Symmetrical heterocyclic cage skeleton: Synthesis, urease inhibition activity, kinetic mechanistic insight, and molecular docking analyses

Article abstract of DOI:10.3390/molecules24020312

The present study focuses on the design and synthesis of a cage-like organic skeleton containing two triazole rings jointed via imine linkage. These molecules can act as urease inhibitors. The in-vitro urease inhibition screening results showed that the combination of the two triazole skeleton in the cage-like morphology exhibited comparable urease inhibition activity to that of the reference thiourea while the metallic complexation, especially with copper, nickel, and palladium, showed excellent activity results with IC₅₀ values of 0.94 ± 0.13, 3.71 ± 0.61, and 7.64 ± 1.21 (3a–c), and 1.20 ± 0.52, 3.93 ± 0.45, and 12.87 ± 2.11 μM (4a–c). However, the rest of compounds among the targeted series exhibited a low to moderate enzyme inhibition potential. To better understand the compounds’ underlying mechanisms of the inhibitory effect (3a and 4a) and their most active metal complexes (3b and 4b), we performed an enzymatic kinetic analysis using the Lineweaver–Burk plot in the presence of different concentrations of inhibitors to represent the non-competitive inhibition nature of the compounds, 3a, 4a, and 4b, while mixed type inhibition was represented by the compound, 3b. Moreover, molecular docking confirmed the binding interactive behavior of 3a within the active site of the target protein.

Full text of DOI:10.3390/molecules24020312

molecules
Article
Symmetrical Heterocyclic Cage Skeleton: Synthesis,
Urease Inhibition Activity, Kinetic Mechanistic
Insight, and Molecular Docking Analyses
Muhammad Hanif 1,2, Fariha Kanwal , Muhammad Rafiq , Mubashir Hassan
1
3
4
,
5
4
1
1
1,
Muhammad Mustaqeem , Sung-Yum Seo , Yunlong Zhang , Changrui Lu , Ting Chen *
5
,
and Muhammad Saleem *
1
State Key Laboratory for Modification of Chemicals Fibers and Polymer Materials, College of Chemistry,
Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China;
han_qau@yahoo.com (M.H.); farihakaanwal@gmail.com (F.K.); 416033@mail.dhu.edu.cn (Y.Z.);
crlu@dhu.edu.cn (C.L.)
Department of Chemistry, GC University Faisalabad, Sub campus Layyah 31200, Pakistan
Department of Physiology and Biochemistry, Cholistan University of Veterinary and Animal Sciences,
2
3
Bahawalpur 63100, Pakistan; rafiqknu@gmail.com
College of Natural Science, Department of Biology, Kongju National University, Gongju,
4
Chungcheongnam 32588, Korea; mubashirhassan_gcul@yahoo.com (M.H.); dnalove@kongju.ac.kr (S.-Y.S.)
Department of Chemistry, University of Sargodha, Sub-campus Bhakkar 30000, Pakistan;
5
muhammad.mustaqeem@uos.edu.pk
*
Correspondence: 414023@mail.dhu.edu.cn (T.C.); saleemqau@gmail.com (M.S.);
Tel.: +86-132-6225-3798 (T.C.); +92-333-6206179 (M.S.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 7 December 2018; Accepted: 10 January 2019; Published: 16 January 2019
Abstract: The present study focuses on the design and synthesis of a cage-like organic skeleton
containing two triazole rings jointed via imine linkage. These molecules can act as urease inhibitors.
The in-vitro urease inhibition screening results showed that the combination of the two triazole
skeleton in the cage-like morphology exhibited comparable urease inhibition activity to that of the
reference thiourea while the metallic complexation, especially with copper, nickel, and palladium,
showed excellent activity results with IC50 values of 0.94
±
0.13, 3.71
±
0.61, and 7.64
±
1.21 (3a–c),
and 1.20 0.52, 3.93 0.45, and 12.87 2.11 M (4a ). However, the rest of compounds among the
±
±
±
µ
–c
targeted series exhibited a low to moderate enzyme inhibition potential. To better understand the
compounds’ underlying mechanisms of the inhibitory effect (3a and 4a) and their most active metal
complexes (3b and 4b), we performed an enzymatic kinetic analysis using the Lineweaver–Burk plot
in the presence of different concentrations of inhibitors to represent the non-competitive inhibition
nature of the compounds, 3a, 4a, and 4b, while mixed type inhibition was represented by the
compound, 3b. Moreover, molecular docking confirmed the binding interactive behavior of 3a within
the active site of the target protein.
Keywords: heterocyclic cage; urease inhibition; metal complexes; lineweaver–burk plot; molecular
docking
1
. Introduction
The development of competitive and non-competitive enzyme inhibitors based on a
metalloskeleton has attracted wide interest of the scientific community because metal ions are
found in the active sites of a large number of metalloproteins, such as hemocyanin, and also in
metalloenzymes, like ureases, tyrosinase, laccase, and ascorbate oxidase [1–8]. The enzyme inhibition
Molecules 2019, 24, 312; doi:10.3390/molecules24020312
www.mdpi.com/journal/molecules

Molecules 2019, 24, 312
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by the metallocompouds is due to the reaction of the organic skeleton as well as the transition metal
ions with the sulfhydryl group in the active center of the enzyme [ ]. Urease action results in an
9
elevation of pH, which exerts harmful effects where it occurs whether in soil or inside the human
body [10].
Urease inhibitors contain two general classifications, including pure organic based compounds
and organic compounds, in ligation with transition metals, as some transition metals themselves
possess a slight urease inhibition potential [11]. The literature survey showed excellent urease inhibition
potential for candidates exhibiting hydroxamic acids, phosphoramides, and thiols’ skeleton [12].
Compounds with thiol functional groups inhibit urease competitively in their thiolate anion form,
−
R-S
[13]. Imine derivatives of organic compounds have been extensively employed as ligands
for several ions, and have been utilized as pigments, catalysts, drugs, and polymer stabilizers [14].
The interested drug candidate can be designed and synthesized via a Schiff base skeleton either through
insilico/bioinformatics study or by synthesizing analogues that are structurally close to the reference
drug. Some of urease inhibitors cannot be used in vivo because of their toxicity or instability. Thus,
seeking novel urease inhibitors with good bioavailability and low toxicity is our main focus [15]. Here,
we report the synthesis and urease inhibition evaluation of metal complexes of substituted triazole
based Schiff base ligands containing thiols and alcoholic moiety connected to a triazole backbone.
2
. Results and Discussion
2
.1. Synthesis of Schiff Base Ligands 1 and 2
4-Methoxyphenylacetic acid was taken as starting material and then esterified into their
aralkylethanoate in the presence of a catalytic amount of sulfuric acid. First, thin layer chromatographic
analysis confirmed the ester formation. The acid spot remained at the bottom due to acidic protons
in the hydrogen bonding with silica gel while that of ester traveled along with the direction of the
solvent front. Secondly, FT-IR spectral measurement by the absence of a hydroxyl signal in the range of
−
1
3
400–2500 cm further confirmed the esterification reaction. The resulting esters were then converted
to their corresponding acid hydrazides by the treatment with hydrazine hydrate in the presence
−
1
of ethanol as the solvent. The new broad band signal in the range of 3342, 3287, and 3158 cm
corresponds to primary and secondary amino group stretching vibrations. Moreover, the resonance
effect due to amide functionality exerts partial single bond characters in the carbonyl double bond
−
1
compared to that of ester, shifting the carbonyl group signal from 1711 to 1674 cm . The emergence
−
1
of signals at 1628 and 1610 cm confirms imines’ synthesis due to the C=N stretching vibration.
2
.2. Ligand-Metals Complexation
The ligand-metal complexation causes a shift in the physical appearances of the ligand before
and after metal-complexation, corroborated by differences in their melting points. Further, FT-IR
analysis showed that both ligands and contain the labile protons, which can stay inside the triazole
ring nitrogen atom as well as outside the ring to the oxygen in molecule and sulfur in molecule
This protonic shift leads to the existence of the ligand in the tautomeric conformation. Among
3
4
3
4
.
the tautomers, the keto tautomer seems more stable in comparison to its enol form and the same for
compounds exhibiting thiol-thione tautomerism. Therefore, both ligands preferably stayed in the
keto (in case of ligand
3) and thione (in case of ligand 4) conformation as indicated in their FT-IR
−
1
absorption spectral analysis due to the appearance of signals at 1680 and 1272 cm for ligands
3
and 4,
respectively. The spectral position for both these ligands shifts to lower frequencies after complexation,
evidence for these molecular sites’ involvement during the ligation with metals. Moreover, complete
−
1
disappearance of the signal at 2500 cm in ligand 4 after metal complexation further confirms sulfur’s
−
1
participation in metal binding. The similar disappearance of ligand 3’s broad peak at 3468 cm after
metal chelation represents the interaction of undersigned oxygen with the transition metal (Table 1).
The schematic representations of the synthetic route adopted to obtain the target molecule (3a-f and

Molecules 2019, 24, 312
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4
a-f) are given in Scheme 1. The detailed synthetic procedure adopted for the accomplishment of
precursors 1 and 2 is inserted in the supporting information.
Table 1. FT-IR spectroscopic data for ligands 3 and 4 before and after complexation.
S. No.
Compounds
-N-H
C-H
C=N
C=C
C=S
C=O
C-O-C
1
2
3
4
5
6
7
8
9
ligand (3)
3a (ligand+Cu
3b (ligand+Ni
3c (ligand+Pd
3d (ligand+Co
3e (ligand+Fe
3f (ligand+Zn
ligand (4)
4a (ligand+Cu
4b (ligand+Ni
3519–3738
3491–3370
3481–3401
3499–3378
3501–3391
3511–3381
3508–3402
3523–3390
3505–3401
3511–3370
3500–3388
3511–3401
3508–3391
3508–3400
3138–2949
3190–2901
3200–2911
3205–2933
3178–2918
3186–2913
3191–2939
3196–2950
3190–2940
3188–2911
3201–2933
3174–2919
3181–2909
3199–2919
1638–1620
1610, 1584
1612, 1588
1620, 1591
1620, 1588
1618, 1601
1622, 1601
1628, 1610
1618, 1602
1609, 1608
1622, 1599
1613, 1584
1617, 1602
1611, 1602
1511–1400
1521–1411
1505–1431
1505–1430
1508–1428
1515–1429
1511–1401
1504–1424
1511–1438
1521–1411
1518–1421
1515–1409
1511–1434
1505–1419
–
–
–
–
–
1680
1640
1638
1638
1640
1638
1638
–
–
–
–
–
1176
1171
1176
1170
1170
1168
1180
1176
1188
1178
1178
1187
1188
1175
2
+
+
+
)
)
)
2
2
2
+
)
2
+
)
)
–
–
2
+
1272
1231
1241
1238
1232
1240
1236
2
+
)
)
)
2
+
+
1
1
1
1
1
0
1
2
3
4
2
4c (ligand+Pd
4d (ligand+Co
4e (ligand+Fe
4f (ligand+Zn
2
+
)
2
+
)
)
–
–
2
+
Scheme 1. Synthesis of target molecule 3a-f and 4a-f; reagents and conditions: (i) Pyridine-2,6-
dicarbaldehyde, glacial acetic acid (3–4 drops), absolute methanol, reflux 8 h; (ii) chloride salts of
transition metals, ligands (3 and 4), methanol, reflux 2–3 h.
2
.3. Optical Analysis
Ligand synthesis and their ligation with several metals were determined by recording their
photophysical parameters via absorption spectral analysis. Ligand
3 showed two absorption bands at
2
40 and 304 nm while ligand exhibited the maximum absorption at 247 and 292 nm, respectively.
4
Several heteroatoms in the ligands’ red-shifts the absorption wavelength while the pi-electronic
transition blue-shifts the wavelength due to a typical longer energy gap between the ground and
excited state for labile π-electrons systems. Complexation leads to almost negligible variation in the

Molecules 2019, 24, 312
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absorption maxima position due to
π
-electrons systems. However, ligation caused a red-shift due to
non-bonding electrons, which indicates the affiliation of lone pair electrons of heteroatoms toward
metals (Table 2).
Table 2. Physical characteristics and optical analysis results for ligands
3 and 4 before and
after complexation.
◦
(nm) a
abs
ε 10 (M cm ) b
5
−1
−1
S. No.
Compounds
Appearance
m.p.
C
λ
1
2
3
4
5
6
7
8
9
ligand (3)
3a (ligand+Cu
yellow powder
dark yellow
purple
barn red
light green
chocolate
sage
light yellow
dark yellow
purple
211–213
232–240
241–243
240–242
238–240
243–245
230–232
183–185
236–238
235–237
244–246
233–235
239–241
229–231
240, 304
240, 361
240, 350
241, 331
246, 333
240, 340
240, 324
247, 292
247, 321
247, 311
247, 333
247, 338
247, 302
247, 333
6.4, 3.8
6.4, 3.41
2
+
+
+
)
)
)
2
3b (ligand+Ni
6.31, 3.84
6.54, 3.58
6.11, 3.61
6.76, 3.58
6.39, 3.38
5.04, 2.7
5.11, 2.78
5.21, 2.38
5.21, 2.81
5.08, 2.11
5.18, 2.02
5.18, 3.34
3c (ligand+Pd2
2
+
3d (ligand+Co
3e (ligand+Fe2
)
+
)
)
3f (ligand+Zn2+
ligand (4)
2
+
4a (ligand+Cu
)
)
)
2+
10
11
12
13
14
4b (ligand+Ni
4c (ligand+Pd2
+
chili
2
+
4d (ligand+Co
4e (ligand+Fe2
)
dark green
chocolate
sage
+
)
)
4f (ligand+Zn2+
a
UV-visible absorption maxima; b Molar absorption coefficient.
2
.4. Ligand Safety Profiles
The ligand safety was assessed after 4 and 24 h treatment to the cells by MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The results showed no toxicity
for L-929 cells at 50 M ligand concentrations. The safe profile of the ligand towards the living cells
[
µ
might open the ligands’ possibility as bioimaging probes. The safe nature of the triazole skeleton
warrants further characterization of the compound.
2
.5. Bio-Evaluation
Urease Inhibition Activity
We tested our synthesized compounds, 3a-f and 4a-f, for their inhibitory effects on urease. Besides
current urease inhibitors [16], we aimed to investigate the effect of the two triazole ring structure
mutually coupled with each other toward urease inhibition as well as to understand the effect of metal
complexation toward the bio-profile of the designed nucleus. The in-vitro screening results showed that
the combination of the two triazole skeleton in the cage like morphology exhibited comparable urease
inhibition potential to that of the reference. The metallic chelation with copper, nickel, and palladium
showed inhibition with IC50 values of 0.94
±
0.13, 3.71
±
0.61, and 7.64 ± 1.21
(
3a
–
c
) and 1.20
±
0.52,
3
.93 0.45, and 12.87 2.11 M (4a ). Copper chelation produced a maximum inhibitory profile
±
±
µ
–c
possibly due to interactions with the tested protein. The overall bioprofile of oxygen containing the
triazole cage and their metal complexes exhibited higher inhibition than the sulfur containing triazole
cage and their corresponding metal complexes. In general, iron and zinc compounds displayed the
least inhibition, while the cobalt complex fell in the middle (Table 3).

Molecules 2019, 24, 312
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Table 3. The inhibitory effect of synthesized compounds toward urease.
Compounds Code
Urease Inhibition IC50 (µM)
3
18.92 ± 1.81
0.94 ± 0.13
3.71 ± 0.61
7.64 ± 1.21
28.93 ± 3.11
37.46 ± 4.23
45.78 ± 5.24
29.33 ± 3.32
1.20 ± 0.52
3.93 ± 0.45
12.87 ± 2.11
23.72 ± 3.33
58.83 ± 7.31
39.89 ± 6.93
20.7 ± 0.45
3
a
3
b
3
c
3
d
3
e
3
f
4
4
a
4
b
4
c
4
d
4
e
4
f
Thio-Urea
2
.6. Mechanism Underlying Inhibitory Effect of Compounds 3a, 4a, 3b, and 4b
Next, we picked compound 3a 4a 3b, and 4b for further tests against the urease enzyme to
investigate the underlying mechanism. We plotted 1/V versus 1/[S] in the presence of different
concentrations of inhibitors, 3a 4a 3b, and 4b, using the Lineweaver-Burke plot to study the
resulting enzyme kinetics (Figures 1, 2, 3 and 4a,b) show the inhibition constants (Ki) calculated
from the Lineweaver-Burke plots. The results showed that compounds 3a 4a, and 4b behaved as a
non-competitive inhibitor (Figures 1, 2 and 4a), which means that 1/Vmax increased while K remained
constant under increasing concentrations of compounds 3a 4a, and 4b, respectively. This behavior
indicated that compounds 3a 4a, and 4b inhibit urease non-competitively to form enzyme inhibitor
EI) complex [16]. Specifically, data from compound 3b, with an increasing the concentration of the
substrate (urea), all intersected within the second quadrant. This result showed that Vmax decreased
with increasing K with increasing concentrations of 3b. This behavior indicated that compound 3b is
a mixed type inhibitor with respect to the substrate, urea, with a Ki value of 1.2 M and a Ki value
of 3.0 M as shown in Figure 3b,c. The results of the kinetic constants and inhibition constants are
summarized in Table 4. The kinetic data is graphically explained in Figures 1–4.
,
,
,
,
,
m
,
,
(
m
µ
'
µ
Figure 1. Kinetic analysis results for target molecule 3a. (
urease in the presence of compound 3a; concentrations of 3a of 0, 0.25, 0.5, 1, and 2
Substrate urea concentrations were 1.57, 3.12, 6.25, 12.5, 25, and 50 M, used respectively; (
secondary replot of the Lineweaver-Burk plot, slope vs. various concentrations of 3a.
a
) Lineweaver-Burk plots for the inhibition of
M, respectively.
) The
µ
µ
b

Molecules 2019, 24, 312
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Figure 2. Kinetic analysis results for target molecule 4a. (
of urease in the presence of compound 4a. Concentrations of 4a of 0, 0.75, 1.5, 3, and 6
respectively. Substrate urea concentrations were 1.57, 3.12, 6.25, 12.5, 25, and 50 M, used respectively;
b) The secondary replot of the Lineweaver-Burk plot, slope vs. various concentrations of 4a.
a) Lineweaver-Burk plots for the inhibition
µM,
µ
(
Figure 3. Kinetic analysis results for target molecule 3b. (a) Double reciprocal Lineweaver-Burk plots
for the inhibition of Jack bean urease in the presence of compound 3b. Concentrations of 3b were
, 0.25, 0.5, 1, and 2 M, respectively. Substrate urea concentrations were 1.57, 3.12, 6.25, 12.5, 25,
and 50 M, used respectively; ( ) The secondary replot of the Lineweaver-Burk plot, slope vs. various
) The secondary replot of the Lineweaver-Burk plot, Intercept vs. various
0
µ
µ
b
concentrations of 3b; (
c
concentrations 3b.
Figure 4. Kinetic analysis results for target molecule 4b. (
urease in the presence of compound 4b; concentrations of 4b were used as 0, 1, 2, 4, and 6
respectively. Substrate (urea) concentrations, 1.57, 3.12, 6.25, 12.5, 25, and 50 M, were used,
) The secondary replot of the Lineweaver-Burk plot, slope vs. various concentrations
a) Lineweaver-Burk plots for the inhibition of
µM,
µ
respectively; (
b
of 4b.

Molecules 2019, 24, 312
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Table 4. Kinetic analysis of compounds, 3a, 4a, 3b, and 4b.
1
/Vmax
0
Compounds
Dose (µM)
Km (µM)
Inhibition Type
Ki (µM)
Ki (µM)
(
∆A/Sec)
0
.0
.25
0.50
12
2.5
2.5
0
20
Non-competitive
0.6
—
—
3
4
a
a
30
2.5
1
2
0
.0
.0
.0
35
2.5
44
2.5
8
0.47
0.47
0.47
0.47
0.47
7.14
10
0
.75
12
Non-competitive
2.1
1.2
2.4
1.5
18
3
6
0
.0
.0
.0
22
35
50
0.25
0.5
60
3
4
b
b
65
12.5
14.2
16.6
0.41
0.41
0.41
0.41
0.41
Mixed-inhibition
Non-competitive
3.0
—
1
2
0
1
.0
.0
.0
.0
70
80
55
65
2.0
100
130
180
4
6
.0
.0
0
V
max is the reaction velocity; K is the Michaelis-Menten constant; Ki is the EI dissociation constant; Ki is the ESI
m
dissociation constant; —: not determined
2
.7. Structural Assessment of Jack Bean Urease
The metal-containing jack bean urease contains four unique structural domains (Figure 5) [17].
Two nickel atoms coordinate key structural interactions in domain four. Structural data revealed that
copper atoms can directly interact with His545, His519, His409, His407, and Asp633 within the active
binding pocket of jack bean urease. The VADAR analysis showed that the protein contains 27% helices,
31% β sheets, and 41% coils, while the Ramachandran plot indicated that 97.5% of residues fall in
favored regions. The Ramachandran graph is mentioned in the supplementary data.
Figure 5. Crystal structure of jack bean urease.

Molecules 2019, 24, 312
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2
.8. Docking Shows Binding Energy and Conformation
Based on in vitro results, we chose 3a for binding conformation inside the active site of jack bean
urease. Docking and fitting (3a) calculated a binding energy value of 10.40 kcal/mol. The 3a-docked
complex showed that compound 3a was enclosed in the active site of the jack bean urease. Compound
formed four active hydrogen bonds with the protein active site. The carbonyl oxygen atom on the
−
3
a
triazole ring was H-bonds with Arg439 residue with bond lengths of 2.20 and 2.46 Å, respectively.
Similarly, the triazole N2 hydrogen likely interacted with Ala636 through hydrogen bonding, having a
bond length of 2.19 Å. Moreover, the carbonyl oxygen formed another hydrogen bond with Arg609
with a bond length of 2.19 Å (Figure 6). The detailed interactive behavior of 3a and urease showed that
in Arg609 bonding, the oxygen atom of 3a acts as an acceptor whereas the hydrogen atom of Arg609
behave as a donor atom. Similarly, the oxygen and nitrogen atoms act as acceptors and donor atoms
in Ala636 bonding, respectively. The significant binding was observed with Arg439 at two different
positions against 3a, the oxygen atom of compound 3a behaves as an acceptor while the hydrogen
atoms of Arg439 behaves as donor atoms in both bonding. The literature also shows similar results
with other urease inhibitors, which corroborates our docking results [18–20]. These combined results
indicate that the 3a compound may be a potent inhibitor of jack bean urease. Docking results of 3a
complexed with jack bean urease are exhibited in Figure 6.
Figure 6. Docking results of 3a complex with jack bean urease. (
the yellow surface, whereas the binding pocket is highlighted in maroon. (
docking complex with the 3a ligand. Two nickel atoms are labeled in brown. (
protein structure with the interactive ligand. ( ) The interactive residues are highlighted in the dark
A
) the protein molecule is rendered as
) The closer view of the
) The ribbon format
B
C
D
brown color and the ligand (3a) is justified in grey color with different moieties’ colors. The purple
lines represent hydrogen bonding and the distance mentioned in angstrom (Å).
3
. Materials and Methods
3
.1. Substrate and Reagents
The 4-methoxybenzoic acid, pyridine-2,6-dicarbaldehyde, phosphorous oxychloride,
carbohydrazide, isocyanate and isothiocyanates, Hydrazine hydrate (80%), TEA, CS , KOH,
2
sodium hydrogen carbonate, and glacial acetic acid were purchased from Sigma-Aldrich, Darmstadt,

Molecules 2019, 24, 312
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Germany. The chloride salts of the transition metals were obtained from Aldrich and Alfa Aesar.
Ethanol, methanol, chloroform, deionized water, acetonitrile, dimethyl sulfoxide, petroleum ether,
ethyl acetate, n-hexane, toluene (Samchun Chemicals, Seoul, Korea), H SO , acetic acid, and HCl
2
4
(
Jin Chemical and Pharmaceutical Co. Ltd., Seoul, Korea) were used in this experiment. Urease from
jack bean (EC 3.5.1.5), Thio-urea, sodium nitroprusside, and active chloride were purchased from
Sigma (St. Louis, MO, USA). Stock solutions of the reducing substrates were prepared in phosphate
buffer (20 mM, pH 6.8).
3
.2. Instrumentations
The reaction progress was monitored by thin layer chromatography (TLC), and the R values
f
were determined with pre-coated silica gel aluminum plates, Kieselgel 60 F254 from Merck (Darmstadt,
Germany). TLC plates were visualized under a UV lamp (VL–4 LC, Collégien, France). The melting
points were determined on a Fisher Scientific (Waltham, MA, USA) melting point apparatus. The FT-IR
spectra were recorded in KBr pellets on a Shimadzu FTIR–8400S spectrometer (Kyoto, Japan). Proton
1
13
and carbon nuclear magnetic resonance ( H-NMR and C-NMR) spectra were recorded on a Bruker
Avance 400 MHz spectrometer with TMS as an internal standard. The chemical shifts are reported
as
δ values (ppm) downfield from the internal tetramethylsilane of the indicated organic solution.
Peak multiplicities are expressed as follows: S, singlet; d, doublet; and m, multiplet. Mass spectra
were recorded on the AB SCIEX Co. 4000 QTRAP LC/MS/MS System. The UV-visible absorption
measurements were carried out using [SCINCO] UV-Vis Spectrophotometer “S-3100” (SCINCO, Seoul,
Korea). Abbreviations are as follows: CD OD, deuterated methanol; DMSO–d , dimethyl sulfoxide-d ,
3
6
6
and FT-IR spectroscopy, Fourier transform infrared spectroscopy.
3
.3. Synthesis of 4-amino-3-(4-methoxyphenyl)-1H-1,2,4-triazol-5(4H)-one (1 and 2)
The 4-methoxybenzoyl chloride was synthesized by reacting 4-methoxybenzoic acid (1 mmol)
in the presence of 1,2–dichloroethane (12 mL) solvent and phosphorous oxychloride (0.4 mL) as
the chlorinating agent under reflux for 3 h. Then, the resulting solution was cooled to room
temperature, and the solvent was removed under reduced pressure. The 4-methoxybenzoyl chloride
and carbohydrazide were separately dissolved in the dichloromethane and mixed together slowly
at low temperature with continuous stirring followed by reflux for 3 h. The reaction progress was
continuously monitored after every 30 min by using aluminium pre-coated silica gel TLC plates.
The product was extracted by using dichloromethane and anhydrous magnesium sulfate and purified
by column chromatography employing a dichloromethane:hexane solvent system. Compound
prepared according to our previously reported procedure [21].
2 was
3
.4. Synthesis of Schiff Base Derivative 3, 4 and Their Metal Complexes
The Schiff base derivative and were synthesized following our previously reported
methods [21]. The metal complexes 3,4a-f were synthesized by refluxing an equimolar quantity
of the respective metal salts with the Schiff base ligands and , utilizing ethanol as the solvent.
3
4
3
4
The formation of the transition metal complexes was initially indicated by the TLC analysis as well as
by visual detection due to a colorimetric change in the reaction mixture. Further characterization was
performed by the FT-IR, UV-visible, and fluorescence spectroscopic analysis. Anal. Calcd. for target:
3,
C H N O : C, 58.70; H, 4.14; N, 24.65; found: C, 58.66; H, 4.11; N, 24.21. for target: , C H N O S :
4
25
21
9
4
25 21
9
2 2
C, 55.23; H, 3.89; N, 23.19, found: C, 55.18; H, 3.85; N, 23.16.
3
.5. General Procedure for Spectroscopic Measurement
The ligands ( and ) and their metal complexes (3-f and 4a-f) stock solution (1 mM) were prepared
by dissolving 5.11 and 5.43 mg of desired compounds in THF (total volume 10 mL). For spectroscopic
measurements, the test solution of 3 mL was prepared with 2.34 mL of THF, 90 L of ligand stock
solution, 0.3 mL of buffer solution (EtOH/PBS buffer saline, 100 mM), and 90 L of ionic stock
3
4
µ
µ

Molecules 2019, 24, 312
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solution. The resulting solutions were mixed before measurement and the final volume was fixed as
◦
3
mL for UV-visible and fluorescent studies. All recognition studies were performed at 25 C while
the samples were shaken to ensure solution uniformity prior to spectrum recording. The limit of
detection and association constants for the probe on metal binding were found by the absorption
titration experiments while the average Stern-Volmer quenching constants for the probe towards
cobalt, mercury, and copper were calculated by fitting the emission titration results to the Stern-Volmer
plot [22–24].
3
.6. General Procedure for MTT Assay
MTT assay was done following the reported procedure [25]. Briefly, the cells were incubated with
ligand and their metal complexes (
3 and 4; 3a-f and 4a-f) (60 µM) for 24 h. Then, cells were washed with
phosphate buffered saline (PBS), and incubated with Dulbecco’s Modified Eagle’s medium (DMEM
medium, 200
µ
L/well) containing 50
µ
L of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
◦
bromide, 5 mg/mL] solution. Following 2 h of incubation at 37 C, growth medium was removed
gently from the plate and 200 µL/well dimethyl sulfoxide was added to solubilize the produced purple
formazan crystals. Later, the absorbance for each well was measured at 570 nm using microplate
spectrophotometer systems (BioTek, synergy HT) and results were calculated in percentage with
respect to the untreated sample called the control. The same procedure was adopted for all the
compounds and their metal complexes.
3
.7. General Procedure for Urease Inhibition Assay
The urease activity was determined by measuring the amount of ammonia produced with the
indophenols method previously described [26,27]. The reaction mixtures, comprising 20 µL of enzyme
(
jack bean urease, 5 U/mL) and 20 L of test compounds in 50
µ
µ
L buffer (100 mM urea, 0.01 M
◦
K HPO , 1 mM EDTA, and 0.01 M LiCl , pH 8.2), were incubated for 30 min at 37 C in a 96-well plate.
2
4
2
Briefly, 50
µL each of phenol reagents (1%, w/v phenol and 0.005%, w/v sodium nitroprusside) and
50 µL of alkali reagent (0.5%, w/v NaOH and 0.1% active chloride NaOCl) were added to each well.
The absorbance at 625 nm was measured after 10 min, using a microplate reader (OPTI Max, Tunable).
All reactions were performed in triplicate. The urease inhibition activities were calculated according to
the following formula:
Urease inhibition activity (%) = (ODcontrol − ODsample × 100)/ODcontrol
where ODcontrol and ODsample represents the optical densities in the absence and presence of the
sample, respectively. Thiourea was used as the standard inhibitor for urease.
3
.8. Computational Methodology
Retrieval of Jack Bean Urease
The three-dimensional protein structure of jack bean urease (C. ensiformis) was accessed from
the Protein Data Bank (PDB) (www.rcsb.org) with PDBID 4H9M. The selected protein structure was
minimized by employing UCSF Chimera 1.10.1 [28]. Molprobity server [29] was used to predict the
Ramachandran plot and values of the target structure [30]. The protein architecture and statistical
percentage values of receptor proteins, helices, beta-sheets, coils, and turns were predicted from the
online server, VADAR 1.8 [31].
3
.9. Molecular Docking
Based on the in vitro results, 3a was selected for the docking experiment to check the binding
against the target protein. Before the docking experiment, the synthesized ligand (3a) was sketched in
the ACD/ChemSketch tool and accessed in the mol format. Furthermore, the UCSF Chimera 1.10.1

Molecules 2019, 24, 312
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tool was employed for energy minimization of 3a having default parameters. The steepest descent
steps were adjusted to 100 with a step size of 0.02 (Å), conjugate gradient steps also fixed to 100 with a
step size of 0.02 (Å), and the update interval was retrained at 10. Finally, Gasteiger charges were added
using Dock Prep in the ligand structure to obtain the good structure conformation. The molecular
docking experiment was run through the PyRx virtual screening tool with the AutoDock VINA Wizard
approach [32,33]. The grid box center values (X = 10.22, Y = 24.56 and Z = 46.18) were adjusted with
default exhaustiveness value = 8 to maximize the binding conformational analysis. The generated
docked complexes were evaluated based on the lowest binding energy (Kcal/mol) values and structure
activity relationship (SAR) analyses. The three dimensional (3D) graphical depictions of the 3a-docked
complex were accomplished by Chimera 1.10.1.
4
. Conclusions
In this study, we joined two triazole rings to form a series of Schiff base derivatives into a
cage-like structure. Then, we evaluated their urease inhibition activity as well as their underlying
mechanism for inhibition with enzyme kinetics studies. Among the tested series, copper, nickel,
and palladium complexes displayed appreciable enzyme inhibition results, while the rest showed
low to moderate enzyme inhibition potential. The kinetic analysis by the Lineweaver–Burk plot
suggested the non-competitive inhibition nature of the compounds, 3a
inhibition by the compound, 3b. Further, molecular docking studies showed that 3a interacted within
the active region of jack bean urease and exhibited a good binding energy value of 10.40 kcal/mol.
, 4a, and 4b, while mixed type
−
These combined results showed that synthesized molecules can serve as structural templates in the
design of novel drugs against the urease enzyme.
Supplementary Materials: Supplementary Materials are available online.
Author Contributions: Conceptualization, T.C. and M.S.; Data curation, M.M. and M.S.; Formal analysis, M.M.
and Y.Z.; Funding acquisition, C.L.; Investigation, M.R. and Mubashir Hassan; Methodology, Muhammad Hanif;
Project administration, Mubashir Hassan, Y.Z. and T.C.; Resources, C.L.; Software, F.K., C.L. and M.S.; Supervision,
S.-Y.S. and C.L.; Validation, S.-Y.S. and T.C.; Visualization, M.R.; Writing—original draft, Muhammad Hanif;
Writing—review & editing, F.K.
Funding: Research funding and APC was provided by the Natural Science Foundation of China (31300603),
Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning
(
No.2012-28), Fundamental Research Funds for the Central Universities (15D110508, 15D110527, 15D110527,
1
3D110522, 17D210502, 17D310512, 18D210501, 18D310507, 2232014D3-39), the National College Student
Innovation Experiment Program (14T10501), General Financial Grant from the China Postdoctoral Science
Foundation (2015M571455). Work in the Dr. Muhammad Saleem lab was sponsored by the University of
Sargodha, Sub-campus Bhakkar, Pakistan.
Acknowledgments: We thank Dr. Chen, Peiran, and Dr. Ishtiaq Qadri for helpful discussions.
Conflicts of Interest: Authors declare no conflict of interest.
References
1.
2.
3.
4.
Habala, L.; Varényi, S.; Bilková, A.; Herich, P.; Valentová, J.; Kožíšek, J.; Devínsky, F. Antimicrobial Activity
and Urease Inhibition of Schiff Bases Derived from Isoniazid and Fluorinated Benzaldehydes and of Their
Copper(II) Complexes. Molecules 2016, 21, 1742. [CrossRef] [PubMed]
Hassan, S.T.S.; Švajdlenka, E.; Berchová-Bímová, K. Hibiscus sabdariffa L. and Its Bioactive Constituents
Exhibit Antiviral Activity against HSV-2 and Anti-Enzymatic Properties against Urease by an ESI-MS Based
Assay. Molecules 2017, 22, 722. [CrossRef] [PubMed]
Hassan, S.T.S.; Švajdlenka, E. Biological Evaluation and Molecular Docking of Protocatechuic Acid from
Hibiscus sabdariffa L. as a Potent Urease Inhibitor by an ESI-MS Based Method. Molecules 2017, 22, 1696.
[CrossRef] [PubMed]
Xu, Y.P.; Chen, Y.H.; Chen, Z.J.; Qin, J.; Qian, S.S.; Zhu, H.L. Synthesis, Crystal Structures, Molecular Docking,
and urease Inhibitory Activities of Transition-Metal Complexes with a 1,2,4-Triazolecarboxylic Acid Derived
Ligand. Eur. J. Inorg. Chem. 2015, 2015, 2076–2084. [CrossRef]

Molecules 2019, 24, 312
12 of 13
5
.
.
You, Z.; Liu, M.; Wang, C.; Sheng, G.; Zhao, X.; Qua, D.; Niu, F. Inhibition studies of Helicobacter pylori
urease with Schiff base copper(II) complexes. RSC Adv. 2016, 6, 16679–16690. [CrossRef]
You, Z.; Yu, H.; Zheng, B.; Zhang, C.; Lv, C.; Li, K.; Pan, L. Syntheses, structures, and inhibition studies of
Jack bean urease by copper(II) complexes derived from a tridentate hydrazone ligand. Inorg. Chim. Acta
6
2
018, 469, 44–50. [CrossRef]
7
8
.
.
You, Z.L.; Ni, L.L.; Shi, D.H.; Bai, S. Synthesis, structures, and urease inhibitory activities of three
copper(II) and zinc(II) complexes with 2-{[2-(2-hydroxyethylamino)ethylimino]methyl}-4-nitrophenol. Eur. J.
Med. Chem. 2010, 45, 3196–3199. [CrossRef] [PubMed]
Ikram, M.; Rehman, S.; Faridoon; Baker, R.J.; Rehman, H.U.; Khan, A.; Choudhary, M.I.; Rehman, S.U.
Synthesis and distinct urease enzyme inhibitory activities of metal complexes of Schiff-base ligands: Kinetic
and thermodynamic parameters evaluation from TG-DTA analysis. Thermochim. Acta 2013, 555, 72–80.
[CrossRef]
9
1
1
.
Du, N.; Chen, M.; Liu, Z.; Sheng, L.; Xu, H.; Chen, S. Kinetics and mechanism of jack bean urease inhibition
by Hg2+. Chem. Cent. J. 2012, 6, 154–161. [CrossRef] [PubMed]
0. Benini, S.; Rypniewski, W.R.; Wilson, K.S.; Mangani, S.; Ciurli, S. Molecular Details of urease Inhibition by
Boric Acid: Insights into the Catalytic Mechanism. J. Am. Chem. Soc. 2004, 126, 3714–3715. [CrossRef]
1. Habal, L.; Roller, A.; Matušk, M.; Valentová, J.; Rompel, A.; Devínsky, F. Complexes of N-hydroxyethyl-N-
benzimidazolylmethylethylenediaminediacetic acid with copper(II) and cobalt(II): Preparation, crystal
structure and urease inhibitory activity. Inorg. Chim. Acta 2014, 421, 423–426. [CrossRef]
1
2. Dong, X.; Li, Y.; Li, Z.; Cui, Y.; Zhu, H. Synthesis, structures and urease inhibition studies of copper(II)
and nickel(II) complexes with bidentate N,O-donor Schiff base ligands. J. Inorg. Biochem. 2012, 108, 22–29.
[
CrossRef] [PubMed]
3. Gul, S.; Sultana, N.; Arayne, M.S.; Shamim, S.; Akhtar, M.; Khan, A. Sparfloxacin-Metal Complexes as urease
Inhibitors: Their Synthesis, Characterization, Antimicrobial, and Antienzymatic Evaluation. J. Chem. 2013
–12. [CrossRef]
1
,
1
1
1
1
4. Fátima, A.D.; Pereira, C.P.; Olímpio, C.R.S.D.G.; Oliveira, B.G.F.O.; Franco, L.L.F.; Silva, P.H.C. Schiff bases
and their metal complexes as urease inhibitors—A brief review. J. Adv. Res. 2018. [CrossRef]
5. Cui, Y.; Dong, X.; Li, Y.; Li, Z.; Chen, W. Synthesis, structures and urease inhibition studies of Schiff base
metal complexes derived from 3,5-dibromosalicylaldehyde. Eur. J. Med. Chem. 2012, 58, 323–331. [CrossRef]
6. Ashraf, Z.; Rafiq, M.; Seo, S.Y.; Babar, M.M.; Zaidi, N.S.S. Synthesis, kinetic mechanism and docking studies
of vanillin derivatives as inhibitors of mushroom tyrosinase. Bioorg. Med. Chem. 2015, 23, 5870–5880.
[CrossRef]
1
1
1
2
7. Abbasi, M.A.; Raza, H.; Siddiqui, S.Z.; Shah, S.A.; Hassan, M.; Seo, S.Y. Synthesis of novel
N-(1,3-thiazol-2-yl)benzamide clubbed oxadiazole scaffolds: Urease inhibition, Lipinski rule and molecular
docking analyses. Bioorg Chem. 2018, 83, 63–75. [CrossRef] [PubMed]
8. Saeed, A.; Ur-Rehman, S.; Channar, P.A.; Larik, F.A.; Abbas, Q.; Hassan, M.; Raza, H.; Seo, S.Y. Jack Bean
urease Inhibitors, and Antioxidant Activity Based on Palmitic acid Derived 1-acyl-3- Arylthioureas: Synthesis,
Kinetic Mechanism and Molecular Docking Studies. Drug Res. 2017, 67, 596–605. [CrossRef] [PubMed]
9. Channar, P.A.; Saeed, A.; Albericio, F.; Larik, F.A.; Abbas, Q.; Hassan, M.; Raza, H.; Seo, S.Y. Sulfonamide-
Linked Ciprofloxacin, Sulfadiazine and Amantadine Derivatives as a Novel Class of Inhibitors of Jack Bean
urease; Synthesis, Kinetic Mechanism and Molecular Docking. Molecules 2017, 16, 1352. [CrossRef]
0. Abbasi, M.A.; Hassan, M.; Aziz-Ur-Rehman Siddiqui, S.Z.; Raza, H.; Shah, S.A.A.; Seo, S.Y. Synthesis,
in vitro and in silico studies of novel potent urease inhibitors: N-[4-({5-[(3-Un/substituted-anilino-
3
-oxopropyl)sulfanyl]-1,3,4-oxadiazol-2-yl}methyl)-1,3-thiazol-2-yl]benzamides. Bioorg. Med. Chem. 2018
,
3
0, 3791–3804. [CrossRef]
2
2
2
2
1. Saleem, M.; Khang, C.H.; Kim, M.; Lee, K.H. Chromo/Fluorogenic Detection of Co(2+), Hg(2+) and Cu(2+)
by the Simple Schiff Base Sensor. J. Fluoresc. 2016, 26, 11–22. [CrossRef] [PubMed]
2. Muhammad Saleem, Muhammad Rafiq, Muhammad Hanif, Organic material based fluorescent sensor for
Hg2+: A brief review on recent development. J. Fluoresc. 2017, 27, 31–58. [CrossRef]
3. Saleem, M.; Lee, L.P.; Lee, K.H. Photoluminescent Sensor for Acetylcholinesterase Inhibitor Determination.
J. Mater. Chem. B 2014, 2, 6802–6808. [CrossRef]
4. Saleem, M.; Kang, S.K.; Lee, K.H. Microwave assisted synthesis of a novel optical chemosensor for selective
Fe3+ detection. J. Lumin. 2015, 162, 14–24. [CrossRef]

Molecules 2019, 24, 312
13 of 13
2
2
2
5. Saleem, M.; Abdullah, R.; Ali, A.; Park, B.J.; Choi, E.H.; Hong, I.S.; Lee, K.H. Facile synthesis, cytotoxicity and
bioimaging of Fe3+ selective fluorescent chemosensor. Bioorg. Med. Chem. 2014, 22, 2045–2051. [CrossRef]
6. Weatherburn, M.W. Phenol-Hypochlorite Reaction for Determination of Ammonia. Anal. Chem. 1967, 39,
9
71–974. [CrossRef]
7. Mojzych, M.; Tarasiuk, P.; Mojzych, K.K.; Rafiq, M.; Seo, S.-Y.; Nicewicz, M.; Fornal, E. Synthesis of chiral
pyrazolo[4,3-e][1,2,4]triazine sulfonamides with tyrosinase and urease inhibitory activity. J. Enzyme Inhib.
Med. Chem. 2017, 32, 99–105. [CrossRef]
2
8. Pettersen, E.F.; Goddard, T.D.; Huang, C.C.; Couch, G.S.; Greenblatt, D.M.; Meng, E.C.; Ferrin, T.E. UCSF
Chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605–1612.
[CrossRef] [PubMed]
2
9. Chen, V.B.; Arendall, W.B.; 3rd Headd, J.J.; Keedy, D.A.; Immormino, R.M. MolProbity: All-atom structure
validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 2010, 66, 12–21.
[CrossRef]
3
3
3
3
0. Lovell, S.C.; Davis, I.W.; Arendall, W.B., III; de Bakker, P.I.; Word, J.M. Structure validation by Calpha
geometry: Phi, psi and Cbeta deviation. Proteins 2003, 50, 437–450. [CrossRef]
1. Willard, L.; Ranjan, A.; Zhang, H.; Monzavi, H.; Boyko, R.F. VADAR: A web server for quantitative evaluation
of protein structure quality. Nucleic Acids Res. 2003, 31, 3316–3319. [CrossRef] [PubMed]
2. Dallakyan, S.; Olson, A.J. Small-molecule library screening by docking with PyRx. Methods Mol. Biol. 2015
,
1
263, 243–250. [PubMed]
3. Rafiq, M.; Saleem, M.; Jabeen, F.; Hanif, M.; Seo, Su.; Kang, S.K.; Lee, K.H. Facile synthesis, biological
evaluation and molecular docking studies of novel substituted azole derivatives. J. Mol. Struct. 2017, 1138,
1
77–191. [CrossRef]
Sample Availability: Samples of the compounds are available from the authors.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).