Synthesis, antioxidant activities and urease inhibition of some new 1,2,4-triazole and 1,3,4-thiadiazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2010.08.034

New series of 4,5-disubstituted-2,4-dihydro-3H-1,2,4-triazole-3-thiones (8a-j) and 2,5-disubstituted-1,3,4-thiadiazoles (9a-h) were synthesized by dehydrative cyclization of hydrazinecarbothioamide derivatives (7a-k) by refluxing in 4 N aqueous sodium hydroxide and by overnight stirring with polyphosphoric acid, respectively. The structures of the newly synthesized compounds were characterized by IR, ¹H NMR, ¹³C NMR, elemental analysis and mass spectroscopic studies and the synthesized compounds were screened for their antioxidant and urease inhibition activities. N-(2,4-Dimethylphenyl)-5-(4-nitrophenyl)-1,3,4-thiadiazol-2-amine (9h) showed excellent antioxidant activity more than the standard drug whereas 4-(2,4-dimethylphenyl)-5-(3-nitrophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (8d) and 4-(2,3-dimethylphenyl)-5-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (8e) exhibited potent urease inhibitory activities.

Full text of DOI:10.1016/j.ejmech.2010.08.034

European Journal of Medicinal Chemistry 45 (2010) 5200e5207
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antioxidant activities and urease inhibition of some new 1,2,4-triazole
and 1,3,4-thiadiazole derivatives
,
b
Imtiaz Khan a, Sajid Ali a, Shahid Hameed a, Nasim Hasan Rama a , Muhammad Tahir Hussain ,
*
Abdul Wadood c, Reaz Uddin c, Zaheer Ul-Haq c, Ajmal Khan c, Sajjad Ali c, M. Iqbal Choudhary c
a Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
b Department of Applied Sciences, National Textile University, Faisalabad 37610, Pakistan
c Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
New series of 4,5-disubstituted-2,4-dihydro-3H-1,2,4-triazole-3-thiones (8aej) and 2,5-disubstituted-
1,3,4-thiadiazoles (9aeh) were synthesized by dehydrative cyclization of hydrazinecarbothioamide
derivatives (7aek) by refluxing in 4 N aqueous sodium hydroxide and by overnight stirring with poly-
phosphoric acid, respectively. The structures of the newly synthesized compounds were characterized by
IR, 1H NMR, 13C NMR, elemental analysis and mass spectroscopic studies and the synthesized compounds
were screened for their antioxidant and urease inhibition activities. N-(2,4-Dimethylphenyl)-5-(4-
nitrophenyl)-1,3,4-thiadiazol-2-amine (9h) showed excellent antioxidant activity more than the stan-
dard drug whereas 4-(2,4-dimethylphenyl)-5-(3-nitrophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione
(8d) and 4-(2,3-dimethylphenyl)-5-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (8e) exhibited potent
urease inhibitory activities.
Received 3 June 2010
Received in revised form
9 August 2010
Accepted 12 August 2010
Available online 18 August 2010
Keywords:
1,2,4-Triazoles
1,3,4-Thiadiazoles
Antioxidant activities
Urease inhibition
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
vulgaris, and Ureaplsma urealyticum, is involved in the formation of
urinary tract stones, which may lead to the chronic inflammation of
Reactive oxygen species (ROS), capable of causing damage to
DNA, has been associated with carcinogenesis, coronary heart
disease, and many other health problems related to advancing age
[1,2]. In low concentrations, synthetic antioxidants are also in use
for many industrial processes e.g. inhibition of radical formation for
preventing premature polymerization during processing, storage
and transportation of unsaturated monomers. They exert their
effects by scavenging or preventing the generation of ROS [3] which
can protect the formation of free radicals and retard the progress of
many chronic diseases [4] including cancer, neurodegenerative,
inflammation and cardiovascular diseases [5].
Urease (urea amidohydrolase, E.C. 3.5.1.5) catalyzes the hydro-
lysis of urea resulting to the formation of ammonia and carbamic
acid. Spontaneous decomposition of carbamic acid produces
a second molecule of ammonia followed by its transformation to
ammonium, which may be up taken by plants under ideal condi-
tions (soil pH <6e6.5) [6]. However, when the conditions are other
than ideal, ammonia is escaped to the surroundings which may
lead to environmental and economic problems [7]. Ureolytic
activity of several microorganisms, e.g. Proteus mirabilis, Proteus
kidney and its pelvis. Moreover, urinary catheter obstruction in
patients causes colonization of urease-producing microorganisms,
essentially P. mirabilis. Overproduction of ammonia by infectious
microorganisms may contribute to ammonia encephalopathy or
hepatic coma. Another mechanism of urease involvement in
pathogenic bacteria infection relies on the formation of a micro-
environment appropriate for the pathogen existence. Helicobacter
pylori infection of stomach is possible only after local neutralization
of gastric acid by released ammonia [8]. Furthermore high
concentration of ammonia disturbs mucosal permeability, in
particular hydrogen ions passage through mucosal surface and
causes formation of peptic ulcers. In this respect, increasing
attention has been focused on the search for compounds that will
inhibit urease activity for the possible development of highly
needed therapy for urease mediated bacterial infections [8e12].
Azoles, a versatile bunch of heterocycles are known chemo-
therapeutic agents. Among these, 1,2,4-triazole and 1,3,4-thiadia-
zoles possess urease inhibition and antioxidant activities [13,14]. In
the present study, we have synthesized some new 1,2,4-triazole
and 1,3,4-thiadiazole derivatives which were tested against 1,1-
diphenyl-2-picrylhydrazyl (DPPH) radical and superoxide anion
scavenging activities. Moreover, the synthesized compounds were
screened as urease inhibitors.
* Corresponding author: Tel.: þ92 51 90642135; fax: þ92 51 90642241.
E-mail address: nhrama@qau.edu.pk (N.H. Rama).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.034

I. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 5200e5207
5201
2. Results and discussion
intermediates for the synthesis of 1,2,4-triazole and 1,3,4-thiadia-
zole derivatives. The 4,5-disubstituted-2,4-dihydro-3H-1,2,4-tri-
azole-3-thiones (8aej) were synthesized by intramolecular
dehydrative cyclization of carbothioamides (7aek) when refluxed
in 4 N sodium hydroxide solution, followed by neutralization with
concentrated HCl [20], while 2,5-disubstituted-1,3,4-thiadiazoles
(9aeh) were synthesized by overnight stirring of carbothioamides
(7aek) at 70 ꢀC in polyphosphoric acid [21]. All the reactions were
monitored by TLC and the newly synthesized compounds were
purified by column chromatography, if necessary. The structure
elucidations of the newly synthesized compounds were carried out
by modern spectroscopic techniques like IR, 1H NMR and 13C NMR.
Further confirmations of the compounds were carried out by mass
spectrometry and microanalysis.
2.1. Chemistry
The synthetic route for the newly synthesized compounds,1,2,4-
triazole and 1,3,4-thiadiazole derivatives (8aej and 9aeh), is illus-
trated and outlined in Scheme 1. Substituted aromatic esters (2aeh)
were synthesized by the reaction of corresponding acids (1a-h) with
methanol in the presence of catalytic amount of sulfuric acid. Esters
(2aeh) were converted to the corresponding acid hydrazides
(3aeh) by refluxing with hydrazine hydrate (80%) in methanol
[15e18]. Dimethylphenylanilines (4aec) were converted into cor-
responding dithiocarbamate salts (5aec) by stirring with concen-
trated ammonia solution and pure carbon disulphide in methanol.
The intermediate dithiocarbamates were not isolated and converted
directly to substituted phenylisothiocyanates (6aec) by overnight
stirring with lead nitrate solution [19]. The compounds (6aec) were
isolated by steam distillation. Hydrazinecarbothioamide derivatives
(7aek) were synthesized by the condensation of corresponding acid
hydrazides (3aeh) and phenylisothiocyanates (6aec) in methanol.
Due to some unavoidable conditions, only 11 carbothioamides
(7aek) could be prepared. These compounds were used as the key
2.2. Pharmacology
2.2.1. Antioxidant activities
Compounds 8d, 8j, and 9f with the IC50 values 242.49 ꢁ 3.64,
292.38 ꢁ 1.60 and 319.62 ꢁ 2.04, respectively, have shown
hydrogen donating ability on reaction with DPPH radical. It is clear
from the results that the antioxidant potential of compounds is
O
O
NH2
CH3
(a)
OH
'
R
O
R
R
2a-h
(b)
4a-c
1a-h
(c)
O
H
N
+
NH2
NCS
SNH4
N
H
(d)
+
R
'
R
'
R
S
6a-c
3a-h
5a-c
(e)
NH
N
H
H
O
N
N
S
(f)
N
N
H
R
'
R
A
R
S
'
R
B
7a-k
(g)
8a-j
N
N
'
R
N
H
S
R
9a-h
R′ = 2,6-diCH3
R′ = 2,6-diCH3
R′ = 2,3-diCH3
R′ = 2,4-diCH3
R′ = 2,3-diCH3
R′ = 2,6-diCH3
R′ = 2,4-diCH3
R′ = 2,3-diCH3
R′ = 2,6-diCH3
R′ = 2,4-diCH3
R′ = 2,6-diCH3
R′ = 2,6-diCH3
R′ = 2,3-diCH3
R′ = 2,4-diCH3
R′ = 2,3-diCH3
R′ = 2,6-diCH3
R′ = 2,4-diCH3
R′ = 2,4-diCH3
4-6a
b
c
8a R = 4-CH3
9a R = 4-CH3
R′ = 2,3-diCH3
R′ = 2,4-diCH3
R′ = 2,6-diCH3
b
c
d
e
f
R = 2-Cl
R = 4-Cl
R = 3-NO2
R = H
R = 4-Cl
R = 3-Cl
R = 2-CH3
R = 3-Cl
R = 4-Cl
b
c
d
e
f
R = 2-Cl
R = 4-Cl
R = 3-NO2
R = H
R = 4-Cl
R = 3-Cl
R = 4-NO2
R′ = 2,6-diCH3
R′ = 2,6-diCH3
R′ = 2,3-diCH3
R′ = 2,4-diCH3
R′ = 2,3-diCH3
R′ = 2,6-diCH3
R′ = 2,4-diCH3
R′ = 2,4-diCH3
R′ = 2,3-diCH3
R′ = 2,6-diCH3
R′ = 2,4-diCH3
7a R = 4-CH3
b
c
d
e
f
g
h
i
R = 2-Cl
R = 4-Cl
R = 3-NO2
R = H
R = 4-Cl
R = 3-Cl
R = 4-NO2
R = 2-CH3
R = 3-Cl
R = 4-Cl
g
h
i
g
h
j
j
k
Scheme 1. Synthesis of 1,2,4-triazole and 1,3,4-thiadiazole derivatives. (a) Dry methanol, conc. H2SO4, reflux; (b) dry methanol, hydrazine hydrate (80%), reflux; (c) CS2, NH4OH
(33%), dry methanol; (d) Pb(NO3)2; (e) dry methanol, reflux; (f) 4 N NaOH, reflux; and (g) PPA. Stirring.

5202
I. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 5200e5207
associated with the position of the substituents on both the rings A
and B. In compounds 8d, 8j and 9f, meta-nitro and para-chloro
substituents are present at aryl ring A while in 8d and 8j, the
dimethyl substituents are present at ortho and para positions on
aryl ring B which contributed in the radical scavenging ability. The
thiadiazole class of compounds was found to be significantly active
against superoxide anion radical. The compound 9h with an IC50
value 94.84 ꢁ 0.34 was found to be more active than the standard
antioxidant propyl gallate. Since the mode of action of superoxide is
different from DPPH radical, it adds to labile site of the compound
and creates a negative charge on the resulting adduct [22]. Thus
electron withdrawing groups could stabilize the formed negatively
charged adduct. The activity of 9h is enhanced in this class due to
the presence of nitro group which is a strong electron withdrawing
in nature. The compounds 9c, 9d and 9g have shown comparable
antioxidant potential with the standard, whereas the compound 9f
showed a moderate superoxide anion scavenging activity.
under the inert atmosphere of dry nitrogen. Melting points were
determined on Stuart melting point apparatus (SMP3) and are
uncorrected. The IR spectra were recorded on Bruker Optics Alpha
FT-IR Spectrometer. 1H NMR (300 MHz) and 13C NMR (75 MHz)
were recorded on a Bruker AV-300 as DMSO-d6 and CDCl3 solutions
using TMS as an internal standard. Electron impact mass spectra
(EIMS) were recorded on a Finnigan MAT-311A (Germany) mass
spectrometer operating at an ionization potential of 70 eV. CHN
analyses were performed on a Carlo Erba Strumentazion-Mod-1106
Italy. Thin layer chromatography was performed on pre-coated
silica gel plates (Kieselgel 60, 254, E. Merck, Germany) and the
chromatograms were visualized by UV at 254 and 365 nm.
3.1. Synthesis of substituted aromatic esters (2aeh) and aromatic
acid hydrazides (3aeh)
Substituted aromatic acids (1aeh) were esterified (2aeh) and
the later were converted into their corresponding acid hydrazides
(3aeh) by the standard procedure [15e18].
2.2.2. Urease inhibition bioassay
Almost all compounds showed moderate to good activities.
Potent compounds have their activities in the range of 45.60 ꢁ 0.04
3.2. Synthesis of substituted phenylisothiocyanates (6aec)
to 483.55 ꢁ1.99
mM. Among the two series of compounds, triazole
derivatives are relatively more active than the derivatives of thia-
diazole (Table 3). The triazole derivatives may be regarded as
substrate like inhibitors on the basis of their structural similarity
with the natural substrate of urease i.e. urea. The Compounds 8d
and 8e were found to be the most active with IC50 values of
Substituted anilines (4aec) were converted to their dithiocar-
bamates (5aec) which were oxidized to respective phenyl-
isothiocyanates (6aec) by the standard procedure [15e18].
3.3. General procedure for the synthesis of
45.60 ꢁ 0.04 and 86.0 ꢁ 0.04
m
M, respectively. In compound 8e, the
hydrazinecarbothioamide derivatives (7aek)
aromatic ring at position 5 of the triazole nucleus is unsubstituted
whereas in 8d this has a meta-nitro group. This indicates that
a nitro group on the aryl ring enhances the activity. Among the
chlorosubstituted groups, compound 8j with the para-chloro
substituent is the most active, while compounds 8c and 8f also
The corresponding acid hydrazide (3) (0.0068 moles) was dis-
solved in methanol (30 mL) and added dropwise to the solution of
substituted isothiocyanate (0.0066 moles) in methanol (10 mL).
The reaction mixture was refluxed for 10e12 h and monitored by
TLC. After consumption of the starting materials, the mixture was
cooled to room temperature. Evaporation of solvent under reduced
pressure left crude hydrazinecarbothioamide (7) as an oil which
was solidified on cooling. It was purified by recrystallization from
a mixture of ethyl acetate and petroleum ether to yield hydrazi-
necarbothioamide (7).
having
a para-chlorosubstituent were found inactive. In the
compound 8j, 2,4-dimethyl substituents are present at ortho and
para positions of the aryl ring which are perhaps more suited to fit
the enzyme active site as compared to 2,3-dimethyl and 2,6-
dimethyl substituents at aryl ring in compounds 8e and 8f. This
activity trend indicates that the triazole moiety with small lipo-
philic methyl substituent at para position of the aryl ring and
a relatively large and polarizable group like NO2 at meta position is
favorable in terms of activity enhancement against urease.
3.3.1. N-(2,6-Dimethylphenyl)-2-(4-methylbenzoyl)
hydrazinecarbothioamide (7a)
White solid (57%, 1.18 g): m.p 148e149 ꢀC; Rf: 0.53 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3341, 3223 (NH), 1677 (C]
O), 1515, 1498 (C]C), 1245 (C]S); 1H NMR (300 MHz, DMSO-d6):
2.2.3. Conclusion
New series of triazole (8aej) and thiadiazole (9aeh) derivatives
were synthesized. The antioxidant ability of these compounds was
evaluated. Some of the compounds were found to be significant
scavengers of free radicals. The compound (9h) showed excellent
antioxidant activity more than the standard drug. Therefore, the
discovered scavengers with curative values should be further
investigated, especially for their efficacy to prevent or control the
oxidative stress in diseases whose tangible and beneficial alterna-
tives are still insufficient.
The synthesized compounds were also evaluated for urease
inhibition activity which demonstrates that some compounds in
the series are most promising. The identified compounds can be
utilized in further optimization of bioactivity using structural
variations in the parent skeleton. The compounds 8d, 8e and 8j are
potential lead candidates with respect to the urease inhibition
activity whereas compounds 9d and 9f show moderate activity.
d
10.47 (s, 1H, NHeC]O), 9.54 (s, 1H, NHeC]S), 9.49 (s, 1H,
NHeC]S), 7.86 (d, 2H, J ¼ 8.1 Hz, AreH), 7.19 (d, 2H, J ¼ 8.1 Hz,
AreH), 7.08e7.01 (m, 3H, AreH), 2.36 (s, 3H, CH3), 2.15 (s, 6H, 2ꢃ
CH3); 13C NMR (75 MHz, DMSO-d6):
d 181.80, 166.31, 142.14, 141.35,
137.52, 130.00, 129.07, 128.50, 127.86, 127.11, 22.29, 18.45; EI-MS: m/
z (rel. abund. %), 313.14 (Mꢄþ, 13.6), 279.17(41.8), 164.07(38.9),
163.02(93.6), 150.11(52.4), 148.06(33.6), 131.09(39.7), 120.09(41.5),
119.04(100), 105.09(37.7), 91.06(88.0), 77.07(32.9), 65.07(39.8).
Analysis. Calcd for C17H19N3SO: C, 65.15; H, 6.11; N, 13.41; S, 10.23.
Found: C, 65.09; H, 6.07; N, 13.25; S, 10.02.
3.3.2. 2-(2-Chlorobenzoyl)-N-(2,6-dimethylphenyl)
hydrazinecarbothioamide (7b)
White solid (73%, 1.61 g): m.p 155e156 ꢀC; Rf: 0.39 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3326, 3281 (NH), 1644
(C]O), 1593, 1520, 1469 (C]C), 1235 (C]S); 1H NMR (300 MHz,
3. Experimental
DMSO-d6): d 10.50 (s, 1H, NHeC]O), 9.75 (s, 1H, NHeC]S), 9.19 (s,
1H, NHC]S), 7.55e7.38 (m, 4H, AreH), 7.13e7.05 (m, 3H, AreH),
All the common chemicals and solvents were of analytical grade
or dry distilled. Air or moisture sensitive processes were conducted
2.17 (s, 6H, 2ꢃ CH3); 13C NMR (75 MHz, DMSO-d6):
d 182.25, 166.13,
137.49, 132.07, 131.38, 131.29, 130.79, 130.09, 129.63, 127.95, 127.52,

I. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 5200e5207
5203
127.30, 18.41; EI-MS: m/z (rel. abund. %), 334.95 (Mꢄ þ 2, 2.3),
332.99 (Mꢄþ, 4.5), 298.99(25.7), 211.91(29.3), 162.94(100), 151.97
(33.7), 147.99(24.4), 145.03(17.5), 140.95(77.8), 138.90(100), 130.03
(68.1), 121.05(28.0), 105.03(24.5), 91.01(10.5), 77.02(24.9), 75.00
(34.0). Analysis. Calcd for C16H16ClN3SO: C, 57.56; H, 4.83; N, 12.59;
S, 9.61. Found: C, 57.41; H, 4.67; N, 12.43; S, 9.52.
129.32, 128.68, 127.89, 127.18, 21.08; EI-MS: m/z (rel. abund. %),
þ
335.00 (Mꢄ þ 2, 3.2), 332.99 (Mꢄþ, 5.4), 298.96(98.2), 178.98(14.5),
171.00(21.6), 162.99(86.4), 152.01(39.7), 148.03(31.9), 145.06(91.3),
140.97(71.7), 138.94(100), 130.05(66.6), 121.08(26.2), 110.98(77.1),
105.06(42.4), 77.05(37.8), 75.03(41.5), 51.03(16.5). Analysis. Calcd
for C16H16ClN3SO: C, 57.56; H, 4.83; N, 12.59; S, 9.61. Found: C,
57.41; H, 4.59; N, 12.38; S, 9.47.
3.3.3. 2-(4-Chlorobenzoyl)-N-(2,3-dimethylphenyl)
hydrazinecarbothioamide (7c)
3.3.7. 2-(3-Chlorobenzoyl)-N-(2,4-dimethylphenyl)
hydrazinecarbothioamide (7g)
White solid (75%, 1.66 g): m.p 165e166 ꢀC; Rf: 0.44 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3296, 3163 (NH), 1673 (C]
O), 1596, 1512 (C]C), 1268 (C]S); 1H NMR (300 MHz, DMSO-d6):
White solid (69%, 1.52 g): m.p 167e169 ꢀC; Rf: 0.38 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3345, 3165 (NH), 1673 (C]
O), 1555, 1533 (C]C), 1239 (C]S); 1H NMR (300 MHz, DMSO-d6):
d
10.63 (s, 1H, NHeC]O), 9.62 (s, 2H, 2NHeC]S), 7.96 (d, 2H,
J ¼ 8.4 Hz, AreH), 7.58 (d, 2H, J ¼ 8.4 Hz, AreH), 7.07e7.06 (m, 3H,
d 10.66 (s, 1H, NHeC]O), 9.63 (s, 1H, NHeC]S), 9.54 (s, 1H,
AreH), 2.24 (s, 3H, CH3), 2.06 (s, 3H, CH3); 13C NMR (75 MHz,
NHeC]S), 8.01 (s,1H, AreH), 7.87 (d, 1H, J ¼ 7.8 Hz, AreH), 7.65 (dd,
1H, J ¼ 8.1, 1.8 Hz, AreH), 7.53 (t, 1H, J ¼ 7.8 Hz, AreH), 7.03e6.97
(m, 3H, AreH), 2.26 (s, 3H, CH3), 2.13 (s, 3H, CH3); 13C NMR (75 MHz,
DMSO-d6):
d 182.06, 165.06, 137.25, 137.03, 131.91, 130.35, 129.37,
129.33, 128.72, 128.48, 127.09,þ126.71, 125.47, 20.52, 14.56; EI-MS:
m/z (rel. abund. %), 335.01 (Mꢄ þ 2, 5.7), 332.98 (Mꢄþ, 18.4), 324.12
(72.9), 291.12(42.1), 237.03(38.9), 163.00(100), 148.03(24.9), 145.09
(42.9), 131.07(32.9), 130.07(83.7), 121.09(51.2), 105.08(39.5), 91.06
(21.4), 77.06(42.1). Analysis. Calcd for C16H16ClN3SO: C, 57.56; H,
4.83; N, 12.59; S, 9.61. Found: C, 57.51; H, 4.69; N, 12.50; S, 9.49.
DMSO-d6):
d 182.18, 165.29, 136.25, 135.91 135.12, 133.42, 132.02,
131.06, 130.69, 129.11, 128.29, 127.13,þ126.83, 125.60, 21.08, 18.09;
EI-MS: m/z (rel. abund. %), 335.01 (Mꢄ þ 2, 1.0), 332.90 (Mꢄþ þ 2.9),
298.94(30.0), 170.92(21.7), 169.93(52.3), 162.88(99.1), 151.95(14.0),
147.95(53.2), 145.01(19.3), 140.93(92.6), 138.86(100), 129.99(96.9),
121.02(19.5), 110.91(98.7), 105.01(60.3), 77.00(35.7), 62.99(14.2),
50.99(20.6) Analysis. Calcd for C16H16ClN3SO: C, 57.56; H, 4.83; N,
12.59; S, 9.61. Found: C, 57.44; H, 4.65; N, 12.42; S, 9.41.
3.3.4. N-(2,4-Dimethylphenyl)-2-(3-nitrobenzoyl)
hydrazinecarbothioamide (7d)
White solid (59%, 1.27 g): m.p 143e145 ꢀC; Rf: 0.40 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3354, 3130 (NH), 1681 (C]
O), 1586, 1555, 1530 (C]C), 1242 (C]S); 1H NMR (300 MHz, DMSO-
3.3.8. N-(2,4-Dimethylphenyl)-2-(4-nitrobenzoyl)
hydrazinecarbothioamide (7h)
d6):
d
10.92 (s, 1H, NHeC]O), 9.71 (s, 1H, NHeC]S), 9.61 (s, 1H,
Yellow solid (63%, 1.35 g): m.p 160e161 ꢀC; Rf: 0.49 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3309, 3154 (NH), 1668 (C]
O), 1596, 1499 (C]C), 1257 (C]S); 1H NMR (300 MHz, DMSO-d6):
NHeC]S), 8.78 (s, 1H, AreH), 8.43 (dd, 1H, J ¼ 8.1,1.8 Hz, AreH),
8.35 (d, 1H, J ¼ 8.1 Hz, AreH), 7.81 (t, 1H, J ¼ 7.8 Hz, AreH),
7.03e6.98 (m, 3H, AreH), 2.26 (s, 3H, CH3), 2.14 (s, 3H, CH3); 13C
d
10.87 (s, 1H, NHeC]O), 9.70 (s, H, NHeC]S), 9.60 (s, 1H, NHeC]
NMR (75 MHz, DMSO-d6):
d
182.06, 164.65, 148.00, 136.31, 135.85,
S), 8.35 (d, 2H, J ¼ 9.0 Hz, AreH), 8.16 (d, 2H, J ¼ 8.7 Hz, AreH),
134.73, 131.09, 129.10, 126.88, 125.53, 125.41, 125.11, 123.89, 123.15,
21.08, 18.09; EI-MS: m/z (rel. abund. %), 344.27 (Mꢄþ, 2.5), 181.03
(15.3), 163.00(100), 150.00(94.1), 148.01(24.1), 145.07(8.5), 130.05
(37.7), 105.05(25.3), 77.03(15.9), 63.00(11.1). Analysis. Calcd for
C16H16N4SO3: C, 55.80; H, 4.68; N, 16.27; S, 9.31. Found: C, 55.64; H,
4.53; N, 16.14; S, 9.20.
7.03e6.98 (m, 3H, AreH), 2.26 (s, 3H, CH3), 2.14 (s, 3H, CH3); 13C
NMR (75 MHz, DMSO-d6):
d 182.18, 165.06, 149.75, 138.86, 136.30,
135.85, 131.10, 129.93, 129.14, 126.86, 126.63, 123.80, 21.07, 18.08;
EI-MS: m/z (rel. abund. %), 344.17 (Mꢄþ, 2.8), 310.05(23.7), 182.04
(38.0), 181.02(34.6), 162.97(100), 151.03(53.0), 149.98(95.3), 148.02
(49.7), 145.07(11.6), 131.06(21.7), 130.05(78.2), 121.07(27.8), 105.06
(64.9), 104.01(80.6), 91.05(12.0), 77.05(35.1), 63.02(27.4). Analysis.
Calcd for C16H16N4SO3: C, 55.80; H, 4.68; N, 16.27; S, 9.31. Found: C,
55.61; H, 4.55; N, 16.19; S, 9.17.
3.3.5. N-(2,3-Dimethylphenyl)-2-(benzoyl)
hydrazinecarbothioamide (7e)
White solid (61%, 1.20 g): m.p 135e136 ꢀC; Rf: 0.37 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3458, 3183 (NH), 1669
(C]O), 1601, 1580, 1531 (C]C), 1237 (C]S); 1H NMR (300 MHz,
3.3.9. N-(2,3-Dimethylphenyl)-2-(2-methylbenzoyl)
hydrazinecarbothioamide (7i)
DMSO-d6):
d
10.54 (s, 1H, NHeC]O), 9.60 (s, 2H, 2NHeC]S), 7.95
White solid (53%, 1.09 g): m.p 150e151 ꢀC; Rf: 0.47 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3250, 3154 (NH), 1665 (C]
O), 1555, 1526, 1491 (C]C), 1237 (C]S); 1H NMR (300 MHz, DMSO-
(d, 1H, J ¼ 7.5 Hz, AreH), 7.60e7.55 (m, 2H, AreH), 7.51e7.46 (m, 3H,
AreH), 7.06 (d, 2H, J ¼ 4.5 Hz, AreH), 2.24 (s, 3H, CH3), 2.07 (s, 3H,
CH3); 13C NMR (75 MHz, DMSO-d6):
d
182.32, 166.54, 138.43, 137.28,
d6): d 10.20 (s, 1H, NHeC]O), 9.48 (s, 1H, NHeC]S), 9.40 (s, 1H,
135.03, 133.08, 132.21, 128.61, 128.43, 127.08, 126.43, 125.44, 20.50,
19.03; EI-MS: m/z (rel. abund. %), 299.18 (Mꢄþ, 3.1), 265.17(66.2),
195.14(13.5), 163.03(99.8), 148.08(46.6), 145.12(28.9), 130.08(94.3),
105.02(100), 91.08(15.7), 77.05(95.4), 63.03(41.4), 51.04(34.0).
Analysis. Calcd for C16H17N3SO: C, 64.09; H, 5.72; N, 14.04; S, 10.71.
Found: C, 63.94; H, 5.62; N, 13.91; S, 10.56.
NHeC]S), 7.68 (bs, 1H, AreH), 7.36 (dt, 1H, J ¼ 8.4,0.9 Hz, AreH),
7.27e7.21 (m, 3H, AreH), 7.06 (d, 2H, J ¼ 8.1 Hz, AreH), 2.41 (s, 3H,
CH3), 2.26 (s, 3H, CH3), 2.09 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-
d6):
d 182.18, 168.94, 138.46, 137.19, 136.13, 136.10, 134.95, 130.99,
130.85, 129.81, 127.70, 125.92, 125.68, 125.57, 21.63, 20.12, 14.51; EI-
MS: m/z (rel. abund. %), 313.11 (Mꢄþ, 3.4), 280.15(44.5), 279.08(96.3),
164.05(19.1), 163.02(96.4), 159.05(32.8), 148.05(28.9), 131.07(52.6),
120.07(35.5), 119.01(100), 105.06(28.1), 91.04(99.8), 77.06(36.5),
65.06(36.0), 63.02(25.4). Analysis. Calcd for C17H19N3SO: C, 65.15; H,
6.11; N, 13.41; S, 10.23. Found: C, 64.99; H, 6.01; N, 13.21; S, 10.08.
3.3.6. 2-(4-Chlorobenzoyl)-N-(2,6-dimethylphenyl)
hydrazinecarbothioamide (7f)
White solid (72%, 1.59 g): m.p 185e186 ꢀC; Rf: 0.36 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3319, 3121 (NH), 1681
(C]O), 1594, 1525, 1492 (C]C), 1251 (C]S); 1H NMR (300 MHz,
3.3.10. 2-(3-Chlorobenzoyl)-N-(2,6-dimethylphenyl)
hydrazinecarbothioamide (7j)
DMSO-d6): d 10.64 (s, 1H, NHeC]O), 9.61 (s, 1H, NHeC]S), 9.43 (s,
1H, NH-C]S), 7.97 (d, 2H, J ¼ 8.4 Hz, AreH), 7.58 (d, 2H, J ¼ 8.7 Hz,
White solid (71%, 1.57 g): m.p 175e176 ꢀC; Rf: 0.41 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3300, 3143 (NH), 1674
(C]O), 1522, 1497 (C]C), 1236 (C]S); 1H NMR (300 MHz, DMSO-
AreH), 7.07e7.02 (m, 3H, AreH), 2.15 (s, 6H, 2ꢃ CH3); 13C NMR
(75 MHz, DMSO-d6): d 181.78, 165.74, 137.42, 136.99, 131.95, 130.41,

5204
I. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 5200e5207
d6):
d
10.69 (s, 1H, NHeC]O), 9.62 (s, 1H, NHeC]S), 9.44 (s, 1H,
131.02, 130.14,þ128.96, 127.77, 124.69, 18.23; EI-MS: m/z (rel. abund.
%), 317.00 (Mꢄ þ 2, 14.4), 315.00 (Mꢄþ, 37.9), 284.10(32.9), 282.01
(100), 145.05(26.3), 136.90(12.1), 105.02(13.7), 91.08(13.9), 77.00
(26.4), 69.10(43.4). Analysis. Calcd for C16H14ClN3S: C, 60.85; H,
4.47; N, 13.31; S, 10.15. Found: C, 60.69; H, 4.22; N, 13.24; S, 10.05.
NHeC]S), 8.03 (s, 1H, AreH), 7.88 (d, 1H, J ¼ 7.8 Hz, AreH), 7.65
(td, 1H, J ¼ 7.2, 0.9 Hz, AreH), 7.53 (t, 1H, J ¼ 8.1 Hz, AreH),
7.08e7.04 (m, 3H, AreH), 2.16 (s, 6H, 2ꢃ CH3); 13C NMR (75 MHz,
DMSO-d6):
d 181.78, 165.50, 137.39, 136.99, 135.72, 135.19, 133.38,
131.98, 130.65, 128.3þ4, 127.90, 126.11, 18.41; EI-MS: m/z (rel.
abund. %), 334.98 (Mꢄ þ 2, 7.7), 332.98 (Mꢄþ, 14.4), 299.01(18.2),
269.03(50.2), 170.98(53.0), 162.96(98.0), 148.01(30.2), 145.06
(19.1), 140.95(86.8), 138.92(100), 130.04(85.8), 121.08(52.0),
110.95(93.1), 77.04(34.3), 75.02(58.6), 51.02(17.7). Analysis. Calcd
for C16H16ClN3SO: C, 57.56; H, 4.83; N, 12.59; S, 9.61. Found: C,
57.49; H, 4.71; N, 12.47; S, 9.48.
3.4.3. 5-(4-Chlorophenyl)-4-(2,3-dimethylphenyl)-2,4-dihydro-3H-
1,2,4-triazole-3-thione (8c)
White crystalline solid (61%, 0.27 g): m.p 290e291 ꢀC; Rf: 0.55
(n-hexane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3262 (NH), 2961 (sp3
CH), 1555 (C]N), 1501, 1477 (C]C), 1253 (C]S); 1H NMR
(300 MHz, DMSO-d6):
d
14.53 (s, 1H, NH), 7.40 (d, 2H, J ¼ 7.2 Hz,
AreH), 7.28 (d, 1H, J ¼ 7.5 Hz, AreH), 7.24 (d, 2H, J ¼ 8.7 Hz, AreH),
7.18 (t, 1H, J ¼ 7.8 Hz, AreH), 7.02 (d, 1H, J ¼ 7.5 Hz, AreH), 2.26 (s,
3.3.11. 2-(4-Chlorobenzoyl)-N-(2,4-dimethylphenyl)
hydrazinecarbothioamide (7k)
3H, CH3), 1.81 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):
d 167.33,
White solid (66%, 1.46 g): m.p 159e160 ꢀC; Rf: 0.33 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3253, 3140 (NH), 1679
(C]O), 1594, 1509, 1476 (C]C), 1238 (C]S); 1H NMR (300 MHz,
148.57, 138.48, 135.00, 131.27, 129.21, 129.16, 128.10, 127.78, 127.19,
126þ.78, 126.06, 20.34, 14.39; EI-MS: m/z (rel. abund. %), 317.07
(Mꢄ þ 2, 18.6), 315.05 (Mꢄþ, 50.5), 300.05(11.5), 284.10(35.5),
282.11(100), 163.07(10.5), 145.11(23.2), 103.07(7.3), 78.05(27.2),
77.08(15.2), 63.03(25.0). Analysis. Calcd for C16H14ClN3S: C, 60.85;
H, 4.47; N, 13.31; S, 10.15. Found: C, 60.75; H, 4.29; N, 13.13; S, 9.98.
DMSO-d6):
d 10.61 (s, 1H, NHeC]O), 9.60 (s, 1H, NHeC]S), 9.52
(s, 1H, NHeC]S), 7.95 (d, 2H, J ¼ 8.4 Hz, AreH), 7.58 (d, 2H,
J ¼ 8.7 Hz, AreH), 7.02e6.97 (m, 3H, AreH), 2.26 (s, 3H, CH3), 2.14
(s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):
d 182.25, 165.56,
137.03, 136.19, 131.91, 131.09, 130.34, 129.35, 128.90, 128.72,
3.4.4. 4-(2,4-Dimethylphenyl)-5-(3-nitrophenyl)-2,4-dihydro-3H-
1,2,4-triazole-3-thione (8d)
128.11, 126.81, 21.07, 18.09;þEI-MS: m/z (rel. abund. %), 334.98
(Mꢄþ þ 2, 1.3), 332.98 (Mꢄ
,
3.7), 300.06(29.1), 299.05(65.3),
Yellow solid (47%, 0.21 g): m.p 278e281 ꢀC; Rf: 0.57 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3373 (NH), 2955 (sp3 CH),
1529 (C]N), 1478 (C]C), 1261 (C]S); 1H NMR (300 MHz, DMSO-
171.00(36.0), 162.95(100), 148.02(44.9), 145.07(29.9), 140.97
(89.0), 138.92(99.0), 130.05(83.7), 121.07(31.7), 110.97(79.0),
105.05(51.3), 77.03(28.0), 75.01(35.1), 51.01(13.1). Analysis. Calcd
for C16H16ClN3SO: C, 57.56; H, 4.83; N, 12.59; S, 9.61. Found: C,
57.47; H, 4.63; N, 12.50; S, 9.46.
d6):
d
14.38 (s, 1H, NH), 8.08 (s, 1H, AreH), 8.26 (d, 1H, J ¼ 7.8 Hz,
AreH), 7.73 (d, 1H, J ¼ 7.5 Hz, AreH), 7.66 (t, 1H, J ¼ 8.1 Hz, AreH),
7.25 (d, 2H, J ¼ 8.1 Hz, AreH), 7.16 (d, 1H, J ¼ 9.0 Hz, AreH), 2.34 (s,
3H, CH3), 2.00 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):
d 167.16,
3.4. General procedure for the synthesis of 1,2,4-triazol-3-thiones
(8aej)
148.87, 148.11, 140.28, 136.01, 133.86, 132.25, 131.24, 131.09, 129.39,
128.40, 127.27, 125.44, 122.43, 21.21, 17.73; EI-MS: m/z (rel. abund.
%), 326.00 (Mꢄþ, 62.8), 293.00(100), 247.01(38.0), 162.90(12.1),
145.06(14.6), 105.03(19.3), 91.07(21.4), 77.02(30.7), 65.10(10.3),
51.11(11.9). Analysis. Calcd for C16H14N4O2S: C, 58.88; H, 4.32; N,
17.17; S, 9.82. Found: C, 58.72; H, 4.21; N, 16.98; S, 9.64.
The corresponding carbothioamide (7) (0.0014 moles) was
refluxed in aqueous sodium hydroxide solution (4 N, 25 mL). The
reaction was monitored by TLC. After completion of reaction
(12e14 h), the reaction mixture was cooled to room temperature
and filtered. The filtrate was neutralized with hydrochloric acid
(4 N) to precipitate the 1,2,4-triazol-3-thione (8) which was
recrystalized from aqueous ethanol.
3.4.5. 4-(2,3-Dimethylphenyl)-5-phenyl-2,4-dihydro-3H-1,2,4-
triazole-3-thione (8e)
White crystalline solid (53%, 0.20 g): m.p 207e209 ꢀC; Rf: 0.59
(n-hexane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3268 (NH), 2963
(sp3 CH), 1550 (C]N), 1489, 1475 (C]C), 1240 (C]S); 1H NMR
3.4.1. 4-(2,6-Dimethylphenyl)-5-(4-methylphenyl)-2,4-dihydro-
3H-1,2,4-triazole-3-thione (8a)
(300 MHz, DMSO-d6):
7.21 (t, 1H, J ¼ 7.8 Hz, AreH), 7.12 (d, 1H, J ¼ 7.5 Hz, AreH), 2.27 (s,
3H, CH3), 1.87 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):
168.83,
d 14.19 (s, 1H, NH), 7.41e7.27 (m, 6H, AreH),
White crystalline solid (69%, 0.28 g): m.p 262e265 ꢀC; Rf: 0.66
(n-hexane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3355 (NH), 2910,
2846 (sp3 CH), 1533 (C]N), 1489, 1472 (C]C), 1281 (C]S); 1H NMR
d
150.87, 138.64, 135.03, 134.03, 131.64, 130.96, 129.19, 127.96, 127.2þ7,
(300 MHz, DMSO-d6):
d
14.37 (s, 1H, NH), 7.36e7.31 (m, 1H, AreH),
126.93, 126.21, 20.13, 14.38; EI-MS: m/z (rel. abund. %), 281.00 (Mꢄ
,
7.23 (d, 2H, J ¼ 7.8 Hz, AreH), 7.14 (bs, 4H, AreH), 2.25 (s, 3H, CH3),
72.7), 266.01(10.6), 248.12(100), 163.03(7.1), 145.09(13.1), 103.07
(26.3), 77.00(33.4), 51.10(13.5). Analysis. Calcd for C16H15N3S: C,
68.30; H, 5.37; N, 14.93; S, 11.40. Found: C, 68.11; H, 5.15; N, 14.86;
S, 11.25.
1.95 (s, 6H, 2ꢃ CH3); 13C NMR (75 MHz, DMSO-d6):
d 167.82, 150.26,
141.13, 136.40, 133.34, 130.33, 129.97, 129.20, 126.91, 123.28, 21.29,
17.99; EI-MS: m/z (rel. abund. %), 295.12 (Mꢄþ, 56.9), 262.17(100),
145.13(24.8), 105.10(6.9), 78.05(57.1), 63.03(62.3); Analysis. Calcd
for C17H17N3S: C, 69.12; H, 5.80; N, 14.22; S, 10.85. Found: C, 69.05;
H, 5.63; N, 14.09; S, 10.63.
3.4.6. 5-(4-Chlorophenyl)-4-(2,6-dimethylphenyl)-2,4-dihydro-3H-
1,2,4-triazole-3-thione (8f)
White crystalline solid (72%, 0.31 g): m.p 257e260 ꢀC; Rf: 0.62
(n-hexane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3206 (NH), 2984 (sp3
CH), 1565 (C]N), 1503, 1481 (C]C), 1222 (C]S);1H NMR
3.4.2. 5-(2-Chlorophenyl)-4-(2,6-dimethylphenyl)-2,4-dihydro-3H-
1,2,4-triazole-3-thione (8b)
White crystalline solid (63%, 0.28 g): m.p 282e284 ꢀC; Rf: 0.64
(n-hexane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3352 (NH), 2939 (sp3
CH), 1552 (C]N), 1498, 1475 (C]C), 1233 (C]S); 1H NMR
(300 MHz, DMSO-d6):
d
14.43 (s, 1H, NH), 7.40 (d, 2H, J ¼ 9.3 Hz,
AreH), 7.33 (dd, 1H, J ¼ 9.0,9.0 Hz, AreH), 7.24 (d, 2H, J ¼ 8.7 Hz,
AreH), 7.23 (d, 2H, J ¼ 7.2 Hz, AreH), 1.93 (s, 6H, 2ꢃ CH3); 13C NMR
(300 MHz, DMSO-d6):
d
14.40 (s, 1H, NH), 7.60 (d, 1H, J ¼ 7.5 Hz,
(75 MHz, DMSO-d6): d 167.87, 148.12, 136.38, 135.51, 133.51, 130.21,
AreH), 7.46 (dt, 1H, J ¼ 7.8,1.5 Hz, AreH), 7.30e7.20 (m, 2H, AreH),
129.54, 128.47, 125.46, 124.21, 18.05; EI-MS: m/z (rel. abund. %),
7.14e7.09 (m, 3H, AreH) 2.05 (s, 6H, 2ꢃ CH3); 13C NMR (75 MHz,
317.00 (Mꢄ þ 2, 15.3), 315.00 (Mꢄþ, 40.7), 284.10(25.5), 282.16
þ
DMSO-d6):
d
167.00, 148.20, 136.61, 133.34, 133.03, 131.96, 131.72,
(80.2), 145.05(30.3), 91.12(9.1), 78.02(82.4), 63.10(100). Analysis.

I. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 5200e5207
5205
Calcd for C16H14ClN3S: C, 60.85; H, 4.47; N, 13.31; S, 10.15. Found: C,
60.71; H, 4.38; N, 13.25; S, 9.91.
3.5. General procedure for the synthesis of 2,5-disubstituted
thiadiazoles (9aeh)
3.4.7. 5-(3-Chlorophenyl)-4-(2,4-dimethylphenyl)-2,4-dihydro-3H-
1,2,4-triazole-3-thione (8g)
The corresponding carbothioamide (7) (0.0014 moles) in poly-
phosphoric acid was stirred overnight at 70 ꢀC. After completion of
the reaction, the cooled solution was poured onto the crushed ice.
The reaction mixture was extracted with ethyl acetate (3 ꢃ 20 mL)
and the combined extracts were washed with sodium bicarbonate
(5%) and water until the washings were neutral. The organic layer
was dried with anhydrous sodium sulphate and concentrated
under reduced pressure to yield 2,5-disubstituted-1,3,4-thiadiazole
(9) which was purified by recrystallization from ethanol.
White crystalline solid (59%, 0.26 g): m.p 260e261 ꢀC; Rf: 0.51
(n-hexane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3201 (NH), 2943
(sp3 CH), 1515 (C]N), 1496, 1486 (C]C), 1274 (C]S); 1H NMR
(300 MHz, DMSO-d6):
d 14.30 (s, 1H, NH), 7.48 (dd, 1H,
J ¼ 2.1,0.9 Hz, AreH), 7.38 (t, 1H, J ¼ 8.1 Hz, AreH), 7.36 (d, 1H,
J ¼ 2.1 Hz, AreH), 7.23e7.20 (m, 3H, AreH), 7.14 (d, 1H, J ¼ 7.8 Hz,
AreH), 2.33 (s, 3H, CH3), 1.96 (s, 3H, CH3); 13C NMR (75 MHz,
DMSO-d6):
d 167.90, 148.21, 135.89, 135.41, 133.32, 132.52, 131.48,
130.51, 130.24, 130.19, 129.68, 127.40, 12þ6.56, 126.55, 20.91, 17.73;
3.5.1. N-(2,6-Dimethylphenyl)-5-(4-methylphenyl)-1,3,4-
thiadiazol-2-amine (9a)
þ
EI-MS: m/z (rel. abund. %), 317.00 (Mꢄ þ 2, 21.4), 315.01 (Mꢄ
,
57.9), 284.06(35.3), 282.03(100), 162.90(8.2), 145.05(24.2), 105.01
(16.4), 91.00(17.4), 77.00(26.2), 51.10(12.8). Analysis. Calcd for
C16H14ClN3S: C, 60.85; H, 4.47; N, 13.31; S, 10.15. Found: C, 60.77; H,
4.36; N, 13.25; S, 9.97.
White solid (58%, 0.24 g): m.p 245e248 ꢀC; Rf: 0.48 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3124 (NH), 2845 (sp3 CH),
1593 (C]N), 1551 (C]C); 1H NMR (300 MHz, DMSO-d6):
d 9.54 (s,
1H, NH), 7.63 (d, 2H, J ¼ 8.1 Hz, AreH), 7.25 (d, 2H, J ¼ 8.1 Hz, AreH),
7.16 (bs, 3H, AreH), 2.32 (s, 3H, CH3), 2.22 (s, 6H, 2ꢃ CH3); 13C NMR
3.4.8. 4-(2,3-Dimethylphenyl)-5-(2-methylphenyl)-2,4-dihydro-
3H-1,2,4-triazole-3-thione (8h)
(75 MHz, DMSO-d6): d 168.83, 157.04, 140.01, 138.66, 135.95, 130.14,
128.99, 128.46, 126.78, 21.36, 18.36; EI-MS: m/z (rel. abund. %),
295.32 (Mꢄþ, 100), 280.31(39.6), 178.20(93.6), 163.17(16.5), 145.21
(66.5), 135.14(23.0), 119.17(26.6), 91.14(26.6), 77.12(11.7). Analysis.
Calcd for C17H17N3S: C, 69.12; H, 5.80; N, 14.22; S, 10.85. Found: C,
69.08; H, 5.59; N, 14.11; S, 10.69.
White crystalline solid (57%, 0.23 g): m.p 244e247 ꢀC; Rf: 0.63
(n-hexane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3277 (NH), 2905
(sp3 CH), 1563 (C]N), 1500, 1482 (C]C), 1255 (C]S); 1H NMR
(300 MHz, DMSO-d6):
d 14.18 (s, 1H, NH), 7.31e7.21 (m, 2H,
AreH), 7.18 (dd, 2H, J ¼ 6.3,2.4 Hz, AreH), 7.12e7.05 (m, 3H,
AreH), 2.24 (s, 3H, CH3), 2.20 (s, 3H, CH3), 1.90 (s, 3H, CH3); 13C
3.5.2. 5-(2-Chlorophenyl)-N-(2,6-dimethylphenyl)-1,3,4-
thiadiazol-2-amine (9b)
NMR (75 MHz, DMSO-d6):
d 168.14, 150.86, 138.32, 138.07, 134.96,
133.42, 131.29, 130.97, 130.82, 130.64, 127.29, 126.44, 126.04,
125.74, 20.24, 20.06, 14.84; EI-MS: m/z (rel. abund. %), 295.10
(Mꢄþ, 81.1), 262.00(100), 163.02(10.5), 144.90(17.7), 131.05(8.5),
117.04(16.3), 91.00(13.7), 77.01(19.8). Analysis. Calcd for
C17H17N3S: C, 69.12; H, 5.80; N, 14.22; S, 10.85. Found: C, 68.99; H,
5.54; N, 14.17; S, 10.66.
White solid (56%, 0.25 g): m.p 240e241 ꢀC; Rf: 0.45 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3134 (NH), 2846 (sp3 CH),
1592 (C]N), 1554 (C]C); 1H NMR (300 MHz, DMSO-d6):
d 9.65 (s,
1H, NH), 8.05e8.02 (m, 1H, AreH), 7.60e7.57 (m, 1H, AreH),
7.497.45 (m, 2H, AreH), 7.16 (bs, 3H, AreH), 2.22 (s, 6H, 2ꢃCH3); 13
C
NMR (75 MHz, DMSO-d6):
d 170.64, 152.19, 138.28, 135.95, 131.62,
130.98, 130.82, 130.68, 129.63þ, 129.05, 128.23, 127.80, 18.34; EI-MS:
m/z (rel. abund. %), 317.04 (Mꢄ þ 2, 33.1), 315.05 (Mꢄþ, 78.4), 302.02
(15.7), 300.02(36.3), 178.00(100), 163.02(29.1), 154.97(26.5), 145.09
(97.3), 136.99(13.0), 119.06(40.8), 77.03(16.6). Analysis. Calcd for
C16H14ClN3S: C, 60.85; H, 4.47; N, 13.31; S, 10.15. Found: C, 60.59; H,
4.41; N, 13.12; S, 9.88.
3.4.9. 5-(3-Chlorophenyl)-4-(2,6-dimethylphenyl)-2,4-dihydro-3H-
1,2,4-triazole-3-thione (8i)
White crystalline solid (66%, 0.29 g): m.p 224e226 ꢀC; Rf: 0.51
(n-hexane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3296 (NH), 2922
(sp3CH), 1576 (C]N), 1541, 1473 (C]C), 1235 (C]S); 1H NMR
(300 MHz, DMSO-d6):
d
14.42 (s, 1H, NH), 7.53 (dd, 1H, J ¼ 2.7,
1.8 Hz, AreH), 7.41e7.35 (m, 2H, AreH), 7.28e7.19 (m, 4H, AreH),
3.5.3. 5-(4-Chlorophenyl)-N-(2,3-dimethylphenyl)-1,3,4-
thiadiazol-2-amine (9c)
1.97 (s, 6H, 2ꢃ CH3); 13C NMR (75 MHz, DMSO-d6):
d 168.20, 148.77,
136.42, 133.80, 133.35, 132.84, 131.48, 131.19, 130.65, 129.34, 125.65,
125.56, 17.95; EI-MS: m/z (rel. abund. %), 317.00 (Mꢄþ þ 2, 24.7),
314.90 (Mꢄþ, 62.1), 283.91(36.3), 282.02(100), 144.90(26.7), 136.92
(10.8), 105.00(17.6), 91.10(15.8), 77.00(27.9), 51.10(10.5). Analysis.
Calcd for C16H14ClN3S: C, 60.85; H, 4.47; N, 13.31; S, 10.15. Found: C,
60.74; H, 4.26; N, 13.16; S, 9.96.
Yellow solid (39%, 0.17 g): m.p 252e254 ꢀC; Rf: 0.47 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3212 (NH), 2907 (sp3 CH),
1600 (C]N), 1561, 1501 (C]C); 1H NMR (300 MHz, DMSO-d6):
d
9.80 (s, 1H, NH), 7.82e7.79 (m, 2H, AreH), 7.55e7.47 (m, 3H,
AreH), 7.12 (t, 1H, J ¼ 7.8 Hz, AreH), 7.03 (d, 1H, J ¼ 7.5 Hz, AreH),
2.27 (s, 3H, CH3), 2.16 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):
d
168.48, 156.14, 139.60, 138.13, 134.82, 130.28, 129.89, 129.70,
3.4.10. 5-(4-Chlorophenyl)-4-(2,4-dimethylphenyl)-2,4-dihydro-
3H-1,2,4-triazole-3-thione (8j)
127.28, 126.49, 121.57, 20.70, 14.34; EI-MS: m/z (rel. abund. %),
þ
317.24 (Mꢄ þ 2, 29.4), 315.22 (Mꢄþ, 57.7), 302.22(10.4), 300.19
White crystalline solid (75%, 0.33 g): m.p 285e286 ꢀC; Rf: 0.48
(n-hexane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3346 (NH), 2986 (sp3
CH), 1555 (C]N), 1485 (C]C), 1276 (C]S); 1H NMR (300 MHz,
(26.7), 282.24(10.7), 178.15(59.1), 163.13(23.8), 155.07(28.2), 145.17
(100), 137.07(18.3), 119.15(25.4), 111.05(11.5), 91.10(14.1), 77.09
(18.4). Analysis. Calcd for C16H14ClN3S: C, 60.85; H, 4.47; N, 13.31; S,
10.15. Found: C, 60.62; H, 4.35; N, 12.89; S, 10.02.
DMSO-d6):
2H, J ¼ 8.7 Hz, AreH), 7.20e7.11 (m, 3H, AreH), 2.32 (s, 3H, CH3),
1.96 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):
168.85, 149.94,
d
14.24 (s, 1H, NH), 7.44 (d, 2H, J ¼ 8.4 Hz, AreH), 7.30 (d,
d
3.5.4. N-(2,4-Dimethylphenyl)-5-(3-nitrophenyl)-1,3,4-thiadiazol-
2-amine (9d)
140.11, 135.94, 135.84, 132.19, 131.30, 129.78, 129.41, 128.33, 125.49,
125.09, 21.21, 17.71; EI-MS: m/z (rel. abund. %), 317.18 (Mꢄþ þ 2,
37.3), 315.19 (Mꢄþ, 93.7), 300.16(9.2), 284.21(51.5), 282.14(100),
163.11(7.8), 145.17(26.3), 105.13(8.3), 91.10(7.1), 78.08(13.4), 77.09
(9.4), 63.05(13.1). Analysis. Calcd for C16H14ClN3S: C, 60.85; H, 4.47;
N, 13.31; S, 10.15. Found: C, 60.66; H, 4.41; N, 13.11; S, 9.87.
Yellow solid (45%, 0.20 g): m.p 240e243 ꢀC; Rf: 0.40 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3287 (NH), 2953 (sp3 CH),
1602 (C]N), 1580, 1556 (C]C); 1H NMR (300 MHz, DMSO-d6):
d
9.81 (s, 1H, NH), 8.54 (s, 1H, AreH), 8.28 (d, 1H, J ¼ 8.4 Hz, AreH),
8.19 (d, 1H, J ¼ 7.8 Hz, AreH), 7.79e7.59 (m, 2H, AreH), 7.76 (t, 1H,

5206
I. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 5200e5207
Table 1
J ¼ 8.1 Hz, AreH), 7.09e7.04 (m, 1H, AreH), 2.27 (s, 3H, CH3), 2.24
(s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):
168.83, 150.87,
DPPH radical scavenging activities of the synthesized compounds (8aej) and
(9aeh).
d
148.69, 136.98, 134.79, 133.25, 132.43, 131.91, 131.44, 130.99,
127.77, 124.63, 123.22, 120.75, 20.90, 14.35; EI-MS: m/z (rel.
abund. %), 326.10 (Mꢄþ, 4.8), 163.10(10.3), 148.00(25.9), 118.02
(23.2), 102.06(58.5), 77.07(13.3), 75.00(23.0), 64.02(100). Anal-
ysis. Calcd for C16H14N4O2S: C, 58.88; H, 4.32; N, 17.17; S, 9.82.
Found: C, 58.68; H, 4.18; N, 16.93; S, 9.67.
Compounds
Conc. (mM)
% RSA
IC50 ꢁ SEM (
e
e
e
mM)
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
9a
9b
9c
9d
9e
9f
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
e
24.55
23.64
20.29
72.81
41.57
16.82
11.03
15.14
24.46
63.27
46.32
38.59
47.95
45.37
15.28
58.06
34.97
38.42
90.31
242.49 ꢁ 3.64
e
e
e
3.5.5. N-(2,3-Dimethylphenyl)-5-phenyl-1,3,4-thiadiazol-2-amine
(9e)
e
e
White solid (43%, 0.17 g): m.p 235e238 ꢀC; Rf: 0.36 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3178 (NH), 2917 (sp3 CH),
1601 (C]N), 1576, 1556 (C]C); 1H NMR (300 MHz, DMSO-d6):
292.38 ꢁ 1.60
e
e
e
d
9.74 (s, 1H, NH), 7.81e7.76 (m, 2H, AreH), 7.51e7.44 (m, 4H,
e
AreH), 7.13 (t, 1H, J ¼ 7.8 Hz, AreH), 7.03 (d, 1H, J ¼ 7.2 Hz, AreH),
e
2.27 (s, 3H, CH3), 2.17 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):
319.62 ꢁ 2.04
d
168.12, 157.36, 139.70, 138.08, 131.03, 130.40, 130.19, 129.67,
9g
9h
e
e
127.17, 126.96, 126.47, 121.48, 20.72, 14.35; EI-MS: m/z (rel. abund.
%), 280.99 (Mꢄþ, 35.3), 265.95(19.6), 248.04(14.8), 204.00(10.9),
177.98(43.1), 162.97(36.3), 145.03(92.4), 131.02(11.0), 120.98(60.3),
103.00(74.2), 91.02(43.7), 77.05(100), 51.01(27.3). Analysis. Calcd
for C16H15N3S: C, 68.30; H, 5.37; N, 14.93; S, 11.40. Found: C, 68.17;
H, 5.13; N, 14.72; S, 11.19.
Std (n-propyl gallate)
30.46 ꢁ 0.27
e, Not done.
130.25, 129.90, 129.70, 129.35, 126.23, 121.54, 20.71, 14.35; EI-MS:
m/z (rel. abund. %), 326.10 (Mꢄþ, 20.6), 178.00(17.3), 163.02(21.5),
148.06(22.9), 145.11(27.2), 119.90(17.7), 119.00(20.5), 118.08(33.2),
102.07(51.6), 77.11(18.5), 75.05(19.3), 64.00(100), 51.10(20.4).
Analysis. Calcd for C16H14N4O2S: C, 58.88; H, 4.32; N, 17.17; S, 9.82.
Found: C, 58.63; H, 4.14; N, 17.01; S, 9.69.
3.5.6. 5-(4-Chlorophenyl)-N-(2,6-dimethylphenyl)-1,3,4-
thiadiazol-2-amine (9f)
White solid (54%, 0.24 g): m.p 231e234 ꢀC; Rf: 0.43 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3412 (NH), 2857 (sp3 CH),
1577 (C]N), 1484 (C]C); 1H NMR (300 MHz, DMSO-d6):
d 9.69 (s,
3.6. Pharmacological assays
1H, NH), 7.77 (d, 2H, J ¼ 8.7 Hz, AreH), 7.51 (d, 2H, J ¼ 8.7 Hz, AreH),
7.16 (bs, 3H, AreH), 2.22 (s, 6H, 2ꢃ CH3); 13C NMR (75 MHz, DMSO-
The antioxidant activities against DPPH radical and superoxide
anion scavenging activities were measured for new triazole (8aej)
and thiadiazole (9aeh) derivatives which are shown in Tables 1
and 2, respectively. The synthesized compounds were also screened
for their urease inhibition activity which is shown in Table 3. The
compounds were found inhibiting the urease in variable concentra-
tions. The standard thiourea was used as a reference material.
d6):
d 169.50, 155.73, 138.51, 135.95, 134.66, 129.99, 129.65, 129.02,
128.46, 127.77, 18.32; EI-MS: m/z (rel. abund. %), 317.21 (Mꢄþ þ 2,
15.8), 315.19 (Mꢄþ, 52.8), 302.14(7.0), 300.19(26.5), 282.22(11.6),
178.15(97.3), 163.13(25.2), 155.05(34.8), 137.07(100), 119.13(27.7),
91.10(15.1), 77.09(18.2), 64.01(60.8), 47.96(19.0); Analysis. Calcd for
C16H14ClN3S: C, 60.85; H, 4.47; N, 13.31; S, 10.15. Found: C, 60.61; H,
4.33; N, 13.14; S, 9.90.
3.6.1. Antioxidant assay (DPPH scavenging assay)
The free radical scavenging capacity of the compounds was
measured by modified 1,1-diphenyl-2-picrylhydrazyl (DPPH)
3.5.7. 5-(3-Chlorophenyl)-N-(2,4-dimethylphenyl)e1,3,4-
thiadiazol-2-amine (9g)
White solid (67%, 0.30 g): m.p 274e277 ꢀC; Rf: 0.57 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3352 (NH), 2952 (sp3 CH),
1594 (C]N), 1567, 1485 (C]C); 1H NMR (300 MHz, DMSO-d6):
Table 2
Superoxide ion scavenging activities of the synthesized compounds (8aej) and
(9aeh).
d
9.71 (s, 1H, NH), 7.84 (s, 1H, AreH), 7.76e7.72 (m, 1H, AreH), 7.61
(d,1H, J ¼ 8.1 Hz, AreH), 7.51e7.49 (m, 2H, AreH), 7.07e7.03 (m, 2H,
AreH), 2.26 (s, 3H, CH3), 2.24 (s, 3H, CH3); 13C NMR (75 MHz,
Compounds
Conc. (mM)
% RSA
IC50 ꢁ SEM (
mM)
DMSO-d6):
d
168.31, 155.79, 137.09, 134.53, 134.33, 132.96, 131.86,
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
9a
9b
9c
9d
9e
9f
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
131.60, 130.80, 130.09, 127.70, 126.08,þ125.78, 123.07, 20.90, 18.25;
EI-MS: m/z (rel. abund. %), 317.00 (Mꢄ þ 2, 2.2), 315.00 (Mꢄþ, 6.5),
178.02(14.1), 163.10(10.0), 154.90(61.2), 136.90(100), 118.05(41.3),
110.91(34.0), 91.10(36.0), 77.00(38.6), 75.06(40.0), 64.01(83.0),
51.10(28.2). Analysis. Calcd for C16H14ClN3S: C, 60.85; H, 4.47; N,
13.31; S, 10.15. Found: C, 60.81; H, 4.27; N, 13.05; S, 10.01.
e
e
e
e
e
e
e
e
3.5.8. N-(2,4-Dimethylphenyl)e5e(4-nitrophenyl)e1,3,4-
thiadiazol-2-amine (9h)
e
18.35
94.65
98.58
3.24
87.21
95.37
99.32
90.31
Yellow solid (41%, 0.19 g): m.p 263e267 ꢀC; Rf: 0.42 (n-hex-
ane:ethyl acetate, 8:2); IR (pure, cmꢂ1): 3460 (NH), 2937 (sp3 CH),
1585 (C]N), 1557, 1540 (C]C); 1H NMR (300 MHz, DMSO-d6):
138.64 ꢁ 1.74
130.76 ꢁ 2.57
e
255.21 ꢁ 3.89
124.30 ꢁ 1.46
94.84 ꢁ 0.34
106.34 ꢁ 1.70
d
9.80 (s, 1H, NH), 7.81 (d, 2H, J ¼ 8.4 Hz, AreH), 7.54e7.48 (m, 1H,
9g
9h
AreH), 7.53 (d, 2H, J ¼ 8.4 Hz, AreH), 7.15e7.07 (m, 1H, AreH), 7.03
(d, 1H, J ¼ 7.2 Hz, AreH), 2.27 (s, 3H, CH3), 2.17 (s, 3H, CH3); 13C NMR
Std (n-propyl gallate)
(75 MHz, DMSO-d6):
d
168.45, 159.54, 156.11, 138.21, 138.11, 134.81,
e, Not done.

I. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 5200e5207
5207
Table 3
incubated with 5 mL of the test synthesized compounds (0.5 mM
Urease inhibition activities of the synthesized compounds (8aej) and (9aeh).
concentration) at 30 ꢀC for 15 min in 96-well plates. Urease activity
was determined by measuring ammonia production using the
indophenol method as described by weatherburn [27]. Briefly,
Compounds
Conc. (mM)
% Inhibition
IC50 ꢁ SEM (
e
e
e
mM)
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
9a
9b
9c
9d
9e
9f
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
e
6.8
48.3
48.3
97.0
93.4
7.5
53.1
43.4
64.8
88.6
27.1
82.8
52.8
73.4
56.8
91.1
12.4
7.2
45
mL each of phenol reagent (1% w/v phenol and 0.005% w/v
sodium nitroprusside) and 70
m
L of alkali reagent (0.5% w/v NaOH
45.60 ꢁ 0.04
86.0 ꢁ 0.04
and 0.1% active chloride NaOCl) were added to each well. The
increasing absorbance at 630 nm was measured after 50 min, using
a microplate reader (Molecular Device, USA). All the reactions were
e
459.56 ꢁ 2.26
e
performed in triplicate in a final volume of 200 mL. The results
357.03 ꢁ 1.90
170.76 ꢁ 0.23
e
(change in absorbance per min) were processed by using softMax
Pro software (Molecular Device, USA). The entire assays were per-
formed at pH 6.8. Percentage inhibitions were calculated from the
formula (Table 3):
313.2 ꢁ 0.09
483.55 ꢁ 1.99
398.2 ꢁ 0.40
426.1 ꢁ 0.00
214.7 ꢁ 0.16
e
100 ꢂ ðODtestwell=ODcontrolÞ ꢃ 100:
9g
9h
Thiourea was used as the standard inhibitor of urease [28].
e
Std (thiourea)
e
21 ꢁ 0.11
e, Not done.
Acknowledgment
methods described by M. I. Choudhary et al [23]. Test compounds
were allowed to react with stable free radical, 1,1-diphenyl-2-pic-
rylhydrazyl radical (DPPH) for half an hour at 37 ꢀC. The concen-
Financial support from Higher Education Commission (HEC)
Islamabad-45320, Pakistan is gratefully acknowledged.
tration of DPPH was kept as 300 mM. The test samples were
dissolved in DMSO while the DPPH solution was prepared in
ethanol. After incubation, decrease in absorption was measured at
515 nm using multiplate reader (Spectra MAX-384). Percent radical
scavenging activity (RSA) of samples was determined in compar-
ison with a DMSO treated control group [24,25] using the following
formula (Table 1):
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3.6.2. Superoxide anion scavenging assay
Measurement of superoxide radical scavenging activity (Table 2)
was carried out by the modified method used by Ferda [26]. The
reaction mixture comprised of 40
adenine dinucleotide reduced form (NADH), 40
blue tetrazolium (NBT), 20 L phenazine methosulphate (PMS)
10 L of 1 mM sample and 90 L of 0.1 M phosphate buffer (pH 7.4).
mL of 280
m
M
b
-nicotinamide
mL of 80 M nitro
m
m
m
m
Reagents were prepared in buffer solution and the sample in DMSO.
The reaction was performed in 96-well microtitre plate at room
temperature and absorbance was measured at 560 nm. The
formation of superoxide was monitored by measuring the forma-
tion of water soluble blue formazan dye. A lower absorbance of
reaction mixture indicates a higher scavenging activity of the
sample. Percent Radical Scavenging Activity (% RSA) by samples can
be determined in comparison with a control [26].
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3.6.3. Urease inhibition assay
Reaction mixtures comprising 25
mL of enzyme (jack bean
urease) solution and 55 L of buffers containing 100 mM urea were
m