Synthesis and in vitro urease inhibitory activity of benzohydrazide derivatives, in silico and kinetic studies

Article abstract of DOI:10.1016/j.bioorg.2018.09.036

Benzohydrazide derivatives 1–43 were synthesized via “one-pot” reaction and structural characterization of these synthetic derivatives was carried out by different spectroscopic techniques such as ¹H NMR and EI-MS. The synthetic molecules were evaluated for their in vitro urease inhibitory activity. All synthetic derivatives showed good inhibitory activities in the range of (IC₅₀ = 0.87 ± 0.31–19.0 ± 0.25 μM) as compared to the standard thiourea (IC₅₀ = 21.25 ± 0.15 μM), except seven compounds 17, 18, 23, 24, 29, 30, and 41 which were found to be inactive. The most active compound of the series was compound 36 (IC₅₀ = 0.87 ± 0.31 μM) having two chloro groups at meta positions of ring A and methoxy group at para position of ring B. The structure–activity relationship (SAR) of the active compounds was established on the basis of different substituents and their positions in the molecules. Kinetic studies of the active compounds revealed that compounds can inhibit enzyme via competitive and noncompetitive modes. In silico study was also performed to understand the binding interactions of the molecules (ligand) with the active site of enzyme.

Full text of DOI:10.1016/j.bioorg.2018.09.036

Accepted Manuscript
Synthesis and in vitro urease inhibitory activity of benzohydrazide derivatives,
in silico and kinetic studies
Azhar Abbas, Basharat Ali, Kanwal, Khalid Mohammed Khan, Jamshed Iqbal,
Shafiq ur Rahman, Sumera Zaib, Shahnaz Perveen
PII:
S0045-2068(18)30845-9
DOI:
https://doi.org/10.1016/j.bioorg.2018.09.036
YBIOO 2533
Reference:
Bioorganic Chemistry
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Accepted Date:
7 August 2018
27 September 2018
28 September 2018
Please cite this article as: A. Abbas, B. Ali, Kanwal, K. Mohammed Khan, J. Iqbal, S. ur Rahman, S. Zaib, S. Perveen,
Synthesis and in vitro urease inhibitory activity of benzohydrazide derivatives, in silico and kinetic studies,
Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.09.036
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Synthesis and in vitro urease inhibitory activity of benzohydrazide derivatives, in silico
and kinetic studies
Azhar Abbas,a Basharat Ali,a Kanwal,a Khalid Mohammed Khan,a,c Jamshed Iqbal,*b Shafiq
ur Rahman,b Sumera Zaib,b Shahnaz Perveend
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
bCentre for Advanced Drug Research, COMSATS University Islamabad, Abbottabad
Campus, Abbottabad-22060, Pakistan
cDepartment of Clinical Pharmacy, Institute for Research and Medical Consultations
(IRMC), Imam Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam Saudi Arabia
dPCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-
75280, Pakistan
Abstract: Benzohydrazide derivatives 1-43 were synthesized via “one-pot” reaction and
structural characterization of these synthetic derivatives was carried out by different
1
spectroscopic techniques such as H-NMR and EI-MS. The synthetic molecules were
evaluated for their in vitro urease inhibitory activity. All synthetic derivatives showed good
inhibitory activities in the range of (IC50 = 0.87 ± 0.31 - 19.0 ± 0.25 µM) as compared to the
standard thiourea (IC50 = 21.25 ± 0.15 µM), except seven compounds 17, 18, 23, 24, 29, 30,
and 41 which were found to be inactive. The most active compound of the series was
compound 36 (IC50 = 0.87 ± 0.31 μM) having two chloro groups at meta positions of ring A
and methoxy group at para position of ring B. The structure-activity relationship (SAR) of
the active compounds was established on the basis of different substituents and their positions
in the molecules. Kinetic studies of the active compounds revealed that compounds can
inhibit enzyme via competitive and noncompetitive modes. In silico study was also
performed to understand the binding interactions of the molecules (ligand) with the active site
of enzyme.
Keywords: In vitro urease inhibitory activity; Benzohydrazide; In silico studies; Kinetic
studies.
Introduction
Urease (EC 3.5.1.5) is a member of family of amidohydrolase enzymes, it possesses two
nickel atoms in its core structure. The conversion of urea into ammonia and carbamate is
*Corresponding Authors: khalid.khan@iccs.edu, drkhalidhej@gmail.com; Tel.: +922134824910, Fax:
+922134819018; drjamshed@ciit.net.pk, jamshediqb@googlemail.com, Tel: +92-992-383591-96, Fax: +92-
992-383441
1

catalyzed by the action of this enzyme. The excessive amount of ammonia released due to
hyper-activity of urease enzyme leads to alkalinity of stomach which in turn increase the
gastric mucosa permeability [1]. The nitrogen metabolism of cattle and various other animals
is controlled by the action of urease enzyme [2]. The elevated levels of these enzymes leads
to several pathogenic conditions, mostly it helps in the survival of some bacterial pathogens
thus leading to some severe side effects [3]. In humans, the low pH of stomach facilitates the
survival of Helicobacter pylori (HP) which leads to the development of gastric and peptic
ulcer which may eventually cause cancer [4]. The increased level of ammonia is also
responsible for several metabolic disorders and destructs the GIT epithelium. A number of
compounds belonging to different classes are reported as urease inhibitors such as thiolates
that binds with nickel atom of the enzyme, hydroxamic acid, its derivatives which acts as
competitive inhibitor and competes with urea, phosphodiamidates, and few peptides chains
having a ligand which may chelate with nickel of urease. Unfortunately, these molecules
have adverse side effects associated with them. Therefore, it is of crucial importance to
identify more urease inhibitors having significant stability, bioavailability, and low toxicity
[5-6].
Hydrazides are acylated derivatives of hydrazine and constitute a class of organic compounds
which had attained the attention of medicinal as well as synthetic chemists due to the fact that
they contain azomethine group (–NH–N=CH–) connected with carbonyl group. They are well
known for possessing a number of pharmacological activities, i.e. anti-inflammatory,
antitumoral, and antitubercular [7-8]. Hydrazides commonly serves as intermediates and also
the starting materials for the synthesis of surfactants as well as pharmaceutical products [9,
10].
Our research group has contributed in identifying a number of urease inhibitors that belongs
to different classes. In this regard, we had synthesized libraries of compounds having
different functionalities and core structures in order to have a molecule which may serve as
lead compounds for urease inhibition [11-15]. In the recent past we had synthesized
benzohydrazide derivatives and identified them as urease inhibitors [16], therefore, in search
of potentially active compounds we had synthesized benzohydrazide derivatives 1-43 and
evaluated their in vitro urease inhibitory activity and found good results. Further
modifications and studies on these compounds might be helpful in bringing a number of lead
compounds which may serve as potential drug candidates (Figure-1).
2

Figure-1: Rationale of current study
Results and discussion
Chemistry
Substituted N-benzoyl/nicotinoyl and aryl benzohydrazides were synthesized by treating
different hydrazide with various benzoyl chlorides in pyridine. The reaction mixture was
refluxed for 36 h. The products thus obtained were purified via crystallization [17]. The
synthetic molecules were then characterized with various spectral studies such as EI-MS, H1-
NMR (Scheme-1).
3

Table-1: Synthetic derivatives of benzohydrazide/nicotinoydrazide (1-43)
S. No. Structures
IC50 ± SEMa (µM)
6.78 ± 0.60
5.30 ± 0.42
7.57 ± 0.28
9.32 ± 0.36
1
2
3
4
9.48 ± 0.23
12.7 ± 0.57
5
6
13.6 ± 0.66
9.63 ± 0.41
7
8
4

9.64 ± 0.19
14.7 ± 0.19
3.83 ± 0.33
9
10
11
12.3 ± 0.19
10.5 ± 0.83
9.87 ± 0.24
13.3 ± 0.72
12
13
14
15
9.07 ± 0.39
16
17
18
b
-
b
-
5

2.72 ± 0.11
1.64 ± 0.48
19
20
6.23 ± 0.28
13.1 ± 0.68
21
22
b
-
23
b
-
24
25
8.90 ± 0.73
4.62 ± 0.62
26
27
19.0 ± 0.25
6

7.35 ± 0.28
28
29
b
-
b
-
30
31
13.7 ± 0.65
16.7 ± 0.36
32
33
1.82 ± 0.61
2.13 ± 0.55
9.71 ± 0.48
0.87 ± 0.31
34
35
36
7

2.66 ± 0.09
4.65± 0.45
37
38
5.01 ± 0.18
1.97 ± 0.90
39
40
41
b
-
9.12 ± 0.14
9.15 ± 0.24
42
43
Thiourea(std)
21.25 ± 0.15
SEMa (Standard error of mean); -b(Not active);Urea (Standard inhibitor for urease enzyme)
In vitro urease inhibitory activity
All synthetic benzohydrazide derivatives 1-43 were screened for their in vitro urease
inhibitory activity. It is noteworthy that except seven compounds 17, 18, 23, 24, 29, 30, and
41, rest of the derivatives were found to be significantly urease inhibitors with IC50 values in
the range of 0.87 ± 0.31 to 19.0 ± 0.25 µM as compared to the standard thiourea (IC50
=
21.25 ± 0.15 µM). The variable activity of synthetic derivatives was might be due to the
different substituents on rings A and B. To understand the better structure-activity
8

relationship we have divided the synthetic compounds into different categories on the basis of
their substitution on ring A (Table-1) (Figure-2).
Structure-activity relationship (SAR)
Dichloro Substituted Ring A:
Compound 36 (IC50 = 0.87 ± 0.31 μM) having two chloro groups at meta positions of ring A
and methoxy group at para position of ring B was the most active compound of library and
exhibited potential inhibitory activity as compared to the standard thiourea (IC50 = 22.3 ±
1.06 μM). The activity of this compound might be due to the presence of methoxy group at
ring B which may have better binding interactions within the enzyme pocket. Compound 34
(IC50 = 2.13 ± 0.55 μM) bearing methyl group at para position of ring B also exhibited good
inhibitory activity, nonetheless, it was less active as compared to methoxy substituted
analogue 36, this shows that the oxygen of methoxy is binding well with the enzyme active
site through hydrogen bond interactions. The activity was decreased up to 4 folds when the
position of methyl was shifted from para to meta as in compound 35 (IC50 = 9.71 ± 0.48 μM)
which showed that substitution at para position is actively participating in the activity.
Among chloro substituted compounds on ring B, compound 33 (IC50 = 1.82 ± 0.61 μM)
having one chloro group at para position was found to be most active might be due to
mesomeric effect of chloro group. Shifting the position of chloro group from para to meta
resulted in decreased activity as in compound 32 (IC50 = 16.7 ± 0.36 μM). It indicated that the
substituents at para position are mainly contributing in the activity and binds well within the
active site of enzyme (Figure-3).
9

The di-chloro substituted compound 38 (IC50 = 4.65 ± 0.45 μM) having two chloro groups at
ortho and para positions also showed good inhibitory activity. Interestingly, compound 39
(IC50 = 5.01 ± 0.90 μM) having two chloro groups at ortho and para positions of both rings
was slightly less active as compared to compound 38, nevertheless, these compounds showed
potent activity in comparison with standard thiourea (IC50 = 22.3 ± 1.06 μM) (Figure-3).
Figure-3: Structure-activity relationship of compounds 32-40
Mono Chloro Substituted Ring A
Compound 21 (IC50 = 6.23 ± 0.28 μM) bearing unsubstituted aromatic ring B was found to
have good inhibitory activity might be the unsubstituted ring fits well within the enzyme
pocket. Addition of chloro group at meta position of ring B resulted in decreased activity as
in compound 22 (IC50 = 13.1 ± 0.68 μM). Compound 27 (IC50 = 19.6 ± 0.25 μM) bearing two
chloro groups at ortho and para positions of ring B was found to have good inhibitory
activity as compared to the standard thiourea (IC50 = 22.3 ± 1.06 μM). However, changing the
position of one of the chloro group from para to meta of ring B resulted in increased activity
as in compound 28 (IC50 = 7.35 ± 0.28 μM). Compound 26 (IC50 = 4.62 ± 0.62 μM) having
methyl group at para position of ring B showed good inhibitory activity as compared to
chloro substituted analogs. The activity was decreased up to 2 folds when the position of
methyl was shifted from para to meta as in compound 25 (IC50 = 8.90 ± 0.73 μM), it shows
10

that the chloro group on ring B is deactivating the ring thus resulting in the decreased activity
as compared to its methyl analogs (Figure-4).
Figure-4: Structure-activity relationship of compounds 21-29
Unsubstituted Ring A:
Compounds 1-8 have unsubstituted phenyl part as ring A also showed good inhibitory
potential as compared to the standard thiourea (IC50 = 22.3 ± 1.06 μM). Compound 1 (IC50 =
6.78 ± 0.60 μM) having unsubstituted phenyl rings both as ring A and B showed superior
inhibitory activity as compared to the standard. The most active compound among them was
compound 2 (IC50 = 5.30 ± 0.42 μM) having two chloro groups at ortho and para positions of
ring B, respectively. Compound 5 (IC50 = 9.48 ± 0.23 μM) bearing chloro groups at ortho and
meta positions of ring B exhibited less activity as compared to compound 2 which showed
that the particular position of the substituents are helping in the good binding interactions of
compounds with enzyme.
Compound 3 (IC50 = 7.57 ± 0.28 μM) bearing one chloro group at meta position also showed
good inhibition against urease enzyme, however, the activity was decreased when the chloro
group was placed at para position in compound 4 (IC50 = 9.32 ± 0.36 μM). Compound 6 (IC50
= 12.7 ± 0.57 μM) having methyl group at meta position was found to be less active as
11

compared to the chloro substituted compounds of this category. Shifting the position of
methyl group from meta to para resulted in the decreased activity in compound 7 (IC50 = 13.6
± 0.66 μM), nevertheless, both of these compounds were more active as compared to the
standard thiourea (IC50 = 22.3 ± 1.06 μM). Compound 8 (IC50 = 9.63 ± 0.41 μM) having
methoxy group at para position also showed good inhibitory potential.
Concisely, the variation in activity was observed on the basis of substituents and their
particular positions, methyl and chloro substituted compounds were more active among them
and meta and para positions were also actively participating in the activity (Figure-5).
Nicitinohydrazide as Ring A
Amongst them, compound 11 (IC50 = 3.83 ± 0.33 μM) having chloro group at para position
of ring B was most active which again showed that the presence of chloro group is
participating in the activity. Compound 10 (IC50 = 14.7 ± 0.19 μM) bearing chloro group at
meta position of ring B showed good inhibitory activity as compared to standard thiourea
12

(IC50 = 22.3 ± 1.06 μM), nonetheless, it was less active as compared to compound 9. Addition
of another chloro group at ortho position as in compound 12 (IC50 = 12.3 ± 0.19 μM) resulted
in 3 folds decreased activity which again showed that in these compounds substitution of
chloro group at para position is favorable and have more binding interactions within
enzyme’s active site, changing the position of chloro group or addition of another chloro
substituent a decreased activity was observed. Compound 9 (IC50 = 9.64 ± 0.19 μM) having
unsubstituted phenyl ring also showed potential activity as compared to the standard.
Compound 13 (IC50 = 10.5 ± 0.83 μM) having methyl group at meta position exhibited good
inhibitory activity, shifting the position of methyl from meta to para resulted in increased
activity as in compound 14 (IC50 = 9.87 ± 0.24 μM). Compound 15 (IC50 = 13.3 ± 0.72 μM)
having ethoxy group at ortho position was also found to have good inhibition against urease
enzyme as compared to the standard thiourea. It is worth noting that among the
nicotinohydrazide derivatives chloro substituted compounds were more active as compared to
other analogs of this category. It was also observed that para position of the substituents also
resulted in the enhanced activity as in compounds 11 and 14 (Figure-6).
13

Methyl Substituted Ring A
Compound 16 (IC50 = 9.07 ± 0.39 μM) having methyl groups at meta position of both rings A
and B also showed good inhibitory activity as compared to the standard thiourea (IC50 = 22.3
± 1.06 μM). However, compound 17 having methyl group at para position of both rings was
found to be inactive which showed that changing the position of methyl group resulted in the
loss of activity (Figure-7).
Compounds 19, and 20 with dimethyl groups at ortho and meta positions of ring A were also
found to have good inhibitory activity as compared to the standard. Among them compound
20 (IC50 = 1.64 ± 0.48 μM) bearing methoxy group at para position was most active and was
the second most active compound of the series. Interestingly, compound 19 (IC50 = 2.72 ±
0.11 μM) having two chloro groups at ortho and meta positions was less active as compared
to the methoxy containing compound 20, might be the electronegative oxygen in methoxy is
helping in the hydrogen binding interactions (Figure-8).
Iodo Substituted Ring A
Compound 31 (IC50 = 13.7 ± 0.65 μM) having iodo group at ortho position of ring A and
methyl group at meta position of ring B showed good inhibitory activity as compared to the
14

standard thiourea (IC50 = 22.3 ± 1.06 μM). The activity of this compound is less as compared
to other halogenated compounds might be due to the presence of methyl group which may
help in the formation of hydrogen bonds within the enzyme pocket (Figure-9).
Methoxy and Nitro substituted Ring A
Compound 42 (IC50 = 9.12 ± 0.14 μM) having tri-methoxy groups at meta and para positions
of ring A and di-chloro groups at ortho and meta positions of ring B showed good inhibitory
potential as compared to the standard thiourea (IC50 = 22.3 ± 1.06 μM). 3-
Nitrobenzohydrazide derivative, compound 43 (IC50 = 9.15 ± 0.24 μM) exhibited comparable
activity as compound 42 which showed that chloro group at ring B and the oxygen atoms of
nitro and methoxy group are easily binding within the active site of enzyme (Figure-10).
Kinetic Studies
In order to explore the mechanism of inhibition, the detailed kinetics studies were performed
on active compound 40, 20, 36, and 33. The Lineweaver-Burk plot for compound 36 revealed
a noncompetitive mode of inhibition, whereas, compounds 40, 20, and 33 showed
competitive mode of inhibition by illustrating the same y-intercept for uninhibited and
inhibited enzymes (Figure-11). Moreover, Michaelis-Menten kinetic parameters; Km and Vmax
of urease inhibition were also determined for inhibitors 40, 20, 36, and 33. In case of
compound 36, the maximal velocity (Vmax) and Michaelis constant (Km) were found to
decrease with the increasing concentration of inhibitors (Figure-11c). On the other hand in
15

case of compounds 40, 20, and 33, the maximal velocity (Vmax) was found almost constant
even by using different concentrations of respective inhibitors, however, the Michaelis
constant (Km) was increasing with increase in concentration of inhibitors as presented in
Figure 11-a, -b and -d.
(a)
(b)
(c)
(d)
Figure-11: Lineweaver-Burk plot for urease inhibitory activity by compounds 40 (a); 20 (b); 36 (c);
and 33 (d) using different concentrations of substrate (0, 250, 500, 1000 and 15000 μM).
Molecular Docking Studies
To carry out the further investigation of potent inhibitors revealed by in vitro analysis, the
docking studies were carried out. The docking studies were carried out by LeadIT, however,
visualization was done by discovery studio. The protein structure PDB 3LA4 was selected
and docking analysis for the synthetic compounds was performed in the active pocket of jack
bean urease. The rational of docking studies was to justify the binding exhibited by enzyme-
ligand complex in active site and correlate with in vitro assay. The interactions showed by the
potent compounds were similar and all the compounds were found to bind in same orientation
16

in the entrance of active pocket of enzyme. The overlay of the potent and selective
compounds from the series are presented in Figure-12.
Figure-12: The overlap of all the docked inhibitors in the active site of urease, compound 40
(red), 34 (purple), 19 (green), 20 (crimson), 37 (chocolate), 36 (blue), 33 (light brown).
When individual analysis were made for each compound, the similar results were noticed.
The compounds showed same binding interactions in the active site of urease. The active site
of urease possess the amino acid residues Ala436, His593, Arg639 and Arg609 in addition to
Asp494 and nickel ions. The nickel ions are important for the catalytic activity of the enzyme
and compounds were found to bind in near vicinity to the nickel ions in active site of urease
(Figure-12). The diacyl hydrazide group (–NH–N=CH–) in the compounds serves as
stabilizing agent in the active site and prevent the binding of substrate. In addition to stabilize
the inhibitors, the -NH group of diacyl hydrazide was also involved making strong hydrogen
bonds with amino acid Arg439 and Ala636. The interactions were shown in 3D interaction
diagrams of inhibitors inside the binding site of urease (Figure-13a-g).
When detailed investigation was done for the docked poses of inhibitors, it was found that all
the potent and selective compounds exhibited di-chloro or tetra-chloro substitution at
benzohydrazide. The most potent compound 36, possess di-chloro at meta positions of ring
A, in addition to methoxy group at ring B. The replacement of methoxy by chloro or methyl
group cause the decrease in inhibitory activity to some extent. Another compound, 20 (N'-(4′-
methoxybenzoyl)-3,5-dimethylbenzohydrazide) having no chloro group exhibited significant
inhibition as well.
For the exploration of binding site interactions of the active compounds inside enzyme’s
pocket, the comprehensive analysis was done. The compounds having dichloro group at meta
position on ring A represent the hydrogen bond formation, which result in increased
17

inhibition potential. Moreover, the methyl group substitution at meta position on ring A
showed significant inhibitory profile. The reason for increased inhibition might be that the
mobile flap of the active site in jack bean urease was covered and essential structural
modification in the conformation of enzyme occur [18]. These conformational changes are
responsible for the regulation of active site. In figure-13, it was clearly noticed that the ring A
and its substituents are completely fitted in the entrance of active site and thus responsible for
this behavior of compounds. In this way substrate was not available to the enzyme and
therefore, no product formation occur, and thus the inhibition took place. The figure-13 (a-g)
depicted that the hydrogen bonds were noticed between electronegative elements like
chloro/oxygen groups of compounds (40, 34, 19, 20, 37, 36 and 33) with the hydrogen atoms
of amino acid residues present inside the active pocket of Jack bean urease. However, the ππ
interactions were observed between phenyl ring A and B and the active pocket amino acids.
All the selected compounds exhibited the interactions with most important residues of active
site like, Ala440, Arg439, Ala436 and Ala636 in addition to Met637. The compounds 33, 37,
19, 40, and 34 having di-chloro substitution along with methoxy or methyl groups prevent the
binding of substrate by occupying the entrance cavity and forming stable hydrogen bonds in
addition to π-π interactions. The hydrogen bonds and π-π interactions are playing significant
role in stabilizing the enzyme-inhibitor complex. Moreover, the inhibitors were found to be in
contact with nickel ions which is another contributing factor for the stability of enzyme-
inhibitor complex. Therefore, the substrate entry was prevented and hence no enzyme-
substrate complex was formed.
(a)
(b)
18

(c)
(d)
(e)
(f)
(g)
Figure-13: The putative binding modes for potent compounds in the series against urease;
a) 40: b) 34: c) 19: d) 20: e) 37: f) 36: g) 33.
19

HYDE assessment of selective compounds against all the targets
The HYDE affinity evaluation was carried out for the first 30 top ranking docked
conformations inside the active pocket of Jack bean urease. The analysis helped in the
selection of better binding mode as well as the selective and potent compound. The binding
free energy ΔG and FlexX docking score for the active compounds were given in Table-2.
The FlexX docking score presented that the potent compound has lower energy scores as
compared to non-selective inhibitors. Moreover, the binding free energies ΔG showed that
the potent inhibitors exhibited higher affinity towards enzyme. The potent and selective
compounds exhibit better binding affinity and give favorable contributions.
Table-2: Docking and Hyde scores and their corresponding ranks by Hyde affinity estimation
Code
FlexX score of the top Binding free energy
ranking pose
-22.62
ΔG (KJ mol‒1)
‒10
‒22
‒16
‒21
‒29
‒19
‒20
19
20
33
34
36
37
40
-20.98
-22.55
-22.80
-23.46
-23.02
-23.71
ADME Profile of synthesized derivatives (1-43)
In order to determine the ADME (Absorption, Distribution, Metabolism, and Excretion)
properties of the selective and potent compounds (1-43), in silico evaluation has been carried
out with some physiochemical parameters. The results are shown in Table-3. Polar surface
area (PSA) is sum of all the polar atoms in compound. It gives us the estimated ability of
synthetic derivatives to cross the blood brain barrier (BBB). The value of PSA less than 140
is ideal and all compounds showing the values less than 100 [19-21]. Another parameter,
cLogP shows the octanol water partition coefficient (ratio of compound concentration in
octanol to water concentration). The parameter cLogP is responsible to calculate the
compound ability to cross the membrane. Similarly, cLogS is the calculation of water
solubility at 25 ˚C. The number of hydrogen bond donors (HD) and hydrogen bond acceptors
(HA) must be less than 5 and 10, respectively. All the compounds presented favorable
ADME properties and no violation of Lipinski’s rule was observed [22-24].
20

Table-3: In silico calculation of some ADME parameters of the selective and potent
compounds 19, 20, 33, 34, 36, 37, and 40.
Codes
Mol. Wt
PSA
cLogP
cLogS
HD
HA
337.20
298.34
343.59
323.17
339.17
378.04
378.04
58.20
67.43
58.20
58.20
67.43
58.20
58.20
4.405
3.181
4.405
4.111
3.769
4.993
4.878
-5.9998
-4.5816
-5.7863
-5.5259
-5.1024
-6.5206
-6.5206
2
2
2
2
2
2
2
2
3
2
2
3
2
2
19
20
33
34
36
37
40
Conclusion
Benzohydrazide derivatives 1-43 were synthesized and screened for in vitro urease inhibitory
activity. It is worth-mentioning that all synthetic compounds were found to have potent
inhibitory activity in the range of (IC50 = 0.87 ± 0.31 – 16.7 ± 0.36 µM) as compared to the
standard thiourea (IC50 = 22.3 ± 1.06 μM), except compounds 17, 18, 23, 24, 29, 30, and 41
which were found to be inactive. Structure-activity relationship (SAR) was established on the
basis of varying structural features of the molecules. It showed that almost all structural
features are taking part in the inhibition potential but some groups Cl, CH3, and OMe are
contributing an important role. Kinetic studies have been done through various parameters
and showed non-competitive or competitive mode of inhibition. However, in silico study was
performed to confirm the binding interactions of the molecules (ligand) with the active site of
enzyme. The current study had identified a range of chemical spaces which may act as lead
molecules for future research to explore powerful urease inhibitors for the treatment of ulcer.
Experimental
Materials and methods
Reagents were purchased from Sigma-Aldrich, USA. Thin layer chromatography was carried
out on pre-coated silica gel, GF-254 (Merck, Germany). Spots were visualized under
ultraviolet light at 254, 366 nm or iodine vapor. Mass spectra were recorded under on MAT
1
312 and MAT 113D mass spectrometers. The H-NMR were recorded on a Bruker AM
spectrometers, operating at 300 and 400 MHz. The chemical shift values are presented in
ppm (), relative to tetramethylsilane (TMS) as an internal standard and the coupling constant
(J) are in Hz. Melting points of the compounds were determined on a Stuart SMP10 melting
point apparatus and are uncorrected.
21

General procedure for the synthesis of benzohydrazide (1-43)
N-benzoyl/nicotinoyl aryl benzohydrazides were obtained by treating different hydrazides
with various derivatives of benzoyl chlorides in pyridine and the reaction mixture was
refluxed for 36 h. The progress of reaction was monitored by TLC, after completion, the
reaction mixture was poured onto 19% HCl, precipitates formed were filtered further washed
with hot water and hexane. The solid product was crystallized from ethanol. The compounds
thus obtained were characterized via EI-MS and H1-NMR. Among all synthetic derivatives,
compounds 19, 37, and 40 are new while rest of the compounds are reported in the literature
[25-37].
Spectral Data of Synthetic Compounds 1-43
N'-Benzoylbenzohydrazide (1) [CAS # 787-84-8]
˚
1
Yield: 87%; M.p.: 241-242 C; H-NMR (300 MHz, DMSO-d6); 10.49 (s, 2H, 1″-NH, 2″-
NH), 7.93 (d, J2,6/3,5,2′,6′/3′,5′ = 7.2 Hz, 4H, H-2, H-6, H-2′, H-6′), 7.61 (overlapping multiplete,
6H, H-3, H-4, H-5, H-3′, H-4′, H-5′); EI-MS m/z (% rel. abund.): 240 (M+, 89), 105 (100), 77
(68), 51 (22).
N'-Benzoyl-2′,4′-Dichlorobenzohydrazide (2) [CAS # 195066-99-0]
˚
1
Yield: 77%; M.p.: 196-198 C; H-NMR (300 MHz, DMSO-d6):  10.64 (s, 1H, NH), 10.49
(s, 1H, NH), 7.93 (d, J2,6/3,5 = 7.2 Hz, 2H, H-2, H-6), 7.75 (s, 1H, H-3′), 7.59 (overlapping
multiplete, 5H, H-3, H-4, H-5, H-5′, H-6′); EI-MS m/z (% rel. abund.): 308 (M+, 7), 310
(M+2, 5), 173 (49), 105 (100), 77 (20).
N'-Benzoyl-3′-chlorobenzohydrazide (3) [CAS # 316142-01-5]
˚
1
Yield: 87%; M.p.: 215-217 C; H-NMR (300 MHz, DMSO-d6):  10.63 (s, 1H, NH), 10.55
(s, 1H, NH), 7.94 (overlapping multiplete, 3H, H-2′, H-5′, H-6′), 7.69 (d, J4′,5′ = 8.4 Hz, 1H,
H-4′), 7.62 (d, J2,6/3,5 = 8.1 Hz, 2H, H-2, H-6), 7.57 (overlapping multiplete, 3H, H-3, H-4, H-
5); EI-MS m/z (% rel. abund.): 274 (M+,41), 276 (M+2, 14), 139 (55), 111 (23), 105 (100), 77
(50).
N'-Benzoyl-4′-chlorobenzohydrazide (4) [CAS # 6828-55-3]
˚
1
Yield: 69%; M.p.: 222-223 C; H-NMR (400 MHz, DMSO-d6):  10.61 (overlapping
multiplete, 2H, NH, NH), 7.94 (m, 4H, H-2, H-6, H-2′, H-6′), 7.61 (d, J3′,5′/2′,6′ = 8.4 Hz, 2H,
22

H-3′, H-5′), 7.57 (overlapping multiplete, 3H, H-3, H-4, H-5); EI-MS m/z (% rel. abund.):
274 (M+, 17), 276 (M+2, 6), 139 (100), 111 (51), 105 (97), 77 (39).
N'-Benzoyl-2,3-dichlorobenzohydrazide (5) [CAS # 1182292-59-6]
˚
1
Yield: 67%; M.p.: 190-192 C; H-NMR (400 MHz, DMSO-d6):  10.64 (s, 1H, NH), 10.49
(s, 1H, NH), 8.73 (s, 1H, H-2), 7.93 (d, J4′,5′ = 7.6 Hz, 1H, H-4′), 7.78 (m, 1H, H-5′), 7.59 (t,
1H, H-4), 7.53 (overlapping multiplete, 3H, H-5, H-6′), 7.46 (overlapping multiplete, 1H, H-
3); EI-MS m/z (% rel. abund.): 308 (M+, 24), 310 (M+2, 14), 312 (M+4, 2), 173 (84), 145
(26), 105 (100), 77 (76), 51 (15).
N'-Benzoyl-3′-methylbenzohydrazide (6) [CAS # 108013-58-7]
˚
1
Yield: 78%; M.p.: 208-210 C; H-NMR (400 MHz, DMSO-d6):  10.48 (overlapping
multiplete, 2H, NH, NH), 7.93 (d, J2,6/3,5 = 7.2 Hz, 2H, H-2, H-6), 7.73 (overlapping
multiplete, 2H, H-4′, H-6′), 7.59 (m, 3H, H-3, H-4, H-5), 7.40 (d, J5′,4′/6′,5′ = 4.8 Hz, 2H, H-5′,
H-4′), 2.37 (s, 3H, CH3); EI-MS m/z (% rel. abound.): 254 (M+, 15), 119 (100), 105 (50), 91
(33), 77 (20).
N'-Benzoyl-4′-methylbenzohydrazide (7) [CAS # 19338-21-7]
˚
1
Yield: 81%; M.p.: 220-221 C; H-NMR (400 MHz, DMSO-d6):  10.45 (s, 1H, NH), 10.41
(s, 1H, NH), 7.92 (d, J2′,6′/3′,5′ = 7.2 Hz, 2H, H-2′, H-6′), 7.83 (d, J3′,5′/2′,6′ = 8.1 Hz, 2H, H-3′, H-
5′), 7.61 (m, 3H, H-3, H-4, H-5), 7.33 (d, J2,6/3,5 = 8.1 Hz, 2H, H-2, H-6), 2.37 (s, 3H, CH3);
EI-MS m/z (% rel. abound.): 254 (M+, 30), 119 (100), 105 (75), 91 (61), 77 (35), 65 (13).
N'-Benzoyl-4′-methoxybenzohydrazide (8) [CAS # 6781-59-5]
˚
1
Yield: 89%; M.p.: 186-187 C; H-NMR (300 MHz, DMSO-d6):  10.42 (s, 1H, NH), 10.34
(s, 1H, NH), 7.91 (overlapping multiplete, 4H, H-2, H-6, H-2′, H-6′), 7.58 (overlapping
multiplete, 1H, H-4), 7.53 (t, J3,2/5,6/3,4/5,4 = 7.5 Hz, 2H, H-3, H-5), 7.06 (d, J3′,5/2′,6′ = 8.7 Hz,
2H, H-3′, H-5′), 3.82 (s, 3H, OCH3); EI-MS m/z (% rel. abund.): 270 (M+, 48), 135 (100), 105
(64), 92 (16), 77 (47).
N'-Benzoylnicotinohydrazide (9) [CAS # 56352-76-2]
˚
1
Yield: 79%; M.p.: 258-259 C; H-NMR (400 MHz, DMSO-d6):  10.70 (s, 1H, NH), 10.57
(s, 1H, NH), 9.07 (d, J6,3 = 1.6 Hz, 1H, H-2), 8.77 (dd, J4,2 = 1.2, J4,5 = 4.8 Hz, 1H, H-4), 8.26
23

(d, J6,5 = 7.6 Hz, 1H, H-6), 7.92 (d, J2′,6′/3′,5′ = 7.6 Hz, 2H, H-2′, H-6′), 7.60 (overlapping
multiplete, 4H, H-5, H-3′, H-4′, H-5′); EI-MS m/z (% rel. abund.): 241 (M+, 12), 105 (100),
77 (45), 51 (15).
N'-(3′-Chlorobenzoyl)nicotinohydrazide (10) [CAS # 258264-84-5]
˚
1
Yield: 71%; M.p.: 230-232 C; H-NMR (300 MHz, DMSO-d6):  10.78 (overlapping
multiplete, 2H, NH, NH), 10.72 (s, 1H, NH), 9.06 (s, 1H, H-2), 8.78 (d, J6,5 = 3.9 Hz, 1H, H-
6), 8.27 (d, J6,5 = 1.8 Hz, 1H, H-5), 7.89 (s, 1H, H-2′) 7.87 (d, J4,5 = 7.5 Hz, 1H, H-4), 7.67
(J6′,5′ = 8.1 Hz, 1H, H-6′), 7.60 (t, 2H, H-5, H-5′); EI-MS m/z (% rel. abund.): 275 (M+, 41),
277 (M+2, 14), 139 (100), 113 (13), 111 (42), 106 (73), 78 (32), 75 (11), 51 (10).
N'-(4′-Chlorobenzoyl)nicotinohydrazide (11) [CAS # 56352-79-5]
˚
1
Yield: 69%; M.p.: 192-194 C; H-NMR (400 MHz, DMSO-d6):  10.73 (overlapping
multiplete, 2H, NH, NH), 9.06 (s, 1H, H-2), 8.77 (d, J6,5 = 3.6 Hz, 1H, H-6), 8.25 (d, J4,5
=
7.6 Hz, 1H, H-4), 7.94 (d, J2′,6′/3′,5′ = 8.0 Hz, 2H, H-2′, H-6′), 7.61 (overlapping multiplete,
3H, H-5, H-3′, H-5′); EI-MS m/z (% rel. abund.): 275 (M+, 7), 277 (M+2, 3), 139 (100), 111
(19), 78 (12).
N'-(2′,4′-Dichlorobenzoyl)nicotinohydrazide (12) [CAS # 392314-26-0]
˚
1
Yield: 65%; M.p.: 254-256 C; H-NMR (400 MHz, DMSO-d6):  10.87 (s, 1H, NH), 10.58
(s, 1H, NH), 9.06 (s, 1H, H-2), 8.77 (d, J6,5 = 4.4 Hz, 1H, H-6), 8.27 (d, J4,5 = 2.4 Hz, 1H, H-
4), 7.76 (m, 1H, H-5), 7.55 (overlapping multiplate, 3H, H-3′, H-5′ H-6′); EI-MS m/z (% rel.
abund.): 309 (7), 311 (M+2, 4), 173 (100), 145 (13), 106 (37), 78 (24).
N'-(3′-Methylbenzoyl) nicotinohydrazide (13) [CAS # 821018-69-3]
˚
1
Yield: 69%; M.p.: 216-218 C; H-NMR (400 MHz, DMSO-d6):  10.70 (s, 1H, NH), 10.52
(s, 1H, NH), 9.06 (s, 1H, H-2), 8.77 (d, J4/5 = 2.7 Hz, 1H, H-4), 8.26 (d, J6/5 = 7.5 Hz, 1H, H-
6), 7.73 (d, J6′/5′ = 8.7 Hz, 2H, H-6′, H-5), 7.57 (s, 1H, H-2′), 7.40 (s, 2H, H-5′,H-4′); EI-MS
m/z (% rel. abund.): 255 (M+, 34), 119 (100), 106 (23), 91 (84), 78 (22), 65 (16), 51 (11).
N'-(4′-Methylbenzoyl) nicotinohydrazide (14) [CAS # 56352-79-5]
˚
1
Yield: 68%; M.p.: 238-240 C; H-NMR (400 MHz, DMSO-d6):  10.68 (s, 1H, NH), 10.50
(s, 1H, NH), 9.06 (s, 1H, H-2), 8.77 (d, J4,5 = 4.2 Hz, 1H, H-4), 8.26 (d, J6,5 = 7.8 Hz, 1H, H-
24

6), 7.83 (d, J6′,2′/5′ ,3′ = 8.1 Hz, 2H, H-2′, H-6′), 7.58 (m, 1H, H-5), 7.33 (d, J3′,5′/2′,6′ = 7.8 Hz,
2H, H-3′, H-5′), 2.37 (s, 3H, CH3); EI-MS m/z (% rel. abund.): 255 (M+, 10), 119 (100), 106
(10), 91 (57), 78 (14), 78 (14), 65 (13).
N'-(2′-Ethoxybenzoyl) nicotinohydrazide (15) [CAS # 713490-22-3]
˚
1
Yield: 70%; M.p.: 202-202 C; H-NMR (300 MHz, DMSO-d6):  11.03 (s, 1H, NH), 10.17
(s, 1H, NH), 9.06 (s, 1H, H-2), 8.76 (d, J4,5 = 4.2 Hz, 1H, H-4), 8.27 (J6,5 = 6.6 Hz, 1H, H-6),
7.77 (dd, J4,2/4,5 = 1.5 Hz, 1H, H-4), 7.57 (overlapping multiplete, 2H, H-6′, H-4′), 7.19 (d,
J3′,4′ = 8.1 Hz, 1H, H-3′), 7.09 (t, 1H, H-5′), 4.24 (q, JOCH2, CH3 = 6.9 Hz, 2H, OCH2), 1.41 (t,
JCH3 CH2
,
= 7.2 Hz, 3H, CH3). EI-MS m/z (% rel. abund.): 285 (M+, 9), 149 (100), 121 (74), 93
(10).
N'-(3′-Methylbenzoyl)-3-methylbenzohydrazide (16) [CAS # 59646-36-5]
˚
1
Yield: 26%; M.p.: 220-222 C; H-NMR (300 MHz, DMSO-d6):  10.40 (s, 2H, NH, NH),
7.73 (overlapping multiplete, 4H, H-2, H-6, H-2′, H-6′), 7.40 (d, J5,4/4,5/5′,4′/4′,5′ = 4.5 Hz, 4H,
H-5, H-4, H-5′, H-4′), 2.37 (s, 6H, CH3); EI-MS m/z (% rel. abund.): 268 (M+, 18), 119 (100),
91 (62), 65 (16).
N'-(4′-Methylbenzoyl)-4-methylbenzohydrazide (17) [CAS # 1530-73-0]
˚
1
Yield: 70%; M.p.: 253-254 C; H-NMR (300 MHz, DMSO-d6):  10.36 (s, 2H, NH, NH),
7.82 (d, J2,6/3,5/2′,6′/3′,5′ = 8.1 Hz, 4H, H-2, H-6, H-2′, H-6′), 7.32 (d, J3,5/2,6/3′,5′/2′,6′ = 7.8 Hz, 4H,
H-3, H-5, H-3′, H-5′), 2.36 (s, 6H, CH3); EI-MS m/z (% rel. abund.): 268 (M+, 22), 119 (100),
91 (69), 65 (19).
N'-(3′-Chlorobenzoyl)-3,5-dimethylbenzohydrazide (18) [CAS # 1002706-72-0]
˚
1
Yield: 63%; M.p.: 232-234 C; H-NMR (400 MHz, DMSO-d6):  10.59 (s, 1H, NH), 10.41
(s, 1H, NH), 7.94 (s, 1H, H-2′), 7.88 (d, J6′,5′ = 7.6 Hz ,1H, H-6′), 7.68 (d, J4′,5′ = 6.8 Hz, 1H,
H-4′), 7.58 (overlapping multiplete, 1H, H-5′), 7.52 (s, 2H, H-2, H-6), 7.22 (s, 1H, H-4), 2.32
(s, 6H, CH3, CH3 ); EI-MS m/z (% rel. abund.): 302 (M+, 19), 304 (M+2, 8), 139 (80), 133
(100), 105 (59), 77 (19).
25

2′,3′-Dichloro-N'-(3,5-dimethylbenzoyl)benzohydrazide (19)
˚
1
Yield: 67%; M.p.: 208-210 C; H-NMR (400 MHz, DMSO-d6):  10.84 (m, 2H, NH, NH),
7.94 (overlapping multiplete, 2H, H-4′, H-6′), 7.69 (s, 1H, H-4), 7.65 (overlapping multiplete,
3H, H-2, H-6, H-5′), 2.65 (s, 6H, 3-CH3, 5-CH3); EI-MS m/z (% rel. abund.): 336 (M+, 8),
338 (M+2, 5), 173 (76), 145 (12), 133 (100), 105 (21).
N'-(4′-Methoxybenzoyl)-3,5-dimethylbenzohydrazide (20) [CAS # 892004-94-3]
˚
Yield: 66%; M.p.: 195 C; 1H-NMR (400 MHz, DMSO-d6):  10.29 (overlapping multiplete,
2H, NH, NH), 7.90 (d, J2′,4′/3′,5′ = 8.4 Hz, 2H, H-2′,H-6′), 7.51 (s, 2H, H-2, H-6), 7.21 (s, 1H,
H-4), 7.04 (d, J3′,5′/2′,6′ = 8.4 Hz, 2H, H-3′, H-5′), 3.82 (s, 3H, OCH3), 2.34 (s, 6H, CH3, CH3);
EI-MS m/z (% rel. abund.): 298 (M+, 41), 135 (100), 105 (26), 92 (13), 77 (22).
N'-Benzoyl-3′-chlorobenzohydrazide (21) [CAS # 316142-01-5]
˚
1
Yield: 62%; M.P.: 215-217 C; H-NMR (400 MHz, DMSO-d6):  10.62 (s, 1H, NH), 10.55
(s, 1H, NH), 7.94 (overlapping multiplete, 4H, H-2, H-6, H-2′, H-6′), 7.69 (d, J4,5 = 8.0 Hz,
1H, H-4), 7.59 (overlapping multiplete, 4H, H-5, H-3′, H-4′, H-5′); EI-MS m/z (% rel.
abund.): 274 (M+, 22), 276 (M+2, 8), 139 (94), 111 (40), 105 (100), 77 (73), 51 (20).
3-Chloro-N'-(3′-chlorobenzoyl) benzohydrazide (22) [CAS # 38192-14-2]
˚
1
Yield: 68%; M.p.: 234-236 C; H-NMR (400 MHz, DMSO-d6):  10.68 (s, 2H, 2NH), 7.94
(d, J2,4/2′,4′ = 1.6 Hz, 2H, H-2, H-2′), 7.88 (d, J6,5/6′,5′ = 7.6 Hz, 2H, H-6, H-6′), 7.69 (d, J4,5/4′,5′
= 8.0 Hz, 2H, H-4, H-4′), 7.58 (t, J5,4/5,6/5′,4′/5′,6′ = 8.0 Hz, 2H, H-5, H-5′); EI-MS m/z (% rel.
abund.): 308 (M+, 42), 310 (M+2, 30), 312 (M+4, 5), 139 (100), 111 (67), 75 (20)
3-Chloro-N'-(4′-chlorobenzoyl)benzohydrazide (23) [CAS # 316146-11-9]
˚
1
Yield: 71%; M.p.: 206-208 C; H-NMR (400 MHz, DMSO-d6):  10.64 (s, 2H, NH), 7.93
(overlapping multiplete, 4H, H-2, H-6, H-2′ H-6′), 7.69 (d, J4,5 = 8.0 Hz, 1H, H-4), 7.61
(overlapping multiplete, 3H, H-5, H-5′, H-3′); EI-MS m/z (% rel. abund.): 308 (M+, 10), 310
(M+2, 5), 312 (M+4, 1), 139 (100), 111 (44), 75 (13).
4-Chloro-N'-(4′-chlorobenzoyl) benzohydrazide (24) [CAS # 895-84-1]
˚
1
Yield: 75%; M.p.: 220-222 C; H-NMR (400 MHz, DMSO-d6):  10.61 (s, 2H, NH, NH),
7.93 (d, J2,6/3,5/2′,6′/3′,5′ = 8.4 Hz, 4H, H-2, H-6, H-2′, H-6′), 7.61 (d, J3,5/2,6/3′,5′/2′,6′ = 8.4 Hz, 4H,
26

H-3, H-5, H-3′, H-5′); EI-MS m/z (% rel. abund.): 308 (M+, 6), 310 (M+2, 5), 139 (100), 111
(31), 75 (13).
3-Chloro-N'-(3′-methylbenzoyl) benzohydrazide (25) [CAS # 304456-89-1]
˚
1
Yield: 66%; M.p.: 202-204 C; H-NMR (400 MHz, DMSO-d6):  10.60 (s, 1H, NH), 10.48
(s, 1H, NH), 7.94 (s, 1H, H-2), 7.88 (d, J6,5 = 7.6 Hz, 1H, H-6), 7.73 (overlapping multiplete,
3H, H-4, H-2′, H-6′), 7.58 (d, J5,4 = 7.6 Hz ,1H, H-5), 7.40 (overlapping multiplete, 2H, H-4′,
H-5′), 2.37 (s, 3H, CH3); EI-MS m/z (% rel. abund.): 288 (M+, 44), 290 (M+2, 13), 139 (75)
119 (100), 111 (28), 91 (90), 65 (14).
3-Chloro-N'-(4′-methylbenzoyl) benzohydrazide (26) [CAS # 316142-02-6]
˚
1
Yield: 68%; M.p.: 202-202 C; H-NMR (300 MHz, DMSO-d6):  10.58 (s, 1H, NH), 10.45
(s, 1H, NH), 7.93 (s, 1H, H-2), 7.68 (d, J4,5 = 8.0 Hz, 1H, H-4), 7.82 (d, J2′,6′/3′,5′ = 8.0 Hz, 2H,
H-2′, H-6′), 7.58 (t, J5,4/5,6 = 7.6 Hz, 1H, H-5). 7.32 (d, J3′,5′/2′,6′ = 8.0 Hz, 2H, H-3′, H-5′), 2.37
(s, 3H, CH3), EI-MS m/z (% rel. abund.): 288 (M+, 21), 290 (M+2, 7), 139 (57), 119 (100),
111 (26), 91 (70), 75 (10), 65 (13).
2′,4′-Dichloro-N'-(3-chlorobenzoyl)benzohydrazide (27) [CAS # 316143-31-4]
˚
1
Yield: 75%; M.p.: 212-214 C; H-NMR (400 MHz, DMSO-d6):  10.77 (s, 1H, NH), 10.51
(s, 1H, NH), 7.95 (s, 1H, H-3′), 7.89 (d, J6′,5′ = 7.6 Hz, 1H, H-6′), 7.75 (s, 1H, H-2), 7.68 (d,
J5′,6′ = 7.6 Hz, 1H, H-5′), 7.58 (overlapping multiplete, 3H, H-4, H-5, H-6); EI-MS m/z (%
rel. abund.): 344 (M+, 5), 346 (M+2, 1), 173 (100), 139 (58), 111 (25), 83 (15).
2′,3′-Dichloro-N'-(3-chlorobenzoyl)benzohydrazide (28) [CAS # 1182660-05-4]
˚
1
Yield: 74%; M.p.: 186-188 C; H-NMR (400 MHz, DMSO-d6): 10.79 (s, 1H, NH), 10.57
(s, 1H, NH), 7.96 (s, 1H, H-2), 7.89 (d, J4′,5′ = 7.6 Hz, 1H, H-4′), 7.78 (m, 1H, H-5′), 7.68 (d,
J6′,5′ = 8.0 Hz, 1H, H-6′), 7.58 (t, J5,4/5,6 = 8.0 Hz, 1H, H-5) 7.50 (overlapping multiplete, 2H,
H-4, H-6); EI-MS m/z (% rel. abund.): 342 (M+, 9), 344 (M+2, 8), 346 (M+4, 3), 173. (83),
139 (100), 111 (27), 75 (12).
3-Chloro-N'-(4′-methoxybenzoyl)benzohydrazide (29) [CAS # 316151-65-2]
˚
1
Yield: 70%; M.p.: 206-208 C; H-NMR (400 MHz, DMSO-d6):  10.55 (s, 1H, NH), 10.39
(s, 1H, NH), 7.93 (overlapping multiplete, 4H, H-2 H-6, H-4′, H-2′), 7.68 (d, J6′,5′ = 8.0 Hz,
27

1H, H-6′), 7.58 (t, J5′,6′/5′,4′ = 7.6 Hz, 1H, H-5′), 7.05 (d, J3,5/2,6 = 8.4 Hz, 2H, H-3, H-5), 3.82
(s, 3H, OCH3); EI-MS m/z (% rel. abund.): 304 (M+, 11), 306 (M+2, 4), 135 (100), 111 (32),
92 (19), 77 (25).
N'-(3′-Chlorobenzoyl)-2-iodobenzohydrazide (30) [CAS # 812686-07-0]
˚
1
Yield: 68%; M.p.: 202-204 C; H-NMR (300 MHz, DMSO-d6):  10.70 (s, 2H, NH, NH),
7.94 (overlapping multiplete, 2H, H-2′, H-6′), 7.86 (d, J6,5/5,6 = 7.8 Hz, 2H, H-5, H-6), 7.70
(d, J4,3/5,4 = 8.7 Hz, 2H, H-3, H-4), 7.59 (overlapping multiplete, 2H, H-4′, H-5′); EI-MS m/z
(% rel. abund.): 308 (32), 310 (M+2, 20), 231 (19), 139 (100), 111 (56), 75 (17).
N'-(3′-methylbenzoyl)-2-iodobenzohydrazide (31) [CAS # 304480-86-2]
˚
1
Yield: 65%; M.p.: 194 C; H-NMR (300 MHz, DMSO-d6):  10.53 (s, 1H, NH), 10.30 (s,
1H, NH), 7.94 (d, J6,5 = 7.8 Hz, 1H, H-6), 7.70 (overlapping multiplete, 2H, H-3, H-6′), 7.40
(overlapping multiplete, 2H, H-5, H-4), 7.25 (m, 1H, H-5′), 7.23 (overlapping multiplete, 3H,
H-2′, H-3′, H-4′), 2.37 (s, 3H, CH3); EI-MS m/z (% rel. abund.): 380 (M+, 32), 231 (78), 202
(12), 119 (100), 91 (28).
3,5-Dichloro-N'-(3′-chlorobenzoyl)benzohydrazide (32) [CAS # 1182308-11-17]
˚
1
Yield: 78%; M.p.: 280-282 C; H-NMR (400 MHz, DMSO-d6):  10.80 (overlapping
multiplete, 2H, NH, NH), 7.93 (overlapping multiplete, 5H, H-2, H-4, H-6, H-2′, H-6′), 7.69
(d, J4′,5′ = 7.6 Hz, 1H, H-4′), 7.58 (t, J5′,6′/5′,4′ = 7.6 Hz, 1H, H-5′); EI-MS m/z (% rel.abund.):
344 (M+, 21), 346 (M+2, 7), 173 (80), 140 (100), 111 (80), 75 (24).
3,5-Dichloro-N'-(4′-chlorobenzoyl)benzohydrazide (33) [CAS # 909221-15-4]
˚
1
Yield: 65%; M.p.: 180 C; H-NMR (400 MHz, DMSO-d6):  10.76 (s, 1H, NH), 10.71 (s,
1H, NH), 7.93 (overlapping multiplete, 5H, H-2, H-4, H-6, H-2′, H-6′), 7.61 (d, J3′,5′/2′,6′ = 8.4
Hz, 2H, H-3′, H-5′); EI-MS m/z (% rel. abund.): 342 (M+, 10), 344 (M+2, 9), 346 (M+4, 3),
173 (39), 139 (100), 111 (50), 75 (22).
3,5-Dichloro-N'-(4′-methylbenzoyl)benzohydrazide (34) [CAS # 909221-07-4]
˚
1
Yield: 70%; M.p.: 214-216 C; H-NMR (400 MHz, DMSO-d6):  10.69 (s,1H, NH), 10.52
(s, 1H, NH), 10.35 (s, 1H, OH), 7.92 (overlapping multiplete, 3H, H-2, H-4, H-6), 7.82 (d,
J2′,6′/3′,5′ = 8.0 Hz, 2H, H-2′, H-6′), 7.32 (d, J3′,5′/2′,6′ = 7.6 Hz, 2H, H-3′ H-5′), 2.37 (s, 3H,
CH3); EI-MS m/z (% rel. abund.): 322 (M+, 3), 324 (M+2, 2), 173(10), 119 (100), 91 (26).
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3,5-Dichloro-N'-(3′-methylbenzoyl)benzohydrazide (35) [CAS # 909220-95-7]
˚
1
Yield: 59%; M.p.: 244-246 C; H-NMR (400 MHz, DMSO-d6):  10.71 (s, 1H, NH), 10.54
(s, 1H, NH), 7.92 (overlapping multiplete, 3H, H-2, H-4, H-6), 7.73 (overlapping multiplete,
2H, H-6′, H-2′), 7.40 (overlapping multiplete, 2H, H-5′, H-4′), 2.37 (s, 3H, CH3); EI-MS m/z
(% rel. abund.): 322 (M+, 16), 324 (M+2, 8), 326 (M+4, 2), 173 (34), 145 (13), 119 (100), 91
(74), 65 (13).
3,5-Dichloro-N'-(4′-methoxybenzoyl)benzohydrazide (36) [CAS # 909221-09-6]
˚
Yield: 68%; M.p.: 192 C; 1H-NMR (400 MHz, DMSO-d6):  10.66 (overlapping multiplete,
2H, NH, NH), 7.91 (overlapping multiplete, 4H, H-2, H-6, H-2′, H-6′), 7.05 (overlapping
multiplete, 3H, H-4, H-3′, H-5′), 3.82 (s, 3H, OCH3); EI-MS m/z (% rel. abund.): 338 (M+, 9),
340 (M+2, 5), 300 (31), 173 (44), 135 (100), 107 (15), 92 (20), 77 (24).
2′,3′-Dichloro-N'-(3,5-dichlorobenzoyl)benzohydrazide (37)
˚
1
Yield: 69%; M.p.: 240-242 C; H-NMR (400 MHz, DMSO-d6):  10.91 (s, 1H, NH), 10.65
(s, 1H, NH), 7.94 (overlapping multiplete, 2H, H-4, H-4′), 7.79 (overlapping multiplete, 2H,
H-3′, H-6′), 7.50 (d, J2,4/6,4 = 4.8 Hz, 2H, H-2, H-6); EI-MS m/z (% rel. abund.): 378 (M+, 7),
380 (M+2, 3), 173 (100), 145 (19), 109 (6).
2′,4′-Dichloro-N'-(3,5-dichlorobenzoyl)benzohydrazide (38) [CAS # 909221-11-0]
˚
1
Yield: 68%; M.p.: 270-271 C; H-NMR (400 MHz, DMSO-d6):  10.88 (s, 1H, NH), 10.62
(s, 1H, NH), 7.93 (overlapping multiplete, 4H, H-2, H-6, H-5′, H-6′), 7.75 (s,1H, H-3′), 7.56
(s, 1H, H-4); EI-MS m/z (% rel. abund.): 378 (M+, 8), 380 (M+2, 4), 173 (100), 145 (26), 109
(10).
2,4-Dichloro-N'-(2′,4′-dichlorobenzoyl)benzohydrazide (39) [CAS # 76114-78-8]
˚
1
Yield: 74%; M.p.: 264-265 C; H-NMR (400 MHz, DMSO-d6):  10.64 (s, 2H, NH, NH),
7.74 (s, 2H, H-3, H-3′), 7.55 (s, 4H, H-6, H-5, H-6′, H-5′); EI-MS m/z (% rel. abund.): 378
(M+, 7), 380 (M+2, 3), 173 (100), 145 (25), 109 (12).
29