Synthesis and urease inhibitory activities of benzophenone semicarbazones/thiosemicarbazones

Article abstract of DOI:10.1007/s00044-016-1673-0

Twenty-five benzophenone semicarbazones and thiosemicarbazones 3–27 were synthesized starting from benzophenones via hydrazones treated with different aryl isocyanates and isothiocyantes under reflux. All synthetic derivatives were evaluated for their urease inhibitory potential. Good to moderate inhibition trend against urease was observed with the IC₅₀ values in the range of 8.7–119.5 μM, when compared with the standard thiourea (IC₅₀ = 21.2 ± 1.3 μM). Compound 15 showed better inhibition than the standard having the IC₅₀ value of 8.7 ± 0.6 μM. Compounds 3, 4, 8, 11–14, 16, and 17 with the IC₅₀ values within the range of 26.1 to 43.6 μM, demonstrated good to moderate activities while compound 9 (IC₅₀ = 119.5 ± 1.6 μM) displayed very weak activity. The enzyme kinetic studies on the most active compounds 15 and 17 were performed to deduce their modes of inhibition and dissociation constants K_i.

Full text of DOI:10.1007/s00044-016-1673-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1673-0
ORIGINAL RESEARCH
Synthesis and urease inhibitory activities of benzophenone
semicarbazones/thiosemicarbazones
Arshia Arshia1 Ajmal Khan1,2 Khalid Mohammed Khan1
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Syed Muhammad Saad1 Nida Iqbal Siddiqui3 Sumaira Javaid1 Shahnaz Perveen4
M. Iqbal Choudhary1,5
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Received: 19 December 2015 / Accepted: 4 July 2016
© Springer Science+Business Media New York 2016
Abstract Twenty-five benzophenone semicarbazones and
thiosemicarbazones 3–27 were synthesized starting from
benzophenones via hydrazones treated with different aryl
isocyanates and isothiocyantes under reflux. All synthetic
derivatives were evaluated for their urease inhibitory
potential. Good to moderate inhibition trend against urease
was observed with the IC50 values in the range of
8.7–119.5 µM, when compared with the standard thiourea
(IC50 = 21.2 1.3 µM). Compound 15 showed better
inhibition than the standard having the IC50 value of
8.7 0.6 µM. Compounds 3, 4, 8, 11–14, 16, and 17 with
the IC50 values within the range of 26.1 to 43.6 µM,
demonstrated good to moderate activities while compound
9 (IC50 = 119.5 1.6 µM) displayed very weak activity.
The enzyme kinetic studies on the most active compounds
15 and 17 were performed to deduce their modes of inhi-
bition and dissociation constants Ki.
●
●
Keywords Benzophenone Semicarbazone
●
●
Thiosemicarbazone Ureases inhibition Structure-activity
relationship
Introduction
Semicarbazones and thiosemicarbazones are organic com-
pounds having the general formulae R1R2C=N–NH(C=O)
NH2 and R1R2C=N–NH(C=S)NH2, respectively. These
moieties are formally synthesized by condensation of
aldehyde or ketone with semicarbazide and thiosemicarba-
zide, respectively. Benzophenone molecules are extensively
used in the field of agriculture and many fungicides, like
flumorph (Yuan et al., 2006), have been reported to contain
benzophenone as a key skeleton. This skeleton is associated
with many pharmacological activities such as anticancer
(Hsieh et al., 2003), antimicrobial (Sujarani and Ramu,
2013), anti-inflammatory (Khanum et al., 2010), anti-
human immunodeficiency virus (HIV; Ma et al., 2011;
Piccinelli et al., 2005), and antioxidant (Stanojević et al.,
2013). Thiosemicarbazone nuclei have wide spectrum of
applications in coordination chemistry (Singh and Prakash,
1992) and have multiple medicinal features like antimalarial
(de Oliveira et al., 2008), anticonvulsant (Taroua et al.,
1996), antimicrobial (Kasuga et al., 2003), and anti-HIV
(Mishra et al., 1998). Benzophenone semicarbazones and
thiosemicarbazones are classes of pharmacologically active
* Khalid Mohammed Khan
khalid.khan@iccs.edu
iqbal.choudhary@iccs.edu
* M. Iqbal Choudhary
khalid.khan@iccs.edu
iqbal.choudhary@iccs.edu
1
H. E. J. Research Institute of Chemistry, International Center for
Chemical and Biological Sciences, University of Karachi, Karachi
75270, Pakistan
2
Department of Chemistry, COMSATS Institute of Information
Technology, Abbottabad 22060, Pakistan
3
Department of Chemistry, Federal Urdu University of Arts,
Sciences and Technology, Gulshan-e-Iqbal, Science Campus,
Karachi 75300, Pakistan
4
PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr Salimuzza-
man Siddiqui, Karachi 75280, Pakistan
5
Department of Biochemistry, Faculty of Sciences, King Abdulaziz
University, Jeddah 21412, Saudi Arabia

Med Chem Res
compounds, which show broad spectrum of medicinal
properties such as anti-asthmatics (Kim et al., 2003),
cathepsin L inhibitors (Kumar et al., 2010), antimalarial
(Pingaew et al., 2010), and anticonvulsant activities
(Yogeeswari et al., 2005). Benzophenone containing
molecules are also involved in adhesive and coatings,
photo-initiators as well as in optical fibers (Karahan et al.,
2014; Wang et al., 2010; Wang et al., 2014; Wang et al.,
2009). These properties mainly arise due to variation in
stereochemistry and modes of bondings through oxygen
and azomethine nitrogen atoms of benzophenone semi-
carbazones and thiosemicarbazones. As these molecules
coordinate to metal centre, both as neutral or as anionic
ligands, hence study of these molecules is a subject of
interest to the chemists (Reena et al., 2008).
Urease (EC 3.5.1.5), also called urease aminohydrolase,
is a two nickel-containing metalloenzyme. It is found in
bacteria, fungi, and higher plants (Matongo and Nwodo,
2014; Uddin et al., 2013; Weber et al., 2008). Urease cat-
alyzes the degradation of urea into ammonia and carbon
dioxide. This results in localized increase in pH. Helico-
bacter pylori is a ureolytic bacterium that survives in low
pH of human stomach due to urease activity. It is the main
cause of gastric and duodenal ulcers which may lead to
cancer. High secretion of stomach acid in response of
H. pylori colonization destroys mucosal membrane and
causes gastric ulcers (Kato et al., 2012; Millo et al., 2012).
Ureolytic bacteria are also responsible for kidney stones and
pyelonephritis (Mobley, 1996). It also contributes to the
gastroduodenal inflammation (Blaser, 1990).
Fig. 1 Benzophenone hydrazones and benzophenone (thio)semi-
carbazones 3–27
analogs demonstrated the encouraging results (Khan et al.,
2011).
Recently, we have reported benzophenone hydrazones as
a new class of uresae inhibitors (Khan et al., 2015). Incor-
poration of a carboxoamidic (CONH) or carbothioamidic
(CSNH) functional group in these molecules may result in
better urease inhibitory potentials (Fig. 1). Therefore,
in the present study, we synthesized 25 benzophenone
semicarbazones and thiosemicarbazones (3–27) and eval-
uated their inhibitory potential against urease in vitro
(Table 1). Encouraging results were found and are discussed
in forthcoming paragraphs. Compounds 5–9, 12–17, and
19–27 were found to be new compounds; while compounds
2a, 2b, 3, 4, 10, 11, and 18 were identified as previously
reported compounds (Barton et al., 1962; Ferraz et al.,
2012; Glinma et al., 2014; Pearson et al., 1953; Joshi et al.,
1981; Singh et al., 2011).
Results and discussion
Chemistry
In order to resolve urease-hyperactivity based compli-
cations, our research group has been involved in designing
urease inhibitors since last decade. In this connection, we
have synthesized several classes of organic compounds for
their urease inhibitory potential assesment. One of our
synthesized urea derivatives, N-4-nitrophenyl-N′-4′-nitro-
phenylurea showed excellent urease inhibition potential
against the standard thiourea (Perveen et al., 2008). Among
the library of synthetic biscoumarins, 3,3′-methylenebis-(4-
hydroxycoumarin) was found to be the most active analog
(Khan et al., 2004). Working on oxadiazoles, we explored
5-(hydroxybenzyl)-2-(4-methylphenyl)amino-1,3,4-oxadia-
zole as an excellant urease inhibitor (Akhtar et al.,
2010). Some acridine and piperidine analogs were also
found to have promising biological activity for the urease
inhibition (Khan et al., 2007; Khan et al., 2006). Pyridine
and triazole nuclei, such as, 4,6-bis(4-methoxyphenyl)-
5,6-dihydropyridin and 4-amino-3-[1-(4-methylbenzene-
sulfon-amido)-2-methylpropyl]-5-thioxo-1H,4H-1,2,4-tria-
zole displayed the urease inhibitory potential comparable
to the standard thiourea (Hameed et al., 2013; Akhtar
et al., 2008). A class of arylidene barbiturates was also
subjected to urease inhibition. Regioisomeric fluorinated
Twenty-five derivatives of benzophenone semicarbazones/
thiosemicarbazones (3–27) were prepared in two-steps
(Scheme 1). First of all, benzophenones (1a and 1b) were
reacted with hydrazine hydrate to have corresponding
hydrazones (2a and 2b). These hydrazones were later
condensed with different aryl isocyanates and iso-
thiocyantes to build a library of benzophenone semi-
carbazones/thiosemicarbazones. Chemical structures of
1
these derivatives were confirmed by H-nuclear magnetic
resonance (NMR), 13C-NMR, electron impact mass spectra
(EI-MS) and infrared (IR) spectroscopy. All synthesized
compounds gave agreeable elemental analyses and high-
resolution electron ionization mass spectrometry (HREI-
MS) data.
Characteristic spectral feature of representative
compound 15
1H- and 13C-NMR spectra of compound 15 were recorded
in deuterated dimethyl sulfoxide (DMSO) on a Bruker AM
1
400 MHz instrument. In H-NMR spectrum, signals at δH

Med Chem Res
10.05 and 10.41 ppm indicated the presence of amidic
protons in the molecule. The hydroxyl proton showed a
signal at δH 9.03 ppm. Three aromatic rings are present in
the structure; among them the most deshielded singlet at δH
7.85 ppm was due to H-2′′ (Ring C). The electron-with-
drawing effect of neighboring N–C=S substituent is
apparently responsible of downfield chemical shift of H-2′′.
C-4 proton (Ring B) appeared as a doublet at δH 7.72 ppm
(J4,3 = J4,5 = 6.8 Hz). Protons on the aromatic ring A having
the hydroxy substituent at C-4′ appeared at upfield region in
the range of δH 6.78–7.56 ppm. This is because of the
electron-donating effect of hydroxyl group. The remaining
aromatic protons appeared between δH 7.65–7.30 ppm
(Fig. 2).
Table 1 Urease inhibitory activity of benzophenone semicarbazones
and thiosemicarbazones 3–27
Compounds Structure
IC50 SEM
(μM)
X
R
Ar
3
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
O
O
O
O
O
H
H
H
H
H
H
H
H
Phenyl
41.2 0.9
43.6 0.5
NA
In the 13C-NMR spectrum, a total 16 carbons appeared
including 9 methine and 7 quarternary carbons. The most
downfield signals at δC 175.5 and 159.5 ppm corresponded
to iminic and thiocarbonylic carbons, respectively. Aro-
matic carbon directly attached to electronegative oxygen
i.e., C-4′ (ring A) also appeared downfield at δC 150.9 ppm.
The C-3′ and C-5′ adjacent to hydroxy group, resonated
upfield at δC 115.2 ppm due to the mesomeric effect of the
ortho hydroxy group. All remaining aromatic carbons
appeared in the usual aromatic range of δC 140.5–116.4
ppm (Fig. 3).
4
3′′-Chlorophenyl
2′′,3′′-Dichlorophenyl
2′′,5′′-Dichlorophenyl
3′′,4′′-Dichlorophenyl
3′′-Bromophenyl
4′′-Bromophenyl
4′′-Nitrophenyl
5
6
NA
7
NA
8
35.8 0.2
119.5 1.6
NA
9
10
11
12
13
14
15
16
17
18
19
20
21
22
OH Phenyl
28.0 0.2
38.0 1.3
31.1 0.1
29.9 1.7
8.7 0.6
28.7 0.8
26.1 0.2
NA
OH 2′′,3′′-Dichlorophenyl
OH 2′′,4′′-Dichlorophenyl
OH 2′′,5′′-Dichlorophenyl
OH 3′′-Bromophenyl
OH 4′′-Bromophenyl
OH 4′′-Nitrophenyl
H
H
H
H
H
High-resolution EI-MS of compound 15 showed M+ at
m/z 425.0188 with a composition of C20H16BrN3OS (calcd
425.0197). The isotopic [M++2] peak at m/z of 427 indi-
cated the presence of a bromine (98 % isotopic relative
abundance) and sulfur (4.4 % isotopic relative abundance)
atoms in a molecule. Neutral losses in the forms of H2S, 3-
Br-C6H4-NCS, 3-Br-C6H4-NH2, and (3-OH-C6H4)(Ph)
C=NNH2 yielded signals at m/z of 391, 212, 254, and 213,
respectively. Cleavage of N–N bond resulted in nitrenium
ion at m/z of 196 as the base peak. Cleavage of adjacent
Phenyl
3′′-Chlorophenyl
3′′-(Trifluoromethyl)phenyl
4′′-(Trifluoromethyl)phenyl
2′′-Chloro-5′
′-(trifluoromethyl)phenyl
NA
NA
NA
NA
23
O
H
4′′-Chloro-2′
′-(trifluoromethyl)phenyl
NA
24
25
26
O
O
O
OH 3′′-Chlorophenyl
OH 4′′-(Trifluoromethyl)phenyl
NA
NA
NA
OH 2′′-Chloro-5′
′-(trifluoromethyl)phenyl
27
O
OH 4′′-Chloro-2′
NA
′-(trifluoromethyl)phenyl
Standard: Thiourea, IC50 SEM = 21.2 1.3 µM
SEM standard error of the mean, NA not active
Fig. 2 1H-NMR analysis of compound 15
Scheme 1 Synthesis of benzophenone semicarbazones and thiosemicarbazones 3–27

Med Chem Res
N–C bond generated a cation at m/z of 211. Peak at m/z 348
was due to stable aromatic (triazolethione) cation arose
through the removal of phenyl radical, followed by intra-
molecular cyclization. Loss of a mercapto radical from the
parent molecule yielded an oxonium ion at m/z 392 having
diaziridine ring in the middle (Fig. 4).
The presence of the main functionalities of compound 15
was further justified with the help of fourier transform
infrared (FT-IR) spectroscopy. The FT-IR spectrum showed
the stretching vibrations at 3555 and 3283 cm−1 that cor-
responded to the O–H and N–H bonds, respectively. The
iminic (C=N) and aromatic (C=C) bonds vibrated at 1583
and 1533 cm−1, respectively. Vibrational stretching of
thiocarbonylic bond (C=S) appeared at 1277 cm−1 (Fig. 5).
Enzyme inhibitory studies
Survey on limited SAR studies of 25 synthetic derivatives
of benzophenone semicarbazones/thiosemicarbazones (3–
27) suggested that substituting different groups on the aryl,
as well as CONH/CSNH parts of the molecules are
responsible for urease inhibitorty activity of the compounds
(Fig. 6).
Fig. 3 13C-NMR analysis of compound 15
Fig. 4 EI-MS fragmentation
pattern of compound 15

Med Chem Res
Compounds 3,4, 8, 9, and 11–17 displayed varying
inhibitory potential of enzyme having IC50 values in the
range of 8.7 0.6–119.5 1.6 µM, in comparison with the
standard thiourea (IC50 = 21.2 1.3 µM). Nonetheless,
compounds 5–7, 10, and 18–27 were identified as entirely
inactive.
Among the library of synthesized 25 compounds, com-
pound 15 (IC50 = 8.7 0.6 µM) demonstrated the highest
urease inhibition. It is about threefolds more active than the
standard urea. The attachment of bromo group at C-3′′ and
hydroxyl group at C-4′ of aryl thiosemicarbazone provide
the best configuration mode to interact with the active site
of the enzyme. Migration of this bromo group to C-4′′, as in
compound 16 (IC50 = 28.7 0.8 µM), suppressed the inhi-
bition to three times. Similar trend was observed when the
bromo group changed with hydrogen as in compound 11
(IC50 = 28.0 0.2 µM).
Among the dichlorinated analogs, keeping the CSNH part
constant (when OH group at C-4′), different positions of both
chlorine groups on the aryl part of molecule are responsible
for increasing or decreasing inhibitory pattern which is para
(Compound 14; IC50 = 29.9 1.7 µM), meta (Compound 13;
Fig. 5 FT-IR analysis of compound 15
IC50 = 31.1 0.1 µM), and ortho (Compound 12; IC50
=
38.0 1.3 µM). Interestingly, when hydroxyl at C-4′ of
Table 2 Kinetic data of most potent benzophenone thiosemicarbazones
15 and 17
Compounds
Ki SEM (μM)
Type of inhibition
15
17
6.76 0.002
14.18 0.002
Mixed
Mixed
Ki dissociation constant, SEM standard error of the mean
Fig. 6 Benzophenone semicarbazones and thiosemicarbazones 3–27
Fig. 7 The mode of inhibition of
urease by compound 15, a is the
Lineweaver–Burk plot of
A
B
0.1
0.1
reciprocal rate of reaction
0.05
0
(velocities) versus reciprocal of
substrate (urea) in the absence
(▼), and in the presence of 4
0
(▲), 8 (Δ), 16 (■), 32 µM (□)
of compound 15. b Is secondary
-0.05
-0.1
replot of Lineweaver–Burk plot
between the slopes of each line
on Lineweaver–Burk plot versus
different concentrations of
-0.1
-2
-1
0
1
2
-40
-20
0
20
40
compound 15 and c is Dixon
plot of reciprocal rate of reaction
(velocities) versus different
1/Substrate
Inhibitor
C
concentrations of compound 15
0.2
0.1
0
-0.1
-0.2
-30
-15
0
15
30
Inhibitor

Med Chem Res
Fig. 8 The mode of inhibition of
urease by compound 17, a is the
Lineweaver–Burk plot of
A
B
0.15
0.075
0
0.06
0.04
0.02
0
reciprocal rate of reaction
(velocities) versus reciprocal of
substrate (urea) in the absence
(▼), and in the presence of 6.25
(▲), 12.5(Δ), and 25 µM (■)
of compound 17. b Is secondary
replot of Lineweaver–Burk plot
between the slopes of each line
on Lineweaver–Burk plot versus
different concentrations of
-0.02
-0.04
-0.06
-0.075
-0.15
-2 -1.5 -1 -0.5
0
0.5
1
1.5
2
-20
-10
0
10
20
1/Substrate
Inhibitor
compound 17 and c is Dixon
plot of reciprocal rate of reaction
(velocities) versus different
C
0.2
concentrations of compound 17
0.1
0
-0.1
-0.2
-20
-10
0
10
20
Inhibitor
CSNH part was replaced with hydrogen keeping the posi-
tions of chlorines intact as in compounds 5, 6, and 7, the
activitity of compounds was dramatically diminished.
However, among the monohalogenated analogs for the
non-hydroxylated thiosemicarbazones, inhibitory activity
was observed. Compound 8 (IC50 = 35.8 0.2 µM) with
bromo group at C-3′′ was found to be modertaely active.
Bromo group at C-4′′ lowered the activity to threefolds as in
compound 9 (IC50 = 119.5 1.6 µM). A slight decline in
activity was observed when bromo group in compound 8
concentrations of substrate urea (0.5–4.0 mM). Inhibition
type was deduced by Lineweaver–Burk plots, where the
reciprocal of the rates of the reaction were plotted against
the reciprocal of substrate concentration to monitor
the effect of inhibitor on both Km and Vmax. The Ki values
were determined by secondary re-plot of Lineweaver–Burk
plots where the slope of each line in the Lineweaver–Burk
plots was plotted against different concentrations of
inhibitor. The Ki values were deduced by Dixon plot where
the reciprocal of the rate of reaction were plotted against
different concentrations of inhibitors (Table 2). Kinetic
studies showed that compounds 15 and 17 inhibited urease
in mixed modes as both Km and Vmax was found to be
affected (Figs. 7 and 8). This indicates that these com-
pounds could interact at the allosteric site or active site of
the enzyme.
was replaced by a chloro group as in compound 4 (IC50
43.6 0.5 µM).
=
Nitro group at para position was not sufficient enough to
display activity until it is hydroxylated at C-4′ as in com-
pound 17 (IC50 = 26.1 0.2 µM). An inactivity may be seen
in compound 10 which lacks hydroxy group at C-4′ but has
nitro group at C-4′′.
All activities discussed so far must have some connection
with sulphur as well. The clear-cut non-compatibilty with the
enzyme may be seen when the sulphur was replaced with
oxygen (semicarabazone derivatives 18–27). However,
thiosemicarbazone derivatives rendered varied inhibitory
potential that may be correlated with combination of sulphur,
hydroxy at C-4′, and different substitutions on the aryl part.
Conclusion
We have synthesized 25 differently substituted benzophe-
none semicarbazones and thiosemicarbazones and screened
them against urease. Compound 15 was found to have IC50
value below the standard thiourea. While compounds, 3, 4,
8, 9, 11–14, 16, and 17 displayed good to moderate IC50
values within the range of 26.1–43.6 µM. Further studies
may help in the development of these molecules as a
potential leads for the urease-hyperactivity based
complications.
Kinetic studies
Type of inhibition by most active compounds 15 and 17
was determined by kinetic studies using various

Med Chem Res
Experimental
4.0 mM). Production of ammonia was measured con-
tinuously at 630 nm for 50 min on an ELISA plate reader,
after the addition of phenol and alkali reagent.
Material and methods
Acetic acid, hydrazine hydrates, benzophenones, and dif-
ferent aryl isocyanates/isothiocyantes, type IX Jack bean
urease (EC 3.5.1.5), urea, thiourea, di-sodium hydrogen
phosphate, mono-sodium di-hydrogen phosphate, and phe-
nol were purchased from Sigma-Aldrich (USA). Acetoni-
trile was dried by charging it with 3 Å molecular sieves. All
chemicals were used as acquired without purification.
1H-NMR and 13C-NMR spectra were recorded on
Avance Bruker AM 300 and 400 MHz machines. EI-MS
were run on a Finnigan MAT-311A (Germany) Mass
Spectrometer. Melting points of the compounds were
recorded on Stuart® SMP10 melting point apparatus. IR
spectra (KBr discs) were recorded on a FTS 3000 MX, Bio-
RAD Merlin (Excalibur Model) spectrophotometer. CHN
analyses were operated on a Carlo Erba Strumentazione-
Mod-1106, Italy. Thin layer chromatography (TLC) ana-
lyses were performed on pre-coated silica gel aluminum
plates (Kieselgel 60, 254, E. Merck, Germany). TLC
chromatograms were visualized under ultraviolet lamp at
254 and 365 nm.
Statistical analysis
All experiments were carried out in triplicate. The results
were processed via Softmax Pro Software (Molecular
Devices, CA, USA). The percentage of inhibition was
calculated using the formula given below.
% Inhibition ¼100 ꢀ ðAbsorbance of test compound=
Absorbance of controlÞ ´ 100
The IC50 of the active compounds was evaluated by
measuring the effect of different concentrations of inhibitors
on production of ammonia. The IC50 values were deduced
via EZ-Fit enzyme kinetics program (Perellela Scientific,
Inc., Amherst, USA). To determine the mechanism of
inhibitor binding to enzyme, Lineweaver−Burk plot,
secondary plot, and Dixon plot were plotted by using
Grafit 7 software (Erithacus Software Limited, UK).
Grafit 7 program was used to evaluate different kinetic
parameters.
General procedure for the synthesis of compounds 2a
and 2b
Assay for urease inhibitory activity
The urease inhibition assay was performed spectro-
photometrically. In the 96-well plate, 25 µL of urease
solution (1 U/well) was incubated with 5 µL of test com-
pound (250 µM) for 15 minutes, at 30 °C. Thereafter, 55 µL
of urea (substrate) with concentration of 100 mM, was
added and the plate was again incubated for 10 min at
30 °C. After incubation, 45 µL of phenol (1 % w/v phenol
and 0.005 % w/v sodium nitroprusside), and 70 µL of alkali
reagents (0.5 % w/v sodium hydroxide and 0.1 % sodium
hypochlorite) were added to each well. The plate was again
incubated for 50 min at 30 °C. Urease activity was measured
with the rate of production of ammonia continuously fol-
lowing the method of Weatherburn (Weatherburn, 1967),
and change in absorbance was monitored at 630 nm on a
ELISA plate reader (Spectra Max M2, Molecular Devices,
CA, USA). Thiourea was used as a standard compound.
Benzophenone or 4′-hydroxybenzophenone (1 mmol) was
taken in hydrazine hydrate (10 mL) and refluxed for 6 h
catalyzed by acetic acid (2 mL; Scheme1). Progress of
chemical reaction was periodically monitored by TLC
analysis. When the reaction completed, water was added
until precipitation. These precipitates were filtered and dried
under vacuum.
General procedure for the synthesis of compounds 3–27
Benzophenone semicarbazones and thiosemicarbazones 3–
27 were synthesized by refluxing benzophenone hydrazones
(2a or 2b; 2 mmol) and substituted aryl isocyanates or
isothiocyanates (2 mmol) in acetonitrile (15 mL) for 24 h.
When TLC analysis suggested the completion of reaction,
the mixtures were left at room temperature to be cooled
down which resulted in precipitation. The precipitates were
filtered and dried under vacuum at 40 °C to afford good
yields of the title compounds.
Kinetic studies protocol
Mechanistic studies were carried out to determine the
mechanism of inhibitors, binding to enzyme. Inhibitors
could bind enzyme in a competitive, non-competitive,
mixed, or uncompetitive way. Briefly the enzyme solution
(1 U/200 µL) was incubated with different concentrations of
inhibitor for 15 min at 30 °C. The reaction was then
started by adding different concentrations of substrate (0.5–
(Diphenylmethylene)hydrazine (2a)
Yield: 90 %; White solid; m.p. 99–100 °C (lit. 97–98 °C;
Barton et al., 1962); Rf: 0.35 (ethyl acetate/hexanes, 2:8);
1H-NMR (400 MHz, DMSO-d6): δH 7.56 (t, 2H, J3(2,4)
=
J5(4,6) = 7.6 Hz, H-3, H-5), 7.48 (t, 1H, J4(3,5) = 7.2 Hz, H-

Med Chem Res
4), 7.33 (d, 2H, J2,3 = J6,5 = 7.2 Hz, H-2, H-6), 7.28 (t, 2H,
J3′(2′,4′) = J5′(4′,6′) = 7.2 Hz, H-3′, H-5′), 7.22 (m, 3H, H-2′,
H-4′,H-6′), 6.22(s, 2H, NH2); 13C-NMR: (100 MHz,
DMSO-d6): δC 144.4 (C=N), 138.8 (C-1), 133.0 (C-1′),
129.3 (C-2, C-6), 128.6 (C-2′, C-6′), 128.4 (C-4), 128.0 (C-
4′), 127.1 (C-3, C-5), 125.3 (C-3′, C-5′); EI-MS: m/z (rel.
abund. %), 196 [M]+ (72.6), 180 (76.1), 165 (91.4), 105
(26.1), 77 (100.0); HREI-MS: m/z calcd for C13H12N2 [M]+
196.1000 found 196.1003; IR (KBr, cm−1): 3430 (N–H),
1630 (C=N), 1550 (C=C); Anal. calcd for C13H12N2: C,
79.56; H, 6.16; N, 14.27; Found: C, 79.58; H, 6.15; N, 14.29.
N-(3′′-Chlorophenyl)-2-(diphenylmethylene)
hydrazinecarbothioamide (4)
Yield: 72 %; White solid; m.p. 128–130 °C; Rf: 0.37 (ethyl
1
acetate/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): δH
10.47 (s, 1H, NH), 8.98 (s, 1H, NH), 7.72 (m, 3H, H-2, H-6,
H-4), 7.64 (s, 1H, H-2′′), 7.62 (d, 2H, J2′,3′ = J6′,5′ = 7.8 Hz,
H-2′, H-6′), 7.57 (d, 1H, J4′′,5′′ = 8.1 Hz, H-4′′), 7.44 (m,
1H, H-5′′), 7.42 (m, 3H, H-3′, H-4′, H-5′), 7.37 (m, 2H, H-
3, H-5), 7.30 (d, 1H, J6′′,5′′ = 9.0 Hz, H-6′′); 13C-NMR:
(100 MHz, DMSO-d6): δC 176.0 (C=S), 150.4 (C=N),
140.3 (C-3′′), 136.1 (C-1′′), 132.2 (C-2′′), 131.3 (C-1),
130.0 (C-1′), 129.8 (C-4′′), 129.7 (C-2, C-6), 128.8 (C-2′,
C-6′), 128.3 (C-6′′), 128.0 (C-3, C-5), 127.9 (C-3′, C-5′),
125.3 (C-5′′), 125.2 (C-4), 124.2 (C-4′); EI-MS: m/z (rel.
abund. %), 367 [M++2] (5.6), 365 [M]+ (14.0), 238 (24.2),
195 (21.3), 180 (100.0), 166 (66.5); HREI-MS: m/z calcd
for C20H16ClN3S [M]+ 365.0753, found 365.0756; IR (KBr,
cm−1): 3475 (N–H), 1587 (C=N), 1542 (C=C), 1298 (C–
N), 1286 (C=S); Anal. calcd for C20H16ClN3S: C, 65.65; H,
4.41; Cl, 9.69; N, 11.48; S, 8.76; Found: C, 65.67; H, 4.43;
N, 11.45.
(E)-[(4′-Hydroxyphenyl)(phenyl)methylene]hydrazine (2b)
Yield: 82 %; White solid; m.p. 130–132 °C (lit. 150 °C;
Joshi and Hari, 1981); Rf: 0.28 (ethyl acetate/hexanes,
2:8); 1H-NMR (300 MHz, DMSO-d6): δH 9.69 (s, 1H, OH),
7.33 (d, 2H, J2,3 = J6,5 = 7.6 Hz, H-2, H-6), 7.28 (m, 3H, H-
3, H-4, H-5), 7.02 (d, 2H, J2′,3′ = J6′,5′ = 8.4 Hz, H-2′,
H-6′), 6.92 (d, 2H, J3′,2′ = J5′,6′ = 8.4 Hz, H-3′, H-5′), 6.12
(s, 2H, NH2); 13C-NMR: (100 MHz, DMSO-d6): δC 157.4
(C-4′), 144.7 (C=N), 139.3 (C-1), 129.9 (C-2, C-6), 127.9
(C-4), 127.0 (C-3, C-5), 125.5 (C-2′, C-6′), 123.0 (C-1′),
116.0 (C-3′, C-5′); EI-MS: m/z (rel. abund. %), 212 [M]+
(100.0), 196 (25.2), 181 (25.2), 152 (15.6), 77 (38.8);
HREI-MS: m/z calcd for C13H12N2O [M]+ 212.0950 found
212.0952; IR (KBr, cm−1): 3465 (O–H), 3428 (N–H), 1635
(C=N), 1560 (C=C); Anal. calcd for C13H12N2O: C, 73.56;
H, 5.70; N, 13.20; O, 7.54; Found: C, 73.52; H, 5.73; N,
13.21.
N-(2′′,3′′-Dichlorophenyl)-2-(diphenylmethylene)
hydrazinecarbothioamide (5)
Yield: 71 %; Yellow solid; m.p. 178–180 °C; Rf: 0.32 (ethyl
1
acetate/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): δH
10.49 (s, 1H, NH), 9.15 (s, 1H, NH), 7.71 (d, 2H, J2,3 = J6,5
= 6.6 Hz, H-2, H-6), 7.66 (d, 2H, J2′,3′ = J6′,5′ = 7.5 Hz, H-
2′, H-6′), 7.62 (m, 2H, H-3, H-5), 7.45 (m, 7H, H-4′′, H-5′′,
H-6′′, H-3′, H-5′, H-4′, H-4); 13C-NMR: (100 MHz,
DMSO-d6): δC 176.9 (C=S), 150.3 (C=N), 138.5 (C-2′′),
136.2 (C-3′′), 131.6 (C-1′′), 131.3 (C-1), 130.0 (C-1′),
129.8 (C-4′′), 129.7 (C-2, C-6), 128.9 (C-2′, C-6′), 129.6
(C-6′′), 128.4 (C-5′′), 128.3 (C-3, C-5), 127.8 (C-3′, C-5′),
127.7 (C-4, C-4′); EI-MS: m/z (rel. abund. %), 364 [M+-Cl]
(87.0), 238 (19.1), 195 (40.8), 180 (99.0), 133 (29.2), 77
(100.0); IR (KBr, cm−1): 3413 (N–H), 1579 (C=N), 1531
(C=C), 1311 (C–N), 1215 (C=S); Anal. calcd for
C20H15Cl2N3S: C, 60.00; H, 3.78; Cl, 17.71; N, 10.50; S,
8.01; Found: C, 60.02; H, 3.76 N, 10.52.
2-(Diphenylmethylene)-N-phenylhydrazinecarbothioamide
(3)
Yield: 71 %; Brown solid; m.p. 164–166 °C (lit. 160–162 °C;
Singh et al., 2011); Rf: 0.56 (ethyl acetate/hexanes, 2:8); 1H-
NMR (300 MHz, DMSO-d6): δH 10.44 (s, 1H, NH), 8.81 (s,
1H, NH), 7.73 (d, 2H, J2,3 = J6,5 = 6.3 Hz, H-2, H-6), 7.67
(m, 3H, H-2′′, H-6′′, H-4′′), 7.54 (d, 2H, J2′,3′ = J6′,5′ = 7.8
Hz, H-2′, H-6′), 7.43 (m, 1H, H-4), 7.41 (m, 6H, H-3, H-5,
H-3′, H-5′, H-3′′, H-5′′), 7.25 (m, 1H, H-4′); 13C-NMR:
(100 MHz, DMSO-d6): δC 176.1 (C=S), 149.8 (C=N),
138.8 (C-1′′), 136.2 (C-1), 131.3 (C-1′), 130.0 (C-2, C-6),
129.8 (C-2′, C-6′), 128.3 (C-4), 128.1 (C-4′), 127.8 (C-3,
C-5), 125.9 (C-3′, C-5′), 125.7 (C-3′′, C-5′′), 124.3 (C-4′),
123.5 (C-2′′, C-6′′); EI-MS: m/z (rel. abund. %), 331 [M]+
(13.0), 298 (17.6), 254 (45.5), 197 (61.8), 180 (61.1);
HREI-MS: m/z calcd for C20H17N3S [M]+ 331.1143 found
331.1146; IR (KBr, cm−1): 3435 (N–H), 1636 (C=N), 1542
(C=C), 1386 (C–N), 1290 (C=S); Anal. calcd for
C20H17N3S: C, 72.48; H, 5.17; N, 12.68; S, 9.67; Found: C,
72.46; H, 5.19; N, 12.67.
N-(2′′,5′′-Dichlorophenyl)-2-(diphenylmethylene)
hydrazinecarbothioamide (6)
Yield: 75 %; White solid; m.p. 168–170 °C; Rf: 0.39 (ethyl
1
acetate/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): δH
10.42 (s, 1H, NH), 9.27 (s, 1H, NH), 7.86 (d, 1H, J4′′,6′′
2.4 Hz, H-4′′), 7.68 (m, 2H, H-3, H-3′), 7.62 (d, 3H, J2,3
=
=
J
2′,3′ = J3′′,4′′ = 6.3 Hz, H-2, H-2′, H-3′′), 7.57 (s, 1H, H-6′
′), 7.43 (m, 6H, H-6, H-6′, H-5, H-5′, H-4, H-4′); 13C-
NMR: (100 MHz, DMSO-d6):δC 176.6 (C=S), 150.5

Med Chem Res
(C=N), 137.6 (C-2′′), 136.2 (C-5′′), 131.3 (C-1′′), 131.0
(C-6′′), 130.6 (C-1), 130.0 (C-1′), 129.6 (C-3′′), 129.2 (C-
5′′), 128.9 (C-2, C-6), 128.5 (C-2′, C-6′), 128.4 (C-3, C-5),
127.8 (C-3′, C-5′), 127.7 (C-4, C-4′); EI-MS: m/z (rel.
abund. %), 364 [M+-Cl] (87.0), 238 (14.0), 195 (38.5), 180
(94.3), 77 (100.0); IR (KBr, cm−1): 3414 (N–H), 1581
(C=N), 1529 (C=C), 1311 (C–N), 1257 (C=S); Anal. calcd
for C20H15Cl2N3S: C, 60.00; H, 3.78; Cl, 17.71; N, 10.50;
S, 8.01; Found: C, 60.01; H, 3.75 N, 10.51.
N-(4′′-Bromophenyl)-2-(diphenylmethylene)
hydrazinecarbothioamide (9)
Yield: 72 %; White solid; m.p. 240–242 °C; Rf: 0.56 (ethyl
1
acetate/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): δH
10.62 (s, 1H, NH), 9.34 (s, 1H, NH), 7.76 (m, 2H, H-2, H-
6), 7.58 (m, 4H, H-2′, H-6′, H-3′′ ,H-5′′), 7.43 (m, 2H, H-3,
H-5), 7.35 (m, 4H, H-3′, H-5′, H-2′′, H-6′′), 7.30 (m, 1H,
H-4), 6.89 (m, 1H, H-4′); 13C-NMR: (100 MHz, DMSO-
d6): δC 173.8 (C=S), 154.8 (C=N), 139.7 (C-1′′), 136.4 (C-
4′′), 131.0 (C-1), 130.0 (C-1′), 129.7 (C-2′′, C-6′′), 129.5
(C-3′′, C-5′′), 128.5 (C-2, C-6), 128.4 (C-2′, C-6′), 128.3
(C-3, C-5), 128.1 (C-3′, C-5′), 127.2 (C-4), 127.0 (C-4′);
EI-MS: m/z (rel. abund. %), 409 [M]+ (Absent), 238 (24.0),
196 (28.9), 180 (100.0), 77 (67.8); IR (KBr, cm−1): 3473
(N–H), 1625 (C=N), 1531 (C=C), 1321 (C–N), 1288
(C=S); Anal. calcd for C20H16BrN3S: C, 58.54; H, 3.93; Br,
19.47; N, 10.24; S, 7.81; Found: C, 58.53; H, 3.92; N,
10.22.
N-(3′′,4′′-Dichlorophenyl)-2-(diphenylmethylene)
hydrazinecarbothioamide (7)
Yield: 72 %; Yellow solid; m.p. 216–218 °C; Rf: 0.39 (ethyl
1
acetate/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): δH
10.39 (s, 1H, NH), 9.15 (s, 1H, NH), 7.74 (m, 2H, H-2, H-
6), 7.70 (m, 3H, H-3, H-4, H-5), 7.66 (m, 2H, H-2′, H-6′),
7.48 (dd, 1H, J6′′,5′′ = 6.6 Hz, J6′′,2′′ = 1.8 Hz, H-6′′), 7.41
(m, 5H, H-2′′, H-5′′, H-3′, H-4′, H-5′); 13C-NMR: (100
MHz, DMSO-d6):δC 176.9 (C=S), 150.3 (C=N), 136.1 (C-
3′′), 135.6 (C-4′′), 131.8 (C-1′′), 131.4 (C-6′′), 131.2 (C-2′
′), 131.0 (C-1), 130.3 (C-1′), 130.0 (C-5′′), 129.6 (C-2, C-
6), 128.8 (C-2′, C-6′), 128.4 (C-3, C-5), 128.3 (C-3′, C-5′),
127.7 (C-4), 127.3 (C-4′); EI-MS: m/z (rel. abund. %), 364
[M+-Cl] (21.3), 238 (2.4), 195 (12.1), 180 (100.0), 166
(73.0), 77 (36.2); IR (KBr, cm−1): 3415 (N–H), 1631
(C=N), 1525 (C=C), 1363 (C–N), 1255 (C=S); Anal. calcd
for C20H15Cl2N3S: C, 60.00; H, 3.78; Cl, 17.71; N, 10.50;
S, 8.01; Found: C, 60.02; H, 3.73 N, 10.54.
2-(Diphenylmethylene)-N-(4′′-nitrophenyl)
hydrazinecarbothioamide (10)
Yield: 69 %; Yellow solid; m.p. 198–200 °C (lit. 197–199 °C;
Singh et al., 2011); Rf: 0.36 (ethyl acetate/hexanes, 2:8); 1H-
NMR (300 MHz, DMSO-d6): δH 10.71 (s, 1H, NH), 9.36 (s,
1H, NH), 8.26 (d, 2H, J3′′,2′′ = J5′′,6′′ = 6.9 Hz, H-3′′,
H-5′′), 8.03 (d, 2H, J2,3 = J6,5 = 6.9 Hz, H-2, H-6), 7.69 (m,
5H, H-2′, H-6′, H-3, H-5, H-4), 7.45 (d, 2H, J2′′,3′′ = J6′′,5′′
= 6.3 Hz, H-2′′, H-6′′), 7.41 (m, 3H, H-3′, H-5′, H-4′); 13C-
NMR: (100 MHz, DMSO-d6): δC 175.7 (C=S), 151.3
(C=N), 145.1 (C-1′′), 143.6 (C-4′′), 136.2 (C-3′′, C-5′′),
131.4 (C-1), 130.1 (C-1′), 130.0 (C-2, C-6), 129.6 (C-2′, C-
6′), 128.5 (C-2′′, C-6′′), 128.3 (C-3, C-5), 128.0 (C-3′, C-
5′), 124.6 (C-4), 123.7 (C-4′); EI-MS: m/z (rel. abund. %),
376 [M]+ (33.4), 299 (17.6), 238 (53.6) 195 (43.2), 180
(100.0), 77 (14.5); HREI-MS: m/z calcd for C20H16N4O2S
[M]+ 376.0994, found 376.0993; IR (KBr, cm−1): 3415 (N–
H), 1598 (C=N), 1546 (C=C), 1517 (N=O), 1332 (C–N),
1282 (C=S); Anal. calcd for C20H16N4O2S: C, 63.81; H,
4.28; N, 14.88; O, 8.50; S, 8.52; Found: C, 63.82; H, 4.27;
N, 14.88.
N-(3′′-Bromophenyl)-2-(diphenylmethylene)
hydrazinecarbothioamide (8)
Yield: 74 %; White solid; m.p. 226–228 °C; Rf: 0.52 (ethyl
1
acetate/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): δH
10.47 (s, 1H, NH), 8.98 (s, 1H, NH), 7.84 (br. s, 1H, H-2′′),
7.72 (d, 2H, J2,3 = J6,5 = 6.9 Hz, H-2, H-6), 7.67 (t, 2H,
J3(2,4) = J5(6,4) = 7.2 Hz, H-3, H-5), 7.62 (d, 2H, J2′,3′
=
J
6′,5′ = 8.7 Hz, H-2′, H-6′), 7.42 (m, 6H, H-4′′, H-5′′, H-3′,
H-4′, H-5′, H-4), 7.33 (d, 1H, J6′′,5′′ = 7.8 Hz, H-6′′); 13C-
NMR: (100 MHz, DMSO-d6): δC 176.7 (C=S), 156.8
(C=N), 153.2 (C-3′′), 136.5 (C-1′′), 131.9 (C-2′′), 130.2
(C-4′′), 130.0 (C-1, C-1′), 129.6 (C-6′′), 128.4 (C-5′′),
127.8 (C-2, C-6, C-4, C-2′, C-6′, C-4′), 127.6 (C-3, C-5, C-
3′, C-5′); EI-MS: m/z (rel. abund. %), 412 [M++2] (3.4),
410 [M]+ (3.9), 376 (4.7), 332 (7.7), 239 (7.5), 180 (100.0),
166 (95.3), 155 (3.5), 77 (44.9); HREI-MS: m/z calcd for
C20H16BrN3S [M]+ 409.0248, found 409.0245; IR (KBr,
cm−1): 3418 (N–H), 1627 (C=N), 1521 (C=C), 1325 (C–
N), 1245 (C=S); Anal. calcd for C20H16BrN3S: C, 58.54; H,
3.93; Br, 19.47; N, 10.24; S, 7.81; Found: C, 58.54; H,
3.93, N, 10.24.
(E)-2-[(4′-Hydroxyphenyl)(phenyl)methylene]-N-
phenylhydrazine carbothioamide (11)
Yield: 75 %; Yellow solid; m.p. 171–173 °C; Rf: 0.40 (ethyl
1
acetate/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): δH
10.35 (d, 1H, NH), 8.90 (s, 1H, NH), 8.60 (s, 1H, OH), 7.73
(d, J2′,3′ = 7.5 Hz, 1H, H-2′), 7.65 (d, J6′,5′ = 7.8 Hz, 1H, H-
6′), 7.55 (d, J2,3 = J6,5 = 7.2 Hz, 2H, H-2, H-6), 7.39 (m,
4H, H-3, H-5, H-3′′, H-5′′), 7.21 (m, 2H, H-4, H-4′′), 7.02
(d, 2H, J2′′,3′′ = J6′′,5′′ = 8.4 Hz, H-2′′, H-6′′), 6.92 (d, J3′,2′

Med Chem Res
= 8.4 Hz, 1H, H-3′), 6.77 (d, J5′,6′ = 8.7 Hz, 1H, H-5′); 13C-
NMR: (100 MHz, DMSO-d6): δC 159.3 (C=S), 129.9 (C=N),
129.8 (C-4′), 129.7 (C-1′′), 129.6 (C-1), 128.3 (C-2′′, C-6′′),
128.2 (C-2, C-6), 128.1 (C-3′′, C-5′′), 127.9 (C-3, C-5),
125.8 (C-4′′), 124.3 (C-4), 123.5 (C-2′, C-6′), 116.4 (C-1′),
115.3 (C-3′, C-5′); EI-MS: m/z (rel. abund. %): 347 [M]+
(6.7), 254 (31.2), 212 (28.4), 196 (100.0), 181 (19.1), 77
(16.0); HREI-MS: m/z calcd for C20H17N3OS [M]+
347.1092, found 347.1096; IR (KBr, cm−1): 3413 (O–H),
3328 (N–H), 1510 (C=N), 1537 (C=C), 1311 (C–N), 1274
(C=S); Anal. calcd for C20H17N3OS: C, 69.14; H, 4.93; N,
12.09; O, 4.61; S, 9.23; Found: C, 69.12; H, 4.94; N, 12.07.
3473 (O–H), 3413 (N–H), 1531 (C=N), 1537 (C=C), 1321
(C–N), 1288 (C=S); Anal. calcd for C20H15Cl2N3OS: C,
57.70; H, 3.63; Cl, 17.03; N, 10.09; O, 3.84; S, 7.70;
Found: C, 57.72; H, 3.62; N, 10.07.
(E)-N-(2′′,5′′-Dichlorophenyl)-2-[(4′-hydroxyphenyl)
(phenyl)methylene]hydrazine carbothioamide (14)
Yield: 73 %; Yellow solid; m.p. 120–122 °C; Rf: 0.38 (ethyl
1
acetate/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): δH
10.37 (d, 1H, NH), 10.04 (d, 1H, NH), 9.31 (d, 1H, OH),
7.93 (d, 1H, J6′′,4′′ = 2.4 Hz, H-6′′), 7.69 (d, 1H, J3′′,4′′
=
7.5 Hz, H-3′′), 7.64 (d, 1H, J4′′,3′′ = 7.5 Hz, H-4′′), 7.61 (m,
2H, H-3, H-5), 7.52 (d, J2′,3′ = 8.7 Hz, 1H, H-2′), 7.43 (m,
3H, H-2, H-4, H-6), 7.21 (d, J6′,5′ = 8.4 Hz, 1H, H-6′), 7.01
(d, J3′,2′ = 8.4 Hz, 1H, H-3′), 6.78 (d, J5′,6′ = 8.7 Hz, 1H, H-
5′); 13C-NMR: (100 MHz, DMSO-d6): δC 176.3 (C=N),
159.5 (C=S), 158.8 (C-4′), 151.0 (C-2′′), 137.6 (C-5′′),
131.6 (C-1′′), 131.0 (C-6′′), 130.6 (C-1), 130.1 (C-3′′),
129.9 (C-4′′), 129.6 (C-2, C-6), 128.7 (C-3, C-5), 128.4 (C-
4), 128.0 (C-2′, C-6′), 116.3 (C-1′), 115.2 (C-3′, C-5′); EI-
MS: m/z (rel. abund. %), 381 [M+-Cl] (24.7), 254 (70.2),
196 (100.0), 161 (90.5), 77 (41.5); IR (KBr, cm−1): 3413
(O–H), 3319 (N–H), 1581 (C=N), 1532 (C=C), 1357 (C–
N), 1269 (C=S); Anal. calcd for C20H15Cl2N3OS: C, 57.70;
H, 3.63; Cl, 17.03; N, 10.09; O, 3.84; S, 7.70; Found: C,
57.73; H, 3.66; N, 10.05.
(E)-N-(2′′,3′′-Dichlorophenyl)-2-[(4′hydroxyphenyl)
(phenyl)methylene] hydrazine carbothioamide (12)
Yield: 77 %; White solid; m.p. 150–152 °C; Rf: 0.30 (ethyl
1
acetate/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): δH
10.42 (d, 1H, NH), 10.03 (d, 1H, NH) , 9.16 (d, 1H, OH),
7.71 (d, J4′′,5′′ = 7.6 Hz, 1H, H-4′′), 7.66 (m, 3H, H-3, H-5,
H-5′′), 7.60 (d, J6′′,5′′ = 8.4 Hz, 1H, H-6′′), 7.54 (d, J2′,3′
=
8.8 Hz, 1H, H-2′), 7.42 (m, 3H, H-2, H-6, H-4), 7.22 (d,
6′,5′ = 8.4 Hz, 1H, H-6′), 7.02 (d, J3′,2′ = 8.4 Hz, 1H, H-3′),
J
6.77 (d, J5′,6′ = 8.7 Hz, 1H, H-5′); 13C-NMR: (100 MHz,
DMSO-d6): δC 159.4 (C=N), 150.7 (C=S), 138.5 (C-4′),
131.6 (C-2′′), 130.0 (C-3′′), 129.9 (C-1′′), 129.6 (C-1), 128.7
(C-4′′), 128.6 (C-6′′), 128.3 (C-5′′), 128.2 (C-2, C-6), 127.9
(C-3, C-5), 127.7 (C-4), 127.6 (C-2′, C-6′), 116.4 (1′), 115.2
(C-3′, C-5′); EI-MS: m/z (rel. abund. %), 381 [M+-Cl] (11.0),
253 (32.4), 196 (100.0), 161 (87.1), 90 (15.0), 77 (16.6); IR
(KBr, cm−1): 3429 (O–H), 3413 (N–H), 1589 (C=N), 1541
(C=C), 1357 (C–N), 1215 (C=S); Anal. calcd for
C20H15Cl2N3OS: C, 57.70; H, 3.63; Cl, 17.03; N, 10.09; O,
3.84; S, 7.70; Found: C, 57.71; H, 3.61; N, 10.08.
(E)-N-(3′′-Bromophenyl)-2-[(4′-hydroxyphenyl)(phenyl)
methylene]hydrazine carbothioamide (15)
Yield: 69 %; Yellow solid; m.p. 160–162 °C; Rf: 0.42 (ethyl
1
acetate/hexanes, 2:8); H-NMR (400 MHz, DMSO-d6): δH
10.41 (d, 1H, NH), 10.05 (d, 1H, NH), 9.03 (d, 1H, OH),
7.85 (s, 1H, H-2′′), 7.72 (d, J4(3,5) = 6.8 Hz, 1H, H-4), 7.65
(E)-N-(2′′,4′′-Dichlorophenyl)-2-[(4′-hydroxyphenyl)
(phenyl)methylene]hydrazine carbothioamide (13)
(t, J3(4,2) = J5(4,6) = 7.2 Hz, 2H, H-3, H-5), 7.56 (d, J2′,3′ =
8.8 Hz, 1H, H-2′), 7.40 (d, J2,3 = J6,5 = 7.6 Hz, 2H, H-2, H-
6), 7.35 (m, 2H, H-5′′, H-6′′), 7.30 (d, J4′′,5′′= 10.0 Hz, 1H,
H-4′′), 7.20 (d, J6′,5′ = 8.0 Hz, 1H, H-6′), 7.02 (d, J3′,2′ =
Yield: 71 %; White solid; m.p. 170–172 °C; Rf: 0.30 (ethyl
1
acetate/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): δH
8.4 Hz, 1H, H-3′), 6.78 (d, J5′,6′ = 8.8 Hz, 1H, H-5′); 13C-
NMR: (100 MHz, DMSO-d6): δC 175.5 (C=N), 159.5
(C=S), 150.9 (C-4′), 140.5 (C-3′′), 131.7 (C-1′′), 130.0 (C-
2′′), 129.9 (C-1), 129.7 (C-4′′), 129.1 (C-6′′), 128.3 (C-5′′),
128.1 (C-2, C-6), 124.7 (C-3, C-5), 121.3 (C-4), 120.5 (C-
2′, C-6′), 116.4 (C-1′), 115.2 (C-3′, C-5′); EI-MS: m/z (rel.
abund. %), 427 [M++2] (12.1), 425 [M]+ (12.4), 392 (2.9),
391 (1.1), 348 (2.5), 254 (70.3), 213 (15.3), 212 (11.7), 211
(22.4), 196 (100.0); HREI-MS: m/z calcd for
C20H16BrN3OS [M]+ 425.0197, found 425.0188; IR (KBr,
cm−1): 3555 (O–H), 3283 (N–H), 1583 (C=N), 1533
(C=C), 1313 (C–N), 1277 (C=S); Anal. calcd for
C20H16BrN3OS:C, 56.34; H, 3.78; Br, 18.74; N, 9.86; O,
3.75; S, 7.52; Found: C, 56.32; H, 3.79; N, 9.87.
10.32 (d, 1H, NH), 10.03 (d, 1H, NH), 9.18 (d, 1H, OH),
7.72 (m, 3H, H-3′′, H-3, H-5), 7.64 (d, 2H, J2,3 = J6,5 = 7.2
Hz, H-2, H-6), 7.53 (d, J2′,3′ = 8.7 Hz, 1H, H-2′), 7.47
(d, J5′′,6′′ = 8.4 Hz, 1H, H-5′′), 7.40 (d, J6′′,5′′ = 7.8 Hz, 1H,
H-6′′), 7.37 (m, 1H, H-4), 7.21 (d, J6′,5′ = 8.4 Hz, 1H, H-
6′), 7.01 (d, J3′,2′ = 8.4 Hz, 1H, H-3′), 6.77 (d, J5′,6′ = 8.7
Hz, 1H, H-5′); 13C-NMR: (100 MHz, DMSO-d6): δC 176.3
(C=N), 159.5 (C=S), 158.7 (C-4′), 150.8 (C-2′′), 136.7 (C-
4′′), 135.8 (C-3′′), 131.6 (C-1′′), 131.1 (C-1), 131.0 (C-5′′),
130.0 (C-6′′), 129.9 (C-2, C-6), 129.6 (C-3, C-5), 128.8 (C-
4), 128.4 (C-2′, C-6′), 128.3 (C-1′), 127.9 (C-3′, C-5′); EI-
MS: m/z (rel. abund. %), 381 [M+-Cl] (2.8), 254 (17.2), 212
(6.6), 196 (100.0), 161 (68.1), 77 (20.8); IR (KBr, cm−1):

Med Chem Res
(E)-N-(4′′-Bromophenyl)-2-[(4′-hydroxyphenyl)(phenyl)
methylene]hydrazine carbothioamide (16)
J2′′,3′′ = J6′′,5′′ = 7.8 Hz, H-2′′, H-6′′), 7.01 (t, 1H, J4′′(3′′,5′′)
= 7.2 Hz, H-4′′); 13C-NMR: (100 MHz, DMSO-d6): δC
152.2 (C=O), 149.2 (C=N), 137.1 (C-1′′), 135.0 (C-1),
133.2 (C-1′), 132.1 (C-2, C-6), 129.5 (C-2′, C-6′), 129.4
(C-4), 128 .5 (C-4′), 127.6 (C-3, C-5), 126.8 (C-3′, C-5′),
126.0 (C-3′′, C-5′′), 125.0 (C-4′), 121.4 (C-2′′, C-6′′); EI-
MS: m/z (rel. abund.%):315 [M]+ (24.4), 257 (5.0), 195
(100.0), 180 (31.6), 165 (56.5), 77 (33.1); EI-HRMS: m/z
calcd for C20H17N3O [M]+ 315.1372, found 315.1375; IR
(KBr, cm−1): 3354 (N–H), 1687 (C=O), 1593 (C=N), 1533
(C=C), 1307 (C–N); Anal. calcd for C20H17N3O: C, 76.17;
H, 5.43; N, 13.32; O, 5.07; Found: C, 76.15; H, 5.42; N,
13.31.
Yield: 72 %; Yellow solid; m.p. 230–232 °C; Rf: 0.38 (ethyl
1
acetate/hexanes, 2:8); H-NMR (400 MHz, DMSO-d6): δH
10.31 (d, 1H, NH), 9.90 (d, 1H, NH), 8.72 (d, 1H, OH),
7.72 (m, 1H, H-4), 7.65 (d, J2′,3′ = 7.2 Hz, 1H, H-2′), 7.55
(m, 4H, H-3′′, H-5′′, H-2, H-6), 7.53 (m, 2H, H-3, H-5),
7.40 (m, 2H, H-2′′, H-6′′), 7.20 (d, J6′,5′ = 8.4 Hz, 1H, H-
6′), 7.02 (d, J3′,2′ = 8.4 Hz, 1H, H-3′), 6.77 (d, J5′,6′ = 8.7
Hz, 1H, H-5′); 13C-NMR: (100 MHz, DMSO-d6): δC 175.8
(C=N), 159.4 (C=S), 158.7 (C-4′), 150.7 (C-4′′), 138.3 (C-
1′′), 136.7 (C-1), 130.9 (C-3′′, C-5′′), 129.7 (C-2′′, C-6′′),
128.3 (C-2, C-6), 128.0 (C-3, C-5), 127.8 (C-4), 121.3 (C-
3′, C-5′), 116.4 (C-1′), 115.2 (C-2′, C-6′); EI-MS: m/z (rel.
abund. %), 425 [M]+ (Absent), 254 (21.9), 212 (55.6), 196
(100.0), 181 (28.2), 77 (13.8); IR (KBr, cm−1): 3417 (O–H),
3278 (N–H), 1697 (C=N), 1593 (C=C), 1353 (C–N), 1257
(C=S); Anal. calcd for C20H16BrN3OS: C, 58.54; H, 3.93;
Br, 19.47; N, 10.24; S, 7.81; Found: C, 58.53; H, 3.94; N,
10.23.
N-(3′′-Chlorophenyl)-2-(diphenylmethylene)
hydrazinecarboxamide (19)
Yield: 73 %; White solid; m.p. 190–192 °C; Rf: 0.42 (ethyl
1
acetate/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): δH
9.27 (s, 1H, NH), 8.79 (s, 1H, NH), 7.75 (s, 1H, H-2′′), 7.64
(d, 2H, J2,3 = J6,5 = 6.0 Hz, H-2, H-6), 7.57 (m, 3H, H-3, H-
5, H-4), 7.38 (m, 4H, H-2′, H-6′, H-3′, H-5′), 7.32 (m, 3H,
H-4′, H-5′′, H-6′′), 7.05 (d, 1H, J4′′,5′′ = 9.0 Hz, H-4′′);
13C-NMR: (100 MHz, DMSO-d6): δC 138.5 (C-3′′), 135.8
(C-1′′), 132.7 (C-2′′), 131.0 (C-1), 128.3 (C-1′), 128.1
(C-4′′), 127.9 (C-2, C-6), 127.0 (C-2′, C-6′), 126.6 (C-6′′),
126.0 (C-3′, C-5′), 121.4 (C-3′′, C-5′′), 118.1 (C-3′, C-5′),
116.4 (C-4); EI-MS: m/z (rel. abund.%), 351 [M++2] (1.8),
349 [M]+ (7.5), 195 (99.0), 165 (57.4), 77 (100.0); EI-
HRMS: m/z calcd for C20H16ClN3O [M]+ 349.0982, found
349.0984; IR (KBr, cm−1): 3267 (N–H), 1662 (C=O), 1616
(C=N), 1593 (C=C), 1305 (C–N); Anal. calcd for
C20H16ClN3O: C, 68.67; H, 4.61; Cl, 10.13; N, 12.01; O,
4.57; Found: C, 68.65; H, 4.60; N, 12.02.
(E)-2-[(4′-Hydroxyphenyl)(phenyl)methylene]-N-
(4′′-nitrophenyl)hydrazine carbothioamide (17)
Yield: 76 %; Yellow solid; m.p. 212–214 °C; Rf: 0.34 (ethyl
1
acetate/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): δH
10.65 (d, 1H, NH), 10.04 (d, 1H, NH), 9.37 (d, 1H, OH),
8.25 (d, 2H, J3′′,2′′ = J5′′,6′′ = 9.0 Hz, H-3′′, H-5′′), 8.04 (d,
2H, J2′′,3′′ = J6′′,5′′ = 9.0 Hz, H-2′′, H-6′′), 7.70 (m, 3H, H-
2, H-4, H-6), 7.54 (d, J2′,3′ = 8.4 Hz, 1H, H-2′), 7.44 (m,
2H, H-3, H-5), 7.21 (d, J6′,5′ = 8.4 Hz, 1H, H-6′), 7.01 (d,
J
3′,2′ = 8.4 Hz, 1H, H-3′), 6.79 (d, J5′,6′ = 8.7 Hz, 1H, H-5′);
13C-NMR: (100 MHz, DMSO-d6): δC 175.8 (C=N), 159.4
(C=S), 158.7 (C-4′), 150.7 (C-1′′), 138.3 (C-4′′), 136.7 (C-
3′′, C-5′′), 131.7 (C-1), 130.9 (C-2′′, C-6′′), 130.0 (C-2, C-
6), 129.9 (C-3, C-5), 129.7 (C-4), 128.4 (C-2′, C-6′), 128.0
(C-1′), 127.8 (C-3′, C-5′); EI-MS: m/z (rel. abund. %), 392
[M]+ (1.0), 254, (3.6), 196 (100.0), 138 (17.9), 77 (11.7);
HREI-MS: m/z calcd for C20H16N4O3S [M]+ 392.0943,
found 392.0945; IR (KBr, cm−1): 3550 (O–H), 3415 (N–H),
1606 (C=N), 1548 (C=C), 1323 (C–N), 1276 (C=S); Anal.
calcd forC20H16N4O3S: C, 61.21; H, 4.11; N, 14.28; O,
12.23; S, 8.17; Found: C, 61.22; H, 4.13; N, 14.26.
2-(Diphenylmethylene)-N-[3′′-(trifluoromethyl)phenyl]
hydrazinecarboxamide (20)
Yield: 74 %; White solid; m.p. 170–172 °C; Rf: 0.38 (ethyl
1
acetate/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): δH
9.43 (s, 1H, NH), 8.82 (s, 1H, NH), 8.03 (s, 1H, H-2′′), 7.77
(d, 1H, J6′′,5′′ = 7.8 Hz, H-6′′), 7.64 (m, 3H, H-2, H-6, H-4),
7.53 (m, 2H, H-3, H-5), 7.51 (d, 1H, J4′′,5′′ = 7.8 Hz, H-4′′),
7.39 (m, 3H, H-2′, H-6′, H-4′), 7.33 (m, 3H, H-5′′, H-3′, H-
5′); 13C-NMR: (100 MHz, DMSO-d6): δC 152.1 (C=O),
148.8 (C=N), 139.9 (C-1′′), 137.1 (C-3′′), 132.1 (C-1),
129.7 (C-1′), 129.5 (C-2′′), 129.1 (C-6′′), 128.4 (C-4′′),
128.2 (C-5′′), 127.1 (C-2, C-6), 125.5 (C-2′, C-6′), 122.8
(C-3, C-5), 118.6 (C-3′, C-5′), 118.5 (C-4, C-4′), 115.2 (3′-
CF3); EI-MS: m/z (rel. abund.%), 383 [M]+ (49.3), 325
(18.1), 248 (18.9), 195 (100.0), 165 (71.1), 77 (19.1); EI-
HRMS: m/z calcd for C21H16F3N3O [M]+ 383.1245, found
383.1257; IR (KBr, cm−1): 3352 (N–H), 1687 (C=O), 1618
2-(Diphenylmethylene)-N-phenylhydrazinecarboxamide (18)
Yield: 73 %;White solid; m.p. 160–163 °C (lit. 163–164 °C;
Pearson et al., 1953); Rf: 0.45 (ethyl acetate/hexanes, 2:8);
1H-NMR (300 MHz, DMSO-d6): δH 9.09 (s, 1H, NH), 8.69
(s, 1H, NH), 7.62 (m, 3H,H-2, H-6, H-4), 7.55 (m, 4H,
H-3, H-5, H-3′,H-5′), 7.38 (m, 3H, H-4, H-3′′, H-5′′), 7.33
(d, 2H, J2′,3′ = J6′,5′ = 8.1 Hz, H-2′, H-6′), 7.27 (d, 2H,

Med Chem Res
(C=N), 1541 (C=C), 1317 (C–N); Anal. calcd for
C21H16F3N3O: C, 65.79; H, 4.21; F, 14.87; N, 10.96; O,
4.17; Found: C, 65.77; H, 4.20; F, 14.85; N, 10.95.
9.48 (s, 1H, NH), 9.06 (s, 1H, NH), 8.14 (d, 1H, J5′′,6′′ =
7.8 Hz, H-5′′), 7.77 (d, 1H, J3′′,5′′ = 2.7 Hz, H-3′′), 7.73 (m,
1H, H-6′′), 7.61 (m, 3H, H-2, H-6, H-4), 7.46 (m, 2H, H-3,
H-5), 7.40 (m, 3H, H-2′, H-6′, H-4′), 7.34 (m, 2H, H-3′, H-
5′); 13C-NMR: (100 MHz, DMSO-d6): δC 151.9 (C=O),
148.7 (C=N), 140.6 (C-4′′), 137.1 (C-1′′), 132.9 (C-2′′),
132.1 (C-1), 130.1 (C-1′), 129.6 (C-3′′), 129.5 (C-5′′),
129.1 (C-6′′), 128.4 (C-2, C-6), 128.2 (C-2′, C-6′), 127.0
(C-3, C-5), 121.9 (C-3′, C-5′), 118.5 (C-4, C-4′), 117.5 (2′
′-CF3); EI-MS: m/z (rel. abund.%): 419 [M++2] (1.0), 417
[M]+ (13.0), 221 (23.6), 195 (100.0), 180 (31.3), 165 (45.6),
77 (37.6); EI-HRMS: m/z calcd for C21H15ClF3N3O [M]+
417.0856, found 417.0858; IR (KBr, cm−1): 3475 (N–H),
1699 (C=O), 1616 (C=N), 1583 (C=C), 1338 (C–N), 877
(C–F); Anal. calcd for C21H15ClF3N3O: C, 60.37; H, 3.62;
Cl, 8.49; F, 13.64; N, 10.06; O, 3.83; Found: C, 60.36; H,
3.60; N, 10.05.
2-(Diphenylmethylene)-N-[4′′-(trifluoromethyl)phenyl]
hydrazinecarboxamide (21)
Yield: 72 %; White solid; m.p. 174–176 °C; Rf: 0.42 (ethyl
acetate/hexanes, 2:8); 1H-NMR (300 MHz, DMSO-d6):
δH 9.46 (s, 1H, NH), 8.90 (s, 1H, NH), 7.76 (d, 2H,
J
3′′,2′′ = J5′′,6′′ = 8.7 Hz, H-3′′, H-5′′), 7.64 (m, 5H, H-2, H-
6, H-4, H-3, H-5), 7.53 (m, 2H, H-2′, H-6′), 7.39 (m, 3H,
H-3′, H-5′, H-4′), 7.33 (d, 2H, J2′′,3′′ = J6′′,5′′ = 7.5 Hz, 2H,
H-2′′, H-6′′); 13C-NMR: (100 MHz, DMSO-d6): δC 151.8
(C=O), 148.9 (C=N), 142.9 (C-1′′), 137.2 (C-4′′), 132.2
(C-1), 129.5 (C-1′), 129.1 (C-2′′, C-6′′), 128.5 (C-3′′, C-5′
′), 128.2 (C-2, C-6), 127.1 (C-2′, C-6′), 125.9 (C-3, C-5),
125.8 (C-3′, C-5′), 125.5 (C-4), 122.0 (C-4′), 118.7 (4′
′-CF3); EI-MS: m/z (rel. abund.%), 383 [M]+ (25.3), 364
(7.2), 195 (100.0), 165 (96.3), 145 (27.8), 77 (39.0); EI-
HRMS: m/z calcd for C21H16F3N3O [M]+ 383.1245, found
383.1247; IR (KBr, cm−1): 3344 (N–H), 1691 (C=O), 1600
(C=N), 1533 (C=C), 1325 (C–N), 846 (C–F); Anal. calcd
for C21H16F3N3O: C, 65.79; H, 4.21; F, 14.87; N, 10.96; O,
4.17; Found: C, 65.76; H, 4.22; N, 10.94.
(E)-N-(3′′-Chlorophenyl)-2-[(4′-hydroxyphenyl)(phenyl)
methylene]hydrazine carboxamide (24)
Yield: 72 %;White solid; m.p. 176–178 °C; Rf: 0.40 (ethyl
1
acetate/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): δH
9.89 (s, 1H, NH), 9.25 (s, 1H, NH), 8.74 (s, 1H, OH), 7.75
(s, 1H, H-2′′), 7.55 (m, 2H, H-5′′, H-4′′), 7.42 (m, 3H, H-2,
H-6, H-4), 7.31 (t, 2H, J3(2,5) = J5(6,4) = 9.0 Hz, H-3, H-5),
7.12 (d, 2H, J2′,3′ = J6′,5′ = 8.4 Hz, H-2′, H-6′), 7.04 (d, 1H
N-[2′′-Chloro-5′′-(trifluoromethyl)phenyl]-2-
(diphenylmethylene)hydrazine carboxamide (22)
J
4′′,5′ = 8.4 Hz, H-4′′), 6.98 (d, 2H, J3′,2′ = J5′,6′ = 8.4 Hz,
H-3′, H-5′); 13C-NMR: (100 MHz, DMSO-d6):δC 158.3
(C=O), 151.9 (C=N), 149.0 (C-4′), 140.7 (C-3′′), 137.7 (C-
1′′), 133.0 (C-2′′), 130.2 (C-1), 130.0 (C-4′′), 129.0 (C-6′′),
128.2 (C-5′′), 127.2 (C-2, C-6), 122.2 (C-3, C-5), 121.9 (C-
4), 118.4 (C-2′, C-6′), 117.4 (C-1′), 116.3 (C-3′, C-5′); EI-
MS: m/z (rel. abund.%): 367 [M++2] (3.0), 365 [M]+ (10.0),
239 (11.0), 211 (100.0), 181 (75.4), 127 (72.4), 77 (19.4);
IR (KBr, cm−1): 3415 (O–H), 3323 (N–H), 1689 (C=O),
1612 (C=N), 1566 (C=C); EI-HRMS: m/z calcd for
C20H16ClN3O2 [M]+ 365.0931, found 365.0934; Anal.
calcd for C20H16ClN3O2:C, 65.67; H, 4.41; Cl, 9.69; N,
11.49; O, 8.75; Found: C, 65.66; H, 4.42; N, 11.48.
Yield: 70 %; White solid; m.p. 200–202 °C; Rf: 0.31 (ethyl
acetate/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): δH
1
9.67 (s, 1H, NH), 9.30 (s, 1H, NH), 8.55 (s, 1H, H-6′′), 7.76
(d, 1H, J3′′,4′′ = 8.4 Hz, H-3′′), 7.62 (m, 3H, H-2, H-6, H-4),
7.46 (m, 2H, H-3, H-5), 7.40 (m, 4H, H-2′, H-6′, H-3′, H-
5′), 7.35 (m, 2H, H-4′, H-4′′); 13C-NMR: (100 MHz,
DMSO-d6): δC 152.2 (C=O), 149.2 (C=N), 137.1 (C-2′′),
135.0 (C-1′′), 133.2 (C-5′′), 132.1 (C-1), 129.6 (C-1′),
129.5 (C-6′′), 129.4 (C-3′′), 128.5 (C-4′′), 128.4 (C-2, C-6),
127.6 (C-2′, C-6′), 126.8 (C-3, C-5), 126.0 (C-3′, C-5′),
125.9 (C-4), 125.0 (C-4′), 121.4 (5′-CF3); EI-MS: m/z (rel.
abund.%): 419 [M++2] (13.4), 417 [M]+ (36.5), 382 (11.8),
195 (100.0), 165 (52.3), 77 (29.6); EI-HRMS: m/z calcd for
C21H15ClF3N3O [M]+ 417.0856, found 417.0859; IR (KBr,
cm−1): 3415 (N–H), 1699 (C=O), 1666 (C=N), 1552
(C=C), 1330 (C–N), 887 (C–F); Anal. calcd for
C21H15ClF3N3O: C, 60.37; H, 3.62; Cl, 8.49; F, 13.64; N,
10.06; O, 3.83; Found: C, 60.35; H, 3.61; N, 10.04.
(E)-2-[(4′-Hydroxyphenyl)(phenyl)methylene)-N-
(4′′-(trifluoromethyl)phenyl) hydrazinecarboxamide (25)
Yield: 69 %; White solid; m.p. 208–210 °C; Rf: 0.30 (ethyl
1
acetate/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): δH
9.90 (s, 1H, NH), 9.44 (s, 1H, NH), 8.85 (s, 1H, OH), 7.75
(d, 2H, J3′′,2′′ = J5′′,6′′ = 8.4 Hz, H-3′′, H-5′′), 7.63 (d, 2H,
N-[4′′-Chloro-2′′-(trifluoromethyl)phenyl]-2-
(diphenylmethylene)hydrazine carboxamide (23)
J2′′,3′′ = J6′′,5′′ = 8.7 Hz, H-2′′, H-6′′), 7.58 (t, 2H,
J3(2,4) = J5(6,4) = 8.3 Hz, H-3, H-5), 7.37 (d, 3H, J2,3
J6,5 = J4(3,5) = 6.0 Hz, H-2, H-6, H-4), 7.13 (d, 2H, J2′,3′
=
=
Yield: 69 %; White solid; m.p. 185–187 °C; Rf: 0.32 (ethyl
acetate/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): δH
J
6′,5′ = 8.4 Hz, H-2′, H-6′), 6.98 (d, 2H, J3′,2′ = J5′,6′ = 8.4
1
Hz, H-3′, H-5′); 13C-NMR: (100 MHz, DMSO-d6):δC 158.7

Med Chem Res
(C=O), 151.9 (C=N), 149.4 (C-4′), 143.0 (C-4′′), 137.7 (C-
1′′), 132.5 (C-1), 130.0 (C-2′′, C-6′′), 129.5 (C-2, C-6),
129.0 (C-3, C-5), 128.2 (C-4), 127.2 (C-2′, C-6′), 125.9 (C-
1′), 118.7 (C-3′′, C-5′′), 116.3 (4′-CF3), 115.1 (C-3′, C-5′);
EI-MS: m/z (rel. abund.%) 399 [M]+ (4.8), 211 (33.4), 196
(27.1), 181 (100.0), 165 (19.6), 77 (18.9); EI-HRMS: m/z
calcd for C20H16ClN3O2 [M]+ 399.1195, found 399.1198;
IR (KBr, cm−1): 3398 (O–H), 3350 (N–H), 1691 (C=O),
1656 (C=N), 1541 (C=C), 1325 (C–N), 840 (C–F); Anal.
calcd for C21H16F3N3O2: C, 63.16; H, 4.04; F, 14.27; N,
10.52; O, 8.01; Found: C, 63.15; H, 4.02; N, 10.51.
EI-MS: m/z (rel. abund.%): 433 [M]+ (2.2), 416 (1.4), 221
(29.3), 211 (56.7), 195 (100.0), 175 (63.3), 43 (74.4); EI-
HRMS: m/z calcd for C20H16ClN3O2 [M]+ 433.0805, found
433.0808; IR (KBr, cm−1): 3344 (O–H), 3278 (N–H), 1691
(C=O), 1589 (C=N), 1589 (C=C), 1311 (C–N), 839 (C–F);
Anal. calcd for C21H15ClF3N3O2: C, 58.14; H, 3.49; Cl,
8.17; F, 13.14; N, 9.69; O, 7.38; Found: C, 58.13; H, 3.47;
N, 9.67.
Acknowledgements We are thankful to the Higher Education
Commission (HEC) Pakistan for financial support under National
Research Grant Program for Universities to Project No. 20–1910.
(E)-N-[2′′-Chloro-5′′-(trifluoromethyl)phenyl]-2-[(4′-
hydroxyphenyl)(phenyl) methylene]hydrazinecarboxamide
(26)
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interests.
Yield: 70 %; White solid; m.p. 200–202 °C; Rf: 0.31 (ethyl
1
acetate/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): δH
9.88 (s, 1H, NH), 9.57 (s, 1H, NH), 9.28 (s, 1H, OH), 8.56
(s, 1H, H-6′′), 7.75 (d, 1H J4,3 = J4,5 = 8.4 Hz, H-4), 7.58
(m, 2H, H-2, H-6), 7.42 (m, 4H, H-3, H-5, H-2′, H-6′), 7.15
(d, 2H, J3′,2′ = J5′,6′ = 8.4 Hz, H-3′, H-5′), 6.97 (d, 1H J3′′,4′
′ = 8.4 Hz, H-3′′), 6.78 (d, 1H J4′′,3′′ = 8.7 Hz, H-4′′); 13C-NMR:
(100 MHz, DMSO-d6):δC 158.4 (C=O), 152.0 (C=N),
149.9 (C-4′), 137.9 (C-2′′), 136.3 (C-5′′), 130.3 (C-1′′),
130.1 (C-6′′), 129.3 (C-1), 128.6 (C-3′′), 128.5 (C-4′′),
128.3 (C-2, C-6), 127.1 (C-3, C-5), 122.5 (C-4), 122.0 (C-
2′, C-6′), 119.8 (C-1′), 116.1 (5′′-CF3), 115.2 (C-3′, C-5′);
EI-MS: m/z (rel. abund.%): 433 [M]+ (10.4), 212 (51.6),
196 (34.6), 181 (100.0), 152 (28.7), 77 (15.2); EI-HRMS:
m/z calcd for C20H16ClN3O2 [M]+ 433.0805, found
433.0808; IR (KBr, cm−1): 3411 (O–H), 3328 (N–H), 1678
(C=O), 1591 (C=N), 1539 (C=C), 1332 (C–N), 821 (C–F);
Anal. calcd for C21H15ClF3N3O2: C, 58.14; H, 3.49; Cl,
8.17; F, 13.14; N, 9.69; O, 7.38; Found: C, 58.12; H, 3.48;
N, 9.68.
References
Akhtar T, Hameed S, Khan KM, Choudhary MI (2008) Syntheses,
urease inhibition, and antimicrobial studies of some chiral 3-
substituted-4-amino-5-thioxo-1H,4H-1,2,4-triazoles. Med Chem
4:539–543
Akhtar T, Hameed S, Khan KM, Khan A, Choudhary MI (2010)
Design, synthesis, and urease inhibition studies of some 1,3,4-
oxadiazoles and 1,2,4-triazoles derived from mandelic acid.
J Enzyme Inhib Med Chem 25:572–576
Barton DHR, O’Brien RE, Sternhell S (1962) A new reaction of
hydrazones. J Chem Soc 470–476.
Blaser MJ (1990) Helicobacter pylori and the pathogenesis of gas-
troduodenal inflammation. J Infec Dis 161:626–633
de Oliveira RB, de Souza-Fagundes EM, Soares RP, Andrade AA,
Krettli AU, Zani CL (2008) Synthesis and antimalarial activity of
semicarbazone and thiosemicarbazone derivatives. Eur J Med
Chem 43:1983–1988
Ferraz KSO, Silva NF, Da Silva JG, Speziali NL, Mendes IC, Beraldo
H (2012) Structural studies on acetophenone-and benzophenone-
derived thiosemicarbazones and their zinc (II) complexes. J Mol
Struct 1008:102–107
Glinma B, Kpoviessi SD, Gbaguidi FA, Kapanda CN, Bero J, Quetin-
Leclercq J, Moudachirou M, Poupaert J, Accrombessi GC,
Gachomo EW (2014) Trypanocidal and cytotoxic evaluation of
synthesized thiosemicarbazones as potential drug leads against
sleeping sickness. Mol Biol Rep 41:1617–1622
(E)-N-[4′′-Chloro-2′′-(trifluoromethyl)phenyl]-2-[(4′-
hydroxyphenyl)(phenyl) methylene]hydrazinecarboxamide
(27)
Hameed A, Anwar A, Khan KM, Malik R, Shahab F, Siddiq S, Basha
FZ, Choudhary MI (2013) Urease inhibition and anticancer
activity of novel polyfunctional 5,6-dihydropyridine derivatives
and their structure-activity relationship. Eur J Chem 4:49–52
Hsieh H-P, Liou J-P, Lin Y-T, Mahindroo N, Chang J-Y, Yang Y-N,
Chern S-S, Tan U-K, Chang C-W, Chen T-W (2003) Structure–
activity and crystallographic analysis of benzophenone deriva-
tives—the potential anticancer agents. Bioorg Med Chem Lett
13:101–105
Joshi V, Hari MI (1981) Studies in hydrazone rearrangement. 1.
Synthesis of anilides, isoquinolinones and their N-nitroso deri-
vatives. Indian J Chem 20:999–1001
Karahan Ö, Balta DK, Arsu N, Avci D (2014) Synthesis and evalua-
tions of novel photoinitiators with side-chain benzophenone,
derived from alkyl α-hydroxymethacrylates. J Photochem Pho-
tobiol A: Chem 274:43–49
Yield: 69 %; White solid; m.p. 172–174 °C; Rf: 0.37 (ethyl
acetate/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): δH
9.85 (s, 1H, NH), 9.37 (s, 1H, NH), 9.15 (s, 1H, OH), 8.15
(s, 1H, H-3′′), 7.76 (d, 2H, J2,3 = J6,5 = 9.0 Hz, H-2, H-6),
7.56 (m, 5H, H-3, H-5, H-4, H-2′, H-6′), 7.29 (m, 1H, H-5),
7.15 (d, 2H, J3′,2′ = J5′,6′ = 8.7 Hz, H-3′, H-5′), 6.95 (d, 1H
1
J
6′′,5′′ = 8.4 Hz, H-6′′), 6.77 (d, 1H J5′′,6′′ = 8.7 Hz, H-5′′);
13C-NMR: (100 MHz, DMSO-d6): δC 158.4 (C=O), 152.2
(C=N), 149.7 (C-4′), 137.6 (C-4′′), 135.1 (C-2′′), 133.2 (C-
1′′), 130.1 (C-3′′), 129.3 (C-1), 128.7 (C-5′′), 128.5 (C-6′′),
128.3 (C-2, C-6), 127.5 (C-3, C-5), 127.0 (C-4), 125.9 (C-
2′, C-6′), 122.2 (C-1′), 116.1 (2′-CF3), 115.2 (C-3′, C-5′);

Med Chem Res
Kasuga NC, Sekino K, Ishikawa M, Honda A, Yokoyama M, Nakano
S, Shimada N, Koumo C, Nomiya K (2003) Synthesis, structural
characterization and antimicrobial activities of 12 zinc (II) com-
plexes with four thiosemicarbazone and two semicarbazone
ligands. J Inorg Biochem 96:298–310
Kato K, Nishida M, Ito K, Tomita M (2012) Characterization of silica
particles prepared via urease-catalyzed urea hydrolysis and
activity of urease in sol–gel silica matrix. Appl Surf Sci 262:
69–75
Khan KM, Ali M, Wadood A, Khan M, Lodhi MA, Perveen S,
Choudhary MI, Voelter W (2011) Molecular modeling-based
antioxidant arylidene barbiturates as urease inhibitors. J Mol
Graph Model 30:153–156
Piccinelli AL, Cuesta-Rubio O, Chica MB, Mahmood N, Pagano B,
Pavone M, Barone V, Rastrelli L (2005) Structural revision
of clusianone and 7-epi-clusianone and anti-HIV activity of
polyisoprenylated benzophenones. Tetrahedron 61:8206–8211
Pingaew R, Prachayasittikul S, Ruchirawat S (2010) Synthesis, cyto-
toxic and antimalarial activities of benzoyl thiosemicarbazone
analogs of isoquinoline and related compounds. Molecules 15:
988–996
Reena T, Seena E, Kurup MP (2008) Synthesis and spectral
studies of cadmium (II) complexes derived from di-2-pyridyl
ketone and N4-phenylsemicarbazide: First structural report
of a cadmium (II) complex of semicarbazone. Polyhedron 27:
1825–1831
Khan KM, Iqbal S, Lodhi MA, Maharvi GM, Choudhary MI, Perveen
S (2004) Biscoumarin: new class of urease inhibitors; economical
synthesis and activity. Bioorg Med Chem 12:1963–1968
Khan KM, Rao NA, Lodhi MA, Perveen S, Choudhary MI, Voelter W
(2007) Synthesis and in vitro inhibitory potential towards urease
of 9-anilinoacridines and aciridinyl hydrazides. Lett Drug Des
Discov 4:114–121
Khan KM, Saify ZS, Lodhi MA, Butt N, Perveen S, Maharvi GM,
Choudhary MI, Atta-Ur-Rahman (2006) Piperidines: A new class
of urease inhibitors. Nat Prod Res 20:523–530
Khan KM, Rahim F, Khan A, Ali S, Taha M, Saad SM, Khan M,
Najeebullah, Shaikh A, Perveen S, Choudhary MI (2015)
Synthesis of benzophenone hydrazone analogs and their DPPH
radical scavenging and urease inhibitory activities. J Chem Soc
Pak 37:479
Khanum SA, Begum BA, Girish V, Khanum NF (2010) Synthesis and
evaluation of benzophenone-n-ethyl morpholine ethers as anti-
inflammatory agents. Int J Biomed Sci 6:60–65
Kim E, Chun H-O, Jung S-H, Kim JH, Lee J-M, Suh B-C, Xiang MX,
Rhee CK (2003) Improvement of therapeutic index of phospho-
diesterase type IV inhibitors as anti-asthmatics. Bioorg Med
Chem Lett 13:2355–2358
Kumar GK, Chavarria GE, Charlton-Sevcik AK, Arispe WM, Mac-
Donough MT, Strecker TE, Chen S-E, Siim BG, Chaplin DJ,
Trawick ML (2010) Design, synthesis, and biological evaluation
of potent thiosemicarbazone based cathepsin L inhibitors. Bioorg
Med Chem Lett 20:1415–1419
Singh AK, Prakash KS (1992) Synthesis of [ArTe (DTC/XNT)
(NaDTC/XNT)](DTC= dithiocarbamate and XNT= xanthate)
through ligand substitution reactions of arylbis (thiosemicarba-
zone) tellurium (II) chloride. Polyhedron 11:1225–1228
Singh P, Jain J, Sinha R, Samad A, Kumar R, Malhotra M (2011)
Synthesis and screening of substituted thiosemicarbazone deri-
vatives: an approach towards novel anticonvulsant search. Cent
Nerv Syst Agents Med Chem 11:60–65
Stanojević JS, Marković DZ, Zvezdanović JB (2013) Benzophenone
suppression of quercetin antioxidant activity towards lipids under
UV-B irradiation regime: Detection by HPLC chromatography.
J Chem 2013:9
Sujarani S, Ramu A (2013) Synthesis, characterization, antimicrobial
and DNA interaction studies of benzophenone--ethanamine
Schiff base with transition metal (II)[Cu (II), Co (II), Mn (II) and
Ni (II)] complexes. J Chem Pharm Res 5:347–358
Taroua M, Ribuot C, Pera M, Taillandier G, Fatome M, Laval J,
Demenge P, Leclerc G (1996) New α,β and γ semicarbazone and
thiosemicarbazone 1,3-dithiolanes as radioprotectors. Antic-
onvulsant activity. Eur J Med Chem 31:589–595
Uddin N, Siddiqui BS, Begum S, Ali MI, Marasini BP, Khan A,
Choudhary MI (2013) Bioassay-guided isolation of urease and α-
chymotrypsin inhibitory constituents from the stems of Lawsonia
alba Lam.(Henna). Fitoterapia 84:202–207
Wang K, Ma G, Yin R, Nie J, Yu Q (2010) Benzophenone-1,3-
dioxane as a free radial initiator for photopolymerization. Mater
Chem Phys 124:453–457
Ma X-D, Zhang X, Dai H-F, Yang S-Q, Yang L-M, Gu S-X, Zheng Y-
T, He Q-Q, Chen F-E (2011) Synthesis and biological activity of
naphthyl-substituted (B-ring) benzophenone derivatives as novel
non-nucleoside HIV-1 reverse transcriptase inhibitors. Bioorg
Med Chem 19:4601–4607
Matongo F, Nwodo UU (2014) In vitro assessment of Helicobacter
pylori ureases inhibition by honey fractions. Arch Med Res
45:540–546
Wang K, Yang K, Yu Q (2014) Novel polymeric photoinitiators with
side-chain benzophenone: Facile synthesis and photo-
polymerization properties without coinitiator. Prog Org Coat
77:1929–1934
Wang Y, Jiang X, Yin J (2009) Novel polymeric photoinitiators
comprising of side-chain benzophenone and coinitiator amine:
photochemical and photopolymerization behaviors. Eur Polym
J 45:437–447
Millo C, Ader M, Dupraz S, Guyot F, Thaler C, Foy E, Ménez B
(2012) Carbon isotope fractionation during calcium carbonate
precipitation induced by urease‐catalysed hydrolysis of urea.
Chem Geol 330:39–50
Mishra V, Pandeya S, DeClercq E, Pannecouque C, Witvrouw M
(1998) Synthesis of aryl semicarbazone of 4-aminoacetophenone
and their anti-HIV activity. Pharm Acta Helv 73:215–218
Mobley H (1996) The role of Helicobacter pylori urease in the
pathogenesis of gastritis and peptic ulceration. Aliment Pharma-
col Ther 10:57–64
Pearson D, Carter K, Greer C (1953) The rearrangement of hydrazones
and semicarbazones. J Am Chem Soc 75:5905–5908
Perveen S, Khan KM, Lodhi MA, Choudhary MI, Voelter W (2008)
Urease and α-chymotrypsin inhibitory effects of selected urea
derivatives. Lett Drug Des Discov 5:401–405
Weatherburn M (1967) Phenol-hypochlorite reaction for determination
of ammonia. Anal Chem 39:971–974
Weber M, Jones MJ, Ulrich J (2008) Optimisation of isolation
and purification of the jack bean enzyme urease by extraction
and subsequent crystallization. Food Bioprod Process 86:
43–52
Yogeeswari P, Sriram D, Thirumurugan R, Raghavendran JV,
Sudhan K, Pavana RK, Stables
J (2005) Discovery of
N-(2,6-dimethylphenyl)-substituted semicarbazones as antic-
onvulsants: hybrid pharmacophore-based design. J Med Chem
48:6202–6211
Yuan S, Liu X, Si N, Dong J, Gu B, Jiang H (2006) Sensitivity of
Phytophthora infestans to flumorph: In vitro determination of
baseline sensitivity and the risk of resistance. Plant Path 55:
258–263