Copper, zinc, and nickel complexes derived from 3-methyl-2-((pyridin-2-ylmethylene)amino)phenol: syntheses, characterization, crystal structures, and urease inhibitory activity

Abstract Five copper(II), zinc(II) and nickel(II) complexes, [Cu2L2(μ 1,1-N3)2] (1), [Zn2L2(μ 2-η 1:η 1-CH3COO)(NCS)]EtOH (2), [Zn2L2(μ 2-η 1:η 1-CH3COO)(N3)]MeOH (3), [Zn2Cl2L2] (4) and [NiL(HL)]ClO4 (5), were prepared from the Schiff base 3-methyl-2-((pyridin-2-ylmethylene)amino)phenol (HL). The compounds have been characterized by physico-chemical methods including elemental analysis, IR, and UV–Vis spectra. Structures of the five new complexes were determined by single crystal X-ray diffraction. The copper ions in 1 are in square pyramidal, while zinc ions in 2, 3, and 4 are distorted square pyramidal, and the nickel ions in 5 are octahedral. The biological assay indicated that zinc complex 2 has the most effective activity on Jack bean urease with IC50 = 1.7 ± 0.8 μmol·L−1. Molecular docking was performed to study the interaction between the active center of the urease with molecules of the complexes.


Materials and measurements
2-Pyridinecarboxaldehyde, 2-amino-3-methylphenol, copper nitrate, zinc acetate, zinc chloride, nickel perchlorate, sodium azide, and ammonium thiocyanate were purchased from Xiya Chemical Reagent Co. Ltd.CHN elemental analyses were performed on a Perkin-Elmer 240 C elemental analyzer.IR spectra were recorded on a Jasco FT/IR-4000 spectrometer as KBr pellets from 4000 to 400 cm À 1 .UV-Vis spectra were recorded on a Lambda 35 spectrometer. 1H and 13 C NMR data were recorded on a Bruker 500 MHz instrument.Conductivity measurements were performed using a Scheme 1.The Schiff base HL.
Metrohm 712 conductometer at 25 � C. The urease inhibitory activity was measured on a Bio-Tek Synergy HT microplate reader.Single crystal X-ray diffraction was carried out on a Bruker Apex II CCD area diffractometer.

Synthesis of the Schiff base HL
2-Pyridinecarboxaldehyde (0.010 mol, 1.1 g) diluted by methanol (20 mL) and 2-amino-3-methylphenol (0.010 mol, 1.2 g) dissolved in methanol (30 mL) were mixed together and stirred for 30 min at room temperature to give a yellowish solution.The solvent was evaporated yielding a solid product, which was re-crystallized from methanol and dried in air.Yield: 1.8 g (85%

Crystal structure determination
Single crystal X-ray diffraction was performed with a Bruker Apex II CCD diffractometer using graphite-monochromated MoK a radiation (k ¼ 0.71073 Å).Diffraction intensities for the complexes were collected at 298(2) K.The collected data were reduced with SAINT [34] and multi-scan absorption correction was performed using SADABS [35].Structures of 1-5 were solved by direct methods and refined against F 2 by full-matrix least-squares method using SHELXT [36] and SHELXL programs [37].All non-hydrogen atoms were refined anisotropically.All H atoms were placed at geometrical positions with fixed thermal parameters.Crystallographic data of 1-5 are summarized in Table 1.Selected bond lengths are listed in Table 2.

Urease inhibitory activity assay
The urease inhibitory activity was performed with the literature method [38].The assay mixture containing 75 lL of Jack bean urease and 75 lL of tested compounds dissolved in DMSO with various concentrations was preincubated for 15 min on a 96-well assay plate.Acetohydroxamic acid was used as a control drug.75 lL of phosphate buffer at pH ¼ 6.8 containing phenol red at concentration of 0.18 mmol�L À 1 and urea at  400 mmol�L À 1 were added and incubated at room temperature.The time required for enough ammonium carbonate to form to raise the pH of the phosphate buffer from 6.8 to 7.7 was measured by a micro-plate reader at 560 nm with the end-point being determined by the color change of phenol-red indicator.

Docking study
Molecular docking study was performed by AutoDock 4.0 software as implemented through the graphical user interface AutoDockTools (ADT 1.5.2).The complex molecules were docked into the active center of Jack bean urease.
During the docking process, all hydrogens of the enzyme were added, Gasteiger charges were calculated, and non-polar hydrogens were merged to carbon atoms.The parameters for the two Ni ions are set as r ¼ 1.170 Å, q ¼ þ2.0, and van der Waals well depth of 0.100 kcal�mol À 1 [39].The 3D structures of the complex molecules were saved in pdb format with Mercury, and the resulting files were saved as pdbqt format.
In the docking a grid box size of 70 � 72 � 70 points in x, y and z directions was built, and the maps were centered on the original ligand molecule (HAE) in the catalytic site of the protein.A grid spacing of 0.375 Å and distances-dependent functions of the dielectric constant were used for calculation of the energy map. 100 runs were generated by Lamarckian genetic algorithm searches.Default settings were used with an initial population of 50 randomly placed individuals, a maximum number of 2.5 � 10 6 energy evaluations, and a maximum number of 2.7 � 10 4 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.

Chemistry
The Schiff base HL was prepared by condensation of 2-pyridinecarboxaldehyde and 2-amino-3-methylphenol in 1:1 molar ratio in methanol.The 1 H NMR, 13 C NMR, IR and UV-Vis spectra of HL are given as Figures S1-S4.The five metal complexes were synthesized from the Schiff base with copper nitrate and sodium azide (1), zinc acetate and ammonium thiocyanate (2), zinc acetate and sodium azide (3), zinc chloride (4) and nickel perchlorate and ammonium thiocyanate (5), respectively, in methanol or ethanol (Scheme 2).IR and UV-Vis spectra of the complexes are given as Figures S5-S14.All the complexes are soluble in common organic solvents like methanol, ethanol, DMF and DMSO.Single crystals of 1--5 are stable in air at room temperature.Molar conductivities of 1-4 in DMSO are within the values 32-43 X À 1 �cm 2 �mol À 1 , indicating their non-electrolytic nature, and that of 5 is 172 X À 1 �cm 2 �mol À 1 , indicating its 1:1 electrolytic behavior [40].The experimental powder X-ray diffraction (XRD) patterns of the bulk samples of the complexes agree well with simulated patterns calculated from single crystal X-ray diffraction (Figures 1-5).

Complex 1
The molecular structure of the centrosymmetric 1 is shown in Figure 1.The complex contains two [CuL] units connected by two end-on azide bridging ligands with the inversion center located at the midpoint of the two Cu ions separated by 3.264(1) Å.The Cu exhibits a distorted square pyramidal coordination, the basal plane being formed by the pyridine nitrogen (N1), imino nitrogen (N2) and phenolate oxygen (O1) atoms of the Schiff base ligand, and one azide nitrogen (N3).The apical position is occupied by the azide nitrogen (N3A) of the symmetry-related azide.The Cu-O and Cu-N bond lengths are 1.901(4)-2.362(5)Å with the longest value relative to the apical N3 donor.The cis and trans bond angles around copper in the basal plane are 81.2(2)-98.1(2)� and 163.1(2)-164.4(2)� , respectively, while those among the apical and basal donor atoms are 83.1(2)-113.8(2)� , which indicate the distortions from ideal values.The deviation is mainly caused by the strain created by the five-membered chelate rings and the Cu 2 N 2 core.The Schiff base ligand is nearly coplanar, with the dihedral angle formed by the pyridine and benzene rings of 2.9(3) � .In the crystal structure, the complex molecules stack along the c axis via p���p interactions with centroid to centroid distance of 3.687(3) Å among the planes N1-C1-C2-C3-C4-C5 and Scheme 2. The synthetic procedure of 1-5.

Complexes 2 and 3
The molecular structures of 2 and 3 are shown in Figures 3 and 4, respectively.Complex 2 contains a lattice ethanol molecule and 3 a methanol one.Both structures of the dinuclear complex molecules are isostructural, differing for terminal ligand, viz.thiocyanate for 2 and azide for 3.The two Zn ions are bridged by one phenolate  oxygen and one bridging acetate ligand, and are separated by 3.422(2) Å (2) and 3.464(2) Å (3).The Zn ions have distorted square pyramidal coordination, the basal plane being formed by the pyridine nitrogen, imino nitrogen and phenolate oxygen atoms of the Schiff base ligand, and one acetate oxygen atom.For Zn1, the apical position is occupied by the oxygen atom of the symmetry related acetate.For Zn2, the apical position is occupied by the nitrogen of the thiocyanate (2) or azide (3).The distortion is evidenced by the index factor s (0.24 for Zn1 and 0.17 for Zn2 (2), 0.30 for Zn1 and 0.20 for Zn2 (3)) [43].The Zn1 ions deviated from the least-squares planes defined by the four basal donor atoms by 0.564(2) Å for 2 and 0.641(2) Å for 3. Zn2 deviated from the least-squares planes defined by the four basal donor atoms by 0.593(2) Å for 2 and 0.625(2) Å for 3.The cis and trans bond angles around zinc in the   16) � for Zn2 (2), and 98.9(2)-136.4(2)� for Zn1 and 105.8(3) -114.0(3)� for Zn2 (3), which indicate the distortions from ideal values.The dihedral angles between the pyridine and benzene rings of the Schiff base ligands are 9.0-9.1 � for 2 and 10.5-12.0� for 3.In the crystal structures, adjacent molecules are linked through C-H���O hydrogen bonds (Table 3) to form a dimer (Figures 5 and 6).In addition, there are p���p interactions with centroid to centroid distances of 3.6-4.6Å among the planes N1-C1-C2-C3-C4-C5, N3-C14-C15-C16-C17-C18, and C7-C8-C9-C10-C11-C12 for both complexes.The bond lengths of Zn-O/N are comparable to those observed in Schiff base zinc complexes [44,45].

Complex 4
The molecular structure of 4 is shown in Figure 7.The molecule possesses crystallographic inversion center symmetry, with the inversion center located at the midpoint of the two Zn ions.The Zn ions are bridged by two phenolate oxygens, with a separation of 3.139(2) Å.The Schiff base ligand coordinated with Zn ions through the phenolate oxygen, imino nitrogen, and pyridine nitrogen.The Zn exhibits distorted square pyramidal coordination, as evidenced by the index factor s (0.38).The basal plane of Note: Symmetry codes: #1: 1 þ x, y, z; #2: 2 À x, 2 À y, 1 À z; #3: 1 À x, 2 À y, 1 À z.The dihedral angle between the pyridine and benzene rings of the Schiff base ligand is 21.7(3) � .In the crystal structure, the complex molecules stack via p���p interactions with centroid to centroid distances of 3.6-4.5Å among the planes N1-C1-C2-C3-C4-C5 and C7-C8-C9-C10-C11-C12 (Figure 8).The bond lengths of Zn-O/N are comparable to those observed in zinc Schiff base complexes [44,45].

Complex 5
The molecular structure of 5 is shown in Figure 9.The asymmetric unit contains two mononuclear complex cations [NiL(HL)] þ counterbalanced by two perchlorate anions.

IR and UV spectra
The strong absorption bands at 1563-1594 cm À 1 for 1-5 are due to skeleton vibration of the azomethine groups, which are different from the free Schiff base at 1589 cm À 1 .The shifts of the absorbance indicate coordination through the azomethine N atoms.The typical absorption for azide ligands are observed at 2042 cm À 1 for 1 and 2069 cm À 1 for 3.The typical absorption for thiocyanate ligands are observed at 2076 cm À 1 for 2.An intense absorption at 1090 cm À 1 and a medium absorption at 618 cm À 1 for 5 indicate uncoordinated perchlorate anions [48].The spectra of 2 and 3 display characteristic bands of acetate ligands at 1560-1570 cm À 1 for m as (CO 2 ) and 1410 cm À 1 for m s (CO 2 ).The frequency differences (D) between m as (CO 2 ) and m s (CO 2 ) are 150-160 cm À 1 , which are within the D values in bridging acetate coordinated complexes [49].
In UV spectra of the Schiff base and the complexes, the absorption peaks at 235 nm are generated by the p!p � transition of aromatic rings and C ¼ N delocalization system in the Schiff base.The absorption peaks at 280-335 nm are generated by the n!p � transition of lone pair electrons on C ¼ N bonds, which also prove that Schiff bases are generated.

Urease inhibitory activity
The results of urease inhibition are summarized in Table 4. Copper complex 1 and zinc complexes 2, 3, and 4 have from medium to strong inhibitory activities on Jack bean urease, with percentage inhibition of 65-95% at 100 lmol�L À 1 .Nickel complex 5 has weak activity with a percentage inhibition of 22% at 100 lmol�L À 1 .The copper and nickel complexes have weaker activity than the corresponding metal salts copper chloride and nickel acetate.However, the three zinc complexes have higher activity than zinc acetate.The copper and zinc complexes have better activity than HL, while the nickel complex has lower activity than HL.Among the complexes, 2 has the most activity with IC 50 value of 1.7 ± 0.8 lmol�L À 1 , which is more effective than the reference drug acetohydroxamic acid.In general, 1-4 with azide, thiocyanate and chloride as co-ligands have better activity than 5 with no such ligands.Complexes 1-3 with azide and thiocyanate as co-ligands have higher activity than 4 with chloride as co-ligand.The structures of 2 and 3 are similar with the only difference being the terminal ligand, viz.thiocyanate for 2 and azide for 3. From the biological assay, ammonium thiocyanate and sodium azide have no urease inhibition.But the activity for 2 is higher than 3.When compared with the literature results, copper complex 1 and nickel complex 5 have lower activity against urease than the copper and nickel complexes with the Schiff base ligand N,N'-bis(5-fluorosalicylidene)-1,3-propanediamine [50].However, the present zinc complexes have stronger activities than those with Schiff ligands 2-f[1-(5-chloro-2-hydroxyphenyl)methylidene]aminog-2-methylpropane-1,3-diol [51].Thus, complex 2 may be used as an efficient urease inhibitor, which deserves further study.

Molecular docking results
Molecular docking was carried out to study the interaction between the molecules of 1-5 and the active center of the urease.In the structure of the urease, two Ni 2þ ions were coordinated by HIS136, HIS138, KCX219, HIS248, HIS274, ASP362, and water molecules, while in the acetohydroxamic acid inhibited model, the water molecules were replaced by acetohydroxamic acid.The docking scores are À 6.70 for 1, À 7.01 for 2, À 6.87 for 3, À 6.80 for 4, and À 7.45 for 5.As a comparison, the docking score for the acetohydroxamic acid-inhibited model is À 5.01.The negative values indicate that the molecules of the complexes bind well with the urease.The binding models of the complexes with the urease are depicted in Figures S15-S19.The results revealed that molecules of 1, 3, 4, and 5 located at the entry of the active pocket of the urease, while that of 2 fits well with the active site and forms hydrogen bonds with the residues HIS322 and ASN168 (Figure 11).From the docking analysis, the interactions among molecules of 2 and 3 with the urease are different.The slight difference of the molecules of the complexes may result in the distinction of the urease inhibition.Thus, more work needs to be carried out to explore the structure-activity relationship.

Conclusion
The present study reports the synthesis, characterization and crystal structures of five copper, zinc and nickel complexes with the Schiff base ligand 3-methyl-2-((pyridin-2ylmethylene)amino)phenol.Zinc complex 2 has the most activity on Jack bean urease, with IC 50 value of 1.7 ± 0.8 lmol�L À 1 .

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 1 .
Figure 1.Left: A perspective view of the molecular structure of 1 with the atom labeling scheme.Thermal ellipsoids are drawn at the 30% probability level.Atoms with suffix A and those unlabled are related to the symmetry position 2 À x, 1 À y, 1 À z.Right: The experimental and simulated powder XRD patterns of 1.

Figure 2 .
Figure 2. Molecular packing diagram of 1 viewed along the b axis.

Figure 3 .
Figure 3. Left: A perspective view of the molecular structure of 2 with the atom labeling scheme.Thermal ellipsoids are drawn at the 30% probability level.Right: The experimental and simulated powder XRD patterns of 2.

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

Figure 5 .
Figure 5. Left: A perspective view of the molecular structure of 3 with the atom labeling scheme.Thermal ellipsoids are drawn at the 30% probability level.Right: The experimental and simulated powder XRD patterns of 3.

Figure 6 .
Figure 6.Molecular packing diagram of 3 viewed along the a axis.Hydrogen bonds are shown as dashed lines.

Figure 7 .
Figure 7. Left: A perspective view of the molecular structure of 4 with the atom labeling scheme.Thermal ellipsoids are drawn at the 30% probability level.Atoms with suffix A and those unlabled are related to the symmetry position 1 À x, 1 À y, 1 À z.Right: The experimental and simulated powder XRD patterns of 4.

Figure 8 .
Figure 8.Molecular packing diagram of 4 viewed along the b axis.

Figure 9 .
Figure 9. Left: A perspective view of the molecular structure of 5 with the atom labeling scheme.Thermal ellipsoids are drawn at the 30% probability level.Right: The experimental and simulated powder XRD patterns of 5.

Figure 10 .
Figure 10.Molecular packing diagram of 5 viewed along the a axis.Hydrogen bonds are shown as dashed lines.

Table 4 .
Urease inhibition of the compounds.The concentration of the tested material is 100 lmol�L À 1 . a