Synthesis and in-vitro anticancer activity of 3,5-bis(indolyl)-1,2,4- thiadiazoles

Article abstract of DOI:10.1016/j.bmcl.2011.07.089

A series of 3,5-bis(indolyl)-1,2,4-thiadiazoles were synthesized and evaluated for their cytotoxicity against selected human cancer cell lines. The reaction of indole-3-thiocarboxamide 3 with iodobenzene diacetate underwent oxidative dimerization to give 3,5-bis(indolyl)-1,2,4-thiadiazoles 4a-n. Among the synthesized bis(indoly)-1,2,4-thiadiazoles, the compound 4h with 4-chlorobenzyl and methoxy substituents showed the most potent activity.

Full text of DOI:10.1016/j.bmcl.2011.07.089

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5897–5900
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in-vitro anticancer activity of 3,5-bis(indolyl)-1,2,4-thiadiazoles
Dalip Kumar a, , N. Maruthi Kumar a, Kuei-Hua Chang b, Ritika Gupta b, Kavita Shah b,
⇑
⇑
a Department of Chemistry, Birla Institute of Technology and Science, Pilani 333 031, India
b Department of Chemistry and Purdue Cancer Center, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3,5-bis(indolyl)-1,2,4-thiadiazoles were synthesized and evaluated for their cytotoxicity
against selected human cancer cell lines. The reaction of indole-3-thiocarboxamide 3 with iodobenzene
diacetate underwent oxidative dimerization to give 3,5-bis(indolyl)-1,2,4-thiadiazoles 4a–n. Among the
synthesized bis(indoly)-1,2,4-thiadiazoles, the compound 4h with 4-chlorobenzyl and methoxy substit-
uents showed the most potent activity.
Received 24 May 2011
Revised 21 July 2011
Accepted 25 July 2011
Available online 30 July 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Bisindoles
1,2,4-Thiadiazoles
Anticancer agents
Dimerization
Hypervalent iodine regents
In the past, many indole-containing alkaloids have been isolated
from different marine invertebrates including bryozoans, sponges,
coelenterates and tunicates, which are known for their wide range
of pharmacological activities.1,2 Particularly, bis(indole)alkaloids
are one of these classes of alkaloids in which two indole rings con-
nected to each other via a 5 /6-membered heterocyclic ring, are par-
ticularly abundant in sponge of the genera Spongosorites and found
to possess antiviral, antimicrobial, antiinflammatory and anticancer
activities.3–7 For example, Nortopsentins A–C, with its 2,4-bis(30-
indolyl) imidazole skeleton exhibited in-vitro cytotoxicity against
P388 cells and antifungal activity against Candida albicans. Their
N-methylated derivatives improved P388 activity as compared to
the activity of parent compounds.8–10 Due to interesting biological
significance of these classes of marine alkaloids and their low avail-
ability in nature had led to discover new synthetic analogues. A vari-
ety of heterocycles such as thiazole, thiophene, pyrimidine and
pyrazine were replaced with imidazole of Nortopsentin and
screened for their anticancer activity (Fig. 1). These structural mod-
ifications gave encouraging results with improved IC50 values
against various cancer cell lines.11–15
5-(3-indolyl)-1,3,4-thiadiazoles,22 and indolyl-1,2,4-triazoles23 as
potential anticancer agents against human cancer cell lines. In a
process to explore the effect of central heterocycle and to prepare
novel bis(indoly)heterocycles, we have introduced 1,2,4-thiadia-
zole as a spacer. Synthesis of targeted 3,5-bis(indolyl)-1,2,4-
thiadiazoles were accomplished by oxidative dimerization of
indole-3-thiocarboxamides using relatively benign and non-toxic
hypervalent iodine reagent, iodobenzene diacetate (IBD). Due to
the facile preparation and low toxicity when compared with other
transition-metal based oxidants, hypervalent iodine reagents have
been extensively used in the construction of bioactive heterocy-
cles.24–26
In the present work we have synthesized
a series of
3,5-bis(indolyl)-1,2,4-thiadiazoles 4a–n with diverse structural
modifications. Our facile and novel protocol to prepare a series of
5-(3-indolyl)-1,3,4-thiadiazoles involves oxidative dimerization of
readily available 3-indolethiocarboxamides 3 in presence of iodo-
benzene diacetate. In general, synthesis of 1,2,4-thiadiazoles was
achieved using intermolecular cyclization,27 intramolecular cycli-
zation,28 and several oxidants were explored to achieve oxidative
dimerization from corresponding thioamides.29–35 In the process
to find the synthetic applications of hypervalent iodine reagents
iodobenzene diacetate (IBD), o-iodoxybenzoic acid with phase
transfer catalyst tetraethylammonium bromide was also used to
afford 1,2,4-thiadiazoles.36 Synthesis of 3,5-bis(indolyl)-1,2,4-thi-
adiazoles was achieved by three-step synthetic protocol as
outlined in the Scheme 1. Initially, we have synthesized indole-3-
carbonitriles 2 in good yields from corresponding indole-3-carbox-
aldehydes 1 by reacting with hydroxylamine hydrochloride in
1,2,4-Thiadiazoles are known for their wide spectrum of biolog-
ical activity such as antimicrobial, antitubercular, herbicides, and
antibiotics.16–19 Recently we have reported a series of indolylazoles
such as 5-(3-indolyl)-1,3,4-oxadiazoles,20 4-(3-indolyl)oxazoles,21
⇑
Corresponding authors. Tel.: +91 1596 245073 229 (D.K.); +1 765 496 9470
(K.S.).
E-mail addresses: dalipk@bits-pilani.ac.in (D. Kumar), shah23@purdue.edu (K.
Shah).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.089

5898
D. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5897–5900
S
N
N
N
H
X
X
X
X
N
R
N
R
N
H
N
H
NortopsentinA-C
2,4-Bis(3'-indolyl)thiazoles
N
X
N
N
X
X
N
X
X
N
R
N
R
N
R
N
R
2,5-Bis(3'-indolyl)pyrazoles
2,5-Bis(3'-indolyl)pyrimidines
Figure 1. Natural and synthetic bis(indolyl) heterocycles.
CSNH2
CN
CHO
b
a
R1
R1
R1
N
N
N
R2
R2
R2
3a-k
1a-k
2a-k
c
S
N
COOH
n
CSNH2
n
d,e
R1
N
N
R1
c
N
R2
N
N
R2
: R2 = H, n = 0, 2-yl
5b: R2 = CH3, n = 0, 2-yl 3m: R2 = CH3, n = 0, 2-yl
5c: R2 = H, n = 1, 3yl 3n: R2 = H, n = 1, 3-yl
R2
R2
2
5a
: R = H, n = 0, 2-yl
3l
4a-n
Scheme 1. Reagents and conditions: (a) NH2OHꢀHCl, HCOONa, HCOOH, 130 °C; (b) NaSH, MgCl2ꢀ6H2O, DMF, 40 °C; (c) IBD, DCM, 25 °C (10–20 min); (d) EDCI, THF, NH3 (30%)
30 °C; (e) Lawesson0s reagent, THF, 80 °C.
presence of sodium formate and formic acid.23 Subsequently, the
reaction of indole-3-carbonitriles 2 with NaSH and magnesium
chloride in presence of dimethylformamide led to the formation
of indole-3-thiocarboxamides 3.37 The intermediate indole-3-
thiocarboxamides 3l–n were prepared from the corresponding
substituted indole-3-carboxylic acids 5a–c. Finally, treatment of
indole-3-thiocarboxamides 3 with iodobenzene diacetate in anhy-
drous dichloromethane afforded bis(indolyl)-1,2,4-thiadiazoles
4a–n in moderate to good yields (50–70%).38 We have studied
the role of solvent and temperature to accomplish the key oxida-
tive dimerization of indole-3-thiocarboxamides 3. After several at-
tempts in solvents such as acetonitrile, tetrahydrofuran, dioxane,
dichloromethane, and toluene, we found that dichloromethane is
the solvent of choice for this reaction as it ensures rapid product
formation (10–20 min) and resulted in lower impurity profiles
We found the optimum temperature to carry out reaction was
25 °C. With the optimum reaction conditions, a series of diverse
3,5-bis(indolyl)-1,2,4-thiadiazoles 4 were synthesized.39 All the
synthesized compounds were characterized by IR, 1H NMR and
mass spectral analysis.
bis(indolyl)-1,2,4-thiadiazoles 4a–n against cancer cell lines are
expressed in terms of IC50 values (Table 1). Most of the compounds
have shown significant to moderate cancer cell growth inhibition.
The effect of various substitutions on indole ring was examined.
The structure–activity relationship (SAR) study revealed that the
substituents at N-1 and C-6 positions of indole ring are crucial
for inducing cytotoxicity and selectivity against particular cancer
cell lines. Bis(indolyl)-1,2,4-thiadiazole 4a showed modest selec-
tive cytotoxicity against MCF7 breast cancer cell lines
(IC50 = 143.2
N-1 position of indole ring led to compounds 4b and 4c with im-
proved activity and selectivity against DU145 (IC50 = 64 M) and
PaCa2 (IC50 = 100.6 M) cancer cell lines, respectively. Introduction
lM). Methyl and 4-chlorobenzyl substituents at the
l
l
of bromo substituent at C-5 position of the indole ring (compound
4d) increases the activity and showed selective inhibition against
MCF7 (IC50 = 31.3
exclusively selective cytotoxic against MCF7 cancer cell line
(IC50 = 63.6 M). It was observed that compound 4f with 5-meth-
oxyindole was successful to induce selectivity against PaCa2 cancer
cell line (IC50 = 45.8 M) but failed to improve the activity against
lM) cell lines. Its N-methyl derivative 4e is
l
l
A plausible mechanism for the formation of 3,5-bis(indolyl)-
1,2,4-thiadiazoles 4a–n is mentioned in the Figure 2. Initial reac-
tion of thioamide A with IBD generates iminothioamide B which
upon reaction with IBD undergoes intramolecular cyclization to
produce 1,2,4-thiadiazole C.
other cell lines. The N-methylation of 4f led to the compound 4g
with improved activity except against PaCa2 cell line. The N-chlo-
robenzyl derivative 4h demonstrated good inhibitory effects
against all the tested cancer cell lines, especially against LnCaP
(IC50 = 14.6 lM). Introduction of 5-nitro group moderately im-
Synthesized 3,5-bis(indolyl)-1,2,4-thiadiazoles 4a–n were
screened for their in-vitro cytotoxicity against six human cancer
cell lines: prostate (PC3, DU145 and LnCaP), breast (MCF7 and
MDA-MB-231) and pancreatic (PaCa2).40 Anticancer activity of
proved the activity (compounds 4i vs 4a) against breast cancer cell
lines. The compounds 4j and 4k with bromo or methoxy group at
C6 position of indole ring showed better cytotoxicity when com-
pared with its regioisomers 4d and 4f having similar substituent

D. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5897–5900
5899
OAc
I
AcO
S
-OAc
-PhI
-S
I
Ph
H
OAc
OAc
R
R
NH2
NH
S
Ph
S
Ph
R
I
S
N
S
R
NH
R
N
H
R
H
R
NH2
A
N
R
R
R
NH
-AcOH
-PhI
R
PhI(OAc) 2
N
S
NH
AcO
S
- H
NH
S
C
I
B
Ph
Figure 2. A plausible mechanism for the formation of 1,2,4-thiadiazoles.
Table 1
Anticancer activity of bis(indolyl)-1,2,4-thiadiazoles 4a–n against selected human cancer cell lines IC50
(l
M)a
Compound
R1
R2
MDA-MB-231
MCF7
LnCaP
DU145
PC3
PaCa2
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
H
H
H
CH3
4-ClC6H4CH2
H
CH3
H
CH3
4-ClC6H4CH2
H
H
H
H
CH3
H
701.5
236.3
>103
100.3
>103
>103
95.7
67.9
98.6
38.3
44.3
143.2
177.4
>103
31.3
63.6
423.5
79.9
32.1
83.5
39.2
35.3
162.3
66.4
40.5
>103
226.3
413.3
159.2
>103
295.1
111.3
14.6
176.6
80.1
35.8
246.8
206.8
43.3
248.4
64
>103
92.3
>103
185.2
100.6
124.3
>103
45.8
177
21.2
187.3
83
>103
56.7
>103
162.2
>103
224
5-Br
5-Br
5-OCH3
5-OCH3
5-OCH3
5-NO2
6-Br
6-OCH3
H
>103
206.2
137.1
369.8
324.9
30.2
42.3
453
>1000
51.2
269.8
21.4
679.6
132.5
76.1
326.2
432.1
44.2
4j
4k
4lb
4mb
4nc
95.3
102.3
260.2
31.3
>1000
>1000
65
H
H
a
b
c
IC50values were obtained using a dose response curve by non-linear regression using a curve fitting program, GraphPad Prism 5.0.
3,5-(2,20-Bisindolyl)-1,2,4-thiadiazole.
3,5-Bis((indol-3-yl)methyl)-1,2,4-thiadiazole.
at C5 position. Shifting the connectivity of 1,2,4-thiadiazole with
indole from C-3 to C-2 position led to the compounds 4l and 4m
with reduced activity except for compound 4m which displayed
cles, 3,5-bis(indolyl)-1,2,4-thiadiazoles showed comparable anti-
cancer activity. Further study to improve the anticancer activity
and cellular targets of 3,5-bis(indolyl)-1,2,4-thiadiazoles is in pro-
gress and will be reported in due course.
moderate selective cytotoxicity against MCF7 (IC50 = 66.4 lM).
Further, introduction of a methylene unit between indole and
1,2,4-thiadiazole ring resulted in compound 4n with broad-
spectrum of improved activity in all the cell lines employed
Acknowledgment
(IC50 = 31.3 lM). By altering the central heterocyclic ring, antican-
Authors acknowledge financial support from the Council of
Scientific and Industrial Research, New Delhi.
cer activities of bis(indolyl) heterocycles varies against different
cancer cell lines. For example, inhibitory activity against MCF-7
cells of 3,5-bis(indolyl)-1,2,4-thiadiazole 4a is better than reported
2,4-bis(3-indolyl) thiazole.13 The 3,5-bis(6-bromo-3-indolyl)-
1,2,4-thiadiazole 4j is less potent towards MCF-7 cells than the
reported 2,4-bis(6-bromo-3-indolyl)thiazole14 but more selective.
The 3,5-bis(5-methoxy-3-indolyl)-1,2,4-thiadiazole 4f showed
poor activity against MCF-7 cells when compared with 2,5-bis(30-
indolyl)thiophene.15 For exact comparison and to identify the role
of heterocyclic ring in various bis(indolyl) heterocycles more
analogues of 3,5-bis(indolyl)-1,2,4-thiadiazoles need to be
synthesized.
In conclusion, we have reported a simple synthesis of novel 3,5-
bis(indolyl)-1,2,4-thiadiazoles by iodobenzene diacetate mediated
oxidative dimerization of readily available indole-3-thiocarboxa-
mide 3. Most of the 3,5-bis(indolyl)-1,2,4-thiadiazoles exhibited
pronounced antitumor activity against a panel of cancer cell lines.
In particular, the indoly-1,2,4-thiadiazole 4h was identified as the
most potent compound in this series with IC50 values of 14.6, 21.4,
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lM against cancer cell lines LnCaP, PC3 and PaCa2,
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cmꢁ1): 3099, 2926, 1597, 1562, 1492, 1246, 858, 744; MS (FAB) m/z calcd for
32H23Cl2N4S (M+H)+ 565.1, obsd 565.8. Compound 4d: Yield 55%, brown solid,
C
mp 163–165 °C; 1H NMR (400 MHz, DMSO-d6): d 11.48 (s, 2H), 8.68 (s, 2H),
8.05 (d, 2H, J = 2.60 Hz), 7.39–7.25 (m, 4H); IR (KBr, v cmꢁ1): 3124, 2926, 1730,
1579. 1566, 1514, 1469, 1278, 794; MS (FAB) m/z calcd for C18H14Br2N5S
(M+NH4)+ 489.9, obsd 489.1 Compound 4e: Yield 60%, brown solid, mp 160–
161 °C; 1H NMR (400 MHz, DMSO-d6): d 8.58 (s, 2H), 8.03 (d, 2H, J = 2.8 Hz),
7.34–7.30 (m, 4H), 3.81 (s, 6H); IR (KBr, v cmꢁ1): 3265, 1606, 1546, 1485, 1479,
1280, 792; MS FAB (m/z) calcd for C20H14Br2KN4S (M+K)+ 538.89, obsd 539.0.
Compound 4f: Yield 70%, mp white solid, 157–159 °C; 1H NMR (400 MHz,
CDCl3): d 8.62 (s, 2H), 7.68 (d, 2H, J = 3.00 Hz), 7.34 (d, 2H, J = 8.84 Hz), 7.18 (d,
2H, J = 2.40 Hz), 6.97 (dd, 2H, J = 8.84, 2.4 Hz), 3.88 (s, 6H); IR (KBr, v cmꢁ1):
3155, 2998, 1678, 1585. 1558, 1520, 1455, 1245, 1187, 796; MS (FAB) m/z calcd
for C20H16N4NaO2S (M+Na)+ 399.08, obsd 399.08. Compound 4g: Yield 65%,
white solid, mp 142–144 °C; 1H NMR (400 MHz, DMSO-d6): 7.74 (d, 2H,
J = 2.80 Hz), 7.43 (d, 2H, J = 8.00 Hz), 7.29 (d, 2H, J = 3.00 Hz), 6.97 (d, 2H,
J = 8.00 Hz), 3.86 (s, 6H), 3.77 (s, 6H); IR (KBr, v cmꢁ1): 2966, 1672, 1537, 1492,
1462, 1228, 794; MS FAB (m/z) calcd for C22H21N4O2S (M+H)+ 405.10, obsd
405.0. Compound 4h: Yield 60%, brown solid, mp 137–138 °C; 1H NMR
(400 MHz, DMSO-d6): d 7.49 (s, 2H), 7.25–7.22 (m, 4H), 7.11–7.08 (m, 4H), 6.98
(d, 4H, J = 8.00 Hz), 6.87–6.84 (m, 2H), 5.21 (s, 4H), 3.79 (s, 6H); IR (KBr, v
cmꢁ1): 2947, 1580, 1529, 1480, 1445, 1228, 796; MS FAB (m/z) calcd for
C
34H27Cl2N4O2S (M+H)+ 625.1, obsd 625.0. Compound 4i: Yield 52%, brown
35. Cronyn, M. W.; Nakagawa, T. W. J. Am. Chem. Soc. 1952, 74, 3693.
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38. Synthesis of 3,5-bis(indolyl)-1,2,4-thiadiazoles 4: Iodobenzene diacetate
solid, mp 178–179 °C; 1H NMR (400 MHz, DMSO-d6): d 11.45 (s, 2H), 8.45 (d,
2H, J = 8.00 Hz), 7.95 (s, 2H), 6.89–6.75 (m, 4H); IR (KBr, v cmꢁ1): 3356, 3134,
1622, 1585, 1471, 1240, 736; MS (FAB) m/z calcd for C18H12N6O4S (M+2H)+
408.05, obsd 408.06. Compound 4j: Yield 52%, brown solid, mp 190–192 °C; 1
H
NMR (400 MHz, DMSO-d6): d 11.30 (s, 2H), 8.25 (s, 2H), 8.12 (d, 2H, J = 2.90 Hz),
7.42–7.38 (m, 4H); IR (KBr, v cmꢁ1): 3169, 2895, 1612, 1523, 1469, 1240, 796;
MS (FAB) m/z calcd for C18H11Br2N4S (M+H)+ 472.89, obsd 472.90. Compound
4k: Yield 70%, brown solid, mp 180–181 °C; 1H NMR (400 MHz, DMSO-d6): d
10.63 (s, 2H), 7.53–7.50 (m, 4H), 6.70–6.82 (m, 4H), 3.77 (s, 6H); IR (KBr, v
cmꢁ1): 3219, 1631, 1585, 1469, 1265, 808; MS FAB (m/z) calcd for C20H17N4O2S
(M+H)+ 377.09, obsd 377.10. Compound 4l: Yield 62%, brown solid, mp 180–
181 °C; 1H NMR (400 MHz, DMSO-d6): d 10.94 (s, 2H), 7.33–7.27 (m, 4H), 7.09–
7.04 (m, 2H), 6.83–6.80 (m, 4H); IR (KBr, v cmꢁ1): 3053, 2972, 1614, 1579,
1556, 1514, 1454, 1261, 804, 736; MS (FAB) m/z calcd for C18H13N4S (M+H)+
317.07, obsd 317.60. Compound 4m: Yield 68%, brown solid, mp 132–133 °C;
1H NMR (400 MHz, DMSO-d6): d 7.68–7.65 (m, 2H), 7.44 (s, 2H), 7.30–7.19 (m,
6H), 3.75 (s, 6H); IR (KBr, v cmꢁ1): 3026, 1554, 1531, 1469, 1421, 1265, 788; MS
FAB (m/z) calcd for C20H16KN4S (M+K)+ 383.07, obsd 383.10. Compound 4n:
Yield 52%, brown solid, mp 170–171 °C; 1H NMR (400 MHz, CDCl3): d 8.79 (s,
2H), 7.61 (d, 2H, J = 8.00 Hz), 7.39–7.33 (m, 4H), 6.67–6.58 (m, 4H), 3.75 (s, 4H);
IR (KBr, v cmꢁ1): 3245, 2928, 1573, 1460, 1232, 767; MS (FAB) m/z calcd for
(1 mmol) was added to
a stirred solution of indole-3-thiocarboxamide 3
(1 mmol) in dichloromethane (5 mL) at 25 °C. Progress of the reaction was
monitored by thin layer chromatography. After completion of the reaction, the
precipitated sulfur was removed and washed the resulting solution with
saturated sodium bicarbonate (25 mL) and brine (25 mL) solution. Organic
phase was dried over anhydrous sodium sulfate and distilled off at reduced
pressure. The crude product obtained was recrystallized from chloroform–
methanol. The compounds 4d, 4i, 4j, and 4l were purified by column
chromatography on silica gel (100–200 mesh) using ethyl acetate and
hexane as eluent.
39. Spectral data of the 3,5-bis(indolyl)-1,2,4-thiadiazoles (4): Compound 4a: Yield
65%, pale yellow solid, mp 168–170 °C; 1H NMR (400 MHz, DMSO-d6): d 11.68
(s, 2H), 7.78 (d, 2H, J = 2.92 Hz), 7.68 (d, 2H, J = 7.50 Hz), 7.50 (d, 2H,
J = 7.80 Hz), 7.29–7.21 (m, 4H); IR (KBr, v cmꢁ1): 3109, 1660, 1583, 1523,
1433, 1240, 736; MS (FAB) m/z calcd for C18H13N4S (M+H)+ 317.07, obsd
317.08. Compound 4b: Yield 60%, pale brown solid, mp 140–142 °C; 1H NMR
(400 MHz, CDCl3): d 8.33–8.30 (m, 2H), 7.67 (s, 2H), 7.38–7.33 (m, 6H), 3.87 (s,
6H); IR (KBr, v cmꢁ1): 1640, 1566, 1556, 1477, 1440, 1240, 792; MS (FAB) m/z
calcd for C20H17N4S (M+H)+ 345.10, obsd 345.10. Compound 4c: Yield 72%,
brown solid, mp 146–150 °C; 1H NMR (400 MHz, DMSO-d6): d 7.81–7.78 (m,
2H), 7.62 (s, 2H), 7.35–7.32 (m, 10H), 7.10–7.07 (m, 4H), 5.34 (s, 4H); IR (KBr, v
C
20H17N4S (M+H)+ 345.10, obsd 344.90.
40. Two stock solutions, having concentrations of 100 and 500 mM, were prepared
by dissolving the compounds in DMSO. These solutions were further diluted in
cell culture media prior to adding to the cells such that DMSO concentration
was maintained at <0.1%.