A series of 2-arylamino-5-(indolyl)-1,3,4-thiadiazoles as potent cytotoxic agents

Article abstract of DOI:10.1016/j.ejmech.2012.06.047

A series of 2-arylamino-5-(indolyl)-1,3,4-thiadiazoles 6a-v were prepared and studied for their anticancer activity against selected human cancer cell lines. The reaction of indolylhydrazides 3a-h with a variety of aryl isothiocyanates 4 afforded the key intermediate thiosemicarbazides 5a-v, which upon treatment with acetyl chloride produced the 2-arylamino-5-(indolyl)-1,3,4- thiadiazoles 6a-v in good yields. Most of the synthesized compounds showed selective cytotoxicity towards human breast cancer cell line (MDA-MB-231). Of the synthesized 2-arylamino-5-(indolyl)-1,3,4-thiadiazoles, compound 6f is the most potent towards tested cancer cell lines (IC₅₀ = 0.15-1.18 μM).

Full text of DOI:10.1016/j.ejmech.2012.06.047

European Journal of Medicinal Chemistry 55 (2012) 432e438
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
A series of 2-arylamino-5-(indolyl)-1,3,4-thiadiazoles as potent cytotoxic agents
,
a
b
b,
Dalip Kumar a , N. Maruthi Kumar , Brett Noel , Kavita Shah
*
**
a Department of Chemistry, Birla Institute of Technology and Science, Pilani, Rajasthan 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 2-arylamino-5-(indolyl)-1,3,4-thiadiazoles 6aev were prepared and studied for their anti-
cancer activity against selected human cancer cell lines. The reaction of indolylhydrazides 3aeh with
a variety of aryl isothiocyanates 4 afforded the key intermediate thiosemicarbazides 5aev, which upon
treatment with acetyl chloride produced the 2-arylamino-5-(indolyl)-1,3,4-thiadiazoles 6aev in good
yields. Most of the synthesized compounds showed selective cytotoxicity towards human breast cancer
cell line (MDA-MB-231). Of the synthesized 2-arylamino-5-(indolyl)-1,3,4-thiadiazoles, compound 6f is
Received 4 March 2012
Received in revised form
7 June 2012
Accepted 22 June 2012
Available online 3 July 2012
the most potent towards tested cancer cell lines (IC50 ¼ 0.15e1.18
mM).
Keywords:
Ó 2012 Elsevier Masson SAS. All rights reserved.
2-Arylamino-1,3,4-thiadiazoles
Indolyl heterocycles
Cytotoxic agents
1,3,4-Thiadiazoles
1. Introduction
antidepressant [18] and anticancer [19]. Some of the marketed
drugs with 1,3,4-thiadiazole scaffold such as Acetazolamide,
Methazolamide and Globucid are well-known for their therapeutic
applications [20]. 1,3,4-Thiadiazoles, bioisosters of oxadiazoles and
thiazoles, are known to have interesting electro-optical properties
and also act as corrosion and oxidation inhibitors, complexation
reagents for dyes and metal ions. Furthermore, widely explored
2-aminothiadiazoles (ATDA) are in clinical trials for the treatment
of patients with different tumours [21,22]. The 2-(4-
fluorophenylamino)-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole
(FABT) 1c is a promising anticancer compound for malignant
tumours of nervous system. FABT has the ability to decrease the
proliferation of cancer cells and their metastasis [23,24] (Fig. 1).
Naturally occurring antimitotic agents Colchicine (1d), Com-
bretastatin A4 (1e) and their synthetic analogues have been
extensively studied for cancer chemotherapy, however, lacks
anticancer efficacy in-vivo at its maximum tolerated dose (MTD)
[25e27]. In analogy to these natural products, several synthetic
Combretastatin analogues bearing the trimethoxyphenyl moiety
were also screened for their anticancer properties [28e31].
Recently, we have reported series of indolylazoles including 4-
The indole scaffold represents one of the most important
structural units prevalent in various naturally occurring and
bioactive compounds. Recently, there has been an increasing
interest in the indole derivatives to identify new drug candidates,
particularly possessing five-membered heterocycles [1,2]. Many
natural and synthetic bioactive indolyl heterocycles have demon-
strated the critical role of indole scaffold in their biological activi-
ties [3,4]. Indolylazoles (1a) such as 5-(30-indolyl)oxazoles
(Labradorins 1 & 2) and indolylthiazoles are known for their cyto-
toxic activities against NCIeH 460 (lung-NSC) human cancer cell
line [5]. Marine indole alkaloids, Meridianins (1b) and their
synthetic analogues have shown prominent anticancer activities
against various cancer cell lines with best IC50 values against MCF-7
breast cancer cell line (0.25 mg/mL) [6]. A diverse variety of indo-
lylazoles with five/six-membered heterocyclic ring were also
evaluated for their anticancer activities [5,7e10].
1,3,4-Thiadiazoles are an important class of heterocyclic
compounds with various biological activities such as antiviral [11],
antimicrobial [12], fungicidal [13], antiinflammatory [14], antihy-
pertensive [15], antituberculosis [16], antileishmanial [17],
(30-indolyl)oxazoles,
5-(30-indolyl)-1,3,4-oxadiazoles,
5-(30-
indolyl)-1,3,4-thiadiazoles and indolyl-1,2,4-triazoles as potential
anticancer agents against various human cancer cell lines
[8,32e37]. In continuation of our efforts to identify indole-based
potent anticancer agents, in this study we synthesized a variety
of 2-arylamino-5-(indolyl)-1,3,4-thiadiazoles 6aev by combining
bio-active indole and 2-arylamino-1,3,4-thiadiazole scaffolds into
* Corresponding author. Tel.: þ91 1596 245073x279.
** Corresponding author. Tel.: þ1 765 496 9470.
E-mail addresses: dalipk@bits-pilani.ac.in (D. Kumar), shah23@purdue.edu
(K. Shah).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.047

D. Kumar et al. / European Journal of Medicinal Chemistry 55 (2012) 432e438
433
Fig. 1. Rational approach to design 2-arylamino-5-(indolyl)-1,3,4-thiadiazoles 6aev.
single small molecules and tested their anticancer activities
against a selected panel of human cancer cell lines (Fig. 1).
in ethanol afforded the intermediate thiosemicarbazides 5aev in
good yields. Finally, the oxidative cyclization of 5aev was accom-
plished in presence of acetyl chloride to afford 6aev in good yields
(Scheme 1). Optimized reaction conditions were used to synthesize
a series of 2-arylamino-5-(indolyl)-1,3,4-thiadiazoles 6aev from
indolylhydrazides 3aeh and aryl isothiocyanates 4. All the
synthesized compounds were well characterized by their IR, Mass
and NMR (1H & 13C) spectral data.
2. Results and discussion
2.1. Chemistry
General synthetic methods for the construction of 1,3,4-
thiadiazoles involve the use of diacylhydrazide precursors under
various reaction conditions. Symmetrical 2,5-disubstituted-1,3,4-
thiadiazoles were prepared by the condensation reaction of aryl
aldehydes, hydrazine hydrate and sulphur in ethanol under
microwave irradiation [38]. A robust protocol for the solid phase
synthesis of 5-alkyl/aryl-2-alkylamino-1,3,4-thiadiazoles was
described from resin bound thiosemicarbazides [39]. Oruc et al.
prepared 1,3,4-thiadiazoles in four steps utilizing acyl halides and
aryl isothiocyanates [16]. The 1,3,4-thiadiazoles were also achieved
from the reaction of 1,3,4-oxadiazoles with thiourea [40]. Recently,
Kumar et al. reported one-pot convenient synthesis of 1,3,4-
thiadiazoles 1f by reacting aryl aldehydes, hydrazine hydrate and
aryl isothiocyanates and studied their anticancer activities [32].
Rostamizadeh et al. utilizes ionic liquid or thiourea mediated
oxidation of in situ generated thiosemicarbazones for the one-pot
synthesis of 1,3,4-thiadiazoles [41,42].
Our initial attempts to synthesize 2-arylamino-5-(indolyl)-
1,3,4-thiadiazoles 6 using reported protocols [32] involved oxida-
tive cyclization of the corresponding thiosemicarbazone obtained
via the reaction of aryl thiosemicarbazide with indole-3-
carboxaldehyde. The cyclization of thiosemicarbazone 5a using
reagents like FeCl3, ZnCl2, iodobenzene diacetate, ferric ammonium
sulphate and chloramines-T was failed to give the desired 2-
arylamino-5-indolyl-1,3,4-thiadiazole 6a. In an alternative route
to synthesize desired indolyl-1,3,4-thiadiazoles 6aev involved the
initial preparation of indolylhydrazides 3aeh from the corre-
sponding indole carboxylic acids 2a as illustrated in the Scheme 1.
’The reaction of indolylhydrazides 3aeh and aryl isothiocyanates 4
3. In-vitro anticancer activity
All the synthesized 2-arylamino-5-(indolyl)-1,3,4-thiadiazoles
6aev were screened against prostate (DU145 and LnCaP), breast
(MCF-7 and MDA-MB-231), cervical (HeLa) and ovarian (Ovcar-3)
human cancer cell lines (Table 1). Most of the compounds exhibited
high selectivity towards breast cancer cell line (MBA-MB-231) with
IC50 values of less than 1 mM. Activity results proved that substit-
uents at C-2 and C-5 positions of the 1,3,4-thiadiazole ring play
a crucial role in imparting the anticancer activity. Incorporation of
an indole ring at 5-position of 1,3,4-thiadiazole led to significant
improvement in anticancer activity when compared with an aryl
ring as reported in our previous studies on 2-arylamino-5-aryl-
1,3,4-thiadiazoles [32]. For example, the compound 6a
(IC50 ¼ 0.9
compared with the previously reported 2-phenylamino-5-(3,4,5-
trimethoxyphenyl)-1,3,4-thiadizole (IC50 122.4 M) against
mM) showed 100-fold improved cytotoxicity when
¼
m
MDA-MB-231 breast cancer cell line [32]. The compound 6a
exhibited sixty fold selective cytotoxicity against tested breast
cancer cell line, MDA-MB-231 (IC50
(IC50 ¼ 55.58 M) and showed moderate activity against Ovcar-3,
M). The para-
¼
0.9 mM) over MCF-7
m
LnCap and HeLa cancer cell lines (<IC50 ¼ 10
m
substitutions (CH3, Cl, OCH3 and CF3) on C-2 arylamino moiety
led to compounds 6bee without any significant improvement in
cytotoxicity, except the compounds 6c and 6e possessing electron
withdrawing groups (Cl and CF3) exhibited slight increase in
cytotoxicity against LnCap (IC50 ¼ 3.14 and 5.24
mM) and MDA-MB-
Scheme 1. Reagents and conditions: (a) (i) EtOH/Hþ; (ii) R1X/NaH; (b) NH2NH2.H2O, EtOH, reflux; (c) R2NCS (4), EtOH, 60 ꢀC; (d) CH3COCl, 20 ꢀC.

434
D. Kumar et al. / European Journal of Medicinal Chemistry 55 (2012) 432e438
Table 1
In-vitro cytotoxicity data of 2-arylamino-5-(indolyl)-1,3,4-thiadiazoles 6aev against selected human cancer cell lines.
Compound
R
R1
R2
IC50
MCF-7
55.58
(
m
M)
LnCap
MDA-MB-231
DU145
HeLa
8.41
Ovcar-3
6a
H
H
C6H5
7.57
7.37
3.14
10.65
5.24
0.15
10.51
6.63
8.31
18.32
7.53
11.45
12.38
7.58
2.16
3.69
11.38
2.93
8.67
5.92
2.30
5.91
2.76
0.91
0.82
0.55
0.94
0.51
0.44
0.62
0.47
0.44
0.99
0.81
0.90
0.90
0.58
0.93
0.64
0.64
0.23
0.25
0.46
5.94
1.34
19.01
19.70
31.97
22.88
22.17
20.95
22.35
63.92
21.63
19.44
18.04
24.37
21.16
90.33
16.11
19.81
18.95
16.42
28.83
42.02
53.75
27.74
24.46
10.86
3.49
3.25
4.05
3.58
3.70
1.18
3.81
3.85
3.96
3.55
3.69
3.68
3.54
3.43
3.56
3.46
3.30
3.10
2.00
3.75
3.82
3.29
10.53
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
4-CH3C6H4
4-ClC6H4
4-OCH3C6H4
4-CF3C6H4
3,4,5-(OCH3)3C6H2
CH2C6H5
C6H5
4-ClC6H4
C6H5
3,4,5-(OCH3)3C6H2
4-OCH3C6H4
3,4,5-(OCH3)3C6H2
4-OCH3C6H4
4-OCH3C6H4
3,4,5-(OCH3)3C6H2
4-ClC6H4
4-OCH3C6H4
3,4,5-(OCH3)3C6H2
CH2C6H5
21.10
10.19
8.79
16.50
0.91
34.56
11.77
12.81
9.13
10.59
8.88
23.15
21.68
23.38
13.37
7.55
27.14
10.08
19.44
21.58
3.64
>100
47.88
10.60
30.80
40.00
72.87
50.73
24.36
63.16
32.34
21.79
14.39
9.74
4-ClC6H4CH2
4-ClC6H4CH2
4-OCH3C6H4CH2
4-OCH3C6H4CH2
H
H
H
H
H
H
H
H
H
5-OCH3
6-OCH3
6-OCH3
H
H
H
H
H
H
6r
6s
6t
6u
8.04
6.48
9.92
12.72
>100
8.62
86.57
4-OCH3C6H4
3,4,5-(OCH3)3C6H2
>100
>100
20.93
6v
Doxorubicin
231 (IC50 ¼ 0.55 and 0.51
m
M) when compared to compound 6a
resulted in poor cytotoxicity against HeLa cancer cell line
(IC50 100 M). The compound 6v with 3,4,5-
trimethoxyphenylamino substituent induced selective cytotox-
M) cancer cells. The
(MCF-7 activity). Our earlier studies with indolyl-1,2,4-triazoles
and indolyl chalcones delivered potent compounds consisting
>
m
a
3,4,5-trimethoxyphenyl moiety. On similar grounds the
icity against MDA-MB-231 (IC50 ¼ 1.34
m
compound 6f was synthesized with enhanced cytotoxicity against
synthesized indolylthiadiazoles 6aev was found to be more cyto-
all the tested cancer cell lines at sub-micromolar concentrations
toxic against tested cancer cell lines than the control Doxorubicin.
(LnCap (IC50 ¼ 0.15
m
M) and MCF-7 (IC50 ¼ 0.91
mM), MDA-MB-231
M)). Benzylamino group
(IC50 ¼ 0.44 M) and Ovcar-3 (IC50 ¼ 1.18
m
m
4. Conclusion
at C-2 position (compound 6g) has no impact on cytotoxicity. N-
Methylation of indole ring (compounds 6hei) was found to be
beneficial for the anticancer activity when compared with their
parent compounds (6a and 6c). In particular, N-methylindole
analogues 6h and 6i exhibited improved cytotoxicity with IC50
We have synthesized a series of 2-arylamino-5-(indolyl)-1,3,4-
thiadiazoles 6aev which displayed a potent cytotoxicity against
various tested cancer cell lines. Compound 6f with 3,4,5-
trimethoxyphenylamino substituent showed high activity profile
against multiple cancer cell lines, particularly promising results
values of 0.47 and 0.44 mM against MDA-MB-231 breast cancer cell
line. However, introduction of bulkier groups (N-1) such as p-
chlorobenzyl and p-methoxyphenyl (compounds 6jem) is detri-
mental for the activity against tested cancer cell lines. Introduction
of a methoxy group at position-5/6 in the indole ring of 6d led to
compounds 6n and 6o with improved cytotoxicity (IC50 ¼ 7.58 and
against prostate cancer cell line (IC50 ¼ 0.15
2-arylamino-5-(indolyl)-1,3,4-thiadaizoles 6aev exhibited good
cytotoxic (IC50 < 1 M) selectivity against breast cancer cell line
(MDA-MB-231). The anticancer activity results revealed that 2-
arylamino-5-(indolyl)-1,3,4-thiadaizoles are more potent than our
previously reported 2-aryl-5-(indolyl)-1,3,4-thiadiazoles [33]. The
studies to improve cytotoxicity and identify the cellular targets of
2-arylamino-5-(indolyl)-1,3,4-thiadaizoles 6aev are in progress.
mM, LnCaP). Most of the
m
2.16
mM, LnCap) and selectivity. While retaining important trime-
thoxyphenyl moiety, inclusion of
a
methoxy substituent at
position-6 of indole ring led to compound 6p with reduced anti-
proliferative activity. Shifting the connectivity of 1,3,4-thiadiazole
with indole ring from C-3 to C-2 position provided the
compounds 6qet with improved cytotoxicity. The compound
6r found to be more cytotoxic (compounds 6r vs 6d) against LnCap
5. Experimental
5.1. Physical measurements
and MDA-MB-231 cancer cells with IC50 values of 2.93 and 0.23 mM,
respectively. The compounds 6s and 6t with 3,4,5-
trimethoxyphenylamino and benzylamino substituent induced
All the laboratory grade reagents were obtained commercially.
The reaction was monitored by thin layer chromatography, which
selectivity against MDA-MB-231 (IC50 ¼ 0.25 and 0.46
mM) cancer
was performed on Merck pre-coated plates (silica gel. 60 F254
,
cells when compared with their regioisomers (compounds 6f and
6g). Further, introduction of a methylene unit between indole
and 1,3,4-thiadiazole rings (compounds 6u and 6v) showed no
appreciable improvement in the anticancer activity, particularly
0.25 mm) and was visualized by fluorescence quenching under UV
light (254 nm). Recrystallization was done by commercially
available absolute ethanol. Solvents were evaporated using Büchi
rotary evaporator. Melting points were determined with

D. Kumar et al. / European Journal of Medicinal Chemistry 55 (2012) 432e438
435
electrothermal capillary melting point apparatus (E-Z melting). 1H
NMR spectra were recorded on a Bruker Advance II (400 MHz)
spectrometer. The coupling constant (J) values are in Hz. Mass
spectra were measured on a ‘HewlettePackard’ model HP GS/MS
5890/5972.
5.2.1.6. 1-Methyl-indole-3-carbohydrazide (3d). Yield 80%; White
solid, mp 149e150 ꢀC. 1H NMR (400 MHz, DMSO-d6,
ppm): 9.18
d
d
(s, 1H), 8.14 (d, J ¼ 7.80 Hz, 1H), 7.92 (d, J ¼ 7.80 Hz, 1H), 7.37 (d,
J ¼ 8.10 Hz, 1H), 7.20e6.99 (m, 2H), 4.03 (s, 2H), 3.65 (s, 3H). 13C
NMR (100 MHz, DMSO-d6,
d ppm): d 160.88, 153.05, 141.03, 136.82,
130.96, 129.00, 125.32 124.60, 122.62, 121.58, 120.90, 117.21, 110.46,
105.69, 33.07. IR (KBr, v cmꢂ1): 3212, 3098, 1658, 1604, 1582, 1520,
1465, 1235, 740. MS (ESI) m/z calcd for C10H11N3O (M)þ 189.1, obsd
189.1.
5.2. General procedure for the synthesis of 2-arylamino-5-(indolyl)-
1,3,4-thiadiazoles 6aev
5.2.1. Preparation of indole-2(3)-carboxylic acid and indole-3-
acetic acid (2)
Indole-3-carboxylic acid, 5-methoxyindole-3-carboxylic acid
and 6-methoxyindole-3-carboxylic acid were prepared according
to the reported protocol [43]. Indole-2-carboxylic acid and indole-
3-acetic acid were procured form commercial sources.
5.2.1.7. 1-(4-Chlorobenzyl)-indole-3-carbohydrazide
75%; White solid, mp 173e174 ꢀC. 1H NMR (400 MHz, DMSO-d6,
ppm):
J ¼ 6.2 Hz, 1H), 7.15 (d, J ¼ 7.0 Hz, 4H), 6.83 (d, J ¼ 7.45 Hz, 2H), 5.28
(s, 2H), 4.43 (s, 2H). IR (KBr, v cmꢂ1): 3117, 3098, 1648, 1601, 1576,
1518, 1456, 1228, 764. MS (ESI) m/z calcd for C16H14ClN3O (M þ H)þ
300.0, obsd 300.0.
(3e). Yield
d
d
9.08 (s, 1H), 8.19 (d, J ¼ 8.0 Hz, 1H), 7.98 (s, 1H), 7.37 (d,
5.2.1.1. General procedure for the synthesis of indole-2(3)-
carbohydrazides and indole-3-acetohydrazide (3aeh). To a solution
of indole carboxylic acid 2 (1 mmol) in ethanol (20 mL) was
added catalytic amount of concentrated sulphuric acid (0.2 mL)
and allowed to reflux for 20 h. After completion of the reaction,
ethanol was removed and the residue was extracted with ethyl
acetate (30 mL) and washed with saturated sodium bicarbonate
solution (25 mL). Organic layer was dried over sodium sulphate
and evaporated to give corresponding ester in good yields
(85e95%). The solution of an appropriate ester (1 mmol) and
hydrazine hydrate (2 mmol) in ethanol (15 mL) was refluxed for
4 h. Reaction mixture was cooled and the solid obtained was
filtered and recrystallized from ethanol to obtain pure hydrazides
3aec, 3g and 3h.
5.2.1.8. 1-(4-Methoxybenzyl)-indole-3-carbohydrazide
75%; White solid, mp 193e194 ꢀC. 1H NMR (400 MHz, CDCl3,
ppm):
(3f). Yield
d
d
8.20 (d, J ¼ 7.3 Hz, 1H), 7.83 (s, 1H), 7.33e7.23 (m, 6H),
7.06e7.09 (m, 2H), 5.30 (s, 2H), 4.45 (s, 2H), 3.91 (s, 3H). IR (KBr, v
cmꢂ1): 3112, 3098, 1650, 1602, 1580, 1521, 1448, 1223, 750. MS (ESI)
m/z calcd for C17H17N3O2 (M þ H)þ 296.1, obsd 296.1.
5.2.1.9. Indole-2-carbohydrazide (3g). Yield 90%; White solid, mp.
247e248 ꢀC. 1H NMR (400 MHz, CDCl3, d ppm):
d
11.70 (s, 1H), 9.85
(s, 1H), 7.80e7.10 (m, 5H), 4.2 (s, 2H). IR (KBr,
n
cmꢂ1): 3356 3217,
3080, 2900, 1640, 1615, 1550, 780, 740. MS (ESI) m/z calcd for
C9H9N3O (M þ H)þ 176.1, obsd 176.2.
5.2.1.2. General procedure for the synthesis of N-alkylated indole-3-
carbohydrazides (3def). To a solution of ethyl ester (10 mmol) of
indole-3-carboxylic acid 2a in tetrahydrofuran (20 mL) was
slowly added 60% sodium hydride (15 mmol) and stirred the
reaction mixture at 10 ꢀC for 15 min. To this suspension, an
appropriate alkyl halide (11 mmol) was added in one portion and
continued to stir the contents at room temperature for 4 h. The
reaction mixture was carefully poured into ice-cold water and
extracted with ethyl acetate (2 ꢁ 50 mL). Combined organic layer
was dried over anhydrous sodium sulphate and concentrated
under reduced pressure to afford pure N-alkylated indole ester
which was treated with hydrazine hydrate to obtain N-alkylated
indole-3-carbohydrazides 3def.
5.2.1.10. Indole-3-acetohydrazide (3h). Yield 85%; Off-white solid,
mp. 145e146 ꢀC. 1H NMR (400 MHz, CDCl3,
d ppm): d 11.0 (s, 1H),
9.65 (s, 1H), 7.59 (dd, J ¼ 7.8 Hz, J ¼ 1.6 Hz, 1H), 7.30 (d, J ¼ 7.8 Hz,
1H), 7.09 (d, J ¼ 7.8 Hz, 1H), 6.98 (d, J ¼ 7.8 Hz, 2H), 4.45 (s, 2H), 3.40
(s, 2H). IR (KBr, n
cmꢂ1): 3317, 1649, 1623, 1235, 1063, 812. MS (ESI)
m/z calcd for C10H11N3O (M þ H)þ 190.1, obsd 190.1.
5.2.2. General synthesis of 1-(indole-3-carbonyl)-4-
arylthiosemicarbazides (5)
Indole-2(3)-carbohydrazide
3
(10 mmol) and aryl iso-
thiocyanate 4 (10 mmol) were taken into round bottom flask con-
taining 10 mL of ethanol and stirred the reaction mixture at 60 ꢀC
for 2e3 h. The solid was filtered and dried well to obtain thio-
semicarbazides 5 in good yields (80e95%). The products were
sufficiently pure and taken as such for further reaction.
5.2.1.3. Indole-3-carbohydrazide (3a). Yield 85%; White solid, mp.
232e234 ꢀC. 1H NMR (400 MHz, DMSO-d6,
d ppm): d 11.31 (s, 1H),
9.14 (s, 1H), 8.17 (d, J ¼ 7.10 Hz, 1H), 7.95 (d, J ¼ 2.80 Hz, 1H), 7.41 (d,
J ¼ 8.10 Hz, 1H), 7.20e6.99 (m, 2H), 3.95 (s, 2H). IR (KBr, v cmꢂ1):
3256, 3109, 1660, 1607, 1583, 1523, 1433, 1240, 736. MS (ESI) m/z
calcd for C9H9N3O (M)þ 175.1, obsd 175.2.
5.2.3. General procedure for the synthesis of 2-arylamino-5-
(indolyl)-1,3,4-thiadiazoles (6)
The thiosemicarbazide 5 (10 mmol) was charged into 10 mL
round bottom flask containing 4 mL of freshly distilled acetyl
chloride. The reaction mixture was stirred at 20 ꢀC temperature till
the completion of the reaction as monitored by thin layer chro-
matography. The contents were poured onto crushed ice and
neutralized with aqueous ammonia solution. Solid obtained was
filtered and recrystallized from ethanol to get desired arylamino-
(indolyl)-1,3,4-thiadiazoles 6 in good yields.
5.2.1.4. 5-Methoxyindole-3-carbohydrazide (3b). Yield 90%; White
solid, mp. 178e179 ꢀC. 1H NMR (400 MHz, DMSO-d6,
d
ppm):
(s, 1H), 9.64 (s, 1H), 7.61 (s, 1H), 7.49 (s, 1H), 7.40 (d, J ¼ 8.00 Hz, 2H),
4.18 (s, 2H), 3.86 (s, 3H). IR (KBr,
cmꢂ1): 3340, 3290, 3050, 2920,
d 11.68
n
1646, 1605, 1545, 778, 724. MS (ESI) m/z calcd for C10H11N3O2
(M þ H)þ 206.1, obsd 206.2.
5.2.3.1. 5-(1H-Indol-3-yl)-N-phenyl-1,3,4-thiadiazol-2-amine (6a).
Yield 65%; White solid, mp. 210e212 ꢀC. 1H NMR (400 MHz, DMSO-
5.2.1.5. 6-Methoxyindole-3-carbohydrazide (3c). Yield 90%; White
solid, mp 213e214 ꢀC. 1H NMR (400 MHz, DMSO-d6,
d
ppm):
d
11.72
d6,
d
ppm):
d
11.52 (s,1H),10.16 (s,1H), 8.18 (d, J ¼ 7.1 Hz,1H), 7.94 (s,
(s, 1H), 9.58 (s, 1H), 7.64 (s, 1H), 7.53 (s, 1H), 7.38 (d, J ¼ 8.00 Hz, 2H),
1H), 7.65e7.59 (m, 3H), 7.30e7.24 (m, 3H), 7.20e7.11 (m, 2H). IR
4.18 (s, 2H), 3.83 (s, 3H). IR (KBr,
n
cmꢂ1): 3345, 3265, 2935, 1653,
(KBr, n
cmꢂ1): 3377, 3250, 1612, 1570, 1431, 1124, 746, 675. MS (ESI)
1610, 1543, 736, 720. MS (ESI) m/z calcd for C10H11N3O2 (M)þ 205.1,
m/z obsd for C16H12N4S: 117.9, 176.0, 260.7, 292.2 (M)þ, 331.0
(M þ K).
obsd 205.1.

436
D. Kumar et al. / European Journal of Medicinal Chemistry 55 (2012) 432e438
5.2.3.2. 5-(1H-Indol-3-yl)-N-p-tolyl-1,3,4-thiadiazol-2-amine (6b).
Yield 70%; Off-white solid, mp. 205e206 ꢀC. 1H NMR (400 MHz,
C
17H14N4S: 279.4, 307.0 (M þ H)þ, 324.0 (M þ NH4)þ, 329.8
(M þ Na)þ, 345.6 (M þ K)þ.
DMSO-d6,
d ppm): d 11.54 (s, 1H), 10.07 (s, 1H), 8.20 (s, 1H), 7.75
(d, J ¼ 8.1 Hz, 1H), 7.56 (d, J ¼ 8.2 Hz, 1H), 7.46 (d, J ¼ 8.0 Hz, 1H),
5.2.3.8. 5-(1-Methyl-1H-indol-3-yl)-N-phenyl-1,3,4-thiadiazol-2-
7.39e7.26 (m, 2H), 7.21e7.07 (m, 3H), 2.30 (s, 3H). 13C NMR
amine (6h). Yield 75%; White solid, mp. 217e218 ꢀC. 1H NMR
(100 MHz, DMSO-d6,
128.77, 124.61, 122.52, 121.38, 120.97, 120.73, 117.21, 109.99,
d
ppm): 161.22, 153.14, 141.01, 136.82, 130.95,
(400 MHz, DMSO-d6,
(s, 1H), 7.75e7.57 (m, 2H), 7.40 (d, J ¼ 8.0 Hz,1H), 7.34e7.28 (m, 3H),
3.88 (s, 3H). IR (KBr,
cmꢂ1): 3194, 1624, 1546, 1456, 1246, 747, 653.
13C NMR (100 MHz, DMSO-d6,
ppm): 160.88, 153.05, 141.03,
136.82, 130.96, 129.00, 125.32 124.60, 122.62, 121.58, 120.90, 117.21,
110.46, 105.69, 33.07. MS (ESI) m/z obsd for C17H14N4S: 307.0
(M þ H)þ. MALDI-TOF m/z calcd for C17H14N4S 307.1018 (M þ H)þ,
obsd 307.1013.
d ppm): d 10.25 (s,1H), 8.18e8.24 (m, 3H), 7.92
106.07, 32.33. IR (KBr,
n
cmꢂ1): 3236, 3122, 1610, 1534, 1498, 1247,
n
747, 685. MS (ESI) m/z obsd for C17H14N4S: 307.0 (M þ H)þ, 308.0
(Mþ2).
d
d
5.2.3.3. N-(4-Chlorophenyl)-5-(1H-indol-3-yl)-1,3,4-thiadiazol-2-
amine (6c). Yield 58%; White solid, mp. 232e234 ꢀC. 1H NMR
(400 MHz, DMSO-d6,
J ¼ 7.4 Hz,1H), 8.09 (d, J ¼ 7.2 Hz,1H), 8.05 (s,1H), 7.71 (d, J ¼ 8.9 Hz,
2H), 7.46 (d, J ¼ 7.5 Hz, 2H), 7.22e7.10 (m, 2H). IR (KBr,
cmꢂ1):
3329, 3253, 1622, 1546, 1496, 1116, 745, 669. 13C NMR (100 MHz,
DMSO-d6, ppm): 167.46, 165.79, 159.39, 140.78, 138.89, 136.15,
135.38, 135.06, 134.58, 132.80, 129.23, 128.46, 126.64, 124.98,
122.82, 122.28, 121.29, 121.12, 117.23, 110.12, 107.40, 106.88, 49.20.
MS (ESI) m/z obsd for C16H11ClN4S 327.0 (M þ H)þ, 328.0 (Mþ2)þ,
349.3 (M þ Na)þ. MALDI-TOF m/z calcd for C16H11ClN4S 327.0471
(M þ H)þ, obsd 327.0470.
d ppm): d 11.59 (s, 1H), 10.42 (s, 1H), 8.17 (d,
5.2.3.9. N-(4-Chlorophenyl)-5-(1-methyl-1H-indol-3-yl)-1,3,4-thiadi-
n
azol-2-amine (6i). Yield 72%; White solid, mp 188e189 ꢀC. 1H NMR
(400 MHz, DMSO-d6,
7.90 (s, 1H), 7.75e7.57 (m, 3H), 7.44 (d, J ¼ 8.2 Hz, 1H), 7.37e7.20 (m,
3H), 3.89 (s, 3H). IR (KBr,
cmꢂ1): 3255, 1616, 1514, 1428, 1219, 815,
736, 667. 13C NMR (100 MHz, DMSO-d6,
ppm): 163.92, 160.85,
d
ppm):
d
10.23 (s, 1H), 8.20 (d, J ¼ 7.9 Hz, 1H),
d
n
d
157.87, 139.61, 136.93, 130.65, 129.77, 128.54, 125.17, 124.41, 122.61,
120.77, 118.78, 110.16, 105.95, 99.07, 33.06. MS (ESI) m/z obsd for
C17H13ClN4S: 149.0214.1, 325.0, 341.0 (M þ H)þ, 342.0 (Mþ2)þ, 363.0
(M þ Na)þ.
5.2.3.4. 5-(1H-Indol-3-yl)-N-(4-methoxyphenyl)-1,3,4-thiadiazol-2-
amine (6d). Yield 65%; White solid, mp. 200e201 ꢀC. 1H NMR
5.2.3.10. 5-(1-(4-Chlorobenzyl)-1H-indol-3-yl)-N-phenyl-1,3,4-thiadi-
(400 MHz, DMSO-d6,
d
ppm):
d
11.60 (s, 1H), 10.05 (s, 1H), 8.16 (t,
azol-2-amine (6j). Yield 70%; White solid, mp 159e160 ꢀC. 1H NMR
J ¼ 8.6 Hz, 1H), 7.79 (dd, J ¼ 8.0, 2.7 Hz, 1H), 7.57 (d, J ¼ 8.9 Hz, 2H),
(400 MHz, DMSO-d6,
1H), 7.54e7.39 (m, 3H), 7.46e7.10 (m, 8H), 5.42 (s, 2H). 13C NMR
(100 MHz, DMSO-d6, ppm): 167.46, 165.79, 159.39, 140.78, 138.89,
d ppm): d 9.98 (s,1H), 8.24e8.21 (m, 2H), 7.97 (s,
7.52e7.45 (m, 1H), 7.25e7.16 (m, 2H), 6.90e6.87 (m, 2H), 3.84 (s,
3H). IR (KBr,
n
cmꢂ1): 3319, 3182, 1616, 1583, 1450, 1247, 1180, 819,
d
750. MS (ESI) m/z obsd for C17H14N4OS: 148.1, 206, 279.1, 317.1,
136.15, 135.38, 135.06, 134.58, 132.80, 129.23, 128.46, 126.64, 124.98,
122.82, 122.28, 121.29, 121.12, 117.23, 110.12, 107.40, 106.88, 49.20. IR
323.0 (M þ H)þ, 345.0 (M þ Na).
(KBr,
n
cmꢂ1): 3192, 2943, 1608, 1527, 1431, 1246, 738. MS (ESI) m/z
5.2.3.5. 5-(1H-Indol-3-yl)-N-(4-(trifluoromethyl)phenyl)-1,3,4-
thiadiazol-2-amine (6e). Yield 72%; White solid, mp. 239e240 ꢀC.
obsd for C23H17ClN4S: 118, 242, 326, 417.0 (M þ H)þ, 418.1 (Mþ2),
MALDI-TOF m/z calcd for C23H17ClN4S 417.0941 (M þ H)þ, obsd
417.0943.
1H NMR (400 MHz, DMSO-d6,
d ppm): d 11.64 (s, 1H), 10.65 (s,
1H), 8.21 (d, J ¼ 7.3 Hz, 1H), 8.10 (s, 1H), 7.89 (d, J ¼ 8.4 Hz, 2H), 7.60
(d, J ¼ 8.6 Hz, 2H), 7.48 (d, J ¼ 7.5 Hz, 1H), 7.23e7.18 (m, 2H). IR (KBr,
5.2.3.11. 5-(1-(4-Chlorobenzyl)-1H-indol-3-yl)-N-(3,4,5-trimethoxy-
n
cmꢂ1): 3396, 3194, 1614, 1547, 1419, 1247, 1196, 840, 748, 671. 13
C
phenyl)-1,3,4-thiadiazol-2-amine (6k). Yield 65%; White solid, mp
NMR (100 MHz, DMSO-d6,
d
ppm): 160.54, 154.49, 143.98, 138.22,
238e239 ꢀC. 1H NMR (400 MHz, DMSO-d6,
d ppm): d 10.41 (s, 1H),
136.44, 126.66, 125.73, 124.22, 122.37, 121.84, 121.57, 120.60, 120.19,
116.86, 116.56, 111.80, 106.67. MS (ESI) m/z obsd for C17H11F3N4S:
149.0, 186, 346.0, 361.0 (M þ H)þ.
8.23 (d, J ¼ 8.1 Hz, 1H), 7.57 (s, 1H), 7.31e7.29 (m, 5H), 7.09 (d,
J ¼ 8.4 Hz, 2H), 6.72 (s, 2H), 5.33 (s, 2H), 3.89 (s, 6H), 3.85 (s, 3H). IR
(KBr, n
cmꢂ1): 3124, 2937,1597,1537,1429,1234,1182, 819, 769, 738.
MS (ESI) m/z obsd for C26H23ClN4O3S: 464.0, 475.7, 491.8507.3
5.2.3.6. 5-(1H-Indol-3-yl)-N-(3,4,5-trimethoxyphenyl)-1,3,4-thiadiazol
(M þ H)þ, 508.0 (Mþ2), 529.1 (M þ Na)þ.
-2-amine (6f). Yield 75%; White solid, mp. 223e224 ꢀC. 1H NMR
(400 MHz, DMSO-d6,
d
ppm):
d
11.55 (s, 1H), 10.16 (s, 1H), 8.18 (d,
5.2.3.12. 5-(1-(4-Methoxybenzyl)-1H-indol-3-yl)-N-(4-methoxyphe-
7.5 Hz, 1H), 8.01 (s, 1H), 7.75 (d, J ¼ 2.8 Hz, 1H), 7.53e7.41 (m, 1H),
nyl)-1,3,4-thiadiazol-2-amine (6l). Yield 55%; White solid, mp
7.23e7.15 (m, 2H), 7.04 (d, J ¼ 2.2 Hz,1H), 3.86 (s, 6H), 3.72 (s, 3H). 13C
174e175 ꢀC. 1H NMR (400 MHz, DMSO-d6,
d ppm): d 10.33 (s, 1H),
NMR (100 MHz, DMSO-d6,
d
ppm):
d
161.25, 158.19, 152.68, 137.57,
8.22 (d, J ¼ 8.8 Hz, 1H), 7.54 (s, 1H), 7.35 (d, J ¼ 8.8 Hz, 3H), 7.29e7.26
136.38, 135.31, 132.17, 130.69, 127.50, 124.34, 122.38, 120.89, 111.93,
(m, 2H), 7.13 (d, J ¼ 8.5 Hz, 2H), 6.93 (d, J ¼ 8.5 Hz, 2H), 6.85 (d,
106.53, 99.52, 94.88, 59.92, 55.63. IR (KBr,
n
cmꢂ1): 3313, 32,113, 1608,
J ¼ 8.6 Hz, 2H), 5.26 (s, 2H), 3.82 (s, 3H), 3.78 (s, 3H). IR (KBr,
n
cmꢂ1):
1550, 1433, 1238, 1146, 827, 746, 621. MS (ESI) m/z obsd for
C19H18N4O3S: 149.0, 353.2, 367.1, 383.0 (M þ H)þ, 405.0 (M þ Na)þ
MALDI-TOF m/z calcd for C19H18N4O3S 383.1178 (M þ H)þ, obsd
383.1176.
3215, 2856, 1616, 1587, 1497, 1246, 809, 746, 685. MS (ESI) m/z obsd
for C25H22N4O2S: 415.0, 427.8, 443.3 (M þ H)þ, 465.7 (M þ Na)þ.
5.2.3.13. 5-(1-(4-Methoxybenzyl)-1H-indol-3-yl)-N-(3,4,5-trimethoxy-
phenyl)-1,3,4-thiadiazol-2-amine (6m). Yield 65%; Pale yellow solid,
5.2.3.7. N-Benzyl-5-(1H-indol-3-yl)-1,3,4-thiadiazol-2-amine (6g).
Yield 62%; Off-white solid, .150e151 ꢀC. 1H NMR (400 MHz, DMSO-
mp.189e190 ꢀC. 1H NMR (400 MHz, DMSO-d6,
d ppm): d 10.99 (s,1H),
8.23 (d, J ¼ 7.4 Hz, 1H), 7.57 (d, J ¼ 8.5 Hz,1H), 7.37 (d, J ¼ 8.3 Hz, 1H),
7.29e7.26 (m, 2H), 7.13 (d, J ¼ 8.7 Hz, 2H), 6.92e6.82 (m, 2H), 6.77 (s,
2H), 5.28 (s, 2H), 3.90 (s, 6H), 3.86 (s, 3H), 3.78 (s, 3H). 13C NMR
d6,
(d, J ¼ 2.5 Hz, 1H), 7.48e7.42 (m, 3H), 7.36e7.28 (m, 3H), 7.19e7.14
(m, 2H) 4.64 (s, 2H). 13C NMR (100 MHz, DMSO-d6,
ppm): 165.93,
161.60, 152.27, 138.07, 136.37, 128.44, 127.65, 127.27, 124.04, 122.64,
120.73, 120.52, 112.01, 106.28, 48.29. IR (KBr,
cmꢂ1): 3221, 3140,
1645, 1583, 1429, 1244, 1135, 745, 696. MS (ESI) m/z obsd for
d
ppm):
d
11.59 (s, 1H), 10.01 (s, 1H), 8.10 (d, J ¼ 7.7 Hz, 1H), 7.74
d
d
(100 MHz, DMSO-d6, d ppm): 161.69, 158.80, 152.92, 152.45, 137.04,
136.00, 134.84, 132.37, 132.10, 129.28, 128.73, 128.32, 124.86, 124.75,
122.60, 120.61, 116.35, 113.74, 110.44, 106.46, 99.80, 95.34, 60.17,
n
55.62, 55.55, 54.89, 49.07. IR (KBr,
n
cmꢂ1): 3263, 2937, 1608, 1583,

D. Kumar et al. / European Journal of Medicinal Chemistry 55 (2012) 432e438
437
1249, 831, 744, 707. MS (ESI) m/z obsd for C27H26N4O4S: 488.1, 474.0,
503.2 (M þ H)þ, 525.2 (M þ Na)þ.
MS (ESI) m/z obsd for C19H18N4O3S: 368.8, 383.4 (M þ H)þ, 405.1
(M þ Na)þ, obsd MALDI-TOF m/z calcd for C19H18N4O3S 383.1178
(M þ H)þ, obsd 383.1173.
5.2.3.14. 5-(5-Methoxy-1H-indol-3-yl)-N-(4-methoxyphenyl)-1,3,4-
thiadiazol-2-amine (6n). Yield 50%; White solid, mp. 196e197 ꢀC.
5.2.3.20. N-Benzyl-5-(1H-indol-2-yl)-1,3,4-thiadiazol-2-amine (6t).
Yield 60%; White solid, mp. 175e176 ꢀC. 1H NMR (400 MHz, DMSO-
1H NMR (400 MHz, DMSO-d6,
d ppm): d 11.29 (s, 1H), 9.84 (s, 1H),
7.89 (s, 1H), 7.74 (d, J ¼ 2.1 Hz, 1H), 7.60 (dd, J ¼ 9.3, 5.8 Hz, 2H),
d6,
d
ppm):
d
11.52 (s, 1H), 8.17 (s, 1H), 7.52 (d, J ¼ 7.9 Hz, 1H),
7.36e7.30 (m, 2H), 6.85 (d, J ¼ 8.0 Hz, 2H), 3.86 (s, 3H), 3.79 (s, 3H).
7.45e7.40 (m, 3H), 7.36 (t, J ¼ 7.2 Hz, 2H), 7.27 (d, J ¼ 7.2 Hz,1H), 7.14
13C NMR (100 MHz, DMSO-d6,
d ppm): 162.85, 152.31, 149.97,
(t, J ¼ 7.6 Hz, 1H), 7.02 (t, J ¼ 7.2 Hz, 1H), 6.69 (d, J ¼ 1.6 Hz, 1H), 4.60
137.22, 136.35, 132.54, 127.87, 127.61, 123.48, 122.85, 121.25, 119.31,
(d, J ¼ 7.2 Hz, 2H). IR (KBr,
n
cmꢂ1): 3364, 3271, 1610, 1543, 1358,
111.94, 102.98, 95.62, 94.97, 59.47, 55.65. IR (KBr,
n
cmꢂ1): 3415,
1246, 745, 685. MS (ESI) m/z obsd for C17H14N4S: 307.3 (M þ H)þ,
3219, 1619, 1581, 1485, 1257, 1185, 825, 794, 685. MS (ESI) m/z obsd
for C18H16N4O2S: 149.0, 204.0, 337.1, 353.0 (M þ H)þ, 375.0
(M þ Na)þ, 391.0 (M þ K)þ.
329.5 (M þ Na)þ.
5.2.3.21. 5-((1H-Indol-3-yl)methyl)-N-(4-methoxyphenyl)-1,3,4-thia-
diazol-2-amine(6u). Yield 65%; White solid, mp. 193e194 ꢀC.1H NMR
5.2.3.15. 5-(6-Methoxy-1H-indol-3-yl)-N-(4-methoxyphenyl)-1,3,4-
(400 MHz, DMSO-d6, d ppm): d 10.71 (s, 1H), 9.72 (s, 1H), 7.50 (d,
thiadiazol-2-amine (6o). Yield 55%; Off-white solid, mp
J ¼ 7.9 Hz, 1H), 7.45 (d, J ¼ 8.0 Hz, 2H), 7.37 (d, J ¼ 8.1 Hz, 1H), 7.21 (d,
J ¼ 2.4 Hz, 1H), 7.09 (t, J ¼ 8.0 Hz, 1H), 6.99 (t, J ¼ 7.4 Hz, 1H), 6.82 (d,
J ¼ 8.8 Hz, 2H), 4.28 (s, 2H), 3.74 (s, 3H). 13C NMR (100 MHz, DMSO-d6,
182e183 ꢀC. 1H NMR (400 MHz, DMSO-d6,
d ppm): d 11.19 (s, 1H),
9.86 (s, 1H), 8.08 (s, 1H), 7.95 (d, J ¼ 8.8 Hz, 1H), 7.57 (d, J ¼ 8.1 Hz,
2H), 6.95 (d, J ¼ 8.7 Hz, 2H), 6.84 (d, J ¼ 8.0 Hz, 2H), 3.84 (s, 3H), 3.78
d
ppm): d 164.92, 160.00, 154.25, 136.33, 134.29, 126.52, 123.72, 121.21,
(s, 3H). 13C NMR (100 MHz, DMSO-d6,
d
ppm): 161.92, 156.32,
119.03, 118.76, 114.28, 111.51, 110.59, 55.26, 26.08. IR (KBr, n
cmꢂ1):
154.28, 152.88, 137.06, 134.64, 125.07, 121.39, 120.89, 118.99, 118.44,
3344, 3199, 1624, 1566, 1436, 1230, 1126, 829, 734, 648. MS (ESI) m/z
114.54, 114.09, 110.63, 106.96, 94.60, 57.56, 54.76. IR (KBr,
n
cmꢂ1):
obsd for C18H16N4OS: 337.1 (M þ H)þ, 359.0 (M þ Na)þ, 375.0 (M þ K).
3240, 3185, 1614, 1579, 1454, 1246, 1165, 817, 746, 673. MS (ESI) m/z
obsd for C18H16N4O2S: 149.0, 204.0, 353.0 (M þ H)þ, 375.0
(M þ Na)þ.
5.2.3.22. 5-((1H-Indol-3-yl)methyl)-N-(3,4,5-trimethoxyphenyl)-
1,3,4-thiadiazol-2-amine (6v). Yield 65%; Off-white solid, mp.
235 ꢀC. 1H NMR (400 MHz, DMSO-d6,
d ppm): d 10.81 (s, 1H), 9.88 (s,
5.2.3.16. 5-(6-Methoxy-1H-indol-3-yl)-N-(3,4,5-trimethoxyphenyl)-
1H), 7.50 (d, J ¼ 8.0 Hz, 1H), 7.37 (d, J ¼ 8.0 Hz, 1H), 7.21 (d,
J ¼ 2.4 Hz, 1H), 7.09 (t, J ¼ 7.1 Hz, 1H), 6.99 (t, J ¼ 7.8 Hz, 1H), 6.92 (s,
2H), 4.35 (s, 2H), 3.79 (s, 6H), 3.68 (s, 3H). 13C NMR (100 MHz,
1,3,4-thiadiazol-2-amine (6p). Yield 60%; Light brown solid, mp.
201e202 ꢀC. 1H NMR (400 MHz, DMSO-d6,
d ppm): d 11.29 (s, 1H),
10.04 (s, 1H), 8.06 (d, J ¼ 8.7 Hz, 1H), 7.99 (s, 1H), 7.61 (d, J ¼ 2.7 Hz,
DMSO-d6,
130.60, 126.62, 123.80, 121.27, 118.66, 118.30, 111.75, 110.39, 95.52,
60.02, 55.54, 26.08. IR (KBr,
cmꢂ1): 3383, 3221, 1616, 1585, 1421,
d ppm): d 164.30, 160.46, 152.99, 137.17, 136.37, 132.22,
1H), 7.02 (s, 2H), 6.81 (dd, J ¼ 8.7, 2.3 Hz, 1H), 3.86 (s, 6H), 3.84 (s,
3H), 3.72 (s, 3H). 13C NMR (100 MHz, DMSO-d6,
d
ppm): 161.42,
n
158.19, 152.85, 137.22, 136.23, 132.11, 126.55, 124.14, 122.41, 120.59,
112.16, 106.91, 100.68, 99.88, 95.34, 94.72, 60.08, 56.08, 55.64,
1240, 1165, 844, 746, 624. MS (ESI) m/z obsd for C20H20N4O3S:
382.9, 397.3 (M þ H)þ, 419.1 (M þ Na)þ, 435.1 (M þ K)þ
55.59. IR (KBr, n
cmꢂ1): 3329, 3215, 1614, 1577, 1455, 1240, 833, 773,
709. MS (ESI) m/z obsd for C20H20N4O4S: 149, 204.0, 356.1, 383.1,
5.3. MTT assay
413.0 (M þ H)þ.
Six human cancer cell lines (LnCaP, DU145, MCF-7, MDA-MB-
231, HeLa and Ovcar-3) were cultured in RPMI-1640 media sup-
plemented with 10% heat inactivated foetal 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
various concentrations of the compounds ranging from 10 nM to
1 mM. After 48 h, MTT (3-(4,5-dimethyldiazol-2-yl)-2,5-diphe-
nyltetrazoliumbromide) was added to the final concentration of
0.2 mg/ml and incubated for 30 min. The cells were washed twice
5.2.3.17. N-(4-Chlorophenyl)-5-(1H-indol-2-yl)-1,3,4-thiadiazol-2-
amine (6q). Yield 65%; White sold, mp. 238e239 ꢀC. 1H NMR
(400 MHz, DMSO-d6,
J ¼ 8.7 Hz, 2H), 7.50 (d, J ¼ 7.9 Hz, 1H), 7.48e7.39 (m, 3H), 7.11 (t,
cmꢂ1):
d ppm): d 11.57 (s, 1H), 10.39 (s, 1H), 7.65 (d,
J ¼ 7.6 Hz, 1H), 6.98 (t, J ¼ 7.4 Hz, 1H), 6.75 (s, 1H). IR (KBr,
n
3342, 3199, 1624, 1535, 1458, 1230, 798, 734, 648. MS (ESI) m/z obsd
for C16H11ClN4S: 149, 189.1, 215.1, 327.0 (M þ H)þ.
5.2.3.18. 5-(1H-Indol-2-yl)-N-(4-methoxyphenyl)-1,3,4-thiadiazol-2-
with PBS and lyses in 100 mL dimethylsulfoxide, and the absorbance
was measured at 570 nm using Tecan Spectrafluor Plus.
amine (6r). Yield 74%; Pale yellow solid, mp 206 ꢀC. 1H NMR
(400 MHz, DMSO-d6,
(m, 3H), 7.21e7.16 (m, 2H), 7.09e7.05 (m,1H), 6.89 (d, J ¼ 7.2 Hz, 2H),
6.77 (s, 1H), 3.79 (s, 3H). 13C NMR (100 MHz, DMSO-d6,
ppm):
164.23, 159.55, 154.61, 149.92, 136.77, 133.87, 128.24, 127.45, 122.62,
d ppm): d 11.54 (s, 1H), 10.10 (s, 1H), 7.56e7.48
Acknowledgements
d
The authors thank CSIR, New Delhi for the financial support and
SAIF, Panjab University for providing analytical support. NMK also
acknowledge the CSIR, New Delhi for the award of SRF.
120.37, 119.61, 118.62, 113.98, 112.00, 103.02, 99.47, 55.05. IR (KBr,
n
cmꢂ1): 3388, 3317,1606,1539,1413,1234, 833, 795, 676. MS (ESI) m/z
obsd for C17H14N4OS: 291.6, 308.8, 323.4 (M þ H)þ, 345.1 (M þ Na)þ.
References
5.2.3.19. 5-(1H-Indol-2-yl)-N-(3,4,5-trimethoxyphenyl)-1,3,4-thiadia-
zol-2-amine (6s). Yield 78%; White solid, mp. 232e233 ꢀC. 1H NMR
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