound 7d having stronger elec-
tron-withdrawing group in the C-3 aryl ring resulted in complete
loss of activity. Introduction of 3,4-dimethoxyphenyl ring at C-3 led
to 7e with enhanced activity against all the cancer cell lines with
best results against DU145 and MCF7 with IC50 5.8 and 8.1 lM,
respectively. However, 3,5-dimethoxyphenyl analog 7f was inactive
up to 28 lM, indicating that para methoxy substituent is important
for the activity. The 3,4-methylenedioxy analog 7g was found to be
less potent than 7e but showed improved activity and selectivity
against MCF7 (IC50 4.8 lM) cell lines. N-Methylation of 4-benzyloxy-
3-methoxyphenyl analog 6e led to compound 7h with improved
activity and selectivity against PC3 cell line with IC50 value of
4.2 lM but reduced activity was observed against other tested cell
lines. Similarly N-methylation of compound 6f led to 3,4,5-trimeth-
oxyphenyl analog 7i with remarkable improvement in activity
against all the tested cell lines. Introduction of bromine atom on
indole moiety at C-5 or C-6 positions resulted in the compounds 7k
and 7l with lower activity relative to parent analog 7i. Surprisingly,
introduction of lipophilic cyclic amine moiety (piperidinyl) led to
compound 7n that displayed excellent cytotoxicity against all the
tested cell lines with twofold selectivity against MCF7 (IC50 1.6 lM)
cell line. However, 4-pyridyl analog 7m displayed diminished activ-
ity. Compound 7o closely related to known anti-cancer agent Labra-
dorin 1 with an isobutyl group also exhibited poor activity. The
activity results of indolyl-1,2,4-triazoles when compared with our
previously reported 4-(3-indolyl)oxazoles [21], 5-(3-indolyl)-1,3,4-ox-
adiazoles [22] and 5-(3-indolyl)-1,3,4-thiadiazoles [25] suggests that
the five-membered heterocyclic ring is an important factor in the
cytotoxicity and selectivity of indolylazoles against different cancer
cell lines. The indolyl-1,2,4-triazoles exhibited slightly improved
activity and selectivity when compared with other indolylazoles. To
clearly understand the role of substitutes and central heterocyclic
ring in the activity of indolylazoles, a detailed SAR study is
required.
Results and Discussion
Chemistry
5-(3¢-Indolyl)-3-substituted-1,2,4-triazoles 6a-g and 7a-o were pre-
pared as described in the Scheme 1. The indole-3-carboxaldehyde 2
and N-methylindole-3-carboxaldehyde 3 were prepared according to
the literature procedures (32,33). Indole-3-carbonitriles (4 and 5)
were synthesized in good yields from the corresponding indole-3-
carboxaldehydes (2 and 3) by reacting with hydroxylamine hydro-
chloride in the presence of sodium formate and formic acid at
reflux temperature. Reaction of indole-3-carbonitriles 4 or 5
(2 mmol) with appropriate acid hydrazides (1 mmol) in the presence
of potassium carbonate (0.5 mmol) in n-butanol afforded the desired
product indolyl-1,2,4-triazole 6 and 7 in good yields (23). The reac-
tion was attempted in other polar solvents such as N,N-dimethylfor-
mamide and polyethylene glycol at high temperatures, which
resulted in unsatisfactory yields of the products. In an alternate
synthetic protocol to prepare indolyl-1,2,4-triazoles from the reaction
of ethyl imidate ester of indole with acid hydrazide in ethanol also
resulted in lower yields and required more than 36 h to complete
the reaction. All the synthesized 5-(3¢-indolyl)-3-substituted-1,2,4-
triazoles (6 and 7) were characterized by using NMR and mass
spectral data.
Anti-cancer activity
Synthesized 5-(3¢-indolyl)-3-substituted-1,2,4-triazoles 6a-g and 7a-
o were screened against prostate (PC3, DU145 and LnCaP), breast
(MCF7 and MDA-MB-231), and pancreatic (PaCa2) cancer cell lines
(Table 1). Some of the compounds have shown significant antican-
cer activity with IC50 values ranging from 1 lM to 1 mM concentra-
tion. The compound 6a with 4-chlorophenyl was inactive up to IC50
value of 581 lM. Compounds 6b and 6c with benzyl and methoxy-
phenyl groups at C-3 position of 1,2,4-triazole showed selective cy-
totoxicities against DU145 (IC50 57 lM) and PC3 (IC50 58 lM) cell
lines, respectively. Introduction of 4-hydroxy group in C-3 phenyl
ring of 6c led to 4-hydroxy-3-methoxyphenyl analog 6d with
improved activity. Further introduction of a bulkier 4-benzyloxy-3-
methoxyphenyl led to analog 6e that was highly potent with IC50
values 8.5 lM (LnCaP), 8.9 lM (DU145) and 7.1 lM (MCF7), which
suggests that a large group is tolerable in the C-3 phenyl ring. The
3,4,5-trimethoxyphenyl analog 6f was moderately selective cytotoxic
against PaCa2 cell line (IC50 83.3 lM). Replacement of the C-3 aryl
moiety with a heteroaryl ring led to an inactive compound 6g up
to 142.4 lM (MCF7). Our observations are in agreement with the lit-
Conclusion
In conclusion, we have synthesized a new series of indolyl-1,2,4-
triazoles as potential anticancer agents. Compounds 7i and 7n
186
Chem Biol Drug Des 2011; 77: 182–188

Potent and Selective Anticancer Agents
bearing 3,4,5-trimethoxyphenyl and 4-piperidinyl substituents
showed significant inhibitory effects against the tested cancer cell
lines. In general, substituents such as 4-fluorophenyl, 3,4,5-trimeth-
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3,4-dimethoxyphenyl,
4-benzyloxy-3-methoxyphenyl,
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the indolyl-1,2,4-triazoles resulted in selective cytotoxicity against
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