Synthetic nicotinic/isonicotinic thiosemicarbazides: In vitro urease inhibitory activities and molecular docking studies

Article abstract of DOI:10.1016/j.bioorg.2018.04.004

Nicotinic and isonicotinic thiosemicarbazide or hydrazine carbothioamides 3–27 were synthesized and the structures of synthetic compounds were elucidated by various spectroscopic techniques such as EI-MS, ¹H-, and ¹³C NMR. Synthetic derivatives were evaluated for their urease inhibitory activity which revealed that except few all derivatives demonstrated excellent inhibition in the range of IC₅₀ values of 1.21–51.42 μM as compared to the standard thiourea (IC₅₀ = 21.25 ± 0.13 μM). Among the twenty-five synthetic derivatives nineteen 1–5, 7, 8, 10, 12, 14–18, 20–22, 24–27 were found to be more active showing IC₅₀ values between 1.13 and 19.74 μM showing superior activity than the standard. Limited structure-activity relationship demonstrated that the positions of substituent as well as position of nitrogen in pyridine ring are very important for inhibitory activity of this class of compound. To verify these interpretations, in silico study was also performed. A good correlation was obtained between the biological evaluation of active compounds and docking study.

Full text of DOI:10.1016/j.bioorg.2018.04.004

Accepted Manuscript
Synthetic nicotinic/isonicotinic thiosemicarbazides: In vitro urease inhibitory
activities and molecular docking studies
Basharat Ali, Khalid Mohammed Khan, Arshia, Kanwal, Shafqat Hussain,
Safdar Hussain, Muhammad Ashraf, Muhammad Riaz, Abdul Wadood,
Shahnaz Perveen
PII:
S0045-2068(18)30253-0
DOI:
https://doi.org/10.1016/j.bioorg.2018.04.004
YBIOO 2327
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 March 2018
12 April 2018
12 April 2018
Please cite this article as: B. Ali, K. Mohammed Khan, Arshia, Kanwal, S. Hussain, S. Hussain, M. Ashraf, M. Riaz,
A. Wadood, S. Perveen, Synthetic nicotinic/isonicotinic thiosemicarbazides: In vitro urease inhibitory activities and
molecular docking studies, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.04.004
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Synthetic nicotinic/isonicotinic thiosemicarbazides: In vitro urease inhibitory activities and
molecular docking studies
Basharat Ali,a Khalid Mohammed Khan,a,e Arshia,a Kanwal,a Shafqat Hussain,a Safdar Hussain,b
Muhammad Ashraf,b Muhammad Riaz,c Abdul Wadood,c Shahnaz Perveen,d
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
bDepartment of Chemistry, The Islamia University of Bahawalpur, Bahawalpur-63100, Pakistan
cDepartment of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul
Wali Khan University, Mardan, Pakistan
cPCSIR Laboratoires Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280,
Pakistan
eDepartment of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC),
Imam Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam Saudi Arabia
*Corresponding Authors: khalid.khan@iccs.edu, drkhalidhej@gmail.com,Tel.: +922134824910, Fax:
+922134819018
1

2

Research Highlight





Syntheses of nicotinic/isonicotinic thiosemicarbazide derivatives have been carried out.
Structures of all synthetic compounds were elucidated by spectroscopic techniques.
Urease inhibitory activity of synthetic compounds has been carried out.
In silico studies of active analogs have been performed.
Structure-activity relationship has been evaluated.
3

Abstract: Nicotinic and isonicotinic thiosemicarbazide or hydrazine carbothioamides 3-27 were
synthesized and the structures of synthetic compounds were elucidated by various spectroscopic
1
techniques such as EI-MS, H-, and 13C-NMR. Synthetic derivatives were evaluated for their
urease inhibitory activity which revealed that except few all derivatives demonstrated excellent
inhibition in the range of IC50 values of 1.21-51.42 μM as compared to the standard thiourea
(IC50 = 21.25 ± 0.13 μM). Among the twenty-five synthetic derivatives nineteen 1-5, 7, 8, 10, 12,
14-18, 20-22, 24-27 were found to be more active showing IC50 values between 1.13 to 19.74 μM
showing superior activity than the standard. Limited structure-activity relationship demonstrated
that the positions of substituent as well as position of nitrogen in pyridine ring are very important
for inhibitory activity of this class of compound. To verify these interpretations, in silico study
was also performed. A good correlation was obtained between the biological evaluation of active
compounds and docking study.
Keywords: Synthesis; nicotinic/isonicotinic; thiosemicarbazide; urease inhibition; in vitro; in
silico; structure-activity relationship.
4

1. Introduction
Urease (urea amidohydrolase, EC 3.5.1.5) is a well-known nickel containing metalloenzyme that
catalyzes the hydrolysis of urea into ammonia and carbamate [1]. At normal physiological pH
carbamate moiety spontaneously hydrolyses to form bicarbonate and a second molecule of
ammonia [2]. Consequently, these reactions cause significant alkalinity due to release of large
quantities of ammonia. The increase in pH is directly associated with the development of
numerous health complications in humans which depend on colonization site by urease-
producing microorganisms and primarily include urinary and gastrointestinal tract infections.
Increase stomach acid secretion in response of H. pylori destroys colonization of mucosal
membrane and causes gastric ulcers [3]. Development of kidney stone, pyelonephritis, hepatic
coma etc are also associated with urease complication. Therefore, it is necessary to control
urease activity by urease inhibitors. A close structural likeness of thiosemicarbazide with
thiourea which is used as one of the standards in urease inhibitory assay draws considerable
attention of medicinal chemist for screening thiosemicarbazide for evaluating their potential as
urease inhibitors [4].
Thiosemicarbazide or hydrazine carbothioamide 1 (Figure-1) is derived from thiocarbamic acid
with hydrazine moiety. Its chemical behavior is same as semicarbazide which is its oxygen
counterpart, however, due to presence of thio group it has great chemical versatility and varying
behavior then semicarbazide [5].
O
S
S
H
N
NH2
R
N
N
R'
H2N
N
H
H
H
1
2
Figure-1: Structure of thiosemicarbazide 1 and acyl thiosemicarbazide 2 (3-27)
Thiosemicarbazide has a major role in the drug industry due to its wide range of pharmacological
activities such as antitubercular [6], herbicidal [7], antibacterial [8], antiviral [9], antinociceptive
[10], and antioxidant properties. Since our research group is continuously engaged in search of
urease inhibitors [11-16] and we have also previously reported benzophenone semicarbazide
5

(semicarbazone), istain and benzophenone thiosemicarbazide (thiosemicarbazone) derivatives as
a potent urease inhibitors (Figure-2) [17-20].
Newly Synthetic compound
Reported compounds
C2H5
S
N
O
NH
HO
NH
O
S
HN
S
N
NH
O2N
Br
N
N
N
H
H
NH
N
H
N-(2-ethylphenyl)-2-(5-nitro-
2-oxoindolin-3-
NO2
(E)-N-(3-bromophenyl)-2-((4-
hydroxyphenyl)(phenyl)methylene)hydrazine-1-
carbothioamide
ylidene)hydrazine-1-
carbothioamide
12
IC50 = 1.13 ± 0.00 µM
IC50 = 8.7 ± 0.6 µM
IC50 = 16.4 ± 0.8 µM
N
O
HO
NO2
S
S
HN
NH
N
NH
N
N
N NH
Me
O
H
H
S
NH
(E)-2-(1-(benzofuran-2-
yl)ethylidene)-N-
phenylhydrazine-1-
carbothioamide
H3C
CH3
(E)-2-((4-hydroxyphenyl)(phenyl)methylene)-N-(4-
nitrophenyl)hydrazine-1-carbothioamide
24
IC50 = 0.089 ± 0.24 µM
IC50 = 26.1 ± 0.2 µM
IC50 = 1.21 ± 0.00 µM
Figure-2: Rationale of current studies
The current study is an expansion of our previous studies based on optimization of new urease
inhibitors with enhanced efficacy. In this regard, we have synthesized a series of
nicotinic/isonicotinic thiosemicarbazide derivatives 3-27 since nicotinic/isonicotinic
thiosemicarbazide have itself medicinal importance [21-22] by reacting nicotinic/isonicotinic
hydrazides with various substituted phenyl isothiocyanates. To the best of our knowledge
compound 4, 10, 20, and 22 are new while remaining compounds are previously reported [23-
29].
6

2.
Results and Discussion
Chemistry
2.1
Nicotinic/isonicotinic hydrazides were reacted with various substituted phenyl isothiocyanates in
ethanol to get desired nicotinic/isonicotinic thiosemicarbazide derivatives (3-27) Scheme-1. The
reaction mixture containing nicotinic/isonicotinic hydrazides and phenyl isothiocyanates in
absolute ethanol was refluxed at 80 C for 30-50 minutes and monitored through TLC. After the
completion of reaction, it was filtered and the solid part was washed with hot hexane then
ethanol which later dried and collected Table-1. The synthetic compounds were characterized by
using various spectroscopic techniques such as EI-MS, 1H-NMR, and 13C-NMR.
O
O
NCS
H
H
NH2
Ethanol, 80 C
Reflux
N
N
R
N
R
N
+
H
H
R'
S
R'
R =
;
14-27
R =
;
3-13
N
N
Scheme-1: Synthesis of nicotinic and isonicotinic acylthiosemicrabazides
Spectral Characterization of of Most Active Compound 12
1
The H-NMR was recorded in DMSO on a 400 MHz instrument. Compound 12 with nicotinic
moiety possess total eleven protons. The molecule contain three NH, the NH-4 was the most
downfield signal appeared as sharp singlet at δ 10.83. The protons for NH-1 and -2 appeared as
singlet and resonated at δ 10.11. The characteristic proton H-2′ of nicotine ring was appeared at δ
9.10 as a singlet. H-4′ was resonated as doublet at δ 8.76 (J = 5.6 Hz) and showed coupling with
H-5′. H-5′ and H-6′ were appeared at δ 8.00 as doublet (J = 7.2 Hz). The H-2″ of aryl ring next to
nitro group appeared as an overlap and resonated at δ 8.43. H-4″ was appeared at δ 8.29 as
doublet (J = 7.2 Hz) showing coupling with H-5″. Two protons H-5″ and H-6″ were appeared at
δ 7.63 as an overlap (Figure-3).
7

8.00 (d, J5',6'/6',5' = 7.2 Hz)
H
5'
6'
= 3.6 Hz) H
H 8.00 (d, J5',6'/6',5' = 7.2 Hz)
8.76 (d, J4',5'
4'
1'
3'N
O
10.11 (s)
2
NH 10.11 (s)
3
2'
H
HN1
9.10 (s)
S
NH10.83 (s)
4
1''
7.63 (m)
7.63 (m)
H
H
6''
H8.43 (ovp)
2''
3''
5''
NO2
4''
H
8.29 (d, J4",5" = 8.0 Hz)
Figure-3: 1H-NMR signals of compound 12
Mass Spectrometry:
The molecular ion peak (M+) was not observed in the EI-MS spectra of compound 12. However,
a fragment with the loss of H2S was observed at m/z 283. The significant fragment at m/z 180
represented the formation of N-thioformylnicotinohydrazide ion which is also the base peak. The
fragment at m/z 137 was due to the formation of 3-nitroaniline ion. The key fragments are
represented in Figure-3.
Figure-4: Key fragmentation pattern (HREI-MS) of compound 12.
8

2.2 Urease inhibitory activity
Synthetic nicotinic/isonicotinic thiosemicarbazide derivatives 3-27 were screened for their urease
inhibitory activity according to literature protocol [30]. Good inhibitory activities were displayed
by almost all the synthetic compounds. Compounds 12 and 24 were highly active compounds
among the series showing IC50 values of 1.21 ± 0.02 and 1.13 ± 0.01 µM, respectively, as
compared to the standard thiourea (IC50 = 21.25 ± 0.13 µM) Table-1.
The structure-activity relationship was established by comparing the nicotinic, isonicotinic, and
substituted aryl part (R′). To develop a better understanding of structure-activity relationship the
synthetic derivatives 3-27 were divided into two categories, category A with nicotinic
thiosemicarbazide derivatives 3-13, while category
B
comprises of isonicotinic
thiosemicarbazide derivatives 14-27. In order to understand structure-activity relationship, the
molecules were divided in three part nicotin/isonicotin, thiosemicarbazide, and aryl part Figure-
3.
Figure-3: General structure of nicotinic/isonicotinic hydrazide
9

Table-1: In vitro urease inhibitory activity of synthetic derivatives 3-27
IC50 ± SEMa [M]
Compounds
R
R'
17.67 ± 0.14
3
15.28 ± 0.18
12.38 ± 0.16
4
5
29.73 ± 0.16
6
16.25 ± 0.13
8.95 ± 0.15
7
8
49.28 ± 0.19
9.35 ± 0.03
51.42 ± 0.13
1.13 ± 0.01
9
10
11
12
10

31.42 ± 0.12
18.64 ± 0.13
12.16 ± 0.14
19.74 ± 0.13
18.46 ± 0.17
19.42 ± 0.18
47.53 ± 0.12
9.53 ± 0.08
15.38 ± 0.12
11.58 ± 0.13
27.35 ± 0.13
13
14
15
16
17
18
19
20
21
22
23
11

1.21 ± 0.02
24
25
13.27 ± 0.16
16.23 ± 0.14
13.28 ± 0.17
26
27
Thiourea(std)
21.25 ± 0.13
standard inhibitor for urease inhibitory
(std)
SEMa is the standard error of the mean, NAb Not active, Thiourea
activity.
Limited structure-activity relationship (SAR) suggested that aryl part, its substitution and
nicotinic or isonicotic parts are involved in the urease inhibitory activities of these synthetic
molecules.
The unsubstituted aryl part containing compounds 3 (IC50 = 17.67 ± 0.14 µM) and 14 (IC50 =
18.64 ± 0.13 µM) of category A and B, respectively, showed comparable activity and were found
to have superior activity as compared to the standard thiourea (IC50 = 21 ± 0.11 µM) (Figure-4).
12

Figure-4: Comparison of SAR of compound 3 and 14
Compound 12 (IC50 = 1.13 ± 0.01 µM) of category A containing nicotinic moiety with nitro
group at meta position found to be most active compound of the series. However, its structurally
similar compound 23 (IC50 = 27.35 ± 0.13 µM) with isonicotinic part was found to have
moderate activity (Figure-9). It suggests that the nitro group on aryl part as well as position of
nitrogen in pyridine ring are playing a vital role in the urease inhibitory activity .
Figure-9: Comparison of SAR of compounds 12 and 23
The dimethyl substituted derivative 24 of category B having methyl groups meta to each other
showed 18-fold more activity (IC50 = 1.13 ± 0.01 µM) than the standard thiourea and is the
second most active compound in whole library. Similarly, another methyl substituted compound
with isonicotinic moiety, compound 25 (IC50 = 13.27 ± 0.16 µM) with dimethyl groups at ortho
13

positions also showed more activity as compared to the standard. However, its structurally closed
compound 13 (IC50 = 31.42 ± 0.12 µM) with nicotinic moiety showed less activity. This showed
derivatives of category B with methyl substituents are more suitable for urease inhibition
(Figure-10).
Figure-10: Comparison of SAR of compounds 13, 24, and 25
Amongst the bromo substituted derivatives of category A, compound 5 (IC50 = 12.38 ± 0.16 µM)
bearing bromo at meta position showed higher activity as compared to the standard. Compounds
4 (IC50 = 15.28 ± 0.18 µM) and 26 (IC50 = 29.73 ± 0.16 µM) with bromo group at ortho and
para, respectively, were found to have less activity which showed that bromo group at particular
position is contributing in the activity. If we compare isonicotinic derivative 15 (IC50 =12.16 ±
0.14 µM) with similar substitution as in compound 4, exhibited comparable activity (Figure-5).
14

Figure-5: Comparison of SAR of compound 4, 5, 6, and 15
In chloro substituted derivatives with nicotinic moiety, compound 7 (IC50 = 16.25 ± 0.13)
containing chloro group at meta position showed good inhibitory activity as compared to the
standard. However, isonicotinic compound 16 (IC50 = 19.74 ± 0.13 µM) with similar substitution
at aryl part showed slightly lowered activity. The dichloro substituted compounds 17 (IC50
=
18.46 ± 0.17 µM), and 18 (IC50 = 19.42 ± 0.18 µM) were found to have comparable activity to
compound 16 which showed that addition of another chloro group is seemingly influenced the
activity (Figure-6). However, if we compared the activity of compound 5 with compound 7 then
it appeared that decrease in bulkiness of substituent decreases activity.
Figure-6: Comparison of SAR of compounds 7, 16, 17, and 18
Compound 8 (IC50 = 8.95 ± 0.15 µM) with ortho substituted fluoro group was found to have
superior activity than the standard. Among difluoro substituted derivatives, compound 9 (IC50 =
15

49.28 ± 0.19 µM) with fluoro, meta to each other exhibited weak activity, however, compound
10 (IC50 = 9.35 ± 0.03 µM) with di ortho fluoro residues was found to have good activity which
showed that susbstitution at ortho position have a greater impact on the activity of compounds.
Compound 11 (IC50 = 51.42 ± 0.13 µM) bearing trifluoro methyl group showed less activity as
compared to other fluorine containing compounds (Figure-7), which indicates that direct
attachment of fluoro residue at phenyl part favors the urease inhibitory activity than one carbon
far fluoro groups.
Figure-7: Comparison of SAR of compounds 8-11
Similarly, in the fluoro substituted compounds of category B, compound 20 (IC50 = 9.53 ± 0.08
µM) bearing two fluoro groups at ortho positions showed comparable activity to the its
structurally similar analogue 10 of category A. Compound 19 (IC50 = 47.53 ± 0.12 µM) with
single fluoro group at ortho position exhibited lower activity than its counterpart 8 of category A.
Compound 21 (IC50 = 15.38 ± 0.12 µM) with trifluoromethyl group at ortho position exhibited
two folds good inhibitory activity as compared to compound 11 of category A. Compound 22
(IC50 = 11.58 ± 0.13 µM) with fluoro and methyl groups para to each other also showed a good
inhibitory activity (Figure-8).
16

Figure-8: Comparison of SAR of compounds 19-22
Alkoxy substituted isonicotnic derivative 26 and 27 also showed superior inhibitory activity than
the standard thiourea, however, compound 26 (IC50 = 13.28 ± 0.17 µM) with methoxy group was
found to be more active as compared to its ethoxy analog 27 (IC50 = 16.23 ± 0.14 µM) (Figure-
11) here increase in bulkiness of substituent decreases the activity.
Figure-11: Comparison of SAR of compounds 26 and 27
From the above discussion it can be concluded that the positions of substituents at aryl part as
well as position of nitrogen in pyridine rings are playing vital roles in urease inhibitory activity,
however, to verify these interpretations, in silico study was performed and discussed in the
following paragraphs.
17

2.3 Molecular Docking Studies
To explain the inhibition mechanism shown by the in vitro studies, molecular docking of
compound (3-27) was performed into the crystal structure of jack bean urease via MOE program.
For all compounds the most favorable docking conformations were observed inside the active
site with proper orientation. The active site consists of both the hydrophobic and hydrophilic
amino acids.
The hydrophobic part was composed of Ala440, Leu523, Ala636, and Met637 while hydrophilic
amino acids included Arg439, Glu493, Asp494, His593, and His519. The two Ni ions also
played a vital role by interacting with ligand and by linking the key amino acid and ligands. It
has been observed that almost all the conformations of all the ligands showed interactions with
key residues inside the pocket. The most promising docked conformation of each compound was
selected for further evaluation of binding mode. The docked poses were ranked by the scores
from the GBVI/WSA binding free energy calculation in the S field. Detailed docking results are
listed in (Table-2).
The analysis of the binding mode of the most favorite docked conformations revealed that all
ligands interacted well over the active site cavity through NH, N, O, (C=O), S, N---O and π
system which made the introduction of these groups in all the ligands to be a good choice.
Figure-12 showed the 3D interaction of some favorable inhibitors. This showed that ligands
occupy the active site residues, using hydrogen bonds, polar bonds, arene-cation and metal ion
interactions to engage tightly the backbone of the enzyme. The synthetic compounds are of two
types, nicotinic and isonicotinic derivatives and almost all the ligands showed similar pattern of
binding inside the active site.
The nicotinic derivatives (3-13) presented almost similar binding mode using the same active
groups with few exceptional cases (4, 8, 11, and 12). Without any substituent at phenyl moiety as
in case of compound 3 the pyridine part, C=O and NH are found active interacting through
hydrogen bonding with the active site residues Arg439 and Asp494. Arg439 also showed arene-
cation contact with pyridine moiety (Figure-12E). The dimethyl substituted derivative 4 is
observed with lesser interactions as compared to isonicotinic dimethyl derivatives by performing
18

two interactions with Arg609 and Ala636. The pyridine moiety presented an inert behavior in
almost all halogen and nitro substituted nicotinic derivatives which might be attributed to
negative inductive effect of these groups. Among the halogens substituted derivatives, compound
8 with ortho fluoro group showed better interactions than its homologue 19. Similarly,
compound 12 the nitro substituted derivative also presented better results than its counterpart 23.
The nitro (NO2) was observed very active by interacting with Ni ion and His492 as depicted in
(Figure-12F). An additional hydrogen bond was also observed between carbonyl oxygen of the
ligand and Arg439.
In isonicotinic derivatives (14-27) the active residues found to be involved in various types of
interactions which are NH, C=O, C=S, pyridine moiety and halogens. Without any substituent at
phenyl moiety as in case of compound 14, only the pyridine part is found active interacting
through hydrogen bonding with the active site residues Arg609 and Asp494 (Figure-12A).
Among the dimethyl substituted isonicotinic derivatives, (24, and 25) compound 24 showed
better interactions and docking score in accordance to the biological activity. (Figure-12B)
reflects that pyridine moiety in compound 24 perform arene-H bonding with His492 and
metal/ionic contact with Ni and the carbonyl oxygen (C=O) is bonded to Arg609 and His593.
Compound 26 and 27 bearing methoxy and ethoxy substituted ligands, respectively, were found
to be less interactive as compared to 24. These compounds showed almost similar binding mode
to non-substituted derivative 14. The pyridine moiety in most of the halogen substituted
isonicotinic derivative (15-18, 21, and 22) was forced to be inert resulting in mild binding modes
except compound 20. The inertness of pyridine moiety may be attributed to the electron
withdrawing inductive effect of halogens, however, in case of 20 the adjacent sitting (ortho) of
two fluorine groups might be responsible for contrast behavior. Compound 20 was found with
four polar interactions with the key residues, Arg439, Asp494, CME592 and Ala636 as revealed
from (Figure-12C). The docking mode of compounds 21 and 22 showed the inhibition of
catalytic activities of the enzyme almost in similar way by performing arene-H and hydrogen
bonding (Table-2). Like halogen derivatives the nitro (NO2) substituted compound 23 also
showed moderate inhibition mode as illustrated in (Figure-12D). Among the isonicotinic
derivatives compound 20 and 24 showed the best interaction mode in the active site which may
19

favor the presence of groups over the phenyl moiety with positive inductive effect to polarize the
compound for the enhanced inhibitory activity.
The docking results complemented well the experimental results based on the multiple
interactions of ligands with key residues of the jack bean urease enzyme. The docking pose of
the most active compounds, 12 and 24 inhibited the catalytic activities of the urease by binding
firmly through strong hydrogen bonding, hydrophobic, polar interactions with key residues and
metal/ion contact.
Table-2: Docking scores (S) and Interactions Detail of the compounds (3-27)
Comp. No.
Docking Score (S)
Interactions Detail
Ligand Receptor Interaction Distance E(kcal/mol)
-10.2695
C
N
N
O
9
3
OD2 ASP 494 (A) H-donor
NH2 ARG 609 (A) H-acceptor 3.11 -0.8
18 OD2 ASP 494 (A) H-donor 3.27 -1.2
14 NH2 ARG 439 (A) H-acceptor 3.29
6-ring NH1 ARG 439 (A) pi-cation 3.85
2.82
-1.7
14
3
-10.3367
-1.2
-2.6
-10.7294
-10.0247
-11.6831
S
S
20 CB CME 592 (A) H-acceptor 3.93 -0.4
20 NE2 HIS 593 (A) H-acceptor 2.59 4.7
NH2 ARG 609 (A) H-acceptor 2.97 -3.8
14 CB ALA 636 (A) H-acceptor 3.17 -0.8
14 NE2 HIS 593 (A) H-acceptor 3.27 -0.7
14 NH1 ARG 609 (A) H-acceptor 3.64 -1.7
14 NH2 ARG 609 (A) H-acceptor 3.32 -3.6
25
4
N
O
O
O
O
5
24
N
3
NI NI 901 (A) metal
14 NH2 ARG 439 (A) H-acceptor 1.60 -1.7
3.54 -0.8
14 NH2 ARG 439 (A) H-acceptor 3.63 -2.1
21 CA ALA 636 (A) H-acceptor 4.46 -0.7
2.76 -0.8
18 NH2 ARG 439 (A) H-acceptor 4.06 -0.8
BR 31 GLY 550 (A) H-donor 3.42 -1.1
21 OH CME 592 (A) H-acceptor 3.49 -1.1
6-ring NE2 HIS 593 (A) π-H 3.74 -0.9
18 OH CME 592 (A) H-acceptor 3.56
12 ALA 636 (A) H-donor 2.00
2.67
-0.6
-10.5660
O
26
6-ring OH CME 592 (A) pi-H
-11.0744
-11.1034
-10.0749
O
S
27
15
4
BR 31 OH CME 592 (A) H-donor
S
-9.3313
-10.9532
O
6
5
S
S
-10.3628
-10.4385
-1.0
-1.1
16
7
N
O
S
21 NE2 HIS 593 (A) H-acceptor 4.32 -0.6
6-ring CB ALA 440 (A) pi-H
3.94 -1.1
-10.3968
-9.9946
CL 31 OD1 ASP 494 (A) H-donor
6-ring OH CME 592 (A) pi-H
3.34 -0.6
3.46 -0.6
17
18
O
S
17 CA ALA 636 (A) H-acceptor 3.53
18 OH CME 592 (A) H-acceptor 3.26
12 ALA 636 (A) H-donor 3.22
22 OD2 ASP 494 (A) H-donor
17 NH2 ARG 439 (A) H-acceptor 3.23 -1.1
-0.8
-1.4
-2.1
-9.5240
-11.5240
N
O
19
8
C
O
2.99 -1.0
-9.0614
-10.8773
C
C
O
22 OD2 ASP 494 (A) H-donor
OD2 ASP 494 (A) H-donor
14 SG CME 592 (A) H-donor
2.84 -1.5
2.95 -1.6
3.94 -0.5
9
20
4
20

N
S
N
S
S
15
20 NH2 ARG 439 (A) H-acceptor 3.91 -1.4
12 ALA 636 (A) H-donor 2.57 -3.5
21 SG CME 592 (A) H-donor 4.11 -0.2
21 NE2 HIS 593 (A) H-acceptor 4.03 -0.9
17 CA ALA 636 (A) H-acceptor 3.52 -0.9
6-ring CD ARG 439 (A) pi-H 3.94 -0.9
O
ALA 636 (A) H-donor
2.94
-5.1
-11.2481
-11.1741
O
10
21
O
-8.2072
-11.4206
11
22
O
17 NH2 ARG 439 (A) H-acceptor 2.56
6-ring OH CME 592 (A) pi-H 3.65 -0.6
14 ALA 636 (A) H-donor 2.94 -4.2
6.6
-10.9724
-12.8545
N
O
23
12
S
O
O
18 NH2 ARG 439 (A) H-acceptor 4.11 -1.2
14 NH2 ARG 439 (A) H-acceptor 3.53 -2.3
33 NE2 HIS 492 (A) H-acceptor 2.88 -2.2
O
O
32 NI NI 902 (A) metal
33 NI NI 902 (A) metal
2.31
2.60
-1.6
-0.7
Figure-12: Three dimensional interaction of compounds 14, 24, 20, 23, 3, and 12 with active site
residues of urease. Ligands are shown in Green color (stick n ball mode), key residues of the
active site are shown in stick mode, nickle ions are shown as cyan dots, hydrogen bonding, and
other interaction is shown in dark color dotted lines. (A) Hydrogen bonding and polar interaction
of compound 14 with the active site residues Arg609 and Asp494, respectively. (B) 3D
interactions network of compound 24 through C=O (Red), and pyridine ring with the key
residues and Ni ion (C) Hydrogen bonding of C=S (Yellow), NH (Blue), and C=O (Red) of
compound 20 with Asp494, CME592, Arg439, and Ala636 (D) Hydrogen bonding and polar
interaction of NH (Blue) and C=S (Yellow) of compound 23 with Ala636 and Arg439. (E)
Hydrogen bonding and π-interaction of NH (Blue), C=O (Red), and pyridine ring of compound 3
with Asp494 and Arg439. (F) Hydrogen acceptor interactions and metal ion contact of C=O
(Red) and NO2 of compound 12 with Arg439, His492 and Ni ion.
21

3.
Conclusion
This study deals with the synthesis of nicotinic/isonicotinic thiosemicarbazide derivatives and
their urease inhibitory potential (3-27). Amongst synthetic compounds nineteen compounds
exhibited good activity. However, compounds 12 (IC50 = 1.13 ± 0.01 µM) and 24 (IC50 = 1.21 ±
0.02 µM) were found to be most active and showed almost 19-fold more activity than the
standard thiourea (IC50 = 21.25 ± 0.13 µM). Structure-activity relationship revealed that the
position of nitrogen in pyridine ring also contributes towards the activity of these compounds and
the compounds with nicotinic part exhibited more activity. Similarly, different positions of
substituents at aryl part resulted in better urease inhibitory activity. Molecular docking studies
further validated these results which showed efficient binding interaction between the active
compound and urease enzyme proteins. Decisively, many new lead molecules are identified as
urease inhibitors and further fine tuning of these molecules may result better inhibitors.
4. Experimental
4.1 Material and Methods
All reagents and solvents were purchased from Sigma-Aldrich USA and of analytical grade and
were as used as received. Pre-coated silica gel, GF-254 (Merck, Germany) was used for thin
layer chromatography. Spots were visualized under ultraviolet light at 254 and 366 nm. Mass
1
13
spectra were recorded on a Finnigan MAT-311A (Germany) mass spectrometer. The H-, C-
NMR were carried out on Avance Bruker AM spectrometers, operating at 300, 400 and 500
MHz. The chemical shift values are presented in ppm (δ), relative to tetramethylsilane (TMS) as
an internal standard and the coupling constant (J) are in Hz.
4.2 Urease inhibitory assay
Urease inhibitory activity was performed by incubating 5 μL of test compound (250 μM) with
°
urease solution (1 U/well) 25 μL for 15 min at 30 C. After that 100 mM concentration of urea
(substrate) 55 μL was added and then incubation of plate was carried out for second time for 10
°
min at 30 C. On completion of incubation period, 70 μL of alkali reagents (0.5% w/v sodium
hydroxide and 0.1% sodium and 45 μL of phenol (1% w/v phenol and 0.005% w/v sodium
°
nitroprusside hypochlorite) was added. Plates were incubated third time for 50 min at 30 C.
22

Urease activity was calculated with the rate of production of ammonia constantly following the
method of Weatherburn [30], and alteration in absorbance was examined at 630 nm on ELISA
plate reader (Spectra Max M2, Molecular Devices, CA, USA). Thiourea was used as a standard
control [30].
4.3 Docking Methodology
Molecular docking study was performed via MOE (Molecular Operating Environment) [31] to
predict the binding mode of the synthetic derivatives in the active site of jack bean urease
enzyme (PDBID 4h9m). The three dimensional structures of the synthetic compounds were built
by using the builder tool of the MOE software. The generated compounds were 3D protonated
and energy minimized using the default parameters of the MOE (gradient: 0.05, Force Field:
MMFF94X). All the compounds were then saved in mdb (Molecular Data Base) file for further
evaluation in molecular docking.
Three D structure of the target protein jack bean urease enzyme was retrieved from the protein
databank. The retrieved protein was opened in MOE, water molecules were removed, and 3D
protonation was carried out followed by energy minimization for the stability of the protein by
using the default parameters of MOE. For docking studies the default parameters of MOE were
used i.e., Placement: Triangle Matcher, Rescoring 1: London dG, Refinement: Force-field,
Rescoring 2: GBVI/WSA. For each ligand 10 conformations were allowed to be formed and the
top ranked conformations on the basis of docking score were selected for further analysis
4.4 General Experimental Procedure for the Synthesis of Compounds 3-27
Nicotinic/isonicotinic hydrazides (1 mmol) were reacted with various substituted phenyl
isothiocyanates (1 mmol) in absolute ethanol and the reaction mixture was refluxed under at 80
C for 30-50 min. The reaction progress was monitored via TLC. After the completion of
reaction, the precipitates formed were filtered, washed with hot hexane followed by ethanol, it
was later dried and collected. The structures of compounds (3-27) were characterized by using
various spectroscopic techniques such as EI-MS, 1H-NMR, and 13C-NMR.
23

1-(Nicotinoyl)-4-(phenyl)thiosemicarbazide (3)
º
º
Solid; Yield: 74%; M.p. 179-181 C (lit. 181-183 C) [25]; 1H-NMR: (400 MHz, DMSO-d6): δH
10.73 (s, 1H, NH-4), 9.83 (s, 1H, NH-2), 9.75 (s, 1H, NH-1), 9.09 (d, 1H, J2′/4′= 4.0 Hz, H-2′),
8.74 (dd, 1H, J4′/2′ = J4′/5’ = 1.6 Hz, H-4′), 8.27 (m, 1H, H-6’), 7.55 (m, 1H, H-5′), 7.41 (ovp s, 2H,
H-2′’, H-6′’), 7.34 (t, 2H, J3′’/(2′’,4’’) = J5′’/(4′’,6’’) = 7.6 Hz, H-3′’, H-5′’), 7.17 (t, 1H, J4′’/(3′’,5’’) = 7.2
Hz, H-4′’); 13C-NMR (75 MHz, DMSO-d6): 181.3, 164.8, 152.5, 148.9, 139.3, 135.5, 128.4,
128.2, 126.4, 125.3, 123.4; EI-MS: m/z (rel. abund. %), 238.2 [M-H2S]+ (66.3), 135 (100), 106.1
(69.3), 77 (69.9), 51 (5.1).
4-(2-Bromophenyl)-1-(nicotinoyl)thiosemicarbazide (4)
º
1
Solid; Yield: 67%; M.p. 193-195 C; H-NMR: (400 MHz, DMSO-d6): δH 10.79 (s, 1H, NH-4),
9.87 (s, 1H, NH-2), 9.69 (s, 1H, NH-1), 9.09 (s, 1H, H-2′), 8.74 (t, 1H, J4′/(2′,5’)= 3.6 Hz, H-4′),
8.28 (d, 1H, J6′/5′= 7.6 Hz, H-6’), 7.65 (d, 1H, J3′’/4’′ = 8.0 Hz, H-3′’), 7.55 (m, 1H, H-5′), 7.37 (ovp
13
s, 2H, H-4′’,H-5′’), 7.21 (m, 1H, H-6′’); C-NMR (75 MHz, DMSO-d6): 181.3, 164.6, 148.5,
148.3, 138.3, 135.1, 130.5, 129.2, 128.2, 126.3, 125.3; EI-MS: m/z (rel. abund. %), 316 [M-
H2S]+(13.2), 215 (100), 137 (18.3), 134 (6.0), 106 (76.3), 78 (56.7), 51 (17.5).
4-(3-Bromophenyl)-1-(nicotinoyl)thiosemicarbazide (5)
º
º
Solid; Yield: 79%; M.p. 238-239 C (lit. 239-240 C) [26]; 1H-NMR: (400 MHz, DMSO-d6): δH
10.77 (s, 1H, NH-4), 9.94 (s, 1H, NH-2), 9.88 (s, 1H, NH-1), 9.09 (s, 1H, H-2′), 8.75 (dd, 1H,
J4′/2′ = J4′/5’ = 1.3 Hz, H-4′), 8.25 (d, 1H, J6′/5′= 8.0 Hz, H-6’), 7.70 (overlapping multiplet, 2H, H-
5′, H-2′’), 7.47 (d, 1H, J6′’/5′’= 8.0 Hz, H-6’’), 7.35 (t, 1H, J5′’/(4′’,6’’) = 8.0 Hz, H-5′’), 7.27 (d, 1H,
J4′’/5′’= 7.6 Hz , H-4′’); 13C NMR (75 MHz, DMSO-d6):  181.3, 164.6, 148.5, 148.3, 139.1,
135.1, 13.5, 130.3, 127.4, 125.5, 125.3, 125.3, 123.2; EI-MS: m/z (rel. abund. %), 316 [M-
H2S]+(9.9), 215 (100), 137 (18.0), 134 (42), 106 (81.5), 92 (7.5), 78 (67.5), 51 (21.3).
4-(4-Bromophenyl)-1-(nicotinoyl)thiosemicarbazide (6)
º
º
Solid; Yield: 80%; M.p. 196-198 C (lit. 192-193 C) [26]; 1H-NMR: (400 MHz, DMSO-d6): δH
10.76 (s, 1H, NH-4), 9.87 (s, 2H, NH-1, NH-2), 9.09 (s, 1H, H-2′), 8.75 (dd, 1H, J4′/2′ = J4′/5’= 1.2
Hz, H-4′), 8.27 (d, 1H, J6′/5′= 8.0 Hz, H-6’), 7.56 (m, 3H, H-5′, H-2′’, H-6′’), 7.08 (ovp d, 2H, H-
24

3′’, H-6′’); 13C-NMR (75 MHz, DMSO-d6):  181.3, 164.6, 148.5, 148.3, 137.3, 135.1, 131.7,
130.5, 125.3, 122.5; EI-MS: m/z (rel. abund. %), 350.0 [M]+ (2.1), 215.0 (100), 318.1 (26.1),
316.2 (24.2), 373.0 (62.0),171.0 (69.7), 106.1 (52.6), 92.0 (25.7), 78.0 (59.2).
4-(3-Chlorophenyl)-1-(nicotinoyl)thiosemicarbazide (7)
Solid; Yield: 75%; M.p. 176-178 ºC (lit. 179-197.5 ºC) [24]; 1H-NMR: (400 MHz, DMSO-d6): δH
10.77 (s, 1H, NH-4), 9.93 (s, 1H, NH-2), 9.88 (s, 1H, NH-1), 9.09 (s, 1H, H-2′), 8.75 (dd, 1H,
J4′/2′ = J4′/5’ = 1.6 Hz, H-4′), 8.27 (d, 1H, J6′/5′= 8.0 Hz, H-6’), 7.56 (m, 2H, H-5′, H-2′’), 4.45 (d,
1H, J6′’/5′’= 8.0 Hz, H-6’’), 7.37 (t, 1H, J5′’/(4′’,6’’) = 8.0 Hz, H-5′’), 7.22 (d, 1H, J4′’/5′’= 7.6 Hz , H-
13
4′’); C-NMR (75 MHz, DMSO-d6):  181.3, 164.6, 148.5, 148.3, 138.3, 135.2, 134.4, 130.5,
130.6, 128.5, 126.7, 125.3, 124.4; EI-MS: m/z (rel. abund. %), 272 [M-H2S]+(21.5), 169.0 (100),
137.1 (28.2), 111.0 (44.6), 106.1 (77.1), 78.0 (60.1), 51.0 (25.2).
4-(2-Fluorophenyl)-1-(nicotinoyl)thiosemicarbazide (8)
º
1
Solid; Yield: 77%; M.p. 162-164 C; H-NMR: (400 MHz, DMSO-d6): δH 10.80 (s, 1H, NH-4),
9.92 (s, 1H, NH-2), 9.65 (s, 1H, NH-1), 9.08 (s, 1H, H-2′), 8.74 (dd, 1H, J4′/2′ = J4′/5’ = 3.6 Hz, H-
4′), 8.27 (d, 1H, J6′/5′= 8.0 Hz, H-6’), 7.55 (m, 1H, H-5′), 7.55 (m, 2H, H-3′’,H-4′’, H-5′’, H-6′’);
13C-NMR (75 MHz, DMSO-d6):  181.3, 164.6, 163.4, 148.3, 148.1, 135.5, 130.5, 130.2, 129.2,
128.1, 125.3, 120.1, 115.6; EI-MS: m/z (rel. abund. %), 290.0 [M]+ (3.8), 153.0 (100), 256.2
(13.0), 137.1 (37.3), 111.1 (23.1), 106.1 (93.1), 95.1 (44.9), 78.0 (73.2), 51.0 (23.3).
4-(2,4-Difluorophenyl)-1-(nicotinoyl)thiosemicarbazide (9)
º
1
Solid; Yield: 81% ;m.p. 138-140 C; H-NMR: (400 MHz, DMSO-d6): δH 10.81 (s, 1H, NH-4),
9.97 (s, 1H, NH-2), 9.61 (s, 1H, NH-1), 9.08 (s, 1H, H-2′), 8.74 (d, 1H, J4′/5’= 3.6 Hz, H-4′), 8.27
(d, 1H, J6′/5′= 7.6 Hz, H-6’), 7.55 (m, 1H, H-5′), 7.30 (ovp t, 2H, H-5′’, H-6′’), 7.08 (t, 1H, J3′’/(2′’F,
4’’F) = 7.6 Hz, H-3′’); 13C-NMR (75 MHz, DMSO-d6):  181.3, 169.1, 164.6, 160.5, 148.5, 148.3,
135.3, 130.5, 129.3, 125.1, 115.5, 111.2, 105.0; EI-MS: m/z (rel. abund. %), 274 [M-H2S]+(9.4),
171.1 (100), 137.1 (28.7), 113.0 (24.4), 106.1 (87.1), 78.0 (69.1), 51.0 (24.2).
25

4-(2,6-Difluorophenyl)-1-(nicotinoyl)thiosemicarbazide (10)
º
1
Solid; Yield: 82%; M.p. 202-204 C; H-NMR: (400 MHz, DMSO-d6): δH 10.82 (s, 1H, NH-4),
10.09 (s, 1H, NH-2), 9.47 (s, 1H, NH-1), 9.09 (s, 1H, H-2′), 8.78 (d, 1H, J4′/5’= 1.2 Hz, H-4′),
13
8.25 (d, 1H, J6′/5′= 7.6 Hz, H-6’), 7.62 (m, 1H, H-5′), 7.62 (m, 1H, H-3′’, H-4′’,H-5′’); C-NMR
(75 MHz, DMSO-d6):  181.3, 169.1, 164.6, 148.5, 148.3, 135.1, 130.5, 125.7, 125.3, 113.2,
111.2; EI-MS: m/z (rel. abund. %), 274 [M-H2S]+(1.7), 106.1 (100), 290.2 (113.8), 179.1 (56.6),
137.2 (10.1), 119.1 (33.4), 78.1 (89.4), 51.0 (24.0).
1-(Nicotinoyl)-4-(3-trifluoromethylphenyl)thiosemicarbazide (11)
º
1
Solid; Yield: 78%; M.p. 159-160 C; H-NMR: (400 MHz, DMSO-d6): δH 10.80 (s, 1H, NH-4),
10.00 (s, 2H, NH-1, NH-2), 9.10 (s, 1H, H-2′), 8.76 (dd, 1H, J4′/2′ = J4′/5’ = 1.2 Hz, H-4′), 8.28 (d,
1H, J6′/5′= 8.0 Hz, H-6’), 7.83 (ovp d, 2H, H-5′, H-2′’), 7.58 (m, 3H, H-4′’, H-5′’, H-6′’); 13C-NMR
(75 MHz, DMSO-d6):  181.3, 164.6, 148.5, 148.3, 137.2, 131.1, 130.5, 130.4, 129.6, 125.1,
124.3, 121.1; EI-MS: m/z (rel. abund. %), 306 [M-H2S]+(19.4), 203.1 (100), 145.1 (47.6), 137.1
(14.0), 106.1 (51.5), 78.1 (43.1), 51.0 (13.1).
1-(Nicotinoyl)-4-(3-nitrophenyl)thiosemicarbazide (12)
º
1
Solid; Yield: 78%; M.p. 201-203 C; H-NMR: (400 MHz, DMSO-d6): δH 10.83 (s, 1H, NH-4),
10.11 (s, 2H, NH-1, NH-2),9.10 (s, 1H, H-2′), 8.76 (d, 1H, J4′/5′= 3.6 Hz, H-4′), 8.43 (ovp s,
1H,H-2′’), 8.29 (d, 1H, J4′’/5’’= 8.0 Hz, H-4′’), 8.00 (d, 2H, J5′/6′ = J6′/5′ = 7.2 Hz, H-5′, H-6′), 7.63
(m, 2H, H-5′’,H-6′’); 13C-NMR (75 MHz, DMSO-d6):  181.3, 164.6, 148.5, 148.3, 138.2, 135.1,
132.2, 130.3, 129.5, 125.1, 119.3, 119.0; EI-MS: m/z (rel. abund. %), 283.2 [M+-H2S]+ (2.8),
180.0 (100), 134.0 (76.2), 106.2 (82.3), 92.1 (51.1),90.1 (51.0), 78.0 (74.7).
4-(2,6-Dimethylphenyl)-1-(nicotinoyl)thiosemicarbazide (13)
º
1
Solid; Yield: 74%; M.p. 139-141 C; H-NMR: (400 MHz, DMSO-d6): δH 10.74 (s, 1H, NH-4),
9.62 (s, 1H, NH-2), 9.43 (s, 1H, NH-1), 9.08 (d, 1H, J2′/4′= 1.6 Hz, H-2′), 8.73 (dd, 1H, 1H, J4′/2′
=
J4′/5’ = 1.6 Hz, H-4′), 8.27 (d, 1H, J6′/5′= 8.0 Hz, H-6′),7.54 (m, 1H, H-5′), 7.63 (m, 3H, H-3′’,H-4′’
,H-5′’), 2.15 (s, 6H, CH3-2C″, CH3-6C″); 13C-NMR (75 MHz, DMSO-d6):  181.3, 164.4, 148.3,
26

148.1, 135.2, 135.1, 130.1, 127.0, 127.0, 125.1, 18.0; EI-MS: m/z (rel. abund. %), 266.2 [M-
H2S]+(65.3), 163.1 (100), 145.1 (53), 137.0 (27.1), 130.1 (58.2), 106 (90); 78.0 (74.2), 51.0 (5).
1-(isonicotinoyl)-4-(phenyl)thiosemicarbazide (14)
º
º
Solid; Yield: 71%; M.p. 200-202 C (lit. 200-202 C) [27]; 1H-NMR: (400 MHz, DMSO-d6): δH
10.83 (s, 1H, NH-4), 9.83 (s, 1H, NH-2),9.77 (s, 1H, NH-1), 8.76 (d, 2H, J3′/2′ = J5′/6′ = 5.6 Hz, H-
3′, H-5′), 7.84 (d, 2H, J2′/3′ = J6′/5′ = 5.6 Hz, H-2′, H-6′), 7.41 (ovp s, 2H, H-2′’,H-6″), 7.34 ( t, 2H,
13
J3′’/2’’,4’’ = J5′’/4′’,6’’=7.6 Hz, H-3′’, H-5″), 7.17 (t, 1H, J4′’/3′’ = J4′’/5′’ =7.2 Hz, H-4); C-NMR (75
MHz, DMSO-d6):  181.3, 164.6, 149.5, 140.6, 138.3, 129.2, 128.2, 126.3, 121.5; EI-MS: m/z
(rel. abund. %), 271.9 (M+, 4.3), 237.9 (100), 178.9 (12.9), 134.9 (87.2), 119.0 (30.4), 106.0
(60.8), 93.0 (25.9), 78.0 (73.2), 77.0 (64.6).
4-(2-Bromophenyl)-1-(isonicotinoyl)thiosemicarbazide (15)
º
1
Solid; Yield: 68%; M.p. 203-205 C; H-NMR: (300 MHz, DMSO-d6): δH 10.88 (s, 1H, NH-4),
9.90 (s, 1H, NH-2), 9.69 (s, 1H, NH-1), 8.76 (d, 2H, J3′/2′ = J5′/6′ = 6.0 Hz, H-3′, H-5′), 7.85 (d,
2H, J2′/3′ = J6′/5′ = 5.4 Hz, H-2′, H-6′), 7.66 (d, 1H, J3′’/4′’ = 8.1 Hz, H-3′’), 7.38 (ovp s, 2H,H-5′’, H-
13
6′’), 7.22 (m, 1H, H-4′’); C-NMR (75 MHz, DMSO-d6):  181.3, 164.6, 149.5, 140.6, 137.4,
132.4, 131.7, 130.3, 128.2, 122.2, 121.5; EI-MS: m/z (rel. abund. %), 315.9 [M-H2S]+ (5.7),
214.9 (100), 134.0 (38.7), 106.0 (61.0), 93.0 (25.9), 78.0 (46.2).
4-(3-Chlorophenyl)-1-(isonicotinoyl)thiosemicarbazide (16)
Solid; Yield: 77%; M.p. 194-196 ºC (lit. 290-292 ºC); [27]; 1H-NMR: (300 MHz, DMSO-d6): δH
10.87 (s, 1H, NH-4), 9.97 (s, 1H, NH-2), 9.90 (s, 1H, NH-1), 8.77 (d, 2H, J3′/2′ = J5′/6′ = 6.0 Hz,
H-3′, H-5′), 7.84 (d, 2H, J2′/3′ = J6′/5′ = 5.6 Hz, H-2′, H-6′), 7.58 (ovp s, 1H, H-2″), 7.45 (d, 1H,
J6′’/5′’ = 8.4 Hz, H-6′’), 7.37 ( t, 1H, J5′’/4’’,6’’ =8.0 Hz, H-5’’), 7.22 (d, 1H, J4′’/5′’ = 7.6 Hz, H-4″);
13C-NMR (75 MHz, DMSO-d6):  181.3, 164.6, 149.5, 140.6, 138.5 134.4, 13.2, 128.1, 126.7,
124.4, 121.5; EI-MS: m/z (rel. abund. %), 306.0 (M+, 1.6), 169.0 (100), 272.1 (55.3),179.1
(49.4), 137.1 (24.0), 127.1 (59.1), 106.1 (47.0), 78.1 (51.2); 51.0 (23.6).
27

4-(2,3-Dichlorophenyl)-1-(isonicotinoyl)thiosemicarbazide (17)
Solid; Yield: 82%; m.p. 207-208 ºC (lit. 200 ºC) [28]; 1H-NMR: (400 MHz, DMSO-d6): δH 10.92
(s, 1H, NH-4), 10.02 (s, 1H, NH-2), 9.81 (s, 1H, NH-1), 8.76 (d, 2H, J3′/2′ = J5′/6′ = 6.0 Hz, H-3′,
H-5′), 7.84 (ovp d, 2H, J2′/3′ = J6′/5′ = 3.2 Hz, H-2′, H-6′), 7.56 (d, 1H, J4′’/5′’ = 8.4 Hz, H-4′’), 7.38
(m, 2H, H-5′’, H-6′’); 13C-NMR (75 MHz, DMSO-d6):  181.3, 164.6, 149.5, 138.3, 137.1, 132.5,
132.2, 130.2, 128.3, 121.5; EI-MS: m/z (rel. abund. %), 306.2 [M]+ (31.6), 203.1 (100), 271.2
(17.7), 161.1 (32.1), 137.1 (28.2), 106.1 (51.8), 78.1 (47.9), 51.0 (23.9).
4-(2,5-Dichlorophenyl)-1-(isonicotinoyl)thiosemicarbazide (18)
º
1
Solid; Yield: 79%; M.p. 211-213 C; H-NMR: (400 MHz, DMSO-d6): δH 10.93 (s, 1H, NH-4),
10.07(s, 1H, NH-2), 9.75 (s, 1H, NH-1), 8.77 (d, 2H, J3′/2′ = J5′/6′ = 6.0 Hz, H-3′, H-5′), 7.84 (d,
2H, J2′/3′ = J6′/5′ = 4.0 Hz, H-2′, H-6′), 7.55 (d, 1H, J3′’/4′’ = 8.8 Hz, H-3″), 7.42 (s, 1H, H-6″),7.37
13
(d, J4′’/3′’ = 7.6 Hz, H-4″); C-NMR (75 MHz, DMSO-d6):  181.3, 164.6, 149.5, 140.6, 137.1,
132.5, 128.4, 128.1, 126.4, 121.5;EI-MS: m/z (rel. abund. %), 306.2[M-H2S]+(26.7), 203.0 (100),
179.1 (15.5), 161.0 (22.9), 137.0 (20.3), 106.0 (42.5), 78.0 (4552), 51.0 (17.1).
4-(2-Fluorophenyl)-1-(isonicotinoyl)thiosemicarbazide (19)
º
º
Solid; Yield: 76%; M.p. 198-200 C (lit. 202-204 C) [30]; 1H-NMR: (400 MHz, DMSO-d6): δH
10.90 (s, 1H, NH-4), 9.95 (s, 1H, NH-2), 9.67 (s, 1H, NH-1), 8.76 (d, 2H, J3′/2′ = J5′/6′ = 6.0 Hz,
H-3′, H-5′), 7.84 (d, 2H, J2′/3′ = J6′/5′ = 4.8 Hz, H-2′, H-6′), 7.29, H-3′’, H-4′’, H-5′’,H-6′’); 13C NMR
(75 MHz, DMSO-d6):  181.3, 164.6, 163.4, 149.5, 140.6, 130.2, 129.2, 128.3, 121.5, 120.1,
115.6;EI-MS: m/z (rel. abund. %), 290.2 (M+, 3.2), 153.0 (100), 256.0 (90.2), 179.1 (32.3), 106.0
(64.0), 95.0 (26.5),90.1 (51.0), 78.0 (10.0),50.9 (24.4).
4-(2,6-Difluorophenyl)-1-(isonicotinoyl)thiosemicarbazide (20)
º
1
Solid; Yield: 78%; M.p. 230-232 C; H-NMR: (300 MHz, DMSO-d6): δH 10.96 (s, 1H, NH-4),
10.12 (s, 1H, NH-2), 9.48 (s, 1H, NH-1), 8.76 (d, 2H, J3′/2′ = J5′/6′ = 1.6 Hz, H-3′, H-5′), 7.85 (d,
2H, J2′/3′ = J6′/5′ = 4.4 Hz, H-2′, H-6′), 7.39 (m, 1H, H-4″), 7.15 (t, 2H, J3′’(2’F,4′) = J5′’(6’F,4′) = 8.0
Hz, H-3′’, H-5′’); 13C-NMR (75 MHz, DMSO-d6):  181.3, 169.1, 164.6, 149.5, 125.7, 121.5,
28

113.2, 111.2; EI-MS: m/z (rel. abund. %), 308.2 (M+, 2.7), 274.2 (100), 179.1 (37.5), 171.1
(43.5), 129.1 (41.8), 106.1 (24.7), 78.1 (55.8) ), 51.0 (19.0).
1-(Isonicotinoyl)-4-(2-trifluoromethylphenyl)thiosemicarbazide (21)
Solid; Yield: 75%; M.p. 218-220 ºC (lit. 189 ºC) [30]; 1H-NMR: (400 MHz, DMSO-d6): δH 10.86
(s, 1H, NH-4), 9.91 (s, 1H, NH-2), 9.66 (s, 1H, NH-1), 8.75(d, 2H, J3′/2′ = J5′/6′ = 5.2 Hz, H-3′, H-
13
5′), 7.83 (ovp d, 2H, H-2′, H-6′), 7.71 (m, 2H, H-3′’,H-4′’), 7.48(ovp m, 2H, H-5′’, H-6′’); C-
NMR (75 MHz, DMSO-d6):  181.3, 164.4, 149.3, 140.2, 132.1, 132.0, 126.2, 125.4, 125.2,
122.2, 121.1, 119.3; EI-MS: m/z (rel. abund. %), 340.0 (M+, 6.9), 202.9 (100), 306.0 (85.1),145.0
(37.9), 106.0 (60.4), 93.0 (25.9), 78.0 (58.5).
4-(5-Fluor-2-methylphenyl)-1-(isonicotinoyl)thiosemicarbazide (22)
º
1
Solid; Yield: 72%; M.p. 202-204 C; H-NMR: (400 MHz, DMSO-d6): δH 10.85 (s, 1H, NH-4),
9.83 (s, 1H, NH-2), 9.63 (s, 1H, NH-1), 8.76 (d, 2H, J3′/2′ = J5′/6′ = 6.0 Hz, H-3′, H-5′), 7.83 (d,
2H, J2′/3′ = J6′/5′ = 5.6 Hz, H-2′, H-6′), 7.26 (t, 1H, J3′’/2’’F,4’’=7.6 Hz, H-3″), 7.04 (m, 2H, H-4′’,H-
13
6′’); C-NMR (75 MHz, DMSO-d6):  181.3, 164.2, 160.0, 149.3, 140.2, 137.2, 132.1, 130.3,
121.3, 114.1, 111.0, 17.1; EI-MS: m/z (rel. abund. %), 270.2 [M-H2S]+ (83.7), 167.3 (100), 149.1
(52.6), 135.0 (38.6), 124.1 (16.1), 106.1 (39.0), 78.1 (53.7), 77.0 (3.8).
1-(Isonicotinoyl)-4-(3-nitrophenyl)thiosemicarbazide (23)
Solid; Yield: 68%; M.p. 200-202 ˚C;.1H-NMR: (400 MHz, DMSO-d6): δH 10.94 (s, 1H, NH-4),
10.14 (s, 1H, NH-1, NH-2), 8.79 (d, 2H, J3′/2′ = J5′/6′ = 5.6 Hz, H-3′, H-5′), 8.42 (s, 1H, H-2′’),
8.01 (t, 2H, J4′’/(3’’, 5′’) = J6′’/(2’’, 5′’),7.6 Hz, H-4′’, H-6′’), 7.86 (d, 2H, J2′/3′ = J6′/5′ = 5.2 Hz, H-2′, H-
6′), 7.63 (t, 1H, J5′’/4’’,6’’ =8.4 Hz, H-5’’); 13C-NMR (75 MHz, DMSO-d6):  181.3, 164.2, 149.1,
148.0, 140.2, 138.2, 132.2, 129.3, 121.1, 119.3, 119.0; EI-MS: m/z (rel. abund. %), 283.2 [M-
H2S]+ (13.8), 180.1 (100), 137.1 (35.9), 134.0 (67.8), 106 (65.2), 90.0 (22), 78.0 (60.2), 51.0
(3.3).
4-(3,5-Dimethylphenyl)-1-(isonicotinoyl)thiosemicarbazide (24)
º
1
Solid; Yield: 70%; M.p. 210-212 C; H-NMR: (300 MHz, DMSO-d6): δH 10.79 (s, 1H, NH-4),
9.69 (s, 2H, NH-1, NH-2), 8.76 (d, 2H, J3′/2′ = J5′/6′ = 5.4 Hz, H-3′, H-5′), 7.84 (d, 2H, J2′/3′ = J6′/5′
29