One-pot synthesis and anticancer studies of 2-arylamino-5-aryl-1,3,4- thiadiazoles

Article abstract of DOI:10.1016/j.bmcl.2011.02.083

A series of 2-arylamino-5-aryl-1,3,4-thiadiazoles 1a-j were synthesized and screened for their anticancer activity against various human cancer cell lines. The novel one-pot synthesis of 1,3,4-thiadiazoles was achieved by refluxing aryl aldehydes, hydrazi

Full text of DOI:10.1016/j.bmcl.2011.02.083

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2320–2323
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
One-pot synthesis and anticancer studies of 2-arylamino-5-aryl-1,3,4-
thiadiazoles
Dalip Kumar a, , Buchi Reddy Vaddula a, Kuei-Hua Chang b, Kavita Shah b,
⇑
⇑
a Department of Chemistry, Birla Institute of Technology and Science, Pilani 333031, Rajasthan, 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-aryl-1,3,4-thiadiazoles 1a–j were synthesized and screened for their anticancer
activity against various human cancer cell lines. The novel one-pot synthesis of 1,3,4-thiadiazoles was
achieved by refluxing aryl aldehydes, hydrazine hydrate, and aryl isothiocyanates in methanol followed
by oxidative cyclization with ferric ammonium sulfate. The compounds 1g–j with trimethoxyphenyl at
Received 11 December 2010
Revised 22 February 2011
Accepted 23 February 2011
Available online 26 February 2011
the C-5 position displayed extremely potent anticancer activity with at least twofold selectivity (IC50
4.3–9.2
tivity towards a particular cancer cell.
:
l
M). The nature of substituent on the C-2 arylamino ring may be critical in opting for the selec-
Keywords:
1,3,4-Thidiazoles
Anticancer agents
One-pot synthesis
Cancer cell lines
Oxidative cyclization
Ó 2011 Elsevier Ltd. All rights reserved.
1,3,4-Thiadiazoles are five-membered ring systems that have
gained prominence by exhibiting a wide variety of biological activ-
ities as well as producing useful intermediates in several organic
preparations.1–7 They have interesting pharmacophores that dis-
play a broad spectrum biological activity. The lower toxicity and
in vivo stability of thiadiazole nucleus is attributed to its aromatic-
ity.8 Thiadiazoles have exhibited potential antiglaucoma,9 antiin-
flammatory,1 antitumor,7 antiulcer,10 antibacterial,11 antiviral,12
analgesic,13 antiepileptic,5 antifungal,11 and radioprotective
activities.14 The marketed drugs, acetazolamide, methazolamide,
globucid, etc. showcase their therapeutic potential. Thiadiazoles,
bioisosters of thiazoles and oxadiazoles, are known to have
interesting electro-optical properties15 and also act as corrosion
and oxidation inhibitors,16 complexation reagents for dyes and
metal ions.17–20
nyl)-1,3,4-thiadiazole (FABT), a promising anticancer compound
for treating malign tumors of the nervous system, inhibits prolifer-
ation by decreasing cell division and inhibiting metastasis.22,31 It
was shown that the one of the important structural unit in several
known natural antimitotic agents such as Combretastatin A-4,
Colchicine, Podophyllotoxin, and Steganacin, which bind at the
Colchicine site on tubulin, is a trimethoxyaryl moiety (Fig. 1).32,33
The incorporation of crucial structural features of anticancer com-
pounds (3,4,5-trimethoxyphenyl and 4-hydroxy-3-methoxyphenyl
moieties) into 1,3,4-thiadiazoles may lead to a potent anticancer
compound (Fig. 1).
Generally, preparation of 1,3,4-thiadiazoles involve use of
diacylhydrazide precursors under different reaction conditions.
Symmetrical 2,5-disubstituted-1,3,4-thiadiazoles are prepared by
condensation of aryl aldehydes, hydrazine, and sulfur in ethanol
under microwave irradiation.34 The general routes for the prepara-
tion of various 1,3,4-thiadiazoles involve either synthesis of acyl
thiohydrazides and then cyclization or thionation of acyl
hydrazides followed by oxidative cyclization of thiosemicarba-
zones. Gierczyk and Zalas reported the four step synthesis of
The 1,3,4-thiadiazoles, their isomeric forms and bioisosters are
extensively investigated for their anticancer activity due to their
therapeutic potential.7,21–23 In this direction, our research group
has successfully explored various heterocycles to develop
a
potential anticancer compound.24–27 2-Amino-1,3,4-thiadiazole,
2-ethylamino-1,3,4-thiadiazole, 2,20-(methylenediamino)bis-1,3,
4-thiadiazole and their N-substituted derivatives were found to
exhibit very good anticancer activity but suffered from adverse
side-effects.28–30 2-(4-Fluorophenylamino)-5-(2,4-dihydroxy-phe-
2,5-disubstituted-1,3,4-thiadiazoles
from
pentafluorophenyl
esters.35 Also a robust protocol for the solid phase synthesis of
5-alkyl/aryl-2-alkylamino-1,3,4-thiadiazoles was described from
resin bound thiosemicarbazide.36 Oruc et al., prepared 1,3,4-thi-
adiazoles in four steps utilizing acyl halides and aryl isothiocya-
nates.37 The 1,3,4-thiadiazoles were also achieved in good yields
from the reaction of 1,3,4-oxadiazoles with thiourea.38 Recently
reported one-pot convenient synthesis of 1,3,4-thiadiazoles by
⇑
Corresponding authors. Tel.: +91 1596 245073 279; fax: +91 1596 244183
(D.K.); tel.: +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.2011.02.083

D. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2320–2323
2321
OH
O
O
O
OAc
H3CO
H3CO
H3CO
O
NHCOCH3
O
O
O
H3CO
H3CO
H3CO
H3CO
O
O
H3CO
OH
OCH3
H3CO
OCH3
OCH3
H3CO
OCH3
Combretastatin A-4
Podophyllotoxin
Colchicine
Steganacin
N
N
N
OH
N
R1
NH
NH
R = F, Cl, Br, H, OCH3, CH
3
S
S
R1 = R2 = R3 = H, OH, OCH3
HO
R2
R3
F
R
FABT
1
Figure 1. Rational for the design of 2-arylamino-5-aryl-1,3,4-thiadiazoles 1.
Table 1
Rostamizadeh et al., utilizes ionic liquid/thiourea mediated
oxidation of in situ generated thiosemicarbazones.39 In general,
most of the reported protocols are time consuming, laborious
and require multi synthetic steps.
Conditions investigated for the conversion of thiosemicarbazone 5a to 1,3,4-thiadi-
azole 1a
S. No.
Reagent
Solvent
Temp. (°C)
Time (h)
Yield (%)
We have developed an expeditious one-pot synthesis of
1,3,4-thiadiazoles 1 which involve the reaction of aryl aldehydes
2, hydrazine hydrate 3, and aryl isothiocyanates 4 followed by
oxidative cyclization of in situ generated thiosemicarbazones with
ferric ammonium sulfate (FAS) (Scheme 1). Our initial efforts to
prepare 1,3,4-thiadiazoles 1 involved one-pot sequential reaction
of 4-hydroxy-3-methoxybenzaldehyde 2a, hydrazine hydrate 3,
and phenyl isothiocyanate 4a in methanol. The reaction of equimo-
lar quantities of 4-hydroxy-3-methoxybenzaldehyde 2a and
hydrazine hydrate 3 in methanol at 80 °C resulted in the interme-
diate benzylidene hydrazine which was further reacted with phe-
nyl isothiocyanate 4a to afford thiosemicarbazone 5a. Further,
we have attempted to prepare thiosemicarbazone 5a in one-pot
by the simultaneous reaction of 4-hydroxy-3-methoxybenzalde-
hyde 2a, hydrazine hydrate 3, and phenyl isothiocyanate 4a in
methanol. To our delight, we found that the thiosemicarbazone
5a was obtained in 90% yields. It was isolated by simple filtration
and subjected to oxidative cyclization. Taking a cue from the liter-
ature reports, the cyclization of 5a was attempted using various
reagents (Table 1). Of the screened reagents, FAS smoothly
produced the desired 1,3,4-thiadiazole in good yields.
1
2
3
4
5
6
7
8
9
FeCl3 (10% soln)
FeCl3 (4 mmol)
FAS (1 mmol)
FAS (3 mmol)
FAS (6 mmol)
ZnCl2 (excess)
AcOH (excess)
IBD (1 mmol)a
HTIB (1 mmol)a
HgCl2 (1.1 mmol)
C2H5OH
H2O
80
90
80
80
80
80
120
0 to rt
0 to rt
rt
4
4
3
1
1
24
24
4
4
10
10
0
40
85
75
0
0
0
0
0
CH3OH
CH3OH
CH3OH
CH3OH
—
CH2Cl2
CH2Cl2
CH3CN
10
a
IBD: iodobenzenediacetate; HTIB: [Hydroxy(tosyloxy)iodo]benzene.
solvent was decanted and added 3 equiv of finely ground FAS to
the reaction pot and heated at 80 °C for further 1 h to obtain the
1,3,4-thiadiazole 1a in 85% yield.40 The formation of the thiadiazole
1a was confirmed by its 1H NMR and MS data.41–43 The 1H NMR
spectrum of 1a evidenced a characteristic singlet at d 9.57 due to
C–2N–H and the mass spectrum shows the [M+H]+ peak at m/z
300.1 which is in agreement with the calculated value.
The optimized protocol was extended to other aryl isothiocya-
nates and aryl aldehydes. The reaction of 4-hydroxy-3-methoxy-
benzaldehyde 2a with hydrazine hydrate
isothiocyanates 4b–e afforded 1b–e in good yields (75–85%). All
aryl isothiocyanates 4a–e were almost equally reactive to generate
the 1,3,4-thiadiazoles 1a–e. The deactivated aldehyde, 3,4,5-tri-
methoxybenzaldehyde 2c, also reacted as efficiently as 4-hydro-
xy-3-methoxybenzaldehyde 2a affording products 1g–j in good
yields (70–80%). Employing 3-cyclopentyloxy-4-methoxybenzal-
dehye 2b with phenyl isothiocyanate 4a resulted in 1f with rela-
tively low yields (55%) which may be due to the cleavage of
cyclopentyl group during oxidative cyclization.
The overall one-pot protocol was established by the reaction of
equimolar quantities of 4-hydroxy-3-methoxybenzaldehyde 2a,
hydrazine hydrate 3, and phenyl isothiocyanate 4a in 25 mL meth-
anol at 80 °C for 1 h. Upon completion of the reaction, as indicated
by TLC, the solid product was allowed to settle down. The excess
3
and aryl
a
Ar'NCS
+
+
ArCHO
H2NNH2. H2O
2
3
4
The synthesized series of diverse 2-arylamino-5-aryl-1,3,4-thi-
adiazoles 1a–j were screened against prostate (PC3, DU145, and
LnCaP), breast (MCF7 and MDA-MB-231), and pancreatic (PaCa2)
cancer cell lines. All compounds decreased cell viability as deter-
mined by colorimetric MTT assay, with IC50 values ranging from
micromolar to greater than 1 mM (Table 2).44 More importantly,
a few compounds were highly potent and exhibited specificity
towards one cell type relative to others.
H
H
N N
S
Ar'
N
N
b
Ar
N
Ar'
N
H
Ar
S
1a-j
5
Scheme 1. One-pot synthesis of 1,3,4-thiadiazoles 1a–j. Reagents and conditions:
(a) methanol, 80 °C, 1 h; (b) FAS, 80 °C, methanol, 1 h.

2322
D. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2320–2323
Table 2
In vitro cytotoxicity dataa of 1,3,4-thiadiazoles 1a–j against selected human cancer cell lines IC50 M)b
(l
Entry
Ar
Ar0
LnCap
DU145
PC3
MCF7
MDA-MB-231
PaCa2
1a
1b
1c
1d
1e
1f
1g
1h
4-OH-3-OCH3C6H3
4-OH-3-OCH3C6H3
4-OH-3-OCH3C6H3
4-OH-3-OCH3C6H3
4-OH-3-OCH3C6H3
3-OC5H9-4-OCH3C6H3
3,4,5-(OCH3)3C6H2
3,4,5-(OCH3)3C6H2
3,4,5-(OCH3)3C6H2
3,4,5-(OCH3)3C6H2
C6H5
205.5
544.6
204.2
62.3
>1000
111.8
92.8
66.5
102.6
29.5
57.6
30
55.3
175.2
204.7
>1000
>1000
84.7
9.2
39
58.5
449.6
49.1
>1000
>1000
33.6
25.9
242.6
5.8
4-CH3C6H4
4-ClC6H4
4-NO2C6H4
4-OCH3C6H4
C6H5
655.6
435.4
>1000
>1000
177.1
283.9
99.7
130.7
33.3
12.3
>1000
634.7
>1000
>1000
96.9
81.8
71.4
20.2
14.5
124
479.8
386.4
>1000
>1000
433.9
122.4
13.8
32.1
25.2
6.8
C6H5
4-NO2C6H4
4-CH3C6H4
4-OCH3C6H4
6.6
1i
1j
10.2
11.1
66.2
4.3
25.3
Doxorubicin
6.3
IC50 values of about 20
l
M are indicated in bold face; OC5H9: cyclopentyloxy
a
These experiments were conducted in triplicates at three independent times.
b
IC50 values were obtained using a dose–response curve by nonlinear regression using a curve fitting program, GraphPad Prism 5.0.
The anticancer activity profiles of the screened compounds indi-
References and notes
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furnishing the activity. The compounds 1a–e with 4-OH-3-OCH3–
C6H3 and 1f with 3-OC5H9-4-OCH3–C6H3 at C-5 position were
found to be less active than the corresponding compounds 1g–j
with 3,4,5-(OCH3)3C6H2. The variation of C-2 arylamino group
seems to have little effect on the activity of 1,3,4-thiadiazoles. As
it was hypothesized, the trimethoxyphenyl substitution might be
playing a crucial role by means of binding to the Colchicine site
on tubulin. Our earlier studies on 3,5-disubstituted-1,2,4-oxadiaz-
oles delivered a very potent compound (IC50 = 10 nM) with 3-
OC5H9-4-OCH3–C6H3 and piperdin-4-yl.45 The compound 1f
designed following similar pattern resulted in moderate activity.
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very good activity (IC50: 4.3–20.2
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at least threefold selectivity compared to other tested cells. Intro-
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l
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l
cell lines (MCF7, IC50 = 6.6 lM; MDA-MB-231, IC50 = 13.8 lM),
whereas electron-donating group such as CH3 or OCH3 (com-
pounds 1i and 1j) resulted in a significant increase in cytotoxic po-
tency except against MCF7 cell line. In particular, compounds 1g
and 1h were found to be selectively cytotoxic in MCF7 cells. The
4-methoxy analogue 1j possessed potent anticancer activity
against all the tested cell lines while simultaneously exhibiting at
least threefold selectivity towards PaCa2. The compounds 1a–e
have exhibited moderate to poor anticancer activity (though some
of these have achieved good selectivity) indicating that 4-OH-3-
OCH3C6H3 group at 5-position is detrimental for the activity. It
earmarks the prominence of trimethoxyphenyl substitution at 5-
position of 1,3,4-thiadiazole. Doxorubicin was used as a control,
which showed higher cytotoxicity towards LnCAP, DU145, and
MDA-MB-231 cell lines.
In conclusion, the one-pot reaction of aryl aldehyde, hydrazine
hydrate and aryl isothiocyanate led to the expeditious synthesis
of 1,3,4-thiadiazoles 1a–j in good yields. The advantages of the
protocol include simple reaction workup, easily available starting
materials and convenient isolation. The compounds 1g–j exhibited
very good anticancer activity with good selectivity indicating the
importance of 3,4,5-trimethoxyphenyl at C-5 position. The detailed
SAR and molecular target study of these 1,3,4-thiadiazoles are in
progress and will be reported in due course.
Acknowledgment
38. Padmavathi, V.; Reddy, G. S.; Padmaja, A.; Kondaiah, P.; Ali, S. Eur. J. Med. Chem.
2009, 44, 2106.
The authors thank DST, New Delhi for the financial support.

D. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2320–2323
2323
39. (a) Rostamizadeh, S.; Aryan, R.; Ghaieni, H. R.; Amani, A. M. J. Heteroatom.
Chem. 2010, 47, 616; (b) Rostamizadeh, S.; Aryan, R.; Ghaieni, H. R.; Amani, A.
M. Heteroatom Chem. 2008, 19, 320.
1H), 7.29–7.24 (m, 2H), 7.09–7.02 (m, 2H), 6.83–6.77 (m, 2H), 6.72 (d,
J = 8.1 Hz, 1H), 3.82 (s, 4H), 3.55 (s, 3H). HRMS (ESI): m/z [M+H]+ calcd for
C
16H15N3O3S: 330.1; found: 330.1; 2-(3-Cyclopentyloxy-4-methoxyphenyl)-
40. General procedure for the synthesis of 1,3,4-Thiadiazoles 1: A mixture of aryl
5-(phenyl amino)-1,3,4-thiadiazole (1f): %yield: 55. Mp: 223–225 °C. 1H
NMR (400 MHz, DMSO-d6): d 7.53–7.47 (m, 4H), 7.36–7.31 (m, 2H), 6.98 (d,
J = 8 Hz, 1H) 6.78 (d, J = 8 Hz, 1H), 6.71 (s, 1H), 4.35 (s, 1H), 3.81 (s, 3H),
1.70–1.50 (m, 8H). MS (ESI): m/z [M+H]+ calcd for C20H22N3O2S: 368.1433;
found: 368.1604; 2-(3,4,5-Trimethoxyphenyl)-5-(phenylamino)-1,3,4-
thiadiazole (1g): %yield: 78. Mp: 228–230 °C. 1H NMR (400 MHz, DMSO-
d6): d 7.66 (s, 1H), 7.57–7.55 (m, 2H), 7.41 (m, 2H), 7.17 (s, 1H), 7.05 (s, 2H),
3.92(s, 6H), 3.86 (s, 3H). MS (ESI): m/z [M+H]+ calcd for C17H17N3O3S:
aldehyde
2
(3.0 mmol), hydrazine hydrate
3
(3.0 mmol) and aryl
isothiocyanate 4 (3.0 mmol) was refluxed in 25 mL methanol for 1 h. Upon
completion of the reaction, as indicated by TLC, the reaction mixture was
cooled to room temperature and allowed the solid product to settle down. The
excess solvent was decanted and added finely ground FAS (9.0 mmol) to the
reaction pot and heated at 80 °C for further 1 h. The reaction mixture was then
filtered, washed with hot methanol and concentrated the filtrate on a rotary
evaporator to obtain crude 1,3,4-thiadiazole 1 which was further recrystallized
from methanol/ethanol.
344.1069;
found:
344.1816;
2-(3,4,5-Trimethoxyphenyl)-5-(4-
methoxyphenyl amino)-1,3,4-thiadiazole (1j): %yield: 81. Mp: 191–
193 °C. 1H NMR (400 MHz, DMSO-d6): d 7.64 (s, 1H), 7.48 (d, J = 8.80 Hz,
2H), 7.03 (s, 2H), 6.92 (d, J = 8.80 Hz, 2H), 3.91 (s, 6H), 3.85 (s, 3H), 3.81 (s,
3H). MS (ESI): m/z [M+H]+ calcd for C18H19N3O4S: 374.1175; found:
374.1354.
41. Spectral data of the selected 1,3,4-thiadiazoles (1): 2-(3-Methoxy-4-
hydroxyphenyl)-5-(phenylamino)-1,3,4-thiadiazole (1a): %yield: 85. Mp: 246–
247 °C. 1H NMR (400 MHz, DMSO-d6): d 13.98 (s, 1H), 9.57 (s, 1H), 7.58–7.48
(m, 3H), 7.40–7.32 (m, 2H), 6.80 (dd, J = 8.2, 2.0 Hz, 1H), 6.73 (dd, J = 9.8, 5.1 Hz,
2H), 3.50 (s, 3H). MS(ESI): m/z [M+H]+ calcd for C15H13N3O2S: 300.1; found:
42. Noto, R.; Buccheri, F.; Cusmano, G.; Gruttadauria, M.; Werber, G. J. Heterocycl.
Chem. 1991, 28, 1421.
300.1;
2-(3-Methoxy-4-hydroxyphenyl)-5-(4-methylphenylamino)-1,3,4-
thiadiazole (1b): %yield: 82. Mp: 248–249 °C. 1H NMR (400 MHz, DMSO-d6):
d 13.57 (s, 1H), 9.61 (s, 1H), 7.32–7.23 (m, 4H), 6.78–6.73 (m, 3H), 3.53 (s, 3H),
2.38 (s, 3H). MS (ESI): m/z [M+H]+ calcd for C16H15N3O2S: 314.1; found: 314.1;
2-(3-Methoxy-4-hydroxyphenyl)-5-(4-chloro-phenylamino)-1,3,4-thiadiazole
(1c): %yield: 78. Mp: 268–270 °C. 1H NMR (400 MHz, DMSO-d6): d 14.03 (s,
1H), 9.60 (s, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.41 (d, J = 8.6 Hz, 2H), 6.81 (s, 1H),
6.79–6.72 (m, 2H), 3.57 (s, 3H). MS(ESI): m/z [M+H]+ calcd for
43. Meo, P. L.; Gruttadauria, M.; Noto, R. ARKIVOC 2005, 1, 114.
44. Six human cancer cell lines (LnCaP, DU145, PC3, MCF7, MDA-MB-231, and
PaCa2) 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 various concentrations of the compounds ranging from
10 nM to 1 mM. After 48 h, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliumbromide) 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 mL dimethylsulfoxide, and the absorbance was measured at
570 nm using Tecan Spectrafluor Plus.
C
15H12ClN3O2S: 334.0; found: 334.0; 2-(3-Methoxy-4-hydroxyphenyl)-5-
(4-nitrophenylamino)-1,3,4-thiadiazole (1d): %yield: 76. Mp: 280–284 °C.
1H NMR (400 MHz, DMSO-d6): d 14.14 (s, 1H), 9.62 (s, 1H), 8.42–8.33 (m,
2H), 7.74–7.66 (m, 2H), 6.86 (s, 1H), 6.73 (s, 2H), 3.57 (s, 3H). MS(ESI): m/z
[M+H]+ calcd for C15H12N4O4S: 345.0; found: 345.0; 2-(3-Methoxy-4-
hydroxyphenyl)-5-(4-methoxyphenyl amino)-1,3,4-thiadiazole (1e): %yield:
80. Mp: 228–230 °C. 1H NMR (400 MHz, DMSO-d6): d 13.93 (s, 1H), 9.56 (s,
45. Kumar, D.; Patel, G.; Johnson, E. O.; Shah, K. Bioorg. Med. Chem. Lett. 2009, 19,
2739.