4-Oxycoumarinyl linked acetohydrazide Schiff bases as potent urease inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104365

Urease enzyme is responsible to catalyze the hydrolysis of urea into carbamate and ammonia. Then carbamate hydrolyzed to ammonia and carbon dioxide. Excess release of ammonia leads to increase pH in stomach that actually encourages the survival of Helicobacter pylori. H. pylori involves in various disorders most commonly peptic ulcer, pyelonephritis, hepatic coma, kidney stone formation, urolithiasis, and encephalopathy. Apart from many pharmacological properties, coumarin and Schiff bases are known to possess urease inhibitory activity. Therefore, these two pharmacologically important scaffolds are combined into single hybrid molecules to assess their potential as urease inhibitors. For this aim, N′-benzylidene-2-((2-oxo-2H-chromen-4-yl)oxy)acetohydrazide Schiff base derivatives 3–27 were synthesized by following a three step reaction strategy. Structures of all synthetic molecules were characterized by EI-MS, ¹H-, and ¹³C NMR spectroscopic techniques. All molecules were assessed for urease inhibitory activity and found to possess a varying degree of inhibitory potential in the range of IC₅₀ = 12.3 ± 0.69 to 88.8 ± 0.04 μM. Amongst the active analogs, compounds 7 (IC₅₀ = 16.2 ± 0.11 μM), 9 (IC₅₀ = 15.2 ± 0.14 μM), 10 (IC₅₀ = 12.3 ± 0.69 μM), 12 (IC₅₀ = 16.3 ± 0.45 μM), and 15 (IC₅₀ = 17.6 ± 0.28 μM) were identified as potent inhibitors compared to standard urea (IC₅₀ = 21.5 ± 0.47 μM). It is conferred from structure-activity relationship (SAR) that variation in inhibitory activity is due to different substitutions pattern on aryl ring. Moreover, molecular docking studies were carried out to understand the interactions of ligand with the active pocket of urease enzyme.

Full text of DOI:10.1016/j.bioorg.2020.104365

Journal Pre-proofs
4-Oxycoumarinyl Linked Acetohydrazide Schiff bases as Potent Urease In-
hibitors
Fouzia Naz, Kanwal, Mehreen Latif, Uzma Salar, Khalid Mohammed Khan,
Mariya al-Rashida, Irfan Ali, Basharat Ali, Muhammad Taha, Shahnaz
Perveen
PII:
S0045-2068(20)31663-1
DOI:
https://doi.org/10.1016/j.bioorg.2020.104365
YBIOO 104365
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
4 July 2020
7 September 2020
8 October 2020
Please cite this article as: F. Naz, Kanwal, M. Latif, U. Salar, K. Mohammed Khan, M. al-Rashida, I. Ali, B. Ali,
M. Taha, S. Perveen, 4-Oxycoumarinyl Linked Acetohydrazide Schiff bases as Potent Urease Inhibitors,
Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104365
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4-Oxycoumarinyl Linked Acetohydrazide Schiff bases as Potent Urease Inhibitors
Fouzia Naz,a Kanwal,a Mehreen Latif,b Uzma Salar,c Khalid Mohammed Khan,a,e Mariya al-
Rashida,d Irfan Ali,a Basharat Ali,a Muhammad Taha,e Shahnaz Perveenf
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
bDepartment of Chemistry, Forman Christian College (A Chartered University), Ferozepur
Road, Lahore 54600, Pakistan
cDr. Panjwani Center for Molecular Medicine and Drug Research, International Center for
Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
dBahria University Medical and Dental College, Karachi, 75270, Pakistan
eDepartment of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC),
Imam Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam, Saudi Arabia
fPCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280,
Pakistan
Abstract: Urease enzyme is responsible to catalyze the hydrolysis of urea into carbamate and
ammonia. Then carbamate hydrolyzed to ammonia and carbon dioxide. Excess release of ammonia
leads to increase pH in stomach that actually encourages the survival of Helicobacter pylori. H.
pylori involves in various disorders most commonly peptic ulcer, pyelonephritis, hepatic coma,
kidney stone formation, urolithiasis, and encephalopathy. Apart from many pharmacological
properties, coumarin and Schiff bases are known to possess urease inhibitory activity. Therefore,
these two pharmacologically important scaffolds are combined into single hybrid molecules to
assess their potential as urease inhibitors. For this aim, N'-benzylidene-2-((2-oxo-2H-chromen-4-
yl)oxy)acetohydrazide Schiff base derivatives 3-27 were synthesized by following a three step
reaction strategy. Structures of all synthetic molecules were characterized by EI-MS, 1H-, and13C-
NMR spectroscopic techniques. All molecules were assessed for urease inhibitory activity and
found to possess a varying degree of inhibitory potential in the range of IC50 = 12.3 ± 0.69 to 88.8
± 0.04 μM. Amongst the active analogs, compounds 7 (IC50 = 16.2 ± 0.11 μM), 9 (IC50 = 15.2 ±
0.14 μM), 10 (IC50 = 12.3 ± 0.69 μM), 12 (IC50 = 16.3 ± 0.45 μM), and 15 (IC50 = 17.6 ± 0.28 μM)
were identified as potent inhibitors compared to standard urea (IC50 = 21.5 ± 0.47 μM). It is
conferred from structure-activity relationship (SAR) that variation in inhibitory activity is due to
*Corresponding Authors: khalid.khan@iccs.edu, drkhalidhej@gmail.com, Tel.: +922134824910, Fax:
+922134819018
1

different substitutions pattern on aryl ring. Moreover, molecular docking studies were carried out
to understand the interactions of ligand with the active pocket of urease enzyme.
Keywords: Synthesis; chromene/coumarin; urease inhibitory activity; In vitro; structure-activity
relationship; In silico
Introduction
Coumarin also known as 2H-chromen-2one, belongs to the benzopyrone family which consists of
a benzene ring fused to a pyrone ring hence also known as benzopyrane-2-one. It is isolated from
plants, animals, and microorganisms [1, 2]. The role of coumarin is well established as it is
associated with a diverse range of biological activities. It is the privileged scaffold in the domain
of medicinal chemistry and it is amongst the natural products those have been extensively used as
medicines [3-6]. Over the years, a number of natural and synthetic coumarin based compounds
have been reported for their wide range of pharmacological activities such as antimicrobial,
antioxidant [7], anticancer [8, 9], antitubercular [10, 11], and antiproliferative [12]. Coumarin
scaffold reported to cure fibrotic diseases [13] as well as also revealed enzyme inhibitory activities
[14-16]. Apart from various biological applications, coumarin is also used as optical brightening
agents, tunable laser dye, cosmetics, agrochemicals, perfumes, and food additive [17,18].
Coumarin derivatives has been identified as potent urease inhibitors [19-23]. Previously, our
research group has already been engaged to synthesize coumarin based bioactive molecules and
explored their distinct biological activities [19, 21-25].
Schiff bases possess azomethine (-C=N-) group which is very well-known for being active
pharmacophore. Schiff bases are also known to exhibit tremendous biological potential [26]. Schiff
bases with various heterocyclic rings have been extensively reported to exhibit bioactivities
including anticancer, anticonvulsant, antifungal, antiproliferative [27], antiviral, antibacterial, anti-
inflammatory, antimalarial [28], and antitumor [29]. Apart from range of enzyme inhibitory
activities, Schiff bases are also recognized as urease inhibitors [30].
Urease is a nickel containing metalloenzyme belonging to the superfamily of phosphotriesterases
and amidohydrolases. It catalyzes the hydrolysis of urea into carbamate and carbon dioxide which
eventually converts to ammonia [31]. Hence, by increasing the pH due to the excess release of
ammonia, it promotes the survival of Helicobacter pylori by countering the acidic pH of stomach
2

[32]. Helicobacter pylori is harmful for human health as it is involved in various ailments including
pyelonephritis, peptic ulcer, hepatic coma, kidney stone formation, urolithiasis, and
encephalopathy [33]. Therefore, identification of novel and potent urease inhibitors with high
stability and less toxicity is extremely required. During last two decades, our research group is
actively involved in the search of new potent urease inhibitors [19, 21-23, 34-38]. We have
identified biscoumarins as non-cytotoxic and selective urease inhibitors [21]. Since, urease possess
two nickel centers for coordination and Schiff bases are capable to form metal complexes [39, 40]
which encourage us to combine both of these moieties within a single scaffold and to explore
further biological aspects related to them (Figure-1). The present study is covering the multi-step
synthesis of coumarin linked with Schiff base and their evaluation as urease inhibitors.
Azomethine
O
O O
O
O
Coumarin
H
N
R
HO
HO
N
O
O
N
N
OH
O
O
H
OH
OH
Aryl group
OH
OH
IC50 = 27.20 ± 1.2 µM [40]
Schiff Base as Urease Inhibitor
OH
3-27
N'-Benzylidene-2-((2-oxo-2H-chromen-4-
yl)oxy)acetohydrazide Schiff base derivatives
IC50 = 12.3 ± 0.69 to 88.8 ± 0.04 µM
OH
IC50 = 4.4 ± 0.21 µM [21]
Biscoumarin as Urease Inhibitor
Standard Urea (IC50 = 21.5 ± 0.47 µM)
Figure-1: Coumarin and Schiff base as a Potent Urease Inhibitors.
Results and discussion
Chemistry
A mixture of 4-hydroxy coumarin and methyl chloroacetate in DMF was refluxed for two hours in
the presence of K2CO3. Formation of methyl 4-oxycoumarinyl acetate (1) reaction was monitored
by thin layer chromatography (TLC). In second step, intervening intermediate (1) was treated with
hydrazine hydrate (NH2NH2. H2O) in ethanol and stirred at room temperature to get 2-((2-oxo-2H-
chromen-4-yl)oxy)acetohydrazide (2). After that, compound 2 was condensed with different
substituted aldehydes in methanol in the presence of glacial acetic acid to obtain a variety of 4-
oxycoumarinyl linked acetohydrazide Schiff bases 3-27 (Scheme-1). Second and third steps were
also monitored by TLC analysis. As a result of above reaction scheme, two geometrical isomers E
and Z were obtained in the ratio of 75% and 25%, respectively, analyzed by integrations in NMR
3

spectra. Chemical shifts corresponds to minor isomer “Z” were marked by steric (*) symbol. To
the best knowledge, all synthesized compounds are new, except compounds 4 and 18 [41].
OH
NH
+
O
O
NH2
R
O
O
O
O
O
O
DMF
EtOH
O
O
NH2NH2 . H2O
K2CO3
H
O
O
+
(2)
(1)
O
MeOH
AcOH
Cl
Reflux
O
H
N
N
O
O
N
R
O
N
R
Amide-iminol
tautomerism
OH
O
O
O
O
(3-27)
(3-27)
Scheme-1: Synthesis of 4-oxycoumarinyl linked acetohydrazide Schiff bases 3-27
Since, amide group may undergo amide-iminol tautomerism and could leads to geometric
isomerism around single bond. There are four possible isomers including s-cis and s-trans with
E/Z isomerism (Figure-2). s-Cis isomers are less stable than s-trans, due to more steric crowding .
However, there is more chance that molecules attained the s-trans conformation and less chance
to get the mixture of s-trans and s-cis. The single chemical shift of iminol OH (refer NMR data of
all compounds) confirmed the presence of one isomerism around single bond that is s-trans.
(s-trans)
(s-trans)
(s-cis)
(s-cis)
H
R
N
N
N
N
O
O
N
O
N
R
O
N
H
O
O
N
(Z)
(Z)
OH
OH
O
OH
OH
H
(E)
R
(E)
H
R
Steric
hinderance
O
O
O
O
O
s-trans, Z
s-cis, Z
s-cis, E
s-trans, E
Figure-2: Four possible geometrical isomers
Detailed Spectral Study of Compound 14
1H-NMR spectrum was recorded in DMSO-d6 on a 300 MHz Bruker AM instrument. The NH
proton was resonated at H 11.63 as most downfield singlet (s). Imine proton was appeared at H
8.25* (s-trans, Z isomer) and H 7.93 (s-trans, E isomer). H-5 of the basic coumarin skeleton was
4

appeared as doublet of doublet (dd) at H 7.90 and showed ortho and meta coupling with H-6 (J =
7.8 Hz) and H-7 (J = 1.0 Hz). H-7 appeared as triplet (t) at H 7.68, presenting ortho coupling with
H-6 and H-8 (J = 8.4 Hz). H-6 and H-8 were appeared as overlapping multiplet (ovp) at H 7.43.
H-2' of aryl ring was resonated at H 7.43 as doublet (d) with J = 1.5 Hz, due to meta coupling
with H-6'. H-6' and H-5' were resonated at H 7.22 and H 7.01, respectively. Both protons appeared
as doublet (d), showing ortho coupling with each other (J = 8.4 Hz). H-3 was appeared as singlet
at H 5.85. Two protons belongs to -OCH2- were appeared at H 5.47 (E isomer) and 4.95* (Z
isomer) as singlet. Six protons of two OMe groups were appeared at H 3.79 as singlet (Figure-
3A).
Broad-band (BB) decoupled 13C-NMR spectrum (in DMSO-d6 at 100 MHz) showed a total twenty
eight carbon signals, including two methoxy, one methylene, eight methine, and eight quaternary
carbons. Eight signals corresponds to minor isomer (Z). Carbonyl carbons of hydrazide and lactone
moieties were appeared as most downfield signals at C 166 and C 164, respectively. C-4 was also
appeared downfield at C 162* (Z isomer), 161 (E isomer) being part of α-β-unsaturated system.
Quaternary C-9, C-4', C-3', and C-10 were appeared at C 152.7, 150.9, 148.9, and 115.1,
respectively. Iminic carbon was appeared at C 148.4* (Z isomer), 144.3 (E isomer). Other carbons
belong to coumarin ring such as C-3, C-5, C-6, C-7, and C-8, and aryl ring C-1', C-2', C-5', and
C-6' were appeared in the usual aromatic region. Methylene carbon appeared at C 67.0* (Z
isomer), 66.0 (E isomer). Two methoxy carbons were appeared at C 55.5 and C 55.4, respectively
(Figure-3B).
8.25*, 7.93 (s)
7.22 (d), J6',5' = 8.4 Hz
(s)
4.95*H
5.47,
H
H
148.4*, 144.3
121.9*, 121.3
108.8*, 108.4
H
67.0*, 66.0
7.01 (d, J5',6' = 8.4 Hz)
H
6
N
166
7.90 (dd, J5,6 = 7.8 Hz,
5,7 = 1.0 Hz)
1
N
O
N
126
J
5
5.85 (s)
H
O
N
162*, 161
150.6
3.79 (s)
123.2*, 122.9
124
H
55.5
OH
91.2*, 91.1
164
2
7.41 (ovp)H
OH
CH3
4
111.4
5
4
6
7
O
3
115
152
148.9
11.63 (s)
7.43 (d, J2',6' = 1.5 Hz)
H
O
3
O
132.9*, 132.8
3.79 (s)
CH3
O
O
O
2
116.4
55.4
7.68 (t,
J7(6,8) = 8.4 Hz)
H
1O
O
8
H7.41 (ovp)
A
( )
B
( )
Figure-3: 1H- (A) and 13C-NMR (B) of symbolic compound 14
In vitro urease inhibitory activity
5

All 4-oxycoumarinyl linked acetohydrazide Schiff bases 3-27 were screened to check their
inhibitory potential against urease enzyme. Results in terms of IC50 values were compared with
standard thiourea (IC50 = 21.5 ± 0.47 μM). Based on the results listed in Table-1, a number of
Schiff bases including 7, 9-12, and 15-18 exhibited potent urease inhibitory activity with IC50
values less than standard thiourea 21.5 ± 0.47 μM. While rest of the compounds showed good to
moderate inhibitory potential.
Table-1: In vitro urease inhibition of 4-oxycoumarinyl linked acetohydrazide Schiff bases 3-27
IC50 ± SEMa
IC50 ± SEMa
Comp.
3
R
Comp.
16
R
(µM)
(µM)
EtO
Cl
68.9 ± 0.28
64.5 ± 0.47
21.5 ± 0.39
18.9 ± 0.10
OMe
OH
4
5
17
18
NO2
OH
OH
67.8 ± 0.46
65.6 ± 0.64
19.6 ± 0.44
22.6 ± 0.16
OMe
O2N
OMe
Br
6
19
OH
OMe
OMe
OH
7
8
16.2 ± 0.11
25.6 ± 0.74
20
21
27.5 ± 0.07
25.8 ± 0.48
F
MeO
Br
OMe
OH
MeO
O
9
15.2 ± 0.14
12.3 ± 0.69
22
23
88.8 ± 0.04
64.8 ± 0.09
Br
Br
MeO
OMe
OMe
10
HO
OMe
6

MeO
MeO
11
12
13
19.6 ± 0.36
16.3 ± 0.45
61.1 ± 0.08
24
25
87.7 ± 0.48
63.6 ± 0.29
86.7 ± 0.11
Cl
Cl
OMe
Cl
OMe
OMe
OMe
Cl
26
27
OMe
Br
HO
Cl
NO2
OMe
OMe
14
15
66.6 ± 0.16
17.6 ± 0.28
69.8 ± 0.17
21.5 ± 0.47
Cl
MeO
Standard
=
S
H2N
NH2
Thioureab
SEMa (Standard error mean); Thioureab (Standard for urease inhibitory activity)
Structure-activity relationship (SAR)
Worth mentioning that all compounds were found to be active which shows that each structural
feature is taking part in the inhibitory activity. Since 4-oxycoumarin ring, and acetohydrazide
moiety are exactly similar in all compounds thus limited structure-activity relationship (SAR) is
rationalized on the basis of varying features i.e. different substitutions pattern in aryl ring.
Amongst the synthetic twenty-five derivatives, tri-methoxy substituted compound 10 (IC50 = 12.3
± 0.69 µM) was found to be the most potent member of this library. Its potent activity might be
due to the presence of three electron donating groups which make the aryl ring electron rich.
However, di-methoxy substituted analogs 8, 11, 12 and 14 exhibited interesting inhibitory activity
pattern, based on the positions of the methoxy groups. Activity comparison of compound 10 with
compound 8 (IC50 = 25.6 ± 0.74 µM) revealed that lacking a methoxy group at meta position
brought two-fold decreased activity. While absence of methoxy at ortho position as in case of
compound 14 (IC50 = 66.6 ± 0.16 µM) displayed more than six-fold decreased activity.
Furthermore, compounds 11 (IC50 = 19.6 ± 0.36 µM) and 12 (IC50 = 16.3 ± 0.45 µM) with
symmetrical dimethoxy substitutions at ortho and meta positions, respectively, demonstrated
7

potent urease inhibition which suggests that symmetrical substitutions might play role during
interactions with the active site of enzyme. In addition to that ortho-methoxy substituted
compound 15 (IC50 = 17.6 ± 0.28 µM) was also found to be potent which suggests that ortho
position is favorable for methoxy group to take part in urease inhibition. Activity of compound 15
may compared with ortho ethoxy substituted analog 3 (IC50 = 68.9 ± 0.28 µM), displayed moderate
inhibitory activity might be due to elongation of alkyl chain length. This activity comparison
further confirms the active participation of ortho methoxy group in case of compound 15 (Figure-
4).
OMe
N
O
O
N
OH
MeO
OMe
O
N
OMe
N
O
O
N
O
O
N
11
OH
OH
OMe
(IC50 = 19.6 ± 0.36 M)
µ
OMe
O
O
8
12
OMe
(IC50 = 25.6 ± 0.74 M)
µ
(IC50 = 16.3 ± 0.45 M)
µ
N
OMe
OMe
O
O
N
OH
OEt
N
O
O
N
N
O
O
O
N
OH
10
OMe
OH
OMe
(IC50 = 12.3 ± 0.69 M)
µ
O
O
14
(IC50 = 66.6 ± 0.16 M)
3
OMe
(IC50 = 68.9 ± 0.28 M)
µ
µ
N
O
O
N
OH
O
15
(IC50 = 17.6 ± 0.28 M)
µ
Figure-4: Urease inhibitory activities comparison of analogs 3, 8, 10-12, 14 and 15, on the basis
of their structures
A number of compounds bearing combination of methoxy/s and halogen/s exibited interesting
inhibitory potential profile. Amongst them, compound 16 (IC50 = 21.5 ± 0.39 µM) which is
distinctively similar to compound 8 but have ortho-chloro instead of methoxy, showed inhibitory
potential better than compound 8 and exactly similar to standard thiourea. Switching the methoxy
8

group from para to meta position as in case of compound 24 (IC50 = 87.7 ± 0.48 µM), activity
sharply decreased. It clearly indicates that chloro and methoxy at adjacent positions is not a good
combination for the inhibitory activity as they might create steric hindrance for each other to
interact with the active site of urease. Similarly comparing activity of compound 16 with another
compound 25 (IC50 = 63.6 ± 0.29 µM) revealed that replacing para-methoxy with para-chloro,
exhibited three-fold diminished potential. Moreover, compound 20 (IC50 = 27.5 ± 0.07 µM) which
has substitution pattern almost similar to compound 14 but para-fluoro instead methoxy, revealed
much better inhibitory activity which indicates the active participation of fluoro group in
inhibition. Compounds 13, 19, 22, and 23 bearing combination of bromo with methoxy showed
good to moderate inhibitory strength. Amongst them, compound such as residue 19 (IC50 = 22.6 ±
0.16 µM) having para-bromo and two methoxy groups at meta positions presented inhibitory
strength comparable to standard. Changing the positions of bromo and methoxy groups as in case
of compound 13 (IC50 = 61.1 ± 0.08 µM) showed sharp decline in the activity. It suggested that
the symmetrical substitutions pattern in 19 and as observed in previous cases 11 and 12 favors the
inhibitory strength of compounds. Besides that, compound 23 (IC50 = 64.8 ± 0.09 µM) having
hydroxy group along with bromo and methoxy was found to be moderately active. In this case
might be positions of the groups are not favorable for the activity. Activity comparison of
compound 23 with 22 (IC50 = 88.8 ± 0.04 µM) showed the decline in the activity is attributed to
the lack of ortho-hydroxy group (Figure-5).
9

Cl
N
OMe
O
O
N
Cl
OH
N
N
OMe
F
O
O
N
O
O
N
OH
OH
O
Cl
24
(IC50 = 87.7 ± 0.48 M)
µ
O
O
25
20
(IC50 = 63.6 ± 0.29 M)
µ
(IC50 = 27.5 ± 0.07 M)
µ
Cl
OMe
Br
Br
N
O
O
N
N
N
O
N
O
N
OH
O
OMe
OH
OH
O
OMe
OMe
16
O
O
O
22
13
(IC50 = 21.5 ± 0.39 M)
µ
(IC50 = 88.8 ± 0.04 M)
µ
(IC50 = 61.1 ± 0.08 M)
µ
Br
N
OMe
N
O
N
O
N
OH
OH
Br
HO
OMe
OMe
O
O
O
O
19
(IC50 = 22.6 ± 0.16 M)
23
µ
(IC50 = 64.8 ± 0.09 M)
µ
Figure-5: Urease inhibitory activities comparison of analogs 13, 16, 19, 20, 22 and 25, on the
basis of their structures
Amongst the hydroxy substituted derivatives, meta hydroxy substituted analog 7 (IC50 = 16.2 ±
0.11 µM) displayed potent activity as compared to standard thiourea (IC50 = 21.5 ± 0.47 μM).
Repositioning of hydroxy group from meta to para as in case of compound 6 (IC50 = 65.6 ± 0.64
µM), gave four-fold decreased activity. It reveals that the change in position of hydroxy group
greatly affects the activity. Di-hydroxy substituted analog 17 (IC50 = 18.9 ± 0.10 µM) having
hydroxy both at meta and para position also displayed potent inhibitory activity. Activity of
compound 17 can compared with compound 21 (IC50 = 25.8 ± 0.48 µM) which showed decreased
activity just due to replacement of hydroxy with bromo at meta position. Similarly, replacing para
hydroxy with methoxy as in case of compound 5 (IC50 = 67.8 ± 0.46 µM) brought sharp decline in
the inhibitory activity (Figure-6).
10

N
N
Br
O
O
N
O
O
N
OH
OH
OH
OH
O
O
21
6
(IC50 = 25.8 ± 0.48 M)
µ
(IC50 = 65.6 ± 0.64 M)
µ
N
OH
O
N
N
N
OH
O
N
O
O
N
OH
OH
O
OH
OH
OMe
O
O
OH
7
O
O
17
5
(IC50 = 16.2 ± 0.11 M)
µ
(IC50 = 18.9 ± 0.10 M)
µ
(IC50 = 67.8 ± 0.46 M)
µ
Figure-6: Urease inhibitory activities comparison of analogs 5-7, 17, 21 and 23, on the basis of
their structures
Compound 18 (IC50 = 19.6 ± 0.44 µM) bearing nitro group at ortho position was found to be a
potent urease inhibitor as it showed better strength than standard thiourea. Switching of nitro group
from ortho to para as in compound 4 (IC50 = 64.3 ± 0.47 µM) leads to more than three-folds
reduced inhibitory activity which shows that ortho position is favorable for nitro substitution.
Another compound 27 (IC50 = 69.8 ± 0.17 µM) having nitro and chloro groups para to each other
showed moderate inhibitory strength (Figure-7).
Cl
NO2
N
N
N
O
O
N
O
O
N
O
O
N
OH
OH
OH
NO2
NO2
O
O
O
27
4
18
(IC = 69.8 ± 0.17 M)
µ
(IC = 64.3 ± 0.47 M)
µ
50
50
(IC = 19.6 ± 0.44 M)
µ
50
Figure-7: Urease inhibitory activities comparison of analogs 4, 18, and 27, on the basis of their
structures
Concisely, limited SAR suggested that almost all substitution/s including methoxy, hydroxy,
bromo, chloro, fluoro, and nitro are taking part in the inhibitions but their positions and numbers
really alter the inhibitory strength of the molecules.
11

In silico study
Molecular docking study was carried out to understand the plausible ligand binding mechanism.
For that purpose, the crystal structure of Jack bean urease (PDB id: 4GY7, 1.49 Å) was
downloaded from the Protein Data Bank (PDB). All steps including standard receptor and ligand
preparation, and subsequent docking studies were carried out according to our previously reported
protocol [42] by using BioSolveIT’s Lead IT software [43]. Discovery Studio Visualizer was used
for the visualization of docked conformation.
The docked conformation of compound 10 is shown in Figure-8. The coumarin ring oxygen was
making hydrogen bond with Arg609, whereas the carbonyl oxygen was making a bond with
Arg609 and His593. The NH group was making a hydrogen bond with carboxymethylated cysteine
amino acid Cme592. Several π-alkyl interactions were observed between the coumarin ring and
Met637, Ala636. The trimethoxy substituted phenyl ring was also making π-alkyl interaction with
Arg439 and Ala436. The pyrone ring of coumarin was also making a π-π T-shaped interaction
with His519 and His492, whereas its adjacent ring was making a π-π stacked interaction with
His545.
Figure-8: 3D and 2D binding site interactions of docked conformation of compound 10
Molecular docking studies for compound 14 were also carried out (Figure-9). The carbonyl oxygen
of coumarin ring and the oxygen atom of the -OH group were making a hydrogen bond with
12

Gln635. The two azomethine nitrogen atoms were making hydrogen bonds with Arg439. A π-alkyl
interaction was observed between the phenyl ring and Ala436. Another π-alkyl interaction was
observed between the coumarin ring and Ala636. Amino acid CMe592 was making π-sulfur
interaction with the phenyl ring and the coumarin ring. Another π-sulfur interaction was found
between the coumarin ring and Met588.
Figure-9: 3D and 2D binding site interactions of docked conformation of compound 14
Conclusion
A series of 4-oxycoumarinyl linked acetohydrazide Schiff bases 3-27 were synthesized and
evaluated for their potential urease inhibitory activity. All compounds were found to be active and
it is worth mentioning that many compounds were identified as potent urease inhibitors as compare
to standard thiourea. Limited structure activity relationship revealed that all substitutions are taking
part in the inhibitory activity but their substitution pattern (number and positions) on aryl ring is
responsible for their varying degree of inhibitory strength. Molecular docking studies was also
performed on analogs 10 and 14 to understand the binding mechanism of synthetic compounds
with active site of enzyme. This study has identified many potent urease inhibitors those may serve
as potential leads to identity new drug therapies for gastric and peptic ulcers.
13

Experimental
Materials and Methods
All analytical grade reagents were purchased from Sigma-Aldrich, USA. For thin layer
chromatography (TLC) silica gel coated aluminum plates GF-254 (Merck, Germany) were used.
Spots were visualized under ultraviolet light at 254 and 366 nm or iodine vapors. Electron impact
mass spectra were recorded on MAT 113D and MAT 312 mass spectrometers. The 1H- and 13C-
NMR spectra were recorded ont 300 and 400 MHz on Bruker AM machines. Chemical shift (δ)
values, relative to (TMS) as an internal standard, are presented in ppm, and the coupling constant
(J) values are presented in Hz.
Synthesis of methyl 4-oxycoumarinyl acetate (1)
4-Hydroxy coumarin (0.163 g) (1 mmol) and K2CO3 (0.13 g) were taken in DMF (10 mL) into a
100 mL round-bottommed flask and stirred to completely solubilized. Then, methyl chloro acetate
(0.1 mL) (1 mmol) was added in the reaction mixture and refluxed for 2 h. Reaction progress was
monitored by TLC analysis. After completion, reaction mixture was poured onto crushed ice.
Precipitates were formed which filtered and washed with distilled water. Structure of compound 1
was confirmed by EI-MS and 1H-NMR.
Methyl 2-((2-oxo-2H-chromen-4-yl)oxy)acetate (1)
White Solid; Yield: 65%; M.p.: 94-96 ºC; 1H-NMR (300 MHz, DMSO-d6):  7.85 (d, J6(5,4) = 7.8
Hz, 1H, H-7), 7.68 (t, J6(5,7) = 7.2 Hz, 1H, H-6), 7.40 (m, 2H, H-5, H-8), 5.94 (s, 1H, H-3), 5.10 (s,
2H, CH2), 3.74 (s, 3H, CH3); EI-MS m/z (% rel. abund.): 234 (M+, 100), 206 (36), 175 (24), 133
(75).
Synthesis of 4-oxycoumarinyl acetohydrazide (2)
Methyl 4-oxycoumarinyl acetate 1 (0.28g) (1 mmol) was taken in ethanol (20 mL) into a 100 mL
round-bottommed flask and dissolved by stirring. Then, hydrazine hydrate (1 mL) was added and
further stirred at room temperature. Formation of the desired product was monitored by TLC. On
completion, precipitates were formed in the reaction which were filtered and washed with cold
14

ethanol (10 mL). Structure of 4-oxycoumarinyl acetohydrazide 2 was elucidated by EI-MS and
1H-NMR.
2-((2-Oxo-2H-chromen-4-yl)oxy)acetohydrazide (2)
White Solid; Yield: 75%; M.p.: 250-252 ºC; 1H-NMR (300 MHz, DMSO-d6):  9.50 (s, 1H, NH),
7.85 (d, J(7,6) = 7.8 Hz, 1H, H-7), 7.68 (t, J6(5,7) = 7.2 Hz, 1H, H-6), 7.40 (m, 2H, H-5, H-8), 5.94
(s, 1H, H-3), 5.10 (s, 2H, CH2), 4.41 (d, 2H, NH2); EI-MS m/z (% rel. abund.): 234 (M+, 38), 163
(85), 120 (100).
General procedure for the synthesis of 4-oxycoumarinyl linked acetohydrazide Schiff bases
(3-27)
4-Oxycoumarinyl acetohydrazide 2 (0.160 g, 1.0 mmol) and different substituted aldehydes (1.0
mmol) were taken in methanol (20 mL) into a 100 mL round-bottommed flask. Then, two drops
of glacial acetic acid was added and refluxed for 2-3 h. Reaction progress was checked by TLC.
After completion of the reaction, solid precipitates were appeared in the reaction mixture which
were separated out through filtration, washed with distilled water and hexane to afford pure
products 3-27. The crude products were crystallized from ethanol. The structural determination of
all compounds was carried out by EI-MS, 1H-, and 13C-NMR spectroscopy.
(E,Z)-N'-(2-Ethoxybenzylidene)-2-((2-oxo-2H-chromen-4-yl) oxy) acetohydrazide (3)
White Solid; Yield: 74%; M.p.: 237-239 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.71 (s, 1H, OH),
8.61*, 8.38 (s, 1H, N=CH), 7.90 (dd, J6',5' = 6 Hz, J6',4' = 3 Hz, 2H, H-6'), 7.68 (t, J7(6,8) = 8.4 Hz,
1H, H-7), 7.43-7.35 (m, 3H, H-6, H-8, H-4'), 7.09 (d, J3',4' = 8.4 Hz, 1H, H-3'), 6.99 (t, J5'(6'',4')
=
7.5 Hz, 1H, H-5'), 5.88 (s, 1H, H-3), 5.44, 4.94* (s, 2H, OCH2), 4.11 (q, 2H, CH2), 1.39 (t, 3H,
13
CH3); C-NMR (400 MHz, DMSO-d6):  166.9, 164.7, 164.5*, 162.1, 157.1*, 156.9, 152.7,
143.5*, 140.2, 132.9*, 132.8, 131.7*, 131.4, 125.8, 125.5*, 124.2, 123.2*, 122.9, 120.6*, 120.5,
116.4, 115.2, 112,.7, 91.1, 66.9*, 66.0, 63.8, 15.8; EI-MS m/z (% rel. abund.): 366 (M+, 18), 234
(27), 188 (34), 177 (75), 162 (63), 147 (53), 133 (100), 119 (84), 105 (306).
(E,Z)-N'-(4-Nitrobenzylidene)-2-((2-oxo-2H-chromen-4-yl) oxy) acetohydrazide (4)
White Solid; Yield: 75%; M.p.: 320-322 ºC; 1H-NMR (300 MHz, DMSO-d6):  12.02 (s, 1H, OH),
8.41*, 8.12 (s, 1H, N=CH), 8.28 (d, J2',3'/6',5' = 6.6 Hz, 2H, H-2', H-6'), 8.04 (d, J3',2'/5',6' = 9 Hz, 2H,
15

H-3', H-5'), 7.90 (d, J5,6 = 6 Hz, 1H, H-5), 7.68 (t, J7(6,8) = 6 Hz, 1H, H-7), 7.43-7.38 (m, 2H, H-6,
H-8), 5.95 (s, 1H, H-3), 5.51, 5.01* (s, 2H, OCH2); EI-MS m/z (% rel. abund.): 367 (M+, 51), 309
(64), 188 (22), 177 (52), 120 (100), 89 (44).
(E,Z)-N'-(3-Hydroxy-4-methoxybenzylidene)-2-((2-oxo-2H-chromen-4-yl)oxy)
acetohydrazide (5)
White Solid; Yield: 72%; M.p.: 313-316 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.55 (s, 1H, OH),
9.19 (s, 1H, OH), 8.12*, 7.95 (s, 1H, N=CH), 7.87 (dd, J5,6 = 8.4, J5,7 = 2.7 Hz, 1H, H-5), 7.68 (m,
1H, H-7), 7.42-7.37 (m, 2H, H-6, H-8), 7.22 (d, J2',6' = 3 Hz, 1H, H-2'), 7.07 (dd, J6',5' = 6.3 Hz,
J6',2' = 1.2 Hz, 1H, H-6'), 6.96 (d, J5',6' = 8.4 Hz, 1H, H-5'), 5.84 (s, 1H, H-3), 5.42, 4.93* (s, 2H,
13
OCH2), 3.79 (s, 3H, OCH3); C-NMR (400 MHz, DMSO-d6):  166.7, 164.7, 164.5*,161.9,
161.4, 161.3*, 152.7, 149.9*,149.7, 148.4*, 146.8*, 146.7, 144.5, 132.9*, 132.8, 126.6, 124.2,
123.3*, 123.0, 120.4*, 120.1, 116.4, 115.2, 114.9*, 112.4, 112.3*, 111.8*, 111.7, 91.2*, 91.1,
67.0*, 65.9, 55.5; EI-MS m/z (% rel. abund.): 368 (M+, 93), 310 (27), 177 (24), 162 (53), 149
(100), 136 (26), 120 (50).
(E,Z)-N'-(4-Hydroxybenzylidene)-2-((2-oxo-2H-chromen-4-yl)oxy) acetohydrazide (6)
White Solid; Yield: 69%; M.p.: 269-272 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.56 (s, 1H, OH),
9.92(s, 1H, OH), 7.92*, 7.90 (s, 1H, N=CH), 7.88 (d, J5,6 = 6 Hz,1H, H-5), 7.68 (m, 1H, H-7), 7.57
(d, J2',3'/6',5' = 8.4 Hz, 2H, H-2', H-6'), 7.43-7.37 (m, 2H, H-6, H-8), 6.82 (d, J3',2' /5',6' = 8.7 Hz, 2H,
13
H-3', H-5'), 5.86 (s, 1H, H-3), 5.41, 4.95* (s, 2H, OCH2); C-NMR (400 MHz, DMSO-d6): 
166.7, 164.7, 164.5*, 161.9*, 161.5, 161.3*, 159.5*, 159.3, 152.7, 148.5*, 144.5, 132.9*, 132.8,
128.9*, 128.7, 124.8, 124.2, 123.3*, 123.0, 116.4, 115.6*, 115.5, 115.2, 114.9*, 91.2*, 91.1; EI-
MS m/z (% rel. abund.): 338 (M+, 51), 280 (33), 177 (52), 162 (47), 120 (100), 106 (37), 92 (41).
(E,Z)-N'-(3-Hydroxybenzylidene)-2-((2-oxo-2H-chromen-4-yl) oxy) acetohydrazide (7)
White Solid; Yield: 80%; M.p.: 312-314 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.68 (s, 1H, OH),
9.61 (s, 1H, OH), 8.38*, 7.93 (s, 1H, N=CH), 7.91 (d, J5,6 = 7.8 Hz, 1H, H-5), 7.71 (t, J7(6,8) = 7.8
Hz, 1H, H-7), 7.43 (m, 2H, H-6, H-8), 7.25 (t, J5'(4',6') = 7.5 Hz, 1H, H-5'), 7.17-7.07 (m, 2H, H-2',
13
H-6'), 6.84 (d, J4',5' = 7.2 Hz,1H, H-4'), 5.86 (s, 1H, H-3), 5.44, 4.94* (s, 2H, OCH2); C-NMR
(400 MHz, DMSO-d6):  167.0, 164.7, 164.5*, 162.3*, 161.5, 161.3*, 157.6*, 157.5, 152.7,
16

148.3*, 144.4, 135.1*, 135.0, 132.9*, 132.8, 129.8*, 128.7, 124.2, 123.3*, 123.0, 118.9*,118.4,
117.6*, 117.3, 116.4, 115.2, 112.9, 112.6*, 91.3*, 91.1, 66.9*, 65.9; EI-MS m/z (% rel. abund.):
338 (M+, 85), 280 (39), 177 (96), 120 (100).
(E,Z)-N'-(2,4-Dimethoxybenzylidene)-2-((2-oxo-2H-chromen-4-yl) oxy) acetohydrazide (8)
White Solid; Yield: 73%; M.p.: 277-279 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.56 (s, 1H, OH),
8.55*, 8.25 (s, 1H, N=CH), 7.90 (dd, J5,6 = 7.5 Hz, J5,7 = 0.6 Hz, 1H, H-5), 7.84 (d, J6',5' = 8.1 Hz,
1H, H-6'), 7.70 (t, J7 (6,8) = 7.2 Hz, 1H, H-7), 7.43 (m, 2H, H-6, H-8), 6.61 (s, 1H, H-3'), 6.59 (d,
J5',6' = 8.7 Hz, 1H, H-5'), 5.85 (s, 1H, H-3), 5.41, 4.96* (s, 2H, OCH2), 3.84 (s, 3H, OCH3), 3.80
(s, 3H, OCH3); 13C-NMR (400 MHz, DMSO-d6):  166.7, 164.7, 164.5*, 162.6*, 162.3, 161.8*,
161.5, 161.4*, 159.2*, 159.0, 152.7, 143.7*, 140.0, 132.9*, 132.8, 126.9, 126.7*, 124.2, 123.3*,
123.0, 116.4, 115.2, 114.9*, 114.6, 106.4, 98.2*, 98.0, 91.2*, 91.1, 67.0*, 66.0, 55.7, 55.4; EI-MS
m/z (% rel. abund.): 382 (M+, 85), 293 (10), 207 (18), 188 (26), 177 (24), 163 (100), 149 (86), 121
(50).
(E,Z)-N'-(3-(Benzyloxybenzylidene)-2-((2-oxo-2H-chromen-4-yl)oxy) acetohydrazide (9)
White Solid; Yield: 73%; M.p.: 280-282 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.7 (s, 1H, OH),
8.35*, 7.97 (s, 1H, N=CH), 7.91 (d, J5,6 = 7.8 Hz, 1H, H-5), 7.71 (t, J7,6/7,8 = 7.5 Hz, 1H, H-7),
7.47-7.30 (m, 10H, H-6, H-8, H-2', H-5', H-6', H-2'', H-3'', H-4'', H-5'', H-6''), 7.08 (d, J4'/5' = 7.8
Hz, 1H, H-4'), 5.88 (s, 1H, H-3), 5.46 (s, 2H, OCH2), 5.14, 4.96* (s, 2H, OCH2); 13C-NMR (400
MHz, DMSO-d6):  167.2, 164.7, 161.5, 158.5, 152.7, 148.0*, 143.9, 136.9, 135.2, 132.8, 129.9,
128.4, 127.8, 127.7, 127.6*, 124.2, 123.3*, 123.0, 120.1, 116.7, 116.4, 112.7*, 112.4, 91.3*, 91.2,
69.2, 66.0; EI-MS m/z (% rel. abund.): 428 (M+, 15), 370 (5), 162 (22), 91 (100).
(E,Z)-2-((2-Oxo-2H-chromen-4-yl) oxy)-N'-(2,3,4-trimethoxybenzylidene) acetohydrazide
(10)
White Solid; Yield: 78%; M.p.: 278-280 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.63 (s, 1H, OH),
8.44*, 8.19 (s, 1H, N=CH), 7.90 (d, J5,6 =7.5 Hz, 1H, H-5), 7.71-7.62 (m, 2H, H-7), 7.58 (d, J6', 5'
= 9 Hz, 1H, H-6'), 7.43-7.37 (m, 2H, H-6, H-8), 6.92 (d, J5',6' = 9 Hz, 1H, H-5'), 5.86 (s, 1H, H-3),
5.43, 4.93* (s, 2H, OCH2), 3.83 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.79 (s, 3H, OCH3); 13C-NMR
(400 MHz, DMSO-d6):  166.7, 164.7, 164.5, 161.5, 155.1, 152.7, 143.6*, 141.4, 140.1, 132.9*,
17

132.8, 124.2, 123.3*, 122.9, 120.8, 120.6*, 119.9, 116.4, 115.1, 108.6, 91.2*.91.1, 67.0*, 66.0,
61.7, 60.4, 56.0; EI-MS m/z (% rel. abund.): 412 (M+, 40), 193 (100), 179 (74), 162 (21), 120 (27).
(E,Z)-N'-(2,6-Dimethoxybenzylidene)-2-((2-oxo-2H-chromen-4-yl)oxy) acetohydrazide (11)
White Solid; Yield: 81%; M.p.: 285-287 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.56 (s, 1H, OH),
8.42*, 8.25 (s, 1H, N=CH), 7.90 (d, J5,6 = 7.5 Hz, 1H, H-5), 7.68 (t, J7 (6,8) = 8.1 Hz, 1H, H-7),
7.43-7.31 (m, 2H, H-6, H-8, H-4'), 6.72 (d, J3',4'/5',4' = 8.4 Hz, 2H, H-3', H-5'), 5.71 (s, 1H, H-3),
5.33, 4.92* (s, 2H, OCH2), 3.81 (s, 6H, OCH3); 13C-NMR (400 MHz, DMSO-d6):  166.8, 164.8,
161.8*, 161.4, 158.8, 158.7*, 152.7, 143.7*, 139.4, 132.8, 131.4*, 131.3, 124.2, 123.2*, 123.0,
116.4, 115.1, 110.4, 104.4, 104.3*, 91.2*, 90.8, 66.9*, 65.9, 56.0, 55.9; EI-MS m/z (% rel. abund.):
382 (M+, 6), 188 (16), 177 (19), 163 (100), 149 (38), 121 (24).
(E,Z)-N'-(3,5-Dimethoxybenzylidene)-2-((2-oxo-2H-chromen-4-yl)oxy) acetohydrazide (12)
White Solid; Yield: 80%; M.p.: 310-312 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.77 (s, 1H, OH),
8.21*, 7.96 (s, 1H, N=CH), 7.90 (d, J5,6 = 9 Hz, 1H, H-5), 7.68 (t, J7 (6,8) = 8.4 Hz, 1H, H-7), 7.43-
7.37 (m, 2H, H-6, H-8), 6.89 (m, 2H, H-2', H-6'), 6.56 (d, J4',2' = 2.1 Hz, 1H, H-4'), 5.87 (s, 1H, H-
13
3), 5.48, 4.96* (s, 2H, OCH2), 3.77 (s, 6H, OCH3); C-NMR (400 MHz, DMSO-d6):  167.2,
164.7, 162.4*, 161.4, 161.3*, 160.6, 152.7, 148.1*, 144.0, 135.8, 132.9*, 132.8, 124.2, 123.2*,
122.9, 116.4, 115.1, 114.9*, 104.9, 102.4*, 102.0, 91.3*, 91.2, 66.9*, 66.0, 55.3; EI-MS m/z (%
rel. abund.): 382 (M+, 80), 219 (12), 177 (42), 163 (100), 149(17), 133 (16), 121 (43).
(E,Z)-N'-(2-Bromo-4,5-dimethoxybenzylidene)-2-((2-oxo-2H-chromen-4-yl)oxy)
acetohydrazide (13)
White Solid; Yield: 77%; M.p.: 304-306 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.84 (s, 1H, OH),
8.54*, 8.26 (s, 1H, N=CH), 7.89 (d, J5,6 = 6.9 Hz, 1H, H-5), 7.68 (t, J7(6,8) = 8.4 Hz, 1H, H-7), 7.50
(s, 1H, H-3'), 7.43-7.37 (m, 2H, H-6, H-8), 7.19 (s, 1H, H-6'), 5.87 (s, 1H, H-3), 5.52, 4.96* (s,
2H, OCH2), 3.81 (s, 3H, OCH3), 3.80 (s, 3H, OCH3); 13C-NMR (400 MHz, DMSO-d6):  167.1,
164.7, 164.5*, 161.4, 152.7, 151.4*, 151.1, 148.5, 146.6*, 142.6, 132.9*, 132.8, 124.7, 124.5*,
124.2, 123.2*,122.9, 116.4, 115.4*, 115.3, 115.1, 114.8, 109.0, 108.4*, 91.3*, 91.2, 66.9*, 66.1,
56.0, 55.6; EI-MS m/z (% rel. abund.): 460 (M+, 17), 462 (M+2, 18), 382 (17), 338 (8), 323 (12),
243 (100), 177 (45), 163 (57).
18

(E,Z)-N'-(3,4-Dimethoxybenzylidene)-2-((2-oxo-2H-chromen-4-yl) oxy) acetohydrazide (14)
White Solid; Yield: 65%; M.p.: 245-247 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.63 (s, 1H, OH),
8.25*, 7.93 (s, 1H, N=CH), 7.90 (dd, J5,6 = 7.8 Hz, J5,7 = 1.0 Hz, 1H, H-5), 7.68 (t, J7(6,8) = 8.4 Hz,
1H, H-7), 7.43 (d, J2',6' = 1.5 Hz, 1H, H-2'), 7.41-7.30 (m, 2H, H-6, H-8), 7.22 (d, J6',5' = 8.4 Hz,
1H, H-6'), 7.01 (d, J5',6' = 8.4 Hz, 1H, H-5'), 5.85 (s, 1H, H-3), 5.47, 4.95* (s, 2H, OCH2), 3.79 (s,
6H, OCH3); 13C-NMR (400 MHz, DMSO-d6):  166.9, 164.7, 162.0*, 161.4, 152.7, 150.6, 148.9,
148.4*, 144.3, 132.9*, 132.8, 126.5, 124.2, 123.2*, 122.9, 121.9*, 121.3, 116.4, 115.1, 111.4,
108.8, 108.4*, 91.2*, 91.1, 67.0*, 66.0, 55.5, 55.4; EI-MS m/z (% rel. abund.): 382 (M+, 100), 324
(6), 177 (10), 163 (73).
(E,Z)-N'-(2-Methoxybenzylidene)-2-((2-oxo-2H-chromen-4-yl) oxy) acetohydrazide (15)
White Solid; Yield: 72%; M.p.: 275-277 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.70 (s, 1H, OH),
8.64*, 8.35 (s, 1H, N=CH), 7.91-7-88 (m, 2H, H-5, H-6'), 7.67 (m, 1H, H-7), 7.42-7.38 (m, 3H,
H-6, H-8, H-4'), 7.10 (d, J3',4' = 7.6 Hz, 1H, H-3'), 7.00 (t, J5'(4',6') = 7.6 Hz, 1H, H-5'), 5.88 (s, 1H,
H-3), 5.44, 4.93* (s, 2H, OCH2), 3.84 (s, 3H, OCH3); EI-MS m/z (% rel. abund.): 352 (M+, 24),
234 (19), 177 (100), 163 (15), 133 (59), 121 (62), 91 (13).
(E,Z)-N'-(2-Chloro-3-methoxybenzylidene)-2-((2-oxo-2H-chromen-4-yl)oxy) acetohydrazide
(16)
White Solid; Yield: 73%; M.p.: 319-321 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.94 (s, 1H, OH),
8.75*, 8.42 (s, 1H, N=CH), 7.90 (d, J5,6 = 7.8 Hz, 1H, H-5), 7.71-7.65 (m, 2H, H-7, H-6'), 7.44-
7.34 (m, 3H, H-6, H-8, H-3'), 7.21 (d, J4',5' = 9 Hz, 1H, H-5'), 5.92 (s, 1H, H-3), 5.47, 4.92* (s, 2H,
OCH2), 3.87 (s, 3H, OCH3); EI-MS m/z (% rel. abund.): 386 (M+, 4), 388 (M+2, 2), 293 (31), 182
(28), 177 (100), 162 (62), 155 (31), 120 (59).
(E,Z)-N'-(3,4-Dihydroxybenzylidene)-2-((2-oxo-2H-chromen-4-yl)oxy) acetohydrazide (17)
White Solid; Yield: 71%; M.p.: 325-327 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.52 (s, 1H, OH),
9.30 (s, 2H, OH), 8.15*, 7.8 (s, 1H, N=CH), 7.90 (d, J5,6 =7.5 Hz, 1H, H-5), 7.71 (t, J7,6/7,8 = 8.1
Hz, 1H, H-7), 7.43 (ovp, 2H, H-6, H-8), 7.16 (s, 1H, H-2'), 6.95 (ovp, 1H, H-6'), 6.77 (d, J5',6' =
8.1 Hz, 1H, H-5'), 5.83 (s, 1H, H-3), 5.40, 4.95* (s, 2H, OCH2); 13C-NMR (400 MHz, DMSO-d6):
 166.6, 164.7, 164.5*, 161.8*, 161.4, 161.3*, 152.7, 148.7, 148.2*, 147.9, 145.7*, 145.6, 144.9,
132.9*, 132.8, 125.2, 124.2, 123.3*, 123.0, 120.7*, 120.2, 116.4, 115.5, 115.2, 114.9, 112.9,
19

112.6*, 91.2*, 91.1, 67.0*, 65.9; EI-MS m/z (% rel. abund.): 354 (M+, 15), 294 (15), 176 (37), 120
(100).
(E,Z)-N'-(2-Nitrobenzylidene)-2-((2-oxo-2H-chromen-4-yl)oxy) acetohydrazide (18)
White Solid; Yield: 76%; M.p.: 312-314 ºC; 1H-NMR (300 MHz, DMSO-d6):  12.04 (s, 1H, OH),
8.69*, 8.39 (s, 1H, N=CH), 8.19 (d, J6',5' = 7.7 Hz, 1H, H-6'), 8.07 (d, J3',4' = 9 Hz, 1H, H-3'), 7.90
(d, J5,6 = 6.9 Hz, 1H, H-5), 7.82 (t, J7(6,8) = 7.5 Hz, 1H, H-7), 7.71-7.64 (m, 2H, H-6, H-8), 7.43-
7.37 (m, 2H, H-4', H-5'), 5.93 (s, 1H, H-3), 5.46, 5.00* (s, 2H, OCH2); EI-MS m/z (% rel. abund.):
367 (M+, 19), 337 (9), 203 (20), 177 (100), 162 (36).
(E,Z)-N'-(4-Bromo-3,5-dimethoxybenzylidene)-2-((2-oxo-2H-chromen-4-yl)oxy)
acetohydrazide (19)
White Solid; Yield: 77%; M.p.: 331-334 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.89 (s, 1H, OH),
8.35*, 7.98 (s, 1H, N=CH), 7.90 (d, J5,6 = 7.5 Hz, 1H, H-5), 7.68 (t, J7(6,8) = 8.1 Hz, 1H, H-7), 7.43-
7.37 (m, 2H, H-6, H-8), 7.09 (s, 1H, H-2'), 7.06 (s, 1H, H-6'), 5.87 (s, 1H, H-3), 5.52, 4.98* (s, 2H,
OCH2), 3.88 (s, 6H, OCH3); EI-MS m/z (% rel. abund.): 460 (M+, 2), 462 (M+2, 2), 279 (3), 243
(11), 178 (100), 103 (05), 44 (14).
(E,Z)-N'-(4-Fluoro-3-methoxybenzylidene)-2-((2-oxo-2H-chromen-4-yl)oxy) acetohydrazide
(20)
White Solid; Yield: 76%; M.p.: 308-310 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.77 (s, 1H, OH),
8.25*, 7.95 (s, 1H, N=CH), 7.88 (d, J5,6 = 9 Hz, 1H, H-5), 7.66 (t, J7(6,8) = 8.4 Hz, 1H, H-7), 7.51
(d, J6',5' = 9 Hz, 1H, H-6'), 7.41-7.35 (m, 2H, H-6, H-8), 7.28 (s, 1H, H-2'), 7.24 (d, J5',6' = 9 Hz,
1H, H-5'), 5.86 (s, 1H, H-3), 5.47, 4.95* (s, 2H, OCH2), 3.86 (s, 3H, OCH3); EI-MS m/z (% rel.
abund. ): 370 (M+, 51), 312 (8), 188 (15), 177 (100), 152 (50), 138 (30), 121 (49).
(E,Z)-N'-(3-Bromo-4-hydroxybenzylidene)-2-((2-oxo-2H-chromen-4-yl) oxy) acetohydrazide
(21)
White Solid; Yield: 79%; M.p.: 320-324 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.64 (s, 1H, OH),
10.75 (s, OH), 8.15*, 7.89 (s, 1H, N=CH), 7.89 (m, 2H, H-5, H-2') 7.68 (t, J7(6,8) = 8.4 Hz, 1H,
H-7), 7.56 (dd, J6',2' = 3 Hz, J6',5' = 8.4 Hz, 1H, H-6'), 7.43-7.38 (m, 2H, H-6, H-8),7.00 (d, J5',6' =
8.4 Hz, 1H, H-5'), 5.88 (s, 1H, H-3), 5.44, 4.93* (s, 2H, OCH2); 13C-NMR (400 MHz, DMSO-d6):
 166.9, 164.7, 161.5, 155.7, 152.7, 164.9*, 143.0, 132.9*, 132.8, 131.6*, 131.2, 128.0, 127.8*,
20

126.5, 124.2, 123.2*, 122.9, 116.4, 116.3*, 115.2, 109.8, 91.2, 66.9*, 66.0; EI-MS m/z (% rel.
abund.): 416 (M+, 51), 418 (M+2, 49), 358 (13), 199 (37), 177 (100), 134 (35), 121 (52), 105 (58).
(E,Z)-N'-(5-Bromo-2-methoxybenzylidene)-2-((2-oxo-2H-chromen-4-yl)oxy) acetohydrazide
(22)
White Solid; Yield: 75%; M.p.: 274-276 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.79 (s, 1H, OH),
8.56*, 8.26 (s, 1H, N=CH), 8.01 (d, J6',4' = 2.7 Hz, 1H, H-6'), 7.90 (d, J5,6 = 9 Hz, 1H, H-5), 7.68
(t, J7(6,8) = 8.1 Hz, 1H, H-7), 7.57 (dd, J4',6' = 2.4 Hz, J4',3' = 9 Hz, 1H, H-4'), 7.43-7.37 (m, 2H, H-
6, H-8), 7.11 (d, J3',4' = 9 Hz, 1H, H-3'), 5.90 (s, 1H, H-3), 5.50, 4.98* (s, 2H, OCH2), 3.84 (s, 3H,
OCH3); 13C-NMR (400 MHz, DMSO-d6):  167.2, 164.7, 162.4, 161.5, 161.3*, 156.7, 152.7,
142.0*, 138.3, 133.9*, 133.8, 132.9*, 132.7, 127.7, 127.4*, 124.2, 124.0*, 123.3*, 122.9, 116.4,
115.2, 114.4*, 114.2, 112.5, 91.2, 67.0*, 66.1, 56.1; EI-MS m/z (% rel. abund.): 430 (M+, 25), 432
(M+2, 21), 341 (8), 234 (20), 211 (46), 188 (30), 177 (100), 121 (32), 91 (26).
(E,Z)-N'-(6-Bromo-2-hydroxy-3-methoxybenzylidene)-2-((2-oxo-2H-chromen-4-yl)oxy)
acetohydrazide (23)
White Solid; Yield: 84%; M.p.: 322-324 ºC; 1H-NMR (300 MHz, DMSO-d6):  12.43 (s, 1H, OH),
12.32 (bs, 1H, OH), 8.84, 8.49* (s, 1H, N=CH), 8.00 (dd, J5,6 = 7.2 Hz, J5,7 = 1.6 Hz, 1H, H-5),
7.71 (ovp, 1H, H-7), 7.44 (ovp, 2H, H-6, H-8), 7.15 (d, J5',4' = 8.8 Hz, 1H, H-5'), 6.99 (d, J4',5' = 8.8
Hz, 1H, H-4'), 5.95 (s, 1H, H-3), 5.44*, 5.03 (s, 2H, OCH2), 3.84 (s, 3H, OCH3); 13C-NMR (400
MHz, DMSO-d6):  166.5*, 164.6*, 164.3, 162.6, 161.4*, 161.3, 152.7, 149.9, 149.6, 148.1*,
147.9, 145.2*, 132.9, 132.8*, 124.2, 123.4, 122.9*, 122.7, 116.7*, 116.4, 115.4, 115.1, 114.8,
114.7, 113.7, 113.1*, 91.5*, 91.3, 66.8, 65.9*, 56.0*, 55.9; EI-MS m/z (% rel. abund.): 446 (M+,
100), 448 (M+2, 98), 309 (29), 177 (45), 163 (74), 135 (30).
(E,Z)-N'-(2-Chloro-3-methoxybenzylidene)-2-((2-oxo-2H-chromen-4-yl)oxy) acetohydrazide
(24)
White Solid; Yield: 83%; M.p.: 323-325 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.92 (s, 1H, OH),
8.71*, 8.42 (s, 1H, N=CH), 7.90 (dd, J5,6 = 6.8 Hz, J5,7 = 1.2, 1H, H-5), 7.70-7.65 (m, 2H, H-7, H-
6'), 7.43-7.34 (m, 3H, H-6, H-8, H-5'), 7.22 (d, J4',5' = 8.8 Hz, 1H, H-4'), 5.92 (s, 1H, H-3), 5.47,
21

4.95* (s, 2H, OCH2), 3.87 (s, 3H, OCH3); EI-MS m/z (% rel. abund.):386 (M+, 10), 388 (M+2, 5),
293 (33), 182 (26), 177 (100), 167 (45), 155 (28), 121 (32).
(E,Z)-N'-(2,4-Dichlorobenzylidene)-2-((2-oxo-2H-chromen-4-yl)oxy) acetohydrazide (25)
White Solid; Yield: 76%; M.p.: 338-340 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.97 (s, 1H, OH),
8.63*, 8.34 (s, 1H, N=CH), 8.12 (d, J6',5' = 8.7 Hz, 1H, H-6'), 7.90 (d, J5,6 = 6.9 Hz, 1H, H-5), 7.72
(d, J6',5' = 3 Hz, 1H, H-3'), 7.66 (t, J7(6,5) = 6 Hz, 1H, H-7), 7.52 (dd, J5',6' = 6.9, J5',3' = 1.8, Hz, 1H,
H-5'), 7.43-7.37 (m, 2H, H-6, H-8), 5.94 (s, 1H, H-3), 5.48, 4.98* (s, 2H, OCH2): 13C-NMR (400
MHz, DMSO-d6):  167.3, 164.6, 162.7*, 161.5, 152.7, 143.0*, 139.1, 135.0, 133.6, 132.9*, 132.8,
130.2, 129.3, 128.4, 128.1*, 128.0*, 127.8, 124.2, 123.3*, 122.9, 116.4, 115.1, 91.3, 66.9*, 66.0;
EI-MS m/z (% rel. abund.): 390 (M+, 11), 392 (M+2, 8), 394 (M+4, 3), 297 (10), 219 (13), 188
(19), 177 (100), 162 (14), 121 (37).
(E,Z)-N'-(3,5-Dichloro-2-hydroxybenzylidene)-2-((2-oxo-2H-chromen-4-yl)oxy)
acetohydrazide (26)
White Solid; Yield: 75%; M.p.: 309-311 ºC; 1H-NMR (300 MHz, DMSO-d6):  11.90 (s, 1H, OH),
10.33 (s, 1H, OH), 8.48*, 8.26 (s, 1H, N=CH), 7.89 (d, J5,6 = 7.2 Hz, 1H, H-5), 7.78 (d, J6',4' = 2.4
Hz, 1H, H-4'), 7.68-7.62 (m, 2H, H-6, H-7), 7.59 (d, J6',4' = 2.4 Hz, 1H, H-6'), 7.44 (d, J8,7 = 7.5
Hz, 1H, H-8'), 5.94 (s, 1H, H-3), 5.51, 5.04* (s, 2H, OCH2); EI-MS m/z (% rel. abund.): 406 (M+,
73), 408 (M+2, 52), 410 (M+4, 10), 348 (15), 219 (34), 187 (65), 177 (37), 163 (100), 120 (78),
92 (37).
(E,Z)-N'-(2-Chloro-5-nitrobenzylidene)-2-((2-oxo-2H-chromen-4-yl)oxy)
acetohydrazide
(27)
White Solid; Yield: 76%; M.p.: 298-300 ºC; 1H-NMR (300 MHz, DMSO-d6):  12.11 (s, 1H, OH),
8.74 (d, J6',4' = 2.7 Hz, 1H, H-6'), 8.66*, 8.41 (s, 1H, N=CH), 8.24 (dd, J4',3' = 9 Hz, J4',6' = 2.7 Hz,
1H, H-4'), 7.90 (d, J5,6 = 7.5 Hz, 1H, H-5), 7.85 (d, J = 8.7 Hz, 1H, H-3'), 7.68 (t, 1H, J7(6,8) = 8.4
13
Hz, H-7), 7.44-7.37 (m, 2H, H-6, H-8), 5.93 (s, 1H, H-3), 5.57, 5.02* (s, 2H, OCH2); C-NMR
(400 MHz, DMSO-d6):  167.4, 164.6, 164.4*, 162.8*, 161.5, 161.3*, 152.7, 148.1*, 148.0,
143.7*, 139.7, 133.8*, 133.4, 132.9*, 132.8, 130.8,*, 130.6, 128.6, 128.4*, 128.1*, 128.0, 124.6*,
124.4, 124.5, 123.2*, 122.9, 116.4, 115.1, 114.9*, 91.3*, 91.2, 66.8*, 66.0; EI-MS m/z (% rel.
abund.): 401 (M+, 80), 403 (M+2, 27), 365 (69), 308 (47), 203 (50), 176 (100).
22

Urease inhibitory assay
Urease inhibitory activity was performed by following the method of Weatherburn (1967). Test
compound (250 μM, 5 μL) was incubated with urease solution (1 U/well) 25 μL for 15 min at 30
°C. After that, urea (substrate) (100 mM, 55 μL) was added and then plate was incubated for 10
°
min at 30 C. Then, 70 μL of alkali reagents (0.5% w/v sodium hydroxide and 0.1% sodium
hypochlorite, and 45 μL of phenol (1% w/v phenol and 0.005% w/v sodium nitroprusside)) was
added. Plate was again incubated for 50 min at 30 °C. Urease activity was calculated with the rate
of production of ammonia and alteration in absorbance was examined at 630 nm on ELISA plate
reader (Spectra Max M2, Molecular Devices, CA, USA). Thiourea was used as a standard control
[44].
Docking methodology
MOE (Molecular Operating Environment) [45] was used to perform molecular docking study in
order to predict the binding mode of the synthetic compounds in the active site of urease enzyme.
Using the builder tool implemented in MOE software, the three dimensional structures of the
synthetic derivatives were generated. The generated compounds were 3D protonated and energy
minimized using the default parameters of the MOE (gradient: 0.05, Force Field: MMFF94X). For
further evaluation of compounds in molecular docking study, all the compounds were then saved
in mdb (Molecular Data Base) file format.
3D structure of the target protein was retrieved from the protein databank (PDB ID 4ubp). The
retrieved protein was opened in MOE, water molecules were removed and 3D protonation was
carried. After 3D protonation, the protein was energy minimized to get a stable conformation of
the protein using the default parameters of MOE. For docking studies, the default parameters of
MOE were used i.e. Placement: Triangle Matcher, rescoring 1: London dG, Refinement: Force
field, Rescoring 2: GBVI/WSA. For each ligand ten conformations were allowed to be formed and
the top ranked conformations on the basis of docking score were selected for further analysis.
Acknowledgement: The authors are thankful to the Pakistan Academy of Sciences for providing
financial support to Project No. (5-9/PAS/440).
23

References
1.
K. Rajanna, F. Solomon, M.M. Ali, P.S. Prakash, Vilsmeier-Haack formylation of
coumarin derivatives, A solvent dependent kinetic study, Int. J. Chem. Kinet. 28 (1996)
865-872.
2.
3.
4.
P. Jain, H. Joshi, Coumarin: chemical and pharmacological profile, J. Appl. Pharm. Sci. 2
(2012) 236-240.
M.V. Kulkarni, G.M. Kulkarni, C.H. Lin, C.M. Sun, Recent advances in coumarins and 1-
azacoumarins as versatile biodynamic agents, Curr. Med. Chem. 13 (2006) 2795-2818.
U. Salar, K.M. Khan, J. Iqbal, S.A. Ejaz, A. Hameed, M. Al-Rashida, S. Perveen, M.N.
Tahir, Coumarin sulfonates: new alkaline phosphatase inhibitors; In vitro and in silico
studies, Eur. J. Med. Chem. 131 (2017) 29-47.
5.
U. Salar, K.M. Khan, A. Jabeen, A. Faheem, M.I. Fakhri, S.M. Saad, S. Perveen, M. Taha,
A. Hameed, Coumarin sulfonates: As potential leads for ROS inhibition, Bioorg. Chem.
69 (2016) 37-47.
6.
7.
8.
S. Emami, S. Dadashpour, Current developments of coumarin-based anti-cancer agents in
medicinal chemistry, Eur. J. Med. Chem. 102 (2015) 611-630.
K. Kasumbwe, K.N. Venugopala, V. Mohanlall, B. Odhav, Antimicrobial and antioxidant
activities of substituted halogenated coumarins, J. Med. Plant Res. 8 (2014) 274-28.
J.l. Li, D.C. Hu, X.P. Liang, Y.C. Wang, H.S. Wang, Y.M. Pan, Praseodymium (III)-
catalyzed regioselective synthesis of C3-N-substituted coumarins with coumarins and
azides, J. Org. Chem. 82 (2017) 9006-9011.
9.
A.H. Halawa, E.M. Eliwa, A.A. Hassan, H.S. Nassar, R. El-Eisawy, M. Ismail, M. Frese,
M. Shaaban, A.M. El-Agrody, A.H. Bedair, Synthesis, in vitro cytotoxicity activity against
the human cervix carcinoma cell line and in silico computational predictions of new 4-
arylamino-3-nitrocoumarin analogues, J. Mol. Struct. 1200 (2020) 127047.
24

10.
11.
12.
13.
14.
15.
S.H. Cardoso, M.B. Barreto, M.C. Lourenço, M. das G. M. de. O. Henriques, A.L.P.
Candéa, C. R. Kaiser, M. V. N. de Souza, Antitubercular activity of new coumarins, Chem.
Biol. Drug Des. 77 (2011) 489-493.
R.S. Keri, B. Sasidhar, B.M. Nagaraja, M.A. Santos, Recent progress in the drug
development of coumarin derivatives as potent antituberculosis agents, Eur. J. Med. Chem.
100 (2015) 257-269.
P. Govindaiah, N. Dumala, I. Mattan, P. Grover, M.J. Prakash, Design, synthesis,
biological and in silico evaluation of coumarin-hydrazone derivatives as tubulin targeted
antiproliferative agents, Bioorg. Chem. 91 (2019) 103143.
D. Deng, H. Pei, T. Lan, J. Zhu, M. Tang, L. Xue, Z. Yang, S. Zheng, H. Ye, L. Chen,
Synthesis and discovery of new compounds bearing coumarin scaffold for the treatment of
pulmonary fibrosis, Eur. J. Med. Chem. 185 (2020) 111790.
F. Carta, A. Maresca, A. Scozzafava, C.T. Supuran, Novel coumarins and 2-thioxo-
coumarins as inhibitors of the tumor-associated carbonic anhydrases IX and XII, Bioorg.
Med. Chem. 20 (2012) 2266-2273.
A. Asadipour, M. Alipour, M. Jafari, M. Khoobi, S. Emami, H. Nadri, A. Sakhteman, A.
Moradi, V. Sheibani, F.H. Moghadam, Novel coumarin-3-carboxamides bearing N-
benzylpiperidine moiety as potent acetylcholinesterase inhibitors, Eur. J. Med. Chem. 70
(2013) 623-630.
16.
17.
R.Z. Batran, M.A. Khedr, N.A.A. Latif, A.A. El Aty, A.N. Shehata, Synthesis, homology
modeling, molecular docking, dynamics, and antifungal screening of new 4-
hydroxycoumarin derivatives as potential chitinase inhibitors, J. Mol. Struc. 1180 (2019)
260-271.
M. Taha, S.A.A. Shah, M. Afifi, S. Imran, S. Sultan, F. Rahim, N.H. Ismail, K.M. Khan,
Synthesis, molecular docking study and thymidine phosphorylase inhibitory activity of 3-
formylcoumarin derivatives, Bioorg. Chem. 78 (2018) 17-23.
25

18.
19.
K. Rohini, P. Srikumar, Therapeutic role of coumarins and coumarin-related compounds,
J. Thermodyn. Catal. 5 (2014) 1-3.
M. Alomari, M. Taha, S. Imran, W. Jamil, M. Selvaraj, N. Uddin, F. Rahim, Design,
synthesis, in vitro evaluation, molecular docking and ADME properties studies of hybrid
bis-coumarin with thiadiazole as a new inhibitor of urease, Bioorg. Chem. 92 (2019)
103235.
20.
I. Khan, A. Khan, S.A. Halim, A. Saeed, S. Mehsud, R. Csuk, A. Al-Harrasi, A. Ibrar,
Exploring biological efficacy of coumarin clubbed thiazolo [3,2-b][1,2,4] triazoles as
efficient inhibitors of urease: A biochemical and in silico approach, Int. J. Biol. Macromol.
142 (2020) 345-354.
21.
22.
23.
24.
25.
26.
U. Salar, A. Nizamani, F. Arshad, K.M. Khan, M.I. Fakhri, S. Perveen, N. Ahmed, M.I.
Choudhary, Bis-coumarins; non-cytotoxic selective urease inhibitors and antiglycation
agents, Bioorg. Chem. 91 (2019) 103170.
K.M. Khan, S. Iqbal, M.A. Lodhi, G.M. Maharvi, Z. Ullah, M.I. Choudhary, Atta-ur-
Rahman, S. Perveen, Bis-coumarin: new class of urease inhibitors; economical synthesis
and activity, Bioorg. Med. Chem. 12 (2004) 1963-1968.
K.M. Khan, S. Iqbal, M.A. Lodhi, G.M. Maharvi, S. Perveen, M.I. Choudhary, Atta-ur-
Rahman, Z.H. Chohan, C.T. Supuran, Synthesis and urease enzyme inhibitory effects of
some dicoumarols, J. Enzy. Inhib. Med. Chem. 19 (2004) 367-371.
K.M. Khan, F. Rahim, A. Wadood, N. Kosar, M. Taha, S. Lalani, A. Khan, M.I. Fakhri,
M. Junaid, W. Rehman, Synthesis and molecular docking studies of potent α-glucosidase
inhibitors based on biscoumarin skeleton, Eur. J. Med. Chem. 81 (2014) 245-252.
U. Salar, M. Taha, K.M. Khan, N.H. Ismail, S. Imran, S. Perveen, S. Gul, A. Wadood,
Syntheses of new 3-thiazolyl coumarin derivatives, in vitro α-glucosidase inhibitory
activity, and molecular modeling studies, Eur. J. Med. Chem. 122 (2016) 196-204.
A. Hameed, M. al-Rashida, M. Uroos, S. Abid Ali, K.M. Khan, Schiff bases in medicinal
chemistry: A patent review (2010-2015), Expert Opin. Ther. Pat. 27 (2017) 63-79.
26

27.
28.
29.
30.
M.S. Karthikeyan, D.J. Prasad, B. Poojary, K.S. Bhat, B.S. Holla, N.S. Kumari, Synthesis
and biological activity of Schiff and Mannich bases bearing 2,4-dichloro-5-fluorophenyl
moiety, Bioorg. Med. Chem. 14 (2006) 7482-7489.
C.M. Da-Silva, D.L. da-Silva, L.V. Modolo, R.B. Alves, M.A. de Resende, C.V. Martins,
Â. de Fátima, Schiff bases: A short review of their antimicrobial activities, J. Adv. Res. 2
(2011) 1-8.
D. Sinha, A.K. Tiwari, S. Singh, G. Shukla, P. Mishra, H. Chandra, A.K. Mishra, Synthesis,
characterization and biological activity of Schiff base analogues of indole-3-
carboxaldehyde, Eur. J. Med. Chem. 43 (2008) 160-165.
M.A.S. Aslam, S.U. Mahmood, M. Shahid, A. Saeed, J. Iqbal, Synthesis, biological assay
in vitro and molecular docking studies of new Schiff base derivatives as potential urease
inhibitors, Eur. J. Med. Chem. 46 (2011) 5473-5479.
31.
32.
B. Krajewska, Ureases I. Functional, catalytic and kinetic properties: A review, J. Mol.
Cat. B: Enz. 59 (2009) 9-21.
S.G. Jadhav, R.J. Meshram, D.S. Gond, R.N. Gacche, Inhibition of growth of Helicobacter
pylori and its urease by coumarin derivatives, Molecular docking analysis. J. Pharm. Res.
7 (2013) 705-711.
33.
34.
C. Belzer, J. Kusters, E. Kuipers, A.V. Vliet, Urease induced calcium precipitation by
Helicobacter species may initiate gallstone formation, Gut 55 (2006) 1678-1679.
B. Ali, K.M. Khan, S. Hussain, S. Hussain, M. Ashraf, M. Riaz, A. Wadood, S. Perveen,
Synthetic nicotinic/isonicotinic thiosemicarbazides: In vitro urease inhibitory activities and
molecular docking studies, Bioorg. Chem. 79 (2018) 34-45.
35.
B. Ali, K.M. Khan, U. Salar, S. Hussain, M. Ashraf, M. Riaz, A. Wadood, M. Taha, S.
Perveen, 1-[(4′-Chlorophenyl) carbonyl-4-(aryl) thiosemicarbazide derivatives as potent
urease inhibitors: Synthesis, in vitro and in silico studies, Bioorg. Chem. 79 (2018) 363-
371.
27

36.
37.
38.
39.
A. Abbas, B. Ali, K.M. Khan, J. Iqbal, S. Ur Rahman, S. Zaib, S. Perveen, Synthesis and
in vitro urease inhibitory activity of benzohydrazide derivatives, in silico and kinetic
studies, Bioorg. Chem. 82 (2019) 163-177.
M. Khan, K.M. Khan, S. Parveen, M. Shaikh, N. Fatima, M.I. Choudhary, Syntheses, in
vitro urease inhibitory activities of urea and thiourea derivatives of tryptamine, their
molecular docking and cytotoxic studies, Bioorg. Chem. 83 (2019) 595-610.
G. Mohiuddin, K.M. Khan, U. Salar, M.A. Lodhi, A. Wadood, M. Riaz, S. Perveen,
Biology-oriented drug synthesis (BIODS), in vitro urease inhibitory activity, and in silico
study of S-naproxen derivatives, Bioorg. Chem. 83 (2019) 29-46.
M. Ikram, S. Rehman, F. Subhan, M.N. Akhtar, M.O. Sinnokrot, Synthesis,
characterization, thermal degradation and urease inhibitory studies of the new hydrazide
based Schiff base ligand 2-(2-hydroxyphenyl)-3-{[(E)-(2-hydroxyphenyl) methylidene]
amino}-2, 3-dihydroquinazolin-4 (1H)-one, Open Chem. 15 (2017) 308-319.
40.
41.
42.
M. Taha, S.A.A. Shah, A. Khan, F. Arshad, N.H. Ismail, M. Afifi, S. Imran, M.I.
Choudhary, Synthesis of 3,4,5-trihydroxybenzohydrazone and evaluation of their urease
inhibition potential, Arabian J. Chem. 12 (2019) 2973-2982.
O.M.A. Elhafez, E.E.D.A.M. El, F. Badria, A.E.D.M. Fathy, Synthesis and biological
investigations of new thiazolidinone and oxadiazoline coumarin derivatives, Arch. Pharm.
Res. 26 (2003) 686-696.
S.U. Qazi, S.U. Rahman, A.N. Awan, M. al-Rashida, R.D. Alharthy, A. Asari, A. Hameed,
J. Iqbal, Semicarbazone derivatives as urease inhibitors: Synthesis, biological evaluation,
molecular docking studies and in-silico ADME evaluation, Bioorg. Chem. 79 (2018) 19-
26.
43.
44.
LeadIT version 2.3.2; BioSolveIT GmbH, Sankt Augustin, Germany, (2017),
www.biosolveit.de/LeadIT
M.K. Rauf, A. Talib, A. Badshah, S. Zaib, K. Shoaib, M. Shahid, U. Floerke, J. Iqbal,
Solution-phase microwave assisted parallel synthesis of N,N′-disubstituted thioureas
28

derived from benzoic acid: biological evaluation and molecular docking studies, Eur. J.
Med. Chem. 70 (2013) 487-496.
45.
Molecular Operating Environment (MOE), 2016.08; Chemical Computing Group Inc.,
1010 Sherbrooke St. West, Suite #910, Montreal, QC, Canada. H3A 2R7. 2016.
29