Synthesis, crystal structures and xanthine oxidase inhibitory activity of benzohydrazone compounds

Article abstract of DOI:10.14233/ajchem.2014.17602

A series of three benzohydrazone compounds, 2N′-(5-hydroxy-2-nitrobenzylidene)-4-methylbenzohydrazide (1), N′-(5-chloro-2-hydroxybenzylidene)-3-methylbenzohydrazide (2) and N′-(2,4-dichlorobenzylidene)-3-methylbenzohydrazide (3 ), were prepared and structurally characterized by elemental analysis, IR spectra and single crystal X-ray determination. Xanthine oxidase inhibitory activities of the compounds were studied. Among the compounds, N′-(5-hydroxy-2-nitrobenzylidene)-4-methylbenzohydrazide (1) shows the most effective activity with IC₅₀ value of 15.2 ± 2.3 μM. Docking simulation was performed to insert the compound into the crystal structure of xanthine oxidase at the active site to investigate the probable binding modes.

Full text of DOI:10.14233/ajchem.2014.17602

Asian Journal of Chemistry; Vol. 26, No. 23 (2014), 8118-8122
ASIAN JOURNAL OF CHEMISTRY
http://dx.doi.org/10.14233/ajchem.2014.17602
Synthesis, Crystal Structures and Xanthine Oxidase Inhibitory Activity of Benzohydrazone Compounds
1,*
2
3
WEI-MING ZHANG , GUI-HUA SHENG and ZHONGLU YOU
1Nanjing Institute for the Comprehensive Utilization of Wild Plant, Nanjing 210042, P.R. China
2School of Life Sciences, Shandong University of Technology, Zibo 255049, P.R. China
3Department of Chemistry and Chemical Engineering, Liaoning Normal University, Dalian 116029, P.R. China
*Corresponding author: E-mail: youzhonglu@126.com
Received: 14 March 2014;
Accepted: 21 May 2014;
Published online: 15 November 2014;
AJC-16308
A series of three benzohydrazone compounds, 2N'-(5-hydroxy-2-nitrobenzylidene)-4-methylbenzohydrazide (1), N'-(5-chloro-2-
hydroxybenzylidene)-3-methylbenzohydrazide (2) and N'-(2,4-dichlorobenzylidene)-3-methylbenzohydrazide (3), were prepared and
structurally characterized by elemental analysis, IR spectra and single crystal X-ray determination. Xanthine oxidase inhibitory activities
of the compounds were studied. Among the compounds, N'-(5-hydroxy-2-nitrobenzylidene)-4-methylbenzohydrazide (1) shows the most
effective activity with IC50 value of 15.2 2.3 µM. Docking simulation was performed to insert the compound into the crystal structure of
xanthine oxidase at the active site to investigate the probable binding modes.
Keywords: Benzohydrazone, Xanthine oxidase, Inhibition, Crystal structure, Molecular docking study.
of great interest in biological chemistry for a long time10-12. Leigh
INTRODUCTION
and co-workers have reported some Schiff bases as novel
Enzyme inhibitors can interact with enzymes and block
their activity towards natural substrates. The importance of
enzyme inhibitors as drugs is enormous since these molecules
have been used for treating a number of pathophysiological
conditions1,2. Xanthine oxidase (XO; EC 1.17.3.2), a molybdenum
hydroxylase, catalyzes the hydroxylation of hypoxanthine and
xanthine to yield uric acid and superoxide anions. These
superoxide anions have been linked to post ischaemic tissue
injury and edema as well as to vascular permeability3. Xanthine
oxidase can oxidize synthetic purine drugs, such as anti-
leukaemic 6-mercaptopurine, with the loss of their pharmaco-
logical properties4. Xanthine oxidase has also been linked to
conditions such as hepatic and kidney damage, atherosclerosis,
chronic heart failure, hypertension and sickle-cell disease due
to the production of reactive oxygen species (ROS) alongside
uric acid5,6. Then, control of the action of xanthine oxidase
may help the therapy of some diseases. Nowadays, the
treatment of gout makes use of allopurinol, a potent inhibitor
of xanthine oxidase known for a long time7. The mode of action
of allopurinol involves the direct coordination of its active
metabolite, oxypurinol (alloxanthine), to the molybdenum
centre in the active site of the enzyme8. However, given its
side effects, toxicity and its inability to prevent the formation
of free radicals by the enzyme9, the research on new xanthine
oxidase inhibitors is needed. Schiff base compounds have been
xanthine oxidase inhibitors13. Benzohydrazone compounds are
a kind of Schiff bases, which show particular interesting biolo-
gical activities. As an extension of the work on the exploration
of effective xanthine oxidase inhibitors related to Schiff bases,
in this paper, a series of benzohydrazone compounds, N'-(5-
hydroxy-2-nitrobenzylidene)-4-methylbenzo-hydrazide (1),
N'-(5-chloro-2-hydroxybenzylidene)-3-methyl-benzohy-
drazide (2) and N'-(2,4-dichlorobenzylidene)-3-methylbenzo-
hydrazide (3), were synthesized and structurally characterized.
The xanthine oxidase inhibitory activities of the compounds
were investigated from both experimental and molecular
docking study.
EXPERIMENTAL
Starting materials, reagents and solvents with AR grade
were purchased from commercial suppliers and used without
further purification. Elemental analyses were performed on a
Perkin-Elmer 240C elemental analyzer. IR spectra were
recorded on a Jasco FT/IR-4000 spectrometer as KBr pellets
in the 4000-400 cm-1 region.
Synthesis of N'-(5-hydroxy-2-nitrobenzylidene)-4-
methylbenzohydrazide (1): 5-Hydroxy-2-nitrobenzaldehyde
(1 mmol, 0.167 g) and 4-methylbenzohydrazide (1 mmol,
0.150 g) were mixed in methanol and stirred at room tempe-
rature for 1 h. The methanol was evaporated to obtain yellow

Vol. 26, No. 23 (2014)
Synthesis, Crystal Structures and Xanthine Oxidase Inhibitory Activity of Benzohydrazone Compounds 8119
crystalline product, which was washed with methanol and dried
in air.Yield: 95 %; single crystals of compound 4 suitable for
X-ray diffraction were obtained by recrystallization of the
product in methanol. Anal. Calcd. for C15H13N3O4: C, 60.2; H,
were carried out to evaluate the binding free energy of the
inhibitor within the macromolecules. The GALS search
algorithm (genetic algorithm with local search) was chosen to
search for the best conformers. The parameters were set using
the software ADT (AutoDockTools package, version 1.5.4)
on PC which is associated withAutoDock 4.2. Default settings
were used with an initial population of 100 randomly placed
individuals, a maximum number of 2.5 × 106 energy evalua-
tions and a maximum number of 2.7 × 104 generations. A
mutation rate of 0.02 and a crossover rate of 0.8 were chosen.
Give overall consideration of the most favorable free energy
of biding and the majority cluster, the results were selected as
the most probable complex structures.
Data collection, structural determination and refinement:
Diffraction intensities for the compounds were collected at
298(2) K using a Bruker D8 VENTURE PHOTON diffracto-
meter with MoKα radiation (λ = 0.71073 Å). The collected
data were reduced using the SAINT program15 and multi-scan
absorption corrections were performed using the SADABS
program16. The structures were solved by direct methods and
refined against F2 by full-matrix least-squares methods using
the SHELXTL17. All of the non-hydrogen atoms were refined
anisotropically. The amino and water H atoms were located in
difference Fourier maps and refined isotropically, with N-H,
O-H and H···H distances restrained to 0.90(1), 0.85(1) and
1.37(2) Å, respectively. All other H atoms were placed in
idealized positions and constrained to ride on their parent
atoms. The crystallographic data for the compounds are
summarized in Table-1. Hydrogen bonding information is
given in Table-2.
4.4; N, 14; found: C, 60; H, 4.3; N, 14.2 %. IR data (KBr, νmax
,
cm-1): 3429 (m), 3320 (w), 1649 (s), 1557 (m), 1493 (m), 1316
(s), 1225 (m), 1127 (w), 1081 (w), 944 (w), 898 (w), 833 (w),
743 (w), 553 (w).
Synthesis of N'-(5-chloro-2-hydroxybenzylidene)-3-
methylbenzohydrazide (2): 5-Chlorosalicylaldehyde (1
mmol, 0.156 g) and 3-methylbenzohydrazide (1 mmol, 0.150
g) were mixed in methanol and stirred at room temperature
for 1 h. The methanol was evaporated to obtain colorless
crystalline product, which was washed with methanol and dried
in air.Yield: 87 %; single crystals of compound 5 suitable for
X-ray diffraction were obtained by recrystallization of the
product in methanol.Anal. Calcd. for C15H13N2O2Cl: C, 62.4;
H, 4.5; N, 9.7; found: C, 62.4; H, 4.6; N, 9.5 %. IR data
(KBr, νmax, cm-1): 3417 (m), 3208 (w), 1649 (s), 1557 (s),
1473 (m), 1356 (w), 1290 (s), 1219 (m), 1094 (w), 951
(w), 873 (w), 814 (m), 650 (w), 559 (w), 468 (w).
Synthesis of N'-(2,4-dichlorobenzylidene)-3-methyl-
benzohydrazide (3): 2,4-Dichlorobenzaldehyde (1 mmol,
0.175 g) and 3-methylbenzohydrazide (1 mmol, 0.150 g) were
mixed in methanol and stirred at room temperature for 1 h.
The methanol was evaporated to obtain colorless crystalline
product, which was washed with methanol and dried in air.
Yield: 95 %; single crystals of compound 6 suitable for X-ray
diffraction were obtained by recrystallization of the product
in methanol. Anal. Calcd. for C15H12N2OCl2: C, 58.7; H, 3.9;
N, 9.1; Found: C, 58.5; H, 3.9; N, 9.2 %. IR data (KBr, νmax
,
cm-1): 3212 (w), 1647 (s), 1562 (s), 1461 (m), 1353 (w), 1288
(s), 1219 (m), 956 (w), 872 (w), 837 (m), 717 (w), 535 (w),
479 (w).
RESULTS AND DISCUSSION
Compounds 1-3 were readily synthesized by reaction of
1:1 molar ratio of aldehydes with benzohydrazides in methanol
at room temperature, according to the literature method18, with
high yields (over 90 %) and purity. Single crystals suitable for
X-ray diffraction were obtained by slow evaporation of the
solutions containing the compounds in air. The compounds
have been characterized by elemental analyses and IR spectra.
Structures of the compounds were further confirmed by single
crystal X-ray crystallography.
Measurement of the xanthine oxidase inhibitory
activity: The xanthine oxidase activities with xanthine as the
substrate were measured spectrophotometrically, based on the
procedure reported by Kong et al.14, with modification. The
activity of xanthine oxidase is measured by uric acid formation
monitored at 295 nm. The assay was performed in a final
volume of 1 mL 50 mM K2HPO4 pH 7.8 in quartz cuvette.
The reaction mixture contains 200 mL of 84.8 mg/mL xanthine
in 50 mM K2HPO4, 50 mL of the various concentrations tested
compounds. The reaction is started by addition of 66 mL 37.7
mU/mL xanthine oxidase. The reaction is monitored for 6 min
at 295 nm and the product is expressed as mmol uric acid per
minute. The reactions kinetic were linear during these 6 min
of monitoring.
Docking simulations: Molecular docking study of the
compounds into the 3D X-ray structure of xanthine oxidase
(entry 1FIQ in the Protein Data Bank) was carried out by using
the AutoDock version 4.2. First, Auto Grid component of the
program precalculates a 3D grid of interaction energies based
on the macromolecular target using the AMBER force field.
The cubic grid box of 60 × 70 × 60 Å3 points in x, y and z
direction with a spacing of 0.375 Å and grid maps were created
representing the catalytic active target site region where the
native ligand was embedded. Then automated docking studies
Structure description of the compounds: Figs. 1-3 give
perspective views of compounds 1-3 with atomic labeling
systems. X-ray crystallography reveals that the compounds
are similar benzohydrazone derivatives. All the benzo-
hydrazone molecules of the compounds adopt E configuration
with respect to the methylidene units. The distances of the
methylidene bonds, ranging from 1.26 to 1.29 Å, confirm them
as typical double bonds. The shorter distances of the C-N bonds
and the longer distances of the C=O bonds for the -C(O)-NH-
units than usual, suggests the presence of conjugation effects in
the molecules. The remaining bond lengths in the compounds
are comparable to each other and are within normal values18-20
.
The dihedral angles between the two aromatic rings are 2.3(3)°
for compound 1, 14.2(4)° for compound 2 and 9.5(3)° for
compound 3. The crystal structures of the compounds are
stabilized by intermolecular hydrogen bonds (Figs. 4-6).

8120 Zhang et al.
Asian J. Chem.
TABLE-1
CRYSTALLOGRAPHIC AND
EXPERIMENTAL DATA FOR THE COMPOUNDS
Compound
1
C15H13N3O4
299.3
2
3
m.f.
m.w.
C15H13N2O2Cl C15H12N2OCl2
288.7
307.2
T (K)
298(2)
298(2)
298(2)
Crystal shape/color Block/yellow Block/colorless Bock/colorless
Crystal size (mm3) 0.17×0.15×0.15 0.17×0.13×0.10 0.20×0.18×0.17
Crystal system
Space group
a (Å)
b (Å)
c (Å)
α (°)
β (°)
Triclinic
P-1
Monoclinic
P21/n
7.129(4)
27.362(16)
7.742(4)
Monoclinic
P21/n
9.674(1)
4.799(1)
31.028(3)
Fig. 2. A perspective view of the molecular structure of compound 2 with
the atom labeling scheme. Thermal ellipsoids are drawn at the 30
% probability level. Hydrogen bonds are shown as dashed lines
7.689(2)
9.068(2)
10.726(2)
95.539(3)
108.625(3)
99.602(3)
689.7(3)
2
111.263(6)
92.594(2)
γ (°)
V (Å3)
Z
1407.4(14)
4
1.363
0.274
600
1439.1(4)
4
1.418
0.447
632
Dc (g cm–3)
1.441
0.107
312
µ (MoKα) (mm-1)
F(000)
Reflections
collected
Unique reflections 2890
Observed reflections 1922
(I ≤ 2σ (I))
5159
7216
6971
Fig. 3. A perspective view of the molecular structure of compound 3 with
the atom labeling scheme. Thermal ellipsoids are drawn at the 30 %
probability level. Hydrogen bond is shown as a dashed line
3020
1788
3081
1781
Parameters
Restraints
204
1
185
1
184
1
Min. and max.
transmission
Goodness-of-
fit on F2
0.982, 0.984
0.955, 0.973
0.916, 0.928
1.066
1.022
1.032
R1, wR2 [I ≤ 2σ (I)]a
0.0501, 0.1336 0.0567, 0.1121 0.0565, 0.1132
R1, wR2 (all data)a 0.0789, 0.1634 0.1080, 0.1334 0.1052, 0.1312
Large diff. peak and 0.226, –0.255 0.194, –0.201 0.198, –0.247
hole (eÅ–3)
aR1 = Fo-Fc/Fo, wR2 = [Σw(Fo2-Fc2)/Σw(Fo2)2]1/2
TABLE-2
HYDROGEN BOND DISTANCES (Å) AND
BOND ANGLES (°) FOR THE COMPOUNDS
D–H···A
d(D–H) d(H···A) d(D···A) Angle (D-H···A)
1
N2–H2···O3#1
O1–H1···O4#2
0.90(1)
0.82
2.36(2)
1.93
2
3.247(2)
2.743(2)
171(3)
168(3)
O1–H1···N1
0.82
0.90(1)
1.88
1.98(2)
3
2.595(3)
2.876(3)
146(3)
168(3)
Fig. 4. Molecular packing diagram of compound 1. Hydrogen bonds are
N2–H2···O2#3
shown as dashed lines
N2–H2···O1#4
0.90(1)
1.92(2)
2.776(3)
159(3)
Symmetry codes: #1) 1 – x, 1 – y, 1 – z; #2) – x, 2 – y, 1 – z; #3) –1/2
+ x, 3/2 – y, –1/2 + z ; #4) x, –1 + y, z
Fig. 1. A perspective view of the molecular structure of compound 1 with
the atom labeling scheme. Thermal ellipsoids are drawn at the 30
% probability level
Fig. 5. Molecular packing diagram of compound 2. Hydrogen bonds are
shown as dashed lines

Vol. 26, No. 23 (2014)
Synthesis, Crystal Structures and Xanthine Oxidase Inhibitory Activity of Benzohydrazone Compounds 8121
Fig. 6. Molecular packing diagram of compound 3. Hydrogen bonds are
shown as dashed lines
Pharmacology: The measurement of xanthine oxidase
inhibitory activity was carried out for three parallel times. The
percents of inhibition at the concentration of 100 µM and IC50
values for the compounds against xanthine oxidase are summa-
rized in Table-3.
TABLE-3
INHIBITION OF XO BY THE TESTED MATERIALS
Tested materials
Percent of inhibitionb
IC50(µM)
67.2 3.1
35.9 2.1
20.5 2.0
80.7 4.3
15.2 2.3
1
-
-
2
3
Allopurinol
8.7 2.3
bThe concentration of the tested material is 100 µM
Allopurinol was used as a reference with the per cent of
inhibition of 80.7 4.3 and with IC50 value of 8.7 2.3 µM.
Compound 1 shows the most effective activity with the percent
of inhibition of 67.2 3.1 and with IC50 value of 15.2 2.3 µM.
Although the number of tested compounds is limited, some
structural features, important to the xanthine oxidase inhibitory
effect, can be inferred. Compound 1, bearing a nitrate group,
has the most effective activity. These findings are coherent
with the results reported in the literature that the existence of
electron-withdrawing groups in the benzene rings can enhance
the activities21.
Molecular docking study: In order to give an explanation
and understanding of potent inhibitory activity observed from
the experiment, molecular docking study was performed to
investigate the binding effects between the compound 1 and
the active sites of xanthine oxidase (entry 1FIQ in the Protein
Data Bank). Allopurinol was used to verify the model of
docking and gave satisfactory results. Fig. 7 is the binding
models for compound 1 in the enzyme active site of xanthine
oxidase. The docking score is -5.73. As a comparison, the
docking score for Allopurinol is -6.27.
Fig. 7. 3D (above) and 2D (below) binding mode of compound 1 with the
active site of XO. Hydrogen bonds are shown as dashed lines
the molecular docking study could explain the inhibitory
activity of the compound on xanthine oxidase.
Supplementary information: CCDC-892366 for com-
pound 1, 892368 for compound 2 and 892369 for compound 3
contain the supplementary crystallographic data for this
paper. These data can be obtained free of charge at http://
www.ccdc.cam.ac.uk/const/retrieving.html or from the
Cambridge Crystallographic Data Centre (CCDC), 12 Union
Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223-336033 or
e-mail: deposit@ccdc.cam.ac.uk.
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