Solution-phase microwave assisted parallel synthesis, biological evaluation and in silico docking studies of 2-chlorobenzoyl thioureas derivatives

Article abstract of DOI:10.1016/j.molstruc.2018.03.056

An efficient and facile microwave-assisted solution phase parallel synthesis for a 38-member library of N-aroyl-N′-aryl thioureas was accomplished successfully. These analogues (1–38) were synthesized under identical set of conditions. It has been observe

Full text of DOI:10.1016/j.molstruc.2018.03.056

Accepted Manuscript
Solution-phase microwave assisted parallel synthesis, biological evaluation and in
silico docking studies of 2-chlorobenzoyl thioureas derivatives
Muhammad Riaz Khan, Sumera Zaib, Muhammad Khawar Rauf, Masahiro Ebihara,
Amin Badshah, Muhammad Zahid, Muhammad Arif Nadeem, Jamshed Iqbal
PII:
S0022-2860(18)30345-4
10.1016/j.molstruc.2018.03.056
MOLSTR 24995
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 9 October 2017
Revised Date: 8 March 2018
Accepted Date: 14 March 2018
Please cite this article as: M.R. Khan, S. Zaib, M.K. Rauf, M. Ebihara, A. Badshah, M. Zahid, M.A.
Nadeem, J. Iqbal, Solution-phase microwave assisted parallel synthesis, biological evaluation and in
silico docking studies of 2-chlorobenzoyl thioureas derivatives, Journal of Molecular Structure (2018),
doi: 10.1016/j.molstruc.2018.03.056.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Solution-phase microwave assisted parallel synthesis, biological evaluation and
in silico docking studies of of 2-chlorobenzoyl thioureas derivatives
Muhammad Riaz Khan, Sumera Zaib, Muhammad Khawar Rauf, Masahiro Ebihara, Amin
Badshah, Muhammad Zahid, Muhammad Arif Nadeem, Jamshed Iqbal*
Cl
O
S
N
N
Cl
H
H
Cl
Compound 6 (IC50 = 0.18 ± 0.1 µM)
37 additional examples
Putative binding mode of 7 bound to the active site of Jack bean urease

ACCEPTED MANUSCRIPT
Solution-phase microwave assisted parallel synthesis, biological evaluation
and in silico docking studies of 2-chlorobenzoyl thioureas derivatives
Muhammad Riaz Khana, Sumera Zaibb, Muhammad Khawar Rauf c,*, , Masahiro Ebiharad,
Amin Badshahc, *, Muhammad Zahid c, Muhammad Arif Nadeemb, Jamshed Iqbalb, *
a Department of Chemistry, Gomal University, Dera Ismail Khan-29050, Pakistan
bCentre for Advanced Drug Research, COMSATS Institute of Information Technology,
Abbottabad-22060, Pakistan.
cDepartment of Chemistry, Quaid-i-Azam University, Islamabad-45320, Pakistan.
dDepartment of Chemistry, Faculty of Engineering, Gifu University, Yanagido, Gifu 501-
1193, Japan
*Correspondance
Prof. Dr. Muhammad Khawar Rauf
:0000-0001-9994-4033
Department of Chemistry, Quaid-i-Azam University, Islamabad-45320, Pakistan.
Email: drjamshed@ciit.net.pk (J. Iqbal), mkhawarrauf@yahoo.co.uk (M.K.Rauf);
aminbadshah@yahoo.com (A. Badshah)
Abstract
An efficient and facile microwave-assisted solution phase parallel synthesis for a 38-
member library of N-aroyl-N´-aryl thioureas was accomplished successfully. These
analogues (1-38) were synthesized under identical set of conditions. It has been observed
that the reaction time was drastically reduced from 8-12 h for conventional methods to only
10-15 mins. Products obtained were more than 98% pure, as characterized by elemental
1
analysis along with FT-IR and H, 13C-NMR. The solid-phase structural analysis was
accomplished by single crystal XRD analysis. The urease inhibitory potential of synthetic
compounds was tested and compounds were found to inhibit urease in moderate to
significant manner. Compound 17 was the most potent inhibitor of urease having an IC50
value of 0.17 ± 0.1 µM. To check the cytotoxic profile of the derivatives, lungs cancer cell
lines were used. Cytotoxicity analysis revealed remarkable toxicity of the compounds
against tested lungs carcinoma and compounds showed variation in inhibition activity due to
1

ACCEPTED MANUSCRIPT
the substituents attached. The molecular docking studies were carried out to identify the
possible binding modes of potent inhibitors in the active site of enzyme. The results
suggested that the compounds can be further investigated and used against different cancers.
Keywords: Microwave Synthesis, N-aroyl-N´-aryl thioureas, Single Crystal XRD, Urease
Inhibition, Cytotoxicity.
Introduction
Cancer is a multifaceted disease initiated by abnormal cellular proliferation linked with
uncontrolled activation of growth signals, impairment of apoptosis, invasion of surrounding
tissues and metastasis, resistance to growth inhibitory signals, and promotion of
angiogenesis in the growing tumor [1]. Moreover, these cells are not easily detected by the
immune system [2] and take up glucose more rapidly than normal cells [2,3]. Almost 20%
cancers in humans are due to the microbe-related diseases [4] and bacterium residing in
stomach causes the gastric cancer as it multiplies rapidly in the acidic pH. Therefore, the
level of urease is increased and simultaneously urea is converted to ammonia. Due to
excessive production of ammonia the acidity of stomach is neutralized, as a result making
stomach more hospitable for the bacterium. This infection is a main cause of gastric
(stomach) cancer, particularly non-cardia gastric cancer [5,6]. Resistance by previously used
antibiotics and compliance related problems notably minimizes the treatment efficacy [7].
Therefore, suggesting that microbial associated studies will lead to potential cancer
prevention strategies [4].
The quest for novel bioactive materials with higher selectivity and minimum side
effects continues to be an area of intensive research in synthetic medicinal chemistry. On the
path of recognizing various chemical substances that may prove as foundations for
designing novel nitrogen, sulfur and oxygen containing bioactive agents are of particular
interest. N-aroyl-N´-aryl thioureas being multipurpose intermediates for various structural
developments and modifications have diverse applications. Some of the N-aroyl-N´-aryl
thioureas exhibited significant pharmacological properties [8-14]. In vitro studies have
shown that a variety of thioureas are potential antimalarial and anticancer agents [8],
transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor
2

ACCEPTED MANUSCRIPT
antagonists [9], herbicides, insecticides [10-11], plant-growth regulators [12,13] and
antihypertensive agents [14].
N-aroyl-N´-aryl thioureas as precursors react with other reagents to yield
pharmacologically active compounds. For example, thioureas condense with a-halogen
substituted carbonyl compounds to afford 2-amino-1,3-thiazoles [15,16] and benzothiazole
derivatives can be prepared from arylthioureas by using bromine as a catalyst[17]. The role
of thioureas to synthesize iminothiazolines [18], thiohydantoins [19, 20], 1,3,5-triazines [21]
and oxazolidines [22] has been reported. N-aroyl-N´-aryl thioureas are generally produced
by the reaction of amines with Na/K/NH4 thiocyanates in the presence of some mineral acids
[14, 23-25]. Synthesis of unsubstituted, mono- and N,N′-disubstituted thioureas through
primary alkyl amines [26] and debenzoylation of N-substituted and N,N′-disubstituted-N′-
benzoylthioureas by hydrazine hydrate under solvent-free conditions has also been reported
[27]. A recent studies on disubstituted thiourea derivatives as urease inhibitors have been
reported [28]. In current decades, several new methods have also been reported for the
synthesis of substituted thioureas [29-31]. But, these methods have some detriments,
specifically, long reaction time, high temperature, the use of toxic reagents and solvents. The
developments of environmentally friendly methods are still anticipated. The microwave
assisted synthesis has gained considerable attention in the present days that helped to
increase the purity of the products and shortened the time. An efficient microwave assisted
synthesis of N,N′-di- and trisubstituted thioureas have been reported earlier as well [32-34].
Taking into account, the synthetic route for aryl thioureas and the related chemistry,
we have synthesized N-aroyl-N´-aryl thioureas and screened against antiureas as well as
anticancer activities. Furthermore, the docking studies were carried out to investigate the
binding site interactions of ligand-protein complex. We expanded the SAR studies on N-
aroyl-N´-aryl thioureas and confirmed their pharmacological potential to inhibit the urease
due to its diverse structural features.
Material and methods
Experimental
Synthesis
(Added in the supporting information file.)
3

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Data collection and structural refinement of 15, 17, 22, 34 and 38
Crystals of the compounds 15, 17, 22, 34 and 38 were grown by the slow evaporation
ethanol/dichloromethane (1:1) solutions at room temperature. The colorless thiourea crystals
for 15, 17, 22 and 34, and yellow for 38 were mounted in random orientation on a glass fiber
and the reflection data were collected on a Rigaku AFC7R Mercury CCD diffractometer
monochromated MoKα radiation (λ = 0.71073Å). The structures was solved by using direct
methods, Sir 97 [35] and refined with Shelxl 97 [36]. 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. Table 1 summarizes the crystal
data and refinement description.
Biological Assays
Measurement of urease activity
Urease inhibitory activity was determined by protocol given in our published research
articles [37-41].
Cytotoxicity assay by sulforhodamine B (SRB)
Cytotoxic activity of synthesized derivatives (1-38) was determined in accordance with
literature published in recent research paper [37, 38, 40, 42].
Docking studies
Docking studies were carried out according to previous protocols [40, 41, 43-48].
Supplementary data
Crystallographic data (including structure factors) for the structures reported in this paper
have been deposited to the Cambridge Crystallographic Data Centre as supplementary
publication numbers CCDC 1501414-1501418 for 15, 17, 22, 34 and 38, respectively.
Copies of available materials can be obtained, free of charge, on application to the director,
CCDC, 12 Union Road, Cambridge CB21EZ, UK, (Fax: +44-(0)-1223-336033 or e-mail:
deposit@ccdc.cam.ac.uk).
Results and Discussion
Chemistry
4

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Solution-phase microwave assisted parallel synthesis of a library of 38 N-aroyl-N´-aryl
thioureas (1-38) was performed using CEM MARS 6 microwave reactor for organic
synthesis. These compounds were successfully characterized by various techniques
including 1H- and 13C-NMR spectroscopy and single crystal XRD analysis. The purity of the
compounds was checked by TLC with Merck Kieselgel GF 254 plates and elemental
analysis. Stoichiometric amounts of 2-cholorobenzoyl isothiocyanate (obtained by
conventional manner and purified by steam distillation) and respective primary/secondary
amines (HNRR´) were subjected to glass vials along with THF as solvent and then irradiated
the vials under same set of conditions (THF, 1.5 min, 60-65 °C and 1 atm pressure). The
yield of the final products obtained through microwaves assisted reactions is strongly
influenced by the nature of substrates HNRR´ (Scheme 1) and is based on the resultant
nucleophillicity of the aromatic amines due to the mesomeric, inductive and electronic
effects of the pendent groups or substituents. All the groups having I(+) and M(+) effect
showed excellent yield unless steric factor play its role. It has been observed that bulky
groups like cyclohexyl although possess I(+) and M(+) effect but steric factor reduces the
relative yield of the products. The lowest yield has been observed for the substrate
containing cyclohexyl group, might be also due to conformational isomerism. Moreover,
inductive effect is affected by the distance and mesomeric effect by position of the
substituents. It has been observed that o-chloroaniline resulted in maximum yield as
compared to its meta and para analogues. The steric factor play its part when both the ortho
positions are substituted as in the case of 2,6-dichloroaniline that resulted low yield with
respect to other mono- and di- substituted anilines/amines. The groups like CF3, NO2 etc on
aniline ring resulted lower yield because of I(–) and M(–) effect. In conclusion all those
groups which were electron donor by any mean showed improved yield while electron with
drawing resulted in reduction of yield for final products. The description of the substituents
for compounds (1-38) is expressed in Scheme 1.
5

ACCEPTED MANUSCRIPT
O
OH
O
Cl
O
N
Cl
C
R'
e
′
S
R'
NH4SCN
THF
Cl
O
Cl
SOCl2
DMF
Cl
HN R
f
N R
THF, MW, 10 min,
60-65 °C, 1 atm
HN
e
d
S
No.
R
R' No.
R
R' No.
R
R'
No.
R
R'
c′
b′
c
′
O2N
Cl
Cl
b
′
b
′
b
′
a′
a
Cl
Cl
1
H
H
H
11
12
13
H
H
H
21
22
23
NO2
H
31
32
33
H
g
a
c
c
c
b
c
c
b
Cl
b
b
Cl
H3CO
b
′
b
′
b′
N
S
a
a
a
2
3
4
5
6
H
H
H
H
H
H
CH3
H
a′
b
c
b
b′
b
Cl
Cl
Cl
Cl
OCH3
c′
c′
c′
b′
a′
a
a
a
c
a
c
b
c
Cl
b
Cl
c
′
c
′
b
′
F3C
b
′
b′
c
′
b
′
H2
a
CH3
Cl
a
H
H
H
14
15
16
H
H
H
24
25
26
34
35
36
C
c
b
c
c
b
b
c
b
Cl
Cl
Cl
H3C
CH3
CF3
b
′
c′
H2
C
a
a
a
a
H
c
b
c
c
b
c
b
b
c
′
Cl
b
′
c
′
b′
b
′
b′
a
CF3
H
Cl
c
b
c
c
b
b
b
c
Cl
′
c
c
′
Br
b′
c
′
b
′
b
c
′
b′
a
a
a
7
8
H
H
17
18
H
H
27
28
H
H
37
38
c
b
c
c
b
b
b
c
Cl
Cl
Cl
Br
b
′
b′
b
′
b′
N
N
a
a
H
Br
b
b
c
b
b
O2N
c
c
′
Cl
b
′
b′
c
′
a
F
9
H
H
19
20
H
H
29
30
H
H
Cl
c
b
b
c
b
Cl
c′
F
c
′
c
′
b′
a
NO2
a
10
c
c
c
b
b
Cl
Scheme 1. Synthesis of N-aroyl-N´-aryl thioureas (1-38).
6

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Spectroscopic studies
The most significant bands recorded in FT-IR spectra of the entire synthesized members of
the library are listed in spectroscopic data. The tentative assignments were made according
to the literature [49]. The neat conversion of isothiocyanate moiety into thiourea (-
NHCSNH-) functionality was confirmed by the absence of ν (S-H) vibration in the range of
2500-2600 cm-1. The characteristic IR absorptions of main functionalities associated with N-
aroyl-N´-aryl thioureas were between/near to; 3120-3400 (N-H), 2960-3100 (C-Haromatic),
2930-2850 (C-Haliphatic), 1650-1690 (C=O), 1530-1625 (C-N), 1240-1280 (C=S) and 1120-
1190 (C-S). The absence of strong band at/around 2000 cm-1 (N=C=S) in the final product is
an indicative of the introduction of thiourea (-HNCSNH-) moiety. The expected normal
carbonyl (C=O) absorption around νmax/cm-1: 1710 appeared as a medium strong band at
1650-1690 cm-1 suggesting a possible hydrogen bond formation between the H-atom of the
NH-group and the O-atom of the carbonyl group as confirmed by the solid state structures.
The band around νmax/cm-1 2500-2600 corresponding to S-H was not found which indicated
the absence of the N=CSH tautomeric form in all the compounds [49-51].
1
The H NMR data for the N-aroyl-N´-aryl thioureas (1-38) showed that the NH
hydrogen resonates considerably downfield. The proton chemical shifts found around 9.00-
9.60 and 10.80-12.90 ppm were assigned for free and hydrogen bonded NH, respectively.
The aromatic protons appeared downfield at 7.23-8.50 ppm. It has also been observed in our
studies that the coordinating or polar solvents like DMSO-d6 had shifted the free NH protons
chemical shift more downfield as compared with the solvents like CDCl3, C6D6 and CD2Cl2.
This shift is possibly due to the hydrogen bonding between the NH and sulfoxide (S=O)
moiety [49].
The 13C NMR data revealed all the signals due to the magnetically different carbons
present in the compounds (1-38). The chemical shifts of the carbons pertaining to CONH
and CSNH groups of the substituted thiourea resonate around 160-170 and 175-185 ppm,
respectively. The aromatic carbon resonances of the N-aroyl-N´-aryl thioureas were assigned
on the basis of signal intensities and then were compared with the reported values [52].
Structural studies
Crystals of the compounds 15, 17, 22, 34 and 38 were grown by the slow evaporation
ethanol/dichloromethane (1:1) solutions at room temperature. Crystallographic and
7

ACCEPTED MANUSCRIPT
refinement details are shown in Table 1. Selected bond lengths and angles are given in Table
S1). The four molecular structures 15, 17, 22 and 34 (Figure 1) resemble similar thiourea
cores with different substitution patterns. The core moieties –(O=C)NH(C=S)NH are mostly
planar with O-C-N-C and S-C-N-C torsion angles of 7.9(2)° & -177.20 (11)° for 15, 1.9(3)°
& 171.35(15)° for 17, -2.2(3)° & -176.02(15)° for 22 and 4.7(2)° & 173.51(12)° for 34
respectively, as well. However, small deviation of S atoms have been observed with respect
to the pseudo six-membered ring OCNCNH stabilized by intramolecular N-H…O hydrogen
bonds. The aromatic ring planes C3-C8 and C9-C14 make dihedral angles of 77.48(3)° &
62.92(3)° for 15, 85.28(5)° & 37.47(5)° for 17 and 51.36(7)° & 5.47(5)° for 22, respectively.
In molecular structure 34, the aromatic ring C3-C8 and benzyl planes subtend dihedral
angles 81.95(3)° and 35.10(5)°, respectively. In general, there are no unexpected geometric
parameters for these structures. The molecular structures 15, 17, 22 and 34 show an
intramolecular N-H…O hydrogen bond stabilizing the thiourea geometry. The structural
conformation and intramolecular H-bonding pattern of 38 (Figure 1) is quite different. The
molecular structure of 38 constitutes intramolecular N-H…N hydrogen bond stabilizing the
pseudo six-membered ring N3C9N2C2N1H1. The crystal structures of 15, 17, 22 and 34
show intermolecular N-H…S {for Si: : (i) -X,-Y,1-Z (15); (i) 1-X,-Y,1-Z (17); (i) 1-X,1-Y,-Z (22);
(i) 1-X,1-Y,1-Z (34)} interactions that connect the molecules into centro-symmetric dimers
(Table S2) [35,36].
(15)
(17)
8

ACCEPTED MANUSCRIPT
(34)
(22)
(38)
Figure 1. Molecular structures of compounds 15, 17, 22, 34 and 38
Table 1. Crystallographic data and structure refinement for compounds 15, 17, 22, 34 and 38
15
C16H15ClN2OS
318.81
34
38
Compound
Formula
FW
17
22
C14H10BrClN2OS C15H13ClN2O2S C15H13ClN2OS C12H9ClN4OS
369.66
320.78
304.78
292.74
Triclinic
P-1
Crystal system
Space group
a (Å)
Triclinic
P-1
Monoclinic
P21/c
Monoclinic
P21/n
Triclinic
P-1
18.484(8)
7.349(4)
9.639(6)
10.990(6)
7.489(3)
9.338(4)
13.274(5)
10.703(3)
9.973(3)
13.796(4)
7.167(3)
8.000(4)
b (Å)
4.2457(19)
18.781(9)
c (Å)
11.252(5)
65.674(13),
69.975(16),
73.639(17)
90.00,
101.197(3),
90.00
90.00,
100.834(6),
90.00,
110.145(6),
90.780(5),
104.165(6)
704.5(7)
81.625(14),
74.580(12),
83.979(15)
613.8(5)
α, β, γ(°)
V (Å3)
Z
783.9(5)
1444.6(7)
1447.6(11)
2
2
2
4
4
T (K)
123(2)
123(2)
123(2)
123(2)
123(2)
9

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DC Mg/m3
µ (mm-1)
1.351
0.376
332.0
1.700
3.170
736.0
1.472
0.413
664.0
1.437
0.415
316.0
1.584
0.477
300.0
F(000)
Crystal size (mm3)
0.50×0.35×0.28 0.45×0.34×0.26
0.40×0.35×0.28 0.45×0.35×0.25 0.30×0.26×0.18
θ range (°)
6.96 to 54.96°
-6<=h<=9,
-9<=k<=12,
-10<=l<=17
6180
6.02 to 54.96°
-13<=h<=9,
-12<=k<=11,
-15<=l<=17
10879
6.68 to 54.94°
-16<=h<=24,
-5<=k<=4,
-22<=l<=24
10482
6.38 to 54.96°
-7<=h<=9,
-12<=k<=12,
-13<=l<=14
5576
6.02 to 54.96°
-8<=h<=9,
-9<=k<=10,
-14<=l<=14
4888
Index ranges
Reflections collected
Indept. Reflect. [R(int)]
3506 [R(int) =
0.0622]
3230 [Rint
0.0308]
97.9%
=
3306 [Rint
0.0272]
99.1%
=
3169 [Rint
0.0563]
97.9%
=
2759 [Rint
0.0320]
97.9%
=
Completeness to θ = 28°
Max. & min. transmission
Data/restraints/parameters
Goodness-of-fit on F2
97.3%
0.8564, 1.0000
3506/0/192
1.081
0.2934, 0.4001
3230/0/181
1.092
0.8419, 1.0000
3306/0/191
1.130
0.7770, 1.0000 0.8680, 1.0000
3169/0/181
1.105
2759/0/172
1.074
R1 = 0.0361,
wR2 = 0.0890
R1 = 0.0380,
wR2 = 0.0903
R1 = 0.0285,
wR2 = 0.0661
R1 = 0.0310,
wR2 = 0.0671
R1 = 0.0458,
wR2 = 0.1047
R1 = 0.0492,
wR2 = 0.1068
0.39/-0.30
R1 = 0.0366,
wR2 = 0.0864
R1 = 0.0396,
wR2 = 0.0882
0.35/-0.24
R1 = 0.0342,
wR2 = 0.0805
R1 = 0.0374,
wR2 = 0.0821
0.29/-0.34
Final R indexes [I>=2σ (I)]
Final R indexes [all data]
Largest peak and hole (eÅ- 0.32/-0.31
0.64/-0.30
3
)
Pharmacology
Urease inhibition assay
The synthesized N-aroyl-N´-aryl thioureas (1-38) were tested for their inhibition profile for
Jack bean urease. The activity results of the active compounds in terms of their IC50 values
are presented in Table 2. Thiourea was used as a standard inhibitor (IC50 = 22.3 ± 1.2 µM).
The results showed that various derivatives in the series exhibited lower IC50 values than the
standard compound, thiourea. Among the evaluated library, compound 17 incorporating a 3-
bromophenyl substitution was the most potent inhibitor in the series having an IC50 value of
0.17 ± 0.1 µM, and emerged to be ~130-folds more active than the standard inhibitor
(thiourea; IC50 = 22.3 ± 1.2 µM). Similar inhibition results were observed for compound 7
bearing a 2,5-dichlorophenyl substitution with an IC50 value of 0.18 ± 0.1 µM. Compound
16 with 2,4,6-trimethylphenyl substitution was also the active inhibitor having an IC50 value
10

ACCEPTED MANUSCRIPT
of 0.47 ± 0.1 µM. The presence of substituent and its nature has considerable effect on the
inhibition shown by compound. The Table 2 showed that electron withdrawing groups
exhibited more inhibition as compare to electron donating groups. Similarly, the position of
substituents had remarkable role in inhibition. Moreover, several compounds with aliphatic
and heterocyclic substituents were also active members of the series with a variable degree
of urease inhibition. It was also noted that compound 18 with a 2,4-dibromophenyl
substitution exhibit 37.2% inhibition, whereas compound 17 with 3-bromophenyl
substitution was the most potent compound. It was depicted that substitution of
dibromophenyl was responsible for the decrease in the urease inhibition. In addition, it was
observed that dichlorophenyl, trichlorophenyl and tetrachlorophenyl thiourea derivatives
were equally significant inhibitors of urease. Similarly, all the mono- and dinitrophenyl
derivatives were inactive against urease in the series. The compound with sterically
demanding naphthyl group was also inactive. The compound 10 with 3,5-dichlorophenyl
moiety was the least active member of the series.
Table 2. In vitro urease inhibition efficacy of all synthetic derivatives (1-38)
Codes
1
IC50 ± SEM (µM)/ %inhibition
4.21 ± 0.3
18.5%
ClogP
2.64
2.96
3.52
3.52
3.62
3.74
3.74
3.18
4.18
4.30
4.35
3.91
4.51
3.14
3.03
3.02
3.67
4.04
2.90
2.90
2.82
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
8.78 ± 0.5
3.51 ± 0.2
4.53 ± 0.3
4.34 ± 0.3
0.18 ± 0.1
20.9%
11.9 ± 0.8
12.9%
19.8%
2.21 ± 0.2
3.11 ± 0.5
3.31 ± 0.4
26.4%
0.47 ± 0.1
0.17 ± 0.1
37.2%
26.5%
22.1%
29.5%
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22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
4.46 ± 0.6
7.3 ± 1.1
36.4%
16.9 ± 0.9
11.7 ± 0.7
18.5 ± 0.8
22.3 ± 0.5
3.75 ± 0.4
17.0%
2.62
2.62
3.04
3.92
3.92
4.73
3.58
2.95
2.95
3.81
3.45
3.53
2.80
3.51
3.38
6.15
1.32
-1.02
21.4%
15.8%
5.61 ± 0.7
1.77 ± 0.2
13.3 ± 0.6
11.3 ± 0.9
11.2 ± 0.9
14.9 ± 0.8
22.3 ± 1.2
Thiourea*
* = standard inhibitor
Octanol-Water partition coefficients (ClogP)
The Modern Drug Data Report (MDDR) and Comprehensive Medicinal Chemistry (CMC),
Derwent World Drug Index (WDI) are among the more commonly referenced data bases for
drug-like compounds [53, 54]. The original rule-of-five (RO5) deals with the four simple
physicochemical parameters associated with the 90% of orally administrable compounds
that have attained phase-II clinical status. A comprehensive computational survey of the
physicochemical properties of more than 15000 established drugs reveal the acceptable
ranges of these RO5 parameters as MWT≤500, logP≤5, H-bond donors≤ 5 and H bond
acceptors≤10 [55]. These physicochemical parameters ensure the acceptable aqueous
solubility and intestinal permeability and are the prerequisite in oral bioavailability. The
RO5 was consciously fashioned to be a conservative predictor in an era where medicinal and
combinatorial chemists produced too many libraries of compounds with very poor
physicochemical properties. If a compound fails to fulfill these requirements then there is a
possibility that oral administration problems will be faced. However, passing the RO5 tests
is no guarantee that a compound is a proven drug-like. Additionally, the RO5 says nothing
about specific chemistry and structural features found in drugs or non-drugs candidates [56,
57]. In the work presented here, the computationally calculated ClogP values (1.32-6.15)
12

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(Table 2) for all the synthesized compounds (1-38) have shown good agreement with the
accepted logP (log P≤5) or ClogP values, making them highly promising drug candidates.
Anticancer activity
Anticancer potential of the synthesized thiourea derivatives (1-38) was evaluated on lung
carcinoma cell lines, using vincristine as a standard drug. The cytotoxic effects were tested
by SRB assay and the results are represented in %inhibition in Figure 2. The compounds
were tested at 100 µM against lung cancer cell lines (H157) and Figure 2 demonstrated their
anticancer activity.
The most potent inhibitor was 16, with 2,4,6-trimethylphenyl substitution, having
66.32% inhibition for H157. Moreover, 15, 22 and 24 possessed > 60% inhibition and had
methyl, methoxy and trifluoro methyl group as substituents, respectively. All the compounds
presented more than 25% inhibition against lung carcinoma which indicates that the
compounds had significant inhibitory effects against cancer cell lines.
Figure 2. Percent inhibition of N-aroyl-N´-aryl thioureas (1-38) and standard drug
vincristine against lung carcinoma (H157) at 100 µM
Molecular docking studies
Molecular docking studies of thiourea derivatives were carried out by LeadIT to investigate
the binding modes of inhibitors as well as interactions with the active sites. The structures of
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the N-aroyl-N´-aryl thioureas were docked into structure of Jack bean urease PDB ID: 3LA4,
determined by X-ray crystallography. A bi-nickel center in the active site of the enzyme was
found. Validation for docking methodology was done by re-docking the reference ligand in
the active site of enzyme. It was observed that most of the compounds in the series exhibited
the similar binding modes within the active site. Amino acid residues found in the active site
are His407, His409, Arg439, Arg609, His492, His519, Asp494, Ala636, His545 and His593.
The synthetic derivatives exhibited notable interactions with the active site Ni atoms. The
docking studies were consistent with in vitro results as the most potent inhibitor fitted in the
catalytic site of enzyme pocket. The interactions within the active pocket of the receptor for
its ligand are very important in the design of inhibitor.
Results of in vitro studies showed that compounds 7 and 17 were most potent among
all the screened derivatives. Docking studies supported the experimental results that potent
inhibitors inhibit the catalytic site of the enzyme. The interactions within the active pocket
of the receptor for its ligand are very important in the design of inhibitor [40, 41, 58]. The
binding interactions of both the inhibitors were indistinguishable into the active pocket. The
possible binding mode was selected for both the inhibitors after HYDE assessment and
shown in Figure 3. It helped in the selection of favorable binding mode of compound within
the active pocket of enzyme. Inside the active site of receptor, compound 7 showed
hydrogen bond interactions with amino acid Ala440, whereas, π-π interactions were
observed by compound 7 with imidazole ring of histidine residues in the active site. Putative
binding pose of compound 7 was selected after Hyde assessment and visualization and
depicted in Figure 3.
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Figure 3. Putative binding mode of 7 (dark blue color) bound inside the active site of Jack bean urease. Carbon
atoms of protein (light blue) while two nickel cations (dark green spheres)
Figure 3 represented that 2-chlorobenzoyl moiety of compound 7 oriented towards the active
pocket and form π-π interactions with imidazole ring of the catalytic histidine residues in the
active pocket. Whereas, 2, 5-dichlorophenyl substituent was found more towards amino
acids Arg609 and His593. The important group C=S of the compound interacted with the
Asp494 by hydrophobic interactions and presented hydrogen bonding with His593. The
group exhibited important interactions within the active site of the urease. Docking studies
revealed that along with the stable hydrogen bonds, hydrophobic interactions were noticed
by compound 17 with the active site residues of enzyme (urease). Figure 4 showed the
favorable binding mode of compound 17, where 2-chlorobenzoyl moiety was directed deep
into the active pocket, while, 3-bromophenyl substituent was oriented towards amino acids
Asp494, His593 and Arg609. In addition, the C=S group interacted with the nickel present
in active site. Moreover, the same group showed hydrogen bonding with imidazole ring of
His492.
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Figure 4. Putative binding mode of 17 bound to the active site of Jack bean urease. Carbon atoms are colored
magenta and that of protein are light blue. Dark green spheres are two nickel cations
Conclusion
A series of N-aroyl-N´-aryl thioureas derivatives analogues were prepared using facile
microwave-assisted solution phase parallel synthesis. These synthetic derivatives were
tested for in vitro urease inhibition profile and found to exhibit excellent urease inhibition.
Compound 17 showed potent urease inhibitory activity having an IC50 value 0.17 ± 0.1 µM.
All the synthetic compounds were also evaluated for the cytotoxic activity. Compound 16
was found potent against Lung carcinoma (H157) possessed 66.32% inhibition. Molecular
docking studies were carried out to find the binding pattern of urease inhibitors. Moreover, it
was interpreted from the results that the compounds can be of use in therapeutic and
preventive purpose against cancer. Therefore, it is suggested to find the appropriate
correlation of anticancer agents with urease inhibition.
Acknowledgements
M.K.R. is thankful to the Quaid-i-Azam University, Islamabad, Pakistan for the grant of
funds for Post-doctoral Fellowship. J.I. is thankful to the Higher Education Commission of
Pakistan for the financial support through Project No. 20-3733/NRPU/R&D/14/520.
16

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Conflicts of Interest
The authors declare no conflict of interest.
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Highlights
•
•
•
•
•
•
Design and synthesis of N-aroyl-N´-aryl thioureas
Characterization by FT-IR, 1H and 13C NMR spectroscopy.
Single crystal X-ray diffraction and NMR studies were performed.
The synthetic compound was active against urease.
Cytotoxicity analysis was also performed.
Molecular docking studies were carried out.