Synthesis, Characterization, Crystal Structures, and Urease Inhibition of Copper(II) and Zinc(II) Complexes Derived from Benzohydrazones

A new copper(II) complex [Cu(L)(NCS)(CH3OH)] (1) and a new zinc(II) complex [ZnCl2(HL)] · CH3OH (2), derived from 4-bromo-N’-(pyridin-2-ylmethylene)benzohydrazide (HL1) and 4-methoxy-N’-(pyridin-2-ylmethylene)benzohydrazide (HL2), were prepared and characterized by elemental analysis, IR and UV-Vis spectroscopy and single crystal X-ray diffraction. The hydrazone HL1 coordinates to the Cu atom in enolate form, while the hydrazone HL2 coordinates to the Zn atom in carbonyl form. Single crystal structural analyses indicate that the hydrazones coordinate to the metal atoms through the pyridine N, imino N, and enolate/carbonyl O atoms. The Cu atom in complex 1 is in square pyramidal coordination, and the Zn atom in complex 2 is in trigonal-bipyramidal coordination. The inhibitory effects of the complexes on Jack bean urease were studied, which show that the copper complex has strong activity on urease.


Introduction
Urease (EC 3.5.1.5; urea amidohydrolase) is a binuclear nickel-dependent hydrolase enzyme, which can be synthesized by numerous organisms, including plants, bacteria, algae, fungi, and invertebrates, and occurs widely in animal and soil. 1 Urease enzyme catalyzes the decomposition of urea into ammonia and carbon dioxide in high efficiency, 2 with the rate of catalyzed reaction 10 14 times higher than the non-catalyzed reaction. 3 The enzyme possesses harmful effects on both human health and fertile soil. 4 In recent years, Schiff base complexes are reported to have interesting urease inhibitory activities, 5 especially the copper complexes with Schiff bases or hydrazones are promising types of lead structures as urease inhibitors. 6 As a continuation of the work on the exploration of new urease inhibitors, a new copper(II) complex [Cu(L 1 )(NCS) (CH 3 OH)] (1) and a new zinc(II) complex [ZnCl 2 (HL 2 )] · CH 3 OH (2), derived from 4-bromo-N'-(pyridin-2-yl-methylene)benzohydrazide (HL 1 ) and 4-methoxy-N'-(pyridin-2-ylmethylene)benzohydrazide (HL 2 ; Scheme 1), were prepared and studied on their urease inhibition activity. Scheme 1. HL 1 and HL 2

1. Materials and Measurements
All reagents and solvents were of commercially available reagent grade quality and were used without further purification. HL 1 was synthesized according to the literature method. 7 Jack bean urease was purchased from Sig-ma-Aldrich. 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. UV-Vis spectra were recorded on a Perkin-Elmer Lambda 900 spectrometer. 1 H NMR and 13 C NMR spectra were recorded on a 500 MHz Bruker Advance instrument. The urease inhibitory activity was measured on a Bio-Tek Synergy HT microplate reader. Single crystal structures were determined by Bruker Smart 1000 CCD area diffraction.

4. Synthesis of the Complex 2
HL 2 (1.0 mmol, 0.26 g) was dissolved in methanol (20 mL), to which ZnCl 2 (1.0 mmol, 0.14 g) dissolved in methanol (20 mL) was added dropwise. The mixture was 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 by filtration. Yield: 0.26 g (61%

5. X-Ray Crystallography
Diffraction intensities for HL 2 and the complexes were collected at 298(2) K using a Bruker Smart 1000 CCD area diffractometer with MoKα radiation (λ = 0.71073 Å). The collected data were reduced with SAINT, 8 and multiscan absorption correction was performed using SAD-ABS. 9 Structures of HL 2 and the complexes were solved by direct methods and refined against F 2 by full-matrix leastsquares method using SHELXTL. 10 All of the non-hydrogen atoms were refined anisotropically. The amino and methanol H atoms in the compounds were located from difference Fourier maps and refined isotropically, with N-H and O-H distances restrained to 0.90(1) and 0.85(1) Å, respectively. The remaining hydrogen atoms were placed in calculated positions and constrained to ride on their parent atoms. Crystallographic data for HL 2 and the complexes are summarized in Table 1. Selected bond lengths and angles are given in Table 2.

6. Urease Inhibitory Activity Assay
The measurement of urease inhibitory activity was carried out according to the literature method. 11 The assay mixture containing 75 μL of Jack bean urease and 75 μL of tested compounds with various concentrations (dissolved in DMSO) was 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 mmol L -1 ) and urea (400 mmol L -1 ) 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.

1. Chemistry
The synthetic procedure of the hydrazones and the complexes is shown in Scheme 2. The hydrazone HL 2 was prepared by the condensation reaction of equimolar quantities of 2-pyridinecarboxaldehyde and 4-methoxybenzohydrazide. The copper complex was prepared by reaction of equimolar quantities of HL 1 , copper perchlorate, and ammonium thiocyanate in methanol. The zinc complex was prepared by reaction of equimolar quantities of HL 2 and zinc chloride in methanol. Single crystals of HL 2 and the complexes were obtained by slow evaporation of the methanolic solution of the compounds.

2. Structure Description of HL 2
The molecular structure of HL 2 is shown in Figure 1. The compound contains two hydrazone molecules and two water molecules of crystallization. The hydrazone molecules adopt E configuration with respect to the methylidene unit. The distances of the methylidene bonds 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 hydrazone molecules. The remaining bond lengths in the compounds are within normal values. 12 The dihedral angles between the pyridine and benzene rings are 61.0 (3) Table 3, and Figure 2).

Structure Description of the Copper Complex
Molecular structure of the copper complex is shown in Figure 3. The Cu atom is in square pyramidal geometry, with the pyridine N, imino N, and enolate O atoms of the hydrazone ligand, and the thiocyanato N atom located at the basal plane, and with the methanol O atom located at the apical position. The Cu atom deviates from the leastsquares plane defined by the four basal donor atoms by 0.242(1) Å. The coordinate bond lengths in the complex Table 3. Hydrogen bond distances (Å) and bond angles (°) for the compounds  (2) 161 (3) Symmetry codes: (i) -1 + x, y, z; (ii) 1 + x, y, z; (iii) -x, -y + 2, -z + 2.    are comparable to those observed in copper(II) complexes with hydrazone ligands. 6a,13 In the crystal structure of the complex, two complex molecules are linked through intermolecular hydrogen bonds of O-H•••N (Table 3), to form a dimer (Figure 4).

4. Structure Description of the Zinc Complex
Molecular structure of the zinc complex is shown in Figure 5. The Zn atom is in trigonal bipyramidal geometry, with the imino atom of the hydrazone ligand, and two chloride atoms located at the basal plane, and with the pyridine N and carbonyl O atoms located at the axial positions. The Zn atom deviates from the least-squares plane defined by the three basal donor atoms by 0.129(1) Å. The coordinate bond lengths in the complex are comparable to those observed in zinc(II) complexes with hydrazone ligands. 14 In the crystal structure of the complex, the complex molecules are linked through intermolecular hydrogen bonds of O-H•••O (Table 3), to form chains along the b axis ( Figure 6).

5. Biological Study
The percent inhibition of the compounds at concentration of 100 μmol L -1 on Jack bean urease is summarized in Table 4. The hydrazones and the zinc complex have weak activity. However, the copper complex showed strong  urease inhibitory activity, with IC 50 value of 1.4 ± 0.8 μmol L -1 . As a comparison, acetohydroxamic acid (AHA) was used as a reference drug with the percent inhibition of 84.3 ± 3.9, and with IC 50 value of 37.2 ± 4.0 μmol L -1 . Copper perchlorate can inhibit urease activity, with IC 50 value of 8.8 ± 1.4 μmol L -1 . Thus, the present copper complex is a good model for urease inhibition.

Conclusion
In summary, a new hydrazone compound 4-methoxy-N'-(pyridin-2-ylmethylene)benzohydrazide was prepared and structurally characterized. With the hydrazones, a new copper(II) complex and a new zinc(II) complex were obtained. The complexes were characterized by physico-chemical method, and their structures were confirmed by single crystal X-ray determination. The copper complex has strong urease inhibitory activity, which deserves further study to explore novel and efficient urease inhibitors.