New 5-(nitroheteroaryl)-1,3,4-thiadiazols containing acyclic amines at C-2: Synthesis and SAR study for their antileishmanial activity

Article abstract of DOI:10.3109/14756366.2012.689297

A novel series of 5-(5-nitrofuran-2-yl)-and 5-(5-nitrothiophen-2-yl)-1,3,4- thiadiazole-2-amines bearing acyclic amine at C-2 position of thiadiazole ring were synthesized and evaluated in vitro against promastigote and amastigote forms of Leishmania major. The structure-activity of series was investigated by studying 40 compounds. The most active derivatives were hydroxypropylamino-and methoxypropylamino-analogs of 5-(5-nitrothiophen-2-yl)-1,3,4-thiadiazole (compounds 29 and 32, respectively) with highest selectivity index (SI >12).

Full text of DOI:10.3109/14756366.2012.689297

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(4): 843–852
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.689297
research arTIcLe
New 5-(nitroheteroaryl)-1,3,4-thiadiazols containing
acyclic amines at C-2: synthesis and SAR study for their
antileishmanial activity
Azar Tahghighi1, Saeed Emami2, Sepide Razmi3, Farzane Rezazade Marznaki3, Sussan Kabudanian
Ardestani3, Siavoush Dastmalchi1, Farzad Kobarfard4, Abbas Shafiee5, and Alireza Foroumadi5
1Department of Medicinal Chemistry, School of Pharmacy and Biotechnology Research Center, Tabriz University of
Medical Sciences, Tabriz, Iran, 2Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center,
Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran, 3Institute of Biochemistry and Biophysics,
Department of Biochemistry, University of Tehran, Tehran, Iran, 4Department of Medicinal Chemistry, School of
Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran, and 5Department of Medicinal Chemistry,
Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, Iran
abstract
A novel series of 5-(5-nitrofuran-2-yl)-and 5-(5-nitrothiophen-2-yl)-1,3,4-thiadiazole-2-amines bearing acyclic amine
at C-2 position of thiadiazole ring were synthesized and evaluated in vitro against promastigote and amastigote
forms of Leishmania major. The structure-activity of series was investigated by studying 40 compounds. The most
active derivatives were hydroxypropylamino- and methoxypropylamino- analogs of 5-(5-nitrothiophen-2-yl)-1,3,4-
thiadiazole (compounds 29 and 32, respectively) with highest selectivity index (SI >12).
Keywords: Antileishmanial activity, 5-nitrofuran, 5-nitrothiophene, 1,3,4-thiadiazole
Introduction
e chemotherapy of leishmanisis is limited to the use
of sodium stibogluconate (Pentostan), meglumine anti-
moniate (Glucantime), amphotericin B, miltefosin, and
pentamidine6. Due to the prolonged duration of therapy,
adverse effects, and resistance to these compounds, the
discovery of new, efficient, and safe leishmanicidal drugs
is required7. In addition; the increase of leishmaniasis-
AIDS co-infections is a serious problem8.
Substituted five-membered heterocyclic rings
have diverse antimicrobial activities. Recently, several
furanyl, thiophenyl, and tetrahydronaphthyl azoles were
synthesized and evaluated for their in vitro antileishmanial
activity9,10. We have also reported the synthesis and in
vitro antileishmanial activities of some 5-(5-nitrofuran-
2-yl) and 5-(5-nitrothiophen-2-yl)-1,3,4-thiadiazoles (I)
bearing a cyclic amine (such as morpholine, piperidine,
piperazine, and N-substituted piperazine) at C-2 position
Leishmaniasis is a group of parasitic diseases of global
distribution transmitted by the bite of the infected female
phlebotomine sandfly and manifests in three forms: vis-
ceral, cutaneous, and mucocutaneous leishmaniasis1.
Leishmania parasites have a dimorphic life-cycle; promasti-
gotes and amastigotes. Promastigotes are extracellular form
and develop in the gut of female sandflies to infectious form
that are transmitted to mammalian hosts2,3. e amastigote
form of Leishmania is an intracellular stage and survives
and multiplies within parasitophorous vacuoles of macro-
phages4. e control of leishmaniasis as a zoonotic infection
remains a serious public health problem. It is affecting an
estimated 12 million people in 88 countries, with ~2 mil-
lion new cases per year1. ere are currently no vaccines
for leishmaniasis5. It is one of the most neglected parasitic
diseases in terms of drug discovery and development.
Address for Correspondence: Alireza Foroumadi, Faculty of Pharmacy and Pharmaceutical Sciences Research Centre, Tehran University of
Medical Sciences, Tehran, Iran. Tel: +98 21 66954708; Fax: +98 21 66461178. E-mail: aforoumadi@yahoo.com
(Received 07 December 2011; revised 23 April 2012; accepted 23 April 2012)
843

844 A. Tahghighi et al.
(Figure 1) 11–14. Our results demonstrated that the C-2
substituent in 5-(nitroheteroaryl)-1,3,4-thiadiazoles is the
most flexible site for chemical change and is an area where
it determines the potency and physicochemical properties
of 5-(nitroheteroaryl)-1,3,4-thiadiazoles. In continuation
of our work on 5-(nitroheteroaryl)-1,3,4-thiadiazoles,
we report here the synthesis of some 5-(5-nitrofuran-
2-yl)- or 5-(5-nitrothiophen-2-yl)-1,3,4-thiadiazoles (II)
possessing acyclic amine at the C-2 position of thiadiazole
ring (Figure 1). All of these compounds were evaluated in
vitro against promastigote form of Leishmania major.
methanol. e compound was obtained as a yellow
solid (yield: 61%). mp >300°C. IR (KBr, cm−1): 3,169,
1,577, 1,350.1H NMR (400 MHz, DMSO-d6) δ: 8.91 (br
s, 1H, NH), 7.85 (d, 1H, J = 3.2 Hz, furan), 7.36 (d, 1H, J
= 3.2 Hz, furan), 2.74 (br s, 1H, c-Pr), 0.80 (br s, 2H, c-
Pr), 0.62 (br s, 2H, c-Pr). MS (ESI): 252.8 [M + H+]. Anal.
Calcd for C9H8N4O3S: C, 42.85; H, 3.20; N, 22.21. Found:
C, 43.22; H, 3.00; N, 21.91.
2-(5-(5-Nitrofuran-2-yl)-1,3,4-thiadiazol-2-ylamino)
ethanol (6)
e resulting product was purified using a short silica
gel column eluting with CH2Cl2 containing 2% methanol.
e compound was obtained as a yellow solid (yield:
63%). mp 179–182°C; IR (KBr, cm−1): 3,395, 3,207, 1,599,
1,351 .1H NMR (500 MHz, DMSO-d6) δ: 8.46 (br s, 1H,
NH), 7.85 (d, 1H, J = 3.6 Hz, furan), 7.34 (d, 1H, J = 3.6
Hz, furan), 3.61 (t, 2H, J = 5.05 Hz, OCH2), 3.45 (t, 2H, J =
5.05 Hz, NCH2). MS (ESI): 256.8 [M + H+]. Anal. Calcd for
C8H8N4O4S: C, 37.50; H, 3.15; N, 21.87. Found: C, 37.24; H,
2.93; N, 22.25.
experimental
Chemistry
All commercial compounds were purchased from Merck
Chemical (Darmstadt, Germany). e 2-chloro-1,3,4-
thiadiazole intermediate 2 was prepared starting from
5-nitrofurfurilidine diacetate or 5-nitrothiophene-2-car-
boxaldehyde according to the literature methods15–18
.
e melting points of compounds were determined on a
Kofler hot-stage apparatus and are uncorrected. e IR
spectra were obtained on a Shimadzu 470 spectropho-
1
tometer (Tokyo, Japan) using KBr dicks. H NMR spectra
3-(5-(5-Nitrofuran-2-yl)-1,3,4-thiadiazol-2-ylamino)
propan-1-ol (7)
were recorded on a Varian unity 500 or 400 spectrometer
and chemical shifts (δ) are reported in parts per million
(ppm) relative to tetramethylsilane (TMS) as an internal
standard. e mass spectra were run on an Agilent 6410
spectrometer or a Finigan TSQ-70 spectrometer (Finnigan
, San Jose, CA, USA) at 70 eV. Elemental analyses were
carried out on CHN-O rapid elemental analyzer (GmbH,
Hanau, Germany) for C, H and N, and the results are within
0.4% of the theoretical values. Merck silica gel 60 F254
plates were used for analytical thin layer chromatography.
Compounds 3 and 4 have been described in the literature19.
e resulting product was purified using a short silica
gel column eluting with CH2Cl2 followed by CH2Cl2
containing 2% methanol. e compound was obtained as
a yellow solid (yield: 60%). mp 174–175°C; IR (KBr, cm−1):
1
3,386, 3,242, 1,542, 1,355. H NMR (400 MHz, DMSO-d6)
δ: 8.42 (br s, 1H, NH), 7.85 (d, 1H, J = 3.6 Hz, furan), 7.34
(d, 1H, J = 3.6 Hz, furan), 4.56 (br s, 1H, -OH), 3.50 (t, 2H,
J = 6.0 Hz, CH2O), 3.41 (t, 2H, J = 6.0 Hz, NCH2), 1.80 (m,
2H, J = 6.0 Hz, -CH2-). MS (ESI): 270.8 [M + H+]. Anal.
Calcd for C9H10N4O4S: C, 40.00; H, 3.73; N, 20.73. Found:
C, 40.34; H, 3.73; N, 20.99.
General procedure for the synthesis of
compounds 5-42
N-(2-Methoxyethyl)-5-(5-nitrofuran-2-yl)-1,3,
4-thiadiazol-2-amine (8)
A solution of 2-chloro-1,3,4-thiadiazole 2 (1.0 mmol) and
appropriate primary amine (1.2 mmol) in absolute etha-
nol (7 mL) was refluxed until the reaction was completed
(4–48 h). en, the solvent was evaporated under reduced
pressure and the residue was purified using silica gel col-
umn chromatography eluting with appropriate solvent.
e resulting product was purified using a short silica gel
column eluting with CH3COOC2H5. e compound was
obtained as a yellow solid (yield: 74%). mp 172–173°C;
1
IR (KBr, cm−1): 3,234, 1,562, 1,352. H NMR (400 MHz,
CDCl3) δ: 7.43 (d, 1H, J = 5.0 Hz, furan), 7.17 (d, 1H, J = 5.0
Hz, furan), 5.82 (br s, 1H, NH), 3.65 (br s, 4H, CH2-CH2),
3.42 (s, 3H, CH3). MS (ESI): 270.8 [M + H+]. Anal. Calcd for
C9H10N4O4S: C, 40.00; H, 3.73; N, 20.73. Found: C, 39.76;
H, 3.61; N, 20.65.
N-Cyclopropyl-5-(5-nitrofuran-2-yl)-1,3,
4-thiadiazol-2-amine (5)
e resulting product was purified using a short
silica gel column eluting with CHCl3 containing 1%
Figure 1. General structures of previously described compounds (I) bearing a cyclic amine and newly designed compounds (II) containing
acyclic amine at the C-2 position of thiadiazole ring.
Journal of Enzyme Inhibition and Medicinal Chemistry

1,3,4-Thiadiazole-2-amines as antileishmanial agents 845
s, 2H, CH2), 2.66 (br s , 2H, -CH2-), 2.46 (t, 4H, J = 7.0 Hz, 2×
CH2), 1.84 (br s, 2H, CH2), 1.47-1.49 (m, 4H, 2× CH2), 1.32-
1.36 (m, 4H, 2× CH2), 0.93 (t, 6H, J = 7.0 Hz, 2× CH3). MS
(ESI): 381.9 [M + H+]. Anal. Calcd for C17H27N5O3S: C, 53.52;
H, 7.13; N, 18.36. Found: C, 53.39; H, 7.00; N, 18.05.
N-(2,2-Dimethoxyethyl)-5-(5-nitrofuran-2-yl)-1,
3,4-thiadiazol-2-amine (9)
e resulting product was purified using a short silica gel
column eluting with CHCl3 containing 0.5% methanol
and then washed with diethyl ether. e compound was
obtained as a yellow solid (yield: 68%). mp 117–119°C.
1
IR (KBr, cm−1): 3,279, 1,561, 1,355. H NMR (400 MHz,
N-(5-(Diethylamino)pentan-2-yl)-5-(5-nitrofuran-2-yl)-1,
3,4-thiadiazol-2-amine (14)
CDCl3) δ: 7.44 (d, 1H, J = 5.0 Hz, furan), 7.18 (d, 1H, J = 5.0
Hz, furan), 5.82 (br s, NH), 4.61 (t, 1H, J = 6.0 Hz, CH), 3.61
(d, 2H, J = 6.0 Hz, CH2), 3.46 (s, 6H, 2× CH3O). MS (ESI):
322.7 [M + Na+]. Anal. Calcd for C10H12N4O5S: C, 40.00; H,
4.03; N, 18.66. Found: C, 39.86; H, 4.26; N, 18.67.
e resulting product was purified using a short silica gel
column eluting with CH2Cl2 followed by CH2Cl2 contain-
ing 2% ethanol. e compound was obtained as a yellow
solid (yield: 15%). mp >300°C. IR (KBr, cm−1): 3,245, 1,539,
1
1,352. H NMR (500 MHz, DMSO-d6) δ: 8.53 (br s, 1H,
N-(2-(Diethylamino)ethyl)-5-(5-nitrofuran-2-yl)-1,
3,4-thiadiazol-2-amine (10)
NH), 7.84 (d, 1H, J = 3.2 Hz, furan), 7.32 (d, 1H, J = 3.2 Hz,
furan), 3.87 (br s, 1H, CH), 3.02 (br s, 6H, 3× CH2N), 1.69
(br s, 2H,CH2), 1.60 (br s, 2H, CH2), 1.22 (d, 3H, J = 6.0 Hz,
CH3), 1.16 (br s, 6H, 2× CH3). MS (ESI): 353.9 [M + H+].
Anal. Calcd for C15H23N5O3S: C, 50.97; H, 6.56; N, 19.81.
Found: C, 50.74; H, 6.69; N, 20.06.
e resulting product was purified using a short silica gel
column eluting with CHCl3. e compound was obtained
as a yellow solid (yield: 70%). mp >300°C. IR (KBr, cm−1):
3,320, 1,538, 1,353. 1H NMR (400 MHz, CDCl3) δ: 7.43 (d,
1H, J = 4.5 Hz, furan), 7.15 (d, 1H, J = 4.5 Hz, furan), 3.49 (t,
2H, J = 7.0 Hz, -CH2-), 2.81 (t, 2H, J = 7.0 Hz, -CH2-), 2.65
(q, 4H, J = 7.0 Hz, 2× CH2), 1.08 (t, 6H, J = 7.0 Hz, 2× CH3).
MS (ESI): 311.8 [M + H+]. Anal. Calcd for C12H17N5O3S: C,
46.29; H, 5.50; N, 22.49. Found: C, 46.63; H, 5.33; N, 22.31.
2-(5-(5-Nitrofuran-2-yl)-1,3,4-thiadiazol-2-ylamino)
butan-1-ol (15)
e resulting product was purified using a short silica gel
column eluting with CHCl3 containing 5% methanol. e
compound was obtained as a yellow solid (yield: 44%).
N-(3-(Diethylamino)propyl)-5-(5-nitrofuran-2-yl)-1,
3,4-thiadiazol-2-amine (11)
1
mp >300°C. IR (KBr, cm−1): 3,445, 3,224, 1,581, 1,349. H
e resulting product was purified using a short silica gel
column eluting with CHCl3. e compound was obtained
as a yellow solid (yield: 66%). mp >300°C. IR (KBr, cm−1):
3,153, 1,536, 1,354. 1H NMR (500 MHz, CDCl3) δ: 7.43 (d,
1H, J = 3.25 Hz, furan), 7.15 (d, 1H, J = 3.25 Hz, furan),
3.58 (br s, 2H, CH2), 2.71 (brs , 2H, CH2), 2.62 (q, 4H, J =
6.75 Hz, 2×CH2), 1.88 (br s, 2H, CH2), 1.12 (t, 6H, J = 6.75
Hz, 2× CH3). MS (ESI): 325.8 [M + H+]. Anal. Calcd for
C13H19N5O3S: C, 47.99; H, 5.89; N, 21.52. Found: C, 48.09;
H, 5.73; N, 21.51.
NMR (500 MHz, DMSO-d6) δ: 8.29 (d, 1H, J = 7.7 Hz,
NH), 7.84 (d, 1H, J = 3.5 Hz, furan), 7.32 (d, 1H, J = 3.5 Hz,
furan), 4.85 (br s, 1H, CH), 3.68 (br s, 1H, OH), 3.51 (br s ,
2H,CH2O), 1.67-1.71 (m, 1H,CH2), 1.51-1.55 (m, 1H,CH2),
0.92 (t, 3H, J = 7.0 Hz, CH3). MS (ESI): 284.8 [M+ H+]. Anal.
Calcd for C10H12N4O4S: C, 42.25; H, 4.25; N, 19.71. Found:
C, 42.43; H, 4.25; N, 19.49.
2-(2-(5-(5-Nitrofuran-2-yl)-1,3,4-thiadiazol-2-ylamino)
ethoxy) ethanol (16)
e resulting product was purified using a short silica gel
column eluting with CHCl3 containing 5% methanol. e
compound was obtained as a yellow solid (yield: 54%).
N-(2-(Diisopropylamino)ethyl)-5-(5-nitrofuran-2-yl)-1,
3,4-thiadiazol-2-amine (12)
e resulting product was purified using a short silica
gel column eluting with CH3COOC2H5. e compound
was obtained as a yellow solid (yield: 62%). mp >270°C.
1
mp >300°C. IR (KBr, cm−1): 3,445, 3,210, 1,579, 1,346. H
NMR (400 MHz, DMSO-d6) δ: 7.43 (d, 1H, J = 5.0 Hz,
furan), 7.21 (d, 1H, J = 5.0 Hz, furan), 3.79 (t, 4H, J = 4.8 Hz,
CH2O), 3.66 (t, 4H, J = 4.8 Hz, CH2), 2.95 (br s, 1H, OH).
MS (ESI): 300.8 [M + H+]. Anal. Calcd for C10H12N4O5S: C,
40.00; H, 4.03; N, 18.66. Found: C, 40.22; H, 3.82; N, 18.97.
1
IR (KBr, cm−1): 3,243, 1,535, 1,351. H NMR (500 MHz,
CDCl3) δ: 7.44 (d, 1H, J = 3.3 Hz, furan), 7.17 (d, 1H, J = 3.3
Hz, furan), 3.41 (br s, 2H, CH2), 3.10 (br s , 2H, CH2), 2.80
(br s , 2H, 2× CH), 1.07 (br s, 12H, 4× CH3). MS (ESI): 339.8
[M + H+]. Anal. Calcd for C14H21N5O3S: C, 49.54; H, 6.24; N,
20.63. Found: C, 49.83; H, 6.15; N, 20.40.
N-Allyl-5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-amine (17)
e resulting product was purified using a short silica gel
column eluting with CHCl3 followed by CH3COOC2H5.
e compound was obtained as a yellow solid (yield:
81%). mp 168–169°C. IR (KBr, cm−1): 3,207, 1,640, 1,535,
N-(3-(Dibutylamino)propyl)-5-(5-nitrofuran-2-yl)-1,
3,4-thiadiazol-2-amine (13)
1
e resulting product was purified using a short silica gel
column eluting with CH2Cl2. e compound was obtained
as a yellow solid (yield: 73%). mp >300°C. IR (KBr, cm−1):
1,353. H NMR (400 MHz, CDCl3) δ: 7.45 (d, 1H, J = 3.2
Hz, furan), 7.15 (d, 1H, J = 3.2 Hz, furan), 5.90-6.01 (m,
1H, CH), 5.38-5.27 (m, 2H, CH2), 4.06 (br s, 2H, CH2). MS
(ESI): 252.8 [M + H+]. Anal. Calcd for C9H8N4O3S: C, 42.85;
H, 3.20; N, 22.21. Found: C, 43.03; H, 3.02; N, 22.44.
1
3,186, 1,582, 1,349. H NMR (500 MHz, CDCl3) δ: 7.44 (d,
1H, J = 3.6 Hz, furan), 7.15 (d, 1H, J = 3.6 Hz, furan), 3.58 (br
© 2013 Informa UK, Ltd.

846 A. Tahghighi et al.
(KBr, cm−1): 3,445, 1,579, 1,530, 1,498, 1,350. 1H NMR (400
MHz, CDCl3) δ: 7.42 (d, 1H, J = 4.5 Hz, furan), 7.40-7.37
(m, 5H, phenyl), 7.16 (d, 1H, J = 4.5 Hz, furan), 6.10 (br s,
1H, NH), 4.62 (s, 2H, CH2). MS (ESI): 302.8 [M + H+]. Anal.
Calcd for C13H10N4O3S: C, 51.65; H, 3.33; N, 18.53. Found:
C, 51.37; H, 3.44; N, 18.24.
5-(5-Nitrofuran-2-yl)-N-(prop-2-ynyl)-1,
3,4-thiadiazol-2-amine (18)
e resulting product was purified using a short silica gel
column eluting with CHCl3 followed by CH3COOC2H5.
e compound was obtained as a yellow solid (yield:
75%). mp 204–206°C. IR (KBr, cm−1): 3,299, 3,207, 2,121,
1,566, 1,345. 1H NMR (400 MHz, CDCl3) δ: 7.45 (d, 1H, J =
4.0 Hz, furan), 7.16 (d, 1H, J = 4.0 Hz, furan), 4.27 (s, 2H,
CH2), 2.35 (s, 1H, ≡ CH). MS (ESI): 250.8 [M + H+]. Anal.
Calcd for C9H6N4O3S: C, 43.20; H, 2.42; N, 22.39. Found: C,
43.53; H, 2.54; N, 22.12.
N-(4-Methoxybenzyl)-5-(5-nitrofuran-2-yl)-1,
3,4-thiadiazol-2-amine (23)
e resulting product was purified using a short silica
gel column eluting with CHCl3. e compound was
obtained as a yellow solid (yield: 74%). mp 168–169°C. IR
(KBr, cm−1): 3,209, 1,610, 1,560, 1,494, 1,352. 1H NMR (400
MHz, CDCl3) δ: 7.43 (d, 1H, J = 5.0 Hz, furan), 7.32 (d, 2H,
J = 9.0 Hz, phenyl), 7.16 (d, 1H, J = 5.0 Hz, furan), 6.92 (d,
2H, J = 9.0 Hz, phenyl), 6.10 (br s, 1H, NH), 4.54 (s, 2H,
CH2), 3.82 (s, 3H, CH3). MS (ESI): 332.8 [M + H+]. Anal.
Calcd C14H12N4O4S: C, 50.60; H, 3.64; N, 16.86. Found: C,
50.89; H, 3.69; N, 16.95.
5-(5-Nitrofuran-2-yl)-N-(2-(pyrrolidin-1-yl)ethyl)-1,
3,4-thiadiazol-2-amine (19)
e resulting product was purified using a short silica
gel column eluting with CH3COOC2H5 followed by
CH3COOC2H5 containing 2% methanol. e compound
was obtained as a yellow solid (yield: 64%). mp 180–
181°C. IR (KBr, cm−1): 3,179, 1,580, 1,351. H NMR (500
1
MHz, DMSO-d6) δ: 8.53 (br s, 1H, NH), 7.84 (d, 1H, J = 4.0
Hz, furan), 7.34 (d, 1H, J = 4.0 Hz, furan), 3.61 (br s, 2H,
CH2), 2.96 (br s, 2H, CH2), 2.83 (br s, 4H, 2× CH2), 1.79 (br
s, 4H, 2× CH2). MS (ESI): 309.8 [M + H+]. Anal. Calcd for
C12H15N5O3S: C, 46.59; H, 4.89; N, 22.64. Found: C, 46.36;
H, 4.95; N, 22.97.
5-(5-Nitrofuran-2-yl)-N-((pyridin-2-yl)methyl)-1,3,
4-thiadiazol-2-amine (24)
e resulting product was purified using a short silica gel
column eluting with CH3COOC2H5. e compound was
obtained as a yellow solid (yield: 53%). mp 192–194°C. IR
(KBr, cm−1): 3,224, 1,581, 1,537, 1,497, 1,355. 1H NMR (400
MHz, CDCl3) δ: 8.60 (d, 1H, J = 7.6 Hz, pyridine), 7.74 (m,
1H, pyridine), 7.44 (d, 1H, J = 4.5 Hz, furan), 7.36 (d, 1H, J
= 7.6 Hz, pyridine), 7.18 (d, 1H, J = 4.5 Hz, furan), 7.11 (m,
1H, pyridine), 4.80 (s, 2H, CH2). MS (ESI): 303.8 [M + H+].
Anal. Calcd for C12H9N5O3S: C, 47.52; H, 2.99; N, 23.09.
Found: C, 47.71; H, 3.08; N, 23.38.
N-((Tetrahydrofuran-2-yl)methyl)-5-(5-nitrofuran-2-yl)-1,
3,4-thiadiazol-2-amine (20)
e resulting product was purified using a short silica gel
column eluting with CHCl3 followed by CHCl3 containing
3% methanol. e compound was obtained as a yellow
solid (yield: 59%). mp 179–180°C. IR (KBr, cm−1): 3,182,
1
1,577, 1,349. H NMR (500 MHz, CDCl3) δ: 7.44 (d, 1H, J
= 3.0 Hz, furan), 7.17 (d, 1H, J = 3.0 Hz, furan), 5.99 (s,
1H), 4.20-4.18 (m, 1H, CH2), 3.95-3.92 (m, 1H, CH2), 3.85-
3.82 (m, 1H, CH), 3.73-3.71 (m, 1H, CH2), 3.42-3.38 (m,
1H, CH2), 2.11-2.07 (m, 1H, CH2), 1.99-1.97 (m, 2H, CH2),
1.69-1.65 (m, 1H, CH2). MS (ESI): 296.8 [M + H+]. Anal.
Calcd for C11H12N4O4S: C, 44.59; H, 4.08; N, 18.91. Found:
C, 44.59; H, 3.87; N, 19.21.
5-(5-Nitrofuran-2-yl)-N-((pyridin-3-yl)methyl)-1,
3,4-thiadiazol-2-amine (25)
e resulting product was purified using a short silica
gel column eluting with CH2Cl2 followed by CH2Cl2
containing 0.25% methanol followed by CH3COOC2H5.
e compound was obtained as a yellow solid (yield:
61%). mp 190–192°C. IR (KBr, cm−1): 3,187, 1,554, 1,527,
1
1,491, 1,369. H NMR (400 MHz, CDCl3) δ: 8.67 (s, 1H,
N-((Furan-2-yl)methyl)-5-(5-nitrofuran-2-yl)-1,
3,4-thiadiazol-2-amine (21)
pyridine), 8.55 (br s, 1H, pyridine), 8.35 (br s, 1H, NH),
7.80 (d, 1H, J = 8.0 Hz, pyridine), 7.46 (d, 1H, J = 4.0 Hz,
furan), 7.33-7.29 (m, 1H, pyridine), 7.14 (d, 1H, J = 4.0 Hz,
furan), 4.67 (s, 2H, CH2). MS (ESI): 303.8 [M + H+]. Anal.
Calcd for C12H9N5O3S: C, 47.52; H, 2.99; N, 23.09. Found:
C, 47.69; H, 2.75; N, 23.21.
e resulting product was purified using a short silica gel
column eluting with CHCl3 containing 1% methanol. e
compound was obtained as a yellow solid (yield: 36%).
mp 166–168°C. IR (KBr, cm−1): 3,161, 1,566, 1,351. 1H
NMR (400 MHz, DMSO-d6) δ: 8.11 (s, 1H, NH), 7.30-7.28
(m, 2H, J = 4.0 Hz, furan), 6.94 (d, 1H, J = 4.0 Hz, furan),
6.20-6.16 (m, 2H, furan), 4.43 (br s, 2H,CH24). MS (ESI):
292.8 [M + H+]. Anal. Calcd for C11H8N4O4S: C, 45.20; H,
2.76; N, 19.17. Found: C, 45.46; H, 2.91; N, 18.85.
N-Methyl-5-(5-nitrothiophen-2-yl)-1,3,
4-thiadiazol-2-amine (26)
e resulting product was purified using a short silica gel
column eluting with CHCl3 followed by CH3COOC2H5.
e compound was obtained as a yellow solid (yield:
35%). mp 260–262°C; IR (KBr, cm−1): 3,167, 1,569, 1,342.
1H NMR (400 MHz, DMSO-d6) δ: 8.33 (br s, 1H, NH), 8.14
(d, 1H, J = 5.5 Hz, thiophene), 7.54 (d, 1H, J = 5.5 Hz, thio-
phene), 2.96 (s, 3H, CH3). MS (ESI): 242.8 [M + H+]. Anal.
N-Benzyl-5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-amine (22)
e resulting product was purified using a short silica
gel column eluting with CHCl3. e compound was
obtained as a yellow solid (yield: 69%). mp 189–190°C. IR
Journal of Enzyme Inhibition and Medicinal Chemistry

1,3,4-Thiadiazole-2-amines as antileishmanial agents 847
Calcd for C7H6N4O2S2: C, 34.70; H, 2.50; N, 23.12. Found:
C, 34.41; H, 2.86; N, 23.01.
N-(2,2-Dimethoxyethyl)-5-(5-nitrothiophen-2-yl)-1,
3,4-thiadiazol-2-amine (31)
e resulting product was purified using a short silica gel
column eluting with CHCl3 followed by CH3COOC2H5.
e compound was obtained as a yellow solid (yield:
61%). mp 173–174°C; IR (KBr, cm−1): 3,243, 1,567, 1,342.
1H NMR (400 MHz, CDCl3) δ: 7.86 (d, 1H, J = 4.5 Hz,
thiophene), 7.17 (d, 1H, J = 4.5 Hz, thiophene), 4.60 (t,
1H, J = 6.5 Hz, CH), 3.60 (d, 2H, J = 6.5 Hz, CH2), 3.46 (s,
6H, 2× CH3). MS (ESI): 338.7 [M + Na+]. Anal. Calcd for
C10H12N4O4S2: C, 37.97; H, 3.82; N, 17.71. Found: C, 37.62;
H, 4.12: N, 17.91.
N-Ethyl-5-(5-nitrothiophen-2-yl)-1,3,
4-thiadiazol-2-amine (27)
e resulting product was purified using a short silica gel
column eluting with CH2Cl2 followed by CH3COOC2H5.
e compound was obtained as a yellow solid (yield:
69%). mp 201–203°C; IR (KBr, cm−1): 3,185, 1,578, 1,342.
1H NMR (400 MHz, CDCl3, DMSO-d6) δ: 7.88 (d, 1H, J =
4.8 Hz, thiophene), 7.52 (br s, 1H, NH), 7.15 (d, 1H, J = 4.8
Hz, thiophene), 3.45 (q, 2H, J = 6.4 Hz, CH2), 1.32 (t, 3H,
J = 6.4 Hz, CH3). MS (ESI): 256.8 [M + H+]. Anal. Calcd for
C8H8N4O2S2: C, 37.49; H, 3.15; N, 21.86. Found: C, 37.69;
H, 3.31; N, 22.01.
N-(3-Methoxypropyl)-5-(5-nitrothiophen-2-yl)-1,
3,4-thiadiazol-2-amine (32)
e resulting product was purified using a short silica gel
column eluting with CHCl3 containing 0.5% methanol
and then washed with diethylether. e compound was
obtained as a yellow solid (yield: 73%). mp 146–147°C.
N-Cyclopropyl-5-(5-nitrothiophen-2-yl)-1,3,
4-thiadiazol-2-amine (28)
e resulting product was purified using a short silica gel
column eluting with CHCl3 containing 1% methanol. e
compound was obtained as a yellow solid (yield: 73%).
mp 247–248°C. IR (KBr, cm−1): 3,181, 1,564, 1,344. 1HNMR
(500 MHz, DMSO-d6) δ: 8.83 (br s, 1H, NH), 8.13 (d, 1H, J
= 4.0 Hz, thiophene), 7.57 (d, 1H, J = 4.0 Hz, thiophene),
2.74-2.72 (m, 1H, CH), 0.82-0.80 (m, 2H, c-Pr), 0.63-0.61
(m, 2H,c-Pr). MS (ESI): 268.8 [M + H+]. Anal. Calcd for
C9H8N4O2S2: C, 40.29; H, 3.01; N, 20.88 Found: C, 40.22;
H, 2.61: N, 20.66.
1
IR (KBr, cm−1): 3,188, 1,593, 1,342. H NMR (400 MHz,
CDCl3) δ: 7.86 (d, 1H, J = 5.0 Hz, thiophene), 7.16 (d, 1H,
J = 5.0 Hz, thiophene), 6.39 (br s, 1H, NH), 3.59-3.54 (m,
4H, 2× CH2), 3.38 (s, 3H, CH3), 1.99-1.97 (m, 2H, -CH2-).
MS (m/e, %): 300 (M+, 47), 285 (100), 255 (27), 241 (85),
229 (33), 212 (12), 195 (10), 169 (14), 154 (8), 126 (13),
116 (9), 100 (7), 69 (9), 57 (7), 45 (12). Anal. Calcd for
C10H12N4O3S2: C, 39.99; H, 4.03; N, 18.65. Found: C, 40.18;
H, 4.28; N, 18.96.
3-(5-(5-Nitrothiophen-2-yl)-1,3,
4-thiadiazol-2-ylamino)propan-1-ol (29)
N-(2-(Diethylamino)ethyl)-5-(5-nitrothiophen-2-yl)-1,
3,4-thiadiazol-2-amine (33)
e resulting product was purified using a short silica gel
column eluting with CHCl3 followed by CH3COOC2H5.
e compound was obtained as a yellow solid (yield:
61%). mp >300°C. IR (KBr, cm−1): 3,377, 1,541, 1,344. H
NMR (400 MHz, CDCl3) δ: 7.87 (d, 1H, J = 4.0 Hz, thio-
phene), 7.15 (d, 1H, J = 4.0 Hz, thiophene), 3.55-3.52 (m,
2H, CH2), 2.86-2.82 (m, 2H, CH2), 2.68 (q, 4H, J = 6.8 Hz,
CH2NCH2), 1.10 (t, 6H, J = 6.8 Hz, 2× CH3). MS (ESI): 327.8
[M + H+]. Anal. Calcd for C12H17N5O2S2: C, 44.02; H, 5.23;
N, 21.39. Found: C, 44.26; H, 5.38; N, 21.58.
e resulting product was purified using a short silica
gel column eluting with CH2Cl2 followed by CH2Cl2 con-
taining 2% methanol. e compound was obtained as a
yellow solid (yield: 62%). mp 170–172°C; IR (KBr, cm−1):
1
1
3,344, 3,192, 1,550, 1,339. H NMR (400 MHz, DMSO-d6)
δ: 8.38 (br s, 1H, NH), 8.12 (d, 1H, J = 4.0 Hz, thiophene),
7.51 (d, 1H, J = 4.0 Hz, thiophene), 4.55 (br s, 1H, OH),
3.49 (t, 2H, J = 6.0 Hz, CH2O), 3.40 (t, 2H, J = 6.0 Hz, CH2N),
1.75-1.73 (m, 2H,-CH2-). MS (m/e, %): 286 (M+, 63), 255
(21), 242 (100), 228 (23), 212 (13), 195 (10), 169 (34), 154
(10), 132 (10), 126 (19), 99 (16), 69 (13). Anal. Calcd for
C9H10N4O3S2: C, 37.75; H, 3.52; N, 19.57. Found: C, 37.39;
H, 3.32; N, 19.32.
N-(3-(Dimethylamino)propyl)-5-(5-nitrothiophen-2-yl)-1,
3,4-thiadiazol-2-amine (34)
e resulting product was purified using a short silica
gel column eluting with CHCl3 followed by CHCl3
containing 3% methanol. e compound was obtained
as a yellow solid (yield: 60%). mp >300°C. IR (KBr, cm−1):
N-(2-Methoxyethyl)-5-(5-nitrothiophen-2-yl)-1,3,
4-thiadiazol-2-amine (30)
e resulting product was purified using a short silica gel
column eluting with CH3COOC2H5 and again crystallized
with n-Hexan. e compound was obtained as a yellow
solid (yield: 70%). mp 182–183°C; IR (KBr, cm−1): 3,281,
1
3,178, 1,587, 1,344. H NMR (400 MHz, DMSO-d6) δ:
8.45 (br s, 1H, NH), 8.13 (d, 1H, J = 4.0 Hz, thiophene),
7.52 (d, 1H, J = 4.0 Hz, thiophene), 3.37 (t, 2H, J = 6.4
Hz, CH2), 2.29 (t, 2H, J = 6.4 Hz, CH2), 2.14 (s, 6H, 2×
CH3), 1.74-1.71 (m, 2H, -CH2-). MS (ESI): 313.8 [M+ H+].
Anal. Calcd for C11H15N5O2S2: C, 42.16; H, 4.82; N, 22.35.
Found: C, 42.47; H, 4.64; N, 22.08.
1
1,543, 1,323. H NMR (400 MHz, CDCl3) δ: 7.86 (d, 1H, J
= 5.0 Hz, thiophene), 7.16 (d, 1H, J = 5.0 Hz, thiophene),
3.65 (br s, 4H, 2× CH2), 3.42 (s, 3H, CH3). MS (ESI): 286.7
[M + H+]. Anal. Calcd for C9H10N4O3S2: C, 37.75; H, 3.52; N,
19.57. Found: C, 37.89; H, 3.76; N, 19.29.
© 2013 Informa UK, Ltd.

848 A. Tahghighi et al.
1
cm−1): 3,356, 1,531, 1,341. H NMR (400 MHz, CDCl3) δ:
2-(2-(5-(5-Nitrothiophen-2-yl)-1,
3,4-thiadiazol-2-ylamino)ethoxy) ethanol (35)
7.86 (d, 1H, J = 4.5 Hz, thiophene), 7.40-7.37 (m, 5H, phe-
nyl), 7.19 (d, 1H, J = 4.5 Hz, thiophene), 4.64 (s, 2H, CH2).
MS (ESI): 318.7 [M+ H+]. Anal. Calcd for C13H10N4O2S2: C,
49.04; H, 3.17; N, 17.60. Found: C, 48.75; H, 3.40: N, 17.86.
e resulting product was purified using a short silica
gel column eluting with CH3COOC2H followed by
CH3COOC2H5 containing 5% methanol. e compound
was obtained as a yellow solid (yield: 62%). mp 159–
160°C. IR (KBr, cm−1): 3,386, 3,185, 1,550, 1,344. H NMR
1
N-(4-Methoxybenzyl)-5-(5-nitrothiophen-2-yl)-1,
(400 MHz, DMSO-d6) δ: 8.50 (br s, 1H, NH), 8.12 (d, 1H, J
= 4.5 Hz, thiophene), 7.52 (d, 1H, J = 4.5 Hz, thiophene),
4.59 (br s, 1H, OH), 3.61-3.46 (m, 8H, 4× CH2). MS (ESI):
316.8 [M+ H+]. Anal. Calcd for C10H12N4O4S2: C, 37.97; H,
3.82; N, 17.71. Found: C, 38. 21; H, 4.00: N, 18.01.
3,4-thiadiazol-2-amine (40)
e resulting product was purified using a short silica gel
column eluting with CHCl3 followed by CH3COOC2H5.
e compound was obtained as a yellow solid (yield:
74%). mp 168–169°C. IR (KBr, cm−1): 3,171, 1,551, 1,510,
1,461, 1,339. 1H NMR (400 MHz, CDCl3) δ: 7.84 (d, 1H, J =
6.0 Hz, thiophene), 7.31 (d, 2H, J = 9.0 Hz, phenyl), 7.15 (d,
1H, J = 6.0 Hz, thiophene), 6.91(d, 2H, J = 9.0 Hz, phenyl),
6.10 (br s, 1H, NH), 4.26 (s, 2H, CH2), 3.82 (s, 3H, CH3).
MS (ESI): 348.7 [M+ H+]. Anal. Calcd for C14H12N4O3S2: C,
48.26; H, 3.47; N, 16.08. Found: C, 47.97; H, 3.47: N, 16.47.
N-Allyl-5-(5-nitrothiophen-2-yl)-1,3,4-thiadiazol-2-amine (36)
e resulting product was purified using a short silica
gel column eluting with CH2Cl2. e compound was
obtained as a yellow solid (yield: 70%). mp 173–174°C. IR
(KBr, cm−1): 3,441, 1,535, 1,496, 1,353. 1H NMR (400 MHz,
CDCl3) δ: 7.86 (d, 1H, J = 4.4 Hz, thiophene), 7.18 (d, 1H, J
= 4.4 Hz, thiophene), 5.98-5.88 (m, 1H, CH); 5.40-5.29 (m,
2H, CH2); 4.04 (br s, 2H, CH2). MS (ESI): 268.8 [M + H+].
Anal. Calcd for C9H8N4O2S2: C, 40.29; H, 3.01; N, 20.88.
Found: C, 40.58; H, 3.12: N, 20.95.
5-(5-Nitrothiophen-2-yl)-N-((pyridin-2-yl)methyl)-1,
3,4-thiadiazol-2-amine (41)
e resulting product was purified using a short silica gel
column eluting with CH3COOC2H5. e compound was
obtained as a yellow solid (yield: 62%). mp 211–213°C.
IR (KBr, cm−1): 3,251, 1,544, 1,339. H NMR (400 MHz,
1
5-(5-Nitrothiophen-2-yl)-N-(prop-2-ynyl)-1,
3,4-thiadiazol-2-amine (37)
CDCl3) δ: 8.95 (br s, 1H, NH), 8.55 (m, 1H,, pyridine),
8.13 (d, 1H, J = 4.1 Hz, thiophene), 7.79 (t, 1H, J = 7.35 Hz,
pyridine), 7.54 (d, 1H, J = 4.1 Hz, thiophene), 7.41 (d, 1H,
J = 7.35 Hz, pyridine), 7.30 (t, 1H, J = 7.35 Hz, pyridine),
4.68 (s, 2H, CH2). MS (ESI): 319.7 [M+ H+]. Anal. Calcd for
C12H9N5O2S2: C, 45.13; H, 2.84; N, 21.93. Found: C, 44.85;
H, 3.05: N, 21.76.
e resulting product was purified using a short silica
gel column eluting with CH2Cl2 followed by CH2Cl2
CH2Cl2 containing 3% methanol. e compound was
obtained as a yellow solid (yield: 45%). mp 218–219°C.
IR (KBr, cm−1): 3,292, 3,235, 2,116, 1,560, 1,329. 1H NMR
(400 MHz, CDCl3) δ: 7.88 (d, 1H, J = 4.0 Hz, thiophene),
7.01 (d, 1H, J = 4.0 Hz, thiophene), 4.28 (s, 2H,CH2), 2.17
(s, 1H, ≡ CH). MS (ESI): 266.7 [M+ H+]. Anal. Calcd for
C9H6N4O2S2: C, 40.59; H, 2.27; N, 21.04. Found: C, 40.21;
H, 2.52: N, 20.84.
5-(5-Nitrothiophen-2-yl)-N-((pyridin-3-yl)methyl)-1,
3,4-thiadiazol-2-amine (42)
e resulting product was purified using a short silica gel
column eluting with CH2Cl2 followed by CH3COOC2H5
containing 5% methanol. e compound was obtained
as a yellow solid (yield: 68%). mp 232–235°C. IR (KBr,
cm−1): 3,178, 1,557, 1,500, 1,464, 1,347. 1H NMR (400
MHz, CDCl3) δ: 8.66 (br s, 1H, pyridine), 8.54 (br s, 1H,
pyridine), 8.35 (br s, 1H, NH), 7.88 (d, 1H, J = 4.0 Hz,
thiophene), 7.79 (d, 1H, J = 8.0 Hz, ,pyridine), 7.33-7.29
(m, 1H, pyridine), 7.16 (d, 1H, J = 4.0 Hz, thiophene),
4.65 (s, 2H, CH2). MS (ESI): 319.7 [M+ H+]. Anal. Calcd for
C12H9N5O2S2: C, 45.13; H, 2.84; N, 21.93. Found: C, 44.78;
H, 2.58: N, 21.72.
N-((Tetrahydrofuran-2-yl)methyl)-5-(5-nitrothiophen-2-yl)-
1,3,4-thiadiazol-2-amine (38)
e resulting product was purified using a short silica
gel column eluting with CHCl3 followed by CHCl3 con-
taining 2% methanol. e compound was obtained as a
yellow solid (yield: 72%). mp 181–182°C. IR (KBr, cm−1):
3,196, 1,556, 1,342. 1H NMR (400 MHz, CDCl3) δ: 7.86 (d,
1H, J = 3.0 Hz, thiophene), 7.16 (d, 1H, J = 3.0 Hz, thio-
phene), 6.25 (s, 1H, NH), 4.21-4.18 (m, 1H,CH2), 3.92-
3.88 (m, 1H, CH2), 3.83-3.80 (m, 1H, -CH-), 3.73-3.69
(m, 1H, CH2), 3.41-3.37(m, 1H, CH2), 2.10-2.07 (m, 1H,
CH2), 1.98-1.96 (m, 2H, CH2-), 1.69-1.65 (m, 1H,CH2).
MS (ESI): 312.8 [M+ H+]. Anal. Calcd for C11H12N4O3S2:
C, 42.30; H, 3.87; N, 17.94. Found: C, 42.59; H, 4.07: N,
17.64.
Antileishmanial activity against L. major
promastigotes
e antileishmanial screening of compounds 3-42
was performed using MTT (3-(4′,5′-dimethylthiazol-
2′-yl)-2,5-diphenyltetrazolium bromide) assay20. e
promastigote form of parasite (vaccine strain MRHO/
IR/75/ER, obtained from Pasteur institute, Tehran,
Iran) was grown in blood agar cultures (Difco, Detroit,
MI) at 25°C. e growth curve of the L. major strain
was determined daily under light microscope and
N-Benzyl-5-(5-nitrothiophen-2-yl)-1,3,
4-thiadiazol-2-amine (39)
e resulting product was purified using a short silica gel
column eluting with CHCl3. e compound was obtained
as a yellow solid (yield: 61%). mp 176–177°C. IR (KBr,
Journal of Enzyme Inhibition and Medicinal Chemistry

1,3,4-Thiadiazole-2-amines as antileishmanial agents 849
counting in a Neubauer’s chamber. en, parasites (2 ×
106/mL) in the logarithmic phase were incubated with
different concentrations (12.5, 25, 50, and 75 µg/mL)
of test compounds for 24 h at 25°C. After incubation,
the media was renewed with 100 μg/well of MTT (0.5
mg/mL) and plates were further incubated for 4 h at
37°C. en, the plates were centrifuged (2,000 rpm ×
5 min) and the obtained pellets were dissolved in 200
μL of DMSO. e samples were read using an ELISA
plate reader at a wavelength of 492 nm. Two or more
independent experiments in triplicate were performed
for each compound. e IC50 were calculated by linear
regression analysis, expressed in mean SD.
triplicate were performed for determination of toxicity
of each compound. e CC50 (cytotoxic concentration
for 50% inhibition) were calculated by linear regression
analysis.
results and discussion
Chemistry
e target compounds 3-42 were synthesized in good
yield by reaction of 2-chloro-1,3,4-thiadiazole 2 and
appropriate primary amines in refluxing absolute etha-
nol (Scheme 1). e intermediate 2-chloro-1,3,4-thiadi-
azole 2 was obtained from 5-nitrofurfurilidine diacetate
or 5-nitrothiophene-2-carboxaldehyde according to the
previously described methods15–18. All of the synthesized
Antileishmanial activity against L. major amastigotes
Selected compounds 6-15, 18, 21, 24, 25, 29, 32–34, 36,
and 38 were evaluated for their activity against amasti-
gote form of L. major in murine peritoneal macrophages.
Briefly, mouse peritoneal macrophages were plated in
RPMI 1640 supplemented with 10% of heat-inactivated
fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin
(Sigma, St Louis, MO, USA) and 100 mg/mL streptomy-
cin. Macrophages were placed on sterile glass cover slips
in 24-well plates (1 × 106/well). After 1 h, nonadherent
cells were removed by washing with RPMI, the stationary
phase promastigotes in RPMI were added (2 × 106 para-
sites/well, three parasites/macrophage) to macrophage
monolayer and the plates were kept at 37°C in a CO2
incubator for 2 h. Extracellular parasites were removed
by washing and then new media containing IC50 concen-
tration of the drug were added. Two sets of experiments
were carried out for each compound at 24 h. Following
these procedures, cells were fixed with methanol, stained
with Giemsa stain (Sigma) and the infectivity index was
determined by multiplying the percentage of macro-
phages that had at least one intracellular parasite by the
average number of intracellular parasites per infected
1
compounds were characterized by IR and H NMR and
mass spectral data.
Biological activity
For assessing the IC50 (50% inhibitory concentrations) of
compounds3-42againstpromastigoteformofL.major,MTT
assay was used20. Two or more independent experiments
in triplicate were performed for each compound. e IC50
were calculated by linear regression analysis, expressed
in mean values and listed in Tables 1 and 2. Meglumine
antimonate (Glucantime®) and fluconazole were used as
a reference drugs. e resulting data revealed that most
compounds exhibited good antipromastigote activity with
IC50 values <50 μM. In 5-nitrofuran series, compounds 9,
11, 12, and 15 showed better activity (IC50 values = 10–13
μM). While, 5-nitrothiophene derivatives 29 and 32 with
IC50 values of 3 μM were the most active compounds in
both series. Although, 5-nitrofurans bearing small alkyl
side chain (Me, Et, c-Pr) showed some activity (IC50 values
≈ 50 μM) while related 5-nitrothiophene analogs were less
active (IC50 values = 64–98.4 μM). In contrast, hydroxypropyl
analog of 5-nitrothiophene (compound 29) was sixfold
more potent than its 5-nitrofuran counterpart (compound
7). is finding indicates that different responses take place
due to O/S replacement in the scaffold. By comparing the
IC50 values of compounds containing simple alkyl side chain
(for example 3, 4, 5, 26, 27, 28) with compounds having
oxyalkyl and animoalkyl substituents (such as, 6-15 and
29–35), it is revealed that the average activity of oxyalkyl
and animoalkyl derivatives is more than that of compounds
containing simple alkyl side chain. us in overall, it could
be concluded that the insertion of oxygen or nitrogen on
alkyl side chain improves antileishmanial activity. e IC50
values of compounds containing bulky groups such as
macrophage (100 cells were examined/well) 21
.
Toxicity against macrophages
e toxicity of compounds 6-15, 18, 21, 24, 25, 29,
32–34, 36, and 38 was assessed against mouse peri-
toneal macrophages plated in 96-well plates at 2 ×
105 cells/well. After cell adherence, the medium was
removed and replaced by the media containing dif-
ferent concentrations of each compounds. e plates
were incubated for 24 h at 37°C in a humidified incuba-
tor with 5% CO2. Cell viability was determined by MTT
colorimetric assay22. Two independent experiments in
Scheme 1. Synthetic route to compounds 3-42. Reagents and conditions: a) NaNO2, HCl, Cu; b) RNH2, EtOH, reflux.
© 2013 Informa UK, Ltd.

850 A. Tahghighi et al.
Table 1. In vitro antileishmanial activity of furan derivatives 3–25 against promastigote form of Leishmania major.
Compound
3
4
R
Me
Et
Antipromastigote activity IC50 (µM)
54 0.17
Cytotoxicity CC50 (µM)a
SIb
—
—
—
50 0.8
—
5
6
7
8
c-Pr
(CH2)2OH
55.5 0.66
21 0.65
—
—
122.11
62.33
131.64
62.46
83.29
104.60
42.18
68.11
367.65
83.29
—
5.81
3.46
4.70
4.80
1.89
8.05
3.24
2.72
11.14
6.94
—
(CH2)3OH
18 0.2
(CH2)2OMe
CH2CH(OMe)2
(CH2)2N(Et)2
(CH2)3N(Et)2
(CH2)2N(i-Pr)2
(CH2)3N(n-Bu)2
CH(Me)(CH2)3N(Et)2
CH(Et)(CH2OH)
(CH2)2O(CH2)2OH
CH2CH=CH2
28 0.94
9
13 0.53
10
11
12
13
14
15
16
17
18
44 0.72
13 0.43
13 0.23
25 0.2
33 0.76
10 0.66
116 0.76
122 0.25
40 0.61
—
141.36
—
3.53
CH2C≡ CH
19
110 0.45
—
—
20
21
22
23
53 0.32
41 0.82
—
88.83
—
—
2.17
—
258 0.45
306 0.65
—
—
24
25
36 0.87
26 0.6
111.71
41.72
3.10
1.60
Glucantime
Fluconazole
68.44c
—
—
—
—
941.1 4.98
CC50, cytotoxic concentration for 50% inhibition; IC50, half-maximal inhibitory concentration.
aCytotoxicity was evaluated against mouse peritoneal macrophages. bSelectivity index (SI) CC50/IC50. c e IC50 of glucantime was in mM.
phenyl, 4-methoxyphenyl, furan-2-yl, tetrahydrofuran-2-yl,
pyrrolidinyl, demonstrated that the introduction of pyridine-
2-yl or 3-yl and furan-2-yl or tetrahydrofuran-2-yl was
well-tolerated and even in the case of pyridinyl, the related
activity was slightly increased (compounds 24 and 25 vs. 3).
Comparison of IC50 values of hydroxyethyl derivatives 6 and
15 revealed that α-branching can moderately improve the
activity. In aminoethyl derivatives, increasing the number
of carbons on distal nitrogen (conversion of diethyl to
diisopropyl or dibutyl) relatively improves the activity. In the
oxyalkyl and animoalkyl series, the length of aliphatic chain
between two heteroatoms was 2-4 carbons which may have
an important effect on antileishmanial activity. Previously,
we reported antileishmanial activity of 5-(nitroheteroaryl)-
1,3,4-thiadiazols containing cyclic amines including
piperazine or morpholine11–13. In piperazine and morpholine
derivatives, the distance between two heteroatoms is two
carbon which could be violated in the acyclic amine analogs
with maintaining the antileishmanial activity. However,
acyclic side chains have more flexibility respect to piperazine
or morpholine for conformational adaptation.
Recently, we have synthesized a novel series of
5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-amines by intro-
ducing N-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] moiety
as a new functionality on the C-2 amine of thiadiazole ring
via click chemistry17. Most compounds exhibited good
antileishmanial activity against the promastigote form
of L. major. In these compounds also the C-2 amine is
acyclic but the distal nitrogen has been incorporated in
the 1,2,3-triazole ring. In another study, to optimize the
antileishmanial activity of piperazinyl-linked 5-(5-nitro-
furan-2-yl)-1,3,4-thiadiazoles, we synthesized a series of
5-(5-nitrofuran-2-y1)-1,3,4-thiadiazoles with piperazinyl-
linked benzamidine substituent as scaffold found in pent-
amidine related antiprotozoals14. e latter compounds
characterized by having bulky benzamidine residue
on distal nitrogen of piperazine ring. e 5-nitrofuran-
2-yl-1,3,4-thiadiazoles having n-propyl, n-butyl and ben-
zyl side chain on benzamidine showed very good activity
against L. major. ese results demonstrate that the distal
nitrogen in the 5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-
2-amines has a more flexibility for chemical manipulation.
Journal of Enzyme Inhibition and Medicinal Chemistry

1,3,4-Thiadiazole-2-amines as antileishmanial agents 851
Table 2. In vitro antileishmanial activity of thiophene derivatives 26–42 against promastigote form of Leishmania major.
Compound
R
Antipromastigote activity IC50 (µM)
Cytotoxicity CC50 (µM)a
SIb
—
—
—
14.05
—
26
27
28
29
30
31
32
33
34
35
36
37
Me
Et
c-Pr
64 0.32
98.4 0.12
97.3 0.98
—
—
—
42.16
—
(CH2)3OH
(CH2)2OMe
CH2CH(OMe)2
(CH2)3OMe
(CH2)2N(Et)2
(CH2)3N(Me)2
(CH2)2O(CH2)2OH
CH2CH=CH2
3
0.41
104 0.7
56 0.74
—
—
3
0.5
37.80
43.80
87.35
—
12.60
1.22
3.49
—
36 0.24
25 0.17
42 0.45
34.3 0.5
61.2 0.22
129.32
—
3.77
—
CH2C≡ CH
38
39
40
35.8 0.62
115.6 0.63
198 0.48
92.4
—
2.58
—
—
—
41
42
228 0.9
—
—
—
—
156 0.16
Glucantime
Fluconazole
68.44c
941.1 4.98
—
—
—
—
CC50, cytotoxic concentration for 50% inhibition; IC50, half-maximal inhibitory concentration.
aCytotoxicity was evaluated against mouse peritoneal macrophages. bSelectivity index (SI) CC50/IC50. ce IC50 of glucantime was in mM.
Compounds 6-15, 18, 21, 24, 25, 29, 32–34, 36, and 38
having good activity against promastigotes were also evalu-
ated for their activity against amastigote form of L. major
in murine peritoneal macrophages. As shown in Figure 2,
all tested compounds significantly decreased the number
of intracellular amastigotes per macrophage, percentage
of macrophage infectivity and infectivity index compared
with the control group. It is notable that compounds 29 and
32 which exhibited potent activity against promastigotes
were the most effective compounds against amastigotes as
showed in the terms of amastigote number per macrophage,
the percentage of macrophage infectivity and infectivity
index (Figure 2A, 2B, and 2C, respectively). Compound 6
which was less potent than compounds 29 and 32 against
promastigotes, also being amongst the most effective com-
pounds against amastigotes in the terms of the percentage
of macrophage infectivity and infectivity index.
e in vitro cytotoxic activity of compounds 6-15, 18,
21, 24, 25, 29, 32–34, 36 and 38 against mouse perito-
neal macrophages was also determined using MTT assay
(Tables 1 and 2). e CC50 values for tested compounds
demonstrated that, most of them had toxicity for macro-
phages (CC50 <100 µM). Compounds 6, 8, 11, 14, 18, 24,
and 36 showed low level of toxicity against macrophages
(CC50 >100 µM). Although, the most active antileishman-
ial compounds 29 and 32 showed significant toxicity
against macrophages (CC50 values of 42.16 and 37.8 µM,
respectively), but also showed the highest selectivity index
(SI = 14.05 and 12.60, respectively).
In conclusion, we have synthesized a new series of
5-(5-nitrofuran-2-yl)- and 5-(5-nitrothiophen-2-yl)-
1,3,4-thiadiazole-2-amines bearing acyclic amine at C-2
position of thiadiazole ring and evaluated in vitro against
promastigote and amastigote forms of L. major. Most of
the target compounds exhibited good antileishmanial
activity against both forms of L. major. e structure-
activity relationships study of compounds revealed
that oxyalkyl and aminoalkyl substituents attached
to the nitrogen of 1,3,4-thiadiazole-2-amine improve
antileishmanial activity. e most active derivatives
were hydroxypropylamino- and methoxypropylamino-
analogs of 5-(5-nitrothiophen-2-yl)-1,3,4-thiadiazole
(compounds 29 and 32, respectively) with highest
selectivity index. While we were able to increase the
potency of 5-(nitroheteroaryl)-1,3,4-thiadiazols by adding
appropriate acyclic amine substituents to the 2-position
of thiadiazole, the physicochemical properties of these
compounds could be optimized.
acknowledgment
is work was supported by grants from the Research
Council of Tehran University of Medical Sciences and
Iran National Science Foundation (INSF).
© 2013 Informa UK, Ltd.

852 A. Tahghighi et al.
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of
a modified MTT assay for screening antimonial resistant
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