Syntheses, crystal structures, andJack beanurease inhibitory activities of copper(II) complexes derived from 4-tert-butyl-N′-(1-(pyridin-2-yl)ethylidene)benzohydrazide

Two new copper(II) complexes, [CuL(HL)]·ClO₄ (1) and [Cu₂Br₂L₂]·0.85H₂O (2), where L is the monoanionic form of 4-tert-butyl-N′-(1-(pyridin-2-yl)ethylidene)benzohydrazide (HL), have been prepared. The complexes were characterized by infrared and UV–vis spectra, and single crystal X-ray diffraction. Complex 1 is a mononuclear copper(II) species and 2 is a bromido-bridged dinuclear copper(II) species. The Cu ion in 1 is in an octahedral coordination mode and that in 2 is trigonal-bipyramidal. The Jack bean urease inhibitory assay indicated that 2 is active, with IC₅₀ value of 20.6 ± 2.3 μmol L⁻¹, while 1 is inactive. Molecular docking of 2 with Jack bean urease was studied.

Keywords: Hydrazone; copper complex; crystal structure; urease inhibition; molecular docking

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Urease is widely found in a variety of organisms such as plants, fungi, algae and bacteria. This enzyme is regarded as a virulent factor in human and animal infections of the urinary and gastrointestinal tracts. It may cause urolithiasis, pyelonephritis, chronic gastritis, duodenal ulcer, gastric ulcer, and even gastric cancer. Urease catalyzes the decomposition of urea to generate ammonia and carbon dioxide [[1]]. The high concentration of ammonia arising from the reaction, as well as the accompanying pH elevation, has important negative implications in medicine and agriculture [[3]]. A great effort has been made to control the activity of urease. As a result, urease inhibitors seem applicable to counteract the negative effects [[7]]. Inhibitors of urease include acetohydroxamic acid, humic acid and 1,4-benzoquinone [[8]], and heavy metal ions such as Cu2+, Zn2+, Pd2+, and Cd2+ [[11]]. Metal complexes have been known as interesting enzyme inhibitors [[13]]. From the point of structural and coordination chemistry, many organic urease inhibitors are suitable ligands, which can coordinate to inorganic urease inhibitors. The complexes bearing both organic and inorganic components may possess higher urease inhibitory activities than their precursors. In recent years, we have reported a number of metal complexes with urease inhibitory activities [[14]]. Taha and coworkers reported that some hydrazones have effective urease inhibitory activities [[19]]. Khan and coworkers also reported that hydrazone derivatives have effective urease inhibitory activities [[20]]. However, the relationship between structures and urease inhibitory activities is not clear. In this paper, two new copper(II) complexes, [CuL(HL)]·ClO4 (1) and [Cu2Br2L2]·0.85H2O (2), where L is the monoanionic form of 4-tert-butyl-N′-(1-(pyridin-2-yl)ethylidene)benzohydrazide (HL), were synthesized and structurally characterized. The urease inhibitory activities of the complexes were studied, and molecular docking analysis was performed.

2-Acetylpyridine and 4-tert-butylbenzohydrazide were purchased from Lancaster. Jack bean urease was purchased from Toyobo (Japan). Copper perchlorate hexahydrate, potassium bromide, acetohydroxamic acid, phenol red, urea, and solvents were obtained from Xiya Reagent Co. Ltd. The hydrazone was prepared according to the literature method [[21]]. 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 from 4000–400 cm−1. UV–vis spectra were recorded on a Perkin-Elmer Lambda 35 spectrometer. The urease inhibitory activity was measured on a Bio-Tek Synergy HT microplate reader. Single crystal structures were determined using a Bruker D8 Venture single crystal diffractometer.

Caution: Perchlorate salts are potentially explosive; only small quantities should be used and handled with great care.

Bis[4-tert-butyl-N′-(1-(pyridin-2-yl)ethylidene)benzohydrazido]copper(II) perchlorate (1). HL (1.0 mmol, 0.295 g) was dissolved in methanol (15 mL), to which was added dropwise Cu(ClO4)2·6H2O (1.0 mmol, 0.371 g) dissolved in methanol (20 mL). The mixture was stirred magnetically for 10 min at room temperature and filtered. The filtrate was kept in air for a few days to form blue single crystals suitable for X-ray diffraction. The crystals were isolated and dried in air. Yield: 0.171 g (45%). IR data (KBr, cm−1): 3458w, 3216w, 1660 m, 1607s, 1444 m, 1375 m, 1268w, 1164s, 1088s, 945 m, 857s, 780w, 619w, 546 m, 519 m. UV–Vis data [methanol, λ/nm (ε/L mol−1 cm−1)]: 272 (13,860), 370 (21,100). Anal. Calcd for C36H41ClCuN6O6: C, 57.4; H, 5.5; N, 11.2. Found: C, 57.2; H, 5.6; N, 11.1%.

Di-μ-bromido-bis[4-tert-butyl-N′-(1-(pyridin-2-yl)ethylidene)benzohydrazido]dicopper(II) 0.85 hydrate (2). HL (1.0 mmol, 0.295 g) was dissolved in methanol (15 mL), to which was added dropwise CuBr2 (1.0 mmol, 0.223 g) dissolved in methanol (20 mL). The mixture was stirred magnetically for 10 min at room temperature and filtered. The filtrate was kept in air for a few days to form blue single crystals suitable for X-ray diffraction. The crystals were isolated and dried in air. Yield: 0.183 g (40%). IR data (KBr, cm−1): 3441 m, 1603s, 1559 m, 1522 m, 1492 m, 1465s, 1445 m, 1377 m, 1307w, 1290s, 1267w, 1159s, 1126 m, 1072w, 947w, 860 m, 776 m, 709w, 545 m, 517w, 467w. UV–Vis data [methanol, λ/nm (ε/L mol−1 cm−1)]: 270 (14,550), 382 (18,430). Anal. Calcd for C36H41.7Br2Cu2N6O2.8: C, 48.6; H, 4.7; N, 9.4. Found: C, 48.2; H, 4.8; N, 9.3%. The complex can also be prepared by the reaction of 1 with potassium bromide in methanol.

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 [[22]], and multi-scan absorption correction was performed using SADABS [[23]]. Structures of the complexes were solved by direct methods and refined against F2 by full-matrix least-squares using SHELXTL [[24]]. All non-hydrogen atoms were refined anisotropically. The amino H atom (H3) in 1 was located from a difference Fourier map and refined isotropically, with N–H distance restrained to 0.90(1) Å. The remaining hydrogens were placed in calculated positions and constrained to ride on their parent atoms. The disordered water atoms, O3 and O4, were first refined with thermal parameters constrained to 0.5 to calculate the possible occupancies, 0.46 and 0.39. Then, the occupancies were constrained and the thermal parameters refined. Crystallographic data for 1 and 2 are summarized in Table 1. Selected bond lengths and angles are given in Table 2.

Table 1. Crystal data for 1 and 2.

Table 2. Selected bond lengths (Å) and angles (°) for 1 and 2.

The measurement of urease inhibitory activity was carried out according to the literature method [[25]]. 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 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.

The molecular docking study of 2 into the 3-D X-ray structure of the Jack bean urease (entry 4UBP in the Protein Data Bank) was carried out 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 set up the enzymes: 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 a van der Waals well depth of 0.100 kcal mol−1 [[26]]. The 3-D structure of the ligand molecule was saved in pdb format with the aid of the program ChemBio3-D. The resulting file was saved as pdbqt format.

AutoDockTools was used to generate the docking input files. In all docking a grid box size of 90 × 90 × 80 points in x, y, and z directions was built; the maps were centered on the original ligand molecule, hydroxamic 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 2.5 × 106 energy evaluations, and a maximum number of 2.7 × 104 generations. A mutation rate of 0.02 and a crossover rate of 0.8 were chosen. The result of the most favorable free energy of binding was selected as the resultant complex structure.

Mononuclear copper complex 1 was prepared by reaction of equimolar quantities of the hydrazone ligand HL and copper perchlorate in methanol. Dinuclear copper complex 2 was prepared by the reaction of 1 with potassium bromide in methanol. Or, 2 can be prepared directly from the reaction of HL with copper bromide. Single crystals were obtained by slow evaporation of the methanolic solution of the complexes. The complexes are stable in air at room temperature. Molar conductivities of 1 and 2 measured in methanol at a concentration of 10−3 mol L−1 are 127 and 18 Ω−1 cm2 mol−1.

The molecular structure of 1 is shown in Figure 1. The complex contains a mononuclear copper(II) complex cation and a perchlorate anion. The Cu ion is coordinated by the pyridine nitrogen (N1), imino nitrogen (N2) and carbonyl oxygen (O1) atoms of the neutral hydrazone ligand, and by the pyridine nitrogen (N4), imino nitrogen (N5) and enolate oxygen (O2) atoms of the deprotonated hydrazone ligand to form an octahedral coordination. The different coordination mode may be due to the Jahn-Teller effect. The axial bonds Cu1–O1 and Cu1–N1 are much longer than those in the equatorial plane, which is defined by N2, N4, N5, and O2. The Cu atom deviates from the least-squares plane defined by the four equatorial donor atoms by 0.021(1) Å. The distortion of the octahedral geometry can also be observed from the coordinate bond angles (Table 2), which obviously deviate from the ideal values of 90° for the cis angles and 180° for the trans angles. The bond lengths and angles of the complex agree well with the equivalent values of the hexa-coordinated copper(II) complexes derived from the ligands N′-[phenyl(pyridin-2-yl)methylidene]furan-2-carbohydrazide [[27]] and N′-[phenyl(pyridin-2-yl)methylidene]benzohydrazide [[28]].

Graph: Figure 1. The molecular structure of 1 showing the atom-numbering scheme.

The molecular structure of 2 is shown in Figure 2. The complex contains two bromido-bridged dinuclear copper(II) complex molecules and a 0.85 disordered water molecule of crystallization. The Cu⋯Cu distances are 3.43–3.45 Å. Each Cu ion is in a trigonal-bipyramidal coordination, with the imino nitrogen atom of the hydrazone ligand and two Br ions defining the basal plane, and with the pyridine nitrogen and enolate oxygen atoms occupying the axial positions. The Cu1 and Cu2 atoms deviate from the corresponding least-squares plane defined by the three basal donor atoms by 0.003(1) Å and 0.008(1) Å, respectively. The distortion of the trigonal-bipyramidal geometry can be observed from the coordinate bond angles (Table 2), which deviate from the ideal values of 120° for the basal angles and 180° for the diagonal angles. The bond lengths and angles of the complex agree well with the equivalent values of the penta-coordinated copper(II) complexes derived from the ligands acetylpyridine benzoyl hydrazone [[29]] and alkoxy diazine hydrazones [[30]].

Graph: Figure 2. The molecular structure of 2 showing the atom-numbering scheme.

For the free aroylhydrazone HL, a sharp band indicative of the N–H vibration is located at 3251 cm−1, and the typical strong ν(C=O) absorption is observed at 1655 cm−1. The strong absorption band at 1612 cm−1 for HL is assigned to ν(C=N) of the azomethine group [[31]], which is located at 1607 cm−1 for 1 and at 1603 cm−1 for 2. The sharp and weak absorption at 3216 cm−1 in the spectrum of 1 substantiates the presence of an N–H group. The broad and medium absorption centered at 3441 cm−1 in the spectrum of 2 substantiates the presence of O–H groups. The intense band indicative of the C=O vibration of the hydrazone ligand in 1 is located at 1660 cm−1, while there is no such absorption in 2. The shift or absence of the absorptions indicated that the aroylhydrazone ligands coordinated to the metal atoms either through carbonyl or through enolate forms. The intense absorption for the perchlorate anion is located at 1088 cm−1 for 1.

Electronic spectra of HL and the complexes were recorded at a concentration of 10−5 mol L−1 in methanol. The bands centered at 250–280 nm may be assigned to π–π* transitions, and those set at 320–360 nm may be assigned to n–π* transitions of the azomethine and pyridine groups. The charge transfer LMCT bands are centered at 370 nm for 1 and 380 nm for 2. The spectra showed weak and broad d–d electronic transitions at 630–720 nm, which are assigned to 2Eg(D) → 2T2 g(D) [[32]]. The broadness of the bands is due to the ligand field and the Jahn–Teller effects.

The percent inhibition of the complexes at a concentration of 100 μmol L−1 on Jack bean urease is summarized in Table 3. Acetohydroxamic acid was used as a reference with the percent inhibition of 85.5 ± 4.3, and with an IC50 value of 35.7 ± 2.6 μmol L−1. Copper perchlorate has strong urease inhibitory activity with an IC50 value of 8.8 ± 1.4 μmol·L−1, while the free hydrazone has no obvious activity. Complex 2 shows strong activity on the inhibition of urease with an IC50 value of 20.6 ± 2.3 μmol·L−1, while 1 has weak activity. Complex 2 has stronger activity than the reference drug acetohydroxamic acid, but weaker than copper perchlorate. The IC50 value of the copper perchlorate is lower than those of the complexes and the reference drug.

Table 3. Inhibition of urease by the tested materials.

1 The concentration of the tested material is 100 μmol·L−1.

The mechanism of inhibition of urease by 2 was investigated in a kinetic inhibition study with a Lineweaver-Burk plot (Figure 3). The plots show that the complex inhibits the urease in a mixed competitive mode.

Graph: Figure 3. Lineweaver–Burk plot for the inhibition of urease by the copper complex.

A molecular docking study was performed to investigate the binding effects between the molecule of 2 and the active site of the Jack bean urease. The binding model of the complex with the urease is depicted in Figure 4. The result revealed that the molecule of 2 fits well with the active pocket of the urease. Additional interactions have been established in a variety of conformations because of the flexibility of the complex molecule and the amino acid residues of the enzyme. The optimized clusters were ranked by energy level in the best conformation of inhibitor–urease modeled structures, where the docking score is −7.26. As a comparison, the docking score for acetohydroxamic acid inhibited model is −5.01. The negative values indicate that the complex molecule binds well with the urease. The molecule of the complex forms hydrogen bonds with the amino-acid residue HIS323. In addition, some hydrophobic interactions exist in the binding model of the inhibitor–urease complex.

Graph: Figure 4. Binding mode of 2 with Jack bean urease.

The present study reports the synthesis, characterization and crystal structures of two new copper(II) complexes derived from 4-tert-butyl-N′-(1-(pyridin-2-yl)ethylidene)benzohydrazide. The bromido-bridged dinuclear copper complex shows strong urease inhibitory activity, with IC50 value of 20.6 ± 2.3 μmol·L−1. Molecular docking study of 2 with Jack bean urease indicated that the molecule of the complex fits well with the active pocket of the urease. However, the urease inhibitory activity of 2 is still less than that of copper perchlorate, which deserves further study to explore more efficient urease inhibitors.

No potential conflict of interest was reported by the authors.

This work was supported by the Natural Science Foundation of China [grant number 21641012].

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CCDC 1548391 (1) and 1548390 (2) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge viahttp://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Center, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: (+44) 1223-336-033; or E-mail: deposit@ccdc.cam.ac.uk.