Synthesis and in vitro urease inhibitory activity of N,N′- disubstituted thioureas

Article abstract of DOI:10.1016/j.ejmech.2014.01.001

Thiourea derivatives (1-38) were synthesized and evaluated for their urease inhibition potential. The synthetic compounds showed a varying degree of in vitro urease inhibition with IC₅₀ values 5.53 ± 0.02-91.50 ± 0.08 μM, most of which are superior to the standard thiourea (IC₅₀ = 21.00 ± 0.11 μM). In order to ensure the mode of inhibition of these compounds, the kinetic study of the most active compounds has been carried out. Most of these inhibitors were found to be mixed-type of inhibitors, except compounds 13 and 30 which were competitive, while compound 19 was identified as non-competitive inhibitor with Ki values between 8.6 and 19.29 μM.

Full text of DOI:10.1016/j.ejmech.2014.01.001

European Journal of Medicinal Chemistry 74 (2014) 314e323
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro urease inhibitory activity of N,N0-disubstituted
thioureas
,
a
b,
Khalid Mohammed Khan a , Farzana Naz , Muhammad Taha d, Ajmal Khan a,
*
Shahnaz Perveen c, M.I. Choudhary a, Wolfgang Voelter e
a H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University Karachi, Karachi 75270, Pakistan
b Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor
D. E., Malaysia
c PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi 75280, Pakistan
d Faculty of Applied Science UiTM, 40450 Shah Alam, Selangor, Malaysia
e Interfakultäres Institut für Biochemie der Universität Tübingen, Hoppe-Seyler Straße 4, D-72076 Tübingen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Thiourea derivatives (1e38) were synthesized and evaluated for their urease inhibition potential. The
synthetic compounds showed a varying degree of in vitro urease inhibition with IC50 values 5.53 ꢀ 0.02
Received 19 November 2013
Received in revised form
27 December 2013
Accepted 2 January 2014
Available online 14 January 2014
e91.50 ꢀ 0.08
m
M, most of which are superior to the standard thiourea (IC50 ¼ 21.00 ꢀ 0.11
mM). In order
to ensure the mode of inhibition of these compounds, the kinetic study of the most active compounds
has been carried out. Most of these inhibitors were found to be mixed-type of inhibitors, except com-
pounds 13 and 30 which were competitive, while compound 19 was identified as non-competitive in-
hibitor with Ki values between 8.6 and 19.29 mM.
Keywords:
Ó 2014 Elsevier Masson SAS. All rights reserved.
Thiourea derivatives
Synthesis
In vitro urease inhibition activity
Kinetic study
1. Introduction
[6]. It is also known to be a major cause of pathologies induced by
Helicobacter pylori which allows the bacteria to survive at the low
pH of the stomach during colonization and therefore plays an
important role in the pathogenesis of gastric and peptic ulcers
which may lead to cancer, Since urease is not only damaging for
humans, but also for animals and in agriculture, various strategies
based on urease inhibition were considered as treatment ap-
proaches for infections caused by urease-producing bacteria [7,8].
Moreover, H. pylori are recognized as a major risk factor for
recurrent gastroduodenal inflammatory diseases and gastric
adenocarcinoma [9]. Ureases play an important role in protecting
the H. pylori from the effects of gastric acid [10]. Medically, ureases
are important virulence factors implicated in the pathogenesis of
many clinical conditions such as pyelonephritis, hepatic coma,
peptic ulceration, and the formation of infection-induced urinary
stones and reactive arthritis [11].
Thiourea is an organic compound with the molecular formula
CSN2H4. It is a versatile reagent in synthetic chemistry. Thioureas
are related to thioamides and can be prepared from ammonium
thiocyanate, but more commonly by the reaction of phenyl iso-
thiocyanate with alkyl or aryl amines. They are important building
blocks in the synthesis of heterocycles [12]. Thioureas manifest
important multiple biological effects and are the basis for target-
Urease (urea amidohydrolase EC 3.5.1.5) is a nickel-containing
enzyme that catalyzes the hydrolysis of urea to ammonia and CO2
or carbamate [1]. A variety of ureases are found in bacteria, fungi,
higher plants and in soil as a soil enzyme [2] Activity of urease (E.C
3.5.1.5) has been shown to be an important virulence determinant
in the pathogenesis of many clinical conditions which are detri-
mental for human and animal health as well as for agriculture. In
agriculture, high urease activity causes significant environmental
and economic problems by releasing abnormally large amounts of
ammonia into the atmosphere during urea fertilization [3]. More-
over, it induces plant damage primarily by depriving plants of their
essential nutrients and secondarily by ammonia toxicity, increasing
the pH of the soil [4].
Urease has been implicated to play a role in the pathogenesis of
many bacteria such as Helicobacter pylori, Proteus mirabilis and
Brucella abortus [5]. Urease has been identified as an immunogenic
modulator in several pathogen-induced inflammatory reactions
* Corresponding author. Tel.: þ92 2134824910; fax: þ92 2134819018.
E-mail addresses: hassaan2@super.net.pk, khalid.khan@iccs.edu (K.M. Khan).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.001

K.M. Khan et al. / European Journal of Medicinal Chemistry 74 (2014) 314e323
315
Table 1
oriented synthesis. Moreover, ureas and thioureas evaluate their
Synthesis of symmetrical and unsymmetrical N,N0-disubstituted thioureas 1e38.
plant growth-regulating activity mainly on the herbicidal, root
growth inhibitory and stimulatory and cytokinin-like activities [13].
Thioureas and ureas (symmetrical or unsymmetrical) have attrac-
ted much attention as drug candidates against a variety of diseases
due to their bioactivities and broad spectrum as pesticides and in
pharmacological activities [14a]. A variety of thiourea derivatives
and their metal complexes exhibit analgesic, anti-inflammatory
[15], anticancer, antitumor [16], antiplatelet [17] antimalarial [18],
and antimicrobial activities [19,20]. Thiourea derivatives also
possess anti-HCV [21], anti-HIV, antituberculosis, and antileukemic
activity [22]. Fluorinated thioureas constitute a novel class of
potent influenza virus neuraminidase inhibitors [23].
Compound
R1
Cl
R2
Compound
R1
Cl
R2
N
1
20
Me
Cl
In continuation of our research work on urea and thiourea de-
rivatives as potential lead compounds in our drug discovery pro-
gram and keeping in mind the urease inhibitory activity of thiourea
[24], we have synthesized a variety of thioureas and screened them
for their urease inhibitory properties.
2
3
4
5
Cl
Cl
Cl
Cl
21
22
23
24
Cl
Cl
Cl
Cl
N
N
Cl
Cl
2. Results and discussion
N
MeO
2.1. Chemistry
Cl
OMe
We have previously reported the synthesis of unsymmetrically-
substituted N,N0-diaryl thioureas and urea derivatives in excellent
yields and their in vitro bioactivities determined as cytotoxic,
phytotoxic, acetylcholinesterase, butyrylcholinesterase [14a], and
anti-glycation activities [14b]. Along with that we have being
working on sulfur containing compounds [25], as well as hydra-
zones in search lead molecules [26]. In the present study thirty-
eight unsymmetrically N,N0-diaryl-substituted thioureas 1e38
were synthesized from commercially available phenyl and 3-
chlorophenyl isothiocyanate with a variety of substituted anilines
and heterocyclic amines in dichloromethane at 0 ꢁC for 45 min
according to literature procedures [27], as shown in Scheme 1
(Table 1). In all experiments, solid materials were formed which
were filtered, triturated with 10 mL of dichloromethane and excess
of hexane, dried under vacuum and recrystallized with methanol.
The structures were determined using different spectroscopic
methods like 1H NMR, EI MS, IR and gave satisfactory CHN analysis.
The compounds 1, 8, 10, 15, 25e32, 34, 37 are reported by various
research groups [28].
N
6
7
Cl
Cl
25
26
H
H
OMe
Me
Br
Br
Me
Me
F
8
Cl
27
H
Me
Me
Cl
F
Me
OMe
9
Cl
Cl
28
29
H
H
MeO
Cl
N
10
2.2. In vitro urease inhibitory evaluation
Cl
N
N
Cl
11
12
Cl
Cl
30
31
H
H
All the synthetic thioureas were screened for their urease
inhibitory assays according to literature protocol [29]. Most of the
38 thiourease 1e38, demonstrated good in vitro urease inhibitory
properties having IC50 values in the range of 5.53 ꢀ 0.02e
Cl
82.70 ꢀ 0.14
mM, whereas standard inhibitor, thiourea, has an IC50
Cl
Cl
value 21.00 ꢀ 0.08
mM, respectively. Compounds 1, 3, 4, 9, 13e16,
18e20, 26, and 30 exhibiting IC50 values 20.00 ꢀ 0.02, 20.33,
20.06 ꢀ 0.02, 20.90 ꢀ 0.02, 11.03 ꢀ 0.02, 20.10 ꢀ 0.00, 19.10 ꢀ 0.00,
16.30 ꢀ 0.02, 15.03 ꢀ 0.02, 18.60 ꢀ 0.04, 15.90 ꢀ 0.00, 18.30 ꢀ 0.14,
N
13
14
Cl
Cl
32
33
H
H
Cl
Cl
Me
Cl
N
Mel
Cl
15
16
Cl
Cl
34
35
H
H
O
F
F
N
Me
Br
F
(continued on next page)
Scheme 1. Synthetic Scheme of Arylthiourea 1e38.

316
K.M. Khan et al. / European Journal of Medicinal Chemistry 74 (2014) 314e323
inactivity found for N-(3-chlorophenyl)-N0-(2-chloro-3-pyridinyl)
Table 1 (continued )
thiourea (24) with a close structure to N-(2-chloro-3-pyridinyl)-N0-
phenylthiourea (36). A general trend in the decline of inhibiting ac-
tivity is observed comparing the N-phenyl derivatives, N-phenyl-N0-
Compound
R1
R2
Compound
R1
H
R2
Cl
17
Cl
36
M), N-phenyl-N0-(3-
M), and N-phenyl-N0-
M) with their
N
(3-pyridinyl) thiourea (30) (IC50 ¼ 8.43 ꢀ 0.02
pyridinyl) thiourea (37) (IC50 ¼ 33.50 ꢀ 0.04
(4-pyridinylmethyl)thiourea (38) (IC50 ¼ 32.60 ꢀ 0.07
chloro analogs N-(3-chlorophenyl)-N0-(3-pyridinyl)thiourea (23)
(IC50 ¼ 24.46 ꢀ 0.02
M), N-(3-chlorophenyl)-N0-(3-pyridinylmethyl)
thiourea (22) (IC50 ¼ 23.03 ꢀ 0.02
M), and N-(3-chlorophenyl)-N0-
M) demon-
m
Me
m
m
Me
Me
N
N
18
19
Cl
Cl
37
38
H
H
m
m
(4-pyridinylmethyl)thiourea (21) (IC50 ¼ 22.70 ꢀ 0.07
m
strating that the chlorine atom attached to the phenyl ring causes
only a minor influence on the chelating efficiency of the inhibitors.
Compounds 20 (IC50
¼
15.90
ꢀ
0.00
mM) and 32
and 8.43 ꢀ 0.02
mM, respectively, were found to be the most active
(IC50 ¼ 25.30 ꢀ 0.08 M) with a 3-methyl residue attached to the
m
members of this series and superior to the standard inhibitor, while
2, 5e9,11,17, 21e23, 25, 27e29, 32e34, 37 and 38 showed still good
inhibiting activity against ureases. Compounds 10,12, 24, 35, and 36
have shown minor or no inhibitory potential (Table 2).
pyridyl moiety were also found to be potent urease inhibitors. As
mentioned above, the chlorine atom attached to the phenyl residue
has only a minor effect on the inhibiting activity. N-(3,5-Dichloro-2-
pyridinyl)-N0-phenylthiourea (33), another pyridine-containing
N-(3-Chlorophenyl)-N0-(2-methoxyphenyl) thiourea (4) bearing
a 2-methoxyphenyl residue at R2 of the thiourea bridge was found
compound, showed an IC50 value of 37.03 ꢀ 0.07
m
M, while N-(5-
bromo-6-methyl-2-pyridinyl)-N0-phenylthiourea
(35)
to be the better active than standard (IC50 ¼ 20.06 ꢀ 0.02
activity of compound 4 as compared to that of its analogs 5 and 6
M, respectively), then it
mM). If the
(IC50 ¼ 82.70 ꢀ 0.14
mM) was found to be the least active compound
among the present series.
(IC50 values 29.56 ꢀ 0.07 and 24.10 ꢀ 0.04
m
N-(3-Chlorophenyl)-N0-(3,4-dichlorophenyl)
thiourea
(13)
appears that the position of the methoxy group at R2 plays a crucial
rule for urease inhibitory activity.
containing a dichlorophenyl residue at the thiourea bridge, showed a
M),
good urease inhibitory potential with an IC50 value 11.03 ꢀ 0.02
m
N-Phenyl-N0-(3-pyridinyl) thiourea (30), bearing a 3-pyridyl
substituent at R2 of the thiourea bridge, was the second most active
however, other dichlorophenyl-containing compounds, like 10, 11,
and 12, had IC50 values of 40.96 ꢀ 0.02, 27.10 ꢀ 0.04, and
compound (IC50 ¼ 8.43 ꢀ 0.02
tivity with those of the isomers N-phenyl-N0-(2-pyridinyl)thiourea
(29) (IC50 ¼ 23.36 ꢀ 0.02
M), and N-phenyl-N0-(4-pyridinyl)thiourea
(31) (IC50 ¼ 43.93 ꢀ 0.14 M), then the inhibiting power decreases
mM) of the series. Comparing its ac-
42.20 ꢀ 0.00
mM, respectively. This activity difference may be
explained on the basis of the different positions of dichloro sub-
stituents on the phenyl ring. In compound 13, the dichloro residues
are present at meta and para position to the thiourea bridge which
may be attend an orientation to block the active site of the urease.
However, in compound 11, the chloro substituents are at meta and
ortho position of the phenyl ring, whereas, in compounds 10 and 12
the chloro atoms are attached atortho, ortho and ortho, para positions,
respectively. Comparing the dichloro substitutions and activity dif-
ferences it appears that the meta chloro substituent is more influ-
ential on the urease inhibiting activity than the ortho and para
substituents: Compound 13 (meta-, para-disubstituted) showed
higher activity than compound 11 (ortho-, meta-disubstituted), and
m
m
from 3-pyridinyl >2-pyridinyl >4-pyridinyl analogs. Increasing the
distance of the 3-pyridinyl residue of 6 from the thiourea moiety by
a methyl spacer causes a dramatic decrease of the inhibiting
activity as demonstrated by N-phenyl-N0-(3-pyridinyl) thiourea (37)
(IC50 ¼ 33.50 ꢀ 0.04
mM). Introduction of a 2-chloro atom into the
pyridine ring of 30 as in N-(2-chloro-3-pyridinyl)-N0-phenylthiourea
(36), causes most probably due to the space-filling and electronic
property of the chlorine atom a complete loss of chelation with the
enzyme’s active site. This suggestion is also confirmed by the total
compounds 10 (IC50 ¼ 40.96 ꢀ 0.02
m
M) and 12 (42.20 ꢀ 0.00
mM),
lacking a meta-chloro substituent, had the least urease inhibitory
activity. A same pattern of activity was observed for the mono-
chlorinated compounds 1, 2, and 3 exhibiting IC50 values of
Table 2
In vitro urease inhibitory activity of compounds 1e38.
Compounds
IC50 ꢀ S.E.M.a
(m
M)
Compounds
IC50 ꢀ S.E.M.a
(mM)
20.00 ꢀ 0.02, 22.20 ꢀ 0.04, and 20.33 ꢀ 0.00
mM in accordance to the
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
20.00 ꢀ 0.02
22.20 ꢀ 0.04
20.33 ꢀ 0.00
20.06 ꢀ 0.02
24.46 ꢀ 0.02
29.56 ꢀ 0.07
24.10 ꢀ 0.04
27.40 ꢀ 0.09
20.90 ꢀ 0.02
40.96 ꢀ 0.02
27.10 ꢀ 0.04
42.20 ꢀ 0.00
11.03 ꢀ 0.02
20.10 ꢀ 0.00
19.10 ꢀ 0.00
16.30 ꢀ 0.02
34.40 ꢀ 0.04
15.03 ꢀ 0.02
18.60 ꢀ 0.04
21.00 ꢀ 0.11
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
e
15.90 ꢀ 0.00
22.70 ꢀ 0.07
23.03 ꢀ 0.02
24.46 ꢀ 0.02
NAb
34.26 ꢀ 0.05
18.30 ꢀ 0.14
24.30 ꢀ 0.00
29.93 ꢀ 0.07
23.36 ꢀ 0.02
8.43 ꢀ 0.02
43.93 ꢀ 0.14
25.30 ꢀ 0.08
37.03 ꢀ 0.07
32.36 ꢀ 0.12
82.70 ꢀ 0.14
NAb
foregoing discussions. Generally, it was observed that halogen sub-
stitutionatmetapositionof the phenylringsignificantlyenhances the
urease inhibitory potential of unsymmetrically-substituted thioureas
as demonstrated by the following comparison: N-(3-Bromophenyl)-
N0-phenylthiourea (26) (IC50
¼
18.30
ꢀ
0.12 mM) >N-(4-
bromophenyl)-N0-phenylthiourea (25). The methyl residue at meta
position of the phenyl ring exerts an enhancing effect on the com-
pound’s inhibiting activity compared to the ortho- and para-phenyl
analogs: Monomethyl substitution 18 (N-(3-chlorophenyl)-N0-(3-
methylphenyl)thiourea (IC50 ¼ 15.03 ꢀ 0.02
chlorophenyl)-N0-(4-methylphenyl)thiourea (IC50
0.04
M)> dimethyl substitution, like9 (N-(3-chlorophenyl)-N0-(2,5-
dimethylphenyl)thiourea (IC50 ¼ 20.90 ꢀ 0.02 M) > 7 (N-(3-
chlorophenyl)-N0-(2,4-dimethylphenyl)thiourea (IC50
24.10
0.04
M) > 8 (N-(3-chlorophenyl)-N0-(2,6-dimethylphenyl)thiourea
(IC50 ¼ 27.40 ꢀ 0.09 M).
mM) > 19 (N-(3-
¼
18.60
ꢀ
m
m
17
18
19
¼
ꢀ
33.50 ꢀ 0.04
32.60 ꢀ 0.07
e
m
Thioureasc
m
The present studies demonstrate that substitutions by func-
tional groups attached to the phenyl or heterocyclic ring R2 to the
thiourea bridge exert a decisive influence on the urease inhibitory
a
S.E.M. the standard error of the mean.
Not active.
Standard inhibitor of the enzyme urease.
b
c

K.M. Khan et al. / European Journal of Medicinal Chemistry 74 (2014) 314e323
317
Table 3
The inhibition and kinetics parameters of urease by thiourea derivatives.
Compounds
IC50
(
m
M)
Km (mM)
Km app (mM)
Vmax
(
m
mol/min)ꢂ1
Vmax app (
m
mol/min)ꢂ1
Ki (
mM) ꢀ S.E.M
Type of inhibition
3
1
9
13
14
15
18
19
20.3
20.0
20.9
11.0
20.1
19.1
15.0
18.6
18.3
8.4
2.25
2.25
2.25
2.25
2.25
2.25
2.25
2.25
2.25
2.25
2.25
2.61
3.24
3
6
2.83
2.9
2.86
2.25
2.64
7.62
7.38
28.16
30.2
30.21
27.32
30.67
30.3
27.62
22.47
30.39
29.15
19.12
11.98
20.04
22.83
27.32
17.87
18.14
19.58
7.56
18.1 ꢀ 0.02
18.07 ꢀ 0.013
19.29 ꢀ 0.026
11.7 ꢀ 0.031
18.48 ꢀ 0.014
16.82 ꢀ 0.024
15.7 ꢀ 0.020
12.1 ꢀ 0.013
15.58 ꢀ 0.0233
8.6 ꢀ 0.024
Mixed
Mixed
Mixed
Competitive
Mixed
Mixed
Mixed
Noncompetitive
Mixed
Competitive
Competitive
26
30
16.44
29.15
19.2
Standard (Thiourea)
21
20.01 ꢀ 0.020
Result represents as mean of triplicate ꢀ standard error of mean (S.E.M.).
activity of this class of compounds. From the series, compound (30)
M) exhibit the strongest inhibiting power
plot by plotting the reciprocal of the rate of reaction against the
different concentrations of compounds.
(IC50 ¼ 8.43 ꢀ 0.02
m
against the enzyme urease and it is striking that for structure, the
lone pair electrons of the nitrogen of 30 in R2 have a distance of
three bond length from the thiourea center and seem to support the
chelating efficiency of the inhibitor attacking the active site of the
enzyme.
In mixed-type of inhibition, both Km and Vmax are affected. The
high Km and low Vmax of these compounds indicated the mixed-
type of inhibition (Fig. 1). In the presence of compound 13, the
Vmax of jack bean urease was not affected, while the Km increased
which indicated a pure competitive type of inhibition (Fig. 2). In the
presence of compound 19, the Km of enzyme was not affected, while
the Vmax of enzyme decreased thus indicated a non-competitive
inhibition (Fig. 3).
Kinetics studies
To check the inhibition mechanism of these compounds, the
kinetics studies of the most active compounds (Compounds 1, 3, 9,
13, 14, 15, 18, 19, 26 and 30) were performed. In the kinetics studies
along with different concentration of test compound the substrates
were also varied. These compounds inhibited the jack bean urease
in a concentration-dependent manner with Ki values between 8.6
3. Conclusion
Summarizingly, a series of substituted thiourea derivatives have
been synthesized successfully in appreciable yields and evaluated
for their in vitro urease inhibition potential. SAR studies carried out
to investigate the role of substitutions by functional groups
attached to the thiourea bridge which exert imperative influence
on the urease inhibitory potential. It is also concluded that sub-
stituents having the lone pair of electrons and are appropriately
oriented from the thiourea center and seem to support the
chelating efficiency of the inhibitor attacking the active site of the
enzyme exhibit the strongest inhibiting power against the enzyme
urease. The easy, at low cost accessible thiourea series resulted in
the discovery of several most efficient urease inhibitions which are
and 19.29 mM. Form the kinetics studies it was clear that most of
these inhibitors were found to be mixed-type of inhibitors, except
compounds 13and 30 which were competitive, while compound 19
was identified as a non-competitive inhibitors Table 3. The type of
inhibition was determined by LineweavereBurk plots, the recip-
rocal of the rate of the reaction were plotted against the reciprocal
of substrate concentration to monitor the effect of inhibitor on both
Km and Vmax. The Ki values were calculated by plotting the slope of
each line in the LineweavereBurk plots against the different con-
centration of compounds. The Ki values were confirmed from Dixon
Fig. 1. The inhibition of urease by compound 30 (A) LineweavereBurk plot of reciprocal of rate of reaction (velocities) vs reciprocal of substrate (urea) in the absence (-), and in
presence of 5 M (,), 10 M (C), and 15 M (B) of compound 30. (B) Secondary replot of LineweavereBurk plot between the slopes of each line on LineweavereBurk plot vs
different concentrations of compound 30. (C) Dixon plot of reciprocal of rate of reaction (velocities) vs different concentrations of compound 30.
m
m
m

318
K.M. Khan et al. / European Journal of Medicinal Chemistry 74 (2014) 314e323
Fig. 2. The inhibition of urease by compound 18 (A) LineweavereBurk plot of reciprocal of rate of reaction (velocities) vs reciprocal of substrate (urea) in the absence (-), and in
presence of 5 M (,), 10 M (C), and 15 M (B) of compound 18. (B) Secondary replot of LineweavereBurk plot between the slopes of each line on LineweavereBurk plot vs
different concentrations of compound 18. (C) Dixon plot of reciprocal of rate of reaction (velocities) vs different concentrations of compound 18.
m
m
m
attractive compounds to be further developed for research and
industrial applications.
at 30 ꢁC for 15 min in 96-well plates. For the kinetics assessment the
urea concentrations were changed from 2 to 24 mM. Urease activity
was determined by measuring ammonia production using the
indophenol method as described by Weatherburn [26]. Briefly,
4. General experimental
45
mL 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 and 0.1%
Melting points were determined on a Büchi 434 melting point
apparatus and are uncorrected. NMR was performed on Bruker AV
300, 400, and 500 MHz instruments, respectively. CHN analyses
were determined on a Carlo Erba Strumentazion-Mod-1106, Italy,
instrument. Infrared (IR) spectra were recorded on a JASCO IR-A-
302 spectrometer. Electron impact mass spectra (EI MS) were
recorded on a Finnigan MAT-311A, Germany, spectrometer. Thin
layer chromatography (TLC) was performed on precoated silica gel
glass plates (Kieselgel 60, 254, E. Merck, Germany).
active chloride NaOCl) were added to each well. The increasing
absorbance at 630 nm was measured after 50min, using a micro-
plate reader (Molecular Device, USA). All reactions were performed
in triplicate in a final volume of 200
mL. The results (change in
absorbance per min) were processed by using SoftMaxPro software
(molecular Device, USA). The entire assays were performed at pH
6.8. Percentage inhibition was calculated from the formula
100 ꢂ (ODtest well/ODcontrol) ꢃ 100. Thiourea was used as the stan-
dard inhibitor for urease.
4.1. Urease assay and inhibition
4.2. General procedure for the synthesis of compounds 1e38
The reaction mixtures, comprising 25
urease) solution and 55 L of buffers containing 100 mM urea, were
incubated with 5 L of the test compounds (0.5 mM concentration)
mL of enzyme (jack bean
m
3-Chlorophenyl isothiocyanate and phenyl isothiocyanate
(1.9 mmol and 3.7 mmol) was dissolved in 10 mL dichloromethane
m
Fig. 3. The inhibition of urease by compound 19 (A) LineweavereBurk plot of reciprocal of rate of reaction (velocities) vs reciprocal of substrate (urea) in the absence (-), and in
presence of 5 M (,), 10 M (C), and 15 M (B) of compound 19. (B) Secondary replot of LineweavereBurk plot between the slopes of each line on LineweavereBurk plot vs
different concentrations of compound 19. (C) Dixon plot of reciprocal of rate of reaction (velocities) vs different concentrations of compound 19.
m
m
m

K.M. Khan et al. / European Journal of Medicinal Chemistry 74 (2014) 314e323
319
at 0 ꢁC. Then aniline (2.7 mmol and 5.5 mmol), was added dropwise
with constant stirring, and completion of reaction is checked by TLC
analysis. After 45 min a white solid appeared, the resultant solid
product was filtered, washed with hexane, triturated with 10 mL of
dichloromethane and excess of hexane and dried under vacuum.
Recrystallization with methanol afforded the desired solid thiourea
1e38.
NMR (500 MHz, CD3OD):
d
7.73 (t, 1H, H-20, J ¼ 3.5 Hz), 7.59 (t, 1H,
H-2, J ¼ 4.0 Hz), 7.40 (ddd, 1H, H-40, J ¼ 8.0, 2.0, 1.5 Hz), 7.35 (dt, 1H,
H-4, J ¼ 8.0, 1.5 Hz), 7.32 (t, 2H, H-50, 60, J ¼ 8.0 Hz), 7.26 (t, 1H, H-5,
J ¼ 8.0 Hz), 7.17 (ddd, 1H, H-6, J6,5 ¼ 7.5, 2.0, 1.5 Hz), 4.85 (s, 3H,
OCH3); 13C NMR (125 MHz, CD3OD):
d 179.10 (C]S), 160.30 (CeO),
138.70 (CeNHAr), 138.10 (CeNHAr), 134.50 (CeCl), 130.60, 130.10,
126.70, 124.50, 118.90, 117.00, 110.30, 55.60; EI MS: m/z (rel. abund.
%) 292 (Mþ, 4.0), 169 (100.0), 165 (42.0), 123 (85.2), 111 (56.4), 94
(52.3); EI MS: m/z (rel. abund. %) 215 (32), 171 (79), 169 (51), 111
(36), 91 (48), 75 (47); Anal. calcd for C13H10Cl2N2S: C, 57.43; H, 4.48;
N, 9.57; Found: C, 57.46; H, 4.51; N, 9.60.
4.2.1. 1,3-Bis(3-chlorophenyl)thiourea (1)
Yield: 74%; M.p.: 161 ꢁC Rf: 0.49 (acetone/hexane, 3:7); IR (KBr):
nmax 3223, 3034, 1587, 1538, 1476, 1307, 1206, 1078 cmꢂ1 1H NMR
;
(500 MHz, CD3OD):
d
7.59 (t, 2H, H-2, 20 J ¼ 3.5 Hz), 7.35 (dt, 2H, H-4,
40 J ¼ 7.0, 3.0 Hz), 7.32 (t, 2H, H-5, 50 J ¼ 8.0 Hz), 7.17 (ddd, 2H, H-6, 60,
4.2.6. N-(3-Chlorophenyl)-N0-(4-methoxyphenyl)thiourea (6)
Yield: 80%; M.p.: 169 ꢁC Rf: 0.49 (acetone/hexane, 3:7); IR (KBr):
nmax 3334, 3160, 3031, 1593, 1545, 1480, 1301, 1248, 1165, 1097,
J ¼ 7.5, 2.0, 2.0 Hz); 13C NMR (125 MHz, CD3OD):
d 177.14 (C]S),
137.50 (CeNHAr), 137.50 (CeNHAr), 133.80 (CeCl), 133.80 (CeCl)
130.20, 130.20, 128.10, 128.10, 126.50, 126.50, 124.20, 124.20; EI MS:
m/z (rel. abund. %) 296 (5.0), 266 (36.0), 264 (100.0), 262 (100.0), 227
(6.0),169(36),127 (37),111 (42), 90 (21); Anal. calcd for C13H10Cl2N2S:
C, 52.54; H, 3.39; N, 9.43; Found: C, 52.57; H, 3.42; N, 9.47.
1037 cmꢂ1 1H NMR (500 MHz, CD3OD):
; d 7.56 (t, 1H, H-2,
J ¼ 4.0 Hz), 7.32 (dt, 1H, H-4, J ¼ 8.5, 2.0 Hz), 7.29 (t, 1H, H-5,
J ¼ 8.0 Hz), 7.27 (d, 2H, H-30, 50, J ¼ 9.0 Hz), 7.16 (ddd,1H, H-6, J ¼ 7.5,
1.5, 2.0 Hz), 6.93 (d, 2H, H-20, 60, J ¼ 7.0 Hz), 3.79 (s, 3H, OCH3); 13C
NMR (125 MHz, CD3OD):
d 178.10 (C]S), 158.90 (CeO), 138.60 (Ce
4.2.2. 1-(2-Chlorophenyl)-3-(3-chlorophenyl)thiourea (2)
NHAr), 134.40 (CeCl), 131.10 (CeNHAr), 130.50, 128.40, 127.60,
127.60, 126.90, 124.60, 114.40, 114.40, 55.70; EI MS: m/z (rel. abund.
%) 292 (Mþ, 36), 259 (8.0), 171 (16), 169 (42), 165 (35), 140 (67), 127
(28), 111 (28), 108 (100.0), 83 (58); Anal. calcd for C13H10Cl2N2S: C,
57.43; H, 4.48; N, 9.57; Found: C, 57.46; H, 4.51; N, 9.60.
Yield: 73%; M.p.: 162 ꢁC Rf: 0.50 (acetone/hexane, 3:7); IR (KBr):
nmax 3295, 3204, 1587, 1535, 1476, 1351, 1203, 1066 cmꢂ1; 1H NMR
(500 MHz, Acetone-d6): d 9.36 (s,1H, NH), 8.87 (s,1H, NH), 7.82 (br.t,
1H, H-2, J ¼ 4.8 Hz), 7.78 (dd, 1H, H-60, J ¼ 8.1, 1.5 Hz), 7.51 (dd, 2H,
H-4, 30, J ¼ 7.8, 1.5 Hz), 7.39 (br.t, 2H, H-5, 50, J ¼ 8.4 Hz), 7.30 (dt, 1H,
H-40, J ¼ 7.8, 1.5 Hz), 7.21 (ddd, 1H, H-6, J ¼ 8.1, 1.8, 1.5 Hz); 13C NMR
4.2.7. 1-(3-Chlorophenyl)-3-(2,4-dimethylphenyl)thiourea (7)
Yield: 75%; M.p.: 193 ꢁC Rf: 0.59 (acetone/hexane, 3:7); IR (KBr):
(125 MHz, CD3OD):
d 178.10 (C]S), 138.20 (CeNHAr), 137.60 (Ce
NHAr), 134.10 (CeCl), 133.20, 131.30, 131.20 (CeCl), 130.60, 130.20,
128.10, 126.50, 124.60, 124.10; EI MS: m/z (rel. abund. %) 266 (14.0),
264 (71), 262 (100), 192 (10), 125 (12), 111 (13), 75 (14); Anal. calcd
for C13H10Cl2N2S: C, 52.54; H, 3.39; N, 9.43; Found: C, 52.56; H, 3.41;
N, 9.45.
nmax 3207, 3038, 1593, 1520, 1474, 1341, 1087 cmꢂ1 1H NMR
;
(400 MHz, DMSO): d 9.65 (s, 1H, NH), 9.42 (s, 1H, NH), 7.71 (br.s, 1H,
H-2), 7.40 (d, 1H, H-4, J ¼ 8.0 Hz), 7.33 (t,1H, H-5, J ¼ 8.0 Hz), 7.14 (d,
1H, H-60, J ¼ 8.0 Hz), 7.10 (d, 1H, H-50, J ¼ 8.0 Hz), 7.05 (s, 1H, H-30),
7.00 (d, 1H, H-6, J ¼ 8.0 Hz), 2.26 (s, 3H, CH3), 2.18 (s, 3H, CH3); 13
C
NMR (125 MHz, CD3OD):
d 179.50 (C]S), 143.90 (CeCH3), 139.90
4.2.3. 1-(3-Chlorophenyl)-3-(4-chlorophenyl)thiourea (3)
(CeCH3),138.60 (CeNHAr),134.50 (CeCl),132.80 (CeNHAr),131.30,
130.50, 128.50, 126.80, 126.40, 124.50, 119.50, 21.50, 18.50; EI MS:
m/z (rel. abund. %) 290 (Mþ, 3), 153 (4), 121 (22), 111 (40), 83 (100),
55 (33); Anal. calcd for C13H10Cl2N2S: C, 61.95; H, 5.20; N, 9.63;
Found: C, 60.57; H, 5.46; N, 9.57.
Yield: 80%; M.p.: 163 ꢁC ꢁC Rf: 0.50 (acetone/hexane, 3:7); IR
(KBr): nmax 3208, 3025, 1590, 1539, 1482, 1316, 1221, 1095 cmꢂ1; 1H
NMR (500 MHz, Acetone-d6):
d 9.22 (s, 2NH), 7.72 (br.s, 1H, H-2),
7.56 (br.d, 2H, H-20, 60, J ¼ 8.7 Hz), 7.46 (d, 1H, H-4, J ¼ 8.1 Hz), 7.38
(d, 2H, H-30, 50, J ¼ 8.7 Hz), 7.37 (t, 1H, H-5, J ¼ 8.4 Hz), 7.19 (ddd, 1H,
H-6, J ¼ 6.0, 2.1, 1.8 Hz); 13C NMR (125 MHz, CD3OD):
d
179.50 (C]
4.2.8. 1-(3-Chlorophenyl)-3-(2,6-dimethylphenyl)thiourea (8)
Yield: 74%; M.p.: 195 ꢁC Rf: 0.59 (acetone/hexane, 3:7); IR (KBr):
S), 138.30 (CeNHAr), 137.10 (CeNHAr), 134.60 (CeCl), 133.20 (Ce
Cl), 131.30, 131.30, 130.20, 129.20, 129.20, 128.10, 126.70, 124.10; EI
MS: m/z (rel. abund. %) 298 (Mþ þ 1, 15), 297 (Mþ, 5), 296 (21), 127
(31), 85 (63), 83 (100); Anal. calcd for C13H10Cl2N2S: C, 52.54; H,
3.39; N, 9.43; Found: C, 52.58; H, 3.43; N, 9.47.
nmax 3331, 3152, 2976, 1591, 1533, 1491, 1349, 1088 cmꢂ1 1H NMR
;
(500 MHz, DMSO): d 9.77 (s, 1H, NH), 9.60 (s, 1H, NH), 7.68 (br.s, 1H,
H-2), 7.39 (t, 1H, H-5, J ¼ 8.4 Hz), 7.31 (br.d, 2H, H-4, 6, J ¼ 8.4 Hz),
7.15 (br.d, 1H, H-40, J ¼ 7.8 Hz), 6.91 (d, 2H, H-30, 50, J ¼ 8.7 Hz), 2.49
(s, 6H, 2CH3); 13C NMR (125 MHz, CD3OD):
d 179.50 (C]S), 138.50
4.2.4. 1-(3-Chlorophenyl)-3-(2-methoxyphenyl)thiourea (4)
Yield: 78%; M.p.: 177 ꢁC Rf: 0.45 (acetone/hexane, 3:7); IR (KBr):
nmax 3266, 3192, 3008, 1590, 1507, 1464, 1363, 1282, 1202, 1117,
(CeNHAr), 135.70 (CeCH3), 135.50 (CeNHAr), 135.40 (CeCH3),
134.70 (CeCl), 130.50, 128.20, 127.80, 127.80, 127.10, 126.80, 124.70,
18.60, 18.60; EI MS: m/z (rel. abund. %) 290 (Mþ, 90), 275 (100), 257
(24), 121 (15), 83 (77); Anal. calcd for C13H10Cl2N2S: C, 61.95; H,
5.20; N, 9.63; Found: C, 61.97; H, 5.22; N, 9.65.
1024 cmꢂ1 1H NMR (500 MHz, CD3OD):
; d 7.58 (t, 1H, H-2,
J ¼ 4.0 Hz), 7.34 (dt, 1H, H-40, J ¼ 7.8, 1.5 Hz), 7.30 (t, 1H, H-50,
J ¼ 8.0 Hz), 7.26 (t,1H, H-5, J ¼ 8.0 Hz), 7.16 (ddd,1H, H-4, J ¼ 7.5, 2.0,
1.5 Hz), 7.09 (t, 1H, H-30, J ¼ 4.5 Hz), 6.95 (dd, 1H, H-60, J ¼ 8.0,
2.0 Hz), 6.77 (ddd, 1H, H-6, J ¼ 6.0, 2.5, 2.5 Hz), 4.83 (s, 3H, OCH3);
4.2.9. 1-(3-Chlorophenyl)-3-(2,5-dimethylphenyl)thiourea (9)
Yield: 77%; M.p.: 194 ꢁC Rf: 0.58 (acetone/hexane, 3:7); IR (KBr):
nmax 3206, 3007, 2916, 1593, 1524, 1475, 1344, 1131, 1083 cmꢂ1; 1H
13C NMR (125 MHz, CD3OD):
d 178.530 (C]S), 154.20 (CeO), 138.10
(CeNHAr), 134.20 (CeCl), 130.20, 129.10, 128.10, 126.60, 125.10; (Ce
NHAr), 124.80, 124.40, 121.80, 113.00, 55.80; EI MS: m/z (rel. abund.
%) 292 (Mþ, 4.0), 169 (100.0), 165 (42.0), 123 (85.2), 111 (56.4), 94
(52.3); Anal. calcd for C13H10Cl2N2S: C, 57.43; H, 4.48; N, 9.57;
Found: C, 57.45; H, 4.50; N, 9.59.
NMR (400 MHz, DMSO): d 9.69 (s, 1H, NH), 9.45 (s, 1H, NH), 7.69 (s,
1H, H-2), 7.39 (d, 1H, H-4, J ¼ 8.4 Hz), 7.33 (t, 1H, H-5, J ¼ 8.0 Hz),
7.15 (s, 1H, H-60), 7.12 (d, 1H, H-30, J ¼ 8.0 Hz), 7.03 (s, 1H, H-40), 6.99
(d, 1H, H-6, J ¼ 8.0 Hz), 2.25 (s, 3H, CH3), 2.17 (s, 3H, CH3); 13C NMR
(125 MHz, CD3OD):
d 179.70 (C]S), 138.60 (CeNHAr), 135.8 (Ce
CH3), 135.70 (CeNHAr), 133.40 (CeCH3), 134.50 (CeCl), 130.30,
129.20, 128.30, 127.00, 124.90, 124.60, 117.60, 23.20, 18.00; EI MS:
m/z (rel. abund. %) 290 (Mþ, 90), 275 (100), 257 (24), 121 (15), 83
(77); EI MS: m/z (rel. abund. %) 290 (Mþ, 23), 275 (12), 163 (17), 138
4.2.5. 1-(3-Chlorophenyl)-3-(3-methoxyphenyl)thiourea (5)
Yield: 79%; M.p.: 168 ꢁC Rf: 0.45 (acetone/hexane, 3:7); IR (KBr):
nmax 3253, 3139, 2949, 1593, 1542, 1469, 1350, 1161, 1041 cmꢂ1; 1H

320
K.M. Khan et al. / European Journal of Medicinal Chemistry 74 (2014) 314e323
(41), 130 (27), 121 (100), 120 (48), 111 (14), 106 (46), 77 (43); Anal.
calcd for C13H10Cl2N2S: C, 61.95; H, 5.20; N, 9.63; Found: C, 61.97; H,
5.22; N, 9.65.
J ¼ 8.4 Hz), 7.18 (dd, 1H, H-6, J ¼ 7.8, 1.8 Hz), 2.29 (s, 3H, CH3); 13
C
NMR (125 MHz, CD3OD):
d 179.90 (C]S), 138.60 (CeNHAr), 137.40
(CeNHAr), 134.70 (CeCH3), 134.60 (CeCl), 131.80 (CeCl), 130.80,
130.40, 128.40, 127.00, 124.80, 124.50, 113.80, 17.90; EI MS: m/z (rel.
abund. %) 331 (Mþ, 3), 203 (35),169 (32),163 (27),161 (43),145 (19),
129 (34), 127 (100),111 (39), 92 (21), 75 (36); EI MS: m/z (rel. abund.
%) 312 (Mþ þ 1, 8), 311 (Mþ, 11),183 (12),169 (12),148 (23),141 (58),
129 (31), 127 (100), 111 (27), 106 (45), 89 (28), 75 (28); Anal. calcd
for C14H12Cl2N2S: C, 54.03; H, 3.89; N, 9.00; Found: C, 54.05; H,
3.91; N, 9.02.
4.2.10. 1-(3-Chlorophenyl)-3-(2,6-dichlorophenyl)thiourea (10)
Yield: 70%; M.p.: 193 ꢁC Rf: 0.43 (acetone/hexane, 3:7); IR (KBr):
nmax 3218, 3005, 1591, 1524, 1433, 1331, 1286, 1244, 1085 cmꢂ1; 1H
NMR (400 MHz, DMSO): d 10.12 (s, 1H, NH), 9.58 (s, 1H, NH), 7.70 (s,
1H, H-2), 7.53 (d, 2H, H-30, 50, J ¼ 8.0 Hz), 7.43 (d,1H, H-4, J ¼ 8.4 Hz),
7.38 (m, 2H, H-40,5), 7.20 (d,1H, H-6, J ¼ 8.0 Hz); 13C NMR (125 MHz,
CD3OD):
d 179.60 (C]S), 138.50 (CeNHAr), 138.20 (CeCl), 138.20
(CeCl), 134.60 (CeCl), 130.80 (CeNHAr), 130.50, 128.40, 128.30,
128.30, 126.80, 126.40, 124.50; EI MS: m/z (rel. abund. %) 300 (32),
298 (98), 296 (100), 125 (11), 111 (9), 75 (10); Anal. calcd for
4.2.15. 1-(3-Chlorophenyl)-3-(4-fluorophenyl)thiourea (15)
Yield: 79%; M.p.: 189 ꢁC Rf: 0.43 (acetone/hexane, 3:7); IR (KBr):
nmax 3224, 3015, 1556, 1509, 1475, 1336, 1235, 1072 cmꢂ1 1H NMR
;
C
8.47.
13H10Cl2N2S: C, 47.08; H, 2.74; N, 8.45; Found: C, 47.10; H, 2.76; N,
(500 MHz, Acetone-d6): d 9.15 (s, 1H, NH), 7.76 (m,1H, H-2), 7.54 (m,
2H, H-30, 50), 7.45 (dd, 1H, H-4, J ¼ 8.1, 4.8 Hz), 7.36 (t, 1H, H-5,
J ¼ 8.1 Hz), 7.19 (dd, 1H, H-6, J ¼ 6.9, 2.7 Hz), 7.15 (dt, 2H, H-20, 60,
J2 ,F ¼ J6 ,F ¼ , 8.7, 2.1 Hz); 13C NMR (125 MHz, CD3OD):
d 179.90 (C]
0
0
4.2.11. 1-(3-Chlorophenyl)-3-(2,3-dichlorophenyl)thiourea (11)
Yield: 74%; M.p.: 171 ꢁC Rf: 0.44 (acetone/hexane, 3:7); IR (KBr):
S), 163.20 (d, J ¼ 205 Hz, CeF), 138.40 (CeNHAr), 134.60 (CeCl),
134.10 (CeNHAr), 131.10, 131.10, 130.30, 128.20, 126.80, 124.50,
115.70, 115.70; EI MS: m/z (rel. abund. %) 283 (Mþ þ 2, 3), 282
(Mþ þ 1, 18), 280 (Mþ, 51), 247 (6), 153 (17), 127 (49), 111 (100), 95
(15); Anal. calcd for C13H10ClFN2S: C, 55.62; H, 3.59; N, 9.98; Found:
C, 55.65; H, 3.62; N, 10.01.
nmax 3219, 1588, 1533, 1501, 1360, 1200, 1154 cmꢂ1 1H NMR
;
(500 MHz, Acetone-d6):
d 9.48 (s, 1H, NH), 8.98 (s, 1H, NH), 7.80 (t,
1H, H-2, J ¼ 3.9 Hz), 7.74 (dd,1H, H-60 J ¼ 8.1,1.5 Hz), 7.50 (dd, 2H, H-
4, 40, J ¼ 8.1, 1.5 Hz), 7.39 (t, 2H, H-5, 50, J ¼ 8.1 Hz), 7.22 (ddd, 1H, H-
6, J ¼ 7.8, 1.8, 1.8 Hz); 13C NMR (125 MHz, CD3OD):
d 179.70 (C]S),
138.60 (CeNHAr), 137.40 (CeNHAr), 136.30 (CeCl), 134.70 (CeCl),
132.60 (CeCl), 132.10, 130.50, 130.30, 128.6, 128.4, 126.80, 124.50; EI
MS: m/z (rel. abund. %) 300 (4), 298 (17), 297 (72), 295 (100), 203
(12), 169 (11), 161 (16), 127 (17); Anal. calcd for C13H10Cl2N2S: C,
47.08; H, 2.74; N, 8.45; Found: C, 47.10; H, 2.76; N, 8.47.
4.2.16. 1-(3-Chlorophenyl)-3-(3,4-difluorophenyl)thiourea (16)
Yield: 75%; M.p.: 184 ꢁC Rf: 0.40 (acetone/hexane, 3:7); IR (KBr):
nmax 3204, 3031, 1594, 1537, 1474, 1345, 1246, 1207, 1151, 1109 cmꢂ1
;
1H NMR (500 MHz, Acetone-d6):
d 9.25 (s, 1H, NH), 7.72 (m, 1H, H-
2), 7.67 (d, 1H, H-20, J2 ,F ¼ 8.1, 1.5 Hz), 7.44 (dd, 1H, H-4, J4,5 ¼ 8.1,
0
4.8 Hz), 7.38 (t, 1H, H-5, J ¼ 8.1 Hz), 7.31 (t, 1H, H-50, J5 (F,6) ¼ ,
0
4.2.12. 1-(3-Chlorophenyl)-3-(2,4-dichlorophenyl)thiourea (12)
Yield: 73%; M.p.: 182 ꢁC Rf: 0.45 (acetone/hexane, 3:7); IR (KBr):
7.50 Hz), 7.28 (t, 1H, H-60, J ¼ 5.1 Hz), 7.21 (dt, 1H, H-6, J ¼ 8.1,
nmax 3296, 3197, 1587, 1532, 1499, 1354, 1202, 1098 cmꢂ1
;
1H NMR
3.0 Hz); 13C NMR (125 MHz, CD3OD):
d 179.90 (C]S), 148.70 (d,
(500 MHz, Acetone-d6):
d
7.80 (d,1H, H-60, J ¼ 8.7 Hz), 7.79 (t,1H, H-
J ¼ 175 Hz, CeF), 143.80 (d, J ¼ 165 Hz, CeF), 138.40 (CeNHAr),
134.60 (CeCl), 134.20 (CeNHAr), 130.40, 128.20, 126.80, 126.50,
124.60, 116.40, 114.80; EI MS: m/z (rel. abund. %) 300 (Mþ þ 2, 12),
299 (Mþ þ 1, 7), 298 (Mþ, 33), 171 (16), 129 (100), 127 (52), 113 (20),
111 (16), 75 (15); Anal. calcd for C13H9ClF2N2S: C, 52.27; H, 3.04; N,
9.38; Found: C, 52.29; H, 3.06; N, 9.40.
2, J ¼ 3.9 Hz), 7.56 (d, 1H, H-30, J ¼ 2.4 Hz), 7.49 (ddd, 1H, H-4, J ¼ 8.1,
1.8, 1.2 Hz), 7.41 (dd, 1H, H-50, J ¼ 8.4, 2.1 Hz), 7.39 (t, 1H, H-5,
J ¼ 8.1 Hz), 7.22 (ddd, 1H, H-6, J ¼ 8.1, 1.8, 1.8 Hz); 13C NMR
(125 MHz, CD3OD):
d 179.90 (C]S), 138.60 (CeNHAr), 134.60 (Ce
Cl), 134.10 (CeNHAr), 133.90 (CeCl), 132.70, 131.60 (CeCl), 130.70,
130.40, 128.40, 126.80, 124.50,121.20; EI MS: m/z (rel. abund. %) 299
(16), 297 (75), 295 (100), 203 (16), 169 (21), 160 (48), 133 (16), 127
(46), 111 (35), 75 (43); Anal. calcd for C13H10Cl2N2S: C, 47.08; H,
2.74; N, 8.45; Found: C, 47.10; H, 2.76; N, 8.47.
4.2.17. 1-(3-Chlorophenyl)-3-(o-tolyl)thiourea (17)
Yield: 77%; M.p.: 168 ꢁC Rf: 0.59 (acetone/hexane, 3:7); IR (KBr):
nmax 1588, 2926, 1789, 1645, 1526, 1448, 1207 cmꢂ1 1H NMR
;
(500 MHz, DMSO): d 9.84 (s, 1H, NH), 9.60 (s, 1H, NH), 7.79 (s, 1H, H-
4.2.13. 1-(3-Chlorophenyl)-3-(3,4-dichlorophenyl)thiourea (13)
Yield: 75%; M.p.: 171 ꢁC Rf: 0.47 (acetone/hexane, 3:7); IR (KBr):
nmax 3175, 3009, 1586, 1537, 1472, 1328, 1245, 1128, 1098 cmꢂ1; 1H
2), 7.44 (d, 1H, H-4, J ¼ 8.1 Hz), 7.33 (t, 1H, H-5, J ¼ 8.1 Hz), 7.26 (t,
2H, H-4’, 50, J ¼ 7.8 Hz), 7.20 (dd, 1H, H-30, J ¼ 7.5 Hz), 7.15 (d, 1H, H-
60, J ¼ 7.5 Hz Hz), 7.10 (s, 1H, H-6), 2.22 (s, 3H, CH3); 13C NMR
NMR (500 MHz, CD3OD):
d
7.77 (d, 1H, H-20, J ¼ 2.5 Hz), 7.59 (t, 1H,
(125 MHz, CD3OD): d 179.80 (C]S), 138.40 (CeNHAr), 135.60 (Ce
H-2, J ¼ 4.0 Hz), 7.46 (d, 1H, H-50, J ¼ 8.5 Hz), 7.38 (dd, 1H, H-60,
J ¼ 8.5, 2.5 Hz), 7.35 (dt, 1H, H-4, J ¼ 8.0, 3.5 Hz), 7.32 (t, 1H, H-5,
J ¼ 8.1 Hz), 7.18 (ddd, 1H, H-6, J6,5 ¼ 8.0, 2.0, 1.5, Hz); 13C NMR
NHAr), 136.40 (CeCH3), 134.50 (CeCl), 130.70, 130.30, 129.60,
128.20, 127.00, 126.80, 126.10, 124.50, 17.80; EI MS: m/z (rel. abund.
%) 278 (Mþ þ 2, 33), 277 (Mþ þ 1, 21), 276 (Mþ, 89), 261 (29), 243
(48), 242 (100), 169 (37), 149 (39), 127 (68), 111 (22), 107 (78), 91
(29); Anal. calcd for C14H13ClN2S: C, 60.75; H, 4.73; N, 10.12; Found:
C, 60.77; H, 4.75; N, 10.14.
(125 MHz, CD3OD):
d 179.80 (C]S), 138.60 (CeNHAr), 136.60 (Ce
NHAr), 135.30, 134.60 (CeCl), 131.30 (CeCl), 130.40, 129.40 (CeCl),
129.10, 128.40, 126.80, 124.70, 121.20; EI MS: m/z (rel. abund. %) 331
(Mþ, 3), 203 (35), 169 (32), 163 (27), 161 (43), 145 (19), 129 (34), 127
(100), 111 (39), 92 (21), 75 (36); Anal. calcd for C13H10Cl2N2S: C,
47.08; H, 2.74; N, 8.45; Found: C, 47.11; H, 2.77; N, 8.48.
4.2.18. 1-(3-Chlorophenyl)-3-(m-tolyl)thiourea (18)
Yield: 79%; M.p.: 155 ꢁC Rf: Rf: 0.57 (acetone/hexane, 3:7); IR
(KBr): nmax 3338, 3155, 2991, 1585, 1535, 1500, 1475, 1346, 1203,
4.2.14. 1-(5-Chloro-2-methylphenyl)-3-(3-chlorophenyl)thiourea
(14)
1083 cmꢂ1; 1H NMR (500 MHz, Acetone-d6):
d 9.14 (s, 1H, NH), 9.06
(s, 1H, NH), 7.75 (t, 1H, H-2, J ¼ 3.9 Hz), 7.46 (ddd, 1H, H-4, J ¼ 8.1,
2.7,1.8 Hz), 7.34 (m, 3H, H-5, 20, 40), 7.26 (dt,1H, H-50, J ¼ 7.5, 2.0 Hz),
7.17 (ddd, 1H, H-60, J ¼ 7.8, 4.8, 2.1 Hz Hz), 7.02 (d, 1H, H-6,
Yield: 80%; M.p.: 185 ꢁC Rf: 0.49 (acetone/hexane, 3:7); IR (KBr):
nmax 3253, 3034, 2838, 1593, 1518, 1475, 1333, 1241, 1081 cmꢂ1; 1H
NMR (500 MHz, Acetone-d6):
d
9.18 (s,1H, NH), 8.87 (s,1H, NH), 7.74
J ¼ 7.2 Hz), 2.86 (s, 3H, CH3); 13C NMR (125 MHz, CD3OD):
d 179.80
(m,1H, H-2), 7.47 (s,1H, H-40), 7.44 (dd,1H, H-4, J ¼ 6.3, 3.3 Hz), 7.36
(C]S), 138.60 (CeCH3), 138.40 (CeNHAr), 137.10 (CeNHAr), 134.50
(CeCleCl), 130.30, 128.80, 128.2, 126.80, 125.20, 125.10, 124.50,
(t, 1H, H-5, J ¼ 8.1 Hz), 7.29 (d, 1H, H-40, J ¼ 8.1 Hz), 7.22 (d, 1H, H-60,

K.M. Khan et al. / European Journal of Medicinal Chemistry 74 (2014) 314e323
321
123.40, 21.2; EI MS: m/z (rel. abund. %) 278 (Mþ þ 2, 25), 277
(Mþ þ 1, 15), 276 (Mþ, 65), 244 (67), 242 (100), 171 (41), 169 (100),
149 (38), 127 (63), 111 (37), 107 (100), 91 (44); Anal. calcd for
4.2.23. 1-(3-Chlorophenyl)-3-(pyridin-3-yl)thiourea (23)
Yield: 74%; M.p.: 167 ꢁC Rf: 0.31 (acetone/hexane, 4.5:5.5); IR
(KBr): nmax 3433, 2995, 1588, 1550, 1477, 1426, 1074 cmꢂ1; 1H NMR
C
14H13ClN2S: C, 60.75; H, 4.73; N, 10.12; Found: C, 60.77; H, 4.75; N,
(500 MHz, Acetone-d6): d 9.41 (s, 1H, NH), 9.32 (s, 1H, NH), 8.65 (d,
10.14.
1H, H-20, J ¼ 2.4 Hz), 8.37 (dd,1H, H-60, J ¼ 4.8,1.5 Hz), 8.04 (ddd,1H,
H-40, J ¼ 5.7 2.7, 2.4 Hz), 7.76 (m, 1H, H-2), 7.47 (d, 1H, H-4,
J ¼ 8.1 Hz), 7.39 (t, 1H, H-5, J ¼ 8.1 Hz), 7.35 (t, 1H, H-50, J ¼ 4.8 Hz),
7.21 (ddd, 1H, H-6, J ¼ 6.3, 1.8, 1.2 Hz); 13C NMR (125 MHz, CD3OD):
4.2.19. 1-(3-Chlorophenyl)-3-(p-tolyl)thiourea (19)
Yield: 80%; m.p.: 178 ꢁC Rf: 0.59 (acetone/hexane, 3:7); IR (KBr):
d
179.40 (C]S), 140.0, 138.70, 138.40 (CeNHAr), 136.20, 134.20 (Ce
nmax 3210, 3072, 3000, 1591, 1545, 1478, 1324, 1223, 1093 cmꢂ1; 1H
NHAr), 134.50 (CeCl) 130.30, 128.20, 127.50, 126.70, 124.50; EI MS:
m/z (rel. abund. %) 263 (Mþ, 3), 229 (4), 169 (66),136 (93),127 (100),
111 (41), 94 (71), 78 (84); Anal. calcd for C13H12ClN3S: C, 56.21; H,
4.35; N, 15.13; Found: C, 56.23; H, 4.37; N, 15.15.
NMR (500 MHz, Acetone-d6): d 9.12 (s,1H, NH), 9.01 (s,1H, NH), 7.77
(m, 1H, H-2), 7.47 (d, 1H, H-4, J ¼ 8.1 Hz), 7.38 (m, 3H, H-5, 30, 50),
7.19 (d, 2H, H-20, 60, J ¼ 7.8 Hz), 7.15 (dd, 1H, H-6, J ¼ 6.6, 1.8 Hz), 2.31
(s, 3H, CH3); 13C NMR (125 MHz, CD3OD):
d 179.80 (C]S), 138.50
(CeNHAr), 137.10 (CeCH3), 135.40 (CeNHAr), 134.50 (CeCl), 130.30,
129.20, 129.20, 128.20, 126.80, 126.30, 126.30, 124.50, 21.20; EI MS:
m/z (rel. abund. %) 278 (Mþ þ 2, 27), 277 (Mþþ1, 16), 276 (Mþ, 70),
244 (36), 242 (100), 169 (32), 127 (42), 111 (18), 107 (84), 91 (52);
Anal. calcd for C14H13ClN2S: C, 60.75; H, 4.73; N, 10.12; Found: C,
60.77; H, 4.75; N, 10.14.
4.2.24. 1-(3-Chlorophenyl)-3-(2-chloropyridin-3-yl)thiourea (24)
Yield: 73%; M.p.: 232 ꢁC Rf: 0.40 (acetone/hexane, 3:7); IR (KBr):
nmax 3243, 3181, 2957,1631,1564,1428,1302,1245,1170,1095 cmꢂ1
;
1H NMR (500 MHz, DMSO):
d 11.19 (s, 1H, NH), 9.52 (s, 1H, NH), 8.30
(dd, 1H, H-60, J ¼ 4.8, 1.2 Hz), 8.07 (t, 1H, H-2, J ¼ 3.9 Hz), 8.00 (dd,
1H, H-40, J ¼ 8.1, 1.2 Hz), 7.66 (dd, 1H, H-4, J ¼ 7.8, 1.2 Hz), 7.42 (t, 1H,
H-50, J ¼ 4.8 Hz), 7.40 (t, 1H, H-5, J ¼ 8.1 Hz), 7.13 (dt, 1H, H-6, J ¼ 8.1,
4.2.20. 1-(3-Chlorophenyl)-3-(3-methylpyridin-2-yl)thiourea (20)
Yield: 80%; M.p.: 178 ꢁC Rf: 0.59 (acetone/hexane, 3:7); IR (KBr):
nmax 3210, 3072, 3000, 1591, 1545, 1478, 1324, 1223, 1093 cmꢂ1; 1H
1.8 Hz); 13C NMR (125 MHz, CD3OD):
d 179.80 (C]S), 145.70 (Ce
NHAr), 139.40 (CeCl), 138.70, 138.40 (CeNHAr), 134.50 (CeCl),
130.30, 128.20, 126.80, 124.50, 123.60, 123.50; EI MS: m/z (rel.
abund. %) 263 (40), 262 (50), 261 (100), 260 (93), 234 (7); Anal.
calcd for C12H9Cl2N3S: C, 48.33; H, 3.04; N,14.09; found: C, 48.35; H,
3.06; N, 14.11.
NMR (500 MHz, Acetone-d6): d 9.12 (s,1H, NH), 9.01 (s,1H, NH), 7.77
(m, 1H, H-2), 7.47 (d, 1H, H-4, J ¼ 8.1 Hz), 7.38 (m, 3H, H-5, 30, 50),
7.19 (d, 2H, H-20, 60, J ¼ 7.8 Hz), 7.15 (dd, 1H, H-6, J ¼ 6.6, 1.8 Hz), 2.31
(s, 3H, CH3); 13C NMR (125 MHz, CD3OD):
d 179.80 (C]S), 154.40
(CeNHAr), 145.60, 138.40 (CeNHAr), 137.30, 134.50 (CeCl), 130.30,
128.20, 126.80, 124.50, 116.80 (CeCH3), 113.70, 16.90; EI MS: m/z
(rel. abund. %) 278 (Mþ þ 2, 27), 277 (Mþ þ 1, 16), 276 (Mþ, 70), 244
(36), 242 (100), 169 (32), 127 (42), 111 (18), 107 (84), 91 (52); Anal.
calcd for C14H13ClN2S: C, 60.75; H, 4.73; N,10.12; Found: C, 60.77; H,
4.75; N, 10.14.
4.2.25. 1-(4-Bromophenyl)-3-phenylthiourea (25)
Yield: 80%; M.p.: 195 ꢁC Rf: 0.59 (acetone/hexane, 3:7); IR (KBr):
nmax 3218, 2993, 1592, 1551, 1449, 1241, 1067, 1010 cmꢂ1 1H NMR
;
(500 MHz, Acetone-d6): d 9.14 (s, 1H, NH), 9.06 (s, 1H, NH), 7.53 (m,
6H, H-2, 6, 20, 30, 50, 60), 7.38 (t, 2H, H-3, 5, J ¼ 8.1 Hz), 7.19 (t,1H, H-4,
J ¼ 7.5 Hz); 13C NMR (125 MHz, CD3OD):
d 179.60 (C]S), 138.40 (Ce
NHAr), 137.40 (CeNHAr), 131.80, 131.80, 131.60, 131.60, 129.10,
129.00, 128.30, 126.50, 126.40, 122.60 (CeBr); EI MS: m/z (rel.
abund. %) 309 (Mþ þ 2, 7), 308 (Mþ þ 1, 46), 307 (Mþ, 12), 306 (39),
274 (100), 272 (94), 173 (66), 171 (72), 93 (60); Anal. calcd for
4.2.21. 1-(3-Chlorophenyl)-3-(pyridin-4-ylmethyl)thiourea (21)
Yield: 80%; M.p.: 178 ꢁC Rf: 0.59 (acetone/hexane, 3:7); IR (KBr):
nmax 3210, 3072, 3000, 1591, 1545, 1478, 1324, 1223, 1093 cmꢂ1; 1H
C13H11BrN2S: C, 50.82; H, 3.61; N, 9.12; found: C, 50.84; H, 3.63; N,
NMR (500 MHz, Acetone-d6): d 9.12 (s,1H, NH), 9.01 (s,1H, NH), 7.77
9.14.
(m, 1H, H-2), 7.47 (d, 1H, H-4, J ¼ 8.1 Hz), 7.38 (m, 3H, H-5, 30, 50),
7.19 (d, 2H, H-20, 60, J ¼ 7.8 Hz), 7.15 (dd, 1H, H-6, J ¼ 6.6, 1.8 Hz), 2.31
4.2.26. 1-(3-Bromophenyl)-3-phenylthiourea (26)
(s, 3H, CH3); 13C NMR (125 MHz, CD3OD):
d 179.40 (C]S), 149.70,
Yield: 77%; M.p.: 157 ꢁC Rf: 0.49 (acetone/hexane, 3:7); IR (KBr):
149.70, 147.50 (CeCH2eNHAr), 138.40 (CeNHAr), 134.50 (CeCl),
130.30,128.20, 126.80,124.50, 122.30,122.30, 50.70; EI MS: m/z (rel.
abund. %) 278 (Mþ þ 2, 27), 277 (Mþ þ 1, 16), 276 (Mþ, 70), 244 (36),
242 (100), 169 (32), 127 (42), 111 (18), 107 (84), 91 (52); Anal. calcd
for C14H13ClN2S: C, 60.75; H, 4.73; N,10.12; Found: C, 60.77; H, 4.75;
N, 10.14.
nmax 3341, 3215, 1591, 1534, 1426, 1359, 1194, 1067 cmꢂ1 1H NMR
;
(500 MHz, Acetone-d6): d 9.19 (s, 1H, NH), 9.09 (s, 1H, NH), 7.90 (m,
1H, H-60), 7.51 (dd, 3H, H-2, 6, 40, J ¼ 8.1, 5.7 Hz), 7.38 (br.t, 2H, H-3,
5, J ¼ 8.1 Hz), 7.31 (t, 1H, H-60, J ¼ 3.9 Hz), 7.29 (t, 1H, H-50,
J ¼ 7.8 Hz), 7.20 (t, 1H, H-4, J ¼ 7.8 Hz); 13C NMR (125 MHz, CD3OD):
d
179.80 (C]S), 139.10 (CeNHAr), 138.40 (CeNHAr), 130.20, 129.00,
129.00, 128.40, 126.50, 127.50, 126.50, 125.50, 125.60, 123.30 (Ce
Br); EI MS: m/z (rel. abund. %) 309 (Mþ þ 2, 3), 308 (Mþ þ 1, 16), 307
(Mþ, 7), 274 (70), 272 (76), 215 (97), 213 (100), 173 (56), 135 (60), 93
(80); Anal. calcd for C13H11BrN2S: C, 50.82; H, 3.61; N, 9.12; found:
C, 50.84; H, 3.63; N, 9.14.
4.2.22. 1-(3-Chlorophenyl)-3-(pyridin-3-ylmethyl)thiourea (22)
Yield: 74%; M.p.: 167 ꢁC Rf: 0.32 (acetone/hexane, 4.5:5.5); IR
(KBr): nmax 3159, 2993, 1589, 1532, 1428, 1382, 1245, 1118,
1025 cmꢂ1 1H NMR (500 MHz, Acetone-d6):
; d 9.14 (s, 1H, NH),
8.59 (br.s, 1H, H-20), 8.45 (dd, 1H, H-60, J ¼ 4.8, 1.5 Hz), 7.93 (s, 1H,
NH), 7.81 (d, 1H, H-40, J ¼ 7.8 Hz), 7.68 (m, 1H, H-2), 7.39 (dt, 1H,
H-4, J ¼ 6.6, 3.6 Hz), 7.35 (t, 1H, H-5, J ¼ 7.8 Hz), 7.32 (t, 1H, H-50,
4.2.27. 1-(2,4-Difluorophenyl)-3-phenylthiourea (27)
Yield: 71.8%; M.p.: 158 ꢁC Rf: 0.49 (acetone/hexane, 3:7); IR
J ¼ 7.8 Hz), 7.17 (dt, 1H, H-6, J ¼ 7.2, 3.6 Hz), 4.92 (s, 2H, CH2); 13
C
(KBr): nmax 3309, 1594, 1449, 1430, 1366, 1260, 1137 cmꢂ1; 1H NMR
NMR (125 MHz, CD3OD):
d
179.40 (C]S), 148.60, 147.20, 138.40
(500 MHz, Acetone-d6): d 9.25 (s, 1H, NH), 8.65 (s, 1H, NH), 7.73 (m,
(CeNHAr), 135.50 (CeCH2eNHAr), 135.40, 134.50 (CeCl), 123.10,
126.80, 128.20, 130.30, 124.50, 50.70; EI MS: m/z (rel. abund. %)
279 (Mþ þ 2, 12), 278 (Mþ þ 1, 6), 277 (Mþ, 30), 171 (31), 169
(87), 127 (56), 111 (32), 92 (100), 80 (37); Anal. calcd for
due to fluorine coupling 1H, H-30), 7.55 (d, 2H, H-2, 6, J ¼ 7.8 Hz),
7.38 (t, 2H, H-3, 5, J ¼ 7.5 Hz), 7.21 (t, 1H, H-4, J ¼ 7.5 Hz), 7.11 (m,
due to fluorine coupling 2H, H-50, 60); 13C NMR (125 MHz, CD3OD):
d
179.90 (C]S),169.30 (d, J ¼ 190 Hz, CeF),160.80 (d, J ¼ 200 Hz, Ce
C
13H12ClN3S: C, 56.21; H, 4.35; N, 15.13; Found: C, 56.23; H, 4.37;
F), 138.40 (CeNHAr), 129.6, 129.00, 129.00, 128.30, 126.4, 126.4,
111.3, 115.80 (CeNHAr), 105.1; EI MS: m/z (rel. abund. %) 266
N, 15.15.

322
K.M. Khan et al. / European Journal of Medicinal Chemistry 74 (2014) 314e323
(Mþ þ 2, 5.7), 265 (Mþ þ 1, 17.6), 264 (Mþ, 81.8), 245 (11.3), 230
7.09 (dd, 1H, H-50, J ¼ 7.2, 2.0 Hz), 2.42 (s, 3H, CH3); 13C NMR
(75.2), 171 (11.8), 129 (100.0), 101 (13.3), 93 (46.7); Anal. calcd for
(125 MHz, CD3OD): d 180.00 (C]S), 154.40 (CeNHAr), 145.70,
C
13H10F2N2S: C, 59.08; H, 3.81; N, 10.60; found: C, 59.10; H, 3.83; N,
137.40 (CeNHAr), 137.30, 129.10, 129.10, 128.30, 126.40, 126.40,
116.80, 113.70, 16.70; EI MS: m/z (rel. abund. %) 245 (Mþ þ 2, 5), 244
(Mþ þ 1, 15), 243 (Mþ, 88), 209 (63), 150 (29), 108 (100), 93 (43);
Anal. calcd for C13H13N3S: C, 64.17; H, 5.38; N, 17.27; Found: C,
64.19; H, 5.42; N, 17.29.
10.62.
4.2.28. 1-(2,5-Dimethoxyphenyl)-3-phenylthiourea (28)
Yield: 78%; M.p.: 166 ꢁC Rf: 0.50 (acetone/hexane, 3:7); IR (KBr):
nmax 3345, 3161, 2973, 1593, 1549, 1446, 1371, 1281, 1207 cmꢂ1; 1H
NMR (500 MHz, Acetone-d6):
d
10.16 (s, 1H, NH), 9.20 (s, 1H, NH),
4.2.33. 1-(3,5-Dichloropyridin-2-yl)-3-phenylthiourea (33)
Yield: 70%; M.p.: 190 ꢁC Rf: 0.47 (acetone/hexane, 3:7); IR (KBr):
nmax 3469, 3294, 1632, 1576, 1475, 1392, 1238, 1125, 1041 cmꢂ1; 1H
7.80 (br.s,1H, H-60), 7.52 (d, 2H, H-2, 6, J ¼ 7.5 Hz), 7.34 (t, 2H, H-3, 5,
J ¼ 7.8 Hz), 7.14 (br.t, 1H, H-4, J ¼ 8.1 Hz), 6.96 (d, 1H, H-30,
J ¼ 8.7 Hz), 6.67 (br.d, 1H, H-40, J ¼ 6.3 Hz), 3.77 (3H, OCH3), 3.67
NMR (300 MHz, Acetone-d6):
d 13.05 (s, 1H, NH), 8.60 (s, 1H, NH),
(3H, OCH3); 13C NMR (125 MHz, CD3OD):
d
180.00 (C]S), 153.10
8.41 (d,1H, H-60, J ¼ 2.4 Hz), 8.18 (d, 1H, H-40, J ¼ 2.4 Hz), 7.78 (d, 2H,
H-2, 6, J ¼ 7.8 Hz), 7.42 (t, 2H, H-3, 5, J ¼ 8.1 Hz), 7.26 (t, 1H, H-4,
(CeOCH3), 146.70 (CeOCH3), 138.40 (CeNHAr), 129.10, 129.00,
128.30, 126.40, 126.40, 126.30 (CeNHAr), 111.20, 110.50, 110.50,
55.90, 55.90; EI MS: m/z (rel. abund. %) 289 (Mþ þ 1, 8), 288 (Mþ,
46), 257 (100), 254 (82), 239 (62), 224 (39), 138 (82); Anal. calcd for
J ¼ 7.5 Hz); 13C NMR (125 MHz, CD3OD):
d 180.00 (C]S), 159.30 (Ce
NHAr), 149.10, 140.20, 138.40 (CeNHAr), 129.10, 129.10, 128.30,
126.40, 126.40, 117.70 (CeCl), 112.50 (CeCl); EI MS: m/z (rel. abund.
%) 300 (Mþ þ 2, 5), 299 (Mþ þ 1, 24), 298 (Mþ, 7), 297 (33), 262
(100), 162 (77), 135 (25), 110 (14), 93 (22); Anal. calcd for
C
15H16N2O2S: C, 62.48; H, 5.59; N, 9.71; Found: C, 62.50; H, 5.61; N,
9.73.
C12H9Cl2N3S: C, 48.33; H,3.04; N, 14.09; Found: C, 48.35; H, 3.06; N,
4.2.29. 1-Phenyl-3-(pyridin-2-yl)thiourea (29)
14.11.
Yield: 70%; M.p.: 202 ꢁC Rf: 0.46 (acetone/hexane, 3:7); IR (KBr):
nmax 3219, 3038, 1598, 1554, 1471, 1427, 1313 cmꢂ1
;
1H NMR
4.2.34. 1-(Furan-2-ylmethyl)-3-phenylthiourea (34)
(500 MHz, Acetone-d6):
d
13.93 (s, 1H, NH), 9.68 (s, 1H, NH), 8.36
Yield: 70%; M.p.: 174 ꢁC Rf: 0.47 (acetone/hexane, 3:7); IR (KBr):
(dd, 1H, H-60, J ¼ 5.1, 1.2 Hz), 7.88 (dd, 1H, H-50, J ¼ 7.8, 2.1 Hz), 7.82
(d, 2H, H-2, 6, J ¼ 7.8 Hz), 7.39 (t, 2H, H-3, 5, J ¼ 8.1 Hz), 7.30 (d, 1H,
H-30, J ¼ 8.4 Hz), 7.21 (t, 1H, H-4, J ¼ 7.5 Hz Hz), 7.13 (dt, 1H, H-40,
nmax 3287, 3167, 3005, 1591, 1549, 1496, 1319, 1249, 1164 cmꢂ1 1H
;
NMR (500 MHz, DMSO):
d 9.56 (s, 1H, NH), 8.05 (s, 1H, NH), 7.60
(br.s, 1H, H-50), 7.44 (d, 2H, H-2, 6, J ¼ 8.7 Hz), 7.33 (t, 2H, H-3, 5,
J ¼ 5.4, 1.8 Hz); 13C NMR (125 MHz, CD3OD):
d 180.00 (C]S),
J ¼ 8.1 Hz), 7.12 (t,1H, H-4, J ¼ 7.2 Hz), 6.41 (br.t,1H, H-40, J ¼ 3.0 Hz),
153.80 (CeNHAr), 148.2, 138.40 (CeNHAr), 138.20, 129.1, 129.10,
128.30, 126.50, 126.50, 117.80, 113.10; EI MS: m/z (rel. abund. %) 231
(Mþ þ 2, 6), 230 (Mþ þ 1, 16), 229 (Mþ, 95), 195 (100), 169 (18), 137
(21), 94 (91), 78 (77); Anal. calcd for C12H11N3S: C, 62.86; H, 4.84; N,
18.33; Found: C, 62.89; H, 4.87; N, 18.36.
6.32 (br.d,1H. H-30, J ¼ 3.3 Hz Hz), 4.71 (br.d, 2H, CH2, J ¼ 5.1 Hz); 13
C
NMR (125 MHz, CD3OD): d 179.40 (C]S), 145.70 (CeNHAr), 142.20,
138.40 (CeNHAr), 129.10, 129.10, 128.30, 126.40, 126.40, 110.50,
110.30; EI MS: m/z (rel. abund. %) 233 (Mþ þ 1, 8), 232 (Mþ, 51), 203
(13), 119 (7), 93 (36), 81 (100); Anal. calcd for C12H12N2OS: C, 62.04;
H,5.21; N, 12.06; Found: C, 62.07; H,5.24; N, 12.09.
4.2.30. 1-Phenyl-3-(pyridin-3-yl)thiourea (30)
Yield: 73%; M.p.: 198 ꢁC Rf: 0.47 (acetone/hexane, 3:7); IR (KBr):
4.2.35. 1-(5-Bromo-6-methylpyridin-2-yl)-3-phenylthiourea (35)
Yield: 72%; M.p.: 239 ꢁC Rf: 0.45 (acetone/hexane, 3:7); IR (KBr):
nmax 3149, 3034, 1586, 1533, 1448, 1378, 1230 cmꢂ1 1H NMR
;
(500 MHz, Acetone-d6):
d
9.28 (s, 1H, NH), 9.10 (s, 1H, NH), 8.64
nmax 3218, 3026, 1600, 1551, 1447, 1370, 1299, 1141, 1033 cmꢂ1 1H
;
(br.s, 1H, H-20), 8.34 (dd, 1H, H-60, J ¼ 4.8, 2.1 Hz), 8.04 (ddd, 1H, H-
40, J ¼ 6.9, 2.7, 2.4 Hz), 7.54 (d, 2H, H-2, 6, J ¼ 8.1 Hz), 7.39 (t, 2H, H-3,
5, J ¼ 7.8 Hz), 7.32 (br.dd, 1H, H-50, J ¼ 8.4, 3.6 Hz), 7.21 (t, 1H, H-4,
NMR (500 MHz, DMSO): d 13.59 (s, 1H, NH), 10.92 (s, 1H, NH), 8.00
(d, 1H, H-40, J4 ,3 ¼ 9.0 Hz), 7.75 (d, 2H, H-2, 6, J ¼ 7.8 Hz), 7.41 (t, 2H,
0
0
H-3, 5, J ¼ 7.8 Hz), 7.22 (t, 1H, H-4, J ¼ 7.5 Hz), 7.06 (d, 1H. H-30,
J ¼ 7.5 Hz); 13C NMR (125 MHz, CD3OD):
d
180.00 (C]S), 140.1,
J ¼ 8.7 Hz), 2.49 (s, 3H, CH3); 13C NMR (125 MHz, CD3OD):
d 180.00
138.40 (CeNHAr), 138.70, 136.20, 134.20 (CeNHAr), 129.10, 129.10,
128.30, 127.50, 126.40, 126.40; EI MS: m/z (rel. abund. %) 230
(Mþ þ 1, 3), 229 (Mþ, 18), 195 (100), 136 (95), 93 (85), 78 (53); Anal.
calcd for C12H11N3S: C, 62.86; H, 4.84; N, 18.33; found: C, 62.89; H,
4.87; N, 18.36.
(C]S), 159.90 (CeCH3), 157.10 (CeNHAr), 147.40, 138.40 (CeNHAr),
129.00, 129.00, 128.30, 126.40, 126.40, 109.70 (CeBr), 108.00; EI MS:
m/z (rel. abund. %) 325 (Mþ þ 2, 17), 323 (Mþ þ 1, 23), 268 (15), 188
(22), 172 (27), 151 (66), 136 (52), 123 (88), 118 (71), 109 (51), 93
(100); Anal. calcd for C13H12BrN3OS: C, 48.46; H, 3.75; N, 13.04;
found: C, 48.48; H, 3.77; N, 13.06.
4.2.31. 1-Phenyl-3-(pyridin-4-yl)thiourea (31)
Yield: 75%; M.p.: 202 ꢁC Rf: 0.48 (acetone/hexane, 3:7); IR (KBr):
4.2.36. 1-(2-Chloropyridin-3-yl)-3-phenylthiourea (36)
nmax 3272, 3121, 1632, 1566, 1532, 1494, 1292 cmꢂ1
;
1H NMR
Yield: 71.8%; M.p.: 230 ꢁC Rf: 0.40 (acetone/hexane, 3:7); IR
(400 MHz, DMSO):
d
7.52 (t, 2H, H-20, 60, J ¼ 8.0 Hz), 7.43 (br.s,1H, H-
(KBr): nmax 3156, 2992, 1593, 1546, 1408, 1235, 1202, 1092 cmꢂ1; 1H
30), 7.36 (m, 5H, H-2, 3, 5, 50, 6), 7.14 (t, 1H, H-4, J ¼ 7.2 Hz); 13C NMR
NMR (500 MHz, DMSO): d 10.25 (s, 1H, NH), 9.54 (s, 1H, NH), 8.26
(125 MHz, CD3OD):
d
180.00 (C]S), 155.20 (CeNHAr), 150.20,
(dd, 1H, H-40, J ¼ 4.5, 1.5 Hz), 8.10 (dd, 1H, H-60, J ¼ 7.8, 1.5 Hz), 7.53
(d, 2H, H-2, 6, J ¼ 7.8 Hz), 7.45 (dd, 1H, H-50, J ¼ 4.5, 7.8 Hz), 7.38 (t,
2H, H-3, 5, J ¼ 8.1 Hz), 7.18 (t, 1H, H-4, J ¼ 7.5 Hz); 13C NMR
150.20, 138.50 (CeNHAr), 129.20, 129.20, 128.30, 126.40, 126.40,
109.10, 109.10; EI MS: m/z (rel. abund. %) 203 (17), 135 (27), 93 (23),
77 (27); Anal. calcd for C12H11N3S: C, 62.86; H, 4.84; N, 18.33;
Found: C, 62.89; H, 4.87; N, 18.36.
(125 MHz, CD3OD):
d 180.00 (C]S), 145.70 (CeNHAr), 139.40 (Ce
Cl), 138.70, 138.40 (CeNHAr), 129.10, 129.10, 128.30, 126.40, 126.40,
123.70, 123.50; EI MS: m/z (rel. abund. %) 263 (Mþ, 6), 262 (24), 228
(47), 226 (100), 128 (18), 92 (10); Anal. calcd for C12H10ClN3OS: C,
54.65; H, 3.82; N, 15.93; Found: C, 54.67; H, 3.84; N, 15.95.
4.2.32. 1-(3-Methylpyridin-2-yl)-3-phenylthiourea (32)
Yield: 72%; M.p.: 188 ꢁC Rf: 0.59 (acetone/hexane, 3:7); IR (KBr):
nmax 3398, 3020, 2769, 1631, 1594, 1514, 1449, 1362 cmꢂ1; 1H NMR
(500 MHz, Acetone-d6):
d
14.08 (s, 1H, NH), 8.24 (d, 1H, H-60,
4.2.37. 1-Phenyl-3-(pyridin-3-ylmethyl)thiourea (37)
J ¼ 5.1 Hz), 7.82 (d, 2H, H-2, 6, J ¼ 7.8 Hz), 7.55 (d, 1H, H-40,
J ¼ 8.1 Hz), 7.40 (t, 2H, H-3, 5, J ¼ 8.1 Hz), 7.22 (t, 1H, H-4, J ¼ 7.5 Hz),
Yield: 80%; M.p.: 175 ꢁC Rf: 0.43 (acetone/hexane, 3:7); IR (KBr):
nmax 3159, 2993, 1589, 1533, 1428, 1295 cmꢂ1 1H NMR (300 MHz,
;

K.M. Khan et al. / European Journal of Medicinal Chemistry 74 (2014) 314e323
323
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DMSO):
d
9.67 (s, 1H, NH), 8.54 (br.s, 1H, H-20), 8.45 (dd, 1H, H-60,
J ¼ 1.5, 4.8 Hz), 8.22 (br.s, 1H, NH), 7.75 (t, 1H, H-50, J ¼ 6.3 Hz), 7.40
(m, 5H, H-2, 3, 4, 5, 6), 7.14 (br.t, 1H, H-40, J ¼ 7.2 Hz), 4.76 (br.d, 2H,
CH2, J ¼ 5.7 Hz); 13C NMR (125 MHz, CD3OD):
d 179.40 (C]S),
148.60, 147.20 (CeNHAr), 138.40 (CeNHAr) 135.50, 135.40, 129.10,
129.10, 128.30, 126.40, 126.40, 123.10, EI MS: m/z (rel. abund. %) 245
(Mþ þ 2, 6), 244 (Mþ þ 1, 18), 243 (Mþ, 100), 209 (15), 168 (9), 107
(18), 93 (78); Anal. calcd for C13H13N3S: C, 64.17; H, 5.38; N, 17.27;
Found: C, 64.20; H, 5.41; N, 17.30.
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566e570.
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1028e1034.
4.2.38. 1-Phenyl-3-(pyridin-4-ylmethyl)thiourea (38)
Yield: 78%; M.p.: 175 ꢁC Rf: 0.43 (acetone/hexane, 3:7); IR (KBr):
nmax 3266, 2996, 1598, 1529, 1420, 1344, 1242 cmꢂ1 1H NMR
;
(300 MHz, DMSO):
d
9.74 (s, 1H, NH), 8.50 (br.d, 2H, H-20, 60,
J ¼ 4.4 Hz), 8.22 (br.s, 1H, NH), 7.41 (d, 2H, H-2, 6, J ¼ 7.6 Hz), 7.35 (t,
2H, H-3, 5, J ¼ 7.6 Hz), 7.28 (d, 2H, H-30, 50, J ¼ 6.0 Hz), 7.15 (t, 1H, H-
4, J ¼ 7.2 Hz), 4.76 (br.d, 2H, CH2, J ¼ 5.6 Hz); 13C NMR (125 MHz,
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Bioorg. Med. Chem. 17 (2007) 6387e6391.
CD3OD):
d 179.40 (C]S), 149.80, 149.80, 147.50 (CeNHAr),
138.40 (CeNHAr), 128.30, 129.00, 129.00, 126.50, 126.50, 122.30,
122.30, 50.70; EI MS: m/z (rel. abund. %) 245 (Mþ þ 2, 3.), 244
(Mþþ1, 10), 243 (Mþ, 58), 210 (8), 107 (13), 93 (100), 77 (20); Anal.
calcd for C13H13N3S: C, 64.17; H, 5.38; N, 17.27; Found: C, 64.20; H,
5.41; N, 17.30.
[25] (a) K.M. Khan, M. Taha, F. Naz, M. Khan, F. Rahim, Samreen, S. Perveen,
M.I. Choudhary, Med. Chem. 7 (2011) 704e710;
(b) K.M. Khan, M. Taha, F. Rahim, M. Ali, W. Jamil, S. Perveen, M.I. Choudhary,
Lett. Org. Chem. 7 (2010) 415e419;
(c) K.M. Khan, M. Taha, M. Ali, S. Perveen, Lett. Org. Chem. 6 (2009) 319e320;
(d) K.M. Khan, M. Ali, M. Taha, S. Perveen, M.I. Choudhary, W. Voelter, Lett.
Org. Chem. 5 (2008) 432e434.
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S. Perveen, M.I. Choudhary, Med. Chem. 7 (2011) 572e580;
(b) K.M. Khan, M. Khan, M. Ali, M. Taha, S. Rasheed, S. Perveen,
M.I. Choudhary, Bioorg. Med. Chem. 17 (2009) 7795e8780;
(c) K.M. Khan, M. Taha, F. Rahim, M.I. Fakhri, W. Jamil, M. Khan, S. Rasheed,
A. Karim, S. Perveen, M.I. choudhary, J. Pak. Chem. Soc. 35 (2013) 929;
(d) K.M. Khan, F. Rahim, N. Ambreen, M. Taha, M. Khan, H. Jahan, Najeebullah,
A. Shaikh, S. Iqbal, S. Perveen, M.I. Choudhary, Med. Chem. 9 (2013) 588;
(e) M. Taha, M.S. Baharudin, N.H. Ismail, K.M. Khan, F.M. Jaafar, Samreen,
S. Siddiqui, M.I. Choudhary, Bioorg. Med. Chem. Lett. 23 (2013) 3463;
(f) K.M. Khan, M. Taha, F. Naz, S. Ali, S. Perveen, M.I. Choudhary, Med. Chem. 8
(2012) 705e7010.
Acknowledgment
The authors are thankful to the Organization for Prohibition of
Chemical Weapons (OPCW), The Netherlands, and Higher Educa-
tion Commission (HEC) Pakistan for their financial support for
projects No. L/ICA/ICB/173681/12 and 20-1910, respectively.
Appendix A. Supplementary data
[27] (a) S.M.A. Hai, S. Perveen, R.A. Khan, K.M. Khan, N. Afza, Nat. Prod. Res. 17
(2003) 351;
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.01.001.
(b) S. Perveen, S.M.A. Hai, R.A. Khan, K.M. Khan, N. Afza, T.B. Sarfaraz, Synth.
Commun. 35 (2005) 1663e1674.
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(c) B.N. Ph, Ng. D. Xuong, Ng. H. Nam, J. Chem. Soc. (1955) 1573e1578;
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