Solution-phase microwave assisted parallel synthesis of N,N′-disubstituted thioureas derived from benzoic acid: Biological evaluation and molecular docking studies

Article abstract of DOI:10.1016/j.ejmech.2013.10.012

An efficient and facile microwave-assisted solution phase parallel synthesis for a 26-member library of N,N′-disubstituted thiourea analogs were accomplished successfully. The reaction time for synthesis of analogs was drastically reduced from a reported 8-12 h to only 10 min. Compounds were more than 95% pure, as characterized by modern analytical techniques, i.e. ¹H & ¹³C NMR and FT-IR. The solid phase structural analysis has also been performed by single crystal XRD analysis. Synthesized compounds were preliminary screened for their in vitro urease inhibition and antifungal activity. Most of the compounds were found to be potent inhibitors of urease. However, the most significant activity was found for 11 with IC ₅₀ of 1.67 μM. The docking scores correlate with the IC ₅₀ values of inhibitors.

Full text of DOI:10.1016/j.ejmech.2013.10.012

European Journal of Medicinal Chemistry 70 (2013) 487e496
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Solution-phase microwave assisted parallel synthesis of N,N0-
disubstituted thioureas derived from benzoic acid: Biological
evaluation and molecular docking studies
,
b
Muhammad Khawar Rauf a, Ammara Talib a, Amin Badshah a , Sumera Zaib ,
**
Khurram Shoaib b, Mohammad Shahid c, Ulrich Flörke d, Imtiaz-ud-Din a, Jamshed Iqbal b
,
*
a Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
b Centre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
c Fraunhofer Institute SCAI, Department of Bioinformatics, Germany
d Department Chemie, Fakultät für Naturwissenschaften, Universität Paderborn, Warburger Str. 100, D-33098 Paderborn, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient and facile microwave-assisted solution phase parallel synthesis for a 26-member library of
N,N0-disubstituted thiourea analogs were accomplished successfully. The reaction time for synthesis of
analogs was drastically reduced from a reported 8e12 h to only 10 min. Compounds were more than 95%
pure, as characterized by modern analytical techniques, i.e. 1H & 13C NMR and FT-IR. The solid phase
structural analysis has also been performed by single crystal XRD analysis. Synthesized compounds were
preliminary screened for their in vitro urease inhibition and antifungal activity. Most of the compounds
were found to be potent inhibitors of urease. However, the most significant activity was found for 11 with
Received 14 March 2013
Received in revised form
25 September 2013
Accepted 7 October 2013
Available online 12 October 2013
Keywords:
IC50 of 1.67
m
M. The docking scores correlate with the IC50 values of inhibitors.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Microwave synthesis
N,N0-Disubstituted thioureas
Single crystal XRD
Urease inhibition
Antifungal
DPPH radical scavenging
1. Introduction
prepared from arylthioureas in the presence of bromine [8]. The use
of thioureas to make iminothiazolines [9], thiohydantoins [10],
1,3,5-triazines [11], and 2-amino-oxazolidines [12] has also been
described.
N,N0-Disubstituted thioureas being versatile precursors, have
been subjected to a various structural modifications in order to
prepare a variety of their derivatives with different biological as-
pects. Some of N,N0-disubstituted thioureas themselves are
remarkable owing to their pharmacological and biological impor-
tance. In vitro studies have revealed that various classes of thioureas
are useful as potential antimalarial and anticancer agents [1],
TRPV1 receptor antagonists [2], herbicides [3], insecticides [4],
plant-growth regulators [5] and antihypertensive activity [6].
Thioureas may react with other reagents having different func-
tionalities to yield active compounds of biological significance. For
instance thioureas condense with a-halocarbonyl compounds to
afford 2-amino-1,3-thiazoles [7] and benzothiazoles can be
N,N0-Disubstituted thioureas are commonly synthesized by the
reaction of amines with alkali metal thiocyanates in the presence of
a strong acid [6], aroyl isothiocyanates with amines followed by
basic hydrolysis [13], isothiocyanates with ammonia or amines [14],
unsubstituted thioureas with primary alkyl amines at high tem-
perature [15] and synthesis of mono- and N,N0-disubstituted thio-
ureas by the debenzoylation of N-substituted and N,N0-
disubstituted-N0-benzoylthioureas with hydrazine hydrate under
solvent-free conditions [16]. In recent decades, several new
methods have also been reported for the preparation of substituted
thioureas [17]. However, these protocols have some disadvantages,
namely, high reaction temperature, long reaction time, the use of
noxious reagents and large quantity of solvents. The development
of mild, efficient, and environmental friendly methods are still
awaited. The microwave assisted organic synthesis has received
considerable attention in present days that helped the chemists to
increase the purity of the resulting products with enhanced
* Corresponding author. Tel.: þ92 992 383591, þ92 992 383596; fax: þ92 992
383441.
** Corresponding author.
E-mail addresses: aminbadshah@yahoo.com (A. Badshah), drjamshed@ciit.net.
pk, jamshediqb@gmail.com (J. Iqbal).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.012

488
M.K. Rauf et al. / European Journal of Medicinal Chemistry 70 (2013) 487e496
chemical yield and shorten the reaction time. A mild and efficient
microwave assisted synthesis of N,N0-di- and trisubstituted thio-
ureas have been reported [18e20]. The reported solvents free
synthesis, however, have some reservations regarding purity of the
products due to the presence of inorganic by-products. These are
because of difficulties in ensuring the uniform heating and mo-
lecular activities in the viscous phase. So the development of pure
and inexpensive novel small drug like agents is a prerequisite to
thwart the menace of multi-drug resistant targets. In continuation
to our previously published work on N,N0-disubstituted thioureas
and their coordination chemistry [21], we report here the micro-
wave assisted solution-phase parallel synthesis of N,N0-disubsti-
tuted thioureas and their urease inhibition both in vitro & in virtual,
DPPH radical scavenging and antifungal activities because of their
inherent biological significance.
inductive effect (ꢀI) that result in increase of nucleophilic character
of the amino group. It is well established that the resonance effect is
stronger than inductive effect and the net result is electron
releasing to rest of the molecule. However the reactivity of di- and
higher substituted substrate has been enhanced owing to their
increased electron releasing capability and of better yield as the
data suggest. This has been reflected in the increase of product yield
in the following order; Cl > R > OR > F > CF3 >cyclohex. The
cyclohexyl containing substrates have shown lowest yield due to its
conformations and bulkiness.
2.2. Spectroscopic studies
The most significant bands observed in the FT-IR spectra of all
the synthesized N,N0-disubstituted thioureas are listed in spectro-
scopic data along with the respective compounds in the experi-
mental section. The tentative assignments for functionalities are
2. Results and discussion
made according to the literature [22]. The absence of
n (SeH) vi-
2.1. Chemistry
bration in the range of 2529e2588 cmꢀ1 confirmed the conversion
of isothiocyanate moiety into thiourea (eNHCSNHe) functionality.
The N,N0-disubstituted thioureas behave both as a monodentate
and bidentate ligands, depending upon the reaction conditions. The
characteristic IR bands of N,N0-disubstituted thioureas are found
around; 3120e3402 (NH), 2960e3090 Ph(CH), 1660e1720 (C]O),
1540e1620 (CN), 1243e1277 (C]S) and 1130e1185 (CeS). The
addition of the amine to the C]N double bond took place by
attacking the carbon atom in the isothiocyanate group resulting in
the formation of the desired compounds. After complete reaction,
the strong band at 2000 cmꢀ1 (N]C]S) in the isothiocyanate
disappeared. Instead of the expected normal carbonyl (C]O) ab-
sorption around nmax/cmꢀ1: 1710 a medium strong band at 1665e
1678 cmꢀ1 suggested a possible hydrogen bond formation between
the H-atom of the NH-group and the O-atom of the carbonyl group.
The band around nmax/cmꢀ1 2500e2600, corresponding to SH, was
not found which indicated the absence of the N]CSH tautomeric
form in all the compounds [22e24].
Microwave assisted solution-phase parallel synthesis of N,N0-
disubstituted thioureas was performed using a microwave oven
which was house modified especially for organic synthesis. A small
library of twenty six candidates (1e26) was developed and char-
acterized successfully. The purity of the compounds was checked by
TLC with Merck Kieselgel GF 254 Plates, elemental analysis, and
their structures were investigated by 1H and 13C NMR spectroscopy.
The synthesis of benzoyl isothiocyanate was accomplished by
conventional manner and a purified sample of benzoyl isothiocy-
anate was obtained by steam distillation. Stoichiometric amounts
of benzoyl isothiocyanate and respective primary amines were
subjected to glass vials along with THF as solvent and then irradi-
ated them under same set of conditions. The complete description
of the substituents for derivatives (1e26) is enunciated in
Scheme 1. The nature of substrates H2NR, strongly influence the
yield of the final products obtained through microwaves assisted
reactions (Scheme 1). The substituents like F, Cl, CF3, OR, etc.
attached at the ortho and para positions on the aromatic ring in
H2NR moiety show strong mesomeric effect (þR) by releasing
electrons through delocalization of lone pairs in spite of the
1
The H NMR data for the synthesized N,N0-disubstituted thio-
ureas showed that the NH hydrogen resonates considerably
downfield from other resonances in the spectra. The proton
chemical shifts were found around 9.00e9.50 and 10.75e
Scheme 1. Synthesis of N,N0-disubstituted thioureas derivatives (1e26).

M.K. Rauf et al. / European Journal of Medicinal Chemistry 70 (2013) 487e496
489
12.80 ppm for free and hydrogen bonded NH, respectively, and
aromatic protons appeared downfield between 7.23 and 8.50 ppm
in their usual regions. It was also observed that the coordinating or
highly polar solvents like DMSO-d6 had profound effect on the free
NH protons chemical shift and appeared more downfield as
compared with the non-coordinating solvents like CDCl3, C6D6 and
CD2Cl2. This shift could be attributed to the possible hydrogen
bonding between the NH and sulfoxide (S]O) moiety [22].
The 13C NMR data explicitly show all the signals due to the
unique carbons present in compounds (1e26). The aromatic carbon
resonances of the thiourea ligands were assigned on the basis of
signal intensities and then were compared with the reported values
[25]. The chemical shifts of the carbons pertaining to CONH and
CSNH moieties of the substituted thiourea ligands resonate around
166e168 and 177e180 ppm, respectively.
compounds 2, 3 and 4 were moderately active and dichloro
substituted compounds 6e10 were significantly active on enzyme.
Compound 24, bearing cyclohexyl substitution was also not very
potent inhibitor of urease. We can establish a structure activity
relationship between the structure of the derivatives 1e26 and the
urease enzyme that the presence of bulky groups on the phenyl ring
of R moiety, particularly at successive ortho and meta positions
keeps the main pharmacophore eCONHCSNHe away from the
binding sites of the enzyme protein. Docking studies fully compli-
ment this structure activity relationship. Most of the compounds
were more potent than the standard inhibitors of urease (thiourea,
IC50 22.3 ꢂ 1.2
compounds have their urease activities in the range of 1.67e
8.23 M (Table 4). The most potent compound 11 was further
mM and acetohydroxamic acid, 41.5 ꢂ 2.3
mM). Potent
m
evaluated to find out the mechanism of urease inhibition. It was
observed that the compounds inhibit the enzyme in a competitive
manner (for graph see Fig. 2).
2.3. Structural studies
The two molecular structures 11 (Fig. 1) and 12 (Fig. S1) with
similar thiourea cores and different substitution patterns are
described here. The cores are mostly planar with SeCeNeC and Oe
CeNeC torsion angles of 165.6(1)ꢁ (molecule I),159.8(1)ꢁ (molecule
II) and 8.8(3)ꢁ (I), 5.4(3)ꢁ (II) for 11, 179.3(3)ꢁ and 1.2(5)ꢁ for 12 as
well. The aromatic ring planes make dihedral angles of
86.78(5)ꢁ(11I) and 36.32(7)ꢁ (11II) and 87.9(1)ꢁ (12) respectively. In
general, there are no unexpected geometric parameters. Both mo-
lecular structures show an intramolecular NeH/O hydrogen bond
stabilizing the thiourea geometry. The crystal structure of 11 de-
picts intermolecular interactions N11/21-H/O (for O11:
x ꢀ 0.5, ꢀy ꢀ 0.5, z ꢀ 0.5; for O21: x þ 0.5, ꢀy ꢀ 0.5, z ꢀ 0.5) and
N12/22-H/S (for S11: x ꢀ 0.5, ꢀy ꢀ 0.5, z ꢀ 0.5; for S21:
x þ 0.5, ꢀy ꢀ 0.5, z ꢀ 0.5) that connect the molecules into endless
rows extended along c-axis (Fig. S2). Crystallographic and refine-
ment details are shown in Table 1. Selected bond lengths and angles
are given in Table 2. The packing of 12 (Figure S3) exhibits C4e
H/O1 (ꢀx þ 1, ꢀy þ 1, ꢀz) (Table 3) bonds that link molecules into
centro-symmetric dimers that are stacked along the a-axis.
2.4.2. Antioxidant study
The free radical scavenging capability of 1-benzoyl-3-
phenylthiourea derivatives (1e26) was measured by DPPH
method. In this assay, propyl gallate and 3-tert-butyl-4-
hydroxyanisole were used as standards. Screening result showed
that some derivatives of 1-benzoyl-3-phenylthiourea had equi-
potent or more active than both of the standards. The thiourea
derivatives exhibited significant activities IC50 values were in the
range of 23.27e30.77 mM (Table 5). Compound 15 having a methyl
group on benzene ring at para position was found to be the most
active among all derivatives. Compounds 10, 5 and 7 having
dichloro substitutions on benzene ring also possessed notable po-
tency. The halogenated rings showed relatively good antioxidant
activity in this series. In general, among mono-substituted com-
pounds, p-substituted compounds had better DPPH radical scav-
enging activity; p-methyl substituted compound 15 was most
active with IC50 value of 23.3 mM. Among di- and tri-substituted
compounds, di-substituted compounds were found to be more
active, probably due to the increased stability of resulting radical Aꢃ
as a result of di-substitution. It is common with the thiourea related
compounds that they scavenge the DPPH radical by donating H.
2.4. Pharmacology
2.4.1. Urease inhibition assay
2.4.3. Antifungal study
The 1-benzoyl-3-phenylthiourea derivatives (1e26) were pre-
liminary screened for their inhibition in vitro against Jack bean
urease. These derivatives were observed as potent inhibitors of
urease enzyme. Compound 11 with 2,4,6-trichloro substitution on
phenyl ring was the most potent derivative with an IC50 of
Antifungal activity of 1-benzoyl-3-phenylthiourea derivatives
(1e26) was analyzed by agar well diffusion method against four
fungal strains Aspergillus niger, Aspergillus flavus, Saccharomyces
cerevisiae and Candida tropicalis the results are shown in Table 5.
The results showed that compounds 11 and 12 containing tri and
tetrachloro substituted benzene rings have equipotent inhibitory
activity as compared to the standards fluconazole and terbinafine.
The tested compounds were relatively less active than fluconazole
1.67 ꢂ 0.51
substitution on phenyl ring was found to be the least active with an
IC50 of 74.91 ꢂ 4.8 M. It was observed that monochloro substituted
mM. However, compound 12, having 2,3,5,6-tetrachloro
m
Fig. 1. Molecular structure of 11 with the two independent molecules I and II showing anisotropic displacement ellipsoids drawn at the 50% probability level.

490
M.K. Rauf et al. / European Journal of Medicinal Chemistry 70 (2013) 487e496
Table 1
Table 3
ꢀ
Crystallographic data for 11 and 12.
The intermolecular and intramolecular hydrogen bonds for 11 and 12. Distance, A;
angle, ꢁ
.
Compound
11
12
DHA (ꢁ)
ꢀ
ꢀ
ꢀ
Compound
D
H
A
DH (A)
HA (A)
DA (A)
Formula
FW
C14H9Cl3N2OS
359.64
C14H8Cl4N2OS
394.08
11
N2 H2N O1 0.870(2) 1.877(19) 2.581 (16) 136.7(17)
Crystal system
Space group
Monoclinic
P2(1)/n
7.6912(4)
36.3243(17)
10.6164(5)
90, 90.732(1), 90
Triclinic
P-1
N5 H6N O2 0.880(2) 1.820(2) 2.551 (17) 139.2(18)
N2 H2N O1 0.870(2) 1.877(19) 2.581 (16) 136.7(17)
N5 H6N O2 0.880(2) 1.820(2) 2.551 (17) 139.2(18)
12
ꢀ
a (A)
8.6312(16)
8.9821(16)
10.9495(19)
76.303(4), 82.455(4),
74.834(4)
793.8(2)
2
120(2)
1.649
0.877
396
ꢀ
b (A)
ꢀ
c (A)
a, b, g
(ꢁ)
in compounds 14, 15, 19 and 21), the antifungal activity decreases
significantly (inhibition zones in the range 1e3 mm). The reason for
this decreased activity is due to the presence of lone pair of elec-
trons on oxygen atom, the substitution of the methoxy group on
benzene ring also decreases the activity as it is electron donating
group and its lone pair of electrons are resonating on benzene ring.
Further, the electron donating effect of methoxy group is lower
than alkyl group, which further confirms the effect of substitution
of electron donating groups on the antifungal activities.
3
ꢀ
V (A )
Z
2965.7(3)
8
120(2)
1.611
0.756
1456
T (K)
DC Mg/m3
m
(mmꢀ1
F(000)
Crystal size (mm3)
range (ꢁ)
)
0.47 ꢄ 0.22 ꢄ 0.20 0.30 ꢄ 0.26 ꢄ 0.18
q
2.00e28.08ꢁ
ꢀ10 ꢅ h ꢅ 10,
ꢀ45 ꢅ k ꢅ 48,
ꢀ14 ꢅ l ꢅ 13
29,693
1.92e28.08ꢁ
Index ranges
ꢀ11 ꢅ h ꢅ 11,
ꢀ11 ꢅ k ꢅ 11,
ꢀ14 ꢅ l ꢅ 13
8179
Reflections collected
Indept. Reflect. [R(int)]
7181
3850 [R(int) ¼ 0.0549]
2.4.4. Molecular docking studies
[R(int) ¼ 0.0295]
The docking scores obtained for each compound were in the
range of ꢀ27.48 to ꢀ21.5 and are summarized in Table 4. This in-
dicates that there is no considerable difference in the docking
scores among the compounds. The correlation between docking
scores and IC50 values was very good. However, none of the com-
pounds are interacting with any Ni atom in the active site. The
metal pharmacophore constraint was leading to no docking solu-
tions for any of the compounds. Therefore, the metal pharmaco-
phore was not used. Moreover, there was a common preference for
residues of the active site to which all of the compounds were
interacting. It was observed that all of the compounds interact with
Ala40, Ala609, Arg494, His409, His492, His519 and His593 (Fig. 3).
Completeness to
q
¼ 28ꢁ
99.6%
99.6%
Max. & min. transmission 0.8634, 0.7175
Data/restraints/parameters 7181/0/379
0.8581 and 0.7788
3850/0/204
1.060
0.0626
0.1731
Goodness-of-fit on F2
1.059
0.0370
0.0906
R1 [I > 2
s(I)]
wR2 [all data]
Largest peak and hole (e Å 0.479 and ꢀ0.281
0.765 and ꢀ0.926
ꢀ3
)
which is a triazole based antifungal drug. The derivatives 13, 17 and
18 containing trifluoromethyl group on benzene ring exhibited
strong antifungal activity. Similarly, compound 20 with fluoro
substituted benzene rings at para position possesses equipotent
antifungal activity as compared to the standards. The derivatives
showed variation in inhibition from simply active to strongly active
nature. Compounds 1 and 23 were the exceptions with no activity
towards all four fungal strains. Compounds 1 and 23, with no
substitutions on the benzene ring did not show any antifungal ac-
tivity. Upon substitution of benzene group with electron with-
drawing groups such as chloro, fluoro and methoxy, the antifungal
activity increases.
Compounds 11, 12, 16, 18 and 20 had significant antifungal ac-
tivity against all four strains of fungi, with inhibition zones in the
range 3e6 mm, which is comparable to the activity of standard
antifungal drugs used, fluconazole and terbinafine. However when
these electronegative groups are replaced by alkyl substituents (as
Table 4
Inhibition of 1-benzoyl-3-phenylthiourea derivatives (1e26) against Jack bean ure-
ase and their corresponding docking scores.
No
IC50 ꢂ SEM (
m
M)
Docking score (kcal/mol)
1
2
3
4
14.1 ꢂ 1.6
41.2 ꢂ 4. 7
27.32 ꢂ 2.7
ꢀ25.4096
ꢀ24.8776
ꢀ25.5442
ꢀ24.4206
ꢀ24.9408
ꢀ25.4436
ꢀ26.7557
ꢀ26.8703
ꢀ26.4381
ꢀ24.1485
ꢀ27.4874
ꢀ21.5821
ꢀ23.7174
ꢀ26.9345
ꢀ26.0900
ꢀ25.1512
ꢀ23.8260
ꢀ25.0731
ꢀ23.2391
ꢀ23.7023
ꢀ25.5481
ꢀ26.9329
ꢀ23.7696
ꢀ22.7353
ꢀ25.9790
ꢀ23.7696
ND
35.28 ꢂ 4.37
32.2 ꢂ 3.8
13.2 ꢂ 1.4
4.54 ꢂ 0.32
4.27 ꢂ 0.41
5.51 ꢂ 0.42
18.4 ꢂ 1.9
1.67 ꢂ 0.51
74.91 ꢂ 4.85
39.6 ꢂ 3.7
3.17 ꢂ 0.22
6.23 ꢂ 0.32
12.8 ꢂ 1.7
38.2 ꢂ 2.8
12.2 ꢂ 1.6
47.2 ꢂ 4.1
41.2 ꢂ 3.4
8.11 ꢂ 0.21
3.52 ꢂ 0.12
27.35 ꢂ 1.29
53.2 ꢂ 4.35
8.21 ꢂ 0.71
35.28 ꢂ 4.38
22.3 ꢂ 1.2
41.5 ꢂ 2.3
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Thiourea
AHA
Table 2
ꢁ
ꢁ
ꢀ
Selected bond lengths (A), angles ( ) and torsion angles ( ) for 11 and 12.
11
Cl(11)eC(110)
S(11)eC(108)
S(21)eC(208)
O(11)eC(101)
O(21)eC(201)
N(11)eC(101)
N(11)eC(108)
12
1.7273(18)
1.6604(18)
1.6593(18)
1.226(2)
1.227(2)
1.378(2)
C(101)eN(11)eC(108)
O(11)eC(101)eN(11)
N(12)eC(108)eN(11)
N(12)eC(108)eS(11)
O(21)eC(201)eN(21)
N(22)eC(208)eN(21)
N(22)eC(208)eS(21)
128.26(15)
122.13(16)
116.20(15)
124.94(13)
122.84(15)
116.07(15)
124.46(13)
1.394(2)
Cl(1)eC(2)
S(1)eC(7)
O(1)eC(8)
N(1)eC(7)
N(1)eH(1)
N(2)eC(7)
1.712(3)
1.646(4)
1.216(4)
1.331(4)
0.8800
C(7)eN(1)eC(1)
C(8)eN(2)eH(2)
C(7)eN(2)eH(2)
N(1)eC(7)eN(2)
N(1)eC(7)eS(1)
N(2)eC(7)eS(1)
121.4(3)
115.6
115.6
115.0(3)
125.1(3)
119.9(3)
ND
ND e not determined.
AHA e acetohydroxamic acid.
1.395(4)

M.K. Rauf et al. / European Journal of Medicinal Chemistry 70 (2013) 487e496
491
450
300
150
0.5
0.05
0
-0.0005
0.0005
1/[Urea]( M)
0.0010
µ
-150
Fig. 3. Common binding modes of all the compounds inside the active site pocket
shown in green surface representation. Dotted lines show hydrogen-bonding in-
teractions with His593 and Arg609. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
Fig. 2. Lineweavere Burk plots of inhibition of urease by compound 11; concentration
of inhibitor used was: 0 mM, 0.5 mM, 0.05 mM.
The hydrogen bonding interaction was observed between the ox-
ygen and hydrogen of the inhibitors with residues Ala609, Arg494
and His593 only as shown in Figs. 4 and 5. Furthermore, the most
potent inhibitor of urease, 11 was docked inside the active site of
the enzyme as depicted in Fig. 6.
Table 5
DPPH radical scavenging and antifungal activities of synthesized compounds (1e
26).
No.
Antioxidant Activity
(IC50 or %age
Antifungal activity
2.4.5. Binding mode analysis
A. niger A. flavus S. cerevisiae C. tropicalis
inhibition)a
Unlike other sulfonamides and thiourea compounds, that form
coordinated interactions with the bi-nickle center in the catalytic
site of urease enzyme, these compounds do not show any inter-
action with the nickel atoms. However, aromatic interactions with
the residues surrounding the nickel ions in the deep shallow pocket
area are common for most of the compounds as shown in Fig. 3.
1
2
3
4
5
6
7
8
769 ꢂ 11
26%
21%
ꢀ
ꢀ
ꢀ
ꢀ
þ
þ
þ
þ
þ
þ
þ
þ
265 ꢂ 10
29.5 ꢂ 0.03
240 ꢂ 15
30.7 ꢂ 0.01
29%
70.0 ꢂ 0.05
27.0 ꢂ 0.01
19%
31%
34%
27%
23.3 ꢂ 0.1
805 ꢂ 19
38%
114 ꢂ 7
676 ꢂ 18
31%
þþ
þ
þþ
þ
þþ
þ
þþ
þ
þþþ þþ
þþþ
þ
þþ
þ
þ
þ
þþ
þ
þþ
þ
þþ
þ
þþ
þ
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
þ
þ
þ
þ
þþþ þþþ
þþþ þþþ
þþþ þþ
þþþ
þþþ
þþþ
þ
þþþ
þþþ
þþ
þ
þ
þ
þþ
þþ
þþ
þþþ
þþ
þþþ
þ
þþ
þþþ
þþþ
þþþ
þ
þþþ þþþ
þþ
þþþ
þþþ þþþ
þ
þ
þþþ þþþ
þþþ
þ
þþþ
þþ
þ
24%
38%
22%
28%
þ
þþ
þ
þþ
ꢀ
þþ
ꢀ
ꢀ
ꢀ
þ
þ
þ
þ
276 ꢂ 16
þ
ꢀ
þ
ꢀ
36%
þ
þ
þ
þ
Fluconazole ꢀ
þþþ þþþ
þþþ þþþ
þþþ
þþþ
ꢀ
þþþ
þþþ
ꢀ
Terbinafine
PG
ꢀ
40.80 ꢂ 1.2
28.20 ꢂ 1.1
ꢀ
ꢀ
ꢀ
ꢀ
BHA
ꢀ
ꢀ
(ꢀ): No activity distance (from growth to sample): nil.
(þ): Active less than 1 mm.
(þþ): Moderately active 1 mme3 mm.
(þþþ): Strongly active 3 mme6 mm.
Fig. 4. Interaction diagram of compound 14. The green curved line indicates hydro-
phobic interactions with the surrounding residues. The dotted lines indicate hydrogen
bonding with residues Asp494, His593 and Arg609. (For interpretation of the refer-
ences to color in this figure legend, the reader is referred to the web version of this
article.)
(þþþþ): Very strongly active more than 6 mm.
a
Values are expressed as mean ꢂ standard error of the mean of at least three
different experiments in triplicate. (% inhibition) at 1 mM concentration of evalu-
ated compounds.

492
M.K. Rauf et al. / European Journal of Medicinal Chemistry 70 (2013) 487e496
further modifications that could give rise to structures with
improved urease inhibitory activity. Selected compounds exhibited
antioxidant activity, such as compound 15 having a methyl group
on benzene ring at para position was found to be the most active
scavenger of free radical. Additionally, compounds 11 and 12
showed equipotent antifungal activity as compared to the
standards.
4. Experimental
4.1. Materials and methods
All experiments were carried out under the specified conditions.
Solvents were distilled from the drying agents, stored over mo-
ꢀ
lecular sieves 4 A and degassed before use. Microwave syntheses
were carried out using a Dawlance DW-293S domestic microwave
oven with variable power settings. The experiments were carried
out in standard Pyrex capable glassware chamber (20 mL). The
temperature of the reaction mixtures was monitored directly by a
microwave-transparent fluoroptic probe.
Fig. 5. Interaction diagram of compound 22. The green curved line indicates hydro-
phobic interactions with the surrounding residues. The dotted lines indicate hydrogen
bonding with residues Asp494, His593 and Arg609. (For interpretation of the refer-
ences to color in this figure legend, the reader is referred to the web version of this
article.)
NMR spectra were recorded on a Bruker ARX, 300 MHz spec-
trometer. 1H NMR (300.13 MHz) and 13C NMR (75.47 MHz) using
internal standard solvent CDCl3 (7.28 ppm from TMS) & internal
standard TMS. The splitting of proton resonances in the reported 1H
NMR spectra are defined as s ¼ singlet, d ¼ doublet, t ¼ triplet,
q ¼ quartet and m ¼ complex pattern; coupling constants are re-
ported in Hz. FT-IR spectra were recorded as KBr pellets on a Bio-
Rad Excalibur FT-IR Model FTS 3000 MX (400-4000 cmꢀ1) and
the elemental analyses were performed using a LECO-932 CHNS
analyzer. The melting points were determined on a Bio Cote
SMP10-UK and are uncorrected. The chemicals like benzoic acid,
ammonium thiocyanate, thionyl chloride, and primary amines,
were purchased from SigmaeAldrich and are used as received.
4.2. Microwave assisted solution-phase parallel synthesis of N,N0-
disubstituted thioureas (1e26)
Freshly prepared benzoyl isothiocyanate was mixed in a 1:1 M
ratio with the respective primary amines in dry THF, in well capped
cylindrical glass vessels. The reaction mixtures were shaked and
subjected to impulse microwave heating (2 min, 5 times with an
interval of 1 min) at 60e65 ꢁC for about 10e15 min. On cooling, the
reaction mixtures were slowly poured into acidified (pH 4e5)
chilled water and stirred well with a glass rod in ditto labeled
beakers (50 mL). The solid products were separated by filtration
and dried at room temperature (Scheme 1).
Fig. 6. Compound 11 docked inside the active site of the enzyme. The nickel atoms are
represented with blue spheres deep inside the pocket. The docked compound is shown
in CPK representation. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
4.3. Spectroscopic data for (1e26)
The spectroscopic data for (1e10) has already been reported
[22]. However the synthetic strategy is different from that of the
reported one to avoid any duplication and the data is given as
electronic Supplementary information. Representative 1H and 13C
NMR spectra of compounds is given in Supplementary information.
Spectroscopic data for rest of the compounds is given below:
This suggests that the compounds possess competitive inhibition
activity by blocking the entrance to the catalytic domain of the
enzyme.
3. Conclusion
4.3.1. 1-Benzoyl-3-(2,4,6-trichlorophenyl)thiourea (11)
White solid; m.p. 198e199 ꢁC; FT-IR (KBr, cmꢀ1) 3382, 3352,
3132, 3050, 1660, 1595, 1514, 1497, 1369, 1263, 1158, 1084, 851, 803,
0
A series of N,N0-disubstituted thiourea analogs were prepared
using facile microwave-assisted solution phase parallel synthesis.
The synthesized compounds were evaluated for in vitro urease
inhibitory activity. Most of the compounds exhibited excellent
urease inhibition, compound 11 showed potent urease inhibitory
688, 592; 1H NMR (300 MHz, CDCl3)
d
7.47 (s, 2H, ArHb¼b ), 7.58 (t,
0
2H, J ¼ 7.8 Hz, ArHe¼e ), 7.70 (t, 1H, J ¼ 7.8 Hz, ArHf), 7.94 (d, 1H,
J ¼ 7.8 Hz, ArHd¼d ) 9.32 (s, CONH), 12.0 (s, CSNH); 13C NMR
0
activity with an IC50 value 1.67 ꢂ 0.51
m
M. The binding patterns of
(75 MHz, CDCl3) d 127.6 (2C), 128.6 (2C), 129.2 (2C), 131.2, 132.3,
the inhibitors were found by molecular docking studies. The pro-
134.0, 134.7, 135.0 (2C) (Aromatic-C), 167 (C]O), 180.7 (C]S); MS
(EI) m/z 322.7 (M ꢀ Cl), 104.9 (base peak); Anal. calcd. for
posed scaffold of compound 11 offers the possibility of convenient

M.K. Rauf et al. / European Journal of Medicinal Chemistry 70 (2013) 487e496
493
C14H9N2OSCl3; C, 46.75 H, 2.52 N, 7.79 S, 8.92 Found: C, 46.73 H,
2.49 N, 7.81 S, 8.90.
670, 541; 1H NMR (300 MHz, CDCl3)
d
7.46 (t, 1H, J ¼ 7.8 Hz, ArHa),
0
7.57 (t, 2H, J ¼ 7.8 Hz, ArHe¼e ), 7.64e7.71 (m, 2H, ArHb,f), 7.75 (d, 1H,
0
J ¼ 7.8 Hz, ArHc), 7.94 (d, 2H, J ¼ 7.8 Hz, ArHd¼d ), 7.99 (d, 1H,
J ¼ 7.8 Hz, ArHb ), 9.16 (s, 1H, CONH), 12.68 (s, 1H, CSNH); 13C NMR
0
4.3.2. 1-Benzoyl-3-(2,3,5,6-tetrachlorophenyl)thiourea (12)
White solid; m.p. 180e182 ꢁC; FT-IR (KBr, cmꢀ1) 3411, 3276,
3109, 1675, 1601, 1501, 1406, 1333, 1251, 1163, 1099, 875, 711, 584;
(75 MHz, CDCl3)
d
121.5, 125.26 & 125.67 (d, 13C-19F, 1J ¼ 31.5 Hz,
CF3), 126.38 & 126.45 (d, 13C-19F, 2J ¼ 3.75 Hz), 127.5, 127.7 (2C),
129.2 (2C), 129.7, 131.3, 132.2, 133.9, 135.5 (AreC), 166.9 (C]O),
180.6 (C]S); Anal. calcd. for C15H11N2OSF3; C, 55.55 H, 3.42 N, 8.64
S, 9.89 Found: C, 55.52 H, 3.41 N, 8.67 S, 9.88.
1H NMR (300 MHz, CDCl3)
d
7.27 (s, 1H, ArHa), 7.57 (t, 2H, J ¼ 7.6 Hz,
0
0
ArHe¼e ), 7.70 (t,1H, J ¼ 7.6 Hz, ArHf), 7.94 (d, 2H, J ¼ 7.6 Hz, ArHd¼d ),
9.35 (s, CONH), 12.0 (s, CSNH); 13C NMR (75 MHz, CDCl3)
d 127.7
(2C), 129.3 (2C), 130.58, 131.0, 132.0 (2C), 132.2, 134.1, 135.9 (2C)
(Aromatic-C), 167.1(C]O), 180.3 (C]S); Anal. calcd. for
4.3.8. 1-Benzoyl-3-(4-triflouromethylphenyl)thiourea (18)
C
14H8N2OSCl4; C, 42.67 H, 2.05 N, 7.11 S, 8.14 Found: C, 42.61 H, 2.01
White solid; m.p. 138e139 ꢁC; FT-IR (KBr, cmꢀ1) 3393, 3184,
3030, 1671, 1602, 1561, 1523, 1329, 1265, 1169, 1111, 1069, 839, 721,
N, 7.14 S, 8.08.
596, 509; 1H NMR (300 MHz, CDCl3)
d
7.58 (t, 2H, J ¼ 8.1 Hz,
0
0
0
0
4.3.3. 1-Benzoyl-3-(3-triflourophenyl)thiourea (13)
ArHe¼e ), 7.67e7.73 (m, 3H, ArHc,c ,f), 7.90e7.96 (m, 4H, ArHd,d ,b,b ),
White solid; m.p. 107e108 ꢁC; FT-IR (KBr, cmꢀ1) 3283, 2999,
9.14 (s, 1H, CONH), 12.8 (s, 1H, CSNH); 13C NMR (75 MHz, CDCl3)
1673, 1599, 1563, 1536, 1474,1345, 1260, 1148, 795, 718, 657, 589; 1H
d
122.0, 123.7 (2C), 126.10 & 126.31 (d, 13C-19F, 1J ¼ 26.2 Hz, CF3),
NMR (300 MHz, CDCl3)
d
7.55e8.07 (m, 9H, AreH), 9.21 (s, CONH),
120.91, 122.96, 124.42 &
127.6 (2C), 128.26 & 128.29 (d, 13C-19F, 2J ¼ 3.9 Hz), 129.4 (2C), 131.3,
12.10 (s, CSNH); 13C NMR (75 MHz, CDCl3)
d
134.0 (2C), 140.6 (AreC), 167.1 (C]O), 178.4 (C]S); Anal. calcd. for
124.62 (d, 13C-19F, 1J ¼ 25 Hz), 127.3, 127.6 (2C), 127.7, 129.0, 129.3
(2C), 129.5,131.4, 134.1, 167.2 (C]O), 178.6 (C]S); MS (EI) m/z 323.7
(Mþ), 104.9 (base peak); Anal. calcd. for C15H11N2OSF3; C, 55.55 H,
3.42 N, 8.64 S, 9.89 Found: C, 55.52 H, 3.41 N, 8.67 S, 9.88.
C15H11N2OSF3; C, 55.55 H, 3.42 N, 8.64 S, 9.89 Found: C, 55.54 H,
3.41 N, 8.65 S, 9.88.
4.3.9. 1-Benzoyl-3-(4-n-butylphenyl)thiourea (19)
White solid; m.p. 159e160 ꢁC; FT-IR (KBr, cmꢀ1) 3289, 3158,
3027, 2923, 1669, 1524, 1437, 1326, 1268, 11,577, 782, 710, 618, 540;
4.3.4. 1-Benzoyl-3-(2,3-dimethylphenyl)thiourea (14)
White solid; m.p. 159e160 ꢁC; FT-IR (KBr, cmꢀ1) 3279, 3153,
1H NMR (300 MHz, CDCl3)
d
0.98 (t, 3H, J ¼ 7.5 Hz, eCaH2eCbH2e
3019, 2932,1671,1526,1447,1328,1258,1155, 783, 712, 616, 542; 1H
CgH2eCdH3), 1.42 (sex, 2H, J ¼ 7.5 Hz, eCgH2e), 1.65 (qnt, 2H,
NMR (300 MHz, CDCl3)
d 2.27 (s, 3H, o-CH3Ar), 2.36 (s, 3H, m-
J ¼ 7.5 Hz, eCbH2e), 2.67 (t, 2H, J ¼ 7.5 Hz, eCaH2e), 7.28 (d, 2H,
0
0
CH3Ar), 7.16e7.23 (m, 2H, ArHc,a), 7.45 (t, 1H, J ¼ 7.6 Hz, ArHb), 7.56
J ¼ 8.4 Hz, ArHc¼c ), 7.44 (d, 2H, J ¼ 8.4 Hz, ArHb¼b ), 7.59 (t, 2H,
0
0
(t, 2H, J ¼ 7.6 Hz, ArHe¼e ), 7.68 (t, 1H, J ¼ 7.6 Hz, ArHf), 7.92 (d, 2H,
J ¼ 7.6 Hz, ArHe¼e ), 7.72 (t, 1H, J ¼ 7.6 Hz, ArHf), 7.94 (d, 2H,
J ¼ 7.6 Hz, ArHd¼d ), 9.25 (s, CONH), 12.18 (s, CSNH); 13C NMR
J ¼ 7.6 Hz, ArHd¼d ), 9.26 (s, CONH), 12.40 (s, CSNH); 13C NMR
0
0
(75 MHz, CDCl3)
d
14.2 (o-CH3Ar), 20.5 (m-CH3Ar), 124.5, 125.8,
(75 MHz, CDCl3) d 13.9, 22.4, 33.5, 35.3 (n-butyl), 124.1, 127.6 (2C),
127.5, 129.2 (2C), 129.4 (2C), 131.6, 132.6, 133.7, 136.3, 138.1 (AreC),
167.1 (C]O), 179.9 (C]S); MS (EI) m/z 283.8 (Mþ), 104.9 (base
peak); Anal. calcd. for C16H16N2OS; C, 67.58 H, 5.67 N, 9.85 S, 11.28
Found: C, 67.55 H, 5.63 N, 9.87 S, 11.26.
128.9 (2C), 130.5(2C), 131.0(2C), 131.2, 133.3, 135.0 (AreC), 166.0
(C]O), 177.6 (C]S); Anal. calcd. for C18H20N2OS; C, 69.20 H, 6.45 N,
8.97 S, 10.26 Found: C, 69.16 H, 6.44 N, 8.99 S, 10.24.
4.3.10. 1-Benzoyl-3-(4-flouroophenyl)thiourea (20)
White solid; m.p. 126e127 ꢁC; FT-IR (KBr, cmꢀ1) 3224, 3133,
3036, 1670, 1607, 1551, 1509, 1331, 1261, 1215, 1153, 837, 701, 672,
0
4.3.5. 1-Benzoyl-3-(4-methylphenyl)thiourea (15)
White solid; m.p. 160e162 ꢁC; FT-IR (KBr, cmꢀ1) 3254, 3001,
1673, 1597, 1557, 1354, 1257, 1148, 817, 698, 612, 505; 1H NMR
610, 511; 1H NMR (300 MHz, CDCl3)
d
7.12 (t, 2H, J ¼ 8.4 Hz, ArHe¼e ),
0
0
(300 MHz, CDCl3)
d
2.39 (s, 3H, p-CH3Ar), 7.45 (d, 2H, J ¼ 8.25 Hz,
7.54 (dd, 2H, J ¼ 7.6 Hz, ArHb¼b ), 7.63e7.71 (m, 3H, ArHc,c ,f), 7.91 (d,
0
0
0
ArHc¼c ), 7.33 (d, 2H, J ¼ 8.25 Hz, ArHb¼b ), 7.53 (t, 2H, J ¼ 8.25 Hz,
1H, J ¼ 8.4 Hz, ArHd¼d ), 9.10 (s, 1H, CONH), 12.56 (s, 1H, CSNH); 13C
115.67 & 115.97 (d, 2C, 13C-19F, 2J ¼ 22.5 Hz),
0
0
ArHe¼e ), 7.67 (t, 1H, J ¼ 8.25 Hz, ArHf), 7.91 (d, 2H, ArHd¼d ), 9.13 (s,
NMR (75 MHz, CDCl3)
d
CONH), 12.50 (s, CSNH); 13C NMR (75 MHz, CDCl3)
d
21.1 (p-CH3Ar),
126.25 & 126.36 (d, 2C, 13C-19F, 3J ¼ 25 Hz), 127.5 (2C), 129.2 (2C),
131.5, 133.57 & 133.61 (d, 13C-19F, 4J ¼ 3.2 Hz), 133.8, 159.37 & 162.64
(d, 13C-19F, 1J ¼ 245.25 Hz) (AreC), 167.0 (C]O), 178.9 (C]S); Anal.
calcd. for C14H11N2OSF; C, 61.30 H, 4.04 N, 10.21 S, 11.69 Found: C,
61.30 H, 4.01 N, 10.23 S, 11.67.
124.2 (2C),127.5 (2C),129.2 (2C),129.5 (2C),131.6,133.7,135.0,136.9
(AreC), 166.9 (C]O), 178.4 (C]S); Anal. calcd. for C15H14N2OS; C,
66.64 H, 5.22 N, 10.36 S, 11.86 Found: C, 66.62 H, 5.20 N, 10.37 S,
11.83.
4.3.6. 1-Benzoyl-3-(3-methoxyphenyl)thiourea (16)
4.3.11. 1-Benzoyl-3-(2, 4, 6-trimethylphenyl)thiourea (21)
White solid; m.p. 67e68 ꢁC; FT-IR (KBr, cmꢀ1) 3253, 3151, 3132,
3085, 1671, 1595, 1528, 1459, 1317, 1277, 1202, 1146, 1032, 843, 718,
White solid; m.p. 162e164 ꢁC; FT-IR (KBr, cmꢀ1) 3329, 3049,
2978, 1671, 1609, 1561, 1526, 1483, 1452, 1350, 1312, 1259, 1182,
687, 647, 453; 1H NMR (300 MHz, CDCl3)
d
3.85 (s, 3H, o-OCH3), 6.84
1142, 1078, 816, 708, 537, 447; 1H NMR (300 MHz, CDCl3)
d 2.30 (s,
0
(d, 1H, J ¼ 8.1 Hz, ArHc), 7.24 (d, 1H, J ¼ 8.1 Hz, ArHa), 7.33 (t, 1H,
6H, 2 (eCH3)), 2.33 (s, 3H, 2 (eCH3)), 6.98 (s, 2H, ArHb¼b ), 7.57 (t,
0
0
0
J ¼ 8.1 Hz, ArHb), 7.50 (s, 1H, ArHc ), 7.56 (t, 2H, J ¼ 8.1 Hz, ArHe¼e ),
2H, J ¼ 7.6 Hz, ArHe¼e ), 7.68 (t, 1H, J ¼ 7.6 Hz, ArHf), 7.94 (d, 2H,
J ¼ 7.6 Hz, ArHd¼d ), 9.27 (s, 1H, CONH), 11.81 (s, 2H, CSNH); 13C NMR
0
0
7.67 (t, 1H, J ¼ 8.1 Hz, ArHf), 7.91 (d, 2H, J ¼ 8.1 Hz, ArHd¼d ), 9.15 (s,
1H, CONH), 12.64 (s, 1H, CSNH); 13C NMR (75 MHz, CDCl3)
d
55.4 (e
(75 MHz, CDCl3) d 18.2 2 (eCH3), 21.1 (eCH3),127.6 (2C), 129.1 (2C),
OCH3), 109.4, 112.8, 116.1, 127.5 (2C), 129.2 (2C), 129.6, 131.6, 133.8,
159.9 (AreC), 166.3 (C]O), 178.0 (C]S); MS (EI) m/z 285.5 (Mþ),
76.8 (base peak); Anal. calcd. for C15H14N2O2S; C, 62.92 H, 4.93 N,
9.78 S, 11.20 Found: C, 62.90 H, 4.90 N, 9.80 S, 11.00.
129.2 (2C), 131.6, 132.8, 133.7, 134.9 (2C), 138.1, 167.0 (C]O), 180.1
(C]S); MS (EI) m/z 298.1 (Mþ), 105.0 (base peak); Anal. calcd. for
C17H18N2OS; C, 68.42 H, 6.08 N, 9.39 S, 10.75 Found: C, 68.40 H, 6.01
N, 9.41 S, 10.73.
4.3.7. 1-Benzoyl-3-(2-triflouromethylphenyl)thiourea (17)
4.3.12. 1-Benzoyl-3-(3-flourophenyl)thiourea (22)
White solid; m.p. 123e124 ꢁC; FT-IR (KBr, cmꢀ1) 3259, 3168,
1676, 1606, 1584, 1509, 1317, 1248, 1169, 1155, 1111, 854, 768, 701,
White solid; m.p. 155e156 ꢁC; FT-IR (KBr, cmꢀ1) 3324, 3088,
2978, 1669, 1609, 1561, 1526, 1483, 1452, 1350, 1258, 1182, 1142,

494
M.K. Rauf et al. / European Journal of Medicinal Chemistry 70 (2013) 487e496
1078, 816, 708, 537, 447; 1H NMR (300 MHz, CDCl3)
d
6.97e7.03 (m,
performed with a Bruker AXS SMART APEX CCD diffractometer
using graphite monochromated MoKa radiation (0.71073 A). Data
ꢀ
0
0
1H, ArHc ), 7.34e744 (m, 2H, ArHb,c), 7.57 (t, 2H, J ¼ 7.8 Hz, ArHe¼e ),
7.68 (t, 1H, J ¼ 7.8 Hz, ArHf), 7.77 (d, 1H, J ¼ 7.8 Hz, ArHa), 7.91 (d, 2H,
reduction and absorption correction with SAINT [26] and SADABS
[27]. Structures were solved by direct and Fourier methods and
refined by full-matrix least-squares based on F2 [28]. All non-
hydrogen atoms were refined anisotropically, hydrogen atoms
were determined from difference Fourier maps and refined at
idealized positions riding on their attached C or N atoms. For 11
there are two crystallographically independent but chemically
equal molecules per asymmetric unit. Compound 12 shows large
anisotropic displacement parameters especially of the C6HCl4 ring,
indicating disorder which, however, could not be modeled further.
J ¼ 7.8 Hz, ArHd¼d ), 9.13 (s, 1H, CONH), 12.24 (s, 1H, CSNH); 13C NMR
0
(75 MHz, CDCl3)
d
111.07 & 111.41 (d, 13C-19F, 2J ¼ 25.5 Hz), 113.55 &
113.83 (d, 13C-19F, 2J ¼ 21 Hz), 119.39 & 119.43 (d, 13C-19F,
4J ¼ 3.22 Hz), 127.5 (2C), 129.3 (2C), 130.00 & 130.12 (d, 13C-19F,
3J ¼ 9.0 Hz), 131.44, 133.94, 138.95 & 139.0 (d, 13C-19F, 3J ¼ 10.4 Hz),
160.93 & 164.20 (d, 13C-19F, 1J ¼ 245.25 Hz) (AreC), 167.05 (C]O),
178.2 (C]S); Anal. calcd. for C14H11N2OSF; C, 61.30 H, 4.04 N,
10.21 S, 11.69 Found: C, 61.31 H, 4.02 N, 10.22 S, 11.68.
4.3.13. 1-Benzoyl-3-benzylthiourea (23)
White solid; m.p. 118e120 ꢁC; FT-IR (KBr, cmꢀ1) 3266, 3169,
2934, 1673, 6101, 1545, 1320, 1254, 1165, 1065, 941, 882, 738, 692,
4.5. Biological assays
613, 515; 1H NMR (300 MHz, CDCl3)
d
4.94 (d, 2H, eCH2e), 7.33e
4.5.1. Measurement of urease activity
0
0
0
7.47 (m, 5H, ArHa,b,b ,c,c ), 7.52 (t, 2H, ArHe¼e ), 7.64 (t, 1H, J ¼ 7.6 Hz,
The indophenol method was used for ammonia quantification
and enzyme activity was determined by absorbance measurement
0
ArHf), 7.85 (d, 2H, J ¼ 7.6 Hz, ArHd¼d ), 9.11 (s, CONH), 11.04 (s,
CSNH); 13C NMR (75 MHz, CDCl3)
d
49.8 (eCH2e), 126.4, 127.4 (2C),
as described by Weatherburn [29]. The assay comprised of 50
buffer (100 mM urea, 1 mM EDTA, 10 mM K2HPO4, 10 mM LiCl2, pH
8.2), 10 L of enzyme (5 U/mL) and 10 L of test compound. All
components were incubated in a 96 well plate for 30 min at 37 ꢁ
and thiourea and acetohydroxamic acid were used as standard in-
hibitors. Solutions of 30 L of phenol reagent (0.005%, w/v sodium
nitroprusside, 1%, w/v phenol) and 30 L of alkali reagent (0.5%, w/v
mL
127.9 (2C), 128.9 (2C), 129.1 (2C), 131.7, 133.6, 136.1 (AreC), 166.8
(C]O), 180.0 (C]S); Anal. calcd. for C15H14N2OS; C, 66.64 H, 5.22 N,
10.36 S, 11.86 Found: C, 66.65 H, 5.21 N, 10.37 S, 11.85.
m
m
C
4.3.14. 1-Benzoyl-3-cyclohexylthiourea (24)
m
White solid; m.p. 74e75 ꢁC; FT-IR (KBr, cmꢀ1) 3323, 3227, 3170,
3048, 2928, 2854, 1672, 1627, 1542, 1341, 1262, 1169, 1022, 778, 707,
m
NaOH, 0.1% active chloride NaOCl) were introduced to each well.
Each experiment was performed in triplicate. The microplate
reader (Bio-TekELx 800Ô, Instruments, Inc. USA) was used to
measure absorbance at 625 nm. The percentage inhibition was
calculated from the equation 100 ꢀ (ODtest well/ODcontrol) ꢄ 100. The
results were analyzed using PRISM 5.0 (GraphPad, San Diego, Cal-
ifornia, USA).
664, 623; 1H NMR (300 MHz, CDCl3)
d 1.23e2.17 (m, 10H, Cyclo-
hexane eCH2e), 4.26e4.35 (m, 1H, Cyclohexane eCHe), 7.51 (t, 2H,
0
J ¼ 7.6 Hz, ArHe¼e ), 7.63 (t, 1H, J ¼ 7.6 Hz, ArHf), 7.84 (d, 2H,
J ¼ 7.8 Hz, ArHd¼d ), 8.94 (s, CONH), 10.72 (s, CSNH); 13C NMR
0
(75 MHz, CDCl3) d 24.3 (2C), 25.4, 33.26 (2C), 57.62 (Cyclohexane-C),
127.3 (2C), 129.1 (2C), 131.8, 133.5 (AreC), 166.6 (C]O), 178.22 (C]
S); Anal. calcd. for C14H18N2OS; C, 64.09 H, 6.91 N, 10.68 S, 12.22
Found: C, 64.00 H, 6.88 N, 10.70 S, 12.20.
4.5.2. Measurement of antioxidant activity
The free radical scavenging capability of 1-benzoyl-3-
phenylthiourea derivatives was measured by 1,1-diphenyl-2-
picrylhydrazyl (DPPH) according to the method of Choudhary
et al. [30] The synthesized derivatives were allowed to react with
stable 1,1-diphenyl-2-picrylhydrazyl in a 96 well microplate for
4.3.15. 1-Benzoyl-3-(pyrimidin-2-yl)thiourea (25)
White solid; m.p. 178e179 ꢁC; FT-IR (KBr, cmꢀ1) 3162, 3050,
2997, 2913, 1720, 1563, 1515, 1331, 1269, 1246, 1175, 905, 807, 710,
538, 490; 1H NMR (300 MHz, CDCl3)
d
7.31 (t, 1H, J ¼ 6.9 Hz, py-
rimidine-Ha), 7.60 (t, 2H, J ¼ 6.9 Hz, ArHe¼e ), 7.69 (t, 1H, J ¼ 6.9 Hz,
30 min at 37 ꢁC. The DPPH concentration was 300
mM and solution
0
0
ArHf), 7.98 (d, 2H, J ¼ 6.9 Hz, ArHd¼d ), 8.79 (d, 2H, J ¼ 6.9 Hz, py-
was prepared in methanol and the test derivatives were dissolved
in DMSO. After incubation, the decrease in absorption was
measured by microplate reader (Bio-TekELx 800Ô, Instruments,
Inc. USA) at 515 nm. The radical scavenging activity of samples was
determined in comparison with DMSO treated as control. In this
experiment, 3-tert-butyl-4-hydroxyanisole and propyl gallate were
used as standards. The results were calculated using PRISM 5.0
(GraphPad, San Diego, California, USA).
rimidine-Hb¼b ), 9.14 (s, 1H, CONH), 12.61 (s, 1H, CSNH); 13C NMR
0
(75 MHz, CDCl3)
d 117.6, 128.5 (2C), 129.4 (2C), 133.5, 133.7 (AreC),
157.5, 158.9 (2C) (pyrimidine), 166.1 (C]O), 178.5 (C]S); Anal.
calcd. for C12H10N2OS; C, 55.80 H, 3.90 N, 21.29 S, 12.41 Found: C,
55.78 H, 3.88 N, 21.30 S, 12.39.
4.3.16. 1-Benzoyl-3-(2-chlorobenzyl)thiourea (26)
White solid; m.p. 101e102 ꢁC; FT-IR (KBr, cmꢀ1) 3402, 3145,
3063, 2932, 1671, 1599, 1551, 1296, 1243, 1155, 1052, 887, 752, 605,
4.5.3. Measurement of antifungal activity
522; 1H NMR (300 MHz, CDCl3)
d
5.04 (d, 2H, eCH2e), 7.25e7.47 (m,
0
The activity of 1-benzoyl-3-phenylthiourea derivatives on
fungal strains A. niger, A. flavus, S. cerevisiae and C. tropicalis were
assayed by slightly modified agar well diffusion method [31] with
sterile cork borer of size 6 mm and spore germination inhibition
assay. The fungal strains were inoculated on cultures of 48 h old
0
4H, ArHa,b,b ,c), 7.52 (t, 2H, ArHe¼e ), 7.64 (t, 1H, J ¼ 7.6 Hz, ArHf), 7.85
(d, 2H, ArHd¼d ), 9.0 (s, CONH), 11.14 (s, CSNH); 13C NMR (75 MHz,
0
CDCl3)
d 47.4 (eCH2e), 127.0 (2C), 127.4 (2C), 129.1, 129.4, 129.7,
130.5, 131.6, 133.6, 133.7, 133.9 (AreC), 166.8 (C]O), 180.2 (C]S);
Anal. calcd. for C15H13N2OSCl; C, 59.11 H, 4.30 N, 9.19 S,10.52 Found:
C, 59.10 H, 4.30 N, 9.21 S, 10.51.
grown potato dextrose agar. An aliquot (20 mL) of inoculum was
introduced to molten potato dextrose agar and poured into a petri
plate by pour plate method. The wells were made on agar plates by
using cork borer on solidification. Slides were then incubated in
4.4. Data collection and structural refinement for 11 and 12
moist chamber at 24e48 h at 28 ꢁC. In assay, 50
mL of test com-
The compounds 11 and 12 were dissolved in minimum quantity
of CH2Cl2 and were recrystallized in a mixture of C2H5OH/CH2Cl2
(1:1) by the slow evaporation method to yield single crystal X-rays
analysis crystals. The crystals were mounted in a random orienta-
tion on a glass fiber and the diffraction data collections were
pounds were introduced serially after completion of one compound
analysis successfully. The experiment was carried out in triplicates
and fungicidal effects of the 1-benzoyl-3-phenylthiourea de-
rivatives were assessed by the inhibition of mycelial growth of the
fungus observed as a zone of inhibition measured with antibiotic

M.K. Rauf et al. / European Journal of Medicinal Chemistry 70 (2013) 487e496
495
zone scale in mm near the disc or the wells. DMSO, 2% in the screen
4.6.4. Mechanism of inhibition
medium served as the negative control, and fluconazole and ter-
binafine in screen medium were used individually as positive
antifungal controls.
The most potent compound, 11 was further investigated to
determine the mechanism of inhibition. Enzyme kinetics was
determined in the absence and in the presence of various con-
centrations of inhibitor. It was found that the Vmax value did not
significantly change in the presence of the inhibitor. However, Km
value increased with increasing inhibitor concentrations indicating
that 11 exhibit a competitive mechanism of inhibition. Fig. 2,
showed Lineweavere Burk plots for compound 11. The both plots
visualize the competitive mechanism of inhibition.
4.6. Molecular modeling & docking studies
4.6.1. Protein structure preparation
In order to study the interactions of the compounds with the
urease enzyme, molecular modeling and docking studies were
performed. The crystal structure of Jack bean urease (PDB ID: 3LA4)
was used. The enzyme structure was prepared by proper proton-
ation of all the residues with special attention to the residues
involved in the binding site/catalytic pocket of the enzyme. An
advanced algorithm Protonate3D [32] implemented in MOE [33]
was used to protonate the protein structure. Protonate3D takes
care of the correct protonation states of the Histidine and other
important residues that occur in the catalytic domain of the
enzyme. The structure preparation also included energy minimi-
zation after the addition of hydrogens by selecting the appropriate
force-field and correct atom-type parameterization. It was also
made sure to set up the correct metal states for the two Ni atoms in
the binding site of the enzyme. During protonation, the four sur-
rounding Histidine residues in the active site pocket were proton-
ated according to the bound state of the two Nickel ions. During
energy minimization, the protein heavy atoms were restrained
during the minimization to allow the flexibility of protein side
chains and hydrogen atoms only. The restrained minimization was
helpful in preventing any distortions in protein conformation,
particularly the active site. Minimization was performed while the
bound phosphate residue and water molecules were present in the
enzyme. After minimization, the water molecules and co-
crystallized bound compounds were removed.
Acknowledgments
The M. Khawar Rauf is thankful to the Quaid-i-Azam University,
Islamabad, Pakistan for the grant of funds for Post-doctoral
Fellowship. This study was financially supported by COMSTECH-
TWAS and German-Pakistani Research Collaboration Programme
to JI.
Abbreviations
DMSO dimethylsulphoxide
DMF
TLC
THF
MW
dimethylformamide
thin layer chromatography
tetrahydrofuran
microwave
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.10.012.
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