Synthesis, in vitro urease inhibitory activity, and molecular docking studies of (perfluorophenyl)hydrazone derivatives

Article abstract of DOI:10.1007/s00044-019-02341-5

A series of (perfluorophenyl)hydrazone derivatives 1–27 were synthesized by the condensation reaction of (perfluorophenyl)hydrazine with a variety of benzaldehydes. Compounds were structurally characterized by various spectroscopic techniques. All compoun

Full text of DOI:10.1007/s00044-019-02341-5

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02341-5
ORIGINAL RESEARCH
Synthesis, in vitro urease inhibitory activity, and molecular docking
studies of (perfluorophenyl)hydrazone derivatives
Momin Khan1 Ghulam Ahad1 Abdul Manaf1 Reshma Naz1 Syed Roohul Hussain1 Farah Deeba1 Sana Shah1
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Ajmal Khan2 Majid Ali2 Khair Zaman1 Salman Zafar3 Uzma Salar4 Abdul Hameed4 Khalid Mohammed Khan4,5
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Received: 10 December 2018 / Accepted: 29 March 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
A series of (perfluorophenyl)hydrazone derivatives 1–27 were synthesized by the condensation reaction of (perfluorophenyl)
hydrazine with a variety of benzaldehydes. Compounds were structurally characterized by various spectroscopic techniques.
All compounds were screened for their urease inhibitory activity which revealed that most of the analogs exhibited
significant urease inhibitory activity in the range of IC50 = 14.09 0.23–78.69 1.56 µM as compare to the standard
thiourea (IC50 = 21.10 0.31 µM). Amongst active compounds, derivatives 2 (IC50 = 14.23 0.21 µM), 5 (IC50 = 16.78
0.33 µM), 7 (IC50 = 15.59 0.60 µM), 9 (IC50 = 20.18 0.78 µM), 10 (IC50 = 16.13 0.93 µM), and 11 (IC50 = 14.09
0.23 µM) showed potent inhibitory activity better than the standard thiourea. A limited structure-activity relationship (SAR)
was established by rationalized the effect of different groups on the inhibitory potential. Molecular docking study was
performed to understand the binding modes of active analogs into the active site of urease enzyme.
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Keywords Synthesis (Perfluorophenyl)hydrazones Schiff bases Urease inhibition Structure-activity relationship
Molecular docking
Introduction
Sander 1997). Jack bean (Canavalia ensiformis) urease
enzyme is well-studied and also crystallized (Sujoy and
Aparna 2012; Taha et al. 2015). It is widely distributed in a
variety of bacteria, fungi, plants, and play an important role
in the circulation of nitrogen in nature. The inhibition of
urease activity has been extensively studied due to its
potential role in disease conditions, such as Helicobacter
pylori-induced peptic ulcer, urinary lithiasis, pyelonephritis,
hepatic coma, and in other infections caused by Proteus
mirabilis and Yersinia enterocolitica. Urease supports the
colony formation of Helicobacter pylori by increasing the
pH of the stomach and therefore plays an important role in
the pathogenesis of gastritis and peptic ulcers, as well as
cancer (Qin and Cabral 1994; Pope et al. 1998; Rodman
1998). Urease inhibitors are also mixed with fertilizers for
controlling the rapid urea degradation by soil bacteria
(Sahrawat 1980; Choudhary et al. 2011). Identification of
new urease inhibitors is an important and appealing area of
research for the medicinal chemist.
Urease (EC 3.5.1.5.) is a nickel containing metalloenzyme,
belongs to the superfamily of amidohydrolases, and phos-
photriestreases. It catalyzes the hydrolysis of urea to
ammonia and carbamate. Than carbamate molecule sud-
denly hydrolyzes to form carbonic acid and additional
molecule of ammonia (Rashid et al. 2013), (Holm and
* Momin Khan
mominkhan@awkum.edu.pk
1
Department of Chemistry, Abdul Wali Khan University,
Mardan 23200, Pakistan
2
UoN Chair of Oman’s Medicinal Plants and Marine Natural
Products, University of Nizwa, Nizwa 616, Oman
3
Institute of Chemical Sciences, University of Peshawar,
Peshawar, Pakistan
4
H. E. J. Research Institute of Chemistry, International Centre for
Schiff bases, an important class of compounds having
azomethine (C=N) functional group also known as imine
(Patai 1970). Basically, Schiff bases are formed by the
condensation reaction of carbonyl compounds (aldehyde
or ketone) with amine to form imine (Da Silva et al. 2011).
Chemical and Biological Science, University of Karachi,
Karachi 75270, Pakistan
5
Department of Clinical Pharmacy, Institute for Research and
Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal
University, P.O. Box 31441, Dammam, Saudi Arabia

Medicinal Chemistry Research
Fig. 1 Rationale of the current
study
Schiff bases have broad spectrum of biological activities
such as antifungal, antibacterial, antimalarial, and antiviral
properties (Bringmann et al. 2004; Salimon et al. 2010;
Hameed et al. 2017). Imine or azomethine groups are
present in different natural, natural-derived, and non-
natural compounds. The presence of reactive imine group
in such types of compounds has been shown to have cri-
tical importance to their biological activities (Prakash and
Adhikari 2011). Beside a diverse range of biological
potential, Schiff bases are reported as significant urease
inhibitors (Aslam et al. 2011). Our group has also reported
a range of hydrazones bearing aryl and heteroaryl rings as
significant urease inhibitors (Fig. 1) (Arshia et al. 2016;
Hameed et al. 2015; Pervez et al. 2008). So, in con-
tinuation of identifying new inhibitors for urease enzyme,
we intended to synthesized a variety of (perfluorophenyl)
hydrazone derivatives to evaluate their potential for urease
inhibition.
In vitro urease inhibitory activity
All synthetic (perfluorophenyl)hydrazone derivatives
1–27 were subjected for urease inhibitory activity by
following the literature protocol (Weatherburn 1967).
Except compounds 12, 21, 26, and 27, all derivatives
showed good to moderate urease inhibitory potential in
the range of IC50 = 14.09 0.23–78.69 1.56 µM as
compared to standard thiourea (IC50 = 21.10 0.31 µM).
Compounds 2 (IC50 = 14.23 0.21 µM), 5 (IC50 = 16.78
0.33 µM), 7 (IC50 = 15.59 0.60 µM), 9 (IC50 = 20.18
0.78 µM), 10 (IC50 = 16.13 0.93 µM), and 11 (IC50
14.09 0.23 µM) were found to be more potent than the
standard thiourea (Table 2).
=
Structure-activity relationship (SAR)
Limited SAR was established by analyzing the effect of
varying groups R1 and R2 on the inhibitory potential.
Bioactivity data presented in Table 2 suggested that most of
the active analogs have hydroxy group/groups in the
structure.
Results and discussion
Chemistry
Amongst the mono-hydroxy substituted analogs, com-
pound 3 (IC50 = 23.54 0.24 µM) and 9 (IC50 = 20.18
0.78 µM) having para and meta hydroxy substituted ben-
zene ring (R1) and hydrogen atom as R2, showed compar-
able activity than standard (IC50 = 21.10 0.31 µM). Ortho
hydroxy substituted analog 24 (IC50 = 36.24 0.44 µM)
and para hydroxy substituted analog 25 (IC50 = 33.88
0.82 µM) with an extra methyl group (R2) showed moderate
activities. The additional non-polar methyl group might be
creating some hindrance for the compounds to bind with the
active site of urease enzyme. Dihydroxy analogs such as 5
(IC50 = 16.78 0.33 µM), 10 (IC50 = 16.13 0.93 µM), and
11 (IC50 = 14.09 0.23 µM), having hydroxy groups at dif-
ferent positions of benzene ring, were found to be the potent
analogs. However, one dihydroxy substituted compound 22
(Perfluorophenyl)hydrazone derivatives 1–27 were pre-
pared by reacting perfluorophenylhydrazine with different
arylaldehydes/acetophenones/arylketones in ethanol in the
presence 2–3 drops of CH3COOH as catalyst. The reac-
tion was refluxed for 25–45 min to afford desired com-
pounds in good yields (85–92%) (Table 1). The
consumption of starting material was monitored through
periodic TLC analysis. On completion, the reaction mix-
ture was cooled to room temperature to get precipitate
which were filtered and dried in vacuum. The precipitates
were crystallized from ethanol. Structure identification
1
was accomplished by using EI-MS and HNMR spectro-
scopic techniques (Scheme 1).

Medicinal Chemistry Research
Table 1 R1 and R2 substitutions of synthetic (perfluorophenyl)hydrazone derivatives 1–27
Compounds
1
R1
R2
Compounds
15
R1
R2
H
H
H
H
H
H
H
2
3
4
5
16
17
18
19
H
H
Me
H
H
Me
Me
6
7
20
21
H
Me
8
22
H
H
H
Me
Me
Me
9
23
24
25
10
11
H
H
H
12
13
14
26
27
-
-
-

Medicinal Chemistry Research
More the number of hydroxy groups more the inhibitory
potential. After hydroxy, nitro group is also playing an
important role in the activity. In addition to that activity
results presented in the Table 2 also suggested that com-
pounds with methoxy, N,N-dimethyl, and halogens sub-
stitutions were found to be moderately active. Since, in
order to get a clear picture of the participation of different
groups in the binding interaction with the active site of
enzyme, molecular docking study was conducted.
Scheme 1 Synthesis of (perfluorophenyl)hydrazone derivatives 1–27
Table 2 In vitro urease inhibitory activity of compounds 1–27
Molecular docking studies
Compounds IC50 + SEM (μM) Compounds
IC50 + SEM (μM)
1
25.93 0.96
14.23 0.21
23.54 0.24
39.43 0.11
16.78 0.33
30.75 0.76
15.59 0.60
23.32 0.15
20.18 0.78
16.13 0.93
14.09 0.23
NAb
15
16
17
18
19
20
21
22
23
24
25
26
27
27.19 0.57
35.61 1.04
49.35 0.56
42.56 0.35
40.76 0.92
33.36 0.27
NA
Top ten compounds with high inhibition were selected for
molecular docking studies with the enzyme urease (3LA4)
through MOE software. It was observed that all the com-
pounds represented excellent binding affinity within the
active site of enzyme as compared to the standard thiourea
(Table 3). From these, compounds 2, 5, 10, and 11 exhibited
outstanding binding affinity with Gibb’s free binding energy
(ΔG) value of −8.72, −7.53, −7.63, and −7.22 kcal mol−1
with inhibition constant (Ki) rate of 0.73, 5.00, 4.25, and
8.26 µM, respectively, as compared to thiourea with Gibb’s
free energy value of only −0.89 kcal mol−1 as shown in
Table 3. Thus, molecular docking studies substantiated that
high ranked compounds in urease inhibition test also
exhibited high affinity for active site of enzyme.
2
3
4
5
6
7
8
29.30 0.35
32.37 0.41
36.24 0.44
33.88 0.82
NA
9
10
11
12
13
14
78.69 1.56
56.67 0.86
NA
Std = Thiourea 21.10 0.310
High ranked compounds in molecular docking studies as
well as in in vitro testing (i.e., compounds 2, 5, 10, and 11)
demonstrated metal interaction with Ni841 and Ni842
through OH groups, conventional hydrogen bonds with
Ala440, KCX490, Asp633, and CME592 also due to OH
groups and π-π stacking with His593 through per-
fluorophenyl ring (Fig. 1). It was observed that the top most
compound in docking studies, i.e., compound 2 interacts
with Ni841 and Ni842 through OH groups at position 3 and
4 simultaneously in a bidentate manner each. It also exhibits
conventional hydrogen bonds with Ala440, KCX490,
His492 and Asp633 and π-π stacking with His593 as shown
in Fig. 4.
Binding poses of compound 2 (pink) and compound 11
(yellow) with the active site of enzyme Jack bean urease
(3LA4) were compared with the binding pose of thiourea
(cyan) in Fig. 5a. Hydrophobic surface and active site cavity
of the enzyme with the docking pose of compound 2 (green)
was also represented in Fig. 5b.
SEM standard error of the mean, NA not active, Std standard inhibitor
for urease enzyme
(IC50 = 29.30 0.35 µM) was exceptionally showed less
potential might be due to not fulfilling the conformational
requirement to fit well into the active site of enzyme. Another
dihydroxy substituted compound 23 (IC50 = 32.37 0.41
µM) with distinctively similar structure to the most active
analog 1 (IC50 = 14.09 0.23 µM) of this series 1, but
having additional methyl group (R2), was found to be
moderately active. Trihydroxy substituted analog 2 (IC50
=
14.23 0.21 µM) was found to be second most active of this
series (Fig. 2). It was observed that compounds bearing
more hydroxy groups found to be more active might be due
to increased number of interactions by the hydroxy group
with the active site of enzyme.
Meta and para nitro substituted analogs 1 (IC50 = 25.93
0.96 µM) and 8 (IC50 = 23.32 0.15 µM), respectively,
showed inhibitory potential comparable to standard. How-
ever, incorporation of hydroxy group with nitro as in com-
pound 7 (IC50 = 15.59 0.60 µM), leads to potent inhibitory
activity. Similarly, incorporation of non-polar methyl group
(R2) as in compound 20 (IC50 = 33.36 0.27 µM) brings out
the decreased activity (Fig. 3).
Physicochemical properties and pharmacokinetics
prediction
Early prediction of physicochemical and ADMET
(Absorption, Distributation, Metabolism, Excretion, and
Toxicity) properties of lead molecules has become very
useful in the drug discovery and development process later
It is rationalized from the limited SAR that compounds
with hydroxy substitutions were found to be more active.

Medicinal Chemistry Research
Fig. 2 SAR of compounds bearing hydroxy substitutions
Fig. 3 SAR of compounds bearing nitro substitutions
Table 3 Gibb’s free binding energy (ΔG) and inhibition constant (ki)
values of top ten compounds studied through MOE
orally administered, 3–4 are transdermal, and high value
produces toxic build up in fatty tissue. According to these
criteria only compound 2 has almost oral absorption, others
have transdermal absorption except compounds 1 and 8 that
represented undesirable bioaccumulation in fatty tissue,
highlighted in Table 4. Another extension in RO5 to
improve the prediction of drug-likeness is MR which should
be 40–130. PSA values are important for the determination
of blood brain barrier (BBB) penetration. According to
Waterbeemd the cutoff value is 90 Å2 or less. Except
compound 7, all the compounds showed BBB penetration.
To determine the toxicity of these compounds ames
mutagenecity test and oral rat LD50 values were estimated
for the selected compounds by using TEST (Toxicity Esti-
mation Software Tool) software. With ames mutagenicity
test, compounds 3, 7, and 9 were found positive, high-
lighted in red (Table 4). According to Hodge and Sterner
scale oral rat LD50 values ranging from 1–50 mg kg−1 are
Compounds MOEa Ki (µM)
Compounds MOEa Ki (µM)
1
−4.99
−8.72
−6.31
−7.53
−6.65
316.23
0.73
8
9
−5.10
−5.95
−7.63
−7.22
256.8
64.74
4.25
2
3
36.12 10
5.00 11
20.80 15
5
8.26
7
−3.81 2078.55
Thiourea
−0.89 236287.03
a
Note: Gibb’s free binding energy values (ΔG) in kcal mol−1
on. Lipinsiki’s rule of five (RO5), molar refractivity (MR),
polar surface area (PSA), octanol water partition coefficient
(logP) were determined through Mcule and PreADMET
online softwares. It was observed that the entire selected
compounds followed RO5 with no violation. Generally,
drugs with logP value below zero are injectable, 0–3 are

Medicinal Chemistry Research
Fig. 4 2D Binding pose representation of compounds 2, 5, 10, and 11 with the active site of enzyme Jack bean urease (3LA4)
Fig. 5 a 3D binding pose
representation of compound 2
(pink), compound 11 (yellow)
and thiourea (cyan) with the
active site of enzyme Jack bean
urease (3LA4) with two nickel
atoms in gray color.
b Hydrophobic surface and the
active site cavity of the enzyme
with two yellow balls
representing nickel atom docked
with compound 2 in green color
highly toxic, 50–500 mg kg−1 are moderately toxic and
500–5000 mg kg−1 are slightly toxic. Referring to this scale
most of the compounds were slightly toxic except com-
pounds 1, 5, and 7 (highlighted in red) which showed
moderate toxicity as shown in Table 4. Keeping all the
properties in view, compounds 2, 10, 11, and 15 were found
to display excellent physicochemical properties that execute
the criteria of drug-likeness as shown in Table 4.
Combining in vitro and in silico studies, we emphasized
that compound 2 (having 2,3,4-trihydroxy benzene ring as
R1) and compound 11 (having 2,5-dihydroxy benzene
as R1) were found to be most efficient and potent inhibitors
of urease enzyme with excellent physicochemical and
drug-like properties. Compound 2 has molecular weight
of 334 g mol−1, IC50 value of 14.23 µM, ΔG value of
−8.72 Kcal mol−1, Ki rate of 0.73 µM, logP value of

Medicinal Chemistry Research
Table 4 Physicochemical
properties and pharmacokinetics
prediction of top ten compounds
Compounds MW RO5a MRb Mutc logPd
Value Bio-availability Value BBB Value Toxicity
PSAe
LD50
f
1
331
334
302
318
347
331
302
318
318
316
0
0
0
0
0
0
0
0
0
0
72.5 −ve 4.2
69.7 −ve 3.0
65.7 +ve 3.6
67.7 −ve 3.3
74.5 +ve 4.0
72.5 −ve 4.3
65.7 +ve 3.6
67.7 −ve 3.3
67.7 −ve 3.3
70.2 −ve 3.9
Accumulate
Oral Abs.
70.2
85.0
44.6
64.8
90.4
70.2
44.6
64.8
64.7
33.6
+ve
+ve
+ve
+ve
−ve
+ve
+ve
+ve
174.8 Moderate
698.6 Slight
515.8 Slight
367.4 Moderate
304.0 Moderate
826.1 Slight
560.0 Slight
920.0 Slight
2
3
Transdermal
Transdermal
Transdermal
Accumulate
Transdermal
Transdermal
Transdermal
Transdermal
5
7
8
9
10
11
15
+ve 1131.2 Slight
+ve 727.3 Slight
aLipinski’s rule of five (RO5) violation
bMolar refractivity
cAmes Mutagenecity test
dGhose-Crippen octanol water coefficient
ePolar surface area
fOral rat LD50 (mg kg−1
)
3.0, LD50 value of 698.6 mg kg−1 and non-mutagenic.
Likewise compound 11 has also exhibited almost similar
properties which signify the consequence of these com-
pounds in pre-clinical and clinical trials.
different substituted aldehydes and acetophenones (1 mmol)
in absolute ethanol (15 mL) with 2–3 drops of CH3COOH
under reflex up to 25–45 min. Reaction completion was
monitored through TLC analysis and the transparent
colored reaction mixture was kept at room temperature for
cooling and dried up to afford crude products 1–27. Com-
pounds were crystallized in ethyl acetate. Reaction yield is
in the range of 85–92%. For structure conformations, the
synthesized compounds were inspected using EI-MS and
1HNMR analysis.
Experimental
Materials and methods
NMR experiments were performed on Bruker Avance AM
300 and 400 MHz machine. Electron impact mass spectra
1-(3-Nitrobenzylidene)-2-(perfluorophenyl)hydrazine (1)
(EIMS) were recorded on
a Finnigan MAT-311A,
Germany. CHN analyses were carried out a Carlo Erba
Strumentazione-Mod-1106, Italy. Thin layer chromato-
graphy (TLC) was performed on pre-coated silica gel alu-
minum plates (Kieselgel 60, 254, E. Merck, Germany).
Chromatograms were visualized by UV at 254 and 365 nm.
2,2-Diphenyl-1-picrylhydrazyl radical (DPPH), ethylene
diaminetetraacetic acid (EDTA), ascorbic acid, tris-HCl
buffer, ferrous sulfate, ferric chloride (FeCl3), sulfuric acid,
O-phenenthroline, potassium phosphate (mono phosphate
and diphosphate), ammonium molybdate, hydrogen per-
oxide (H2O2), and ethanol were of analytical grade and
purchased from Sigma Aldrich, USA.
Yield: 0.30 g (90%); 1H-NMR: (300 MHz, DMSO-d6):
δ 10.66 (s, 1H, –NH), 8.40 (s, 1H, –H-2), 8.20 (s, 1H, Vin.
H), 8.16 (d, 1H, J4,5 = 8.5 Hz, H-4), 8.02 (d, 1H, J6,5 = 8.5
Hz, H-6), 7.70 (t, 1H, J5,(4,6) = 8.5 Hz, H-5); EI-MS: m/z
(rel. abund. %) 331 (M+, 75), 182 (100), 155 (40), 76 (38);
Anal. Calcd for C13H6F5N3O2 (331.20): C, 47.14; H, 1.83;
N, 12.69; Found: C, 47.12; H, 1.82; N, 12.70.
4-((2-(Perfluorophenyl) hydrazono) methyl)benzene-1,2,3-
triol (2)
Yield: 0.26 g (78%); 1H-NMR: (300 MHz, DMSO-d6):
δ 10.49 (s, 1H, –NH), 10.22 (s, 1H, –OH), 9.34 (br.s, 1H,
–OH), 8.44 (br.s, 1H, -OH), 8.20 (s, 1H, Vin. H), 6.67 (d,
1H, J6,5 = 8.5 Hz, H-6), 6.35 (d, 1H, J5,6 = 8.5 Hz, H-5); EI-
MS: m/z (rel. abund. %) 334 (M+, 100), 183 (70), 124 (93),
7946); Anal.Calcd for C13H7F5N2O3 (334.2): C, 46.72; H,
2.11; N, 8.38; Found: C, 46.71; H, 2.13; N, 8.35.
General procedure for the synthesis of compounds
1–27
(Perfluorophenyl)hydrazone derivatives 1–27 were prepared
by reacting perfluorophenylhydrazine (1 mmol) with

Medicinal Chemistry Research
4-((2-(Perfluorophenyl) hydrazono) methyl) phenol (3)
1-(4-Nitrobenzylidene)-2-(perfluorophenyl)hydrazine (8)
Yield: 0.27 g (84%); 1H-NMR: (300 MHz, DMSO-d6):
δ 10.00 (s, 1H, -NH), 9.79 (br.s, 1H, –OH), 8.01 (s, 1H, Vin.
Yield: 0.27 g (82%); 1H-NMR: (300 MHz, DMSO-d6):
δ 10.81 (s, 1H, -NH), 8.24 (d, 2H, J3,2/5,6 = 8.7 Hz, H-3/5),
8.18 (s, 1H, Vin. H), 7.82 (d, 2H, J2,3/6,5 = 8.7 Hz, H-2/6);
EI-MS: m/z (rel. abund. %) 331 (M+, 100), 182 (65), 155
(24), 76 (13); Anal.Calcd for C13H6F5N3O2(331.20): C,
47.14; H, 1.83; N, 12.69; Found: C, 47.17; H, 1.80; N,
9.65.
H), 7.40 (d, 2H, J2,3/6,5 = 8.5 Hz, H-2/6), 6.78 (d, 2H, J3,2/5,6
=
8.5 Hz, H-3/5); EI-MS: m/z (rel. abund. %) 302 (M+, 100),
182 (40), 118 (73), 76 (66); Anal. Calcd for C13H7F5N2O
(302.19): C, 51.67; H, 2.33; N, 9.27; Found: C, 51.66; H,
2.30; N, 9.29.
1-(3,4-Dichlorobenzylidene)-2-(perfluorophenyl)hydrazine
3-((2-(Perfluorophenyl) hydrazono)methyl)phenol (9)
(4)
Yield: 0.26 g (86%); 1H-NMR: (300 MHz, DMSO-d6):
δ 10.23 (s, 1H, –NH), 9.50 (s, 1H, –OH), 8.02 (s, 1H, Vin.
H), 7.18 (t, 1H, J5(4,6) = 8.0 Hz, H-5), 7.04 (br.s, 1H, H-
2),6.98 (d, 1H, J6,5 = 8.0 Hz, H-6), 6.74 (dd, 1H, J4,6 = 1.5,
J4,3 = 8.0 Hz, H-4); EI-MS: m/z (rel. abund. %) 302 (M+,
100), 183 (68), 83 (55); Anal. Calcd for C13H7F5N2O
(302.20): C, 51.67; H, 2.33; N, 9.27; Found: C, 51.69; H,
2.31; N, 9.29.
Yield: 0.33 g (95%) ;1H-NMR: (300 MHz, DMSO-d6):
δ 10.75 (s, 1H, –NH), 8.44 (s, 1H, Vin. H), 7.85 (d, 1H, J5,6
8.7 Hz, H-5), 7.66 (d, 1H, J2,6 = 2.0 Hz, H-2), 7.45 (dd, 1H,
J6,2 = 2.0,J6,5 = 8.7 Hz, H-6); EI-MS: m/z (rel. abund. %)
356 (M+2, 50), 354 (M+, 72), 319 (37), 183 (100), 172
(36), 155 (37); Anal. Calcd for C15H11F5N2O2 (346.25): C,
52.03; H, 3.20; N, 8.09; Found: C, 52.01; H, 3.18; N, 8.06.
=
4-((2-(Perfluorophenyl)hydrazono)methyl) benzene-1,2-diol
3-((2-(Perfluorophenyl)hydrazono)methyl)benzene-1,2-diol
(5)
(10)
Yield: 0.26 g (82%); 1H-NMR: (300 MHz, DMSO-d6):
δ 9.90 (br.s, 2H, –OH), 9.67 (s, 1H, –NH), 7.94 (s, 1H, Vin.
H), 7.09 (d, 1H, J2,6 = 1.5 Hz, H-2), 6.80 (d, 1H, J5,6 = 8.0
Hz, H-5), 6.80 (dd, 1H, J6,2 = 1.5,J6,5 = 8.0 Hz, H-6); EI-
MS: m/z (rel. abund. %) 318 (M+, 100), 183 (40), 137 (64),
109 (60); Anal. Calcd for C13H7F5N2O2 (318.20): C, 49.07;
H, 2.22; N, 8.80; Found: C, 49.04; H, 2.25; N, 8.76.
Yield: 0.28 g (88%); 1H-NMR: (300 MHz, DMSO-d6):
δ 10.42 (s, 1H, –NH), 10.08 (s, 1H, –OH), 10.02 (s, 1H,
-OH), 8.32 (s, 1H, Vin. H), 6.85 (d, 1H, J6,5 = 7.5 Hz, H-6),
6.76 (d, 1H, J4,5 = 7.5 Hz, H-4), 6.70 (t, 1H, J5(4,6) = 7.5 Hz,
H-5); EI-MS: m/z (rel. abund. %) 318 (M+, 100), 183 (65),
135 (74), 108 (71); Anal. Calcd for C13H7F5N2O2 (318.05):
C, 49.07; H, 2.22; N, 8.80; Found: C, 49.04; H, 2.18; N,
8.84.
1-(2-Bromobenzylidene)-2-(perfluorophenyl)hydrazine (6)
4-((2-(Perfluorophenyl)hydrazono)methyl)benzene-1,3-diol
(11)
Yield: 0.33 g (90%); 1H-NMR: (300 MHz, DMSO-d6):
δ 10.70 (s, 1H, –NH), 8.45 (s, 1H, Vin. H), 7.85 (dd, 1H,
J3,5 = 1.2, J3,4 = 8.0 Hz, H-3), 7.64 (d, 1H, J6,5 = 8.0 Hz, H-
Yield: 0.28 g (88%); 1H-NMR: (300 MHz, DMSO-d6):
δ 10.54 (s, 1H, –NH), 10.17 (s, 1H, –OH), 9.80 (s, 1H, –
OH), 8.24 (s, 1H, Vin. H), 7.20 (d, 1H, J6,5 = 7.5 Hz, H-6),
6.32 (dd, 1H, J5,3 = 2.0,J5,6 = 8.5 Hz, H-5), 6.27 (s, 1H, H-
3); EI-MS: m/z (rel. abund. %) 318 (M+, 15), 183 (100),
136 (24); Anal. Calcd for C13H7F5N2O2 (318.20): C, 49.07;
H, 2.22; N, 8.80; Found: C, 49.06; H, 2.25; N, 8.83.
6), 7.39 (t, 1H, J4(3,5) = 8.0 Hz, H-4), 7.24 (t, 1H, J5(4,6)
=
8.0 Hz, H-5); EI-MS: m/z (rel. abund. %) 366 (M+2, 56),
364 (M+, 53), 285 (100), 183 (82), 155 (41); Anal. Calcd
for C13H6BrF5N2 (365.10): C, 42.77; H, 1.66; N, 7.67;
Found: C, 42.75; H, 1.64; N, 7.71.
4-Nitro-2-((2-(perfluorophenyl)hydrazono)methyl)phenol
(7)
4-((2-(Perfluorophenyl)hydrazono)methyl)benzaldehydes
(12)
Yield: 0.32 g (92%); 1H-NMR: (300 MHz, DMSO-d6):
δ 10.61 (s, 1H, –NH), 8.44 (d, 1H, J6,4 = 2.5 Hz, H-6), 8.41
(s, 1H, Vin. H), 8.05 (dd, 1H, J4,6 = 2.5, J4,3 = 9.0 Hz, H-4),
6.98(d, 1H, J3,4 = 9.0 Hz, H-3); EI-MS: m/z (rel. abund. %)
347 (M+, 92), 183 (100), 155 (26); Anal. Calcd for
C13H6F5N3O3 (347.20): C, 44.97; H, 1.74; N, 12.10; Found:
C, 45.01; H, 1.72; N, 12.07.
Yield: 0.26 g (83%); 1H-NMR: (300 MHz, DMSO-d6): δ 10.66
(s, 1H, –NH), 9.98 (s, 1H, –CHO), 7.99 (s, 1H, Vin. H), 7.92
(d, 2H, J2,3/6,5 = 8.7 Hz, H-2/6), 6.79 (d, 2H, J3,2/5,6 = 8.7 Hz,
H-3/5); EI-MS: m/z (rel. abund. %) 314 (M+, 51), 183
(100), 103 (29); Anal. Calcd for C14H7F5N2O(314.21): C,
53.52; H, 2.25; N, 8.92; Found: C, 53.55; H, 2.22; N, 8.90.

Medicinal Chemistry Research
N, N-Dimethyl-4-((2-(perfluorophenyl)hydrazono)methyl)
1-(4-Methoxybenzylidene)-2-(perfluorophenyl)hydrazine
aniline (13)
(18)
Yield: 0.26 g (79%); 1H-NMR: (300 MHz, DMSO-d6):
δ 9.89 (s, 1H, –NH), 7.99 (s, 1H, Vin. H), 7.41 (d, 2H, J2,3/
6,5 = 8.7 Hz, H-2/6), 6.70 (d, 2H, J3,2/5,6 = 8.7 Hz, H-3/5),
2.93 (s, 6H, N(CH3)2); EI-MS: m/z (rel. abund. %) 329 (M+,
100), 147 (62), 120 (20); Anal. Calcd for C15H12F5N3
(329.27): C, 54.72; H, 3.67; N, 12.76; Found: C, 54.70; H,
3.69; N, 12.75.
Yield: 0.25 g (83%); 1H-NMR: (300 MHz, DMSO-d6):
δ 10.10 (s, 1H, –NH), 8.05 (s, 1H, Vin. H), 7.54 (d, 2H,
J2,3/6,5 = 8.5 Hz, H-2/6), 6.96 (d, 2H, J3,2/5,6 = 8.5 Hz,
H-3/5), 3.76 (s, 3H, –OCH3); EI-MS: m/z (rel. abund. %)
316 (M+, 100), 183 (44), 134 (100), 77 (100); Anal. Calcd
for C14H9F5N2O(316.23): C, 53.17; H, 2.87; N, 8.86;
Found: C, 56.04; H, 3.00; N, 9.36.
1-(Perfluorophenyl)-2-(3,4,5-trimethoxybenzylidene)
1-(Perfluorophenyl)-2-(1-phenylethylidene) hydrazine (19)
hydrazine (14)
Yield: 0.133 g (89%); 1H-NMR: (400 MHz, DMSO-d6):
δ 8.78 (s, 1H, –NH), 7.72 (d, 2H, J2,3/6,5 = 7.2 Hz, H-2/6),
7.39-7.33 (m, 3H, H-3/4/5), 2.30 (s, 3H, CH3); EI-MS: m/z
(relative. abundence. %) 300 (M+, 100), 182 (8), 155 (12),
118 (92), 103 (6), 77 (95), 69 (3), 51 (12); Anal. Calcd for
C14H9F5N2 (300.23): C, 56.01; H, 3.02; N, 9.33; Found:
C,56.00, H, 3.05, N, 9.30.
1
Yield: 0.33 g (88%); H-NMR: (300 MHz, DMSO-d6):
δ 10.28 (s, 1H, –NH), 8.00 (s, 1H, Vin. H), 6.67(s, 2H, H-2/
6), 3.78 (s, 6H, 2-OCH3), 3.66 (s, 3H, OCH3); EI-MS: m/z
(rel. abund. %) 376 (M+, 100), 161 (42), 167 (24), 152 (28);
Anal. Calcd for C13H7F5N2O3 (334.2): C, 51.07; H, 3.48; N,
7.44; Found: C, 51.04; H, 3.50; N, 7.45.
2-Methoxy-6-((2-(perfluorophenyl) hydrazono)methyl)
1-(1-(3-Nitrophenyl) ethylidene)-2-(perfluorophenyl)
phenol (15)
hydrazine (20)
Yield: 0.29 g (87%); 1H-NMR: (300 MHz, DMSO-d6):
δ 10.42 (s, 1H, –OH), 10.03 (s, 1H, –NH), 8.36 (s, 1H,
Vin. H), 7.05 (d, 1H, J6,5 = 7.0 Hz, H-6), 6.94 (d, 1H, J4,5
= 7.0 Hz, H-4), 6.80 (t, 1H, J5(4,6) = 7.0 Hz, H-5), 3.78 (s,
3H, OCH3); EI-MS: m/z (rel. abund. %) 332 (M+, 85), 149
(57), 135 (100), 83 (81); Anal. Calcd for C14H9F5N2O2
(332.23): C, 50.61; H, 2.73; N, 8.43; Found: C, 50.58; H,
2.76; N, 8.44.
Yield: 0.162 g (92%); 1H-NMR: (400 MHz, DMSO-d6):
δ 9.12 (s, 1H, –NH), 8.49 (s, 1H, H-2), 8.18 (d, 1H, J4,5 = 8
Hz, H-4), 8.135 (d, 1H, J6,5 = 7.6 Hz, H-6), 7.684
(t, 1H, J5,(4.6) = 8 Hz, H-5), 2.364 s (s, 3H, CH3); EI-MS: m/
z (relative. abundence. %) 345 (M+, 100), 328 (4), 298 (9),
182 (22), 163 (44), 155 (12), 117 (65), 92 (5),76 (23), 50
(4); Anal. Calcd for C14H8F5N3O2, (345.22); C, 48.71; H,
2.34; N, 12.17; Found: C, 48.70; H, 2.35; N, 12.18
1-(4-Methylbenzylidene)-2-(perfluorophenyl)hydrazine (16)
1-(1-(Naphthalen-2-yl) ethylene)-2-(perfluorophenyl)
hydrazine (21)
Yield: 0.25 g (83%); 1H-NMR: (300 MHz, DMSO-d6):
δ 10.20 (s, 1H, –NH), 8.07 (s, 1H, Vin. H), 7.48 (d, 2H, J2,3/
6,5 = 8.0 Hz, H-2/6), 7.20 (d, 2H, J3,2/5,6 = 8.0 Hz, H-3/5),
2.30 (s, 3H, –CH3);MS: m/z (rel. abund. %) 300 (M+, 100),
183 (26), 118 (25), 91 (56); Anal. Calcd for C14H9F5N2
(300.23): C, 56.01; H, 3.02; N, 9.33; Found: C, 56.04; H,
3.00; N, 9.36.
Yield: 0.152 g (86%); 1H-NMR: (400 MHz, DMSO-d6):
δ 8.90 (s, 1H, –NH), 8.16 (s, 1H, H-1), 7.997-7.952 (m, 2H,
H-3/4), 7.894-7.854 (m, 2H, H-5/8), 7.514 (t, 2H, J6,(5,7)/7,
(6,8), = 7.2 Hz, H-6/7), 2.42 (s, 3H, CH3); EI-MS: m/z
(relative. abundence. %) 350 (M+, 86), 335 (2), 222 (2),
181 (7), 168 (71), 153 (15), 127 (100), 101 (3),77 (5), 44
(1); Anal. Calcd for C18H11F5N2 (350.29), C, 61.72; H,
3.17; N, 8.00; Found: C,61.75; H, 3.15; N, 8.03.
1-(3,4-Dimethoxybenzylidene)-2-(perfluorophenyl)
hydrazine (17)
2-(1-(2-(Perfluorophenyl) hydrazono) ethyl) benzene-1,3-
diol (22)
Yield: 0.32 g (92%);1 H-NMR: (300 MHz, DMSO-d6):
δ 10.13 (s, 1H, –NH), 8.03 (s, 1H, Vin. H), 7.20 (br.s, 1H,
H-2), 7.07 (dd, 1H, J6,2 = 1.2, J6,5 = 8.5 Hz, H-6), 6.96 (d,
1H, J5,6 = 7.5 Hz, H-5), 3.76 (s, 6H, (–OCH3)2); EI-MS: m/z
(rel. abund. %) 346 (M+, 100), 164 (17), 137 (17); Anal.
Calcd for C15H11F5N2O2 (346.25): C, 52.03; H, 3.20; N,
8.09; Found: C, 52.05; H, 3.23; N, 8.05.
1
Yield: 0.147 g (88%); H-NMR: (400 MHz, DMSO-d6):
δ 10.03 (s, 2H, –OH-2/6), 8.69 (s, 1H, -NH), 6.96 (t, 1H,
J4,(3,5) = 8 Hz, H-4), 6.25 (d, 2H, J3,4/5,4 = 8 Hz, H-3/5),
2.348 (s, 3H, CH3); EI-MS: m/z (relative. abundence. %)
332 (M+, 100), 315 (26), 222 (6), 181 (14), 150 (42), 122

Medicinal Chemistry Research
(6), 107 (9), 81 (6), 42 (5); Anal. Calcd for C14H9F5N2O2
(332.23), C, 50.61; H, 2.73; N, 8.43; Found: C, 50.60; H,
2.75; N, 8.41
4,4’-((2-(Perfluorophenyl) hydrazono)methylene)bis(N,N-
dimethylaniline) (27)
Yield: 0.208 g (92%); 1H-NMR: (400 MHz, DMSO-d6):
δ 10.66 (s, 1H, –NH), 7.58 (d, 4H, J2,3/6,5/2ˊ,3ˊ/6ˊ,5ˊ = 8.8 Hz,
H-2/6/2′/6′), 6.74 (d, 4H, J3,2/5,6/3ˊ,2ˊ/5ˊ,6ˊ = 8.8 Hz, H-3/5/3′/
5′), 3.08 (s, 12H, -N(CH3)22); EI-MS: m/z (relative. abun-
dence. %) 448 (M+, 10), 268 (100), 251 (42), 224 (59), 148
(92); Anal. Calcd for C23H21F5N4(448.43), C, 61.60; H,
4.72; N, 12.49; Found: C, 61.62; H, 4.70; N, 12.50.
4-(1-(2-(Perfluorophenyl) hydrazono) ethyl) benzene-1,3-
diol (23)
Yield: 0.151 g (90%);1H-NMR: (400 MHz, DMSO-d6):
δ 12.12 (s, 1H, –OH), 9.75 (s, 1H, –OH), 8.867 (s, 1H,
–NH), 7.382 (d, 1H, J6,5 = 8.8 Hz, H-6), 6.32 (dd, 1H,
J5,3/5,6 = 2.4 Hz, 8.4 Hz, H-5), 6.218 (s, 1H, H-3) 2.357
(s, 3H, CH3); EI-MS: m/z (relative. abundence. %) 332
(M+, 100), 315 (14), 222 (6), 181 (16), 116 (7), 150 (46),
135 (8), 81 (8), 69 (4), 42 (8); Anal. Calcd for
C14H9F5N2O2 (332.23), C, 50.61; H, 2.73; N, 8.43;
Found: C, 50.60; H, 2.75;N, 8.40
Urease inhibition assay (In vitro)
Reaction mixture comprising on 25 μL of enzyme (Cana-
valia ensiformis urease) solution and 55 μL of buffers,
containing 100 mM urea, were incubated with 5 μL of test
compounds (0.5 mM concentration) at 30 °C for 15 min in
96-well plates. Urease activity was determined by measur-
ing ammonia production by using the indophenol method,
as described by Weatherburn (Weatherburn 1967). Briefly,
45 μL each phenol reagent (1% w/v phenol and 0.005% w/v
sodium nitroprussside) and 70 μL of alkali reagent (0.5% w/
v NaOH and 0.1% active chloride NaOCl) were added to
each well. The increasing absorbance at 630 nm was mea-
sured after 50 min by using a microplate reader (Spectra
Max, Molecular Devices, CA, USA). All reactions were
performed in triplicate in a final volume of 200 μL. The
results (change in absorbance per min) were processed by
using soft Max Pro software (Molecular Devices, CA,
USA). The entire assays were performed at pH 6.8. Per-
centage inhibitions were calculated from the formula 100-
(ODtestwell/ODcontrol) × 100. Thiourea was used as the stan-
dard inhibitor of urease (Abid et al. 2010).
2-(1-(2-(Perfluorophenyl) hydrazono)ethyl)phenol (24)
Yield: 0.149 g (85%); 1H-NMR: (400 MHz, DMSO-d6):
δ 8.44 (s, 1H, –OH), 8.42 (s, 1H, –NH), 8.092 (d, 1H, J3,4
=
8 Hz, H-3), 7.982 (d, 1H, J6,5 = 8.8 Hz, H-6), 7.76 (t, 1H,
J4,(3,5) = 7.2 Hz, H-4), 7.59 (t, 1H, J5,(4,6) = 8.8 Hz, H-5),
2.90 (s, 3H, CH3); EI-MS: m/z (relative. abundence. %) 348
(M+, 100), 329 (8.6), 306 (2.0), 288 (2.2), 183 (11), 174
(5), 153 (3), 140 (8), 114 (4), 64 (11), 44 (9); Anal. Calcd
for C14H9F5N2O (316.23), C, 53.17; H, 2.87; N, 8.86;
Analyzed: C, 53.20; H, 2.85; N,8.85
4-(1-(2-(Perfluorophenyl) hydrazono)ethyl)phenol (25)
1
Yield: 0.148 g (92%); H-NMR: (400 MHz, DMSO-d6):
δ 10.65 (s, 1H, –NH), 7.82 (d, 2H, J3,2/5,6 = 8.5 Hz, H-3/
5), 6.82 (d, 2H, J2,3/6,5 = 8.5 Hz, H-2/6), 2.35 (s, 3H,
CH3); EI-MS: m/z (relative. abundence. %) 316 (M+, 1.7),
287 (18), 231 (2.4), 211 (1.8), 150 (85), 121 (100), 93
(78), 80 (44.5), 65 (38), 48 (15); Anal. Calcd for
C14H9F5N2O (316.23), C, 53.17; H, 2.87; N, 8.86; Found:
C, 53.20; H, 2.85; N, 8.85
In silico studies
The molecular construction of the compounds was performed
using ChemBioDraw Ultra 14 suite (PerkinElmer Inc.) and
converted into 3D conformations by ChemBio3D (Mills
2006). Crystal structure of the enzymes, Jack bean urease
(PDB: 3LA4) (Balasubramanian and Ponnuraj 2010) was
retrieved from Protein Data Bank (Berman et al. 2000).
Thiourea (PubChem-2723790) was taken as a standard
ligand. The target proteins were prepared by the addition of
hydrogen, removal of water and the removal of co-
crystallized ligands. All other parameters were used with
the default settings. The compounds were docked using MOE
(Molecular operating environment) software (Da Silva et al.
2011). The structures of the compounds were energy mini-
mized using MMFF94x forcefield and gradient: 0.05. The
active sites of the enzymes were obtained by site finder and
1-(1,2-Diphenylethylidene)-2-((perfluorophenyl)methyl)
hydrazine (26)
Yield: 0.176 g (92%); 1H-NMR: (400 MHz, DMSO-d6):
δ 9.18 (s, 1H, -NH), 7.71 (d, 2H, J2ˊ,3ˊ/6ˊ,5ˊ = 7.2 Hz, H-2′/
6′), 7.303 (m, 5H, H-2/3/4/5/6), 7.22 (m, 2H, H-3′/5′), 7.18
(t, 1H, J4ˊ,(3ˊ,5ˊ) = 15.6 Hz, H-4ˊ), 4.309 (s, 2H, CH2); EI-
MS: m/z (relative. abundence. %) 376 (M+, 100), 285 (4),
193 (53), 182 (49), 155 (8), 103 (3), 91 (33), 65 (3); Anal.
Calcd for C21H15F5N2 (390.35), C, 64.62; H, 3.87; N, 7.18;
Found: C, 64.65; H, 3.90; N, 7.20

Medicinal Chemistry Research
the compounds were docked by the default parameters, i.e,
Placement: Trainagular Matcher, Rescoring1: London dG,
Refinement: Forcefield and Rescoring2: Affinity dG. For each
ligand thirty conformations were generated. Topmost inter-
action was selected with lowest Gibb’s free energy value. The
images in 2D were captured through MOE ligand binding
interaction. 3D images were taken using Discovery Studio
Visualizer and Chimera 1.11 software (Pettersen et al. 2004).
Physicochemical properties and pharmacokinetics stu-
dies were also performed through computational tools.
RO5, MR, PSA, and octanol water partition coefficient
(logP) were determined through Mcule, Inc (Moura et al.
2012) and PreADMET online softwares (Menna-Barreto
et al. 2005). Oral rat LD50 values and ames mutagenicity
test were analyzed using TEST (Shalini et al. 2010).
Hameed A, Khan KM, Zehra ST, Ahmed R, Shafiq Z, Bakht SM,
Yaqub M, Hussain M, de León AdlV, Furtmann N (2015)
Synthesis, biological evaluation and molecular docking of N-
phenyl thiosemicarbazones as urease inhibitors. Bioorg Chem
61:51–57
Holm L, Sander C (1997) An evolutionary treasure: unification of a
broad set of amidohydrolases related to urease. Prot.: Struct.,
Function, Bioinform 28:72–82
Menna-Barreto RFS, Henriques-Pons A, Pinto AV, Morgado-Diaz JA,
Soares MJ, De Castro SL (2005) Effect of a β-lapachone-derived
naphthoimidazole on Trypanosoma cruzi: identification of target
organelles. J Antimicrob Chemother 56:1034–1041
Mills N (2006) ChemDraw Ultra 10.0 CambridgeSoft, 100 Cam-
bridgePark Drive, Cambridge, MA 02140. www.cambridgesoft.
com. Commercial Price: 1910fordownload, 2150 for CD-ROM;
Academic Price: 710fordownload, 800 for CD-ROM. J Am
Chem Soc 128:13649-13650
Moura KCG, Carneiro PF, Pinto MdCFR, da Silva JA, Malta VRS, de
Simone CA, Dias GG, Jardim GAM, Cantos J, Coelho TS, da
Silva PEA, da Silva Jr EN (2012) 1,3-Azoles from ortho-naph-
thoquinones: synthesis of aryl substituted imidazoles and oxa-
zoles and their potent activity against Mycobacterium
tuberculosis. Bioorg Med Chem 20:6482–6488
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Patai S (1970) Chemistry of the carbon-nitrogen double bond. John
Wiley & Sons, New York
Pervez H, Iqbal MS, Tahir MY, Nasim F-u-H, Choudhary MI,
Khan KM (2008) In vitro cytotoxic, antibacterial, antifungal
and urease inhibitory activities of some N 4-substituted
isatin-3-thiosemicarbazones. J Enzyme Inhib Med Chem
23:848–854
Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM,
Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization
system for exploratory research and analysis. J Comput Chem
25:1605–1612
Pope AJ, Toseland N, Rushant B, Richardson S, McVey M, Hills J
(1998) Effect of potent urease inhibitor, fluorofamide, on Heli-
cobacter sp. in vivo and in vitro. J Dig Dis Sci 43:109–119
Prakash A, Adhikari D (2011) Application of Schiff bases and their
metal complexes-A Review. Int J Chem Tech Res 3:1891–1896
Qin Y, Cabral JMS (1994) Kinetic studies of the urease-catalyzed
hydrolysis of urea in a buffer-free system. Appl Biochem Biotech
49:217–240
Rashid U, Batool I, Wadood A, Khan A, Chaudhary MI, Ansari FL
(2013) Structure based virtual screening-driven identification of
monastrol as a potent urease inhibitor. J Mol Graphics Modelling
43:47–57
Rodman J (1998) Struvite stones. Nephron 81:50–59
Sahrawat KL (1980) Control of urea hydrolysis and nitrification in soil
by chemicals-Prospects and problems. Plant Soil 57:335–352
Salimon J, Salih N, Ibraheem H, Yousif E (2010) Synthesis of 2-N-
salicylidene-5-(substituted)-1,3,4-thiadiazole as potential anti-
microbial agents. Asian J Chem 22:5289–5296
Shalini K, Sharma PK, Kumar N (2010) Imidazole and its biological
activities: a review. Chem Sin 1:36–47
Sujoy B, Aparna A (2012) Isolation, partial purification, character-
ization and inhibition of urease (EC 3.5. 1.5) enzyme from the
Cajanus cajan seeds. Asian J Bio Sci 7:203–209
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
References
Abid O-u-R, Babar TM, Ali FI, Ahmed S, Wadood A, Rama NH,
Uddin R, Khan A, Choudhary MI (2010) Identification of novel
urease inhibitors by high-throughput virtual and in vitro screen-
ing. ACS Med Chem Lett 1:145–149
Arshia A, Khan A, Khan KM, Saad SM, Siddiqui NI, Javaid S,
Perveen S, Choudhary MI (2016) Synthesis and urease inhibitory
activities of benzophenone semicarbazones/thiosemicarbazones.
Med Chem Res 25:2666–2679
Aslam MAS, Mahmood S-u, Shahid M, Saeed A, Iqbal J (2011)
Synthesis, biological assay in vitro and molecular docking studies
of new Schiff base derivatives as potential urease inhibitors. Eur J
Med Chem 46:5473–5479
Balasubramanian A, Ponnuraj K (2010) Crystal structure of the first
plant urease from jack bean: 83 years of journey from its first
crystal to molecular structure. J Mol Biol 400:274–283
Berman H, Westbrook J, Feng Z, Gilliland G, Bhat T, Weissig H,
Shindyalov I, Bourne P (2000) Protein Data Bank Nucleic Acids
Res 28:235–242
Bringmann G, Dreyer M, Faber JH, Dalsgaard PW, Stærk D, Jar-
oszewski JW, Ndangalasi H, Mbago F, Brun R, Christensen SB
(2004) Ancistrotanzanine C and related 5,1 ‘-and 7, 3 ‘-Coupled
Naphthylisoquinoline Alkaloids from Ancistrocladus t anza-
niensis 1. J Nat Prod 67:743–748
Choudhary MI, Ali M, Wahab A-t, Khan A, Rasheed S, Shyaula SL,
Rahman A-u (2011) New antiglycation and enzyme inhibitors
from Parmotrema cooperi. Sci China Chem 54:1926–1931
Da Silva CM, da Silva DL, Modolo LV, Alves RB, de Resende MA,
Martins CV, de Fátima  (2011) Schiff bases: a short review of
their antimicrobial activities. J Adv Res 2:1–8
Taha M, Ismail NH, Khan A, Shah SAA, Anwar A, Halim SA, Fatmi
MQ, Imran S, Rahim F, Khan KM (2015) Synthesis of novel
derivatives of oxindole, their urease inhibition and molecular
docking studies. Bioorg Med Chem Lett 25:3285–3289
Weatherburn M (1967) Phenol-hypochlorite reaction for determination
of ammonia. Anal Chem 39:971–974
Hameed A, al-Rashida M, Uroos M, Abid Ali S, Khan KM (2017)
Schiff bases in medicinal chemistry: a patent review (2010-2015).
Expert Opin Ther Pat 27:63–79