Synthesis, in vitro urease inhibitory activity, and molecular docking studies of thiourea and urea derivatives

Article abstract of DOI:10.1016/j.bioorg.2018.06.007

The current study deals with the synthesis of urea and thiourea derivatives 1–37 which were characterized by various spectroscopic techniques including FAB-MS, ¹H-, and ¹³C NMR. The synthetic compounds were subjected to urease inhibitory activity and compounds exhibited good to moderate urease inhibitory activity having IC₅₀ values in range of 10.11–69.80 μM. Compound 1 (IC₅₀ = 10.11 ± 0.11 μM) was found to be most active and even better as compared to the standard acetohydroxamic acid (IC₅₀ = 27.0 ± 0.5 μM). A limited structure–activity relationship (SAR) was established and the compounds were also subjected to docking studies to confirm the binding interactions of ligands (compounds) with the active site of enzyme.

Full text of DOI:10.1016/j.bioorg.2018.06.007

Accepted Manuscript
Synthesis, in vitro urease inhibitory activity, and molecular docking studies of
thiourea and urea derivatives
Bilquees Bano, Kanwal, Khalid Mohammed Khan, Arif Lodhi, Uzma Salar,
Farida Begum, Muhammad Ali, Muhammad Taha, Shahnaz Perveen
PII:
S0045-2068(18)30298-0
DOI:
https://doi.org/10.1016/j.bioorg.2018.06.007
YBIOO 2387
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 March 2018
28 May 2018
3 June 2018
Please cite this article as: B. Bano, Kanwal, K. Mohammed Khan, A. Lodhi, U. Salar, F. Begum, M. Ali, M. Taha,
S. Perveen, Synthesis, in vitro urease inhibitory activity, and molecular docking studies of thiourea and urea
derivatives, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.06.007
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Synthesis, in vitro urease inhibitory activity, and molecular docking studies of
thiourea and urea derivatives
Bilquees Bano,a Kanwal,a Khalid Mohammed Khan,a,d Arif Lodhi,b Uzma Salar,a Farida
Begum,b Muhammad Ali,c Muhammad Taha,d Shahnaz Perveene
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
bDepartment of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul
Wali Khan University, Mardan, Pakistan
cDepartment of Chemistry, COMSATS Institute of Information Technology (CIIT) University
Road, Abbottabad-22060, Pakistan
dDepartment of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC),
Imam Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam Saudi Arabia
ePCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280,
Pakistan
Abstract: The current study deals with the synthesis of urea and thiourea derivatives 1-37 which
1
13
were characterized by various spectroscopic techniques including FAB-MS, H-, and C-NMR.
The synthetic compounds were subjected to urease inhibitory activity and compounds exhibited
good to moderate urease inhibitory activity having IC50 values in range of 10.11-69.80 µM.
Compound 1 (IC50 = 10.11 ± 0.11 µM) was found to be most active and even better as compared
to the standard acetohydroxamic acid (IC50 = 27.0 ± 0.5 µM). A limited structure-activity
relationship (SAR) was established and the compounds were also subjected to docking studies to
confirm the binding interactions of ligands (compounds) with the active site of enzyme.
Keywords: Synthesis; urea; thiourea; in vitro urease activity; structure-activity relationship;
molecular docking studies
Introduction
Urease (urea amidohydrolase E C 3.5.1.5) is an enzyme having two nickel atoms in its core
structure and is responsible for the hydrolysis of urea into ammonia and CO2 or carbamate [1-3].
The carbamate decomposes readily into another molecule of ammonia. These reactions causing
the increase in the alkalinity of physiological system which is accountable for numerous adverse
*Corresponding Author: khalid.khan@iccs.edu, drkhalidhej@gmail.com; Tel.: +922134824910, Fax:
+922134819018
1

effects to animal and human health [4]. In addition, excess ammonia production is also harmful
for agriculture and causes the death of cash crops. Ureases are found in fungi, bacteria, higher
plants, and in soil as a soil enzyme. Bacterial ureases are large heteropolymeric metalloproteins
with nickel (II) ions present in their active sites [5-7]. Many microbes use this enzyme in a
reaction, which provides nitrogen for growth [8], and it is also involved in plant nitrogen
metabolism during the germination process [9]. Elevated urease activity causes significant
economic and environmental problems by releasing large amounts of ammonia into the
atmosphere during urea fertilization in agriculture sector [10] which leads to plant damage
primarily by depriving plants from their essential nutrients and secondarily by ammonia toxicity,
thus resulting in the increased soil pH [11]. Urease plays a vital role in many pathogenic
processes in animals as well as in humans. It played a major role in urinary catheter incrustation,
peptic ulceration, kidney stone, pyelonephritis, urolithiasis, hepatic encephalopathy, and reactive
arthritis [12-16]. Urease has also been reported as an immunogenic modulator in a number of
pathogen-induced inflammatory reactions [17].
Urea and thiourea are versatile reagents in synthetic organic chemistry. They are important basic
building blocks in the synthesis of heterocycles [18]. Thioureas and ureas (unsymmetrical or
symmetrical) have drawn considerable attention due to their broad spectrum biological as well as
pharmacological activities, and their application as pesticides [19]. A variety of thiourea and urea
derivatives exhibited antimicrobial [20-21], antimalarial [22], analgesic, antiinflammatory [23],
anticancer, antitumor [24], hypoglycemic [25], antituberculosis [26], and anti-HCV [27]
activities. Despite having a large number of urease inhibitors being reported and marketed, there
is still need of more potent inhibitors having less side effects and more efficacy. Since, our
research group has been engaged in search of lead compounds as urease inhibitors. We have
reported different classes of compounds as urease inhibitors including 5-nitroisatin-3-
thiosemicarbazones, 1,3,4-oxazole-2(3H)-thiones, urea, and thiourea [28-29]. Therefore, in
continuation of our research work and keeping in mind the reported urease inhibitory potential of
urea and thiourea derivatives (Figure-1), we synthesized compounds 1-37 and screened them
against urease enzyme. To the best of our knowledge, six compounds 1, 2, 25, 28, 32, and 37 are
known [30] while rest of the compounds are new.
2

Figure-1: Rationale of the present study
Results and discussion
Chemistry
Urea and thiourea derivatives 1-37 were synthesized by the reaction of commercially available
substituted phenyl isothiocyanate and isocyanate with a variety of substituted anilines in
dichloromethane at 0 ˚C for 45 min according to literature procedure [31] as shown in Scheme-1
(Table-1). In all experiments, precipitates were formed which were filtered and triturated with
10 mL of dichloromethane and excess of hexane. The product thus obtained was dried in vacuum
and crystallized from methanol. The structures were determined using different spectroscopic
methods such as FAB-MS, 1H-, and 13C-NMR.
3

Scheme-1: Synthesis of aryl urea and thiourea derivatives 1-37
Spectral analysis of most active compound 1
NMR Spectroscopy
1
The H-NMR was recorded in deuterated DMSO-d6 on a 400 MHz instrument. A sharp singlet
for NH appeared as the most downfield signal at δH 10.09. The molecule possess eight aromatic
protons in which H-2′ resonated as singlet at δH 8.52. H-4′ resonated at δH 7.95 as doublet with
coupling constant J4′,3′ = 8.0 Hz. H-6′ also appeared as doublet at δH 7.89 with coupling constant
J6',5'= 7.6 Hz showing ortho coupling with H-5′. A triplet of H-5′ appeared at δH 7.61 having
coupling constant J5'(4′,6′) = 8.2 Hz, showing coupling with H-4′ and H-6′, respectively. Another
triplet of H-5 was observed at δH 7.27 having coupling constant J5(4,6) = 8.0 Hz. A sharp singlet
of H-2 appeared at δH 7.13. The doublet of H-6 was resonated at δH 7.01 showing ortho coupling
with H-5 having coupling constant J6,5 = 8.0 Hz. H-4 appeared at δH 6.74 as doublet showed
coupling with H-5 with coupling constant J4,5 = 7.6 Hz. The OCH3 protons resonated at δH 3.73
as singlet as shown in Figure-2A.
Figure-2: Representative 1H- (A) and 13C-NMR (B) chemical shift of compound 1
13C-NMR broad-band decoupled spectrum (CD3OD-d4) showed total 14 carbon signals including
eight methine, five quarternary, and one methoxy carbon. The most downfield signal was of
thiocarbonyl group appeared at C 154.5. Quarternary C-1, -3, -1′, and -3′ were resonated at C
150.6, 149.4, 132.2, and 122.5, respectively. However, methine carbon C-2′ and C-2 appeared at
C 151.6 and C 134.3, respectively. Signals of all remaining aromatic carbons appeared in the
4

usual aromatic range C 130.6-111.3. The most upfield signal belongs to methoxy carbon that
appeared at C 56.7 Figure-2B.
Mass Spectrometry
The compound 1 was unstable in EI-MS, thus was subjected to FAB-MS and showed the M+1 at
m/z 304.
Table-1: Thiourea and urea derivatives (1-37)
Compounds
R1
R2
IC50 ± SEMa (µM)
Thiourea Derivatives
10.11 ± 0.11
11.24 ± 0.31
1
2
13.31 ± 0.15
3
19.48 ± 0.71
39.22 ± 0.61
61.86 ± 2.11
4
5
6
5

15.13 ± 0.71
69.80 ± 0.71
7
8
35.10 ± 2.11
67.12 ± 2.19
9
10
19.28 ± 0.31
22.23 ± 0.16
10.51 ± 0.15
17.32 ± 0.29
11
12
13
14
72.99 ± 3.71
17.11 ± 0.25
15
16
6

18.44 ± 0.61
60.33 ± 2.71
17
18
51.21 ± 0.11
67.42 ± 1.01
14.23 ± 0.31
17.00 ± 0.21
19
20
21
22
15.43 ± 0.36
23
19.16 ± 0.11
15.41 ± 0.15
24
25
7

11.19 ± 0.11
19.25 ± 0.21
17.29 ± 0.31
13.30 ± 0.11
26
27
28
29
18.19 ± 0.16
19.22 ± 0.31
30
31
23.01 ± 0.61
32
39.70 ± 0.71
33
Compounds
R1
R2
IC50 ± SEMa (µM)
Urea Derivatives
8

12.19 ± 0.15
13.34 ± 0.01
34
35
12.78 ± 0.18
17.12 ± 0.31
36
37
Acetohydroxamic acidb
27.0 ± 0.5
SEMa is the standard error of the mean, Acetohydroxamic Acidb is the standard inhibitor for urease enzyme
Structure-activity relationship (SAR)
Synthetic compounds 1-37 were divide into two classes such as thiourea and urea derivatives. In
general, differences in the activity of compounds were observed due to varying substitutions and
positions at rings A and B. Mostly, the substituents at ring B are varied. To understand the better
structure-activity relationship (SAR) of synthetic molecules with the enzyme binding site, the
synthetic compounds were divided in three different categories based on substitutions at ring A,
i.e. category (i) comprises of electron withdrawing substituents (nitro and trifluoromethyl),
category (ii) with electron donating groups (methoxy), and category (iii) having halogen
substituents (F, Cl, and Br), directly attached on the benzene ring).
Figure-3: General structure of compounds
9

Nitro and trifluoromethyl substituted groups (Category i):
The highly active compound 1 (IC50 = 10.11 ± 0.11 μM) with nitro and methoxy groups at meta
position of ring A and B, respectively, was found to be almost three-folds better active than the
standard acetohydroxamic acid (IC50 = 27.0 ± 0.5 μM). The enhanced activity of this compound
might be due to the interaction of nitro group with the active site of enzyme (Figure-4).
Figure-4: Structure-activity relationship of compound 1
Compound 2 (IC50 = 11.24 ± 0.31 μM) having trifluoromethyl substituent at meta position of
ring A and iodo group at para position of ring B also showed potent activity. The variation at
ring B in compound 3 (IC50 = 13.31 ± 0.15 μM) bearing one chloro and two methoxy groups at
ortho, para, and meta positions, respectively, resulted in the decreased activity as compared to
compound 2, but better than the standard acetohydroxamic acid (IC50 = 27.0 ± 0.5 μM). It
indicates that the decline in activity is due to the addition of electron donating groups along with
halogen (Figure-5).
Figure-5: Structure-activity relationship of compounds 2 and 3
Compounds 4 (IC50 = 19.48 ± 0.71 μM) and 5 (IC50 = 39.22 ± 0.61 μM) having CF3 at meta
position of ring A and two methyl groups at different positions of ring B. Amongst them,
compound 4 having methyl groups at meta and para positions, respectively, showed good
activity, however, the change in position of the methyl groups in compound 5 resulted in two
folds decreased activity and it was further lost when two methyl groups were replaced with an
ethyl group in compound 6 (IC50 = 61.86 ± 2.11 μM). Hence, the above activity pattern revealed
10

that the positions and nature of electron donating substituents at ring B matters a lot for good
activity (Figure-6).
Figure-6: Structure-activity relationship of compounds 4, 5, and 6
Compounds 7, 8, 9, and 10 with trifluoromethyl group at ortho position of ring A also displayed
good to moderate activity. Compound 7 (IC50 = 15.13 ± 0.71 μM) bearing thiomethyl group at
meta position of ring B exhibited better potential as compared to the standard acetohydroxamic
acid (IC50 = 27.0 ± 0.5 μM). The para chloro substituted compound 8 (IC50 = 69.80 ± 0.71 μM)
showed lesser activity, however, mixed substitution of chloro with methoxy in compound 9 (IC50
= 35.10 ± 2.11 μM) resulted in good activity as compared to compound 8. Compound 10 (IC50 =
67.12 ± 2.19 μM) having dimethyl aryl part was also weakly active (Figure-7).
Figure-7: Structure-activity relationship of compounds 7, 8, 9, and 10
Among synthetic derivatives with 2-methoxy and 5-trifluoromethyl substitutions at ring A.
Compound 11 (IC50 = 19.28 ± 0.31 μM) with methoxy group at meta position showed good
activity. The replacement of methoxy with bromo at ortho position as in compound 12 (IC50 =
22.23 ± 0.16 μM), a decreased activity was observed in contrast to compound 11, however, it
remains more active than the standard acetohydroxamic acid (IC50 = 27.0 ± 0.5 μM) (Figure-8).
Activities of compounds 11 and 12 can also be compared with compounds 3 (IC50 = 13.31 ± 0.15
μM) which lacks electron donating methoxy group at ring A but have a combination of halogen
with methoxy groups on ring B was found to be more active. Which suggest that combination of
electron donating groups with positive mesomeric effect on ring B such as halogen and methoxy
11

favors the good activity. Similarly, it further confirms by comparing the activities of 11 and 12
with compound 16 which lacks electron withdrawing trifluoromethyl at ring A but having
combination of halogen and methoxy groups, was again found to be more active. Which further
confirms the active participation of electron donating groups with positive mesomeric effect on
ring B.
Figure-8: Structure-activity relationship of compounds 11 and 12
Methoxy substituted compounds (Category ii)
This category of compounds have methoxy group at meta position of ring A, among them
compound 13 (IC50 = 10.51± 0.15 μM) with methyl substitutions at ortho and meta positions of
ring B was the second most active compound of the series, this may be due to the interaction of
methoxy and methyl groups with the active site of enzyme. However, positional difference of
methyl substituents at ring B in compound 14 (IC50 = 17.32 ± 0.29 μM) resulted in decreased
activity. The replacement of both methyl groups with a ethyl at para position of ring B leads to
decreased activity as in compound 15 (IC50 = 72.99 ± 3.71 μM). It showed that methyl groups at
particular positions are positively contributing in the activity (Figure-9).
Figure-9: Structure-activity relationship of compounds 13, 14, and 15
Among halogen substituted compounds at ring B, compound 16 (IC50 = 17.11 ± 0.25 μM) was
found to have potent activity, this may be due to the presence of methoxy substituent along with
12

chloro group which enhanced the activity of compound as compared to its other halogen
substituted analogues. The activity was slightly decreased in compound 17 (IC50 = 18.44 ± 0.61
μM) bearing iodo group, however, a sharp decline in the activity was observed when the position
of iodo was changed from ortho to para as in compound 18 (IC50 = 60.33 ± 2.71 μM).
Compounds 19 (IC50 = 51.21 ± 0.11 μM) and 20 (IC50 = 67.42 ± 1.01 μM) with bromo, fluoro,
and nitro substitutions also showed weak activity as compared to the standard (Figure-10).
Figure-10: Structure-activity relationship of compounds 16, 17, 18, 19, and 20
Halogen substituted compounds (iii)
The synthetic compounds with halogen substitution at ring A showed potential inhibitory activity
and all compounds 21-33 were more active than the standard acetohydroxamic acid (IC50 = 27.00
± 0.5 μM) except compound 33.
The dichloro groups at ring A also enhanced the activity of compounds, among them compound
21 (IC50 = 14.23 ± 0.31 μM) with dimethyl substitutions at ring B was found to have superior
activity. The change in positions of these methyl groups in compound 22 (IC50 = 17.00 ± 0.21
μM) resulted in lesser activity. Interestingly, compound 23 (IC50 = 15.43 ± 0.36 μM) having
ethyl group at para position showed better activity, however, in the previous categories the
substitution of ethyl group at ring B resulted in decreased activity. Compound 24 (IC50 = 19.16 ±
0.11 μM) with two chloro groups at meta and para positions of ring A and methyl and chloro
groups at ortho and meta positions of ring B, respectively, showed better potential as compared
to the standard acetohydroxamic acid (IC50 = 27.00 ± 0.5 μM) (Figure-11).
13

Figure-11: Structure-activity relationship of compounds 21, 22, 23, and 24
Mono fluoro compound 25 (IC50 = 15.41 ± 0.15 μM) with bromo group at the para position of
ring B was found to have good activity. Addition of another fluoro group at ring A and iodo at
para position of ring B in compound 26 (IC50 = 11.19 ± 0.11 μM) resulted in better activity
which may be due to the addition of fluoro and iodo groups. The activity of compound 27 (IC50 =
19.25 ± 0.21 μM) was decreased by changing the position of iodo from para to ortho. The bromo
substituted derivative 28 (IC50 = 17.29 ± 0.31 μM) showed good activity but less active than
compound 26. Compounds 29 (IC50 = 13.30 ± 0.11 μM) and 30 (IC50 = 18.19 ± 0.11 μM)
exhibited good inhibitory activity than standard. Both of these compounds were positional
isomers with respect to the positions of methyl group at ring B, in compound 29 the methyl
groups were at both meta positions whereas in compound 30 with methyl groups at meta and
para positions. This showed that particular positions of substituents at ring B are actively
contributing in the activity (Figure-12).
14

Figure-12: Structure-activity relationship of compounds 25, 26, 27, 28, 29, and 30
Among bromo substituted compounds, bearing bromo at meta position of ring A, compound 31
(IC50 = 19.22 ± 0.31 μM) with thiomethyl group at meta position of ring B was most active
might be due to the interaction of the thiomethyl group with the active site. However, compound
32 (IC50 = 23.01 ± 0.61 μM) with ethyl group at para position exhibited less activity as
compared to compound 31 even though it was more active than the standard acetohydroxamic
acid (IC50 = 27.00 ± 0.5 μM). Compound 33 (IC50 = 39.70 ± 0.71 μM) bearing two methoxy and
one chloro group at ortho, para, and meta positions of ring B, respectively, was found to be less
active than the standard Figure-13.
Figure-13: Structure-activity relationship of compounds 30, 31, and 32
Urea Derivatives:
Urea derivatives also showed good inhibitory potential than standard acetohydroxamic acid (IC50
= 27.00 ± 0.5 μM), among them compound 34 (IC50 = 12.19 ± 0.15 μM) with chloro at meta
15

position of ring B and nitro at meta position of ring A was most active. However, compound 35
(IC50 = 13.34 ± 0.01 μM) having triflouro methyl and chloro group at ring A and meta chloro at
ring B was found to be less active. Compound 36 (IC50 = 12.78 ± 0.18 μM) containing a meta
chloro at ring A as well as meta chloro and two dimethoxy groups at ortho and para positions of
ring B was found to be second most active among urea derivatives probably due to substitutions
at ring B. The replacement of chloro with bromo and ejection of dimethoxy groups from ring B
resulted in decreased activity in compound 37 (IC50 = 17.12 ± 0.31 μM) but it exhibited better
activity than the standard (Figure-14).
Figure-14: Structure-activity relationship of compounds 34, 35, 36, and 37
Molecular Docking Studies
Molecular docking studies were carried out to support the in vitro studies of the synthetic
compounds. Enzyme selected for docking studies was downloaded from RCSB Protein Data
Bank (PDB ID; 4UBP). The size of the grid was selected keeping in view the size of the ligands
and the important residues of the enzyme i.e. H137, H139, A170, K220, H249, H275, G280,
C322, H323, H324, R339, D363, A364 Ni798 and Ni799. The urease enzyme and the
conformation adopted by the ligands in the active site is shown in Figure-15.
16

Figure-15: Urease from Bacillus pasteurii (Ribbon form in grey color), actives site of urease
(enclosed in red color) and zoomed in docked conformation of compound 1 (Lilac colored sticks
along with Nickle atoms colored in red)
The top five most active compounds i.e. 1, 13, 2, 29, and 3 among the series were analyzed for
their interactions with the active site of urease enzyme. Compound 1 with IC50 = 10.11 ± 0.11
µM was found forming different hydrogen bond and hydrophobic interactions with the side chain
residues of active site. Two hydrogen bond interactions with a distance of 3.07 Å and 3.0 Å were
formed by oxygen of nitro group as a hydrogen bond acceptor (HBA) and NH group of thiourea
as a hydrogen bond donor (HBD) with the NH of imidazole and carbonyl oxygen of the side
chain His222 and Lys169, respectively. In addition, the oxygens of nitro group at variable phenyl
ring mediated metal bond interactions with both nickle atoms i.e. Ni 798 (2.66 Å) and Ni 799
(2.72 Å), respectively. Other interactions found in complex are shown in Figure-16a.
Compound 13 (IC50 = 10.51 ± 0.15 µM was found mediating three hydrogen bond interactions
with the side chain amino acids of active site. A hydrogen bond interaction (3.29 Å) was formed
between oxygen of methoxy and NH group of side chain His222 and two hydrogen bond
interactions between both NH groups of thiourea and carboxyl oxygens of side chain Asp224
with a distance of 3.01 Å and 3.32 Å, respectively. The carbon of methoxy group at phenyl ring
mediated metal bond interaction with Ni799 (3.06 Å). In addition, same carbon atom of methoxy
group was found forming carbon bonding with the carboxy oxygens of KCX220 (3.76 Å) and
Asp363 (mean = 3.69 Å). Other interactions found in stabilizing the complex were π-π stacked
interaction between methoxybenzene ring and side chain His323, π-sulfur interaction between
17

sulfur of thiourea and side chain His323, π-cation interaction between methoxybenzene ring and
side chain Arg339 and π-alkyl and alkyl interactions as shown in Figure-16b.
Third most active compound 2 among the series (IC50 = 11.24 ± 0.31 µM) was found forming
two hydrogen bond interactions, first between NH group of thiourea as HBD and carbonyl
oxygen of side chain Lys169 (3.01 Å), and second between fluorine atom of trifluoromethyl
group and NH of imidazole ring as HBD of side chain His222 (2.94 Å) as shown in Figure-16c.
Fluorine atoms of trifluoromethyl group also mediated metal acceptor interactions with Ni798
and Ni799 with a bond distance of 2.61 Å and 2.69 Å, respectively. Other interactions spotted
between compound 2 and side chain residues of the active site are shown in Figure-16c.
Analysis of least energy conformations adopted by fourth most active compound 29 (IC50 =
13.30 ± 0.11 µM) and fifth most active compound 3 (IC50 = 13.31 ± 0.15 µM) in the binding site
of urease showed that both were forming two hydrogen bond interactions (each) with the side
chain residues. Also, both compounds are forming metal interactions with the nickle atoms of the
binding site as shown in Figure-16d and Figure-16e. Summarizing the interactions of five most
active compounds we can conclude that their high activities could be due to formation of
interactions with the nickle atoms which are considered as the catalytic residues as they increase
electrophilicity of carbonyl carbon of urea and nucleophilicity of water molecule during the
hydrolysis of urea [32].
18

19

Figure-16:The ligand-protein interactions of most active compounds (a) 1, (b) 13, (c) 2, (d) 29,
and (e) 3 with the active site of urease from Bacillus pasteurii (4UBP) created by using
Discovery Studio 17.2 and LigPlot+. The left side displays 3D interactions of the compounds in
the binding site. The right side shows the 2D interaction patterns. Dashed lines show the
interactions among the ligand and the amino acids of the protein. Distances are in Angstrom.
Analyzing poses adopted by average active compound 9 (IC50 = 35.10 ± 2.11 µM) and least
active compounds 18 (IC50 = 60.33 ± 2.71 µM) and 8 (IC50 = 69.80 ± 2.71 µM), it was found that
these compounds were also involved in forming hydrogen bond interactions and other
hydrophobic interactions which were observed in case of most active derivatives. The
interactions which were not detected in least active compounds were the interactions of these
compounds with the metal atoms in the active site of urease enzyme as shown in Figure-17.
20

Figure-17: Three-Dimensional ligand-protein interactions of average active (a) 9 and least
active i.e. (b) 18 and (c) 8 compounds with the active site of urease from Bacillus pasteurii
(4UBP) created by using Discovery Studio 17.2.
Concluding computational studies, it can be summarized that the potential of inhibition exhibited
by synthetic compounds depends on the nature and position of substituents on both variable
phenyl ring. Derivatives having substituents in direct interaction with the catalytic nickle atoms
of enzyme’s active site showed the highest inhibition among the series. In our study nitro groups,
and halogens mainly made direct interactions with nickle atoms and it was also found that
substituents at meta and para positions mainly resulted in minimizing the complex energy and
this could be due to the fact that these two positions get closer to the nickle atoms as compared to
substituent at ortho position. The substituents on the second variable ring also influence the
activity and receptor ligand interactions i.e. substituents involved in forming hydrophobic
21

interactions and halogen interactions (particularly large size halogens) were found more active.
Thiourea moiety also played a major role in stabilizing the complex as its NH groups acted as
hydrogen bond donors. In some results, sulfur of thiourea group was also spotted forming π-
sulfur interactions with the side chain amino acids particularly histamines, a well-known
interaction playing important role in protein folding and stabilization.
Conclusion:
Urea and thiourea derivatives 1-37 were synthesized and evaluated for their urease inhibitory
activity. Amongst synthetic compounds twenty-five compounds exhibited good inhibitory
potential. Compounds 1 (IC50 = 1.13 ± 0.01 µM), 2 (IC50 = 11.24 ± 0.31 µM), 3 (IC50 = 13.31 ±
0.15 µM), 13 (IC50 = 10.51 ± 0.15 µM), and 29 (IC50 = 13.30 ± 0.11 µM) were found to be most
active than the standard acetohydroxamic acid (IC50 = 27.0 ± 0.5 µM). Structure-activity
relationship revealed that the different position of substituents mainly the meta and para
substitutions at rings A and B, respectively, resulted in variable urease inhibitory activity. The
docking studies further validated these results and showed efficient binding interaction between
the active compound and urease enzyme proteins. Conclusively, a number of new lead
compounds are identified as urease inhibitors which may be proceeded for further studies in
search for better inhibitors.
Material and Methods:
13
1H- and C-NMR spectra were recorded on Bruker 300, 400, 75, and 100 MHz spectrometers,
respectively. Mass experiments were carried out on a Finnigan MAT-311A (Germany) mass
spectrometer. Thin-layer chromatography (TLC) was monitored on pre-coated silica gel
aluminum plates (Kieselgel 60, 254, E. Merck, Germany). Visualization of TLC chromatograms
was performed by UV light at wavelengths of 254 and 365 nm. Dichloromethane of analytical
grade was used as received from supplier RCI Labscan Limited, Thailand. Aniline and all
isocyanates and isothiocyanates derivatives of analytical grades were used as received from the
suppliers.
22

General procedure for the synthesis of compounds 1-37
Different substituted anilines (1 mmol) were dissolved in dichloromethane at 0 ˚C, reaction
mixture was stirred for 10-15 min then isocyanates or isothiocayanates (1 mmol) were added,
progress of reaction was monitored with TLC. After 45 min, a solid mass was appeared which
was filtered, washed with hexane, triturated with 10 mL of dichloromethane and 50 mL of
hexane and dried under vacuum. Crystallization from ethanol get the desired solid thiourea/urea.
The structural determination was carried out by 1H-, 13C-NMR, and mass spectrometry.
Spectral Data for the Synthetic Compounds 1-37
1-(3-Methoxyphenyl)-3-(3-nitrophenyl)thiourea (1)
Yield: 75%, M.p.: 170-174ºC; 1H-NMR (400 MHz, DMSO-d6): δ 10.09 (s, 2H, NH), 8.52 (s, 1H,
H-2'), 7.95 ( d, J4',5' = 8.0 Hz, 1H, H-4') ,7.89 (d, J6',5' = 7.6 Hz, 1H, H-6'), 7.61 (t, J5'(4',6') = 8.0 Hz
, 1H, H-5'), 7.27 (t, J5(4,3) = 8.0 Hz, 1H, H-5), 7.13 (s, 1H, H-2) ,7.01 (d, J6,5 = 8.0 Hz, 1H, H-6),
13
6.74 (d, J4,5 = 7.6 Hz, 1H, H-4), 3.73 (s, 3H, OCH3 ), C-NMR (100 MHz, DMSO-d6): δ154.5,
151.6, 150.6, 149.4, 134.3, 132.2, 130.6, 128.4, 126.1, 122.5, 122.5, 112.0, 111.3, 56.7. FAB+:
304 (M+1)
1-(4-Iodophenyl)-3-(3-(trifluoromethyl)phenyl)thiourea (2)
º
1
Yield: 65%, M.p.: 231-235 C; H-NMR (400 MHz, DMSO-d6): δ 10.04 (s, 2H, NH), 7.92 (s,
1H, H-2), 7.74 (d, J4,3 = 8.0 Hz, 1H, H-4), 7.68(d, J5',6' = J2',3' = 8.4 Hz, 2H, H-3'/H-5'), 7.57 (t,
13
J6(4,5) = 7.6 Hz, 1H, H-6), 7.31 (d, J6',5' = J2',3' = 8.4 Hz, 2H, H-2'/H-6'), C-NMR (100 MHz,
DMSO-d6): δ 163.2, 158.6, 146.4, 146.6, 141.6, 133.1, 130.5, 129.2, 128.7, 128.2, 127.8, 127.3,
125.1, 122.7, 120.9, 118.7, FAB+: 423 (M+1).
1-(5-Chloro-2,4-dimethoxyphenyl)-3-(3-(trifluoromethyl) phenyl)thiourea (3)
Yield: 55%, M.p.: 172-176 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.95 (s, 1H, NH), 9.31(s, 1H,
NH), 7.99 (s, 1H, H-2), 7.75 (d, J4,5 = 8.0 Hz, 1H, H-4), 7.64 (s, 1H, H-6'), 7.55 (t, J5(4,6) = 7.8
Hz, 1H, H-5), 7.44 (d, J6,5 = 7.6 Hz, 1H, H-6), 6.85 (s, 1H, H-3'), FAB+: 391 (M+1).
23

1-(3,4-Dimethylphenyl)-3-(3-(trifluoromethyl) phenyl)thiourea (4)
Yield: 85%, M.p.: 231-234 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.82 (s, 2H, NH), 7.93 (s, 1H,
H-2), 7.73 (d, J4,5 = 8.4 Hz, 1H, H-4), 7.52 (t, J5(4,3) = 7.8 Hz, 1H, H-5), 7.43 (d, J6,5 = 7.6 Hz, 1H,
H-6), 7.17 (ovp, 3H, H-5/H-6/H-2), 2.19 (s, 6H,CH3), FAB+: 325 (M+1).
1-(2,5-Dimethylphenyl)-3-(3-(trifluoromethyl) phenyl)thiourea (5)
Yield: 64%, 1 M.p.: 166-170 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.95(s, 1H, NH), 9.85 (s, 1H,
NH), 7.94 (s, 1H, H-2), 7.73 (d, J4,5 = 8.5 Hz, 1H, H-4), 7.54 (t, J5(4,6) = 8.0 Hz, 1H, H-5), 7.44 (d,
J6,5 = 8.0 Hz, 1H, H-6), 7.10 (ovp, 1H, H-3'), 7.07 (s, 1H, H-6'), 6.89 (dd, J4',3' = 8.0 Hz, J4',6' =
2.0 Hz, 1H, H-4'), 3.74 (s, 3H, CH3), 2.10 (s, 3H, CH3), FAB+ : 323 (M+1).
1-(4-Ethylphenyl)-3-(3-(trifluoromethyl) phenyl)thiourea (6)
Yield: 65%, M.p.: 249-251 ºC; 1H-NMR (400 MHz, MeOD-d4): δ 7.85 (s, 1H, H-2), 7.68 (d, J4,5
= 8.0 Hz, 1H, H-4), 7.51 (t, J5(4,6) = 7.8 Hz, 1H, H-5), 7.43 (d, J6,5 = 8.0 Hz, 1H, H-6), 7.31 (d, J5,6
= J6,5 = 8.4 Hz, 2H, H-5/H-6), 7.22 (d, J2,3 = J3,2 = 8.4 Hz, 2H, H-2/H-3). EI-MS: m/z (rel. abund.
%) 324 (M+, 67), 275 (49), 204 (15), 203 (37), 161 (61), 121 (68), 106 (100), 75 (24).
1-(3-(Methylthio)phenyl)-3-(2-(trifluoromethyl) phenyl)thiourea (7)
º
1
Yield: 72%, M.p.: 245-249 C; H-NMR (400 MHz, DMSO-d6): δ 10.00 (s, 1H, NH), 9.35 (s,
1H, NH) ,7.73 (d, J3,4 = 8.0 Hz, 1H, H-3), 7.69 (t, J4(5,6) = 7.4 Hz, 1H, H-4), 7.56 (d, J6,5 = 8.0
Hz, 1H, H-6),7.50 (ovp, 2H, H-5/H-2'), 7.27 (ovp, 2H, H-6'/H-5'),7.04 (d, J4',3' = 6.8 Hz, 1H, H-
4’), 2.49 (s, 3H, CH3), FAB+: 343 (M+1).
1-(4-Chlorophenyl)-3-(2-(trifluoromethyl) phenyl)thiourea (8)
º
1
Yield: 82%, M.p.: 220-223 C; H-NMR (400 MHz, DMSO-d6): δ 10.01 (s, 1H, NH), 9.39 (s,
1H, NH), 7.74 (d, J3,4 = 7.6 Hz, 1H, H-3), 7.69 (t, J4(5,6) =7.6 Hz, 1H, H-4), 7.56 (ovp, 3H, H-
6/H-3'/H-5')7.50 (t, J5(4,6) = 7.6 Hz, 1H, H-5), 7.39 (d, J = 8.0 Hz, 2H, H-6'/H-2'), FAB+: 331
(M+1).
1-(5-Chloro-2,4-dimethoxyphenyl)-3-(2-(trifluoro methyl)phenyl)thiourea (9)
Yield: 85%, M.p.: 248-252 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.41 (s, 2H, NH) 7.85 (s, 1H,
H-2’), 7.72 (d, J3,4 = 7.6 Hz, 1H, H-43), 7.67 (t, J5(4,6) =7.6 Hz, 1H, H-5), 7.58 (d, J6,5 = 8.0 Hz,
24

1H, H-6), 7.47 (t, J4(3,2) = 7.4 Hz, 1H, H-4), 6.84 (s, 1H, H-5'), 3.89 (s, 6H, OCH3), FAB+: 391
(M+1).
1-(3,4-Dimethylphenyl)-3-(2-(trifluoromethyl) phenyl)thiourea (10)
Yield: 75%, M.p.: 201-205 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.85 (s, 1H, NH), 9.13 (s, 1H,
NH), 7.72 (d, J3,4 = 8.0 Hz, 1H, H-3), 7.65 (d, J4,3 = 8.0 Hz, 1H, H-4), 7.57 (d, J6,5 = 8.0 Hz, 1H,
H-6), 7.47 (t, J5(4,6) = 7.2 Hz, 1H, H-5), 7.22 (s, 1H, H-2'), 7.20 (d, J6’,5’ = 8.0 Hz, 1H, H-6'), 7.10
(d, J5’,6’ = 8.4 Hz, 1H, H-5'), 2.19 (s, 6H, 2-CH3), FAB+: 325 (M+1).
1-(2-Methoxy-5-(trifluoromethyl)phenyl)-3-(3-methoxyphenyl)thiourea (11)
º
1
Yield: 65%, M.p.: 244-248 C; H-NMR (400 MHz, DMSO-d6): δ 10.18 (s, 1H, NH), 9.28 (s,
1H, NH), 8.45 (s, 1H, H-6'), 7.50 (d, J4',5' = 8.4 Hz, 1H, H-4'), 7.24 (ovp, 3H, H-3'/H-6/H-2 ),
7.05 (d, J5,6 = 7.6 Hz, 1H, H-5), 6.74 (d, J4,5 = 7.2 Hz, 1H, H-4), 3.91 (s, 3H, OCH3), 3.74 (s, 3H,
OCH3), 13C-NMR (100 MHz, DMSO-d6): δ 151.6, 150.5, 149.3, 134.2, 132.2, 130.5, 128.3,
127.3, 126.0, 122.4, 122.4, 112.0, 111.2, 56.6, 56.5. FAB+, 357 (M+1).
1-(2-Bromophenyl)-3-(2-methoxy-5(trifluoro methyl) phenyl)thiourea (12)
Yield: 75%, M.p.: 214-218 ºC; 1H-NMR (300 MHz, DMSO-d6): δ 9.86 (s, 1H, NH), 9.58 (s, 1H,
NH), 8.59 (s, 1H, H-6'), 7.68 (d, J4',5’ = 7.8 Hz, 1H, H-4'), 7.59 (d, J3,4 = 7.5 Hz, 1H, H-3),7.50 (d,
J5,4 = 7.8 Hz, 1H, H-5), 7.41 (t, J4(3,2) = 7.5 Hz, 1H, H-4), 7.26 (ovp, 2H, H-3'/H-6), 3.93 (s, 3H,
OCH3), FAB+: 405(M+1).
1-(2,5-Dimethylphenyl)-3-(3-methoxyphenyl)thiourea (13)
Yield: 55%; M.p.: 200-204ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.59 (s, 1H, NH), 9.26 (s, 1H,
NH), 7.22 (ovp, 2H, /H-2/H-4), 7.10 (d, J6,5 = 7.6 Hz, 1H, H-6),7.03 (d, J5,6 = 5.6 Hz, 1H, H-5),
7.00 (s, 1H, H-2'), 6.97 (d, J4',5' = 7.6 Hz, 1H, H-4'), 6.69 (d, J5',6' = 8.0 Hz, 1H, H-5'), 3.72 (s, 3H,
OCH3), 2.24 (s, 3H, CH3), 2.17 (s, 3H, CH3 ), FAB+: 287(M+1).
1-(3,5-Dimethylphenyl)-3-(3-methoxyphenyl)thiourea (14)
Yield: 75%;1 M.p.: 211-215 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.65 (s, 1H, NH), 9.62 (s, 1H,
NH), 7.22 (t, J5(4,3) = 8.2 Hz, 1H, H-5), 7.15 (s, 1H, H-6'), 7.04 (s, 2H, H-2'/H-4') ,7.00 (d, J6,5 =
25

7.6 Hz, 1H, H-6), 6.76 (s, 1H, H-2), 6.69 (dd, J4,5 = 8.0 Hz, J4,2 = 1.6 Hz, 1H, H-4), 3.72 (s, 3H,
OCH3), 2.23 (s, 6H, CH3); FAB+: 287(M+1).
26

1-(4-Ethylphenyl)-3-(3-methoxyphenyl)thiourea (15)
Yield: 85%, M.p.: 241-245 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.67 (s, 1H, NH), 9.66 (s, 1H,
NH), 7.35(d, J2',3' = J6',5' = 8.0 Hz, 2H, H-2'/H-6'), 7.23 (d, J4,5 = 8.4 Hz ,1H, H-4 ), 7.19 (ovp,
3H, H-3'/H-5'/H-2), 7.02 (d, J6,5 = 8.0 Hz, 1H, H-6), 6.69 (ovp, 1H, H-5), 3.72 (s, 3H, OCH3),
2.59 (q, 2H, CH2), 1.18 (t, 3H, CH3), FAB+: 287(M+1)
1-(5-Chloro-2,4-dimethoxyphenyl)-3-(3-methoxy phenyl)thiourea (16)
Yield: 65%, M.p.: 188-193 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.83 (s, 1H, NH), 9.05 (s, 1H,
NH), 7.74 (s, 1H, H-2'), 7.23 (ovp, 2H, H-6/H-2), 7.02 (ovp, 1H, H-5) ,6.83 (s ,1H, H-5'), 6.70
(dd, J4,5 = 8.0 Hz, J4,2 = 2.0 Hz, 1H, H-4), 3.88 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.73 (s, 3H,
OCH3), FAB+: 353(M+1).
1-(2-Iodophenyl)-3-(3-methoxyphenyl)thiourea (17)
Yield: 65%, M.p.: 127-131 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.92 (s, 1H, NH), 9.31 (s, 1H,
NH), 7.87 (d, J3',4' = 7.6 Hz, 1H, H-3'), 7.39 (ovp, 2H, H-4'/H-5'), 7.27(s, 1H, H-2), 7.24 (d, J6',5'
=
8.4 Hz, 1H, H-6'), 7.03 (d, J4,3 = 8.0 Hz, 1H, H-4), 6.99 (ovp, 1H, H-5), 6.73 (ovp, 1H, H-6), 3.74
(s, 3H, OCH3), 13C-NMR (75 MHz, DMSO-d6): δ 160.3, 150.8, 148.6, 148.3, 132.0, 129.7,
128.3, 126.9, 126.2, 123.6, 121.1, 113.3, 109.8, 56.3. FAB+: 385(M+1).
1-(4-Iodophenyl)-3-(3-methoxyphenyl)thiourea (18)
Yield: 75%, M.p.: 225-229 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.81(s, 2H, NH) ,7.64 (d, J3',4'
= J5',6' = 8.4 Hz, 2H, H-3'/H-5'), 7.31 (d, J2',3' = J6',5' = 8.0 Hz, 2H, H-2'/H-6'), 7.21 (t, J5(4,3) = 8.0
Hz, 1H, H-5), 7.14 (s, 1H, H-2), 7.00 (d, J6,5 = 8.0 Hz, 1H, H-6), 6.70 (br.s, 1H, H-4), 3.72 (s,
3H, OCH3), FAB+: 285(M+1).
1-(4-Bromophenyl)-3-(3-methoxyphenyl)thiourea (19)
Yield: 85%, M.p.: 194-198 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.81 (s, 2H, NH), 7.50 (d, J2',3'
= J3',2' = 8.0 Hz, 2H, H-2'/H-3') ,7.45 (d, J5',6' = J6',5' = 8.8 Hz, 2H, H-5'/H-6'), 7.22 (t, J5(4,3) = 8.2
Hz, 1H, H-5), 7.14 (s, 1H, H-2), 7.00 (d, J6,5 = 8.0 Hz, 1H, H-6), 6.71 (d, J4,3 = 8.4 Hz, 1H, H-4),
3.72 (s, 3H, OCH3), FAB+: 337(M+1).
27

1-(4-Fluoro-3-nitrophenyl)-3-(3-methoxy phenyl) thiourea (20)
º
1
Yield: 55%, M.p.: 249-253 C; H-NMR (400 MHz, DMSO-d6): δ 10.07 (s, 1H, NH), 9.99 (s,
1H, NH), 8.37 (d, J6',2' = 4.4 Hz, 1H, H-6'),7.87 (ovp, 1H, H-2'), 7.56 (t, J5' (4',6') =10.0 Hz, 1H, H-
5'), 7.27 (t, J5(6,4) = 8.2 Hz, 1H, H-5), 7.11 (s, 1H, H-2), 7.00 (d, J4,5 = 8.0 Hz, 1H, H-4), 6.75 (d,
J6,5 = 8.0 Hz, 1H, H-6), FAB+: 322 (M+1).
1-(2,4-Dichlorophenyl)-3-(3,5-dimethylphenyl)thiourea (21)
Yield: 85%, M.p.: 158-162 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.91 (s, 1H, NH), 9.32 (s, 1H,
NH), 7.64 (s, 1H, H-3), 7.60 (d, J5,6 = 8.8 Hz, 1H, H-5), 7.40 (d, J6,5 = 8.0 Hz, 1H, H-6), 7.08 (s,
2H, H-2'/H-4'), 6.79 (s, 1H, H-6'), 2.24 (s, 6H, CH3), FAB+, 325 (M+1).
1-(2,4-Dichlorophenyl)-3-(3,4-dimethylphenyl)thiourea (22)
Yield: 75%, M.p.: 218-222 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.90 (s, 1H, NH), 9.28 (s, 1H,
NH), 7.65 (ovp, 1H, H-5), 7.60 (s, 1H, H-3), 7.41 (ovp, 1H, H-6), 7.21 (ovp, 3H, H-6'/ H-2'/H-
5'), 2.20 (s, 6H, CH3), FAB+: 325 (M+1).
1-(2,4-Dichlorophenyl)-3-(4-ethylphenyl)thiourea (23)
Yield: 55%, M.p.: 162-166 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.96 (s, 1H, NH), 9.35 (s, 1H,
NH), 7.66 (ovp, 1H, H-5), 7.60 (s, 1H, H-3), 7.42 (d, J6,5 = 3.0 Hz, 1H, H-6), 7.39 (d, J6',5' = J2',3'
=
9.0 Hz, 2H, H-2'/H-6'), 7.20 (d, J5',6' = J3',2' = 9.0 Hz, 2H, H-3'/H-5'), 2.62 (q, 2H, CH2), 1.19 (t,
3H, CH3), FAB+: 325 (M+1).
1-(5-Chloro-2-methylphenyl)-3-(3,4-dichlorophenyl)thiourea (24)
Yield: 75%, M.p.: 180-184 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.96 (s, 1H, NH), 9.59 (s, 1H,
NH), 7.89 (d, J6, 2 = 2.0 Hz, 1H, H-6), 7.57 (d J5, 6 = 8.4 Hz, 1H, H-5), 7.46 (dd, J6,5 = 8.8 Hz, J6,2
= 2.4 Hz, 1H, H-6), 7.35 (d, J6',4' = 1.6 Hz, 1H, H-6'), 7.28 (ovp, 2H, H-4'/H-3'), 2.19 (s, 3H,
CH3), FAB+: 345 (M+1).
28

1-(4-Bromophenyl)-3-(3-fluorophenyl)thiourea (25)
Yield: 75%, M.p.: 168-172 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.97 (s, 2H, NH), 7.51 (d, J3',2'
= J5',6' = J4,5 = 8.0 Hz, 3H, H-3'/H-5'/H-4),7.45 (d, J6',5' = J2',3' = 8.8 Hz, 2H, H-6'/H-2'), 7.37 (ovp,
13
1H, H-2), 7.24 (d, J6,5 = 8.4 Hz, 1H, H-6), 6.95 (ovp, 1H, H-5), C-NMR (100 MHz, DMSO-
d6): δ 154.2, 153.6, 152.0, 137.7, 136.9, 135.0, 133.3, 133.2, 129.0, 127.4, 126.0, 124.1, 123.5.
FAB+: 325 (M+1).
1-(2,4-Difluorophenyl)-3-(4-iodo phenyl)thiourea (26)
Yield: 65%, M.p.: 238-242 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.63 (s, 1H, NH), 9.60 (s, 1H,
NH), 7.89(d, J5,6 = 8.0 Hz, 1H, H-5), 7.66 (d, J6,5 = 8.0 Hz, 1H, H-6), 7.52 (ovp, 4H, H-2'/H-3'/H-
5'/H-6'), 7.05 (ovp, 1H, H-3), FAB+: 391 (M+1).
1-(2,4-Difluorophenyl)-3-(2-iodophenyl)thiourea (27)
Yield: 55%, M.p.: 164-170 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.52 (s, 1H, NH), 9.45 (s, 1H,
NH), 7.89 (d , J5,6 = 7.6 Hz, 1H, H-5), 7.66 (ovp, 1H, H-3), 7.42 ( ovp, 2H, H-3'/H-6), 7.33 (ovp,
1H, H-5'), 7.09 (ovp, 2H, H-6'/H-4'), FAB+: 389 (M+1).
1-(4-Bromophenyl)-3-(2,4-difluorophenyl)thiourea (28)
Yield: 65%, M.p.: 218-220 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.97 (s, 1H, NH) 9.45 (s, 1H,
NH), 7.53 (ovp, 5H, H-2'/H-3'/H-5'/H-6'/H-3), 7.32 (ovp, 1H, H-5), 7.09 (t, J6(5,4) = 8.6 Hz, 1H,
H-6), FAB+: 345 (M+1).
1-(2,4-Difluorophenyl)-3-(3,5-dimethylphenyl)thiourea (29)
Yield: 65%, M.p.: 156-160 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.82 (s, 1H, NH), 9.26 (s, 1H,
NH), 7.53 (ovp, 1H, H-3), 7.30 (ovp, 1H, H-5), 7.07 (ovp, 3H, H-6/H-2'/H-4'), 6.78 (s, 1H, H-6'),
13
2.49 (s, 3H, CH3), 2.40 (s, 3H, CH3), C-NMR (100 MHz, DMSO-d6): δ 160.2, 153.7, 151.0,
148.0, 133.2, 132.0, 129.7, 126.7, 125.7, 123.6, 121.3, 117.0, 113.5, 60.3, 56.2. FAB+: 293
(M+1).
1-(2,4-Difluorophenyl)-3-(3,4-dimethylphenyl)thiourea (30)
Yield: 75%, M.p.: 145-149 ºC; 1H-NMR (400 MHz, DMSO-d6): δ 9.78 (s, 1H, NH), 9.20 (s, 1H,
NH), 7.54 (ovp, 1H, H-3),7.30 (ovp, 1H, H-5), 7.18 (ovp, 2H, H-2'/H-6), 7.09 (ovp, 2H, H-5'/H-
6’), 2.19 (s, 3H, CH3), 2.18 (s, 3H,CH3), FAB+: 293 (M+1).
29