Utility of anthranilic acid and diethylacetylenedicarboxylate for the synthesis of nitrogenous organo/organometallic compounds as urease inhibitors

Article abstract of DOI:10.1002/ardp.201800314

Fumarate diester 3 was synthesized upon reacting anthranilic acid with diethylacetylenedicarboxylate. Compound 3 was reacted with different nucleophiles in mild reaction conditions. Selected reaction routes that afforded products 6, 9, 10, 11, and 12 were explained. The estimated mechanism for the reaction of 3 with ethylenediamine to afford 9 was proved by X-ray single-crystal and retro-synthetic reaction. Acetyl anthranilic acid was utilized with zinc and copper to afford the organometallic compounds 14a and 14b, respectively. Three single crystals were afforded for 3, 9 and the organocopper complex 14b. Target compounds were screened for their inhibitory potential against urease enzyme. Most compounds were more potent than thiourea as standard inhibitor, considering that oxopiperazine 9 exhibited double the activity: IC₅₀ = 8.16 ± 0.65 μM (thiourea IC₅₀ = 20.04 ± 0.33 μM). Docking studies were in agreement with the in vitro enzyme assay.

Full text of DOI:10.1002/ardp.201800314

Received: 26 October 2018
Revised: 25 March 2019
Accepted: 31 March 2019
|
|
DOI: 10.1002/ardp.201800314
F U L L P A P E R
Utility of anthranilic acid and diethylacetylenedicarboxylate
for the synthesis of nitrogenous organo/organometallic
compounds as urease inhibitors
1
1
2,3
Heba S. A. El‐Zahabi
Amany M. Hegab
|
Hanan G. Abdulwahab
|
Mastoura M. Edrees
|
4
1
Department of Pharmaceutical Chemistry,
Faculty of Pharmacy (Girls), Al‐Azhar
University, Cairo, Egypt
Abstract
Fumarate diester 3 was synthesized upon reacting anthranilic acid with diethylacety-
lenedicarboxylate. Compound 3 was reacted with different nucleophiles in mild
reaction conditions. Selected reaction routes that afforded products 6, 9, 10, 11, and 12
were explained. The estimated mechanism for the reaction of 3 with ethylenediamine
to afford 9 was proved by X‐ray single‐crystal and retro‐synthetic reaction. Acetyl
anthranilic acid was utilized with zinc and copper to afford the organometallic
compounds 14a and 14b, respectively. Three single crystals were afforded for 3, 9 and
the organocopper complex 14b. Target compounds were screened for their inhibitory
potential against urease enzyme. Most compounds were more potent than thiourea
as standard inhibitor, considering that oxopiperazine 9 exhibited double the activity:
IC50 = 8.16 ± 0.65 μM (thiourea IC50 = 20.04 ± 0.33 μM). Docking studies were in
agreement with the in vitro enzyme assay.
2
Department of Organic Chemistry, National
Organization for Drug Control and Research,
Giza, Egypt
3
Department of Chemistry, Faculty of
Science, King Khalid University, Abha,
Saudi Arabia
4
Developmental Pharmacology Department,
National Organization for Drug Control and
Research, Giza, Egypt
Correspondence
Hanan Gaber Abdulwahab, Department of
Pharmaceutical Chemistry, Faculty of
Pharmacy (Girls), Al‐Azhar University,
Cairo 11754, Egypt.
Email: hanangaber@azhar.edu.eg
K E Y W O R D S
anthranilic acid, diethylacetylenedicarboxylate, docking study, organometallic, single crystal,
urease inhibitor
1
|
INTRODUCTION
and understanding novel aspects of the mechanism of action of
[4,5]
ureases.
Urease is a nickel‐based metalloenzyme that catalyzes the hydrolysis
of urea into carbon dioxide and ammonia. Ureases are widely found
in various organisms including bacteria, fungi, and plants. Although
having different origin, all known ureases share at least 50% identity,
possess common amino acid sequences in the active site and thus
Anthranilic acid has an old history as a chemical precursor for
many organic bioactive compounds; for example, diuretics, antiox-
[6–9]
idants, antiproliferative and antiallergic agents.
Furthermore,
[10]
Rauf et al.
reported the urease inhibitory activity of diverse
substituted aniline based thiobarbiturates, among which the anthra-
nilic acid derivative I displayed potent urease inhibitory activity with
IC50 = 12.96 ± 0.13 μM compared to thiourea as reference standard
(IC50 = 21 ± 0.011 μM) (Figure 1). Also, the importance of
unsaturated carbonyl compounds as urease inhibitors was illustrated
[1]
show same catalytic mechanism. Production of ammonia from
urease is responsible for harmful complications in health fields. The
pathogenesis of many clinical conditions, such as peptic ulcer caused
by Helicobacter pylori, hepatic coma, pyelonephritis, and infection‐
induced urinary stones are related to the ureolytic activity of
[11–13]
[14]
in many literatures.
Recently, Macegoniuk et al.
described
[
2,3]
microbial enzymes.
Thus, searching for urease inhibitor drugs is
the potent urease inhibitory activity of several unsaturated carbonyl
compounds, with acetylenedicarboxylic acid II, and dimethylacetyle-
nedicarboxylate III (Figure 1) being the most potent. Additionally,
an important target in controlling infections caused by urease‐
producing bacteria. These inhibitors play an important role in
controlling ureolytic microorganisms, and also help exploring
[
15,16]
benzimidazole‐based antiulcer drug, rabeprazole,
wileyonlinelibrary.com/journal/ardp © 2019 Deutsche Pharmazeutische Gesellschaft
various
Arch. Pharm. Chem. Life Sci. 2019;e1800314
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EL‐ZAHABI ET AL.
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FIGURE 1 Structural similarities between some reported urease inhibitors (a) and target compounds (b)
[
12,13,17]
[18,19]
carboxylic acids,
piperazine derivatives,
[12,13,20,21]
dioxopyr-
dimethylacetylenedicarboxylate in methanol at room temperature
[
13]
roles,
and metal complexes
displayed potent urease
for 1 hr. Herein, the new diethoxy analog 3 was afforded by stirring
[28]
inhibitory activity (Figure 1).
anthranilic acid 1 with DEAD 2 adopting the reported procedure,
From a chemical point of view, the conjugation between the
diester moiety and the acetylene system of dimethylacetylenedicar-
boxylate (DMAD) explored various chemical reactivities. Several
research groups investigated the reaction of DMAD with numerous
nucleophiles, illustrating different pathways, either attacking the
acetylene moiety or the ester function. These previous reactions
covered broad estimated mechanisms, afforded various heterorganic
compounds, and were noticeably affected by solvent, temperature,
using ethanol as a solvent. Anthranilic acid 1 initiated the reaction by α
[22–27]
and the nature of the basic reactants.
Motivated by these findings, diethylacetylenedicarboxylate
DEAD) was reacted with anthranilic acid, to afford aryl/heteroaryl
(
and alicyclic derivatives of expected urease inhibitory activity.
Additionally, metal complexation reactions of anthranilic acid with
zinc and copper were carried out to explore the antiurease activity of
the afforded organometallic complexes.
2
2
|
RESULTS AND DISCUSSION
|
Chemistry
.1
Synthesis of dimethoxy‐dioxobutenylamino benzoic acid was
FIGURE 2 Molecular structure of compound 3 with anisotropic
displacement ellipsoids drawn at the 50% probability level
[28]
reported by Khetan et al.,
via stirring anthranilic acid with

EL‐ZAHABI ET AL.
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and β nucleophilic addition on the triple bond rather than the
Figure 2, Table 1. To illustrate the reactivity of the diethyl fumarate
analog 3, nucleophilic reactions with different amines were carried out.
The point of the investigation was to detect the position of attack,
either α,β addition on the double bond or direct aminolysis of the ester
function. The reaction proceeded in polar protic solvent for example,
ethanol under reflux with TLC follow‐up. As a starting point,
compound 3 was treated with hydrazine hydrate in ethanol under
reflux in 1:5 mole equivalence, respectively. Interestingly, the
structure of the product 4 was ruled out according to the spectral
data that explored complete disappearance of the signals correspond-
ing for the aromatic protons. Since a variety of products, for example,
aminolysis of the ester function of compound 2. The infrared radiation
1
(
(
IR), mass spectrometry (MS) and H nuclear magnetic resonance
NMR) spectra proved the assigned structure of compound 3. IR
spectrum of 3 showed the presence of two carbonyl absorptions at
−1 1
1
728, 1680 (ester C=O), and 1670 (acid C=O) cm . H NMR spectra
revealed the appearance of four signals δ 1.04 (t), 1.10 (t), 4.08–4.17
m) due to the diethyl ester protons. Also, a singlet appeared at δ
(
5.43 ppm for the vinyl resonance. The mass spectrum showed the
+
presence of m/z at 307 due to the molecular ion peak (M ). Compound
3
was also proved by single crystal X‐ray analysis as depicted in
TABLE 1 Main bond geometries (lengths and angles) for compounds 3, 9, 14b
3
9
14b
Bond length (Å)
O1—C11
O2—C11
O3—C18
O4—C12
O4—C17
O5—C12
O6—C18
N7—C8
1.235 (4)
1.310 (4)
1.211 (4)
1.328 (5)
1.459 (6)
1.204 (4)
1.343 (5)
1.402 (5)
1.375 (4)
1.465 (5)
1.503 (6)
1.343 (5)
1.449 (6)
0.960 (3)
0.960 (3)
O1—C8
1.351 (3)
1.455 (4)
1.240 (3)
1.322 (4)
1.454 (4)
1.225 (4)
1.502 (4)
1.346 (4)
1.353 (4)
1.446 (4)
1.433 (4)
1.469 (5)
1.474 (5)
0.960 (3)
0.960 (4)
Cu1—O2
Cu1—O4
Cu1—O5
Cu1—O6
Cu1—O9
O2—C17
O3—C17
O6—C12
N11—C16
C13—C17
N15—C21
N15—C22
O18—C19
C19—C28
O20—C22
1.905 (2)
1.962 (3)
1.993 (2)
1.928 (2)
2.275 (3)
1.308 (4)
1.237 (4)
1.287 (4)
1.429 (5)
1.482 (5)
1.390 (5)
1.366 (5)
1.204 (5)
1.520 (6)
1.236 (5)
O1—C12
O2—C6
N3—C6
N3—C11
O4—C8
C5—C6
C5—N7
N7—C16
C10—C11
C12—C16
C14—C16
C14—C18
O2—H2
C5—C10
N7—C9
C8—C10
C9—C11
C12—C13
C9—H9A
C11—H11A
N7—H7
o
Bond angle ( )
C12—O4—C17
C8—N7—C16
115.8 (3)
125.8 (3)
121.2 (3)
122.4 (3)
118.9 (3)
121.4 (3)
124.6 (4)
111.5 (3)
123.9 (3)
123.7 (4)
116.2 (3)
109.2 (4)
109.5 (5)
122.6 (4)
111.5 (3)
119.6 (3)
109.5 (6)
109.5 (5)
C8—O1—C12
C6—N3—C11
C6—C5—N7
116.2 (3)
123.3 (3)
118.0 (3)
124.6 (3)
122.5 (3)
117.0 (3)
121.8 (3)
122.5 (3)
110.6 (3)
123.6 (3)
111.2 (3)
107.4 (3)
109.6 (3)
110.4 (3)
109.5 (3)
107.9 (3)
110.5 (4)
109.5 (4)
O2—Cu1—O4
O2—Cu1—O5
O2—Cu1—O6
O2—Cu1—O9
O4—Cu1—O5
O4—Cu1—O6
O4—Cu1—O9
O5—Cu1—O6
O5—Cu1—O9
O6—Cu1—O9
Cu1—O2—C17
Cu1—O6—C12
C12—C10—C21
C16—N11—C19
C21—N15—C22
N11—C19—O18
N15—C22—O20
H26A–C26–H26B
89.15 (11)
92.77 (11)
173.04 (11)
87.36 (10)
157.74 (12)
89.27 (11)
110.51 (11)
91.26 (11)
91.74 (11)
86.84 (11)
128.3 (2)
N7—C8—C9
C8—C10—C11
C8—C10—C13
O1—C11—O2
O4—C12—O5
O4—C12—C16
C16—C14—C18
N7—C16—C14
C12—C16—C14
O4—C17—C20
H17A—C17—H17B
O3—C18—O6
O6—C18—C14
C8—N7—H7
N7—C5—C10
O2—C6—N3
N3—C6—C5
C5—N7—C9
O1—C8—O4
N7—C9—C11
C5—C10—C8
N3—C11—C9
O1—C12—C13
N7—C9—H9B
C11—C9—H9A
H9A—C9—H9B
N3—C11—H11B
C9—C11—H11A
H11A–C11–H11B
124.8 (2)
124.6 (3)
129.3 (3)
131.3 (4)
125.5 (4)
H20A—C20—H20B
H21A—C21—H21B
122.1 (4)
109.5 (5)

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SCHEME 1 Synthesis of compounds 3 and 6
, 6, were suggested, yet the reaction was estimated to proceed via
4
condition.
A
similar mechanistic pathway was assumed via
displacement of the aminobenzoic acid moiety with hydrazinolysis of
one ethoxide moiety, consuming two moles of hydrazine hydrate.
Sequentially, nucleophilic attack of the hydrazinyl moiety neighboring
to carbonyl group in intermediate 5 afforded the pyrazole carbohy-
drazide derivative 6. Suggested product 6 was concomitant to the
displacement of aminobenzoic acid moiety to afford enamine
intermediate 8 that constituted a ready synthesis of piperazine
1
analog 9. IR, MS, and H NMR spectra of the isolated product
greatly supported the assigned structure 9. IR spectrum showed the
−
1 1
presence of two carbonyl absorptions at 1689 and 1658 cm
. H
[
29]
pattern of Heindel et al.
Pyrazole carbohydrazide 6 was preferen-
NMR spectrum revealed the appearance of three signals at δ 1.15
(t), 3.25–3.29 (m) and 4.04 (q) ppm due to the ethyl group of ester
and two methylene protons of piperazine. Finally, the vinyl
resonance appeared at 5.16 (s) ppm. The mass spectrum showed
tially suggested rather than pyridazindione compound 7. The cycliza-
[29]
tion route according to Heindel et al.
was found to afford
1
pyrazolin‐5‐one analog with H NMR signal at δ 5.97 ppm for the
[30]
+
vinyl resonance. Additionally, Wu et al.
reported the existence of
the presence of m/z at 184 due to the molecular ion peak (M ). The
the vinyl resonance of pyridazin‐3‐one ring at lower field, for example,
structure of compound 9 was unequivocally determined by single
crystal X‐ray analysis as depicted in Figure 3, Table 1. Further,
1
7.15 ppm. Herein, H NMR analysis was in complete agreement with
structure 6, illustrating the signal of the vinyl proton at the upper field
applying the retro‐synthetic route of compound 9 that reported by
1
3
[31]
of δ 5.96 ppm. Simultaneously, C NMR spectrum supported the
Iwanami et al.
was an additional confirmative tool for the
1
3
structure of 6 rather than pyridazindione 7. Actually, the observed
NMR spectrum revealed two close signals at δ 161.41 and 161.83 ppm
2CO). Concomitantly, two additional signals appeared at δ 141.47 and
6.70 ppm assigned for C3 and C4 of pyrazoline ring, respectively
Scheme 1).
Simultaneously, diethoxy analog 3 was treated with equimolar
equivalence of ethylenediamine, applying the previously mentioned
C
estimated reaction mechanism between ethylenediamine and
precursor 3 (Scheme 2). Focusing on the oxo‐α,β‐unsaturated ester
entity of 9, two reactive sites were explored, either olefinic bond or
ester moiety. Consequently, nucleophilic addition of p‐aminophenyl
acetic acid on olefinic bond of oxopiperazinylidene acetate 9 was
(
8
(
[
32]
carried out, adopting the study done by Medvedeva et al.
So, the
reaction was carried out in ethanol under reflux to afford the

EL‐ZAHABI ET AL.
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FIGURE 3 Molecular structure of compound 9 with anisotropic displacement ellipsoids drawn at the 50% probability level
enamine isomer (I) rather than imine isomer (II) (Figure 4). Reported
nucleophilic addition on olefinic bond was afforded rather than
[
33]
literature
confirmed the aminolysis of α,β‐unsaturated ester by
aminolysis of the ester function to yield compound 10., Its structure
was proved by elemental and spectral data (see experimental
section). The mass spectrum showed the presence of m/z at 335 due
using excess amine under fusion, herein, the reaction was applied
using double mole equivalence of p‐aminophenyl acetic acid in
ethanol under reflux condition (TLC follow‐up). Interestingly,
+
to the molecular ion peak (M ).
SCHEME 2 Synthesis of compounds 9 and 10

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EL‐ZAHABI ET AL.
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1
structure 11. H NMR spectrum revealed the disappearance of the
protons of the ester functions. Concomitantly, the methylenic protons
of acetic acid moiety appeared at 3.37 ppm. The vinyl resonance
displayed a singlet signal at 5.78 ppm. A multiplet for the aromatic
13
protons appeared at 7.18–7.40 ppm. C NMR spectrum revealed a
signal at 40.59 ppm for methylenic carbon of acetic acid moiety. Also,
four signals appeared at 167.01, 172.00, 173.14, and 173.82 for (CO)
(see Section 4). The mass spectrum showed the presence of m/z at 366
+
FIGURE 4 Estimated isomeric forms I and II of compound 9
due to the molecular ion peak (M ). The reaction of o‐phenylenediamine
with precursor 3 provided aminolysis of one ethoxide group followed by
intramolecular cyclocondensation to afford the benzimidazole deriva-
tive 12. The structure of 12 was established on the basis of its elemental
analysis and spectral data (see Section 4).
In the next step, the diethoxy analog 3 was treated with aromatic
amines (Scheme 3). In contrast to ethylenediamine in Scheme 1, the
applied aromatic amines could not replace the aminobenzoic acid
moiety. So, analog 3 was reacted with equimolar equivalence of either
p‐aminophenyl acetic acid or o‐phenylenediamine in ethanol under
reflux to afford the corresponding heterocyclic analogs 11 and 12,
respectively. Noticeably, the reported reaction of diethyl fumarate with
Moreover, the utility of anthranilic acid scaffold was extended to
apply complexation of the acetyl anthranilic acid with copper and
[
35,36]
zinc metals adopting the reported procedures.
The complexa-
tion proceeded via mixing ethanolic solution of the acetyl anthranilic
acid 13 with aqueous suspension of Zn(OH)2 or solution of CuSO4
to afford the corresponding complex 14a and 14b, respectively
(Scheme 4). The structure of bis(2‐acetylaminobenzoato)Cu(II)
14b was confirmed by single crystal X‐ray analysis as depicted in
Figure 5, Table 1.
[34]
amines proceeded mainly via Micheal addition and aminolysis.
Compound 11 was afforded via aminolysis of the two ethoxide groups,
1
consuming one mole of p‐aminophenyl acetic acid. The IR, MS and
H
NMR spectra of the isolated product greatly supported the assigned
SCHEME 3 Synthesis of compounds 11 and 12
SCHEME 4 Synthesis of compounds 14a,b

EL‐ZAHABI ET AL.
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FIGURE 5 Molecular structure of compound 14b with anisotropic displacement ellipsoids drawn at the 50% probability level
2
.2
|
In vitro urease inhibition assay
14.13 ± 4.16, 14.09 ± 0.49, and 13.68 ± 0.67 μM, respectively. Zinc
complex 14a exhibited almost half the activity of thiourea with IC50
6.91 ± 0.08 μM. On the other hand, copper complex 14b and
Among numerous ureases, jack bean (Canavalia ensiformis) urease, the
4
[37]
[38–40]
first enzyme crystallized and best‐characterized,
has been widely
benzimidazole derivative 12 showed no inhibitory activity against urease
[41,42]
utilized in urease inhibition studies.
Accordingly, the newly
enzyme.
synthesized compounds, namely, 3, 9, 10, 11, 12, and 14a,b were
screened for their inhibitory potential against jack bean urease using
thiourea as a standard inhibitor. Screening results revealed that
compounds 3, 9–11, and 14a demonstrated diverse antiurease activity
having IC50 values in a range of 8.16 ± 0.65 to 46.91 ± 0.08 μM compared
to thiourea of IC50 value 20.04 ± 0.33 μM. Compounds 3, 9–11 were
found to be the most active and superior to the standard inhibitor
2
.3
|
Structure‒activity relationships
Herein, we have synthesized some new compounds in which
different antiurease scaffolds were incorporated. Also, the
molecular hybridization approach was considered. Thus, anthra-
nilic acid scaffold was hybridized with fumarate diester in
compound 3. It was also linked to pyrrole and benzimidazole
scaffolds in 11 and 12, respectively. Piperazine scaffold was
hybridized with fumarate diester in compound 9. Based on the
design of our target compounds (Figure 1), the test compounds 3,
(Table 2). Among the tested compounds, oxopiperazine 9 was the most
potent, compared to the reference standard. It exhibited double the
activity of thiourea with IC50 8.16 ± 0.65 and 20.04 ± 0.33 μM,
respectively. Compounds 3, 10, and 11 were almost equipotent with IC50
9
–11, 14a,b could be structurally classified as anthranilic acid
analogs 3, 11, 12, oxopiperazines 9 and 10, and metalloanthra-
nilic acid complexes 14a,b. Structurally, replacing one sided
amino group of thiourea by oxopiperazinylidene moiety afforded
compound 9, which greatly exceeded thiourea as urease inhibitor.
Focusing on anthranilic acid analogs 3, 11, 12, the fumarate ester
TABLE 2 In vitro urease inhibitory activity of tested compounds
compared to thiourea
Compound no.
IC50 ± SEM (µM)
14.13 ± 4.16
8.16 ± 0.65
14.09 ± 0.49
13.68 ± 0.67
NA
3
9
3
, and the dioxopyrrole 11 almost showed the same activity. The
1
1
1
1
1
0
rigid diethoxyfumarate moiety in 3 was replaced by N‐phenyl
dioxopyrrole ring in 11, considering the hydrophilic carboxy-
methyl moiety in the para‐position. Noticeably, this structural
modification did not affect urease inhibitory activity. However,
replacing the carboxyethyl moiety in 3 by benzimidazole ring in
1
2
4a
4b
46.91 ± 0.08
NA
[
43]
a
12 afforded complete loss of antiurease activity. Khan et al.
Thiourea
20.04 ± 0.33
reported weak antiurease activity of some benzimidazole iso-
steres and this could be attributed to the steric hindrance effect
of the large planer aromatic ring. Similarly, replacing the olefinic
Note. NA: no activity; SEM: standard error of the mean.
Result represented as mean of triplicate ± SEM.
a
Thiourea standard inhibitor for antiurease activity.

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hydrogen in oxopiperazine 9 with p‐aminophenyl acetic acid in
0 diminished the urease‐inhibitory activity to its half value.
TABLE 3 The docking scores of compounds 3, 9–12, with
interacting residues in the active site of jack bean urease compared
to thiourea and acetohydroxamic acid.
1
Again, the remarkable decrease of the activity between oxopi-
perazine analogs 9 and 10 could be attributed to the steric
hindrance effect. Noticeably, oxopiperazine moiety was preferred
than anthranilic acid moiety in a small nonsterically hindered
molecule for the structural requirements of urease inhibition.
This proposal could be supported by the poor urease inhibitory
activity of the large organometallic complexes 14a,b carrying bis‐
anthranilic acid moiety.
Docking score
(kcal/mol)
Interacting residues
(distance in Å)
Compound no.
3
−5.73
Gly 550 (2.5)
Arg 609 (arene‐cation)
Ni 901 (2.6)
Ni 902 (2.0)
9
−3.07
Asp 494 (2.7)
His 593 (2.8)
Arg 609 (2.5)
Ni 901 (2.5)
2
.4 Molecular modeling study
|
In an effort to understand the obtained biological data on a structural
basis, considering the potent urease inhibitory activity of the target
compounds 3 and 9–11 compared to thiourea, molecular modeling
simulation was performed for all of them using molecular operating
environment. Further, a comparative modeling was carried out for
the inactive compound 12. Urease cocrystallized with acetohydroxa-
mic acid (Protein Data Bank ID: 4h9m) has been taken as a reference
to control the performance of the docking approach.
Ni 902 (2.6)
1
0
1
−8.12
−7.69
Asp 494 (3.1)
Cys 592 (2.8)
Ni 901 (2.2)
Ni 902 (2.1, 2.3)
Arg 439 (arene‐cation)
Cys 592 (2.8, 2.9)
His 594 (2.7)
1
Obviously, compounds 3 and 9–11, which were superior to
the standard urease inhibitor thiourea in enzyme assay, revealed
much better docking scores (−8.12 to −3.07 kcal/mol), interacted
strongly with both nickel atoms, Ni 901 and Ni 902, at distances
less than 3 Å (2.0–2.8 Å) and were also hydrogen‐bonded to
amino acid residues located in the binding pocket of the enzyme.
However, thiourea revealed a docking score of −0.86 kcal/mol,
through only one coordination bond with nickel atom Ni 902
Ni 901 (2.0, 2.8)
Ni 902 (2.0)
1
2
−6.7
Asp 494 (2.9)
His 593 (2.9)
His 593 (arene‐arene)
Ni 902 (2.6)
a
Thiourea
−0.86
−6.05
b
Acetohydroxamic acid
His 409 (2.6)
(
2.6 Å) (Table 3).
Leu 490 (2.4)
His 492 (2.9)
Focusing on the binding pose of the most potent oxopiperazine 9,
the carbonyl oxygen of the ester moiety illustrated two strong
coordination bonds with Ni 901 (2.5 Å) and Ni 902 (2.6 Å). In
addition, the carbonyl oxygen of the oxopiperazine ring accepted one
Asp 633 (2.2, 2.6)
Ni 901 (1.3)
2
hydrogen from the NH group of Arg 609 (2.5 Å). Also, the two NHs
Ni 902 (2.7)
of oxopiperazine ring were hydrogen‐bonded to Asp 494 and His 593
at distances 2.7 and 2.8 Å, respectively (Figure 6).
a
Thiourea reference standard for in vitro urease assay.
b
Acetohydroxamic acid cocrystallized ligand with jack bean urease
In turn, the aromatic carboxylate of compound 3 displayed two
coordination bonds with the two nickel atoms, Ni 901 (2.6 Å) and
Ni 902 (2.0 Å). Also, a strong hydrogen bond (2.5 Å) was observed
between carboxylate OH moiety and carbonyl oxygen of Gly 550, in
addition to arene‐cation interaction between the phenyl moiety of
compound 3 and the guanidine group of Arg 609 (Figure 6).
Noticeably, the anthranilic acid analog 3 and the phenyl acetic
acid analogs 10 and 11 exhibited similar binding modes. The
carboxylate group of anthranilic acid moiety in 3 and that of phenyl
acetic acid moieties in 10 and 11 were coordinated to the two nickel
atoms. Additionally, the two NHs of oxopiperazine ring in compound
enzyme in Protein Data Bank (4h9m).
may account for the equipotency of compounds 3, 10, and 11 as
urease inhibitors (Figures 6, 7).
Focusing on benzimidazole 12, despite having docking score
much better than thiourea, it revealed no interaction with nickel
atoms. The bulky benzimidazole ring was anchored to the
entrance of the binding pocket, showing arene‐arene interaction
with the imidazole ring of His 593, and subsequently withdrawing
the carboxylate group of the anthranilic acid moiety away from
nickel atoms by more than 8 Å. Instead, the carboxylate group
formed two hydrogen bonds with Asp 494 and His 593 (Figure 7).
This could explain the inactivity of benzimidazole 12 against
urease enzyme.
10 formed two hydrogen bonds with Asp 494 and Cys 592 (CME
592). Similarly, compound 11 formed three hydrogen bonds with Cys
592, and His 594 by virtue of the NH and the carboxylate of
anthranilic acid moiety, respectively. This similarity in binding mode

EL‐ZAHABI ET AL.
9 of 13
|
FIGURE 6 The binding mode of compounds 3 (a), 9 (b), 10 (c), and 11 (d) docked into the active site of jack bean urease enzyme (colored by
atom type, light blue dashed lines for interaction with nickel atoms, purple dashed lines for hydrogen bonding, light blue balls for nickel atoms)
3
|
CONCLUSION
During this study, anthranilic acid and diethylacetylenedicarbox-
ylate were used in the synthesis of new nitrogenous organo
compounds under mild reaction conditions. The newly synthesized
compounds were evaluated for their in vitro inhibitory potential
against urease enzyme. Most compounds were identified as potent
urease inhibitors. Docking studies of active compounds into the
binding pocket of urease enzyme revealed a common strong
interaction with nickel atoms. The docking scores, as well as the
binding modes, were in agreement with the in vitro urease assay.
4
|
EXPERIMENTAL
4
4
.1 Chemistry
|
.1.1 | General
All melting points were measured on a Gallenkamp electrothermal
melting point apparatus. The infrared spectra were recorded for
potassium bromide pellets on a PyeUnicam SP 3‐300 and FT IR 8101
1
PC Shimadzu infrared spectrophotometers. The H NMR spectra
were recorded in dimethylsulfoxide‐d6 (DMSO‐d6) at 400 MHz on
Agilent Technologies Mercury NMR spectrometers. Mass spectra
were recorded at 70 eV on a GCMSeQP 1000 EX Shimadzu mass
spectrometer (for gas chromatography (GC)/MS) and on Agilent GC
FIGURE 7 (a) The binding mode of compound 12 docked into the
active site of jack bean urease enzyme. (b) Overlay of compounds
3
(purple), 9 (white), 10 (red), 11 (blue), 12 (green), and
6
890 N coupled with an Agilent Mass Selective Detector 5973 (for
acetohydroxamic acid (light blue) docked into the active site of jack
bean urease enzyme
ESI‐MS). Elemental analyses were carried out at the Microanalytical

10 of 13
EL‐ZAHABI ET AL.
|
Center of Cairo University. Analyses indicated by the symbols of the
elements or functions were within ± 0.4% of the theoretical values.
X‐ray crystallography was carried out on a Kappa CCD FR 590
diffractometer (Enraf Nonius), National Research Center, Dokki,
Cairo, Egypt. DEAD and anthranilic acid were obtained from Aldrich
and used without further purification. The tested compounds have a
range between 95% and 100% purity.
The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK
(http://www.ccdc.cam.ac.uk).
5‐Oxo‐2,5‐dihydro‐1H‐pyrazole‐3‐carbohydrazide (6)
Compound (6) was prepared by mixing compound 3 (1 mmol) with
excess NH NH .H O in ethanol (10 ml) under reflux for 3 hr. The
2
2
2
reaction mixture was cooled. The solid formed was collected by
filtration, washed with ethanol and diethylether, dried and finally
recrystallized from ethanol to afford 6: as faint yellow powder
The original spectra of the investigated compounds and detailed
structural data of compounds 3, 9, and 14b are provided as
Supporting Information. The InChI codes of the investigated
compounds together with some biological activity data are also
provided as Supporting Information.
−
1
(C H OH), yield 40%, mp. 243–245°C; IR (KBr) ν (cm ): 3371,
2
5
1
3275 (NH), 1716, 1681 (C=O); H NMR (DMSO‐d6) δ 4.45 (brs, 2H,
D O exchangeable NH ), 5.96 (s, 1H, vinylic CH), 9.49, 9.79, and
2
2
1
3
1
2.29 (3s, 3H, D O exchangeable NH); C NMR (DMSO‐d6) δ
2
2
‐((1,4‐Diethoxy‐1,4‐dioxobut‐2‐en‐2‐yl)amino)benzoic
86.70 (C4‐pyrazoline ring), 114.47 (C3‐pyrazoline ring), 161.41,
161.83 (C5‐pyrazoline ring and C3‐carbohydrazide); MS m/z (%):
acid (3)
+
1
+
Compound 3 was prepared by stirring anthranilic acid 1 (1 mmol)
with DEAD 2 (1 mmol) in ethanol (5 ml) at rom temperature for
143 (M , 1.87), 142 (M , 19.54), 111 (85.21), 105 (3.29), 83
(18.46), 68 (16.95), 57 (11.59), 55 (81.54), 53 (100), 44 (20.56),
2
hr. The solid formed was collected by filtration, washed with diethyl
43.13 (32.12). Anal. calcd. for C
4 6 4 2
H N O (142.05) C, 33.81;
ether, dried and finally recrystallized from acetic acid to afford 3: as
H, 4.26; N, 39.42. Found: C, 34.10; H, 4.66; N, 39.10.
yellow plates (CH
3
COOH), yield (95%), mp. 158–160°C. IR (KBr) ν
−1
1
(
cm ): 3275 (NH), 1728, 1670 (ester C=O), 1680 (acid C=O); H NMR
4
2
.1.3 | Synthesis of ethyl
‐(3‐oxopiperazin‐2‐ylidene)acetate (9): Method A
(
DMSO‐d6) δ 1.041 (t, 3H, CH
3
CH2, J = 7.2 Hz), 1.10 (t, 3H, CH
CH )), 5.43 (s, 1H, vinylic CH),
.65 (d, 1H, ArH, J = 8.4 Hz), 7.04–7.07 (m, 1H, ArH), 7.42–7.46 (m, 1H,
ArH), 7.87 (d. 1H, ArH, J = 8 Hz), 8.1, 11.03 (2s, 2H, D O exchangeable
NH, COOH). MS m/z (%): 308 (M , 1.09), 307 (M , 5.53), 261 (2.23),
34 (80.67), 188 (52.95), 170 (14.73), and 146 (100); Anal. calcd. for
17NO (307.10) C, 58.63; H, 5.58; N, 4.56. Found: C, 58.11; H,
.08; N, 5.03.
3
CH2,
J = 5.2 Hz), 4.08–4.17 (m, 4H, 2(CH
3
2
6
Compound 9 was prepared by mixing compound 3 (1 mmol) with
ethylenediamine (1 mmol) in ethanol (5 ml) under reflux for 2 hr.
The reaction mixture was cooled. The solid formed was collected
by filtration, washed with ethanol and diethylether, dried, and
finally recrystallized from ethanol to afford 9: as yellow cubes,
2
+
1
+
2
C
15
H
6
5
−1
yield 55%, mp. 167–169°C. IR (KBr) ν (cm ): 3325, 3224 (NH),
1
1
689, 1658 (C=O). H NMR (DMSO‐d6) δ 1.15 (t, 3H, CH
3
CH
2
, J =
4
.1.2 | X‐ray structure determination of
7.2 Hz), 3.25–3.29 (m, 4H, CH
2
–CH
2
piprazine), 4.04 (q, 2H, CH –
3
compound 3
2
CH2, J = 7.2 Hz), 5.16 (s, 1H, vinylic CH), 8.33, 8.40 (2s, 2H, D O
+
exchangeable NH). MS m/z (%): 184 (M , 10.42), 177 (33.50), 158
A single crystal of compound 3 was obtained by slow evaporation
(
(
(
11.73), 140 (31.16), 139 (100), 124 (29.87), 111 (40.54), 102
42.21), 84 (72.44), and 75 (41.50). Anal. calcd. for C
184.08) C, 52.17; H, 6.57; N, 15.21. Found: C, 51.99; H, 6.10;
6
from acetic acid. Compound 3: C15H17NO , Mr = 307.302, yellow
8
12 2 3
H N O
plates, triclinic space group P¯1 with Z = 2, a = 8.8202(4),
b = 9.6161(4), c = 10.0404(6) Å, alpha = 78.0398(14)°, beta =
N, 15.55.
3
6
9.722(2)°, gamma = 75.802(2)°, V = 767.38(7) Å ; Dx = 1.330
−
1
Mg/m³, μ = 0.10 mm . The intensity data were recorded using a
[
44]
Bruker Noniu CCD FR 590 area‐detector diffractometer,
with
4
.1.4 | X‐ray structure determination of
graphite monochromated Mo Kα radiation (λ = 0.71073 Å) at
T = 298 K. 1657 observed reflections, θmax = 27.49°; 3650
independent reflections I > 3 sigma(I), Rint = 0.024. Structure
solution by direct methods full‐matrix least squares refinement
based on F2 and 199 parameters. All but H‐atoms were refined
anisotropiclly. Hydrogen atoms were clearly located from differ-
ence Fourier maps, refined at idealized positions riding on the
carbon atoms with isotropic displacement. Refinement converged
at R(all) = 0.109, wR(all) = 0.110, S(all) = 0.782; min./max. deltaF
compound 9
A single crystal of compound 9 was obtained by slow evaporation from
8 12 2 3
ethanol. Compound 9: C H N O , Mr = 184.195, yellow cube,
1
monoclinic space group P2 /a with Z = 4, a = 12.0301(8), b =
4.5470(3), c = 17.0770(13) Å, alpha = 90.00°, beta = 94.619(3)°,
3
gamma = 90.00°, V = 931.09(11) Å ; Z = 4; Dc = 1.314 Mg/m³, μ = 0.10
−1
mm . The intensity data were recorded using a Bruker Nonius CCD
[44]
FR 590 area‐detector diffractometer with graphite monochromated
Mo Kα radiation (λ = 0.71073 Å) at T = 298 K. 533 reflections
collected θmax = 30.0°; 3257 independent reflections I > 3 sigma(I),
Rint = 0.081. Structure solution by direct methods, full‐matrix least
squares refinement based on F2 and 118 parameters. All but H‐atoms
−
0.35/0.49 e/ų. Crystallographic data for the structural analysis
of compound 3 has been deposited with the Cambridge Crystal-
lographic Data Center (CCDC) under the number 1498175. Copies
of the information may be obtained free of charge from

EL‐ZAHABI ET AL.
11 of 13
|
were refined anisotropiclly. Hydrogen atoms were clearly located from
difference Fourier maps, refined at idealized positions riding on the
carbon atoms with isotropic displacement parameters. Refinement
converged at R(all) = 0.246, wR(all) = 0.111, S(all) = 0.632; min./max.
deltaF −0.60/0.56 e/ų. Crystallographic data for the structural
analysis of compound 9 has been deposited with the CCDC under
the number 1498175. Copies of the information may be obtained free
of charge from The Director, CCDC, 12 Union Road, Cambridge CB2
δ 3.37 (s, 2H; CH
5.78 (s, 1H, vinylic CH), 6.50 (d, 2H, ArH, J = 8.4 Hz), 6.87 (d, 2H, ArH,
J = 8 Hz), 7.18–7.40 (m, 4H, ArH), 9.8 (s, 1H, D O exchangeable NH).
–COOH), 114.28, 120.08, 120.54,
2 2
–COOH), 3.5 (brs, 1H, D O exchangeable COOH),
2
1
3
C NMR (DMSO‐d6) δ 40.59 (CH
2
122.31, 126.95, 130.07, 130.14, 130.27, 130.42, 130.73, 131.11,
138.33, 147.55 (Ar C and vinylic C), 167.01, 172.00 (CO imide),
+
173.14, 173.82 (CO, COOH), MS m/z (%): 366 (M , 6.34), 365 (45.77),
335 (100), 319 (41.73), 313 (15), 311 (11.33), 111 (19.00), 302
(18.10), 301 (10.73), 273 (10.72), 254 (16.51), 231 (11.26), 150
1EZ, UK (http://www.ccdc.cam.ac.uk).
14 2 6
(17.75), 122 (10.88). Anal. calcd. for C19H N O (366.09) C, 62.30;
H, 3.85; N, 7.65. Found: C, 62.50; H, 3.96; N, 7.42.
4
2
.1.5 | Synthesis of ethyl
‐(3‐oxopiperazin‐2‐ylidene)acetate (9): Method B
The second method for preparation of compound 9 adopted the
2‐((1‐(1H‐Benzo[d]imidazol‐2‐yl)‐3‐ethoxyprop‐1‐en‐1‐yl)ami-
no)benzoic acid (12)
[
31]
reported literature.
Compound 9 was prepared by warming DEAD
2
(1 mmol) with ethylenediamine (1 mmol) in ethanol (5 ml) with
Compound 12 was prepared by mixing compound 3 (1 mmol) with
o‐phenylenediamine (1 mmol) in ethanol (5 ml) under reflux for 3 hr
(TLC‐control). The reaction mixture was cooled. The solid formed was
collected by filtration, recrystallized from ethanol to afford 12 as
stirring for 2 hr. The solid formed was collected by filtration, washed
with ethanol and diethylether, dried and finally recrystallized from
ethanol to afford compound 9: as faint yellow flakes, yield 65%, mp.
−1
167–169°C.
yellow crystals: yield 88%, mp. 210–212°C. IR (KBr) ν (cm ): 3367,
1
3
265 (NH), 1685, 1651 (C=O). H NMR (DMSO‐d6) δ 1.18 (t, 3H,
3 3 2
CH CH2, J = 7.2 Hz), 4.10 (q, 2H, CH CH , J = 7.2 Hz), 5.47 (s, 1H,
2
‐(4‐((2‐Ethoxy‐2‐oxo‐1‐(3‐oxopiperazin‐2‐yl)ethyl)amino)phe-
vinylic CH), 6.99–7.06 (m, 4H, ArH), 7.25–7.31 (m, 2H, ArH), 7.51
(t, 1H, ArH, J = 1.6 Hz), 7.71 (d, 1H, ArH, J = 8.8 Hz). MS m/z (%): 352
nyl)acetic acid (10)
+
1
+
Compound 10 was prepared by mixing compound 9 (1 mmol) with
p‐aminophenyl acetic acid (2 mmol) in ethanol (5 ml) under reflux for
(M , 1.66), 351 (M , 1.81), 322 (1.75), 308 (2.20), 306 (1.87), 237
(1.60), 204 (2.20), 186 (7.92), 160 (10.75), 131 (19.03), 80 (64.59), 64
6
hr (TLC‐control). The reaction mixture was cooled. The solid
17 3 4
(100). Anal. calcd. for C19H N O (351.12) C, 64.95; H, 4.88; N,
formed was collected by filtration, recrystallized from ethanol to
11.96. Found: C, 64.82; H, 4.38; N, 12.46.
afford 10 as brown powder; yield 80%, mp. > 350°C. IR (KBr) ν
−
1
1
(
cm ): 3367, 3336, 3305 (OH, NH), 1705, 1689 and 1658 (C=O). H
NMR (DMSO‐d6) δ 1.03 (t, 3H, CH CH , J = 8 Hz), 1.13 (brs, 1H, D
exchangeable OH), 3.25–3.42 (m, 10H; 4H, CH ‐CH piprazine, 1H,
CH ethyl, 1H, CH piperazinyl, 2H, CH COOH and 2H, CH CH ), 6.46
d, 2H, ArH, J = 8.4 Hz), 6.85 (d, 2H, ArH, J = 8.4 Hz), 6.99, 7.00 (2s,
H, D O exchangeable NH), 8.50 (brs, 1H, D O exchangeable
COOH). C NMR (DMSO‐d6) δ 19 (COOCH CH ), 40.59, 40.63
‐COOH), 56.46 (COOCH CH , C1 ethyl),
14, 126, 129, 130 (Ar C), 148 (C–OH, piperazine C3), 160
9 8 3 2
Tri‐aqua bis(2‐acetylaminobenzoato)Zn(II), [Zn(C H NO )
3
2
2
O
2 3 2 2
(H O) ](H O) (14a)
2
2
3 2 2
The complex 14a was prepared by dissolving Zn(NO ) .4H O
2
3
2
(1 mmol) in distilled water (10 ml). Then, solution of 1 M NaOH
was added portionwise until the formation of the gelatinous hydrated
zinc hydroxide was completed. The resulting mixture was centrifuged
and the solid was washed thoroughly with distilled water (6 × 5 ml).
The wet gelatinous solid was suspended in distilled water (25 ml).
Then it was added to a solution of acetyl anthranilic acid (1 mmol) in
ethanol (10 ml) with stirring. A glossy white precipitate of zinc
complex appeared after a few seconds, filtered and washed several
times with water and ethanol to afford 14a as fine white crystal: yield
(
2
2
2
1
3
2
3
(
C2,5,6 piperazine, CH
2
2
3
1
+
1
(
(
(
CO, CH
2
COOH and COOC
, 1.72), 313 (100), 305 (6.01), 290 (2.25), 285 (26.84), 264
17.18), 262 (11.79), 184 (3.01), 150 (11.17), 109 (15.14). Anal. calcd.
(335.15) C, 57.30; H, 6.31; N, 12.53. Found:
C, 57.00; H, 6.10; N, 12.63.
2 5
H ), MS m/z (%): 336 (M , 25.67), 320
+
M –CH
3
for C16
H
21
N O
3 5
26 2
50%, mp. > 300°C. Anal. calcd. for C18H N O11Zn; (510.08) C,
42.24; H, 5.12; N, 5.47. Found: C, 42.74; H, 5.32; N, 5.07.
Tri‐aqua bis(2‐acetylaminobenzoato)Cu(II), [Cu(C
(H O) ](H O) (14b)
The complex 14b was prepared by dissolving CuSO
9
H
8
NO
3 2
)
2
‐((1‐(4‐(Carboxymethyl)phenyl)‐2,5‐dioxo‐2,5‐dihydro‐1H‐
2
3
2
2
pyrrol‐3‐yl)amino) benzoic acid (11)
4
2
.5H O (1.5
Compound 11 was prepared by mixing compound 3 (1 mmol) with
mmol) in distilled water (10 ml). The salt solution was added to a
solution of acetyl anthranilic acid (1 mmol) in ethanol (10 ml) with
stirring. The metal complex was precipitated after addition of small
aliquots of 1 M NaOH to afford 14b as green crystals: yield 30%, mp.
p‐aminophenyl acetic acid (1 mmol) in ethanol (5 ml) under reflux for
4
hr (TLC‐control). The reaction mixture was cooled. The solid
formed was collected by filtration, recrystallized from ethanol to
afford 11: as yellow powder, yield 83%, mp. 184–186°C. IR (KBr)
26 2
> 300°C. Anal. calcd. for C18H N O11Cu; (509.08) C, 42.39; H, 5.14;
−
1
1
ν (cm ): 3394, 3367 (NH), 1705, 1651 (C=O). H NMR (DMSO‐d6)
N, 5.49. Found: C, 42.21; H, 4.82; N, 6.05.

12 of 13
EL‐ZAHABI ET AL.
|
4
.1.6 | X‐ray structure determination of copper
ACKNOWLEDGMENT
complex 14b
The authors express deep thanks to Dr. Abdel‐Sattar S. Hamad
Elgazwy for his valuable instructions to get the single crystal of
compound 14b with CCDC under the number 766987.
Here the single crystal of 14b proved a mononuclear geometrical
structure. The copper atom existed in a central symmetrical position
in pentadentate coordination system. There are two coordination
bonds shared by two oxygen atoms of the carboxylate ions. So, each
carboxylate group bound in a mono dentate manner and the other
three coordination bonds shared by three water molecules. In this
case the geometry was best described as tetrahedral. The arrange-
ment was completed by existence of another two water molecules at
the top of the crystal geometry.
CONFLICT OF INTERESTS
The authors have declared that there is no conflict of interest.
ORCID
A single crystal of compound 14b was obtained by slow
evaporation from a mixture of ethanol/water (1:1). Compound
Heba S.A. El‐Zahabi
http://orcid.org/0000-0002-7023-6364
http://orcid.org/0000-0003-3035-2624
Hanan G. Abdulwahab
1
4b: C18
26 2
H N O11Cu, Mr = 419.874, green prismatic crystal,
orthorhombic space group P2
1
2
1
2
1
with Z = 4; a = 7.1674(2), b =
REFERENCES
1
7.3033(6), c = 18.3535(9) Å, alpha = 90.00°, beta = 90.00°,
3
3
gamma = 90.00°. V = 2276.2(2) Å ; D
x
= 1.225 Mg/m , μ = 0.99
[
1] H. L. Mobley, R. P. Hausinger, Microbiol. Rev. 1989, 53, 85.
−
1
mm , The intensity data were recorded using a Bruker Nonius
[2] C. R. Carlini, R. Ligabue‐Braun, Toxicon 2016, 110, 90. https://doi.
[
44]
org/10.1016/j.toxicon.2015.11.020
CCD FR 590 area‐detector diffractometer
monochromated Mo Kα radiation (λ = 0.71073 Å) at T = 298 K.
726 observed reflections, θ = 2.910–27.485°; 3185 independent
with graphite
[
[
[
[
3] R. A. Burne, Y. Y. M. Chen, Microbes Infect. 2000, 2, 533. https://doi.
org/10.1016/S1286-4579(00)00312-9
1
4] C. Follmer, J. Clin. Pathol. 2010, 63, 424. https://doi.org/10.1136/jcp.
2009.072595
reflections I > 3 sigma (I), Rint = 0.042. Structure solution by direct
methods, full‐matrix least squares refinement based on F2 and 289
parameters. All but H‐atoms were refined anisotropiclly. Hydrogen
atoms were clearly located from difference Fourier maps, refined
at idealized positions riding on the carbon atoms with isotropic
displacement parameters. Refinement converged at R(all) = 0.097,
wR(all) = 0.107, S(all) = 2.025; min./max. deltaF −0.78/0.72 e/ų.
Crystallographic data for the structural analysis of compound 14b
has been deposited with the CCDC under the number 766987.
Copies of the information may be obtained free of charge from The
Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (http://
www.ccdc.cam.ac.uk).
5] P. Kosikowska, Ł. Berlicki, Expert Opin. Ther. Pat. 2011, 21, 945.
https://doi.org/10.1517/13543776.2011.574615
6] A. M. Felix, L. B. Czyzewski, D. P. Winter, R. I. Fryer, J. Med. Chem.
1
969, 12, 384.
[7] A. Eudes, E. E. K. Baidoo, F. Yang et al., Appl. Microbiol. Biotechnol.
2011, 89, 989. https://doi.org/10.1007/s00253-010-2939-y
[
8] S. Tayyab, M. S. Zaroog, S. R. Feroz, S. B. Mohamad, S. N. A. Malek,
Int. J. Pharm. 2015, 491, 352. https://doi.org/10.1016/j.
ijpharm.2015.06.042
[
9] M. Isaji, H. Miyata, Y. Ajisawa, Y. Takehana, N. Yoshimura, Br.
J. Pharmacol. 1997, 122, 1061. https://doi.org/10.1517/13543776.
2
011.574615
10] A. Rauf, S. Shahzad, M. Bajda et al., Bioorg. Med. Chem. 2015,
3, 6049.
[
2
[
11] T. Tanaka, M. Kawase, S. Tani, Life Sci. 2003, 73, 2985.
12] P. Kafarski, M. Talma, J. Adv. Res. 2018, 13, 101. https://doi.org/10.
1016/j.jare.2018.01.007
[
4
.2 Urease inhibition assay
|
[
13] A. Ibrar, I. Khan, N. Abbas, Arch. Pharm. (Weinheim) 2013, 346, 423.
https://doi.org/10.1002/ardp.201300041
The urease activity was determined by measuring the amount of
ammonia being produced using indophenol method described by
[14] K. Macegoniuk, R. Kowalczyk, A. Rudzińska, M. Psurski, J. Wietrzyk,
Ł. Berlicki, Bioorg. Med. Chem. Lett. 2017, 27, 1346.
[
45,46]
Weatherburn.
Briefly, the assay mixture, containing 10 μL
[
15] M. Tsuchiya, L. Imamura, J. Park, K. Kobashi, Biol. Pharm. Bull. 1995,
8, 1053.
16] J. B. Park, L. Imamura, K. Kobashi, Biol. Pharm. Bull. 1996, 19, 182.
of urease Canavalia ensiformis (jack bean urease, Sigma) and 10 μL
of test compound in 40 μL phosphate buffer containing 100 mM
urea were incubated for 30 min at 37°C in 96‐well plates. 40 μL
each of phenol reagents (1%, w/v phenol and 0.005%, w/v sodium
nitroprusside) and 40 μL of alkali reagent (0.5%, w/v NaOH and
1
[
[17] S. T. S. Hassan, M. Žemlička, Arch. Pharm. (Weinheim) 2016, 349, 507.
[
[
18] M. Taha, A. Wadood, Bioorg. Chem. 2018, 78, 411.
19] D. M. S. Vardhan, H. K. Kumara, H. P. Kumar, D. C. Gowda, Int.
J. Pharm. Pharm. Sci. 2017, 9, 92.
0
.1% active chloride NaOCl) were added to each well. The
[
20] Z. ‐L. You, L. ‐L. Ni, D. ‐H. Shi, S. Bai, Eur. J. Med. Chem. 2010, 45, 3196.
absorbance at 625 nm was measured after 30 min, using a
microplate reader (BioTekELx 800, Instruments, Inc.). All reac-
tions were performed in triplicate. Thiourea was used as the
standard inhibitor of urease. The IC50 values were determined by
the nonlinear curve fitting program PRISM 5.0 (GraphPad, San
Diego, CA).
[21] Y. M. Cui, X. W. Dong, W. Chen, W. J. Wang, Y. G. Li, H. L. Zhu,
J. Enzyme Inhib. Med. Chem. 2012, 27, 528.
[
[
22] D. Ali, S. Kazem, R. I. Mohammad, ARKIVOC 2007, i, 180.
23] F. I. Zubkov, V. P. Zaitsev, A. A. Orlova, A. S. Peregudov, N. M.
Mikhailova, A. V. Varlamov, Russ. J. Org. Chem. 2007, 43, 1202.
[24] A. Komaraiah, K. Ramakrishna, B. Sailu, P. S. N. Reddy, ARKIVOC
2007, xiv, 110.

EL‐ZAHABI ET AL.
13 of 13
|
[
25] O. S. Eltsov, M. V. Smirnova, Y. Yu. Morzherin, N. P. Belskaiya, V. S.
[42] S. Shabana, A. Kawai, K. Kai, K. Akiyama, H. Hayashi, Biosci.
Biotechnol. Biochem. 2010, 74, 878.
Mokrushin, ARKIVOC 2008, xvii, 306.
[
26] J. Crosby, M. C. McKie, R. M. Paton, J. F. Ross, ARKIVOC 2000, v, 720.
27] J. F. Ross, C. Delipala, M. C. Watson, J. Crosby, R. M. Paton, ARKIVOC
[43] K. M. Khan, F. Rahim, A. Khan et al., Bioorg. Med. Chem. 2014, 22, 4119.
[44] S. Mackay, C. J. Gilmore, C. Edwards, N. Stewart, K. Shankland,
maXus Computer Program for the Solution and Refinement of Crystal
Structures, MacScience, Japan and The University of Glasgow, Bruker
Nonius, The Netherlands 1999.
[
2013, iii, 372.
[28] S. K. Khetan, M. V. George, Can. J. Chem. 1969, 47, 3545.
[29] N. D. Heindel, P. D. Kennewell, M. Pfau, J. Chem. Soc. D 1969, 757.
[30] J. Wu, S. Kang, Q. Yuan et al., Molecules 2012, 17, 9413.
[31] Y. Iwanami, Y. Kenjo, K. Nishibe, M. Kajiura, S. Isoyama, Bull. Chem.
Soc. Jpn. 1964, 37, 1740.
[45] M. W. Weatherburn, Anal. Chem. 1967, 39, 971.
[46] M. A. S. Aslam, S. U. Mahmood, M. Shahid, A. Saeed, J. Iqbal, Eur.
J. Med. Chem. 2011, 46, 5473.
[
32] S. M. Medvedeva, K. S. Shikhaliev, J. Mater. Sci. Eng. 2015, 5, 310.
33] Y. A. Ammar, M. M. F. Ismail, M. K. Ibrahim, H. S. A. El‐Zahaby,
Afinidad 2005, 62, 151.
[
SUPPORTING INFORMATION
[
[
[
34] O. V. Shekhter, N. L. Sergovskaya, Yu. S. Tsizin, Chem. Heterocycl.
Compd. 1985, 21, 665.
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
35] I. Vlahou, N. Kourkoumelis, A. Michaelides, S. Skoulika, J. C.
Plakatouras, Inorg. Chim. Acta 2006, 359, 3540.
36] L. Naso, A. C. González Baró, L. Lezama, T. Rojo, P. A. M. Williams,
E. G. Ferrer, J. Inorg. Biochem. 2009, 103, 219.
[
[
37] J. B. Sumner, J. Biol. Chem. 1926, 69, 435.
How to cite this article: El‐Zahabi HSA, Abdulwahab HG,
Edrees MM, Hegab AM. Utility of anthranilic acid and
diethylacetylenedicarboxylate for the synthesis of
nitrogenous organo/organometallic compounds as urease
inhibitors. Arch. Pharm. Chem. Life Sci. 2019;e1800314.
https://doi.org/10.1002/ardp.201800314
38] S. Ciurli, S. Benini, W. R. Rypniewski, K. S. Wilson, S. Miletti,
S. Mangani, Coord. Chem. Rev. 1999, 190–192, 331.
[
[
[
39] C. Follmer, F. V. Pereira, N. P. da Silveira, C. R. Carlini, Biophys. Chem.
2004, 111, 79.
40] L. Sheridan, C. M. Wilmot, K. D. Cromie, S. Van der Logt, S. Phillips,
Acta Crystallogr. Sect. D 2002, 58, 374.
41] C. Follmer, Phytochemistry 2008, 69, 18.