Syntheses, structures, and inhibition studies of Jack bean urease by copper(II) complexes derived from a tridentate hydrazone ligand

Article abstract of DOI:10.1016/j.ica.2017.09.011

Urease inhibitors can counteract the negative effects of urease. In this paper, three new copper(II) complexes derived from 3-methyl-N′-(pyridin-2-ylmethylene)benzohydrazide (HL) were prepared. They are [CuBr(CH₃OH)L] (1), [Cu(CH₃OH)L(NCS)] (2), and [CuL(HL)]·ClO₄ (3). The complexes were characterized by infrared and UV–Vis spectra, and single crystal X-ray determination. The Cu atoms in complexes 1 and 2 display square pyramidal coordination, and in complex 3 the Cu atom displays octahedral coordination. Complex 1 shows effective urease inhibitory activity, with IC₅₀ value of 1.46 ± 0.83 μM. Molecular docking study of the complexes with Jack bean urease was performed.

Full text of DOI:10.1016/j.ica.2017.09.011

Accepted Manuscript
Research paper
Syntheses, structures, and inhibition studies of Jack bean urease by copper(II)
complexes derived from a tridentate hydrazone ligand
Zhonglu You, Huiyuan Yu, Boyang Zheng, Chenglu Zhang, Chengwei Lv, Kun
Li, Lin Pan
PII:
S0020-1693(17)31232-X
http://dx.doi.org/10.1016/j.ica.2017.09.011
ICA 17867
DOI:
Reference:
To appear in:
Inorganica Chimica Acta
Received Date:
Revised Date:
Accepted Date:
7 August 2017
5 September 2017
5 September 2017
Please cite this article as: Z. You, H. Yu, B. Zheng, C. Zhang, C. Lv, K. Li, L. Pan, Syntheses, structures, and
inhibition studies of Jack bean urease by copper(II) complexes derived from a tridentate hydrazone ligand,
Inorganica Chimica Acta (2017), doi: http://dx.doi.org/10.1016/j.ica.2017.09.011
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Syntheses, structures, and inhibition studies of Jack bean urease by copper(II)
complexes derived from a tridentate hydrazone ligand
∗
Zhonglu You , Huiyuan Yu, Boyang Zheng, Chenglu Zhang, Chengwei Lv, Kun Li,
Lin Pan
Department of Chemistry and Chemical Engineering, Liaoning Normal University,
Dalian 116029, P. R. China
ABSTRACT
Urease inhibitors can counteract the negative effects of urease. In this paper, three
new
-methyl-N’-(pyridin-2-ylmethylene)benzohydrazide (HL) were prepared. They are
CuBr(CH OH)L] (1), [Cu(CH OH)L(NCS)] (2), and [CuL(HL)]·ClO (3). The
copper(II)
complexes
derived
from
3
[
3
3
4
complexes were characterized by infrared and UV-Vis spectra, and single crystal
X-ray determination. The Cu atoms in complexes 1 and 2 display square pyramidal
coordination, and in complex 3 the Cu atom displays octahedral coordination.
Complex 1 shows effective urease inhibitory activity, with IC50 value of 1.46 ± 0.83
µM. Molecular docking study of the complexes with Jack bean urease was performed.
Keywords: Hydrazone; Copper complex; Crystal structure; Urease inhibition;
Molecular docking
1. Introduction
∗
Corresponding author. Zhonglu You, Department of Chemistry and Chemical Engineering, Liaoning Normal
University, Dalian 116029, P. R. China. Tel.: +8641182156989. Fax.: +8641182159660. E-mail address:
youzhonglu@126.com
-
1 -

Urease (E.C.3.5.1.5) plays an important role in plant nitrogen metabolism [1-3].
However, excessive levels of urease in soil can degrade the fertilizer urea quickly, and
result in phytopathic effects and loss of ammonia [4]. Moreover, urease is regarded as
a virulent factor in human and animal infections of the urinary and gastrointestinal
tracts [5]. Studies indicated that control of the activity of urease through the use of
inhibitors could counteract the negative effects [6-14].
Metal complexes are well established enzyme inhibitors [15]. In recent years, we
have reported a number of metal complexes with urease inhibitory activities [16-20].
Among the compounds, copper(II) complexes have the most effective urease
inhibitory activities. Li and coworkers have reported some Schiff base copper(II)
complexes with potent urease inhibitory activities [21,22]. However, the relationship
between structures and urease inhibitory activities is not clear. Hydrazone is a kind of
special Schiff base, which possess the typical functional group –CH=N–NH–C(O)–. It
has been shown that hydrazones have interesting biological activities, including
urease inhibitory activity [23]. In this paper, three new copper(II) complexes,
[
CuBr(CH
3
OH)L] (1), [Cu(CH
3
OH)L(NCS)] (2), and [CuL(HL)]·ClO (3), where L is
4
the monoanionic form of 3-methyl-N’-(pyridin-2-ylmethylene)benzohydrazide (HL),
were synthesized and structurally characterized. The urease inhibitory activity of the
complexes was investigated both from experimental and molecular docking analysis.
HL
2. Experimental section
-
2 -

2.1. Materials and measurements
Starting materials, reagents and solvents were purchased from commercial
suppliers with AR grade, and used without purification. Elemental analyses were
performed on a Perkin-Elmer 240C elemental analyzer. IR spectra were recorded on a
–1
Jasco FT/IR-4000 spectrometer as KBr pellets in the 4000–400 cm region. UV-Vis
spectra were recorded on a Perkin-Elmer Lambda 900 spectrometer. The urease
inhibitory activity was measured on a Bio-Tek Synergy HT microplate reader. Single
crystal structures were determined by Bruker D8 Venture single crystal diffraction.
2.2. Synthesis of HL
Pyridine-2-carboxaldehyde (1.0 mmol, 0.107 g) and 3-methylbenzohydrazide (1.0
mmol, 0.150 g) were dissolved in methanol (30 mL). The mixture was stirred at room
temperature for 1 h to give a clear solution. The solvent was evaporated to give
colorless solid product, which was re-crystallized from methanol. Yield: 212 mg
-1
(
89%). Characteristic IR data (KBr, cm ): 3453 (OH), 1617 (CH=N). Anal. Calcd for
1
C
14
H
13
N
3
O: C, 70.3; H, 5.5; N, 17.6. Found: C, 70.1; H, 5.5; N, 17.5%. H NMR
6
(
(
d -DMSO): δ: 2.33 (s, 3H, CH
s, 1H, NH).
3
), 7.35–7.90 (m, 8H, ArH), 8.71 (d, 1H, ArH), 11.82
2
.3. Synthesis of the complexes
CuBr(CH OH)L] (1)
The hydrazone HL (1.0 mmol, 0.239 g) was dissolved in methanol (15 mL), to
which was added dropwise CuBr (1.0 mmol, 0.223 g) dissolved in methanol (10 mL).
[
3
2
The mixture was magnetic stirred for 10 min at room temperature, and filtered. The
filtrate was kept in air for a few days, to form crystals suitable for single crystal X-ray
diffraction. The crystals were isolated, washed three times with cold methanol and
-1
dried in air. Yield: 175 mg (42%). Characteristic IR data (KBr, cm ): 3441 (OH),
-
3 -

–
1
–1
1
603 (CH=N). UV–Vis data [methanol, λ/nm (ε/L·mol ·cm )]: 272 (5,850), 380
(
7,980), 657 (110). Anal. Calcd for C15 : C, 43.5; H, 3.9; N, 10.2. Found:
3 2
H16BrCuN O
–
3
–1
2
–1
M
C, 43.7; H, 4.0; N, 10.0%. Λ (10 M in acetonitrile): 21 Ω ·cm ·mol .
[
Cu(CH
The hydrazone HL (1.0 mmol, 0.239 g) was dissolved in methanol (15 mL), to
which was added dropwise CuBr (1.0 mmol, 0.223 g) and ammonium thiocyanate
1.0 mmol, 0.076 g) dissolved in methanol (15 mL). The mixture was magnetic stirred
3
OH)L(NCS)] (2)
2
(
for 10 min at room temperature, and filtered. The filtrate was kept in air for a few
days, to form crystals suitable for single crystal X-ray diffraction. The crystals were
isolated, washed three times with cold methanol and dried in air. Yield: 203 mg (52%).
-
1
Characteristic IR data (KBr, cm ): 3450 (OH), 2093 (NCS), 1604 (CH=N). UV–Vis
–
1
–1
data [methanol, λ/nm (ε/L·mol ·cm )]: 269 (4,450), 392 (15,270), 646 (125). Anal.
Calcd for C16 S: C, 49.0; H, 4.1; N, 14.3. Found: C, 48.9; H, 4.2; N, 14.3%.
4 2
H16CuN O
–
3
–1
2
–1
Λ
M
(10 M in acetonitrile): 18 Ω ·cm ·mol .
CuL(HL)]·ClO (3)
The hydrazone HL (1.0 mmol, 0.239 g) was dissolved in methanol (15 mL), to
which was added dropwise Cu(ClO ·7H O (1.0 mmol, 0.388 g) dissolved in
[
4
4
)
2
2
methanol (15 mL). The mixture was magnetic stirred for 10 min at room temperature,
and filtered. The filtrate was kept in air for a few days, to form crystals suitable for
single crystal X-ray diffraction. The crystals were isolated, washed three times with
cold methanol and dried in air. Yield: 231 mg (36%). Characteristic IR data (KBr,
-
1
cm ): 3190 (NH), 1609 (CH=N), 1162 (ClO
4
). UV–Vis data [methanol, λ/nm
ε/L·mol ·cm )]: 275 (5,135), 378 (8,230), 670 (143). Anal. Calcd for
: C, 52.5; H, 3.9; N, 13.1. Found: C, 52.3; H, 4.1; N, 13.1%. Λ
–
1
–1
(
C
28
H
25ClCuN
6
O
6
M
–3
–1
2
–1
(
10 M in acetonitrile): 135 Ω ·cm ·mol .
-
4 -

2.4. X-ray crystallography
Diffraction intensities for the complexes were collected at 298(2) K using a Bruker
D8 Venture diffractometer with MoKα radiation (λ = 0.71073 Å). The collected data
were reduced with SAINT [24], and multi-scan absorption correction was performed
using SADABS [25]. Structures of the complexes were solved by direct methods and
2
refined against F by full-matrix least-squares method using SHELXTL [26]. All of
the non-hydrogen atoms were refined anisotropically. The methanol H atoms in 1 and
2, and the amino H atom in 3 were located from difference Fourier maps and refined
isotropically, with O–H and N–H distances restrained to 0.85(1) and 0.90(1) Å,
respectively. The remaining hydrogen atoms were placed in calculated positions and
constrained to ride on their parent atoms. Crystallographic data for the complexes are
summarized in Table 1. Selected bond lengths and angles are given in Table 2.
2.5. Urease inhibitory activity assay
The measurement of urease inhibitory activity was carried out according to the
literature method [27]. The assay mixture containing 75 µL of Jack bean urease and
5 µL of tested compounds with various concentrations (dissolved in DMSO) was
7
pre-incubated for 15 min on a 96-well assay plate. Acetohydroxamic acid was used as
a reference. Then 75 µL of phosphate buffer at pH 6.8 containing phenol red (0.18
mM) and urea (400 mM) were added and incubated at room temperature. The reaction
time required for enough ammonium carbonate to form to raise the pH phosphate
buffer from 6.8 to 7.7 was measured by micro-plate reader (560 nm) with end-point
being determined by the color change of phenol-red indicator.
2.6. Inhibition kinetic study
The maximum velocity (vmax) values were determined by means of
Lineweaver–Burk plots, using initial velocities obtained over substrate concentrations
-
5 -

–
1
of 41.4, 20.7, 10.35, and 5.17 µg·mL , respectively. Inhibitory constant (K
i
) value
was calculated from the Dixon plot. Alternatively, K value was determined from
i
abscissa of the plots of slopes vs. different concentrations of the complex, in which
slope was obtained from the Lineweaver–Burk lines [28].
2.7. Docking study
Molecular docking study of the complexes into the 3D X-ray structure of the Jack
bean urease (entry 4UBP in the Protein Data Bank) was carried out by using the
AutoDock 4.0 software as implemented through the graphical user interface
AutoDockTools (ADT 1.5.2). The graphical user interface AutoDockTools was
employed to setup the enzymes: all hydrogens were added, Gasteiger charges were
calculated and nonpolar hydrogens were merged to carbon atoms. The Ni initial
parameters are set as r = 1.170 Å, q = +2.0, and van der Waals well depth of 0.100
–
1
kcal·mol [29]. The 3D structures of the ligand molecules were saved in pdb format
with the aid of the program ChemBio3D. The resulting files were saved as pdbqt
format.
The AutoDockTools was used to generate the docking input files. The maps were
centered on the original ligand molecule, acetohydroxamic acid, in the catalytic site of
the protein. A grid spacing of 0.375 Å and a distances-dependent function of the
dielectric constant were used for the calculation of the energetic map. 100 runs were
generated by using Lamarckian genetic algorithm searches. Default settings were used
with an initial population of 50 randomly placed individuals, a maximum number of
6
4
2
.5 × 10 energy evaluations, and a maximum number of 2.7 × 10 generations. A
mutation rate of 0.02 and a crossover rate of 0.8 were chosen. The results of the most
favorable free energy of binding were selected as the resultant complex structures.
3. Results and discussion
-
6 -

3.1. Chemistry
The copper complexes were readily prepared by the reaction of equimolar
quantities of the hydrazone ligand, copper salts, and/or secondary ligand (NCS) in
methanol. Single crystals of the complexes were obtained by slow evaporation of the
methanolic solution of the complexes. The hyrazone ligands in complexes 1 and 2
adopt enolate form, while those in complex 3 adopt both enolate and keto forms. The
three complexes are stable in air at room temperature. Molar conductivities of
–
3
–1
complexes 1 and 2 measured in methanol at concentration of 10 M are about 20 Ω
2
–1
cm mol , indicating the non-electrolytic nature of them in such solution [30]. Molar
–
3
conductivity of complex 3 measured in methanol at concentration of 10 M is 135
–
1
2
–1
Ω cm mol , which indicates the 1:1 electrolytic nature [30].
3.2. Structure description of the complexes
Structure description of 1
Molecular structure of complex 1 is shown in Figure 1. The Cu atom in the
complex is in square pyramidal geometry, with the pyridine N, imino N and enolate O
atoms of the hydrazone ligand, and the Br atom located at the basal plane, and with
the methanol O atom located at the apical position. The Cu atom deviates from the
least-squares plane defined by the four basal donor atoms by 0.212(1) Å. The
coordinate bond lengths in the complex are comparable to those observed in copper(II)
complexes with hydrazone ligands [31,32]. The distortion of the square pyramidal
geometry can be observed from the deviation of the coordinate bond angles from the
ideal square pyramid (Table 2), which are caused by the strain created from the
five-membered chelate rings Cu1-N1-C5-C6-N2 and Cu1-N2-N3-C7-O1. The
dihedral angle between the pyridine and the benzene rings of the hydrazone ligand is
3.1(3)°.
-
7 -

In the crystal structure of the complex, molecules are linked by methanol ligands
through intermolecular hydrogen bonds (Table 3), to form dimers (Figure 2).
Structure description of complex 2
Molecular structure of complex 2 is shown in Figure 3. The Cu atom in the
complex is in square pyramidal geometry, with the pyridine N, imino N and enolate O
atoms of the hydrazone ligand, and the N atom of the thiocyanate ligand located at the
basal plane, and with the methanol O atom located at the apical position. The Cu atom
deviates from the least-squares plane defined by the four basal donor atoms by
0
.118(1) Å. The coordinate bond lengths in the complex are similar to complex 1 and
also comparable to those observed in copper(II) complexes with hydrazone ligands
31,32]. The distortion of the square pyramidal geometry can be observed from the
[
deviation of the coordinate bond angles from the ideal square pyramid (Table 2),
which are caused by the strain created from the five-membered chelate rings
Cu1-N1-C5-C6-N2 and Cu1-N2-N3-C7-O1. The dihedral angle between the pyridine
and the benzene rings of the hydrazone ligand is 15.6(4)°.
In the crystal structure of the complex, molecules are linked by methanol ligands
through intermolecular hydrogen bonds (Table 3), to form dimers (Figure 4).
Structure description of complex 3
Molecular structure of complex 3 is shown in Figure 5. The asymmetric unit of the
complex contains a mononuclear copper(II) complex cation and a perchlorate anion.
The Cu atom is coordinated in an octahedral geometry by two hydrazone ligands, one
adopts monoanionic enolate form, and the other one adopts neutral keto form. It is
clear that the coordinate bond lengths of the neutral hydrazone ligand are longer than
the corresponding bonds in the monoanionic hydrazone ligand. Thus, the equatorial
plane of the octahedral geometry can be defined by the three donor atoms (O2, N5,
-
8 -

N4) of the monoanionic hydrazone ligand, and the imino N atom (N2) of the neutral
hydrazone ligand, and the apical positions can be defined by the remaining two donor
atoms of the neutral hydrazone ligand. The Cu atom deviates from the least-squares
plane defined by the equatorial donor atoms by 0.041(1) Å. The distortion of the
octahedral geometry can be observed from the deviation of the coordinate bond angles
from the ideal values (Table 2).
In the crystal structure of the complex, molecules are linked by intermolecular
hydrogen bonds (Table 3), to form chains along the a axis (Figure 6).
3
.3. IR and UV-Vis spectra
The medium and broad absorption centered at about 3450 cm in the spectra of
complexes 1 and 2 substantiate the presence of O–H groups. The sharp bands
–1
–
1
indicative of the N–H vibration of complex 3 is located at 3190 cm . The strong
–
1
absorption bands in the region 1600–1610 cm for the complexes are assigned to the
–
1
azomethine ν(C=N) [33]. The intense absorption at 2093 cm for complex 2 can be
assigned to the vibration of the NCS ligand [34]. The band indicative of the
–
1
perchlorate anion in complex 3 is observed at 1162 cm .
Electronic spectra of the complexes were recorded in methanol with concentration
-5
of 10 M. The complexes displayed strong bands centered at about 270 nm, which
can be assigned to the intra-ligand π–π* transition of the aromatic rings. The charge
transfer LMCT bands are located in the range 370–390 nm. The spectra showed weak
and broad d-d electronic transitions in the range 640-670 nm, which are assigned to
2
2
E
g(D)→ T2g(D) [35].
3.4. Pharmacology study
The percent inhibition of the complexes at concentration of 12.5 µM on Jack bean
urease and IC50 values are listed in Table 4. Complex 1 shows excellent urease
-
9 -

inhibitory activity with IC50 value of 1.46 ± 0.83 µM. Complexes 2 and 3 show from
medium to strong activities with IC50 values of 10.8 ± 2.1 and 15.4 ± 1.8 µM,
respectively. As a comparison, AHA was used as a reference with the percent
inhibition of 64.0 ± 2.7, and with IC50 value of 36.3 ± 2.5 µM. Copper perchlorate can
inhibit urease activity, with IC50 value of 8.5 ± 1.7 µM. The present copper complexes
have
N-hydroxyethyl-N-benzimidazolylmethylethylenediaminediacetic acid (IC50 = 35 µM)
36], and also stronger than the copper(II) complexes with Schiff base ligands (IC50
9 and 39 µM) [37]. However, the complexes have a little weak activity than the
stronger
activities
than
the
copper(II)
complex
with
[
=
1
copper(II) complexes with hydrazone ligands derived from salicylaldehyde and its
analogues [38].
3.5. Kinetic study of the urease inhibitory activity by complex 1
Complex 1 has the most effective urease inhibitory activity. Thus, the inhibition
mechanism of the complex was studied with Lineweaver-Burk plots (Figure 7a). The
type of inhibition was elucidated from analysis of Lineweaver-Burk plots. For
Lineweaver-Burk plot, the slope of the resulting line is K
/vmax, and the x-intercept is 1/K . The figure shows a series of lines intersect one
with another in the third quadrant while the y-intercept of the plots increased with the
m
/vmax, the y-intercept is
1
m
–
1
increase of the concentration (5.17, 10.35, 20.7, 41.4 µg·mL ) of the complex. This
illustrated the inhibition of the urease by the complex caused a decrease in vmax with
change of K
4.5 was calculated from the slope of the Lineweaver-Burk plot vs. the concentration
of inhibitor (Figure 7b).
.6. Molecular docking study
Molecular docking study was performed to investigate the binding effects between
m i
values, suggesting a mixed-competitive inhibition type. The K value of
–
3
-
10 -

the molecules of the complexes and the active site of the Jack bean urease. The
binding models of complexes 1, 2 and 3 with the urease were depicted in Figures 8-10,
respectively. The results revealed that the molecule of complex 1 fits well with the
active pocket of the urease, while complexes 2 and 3 cannot enter the active pocket.
The size of the molecules might be the basal principle of the inhibition. Additional
interactions have been established in a variety of conformations because of the
flexibility of the complex molecules and the amino acid residues of the enzyme. The
optimized clusters (100 occurrences each) were ranked by energy level in the best
conformation of inhibitor–urease modeled structures, where the docking scores are
–5.98 (1), –5.83 (2), and –7.88 (3), respectively. As a comparison, the docking score
for the AHA inhibited model is –5.01. The negative values indicate that the complex
molecules bind well with the urease.
The mechanism of urease inhibition by complex 1 was considered to be embedded
in the active pocket of the urease and interact with the residues MET366, HIS322,
ALA365 and GLN364. The Br ligand of complex 1 is close to the Ni atom of the
enzyme. In addition, there are some hydrophobic interactions between the complex
and the residues of the urease. The mechanism of urease inhibition by complexes 2
and 3 was considered to be partial blockage of the entrance of the urease active pocket,
which hindered the urea to access to the urease active center.
4. Conclusion
The present study reports the syntheses, characterization and crystal structures of
three
-methyl-N’-(pyridin-2-ylmethylene)benzohydrazide as ligand. Complex 1 shows
excellent urease inhibitory activity, with IC50 value of 1.46 ± 0.83 µM. The complex
new
copper(II)
complexes
with
3
-
11 -

has a mixed competitive inhibition mechanism. Molecular docking study indicated
that suitable size and conformations of the complexes are required for the inhibition
of the urease. The biological evaluation and mechanism study of the compounds
reveal that complex 1 is a prospective urease inhibitor.
Supplementary data
CCDC 1553134 (1), 1553135 (2) and 1553157 (3) contain the supplementary
crystallographic data for this paper. These data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html,
Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44)
223-336-033; or e-mail: deposit@ccdc.cam.ac.uk.
or
from
the
Cambridge
1
Acknowledgments
This work was financially supported by the Natural Science Foundation of China
(
Project No. 21641012).
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[
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26]G.M. Sheldrick, Acta Crystallogr. A64 (2008) 112-122.
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33]Z.-L. You, M. Zhang, D.-M. Xian, Dalton Trans. 41 (2012) 2515-2524.
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[
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You, J. Inorg. Biochem. 159 (2016) 22-28.
-
15 -

Figure 1. Molecular structure of 1, showing the atom-numbering scheme.
Displacement ellipsoids are drawn at the 30% probability level and H atoms are
shown as small spheres of arbitrary radii.
Figure 2. Molecular packing diagram of 1, viewed along the b axis. Hydrogen bonds
are shown as dashed lines.
-
16 -

Figure 3. Molecular structure of 2, showing the atom-numbering scheme.
Displacement ellipsoids are drawn at the 30% probability level and H atoms are
shown as small spheres of arbitrary radii.
-
17 -

Figure 4. Molecular packing diagram of 2, viewed along the c axis. Hydrogen bonds
are shown as dashed lines.
-
18 -

Figure 5. Molecular structure of 3, showing the atom-numbering scheme.
Displacement ellipsoids are drawn at the 30% probability level and H atoms are
shown as small spheres of arbitrary radii.
-
19 -

Figure 6. Molecular packing diagram of 3, viewed along the c axis. Hydrogen bonds
are shown as dashed lines.
-
1
100
80
5
1
2
4
.17 µg⋅mL
800
600
-1
0.35 µg⋅mL
-
1
0.7 µg⋅mL
-
1
1.4 µg⋅mL
60
40
20
0
4
00
00
2
-
0.06 -0.04 -0.02 0.00 0.02 0.04 0.06 0.08
1
-10
0
10
20
30
40
-
1/urea mM
Complex 1 (mM)
(
a)
(b)
Figure 7. (a) Lineweaver–Burk plot of the reciprocal of initial velocities vs. the
reciprocal of substrate concentration in the presence of 41.4, 20.7, 10.35, and 5.17 µM
of complex 1. (b) Plot of the slopes from the Lineweaver-Burk plots vs. various
concentrations of complex 1.
-
20 -

Figure 8a. Binding mode of 1 with Jack bean urease. The enzyme is shown as surface,
and the complex is shown as sticks.
Figure 8b. Binding mode of 1 with Jack bean urease. The residues of the enzyme are
shown as ribbon and sticks, and the complex is shown as sticks.
-
21 -

Figure 9. Binding mode of 2 with Jack bean urease. The enzyme is shown as surface,
and the complex is shown as sticks.
Figure 10. Binding mode of 3 with Jack bean urease. The enzyme is shown as surface,
and the complex is shown as sticks.
-
22 -

Table 1 Crystal data for complexes 1, 2 and 3
1
2
3
Formula
C
15
H16BrCuN
3
O
2
C
16
H16CuN
4
O
2
S
28 6 6
C H25ClCuN O
FW
413.76
391.93
640.53
Crystal shape/colour
Crystal size /mm
block/blue
block/blue
block/blue
0.31×0.27×0.27
Triclinic
0.27×0.26×0.23
Triclinic
0.27×0.23×0.23
Triclinic
Crystal system
Space group
a (Å)
P-1
P-1
P-1
7.4561(6)
8.7736(7)
12.4435(10)
87.286(2)
76.280(2)
8.0667(17)
9.3414(19)
13.049(2)
72.161(2)
86.056(2)
8.6664(4)
11.5419(6)
15.6152(8)
105.408(2)
96.761(2)
b (Å)
c (Å)
α (º)
β (º)
γ (º)
86.661(2)
788.97(11)
2
66.940(2)
859.8(3)
2
102.840(2)
1441.95(12)
2
3
V (Å )
Z
λ (MoKα) (Å)
T (K)
0.71073
298(2)
3.926
0.71073
298(2)
1.407
0.71073
298(2)
0.903
–
1
µ (MoKα) (cm )
T
T
min
max
0.3758
0.4171
0.7025
0.7379
3591/223
3178
0.7926
0.8193
13738/384
5342
Reflections/parameters 7582/204
Unique reflections 2920
Observed reflections [I 2455
2807
4447
-
23 -

≥
2σ(I)]
Restraints
1
1
1
2
Goodness of fit on F
1.037
1.031
1.041
R
R
1
1
, wR
, wR
2
2
[I ≥ 2σ(I)]
0.0272, 0.0614
0.0367, 0.0657
0.355, –0.288
0.0314, 0.0827
0.0375, 0.0873
0.425, –0.474
0.0483, 0.1195
0.0605, 0.1295
0.959, –0.504
(all data)
–
3
∆ρmax, ∆ρmin, e Å
-
24 -

Table 2 Selected bond lengths (Å) and angles (º) for the complexes
1
Cu1–N1
2.037(2)
Cu1–N2
Cu1–O2
1.944(2)
2.296(2)
Cu1–O1
1.9874(19)
2.3586(4)
78.24(8)
Cu1–Br1
N2–Cu1–O1
O1–Cu1–N1
O1–Cu1–O2
N2–Cu1–Br1
N1–Cu1–Br1
2
N2–Cu1–N1
N2–Cu1–O2
N1–Cu1–O2
O1–Cu1–Br1
O2–Cu1–Br1
79.97(9)
98.98(8)
90.40(8)
99.92(5)
98.71(5)
158.15(8)
94.67(8)
162.31(7)
100.30(7)
Cu1–N1
2.045(2)
1.913(2)
2.3245(18)
175.47(9)
78.57(7)
80.04(8)
93.16(9)
96.23(7)
Cu1–N2
Cu1–O1
1.9403(19)
1.9949(17)
Cu1–N4
Cu1–O2
N4–Cu1–N2
N2–Cu1–O1
N2–Cu1–N1
N4–Cu1–O2
O1–Cu1–O2
3
N4–Cu1–O1
N4–Cu1–N1
O1–Cu1–N1
N2–Cu1–O2
N1–Cu1–O2
100.52(9)
100.14(9)
156.98(8)
91.35(7)
92.66(7)
Cu–O1
2.640(2)
Cu1–O2
2.018(2)
Cu1–N1
2.255(3)
Cu1–N2
2.041(2)
Cu1–N4
2.079(3)
Cu1–N5
1.920(2)
O1–Cu1–N1
O1–Cu1–N4
143.49(10)
93.94(10)
O1–Cu1–N2
O1–Cu1–N5
68.09(10)
109.14(10)
-
25 -

O1–Cu1–O2
N5–Cu1–N2
N5–Cu1–N4
N2–Cu1–N4
O2–Cu1–N1
N4–Cu1–N1
92.01(10)
176.97(10)
79.53(10)
99.29(10)
93.82(10)
93.98(10)
N5–Cu1–O2
O2–Cu1–N2
O2–Cu1–N4
N5–Cu1–N1
N2–Cu1–N1
78.41(9)
102.69(9)
157.89(9)
107.35(10)
75.47(9)
-
26 -

Table 3 Hydrogen bond distances (Å) and bond angles (°) for the complexes
D–H···A
d(D–H)
0.84(1)
0.84(1)
d(H···A)
2.76(3)
1.97(1)
d(D···A)
3.449(3)
2.800(3)
Angle (D–H···A)
1
i
O2–H2···N2
142(4)
2
ii
iii
O2–H2···N3
3
173(3)
N3–H3···O5
0.90(1)
0.930
0.930
0.930
2.14(3)
2.43(3)
2.48(3)
2.52(3)
2.41(3)
2.902(4)
3.217(5)
3.391(5)
3.132(5)
3.224(5)
143(4)
142(4)
166(4)
124(5)
146(4)
ii
C1–H1···N6
C3–H3A···O4
iii
C6–H6···O5
iv
C20–H20···O1 0.930
Symmetry codes: (i) – x, 1 – y, – z; (ii) 1 – x, 1 – y, 1 – z; (iii) 1 – x, 1 – y, – z; (iv) – x,
– y, 1 – z.
1
-
27 -

Table 4 Inhibition of urease by the tested materials
Tested materials
Percentage Inhibition IC50 (µM)
#
rate
1
97.9 ± 3.8
73.8 ± 3.1
61.5 ± 4.0
70.2 ± 3.3
1.46 ± 0.83
10.8 ± 2.1
15.4 ± 1.8
8.5 ± 1.7
2
3
Copper perchlorate
Acetohydroxamic acid 64.0 ± 2.7
36.3 ± 2.5
#
The concentration of the tested material is 12.5 µM.
-
28 -

Graphical abstract (picture)
-
29 -