FULL PAPER
for all procedures. IR spectra were recorded with a FTIR Nicolet
5700 Spectrometer from 4000 to 400 cm–1. The crystal data of
trizole ligand and complexes were collected with a Bruker D8
VENTURE PHOTON diffractometer. Enzyme inhibitory activity
was measured with a BioTek Synergy HT microplate reader.
found C 36.49, H 3.39, N 23.65. IR (KBr): ν = 3291, 3184, 1609,
˜
1588, 1445, 1365, 1243, 985, 742, 685, 607, 532 cm–1.
Crystal Structure Determinations: X-ray crystallographic data were
collected with a Bruker D8 VENTURE PHOTON diffractometer
with graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å)
using the Genenic omega scan technique. The structure was solved
by direct methods and refined on F2 by full-matrix least-squares
with Bruker’s SHELXL-97 program.[22] All of the non-hydrogen
atoms were refined anisotropically. All other hydrogen atoms were
placed in geometrically ideal positions and constrained to ride on
their parent atoms.
Synthesis of the Ligand 2-{[4-Amino-3-(pyridin-4-yl)-4,5-dihydro-
1H-1,2,4-triazol-5-yl]thio}acetic Acid (HL): Prepared by nucleo-
philic substitution reaction between chloroacetic acid and 4-amino-
3-(pyridin-4-yl)-4,5-dihydro-1H-1,2,4-triazole-5-thiol, which was
prepared through multistep reaction with isonicotinohydrazide by
using the method of Reid and Heindel[21] with modifications.
Molecular Docking: Molecular docking of the inhibitor with the
3D structure of jack bean urease (entry 3LA4 in the Protein Data
Bank) was carried out by using the Autodock 4.2 program suite.
The crystal structures of ligands were used in the docking protocol.
The graphical user interface AutoDockTools was performed to
setup every inhibitor–enzyme interaction; where all hydrogen atoms
were added, Gasteiger charges were calculated and nonpolar
hydrogen atoms were merged to carbon atoms. The Ni initial
parameters were set as r = 1.170 Å, q = +2.0, and van der Waals
well-depth of 0.100 kcal/mol.[23] The 3D structures of ligand mo-
lecule were saved in Mol2 format with the aid of the program
MERCURY 3.0. The partial charges of Mol2 file were further
modified by using the AutoDockTools package (version 1.5.4) so
that the charges of the nonpolar hydrogen atoms would be assigned
to the atom to which the hydrogen is attached. The choice of the
flexible bonds in the ligands was in accordance with SP3 hybridiza-
tion. The resulting file was saved as pdbqt file.
Isonicotinohydrazide (13.7 g, 0.1 mol) reacted with carbon disulf-
ide (9.04 mL, 0.15 mol) and potassium hydroxide (8.4 g, 0.15 mol)
in absolute ethyl alcohol to give potassium dithiocarbazinate,
which was then cyclized to 4-amino-3-(pyridin-4-yl)-4,5-dihydro-
1H-1,2,4-triazole-5-thiol by reacting with hydrazine hydrate
(10 mL, 0.16 mol), neutralized with hydrochloric acid to form the
precipitate and then HL was synthesized with chloroacetic acid
(14.25 g, 0.15 mol) and potassium hydroxide (8.4 g, 0.15 mol). The
resulting mixture was heated to reflux for 12 h at 105 °C and, after
cooling, the solvent was neutralized with hydrochloric acid to form
a precipitate, which was isolated by filtration and purified by
recrystallization from ethanol to give pure HL (15.891 g, 63%).
C9H10N5O2S (252.27): calcd. C 42.85, H 3.99, N 27.76; found C
42.84, H 4.00, N 27.76. IR (KBr): ν = 3464, 3295, 3184, 1645, 1578,
˜
1445, 1388, 1224, 993, 727, 687, 652, 551 cm–1.
[Cu(L)2(NH3)2(H2O)2] (1): Ligand HL (0.288 g, 1 mmol) was dis-
solved in a solvent mixture of methanol and N,N-dimethylform-
amide (1:1 v/v, 10 mL), and added to Cu(CH3COO)2·H2O (0.200 g,
1 mmol) in methanol (5 mL). NH3·H2O (17%; 1 mL) was added
and the resulting solution was stirred for 30 min at room tempera-
ture and then filtered. The filtrate was kept in air for about 7 d,
forming crystals of 1 (0.295 g, 60%). C18H26CuN12O6S2 (634.15):
calcd. C 34.09, H 4.13, N 26.50; found C 34.08, H 4.14, N 26.51.
The A program was used to generate the docking input files. In all
docking, a grid box size of 60ϫ60ϫ60 pointing in x, y and z
directions was built, the maps were centered on the Ni842 atom in
the catalytic site of the protein. The nickel bridging hydroxide was
retained in the calculations. A grid spacing of 0.508 Å and a dis-
tance-dependent function of the dielectric constant were used to
calculate the energetic map. 50 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 2.5ϫ106 energy evaluations, and a maximum
number of 2.7ϫ104 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 struc-
tures. Usually, the first docking conformation of the docking re-
sults had the lowest energy, which indicated the stablest system and
thus a likely binding interaction. The docking procedure of com-
plex 1 with the enzyme active site of jack bean urease was per-
formed as described.
IR (KBr): ν = 3284, 3178, 1617, 1591, 1450, 1360, 1231, 996, 740,
˜
689, 603, 524 cm–1.
{[Cu(L)2(H2O)2]·2H2O}n (2): Ligand HL (0.288 g, 1 mmol) was dis-
solved in a solvent mixture of methanol and N,N-dimethylform-
amide (1:1 v/v, 10 mL), and added to Cu(CH3COO)2·H2O (0.200 g,
1 mmol) in methanol (5 mL). The resulting solution was stirred for
15 min at room temperature and then filtered. The filtrate was kept
in air for about 20 d, forming crystals of 2 (0.214 g, 43%).
C18H20CuN10O6S2 (600.09): calcd. C 36.03, H 3.36, N 23.34; found
C 36.02, H 3.35, N 23.33. IR (KBr): ν = 3285, 3178, 1617, 1590,
˜
1451, 1356, 1230, 995, 734, 688, 600, 523 cm–1.
{[Zn(L)2(H2O)2]·2H2O}n (3): Ligand HL (0.288 g, 1 mmol) was dis-
solved in a solvent mixture of methanol and N,N-dimethylform-
amide (1:1 v/v, 10 mL), and added to Zn(CH3COO)2·2H2O
(0.220 g, 1 mmol) in methanol (5 mL). The resulting solution was
stirred for 15 min at room temperature and then filtered. The fil-
trate was kept in air for about 20 d, forming crystals of 3 (0.307 g,
63%). C18H20N10O6S2Zn (601.92): calcd. C 35.91, H 3.35, N 23.26;
Measurement of Jack Bean Urease Inhibitory Activity: The
measurement of urease activity was carried out according to the
procedure reported by Tanaka.[24] The assay mixture, containing
25 μL of jack bean urease (40 kU/L) (dissolved in distilled water)
and 25 μL of the tested complexes of different concentrations (dis-
solved in DMSO/H2O mixture (1:1 v/v)) was preincubated for 1 h
at 37 °C in a 96-well assay plate. After preincubation, 200 μL of
100 mm HEPES (N-[2-hydroxyethyl] piperazine-NЈ-[2-ethanesul-
fonic acid]) buffer pH 6.8 containing 500 mm urea and 0.002%
phenol red were added and incubated at 37 °C.[25] The reaction,
which was measured with a microplate reader (570 nm), was re-
quired to produce enough ammonium carbonate to raise the pH
of a HEPES buffer from 6.8 to 7.7, with the endpoint being deter-
mined by the color of phenol red indicator.[26]
found C 35.92, H 3.34, N 23.27. IR (KBr): ν = 3311, 3191, 1624,
˜
1458, 1381, 1228, 977, 730, 695, 593, 522 cm–1.
Synthesis of {[Fe(L)2(H2O)2]·2H2O}n (4): Ligand HL (0.288 g,
1 mmol) was dissolved in a solvent mixture of methanol and N,N-
dimethylformamide (1:1 v/v, 10 mL), and added to a solution of
FeCl2 (0.126 g, 1 mmol) in methanol (5 mL). The resulting solution
was stirred for 30 min at room temperature and then filtered. The
filtrate was kept in air for about 40 d, forming crystals of 4 (0.302 g, Kinetics Study: Lineweaver–Burk plots of 1/absorbance vs. 1/urea
73%). C18H20FeN10O6S2 (592.39): calcd. C 36.50, H 3.40, N 23.64;
were used to reveal the mechanism of inhibition. Urease inhibition
Eur. J. Inorg. Chem. 2015, 2076–2084
2083
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Xu, Yong-Peng
