Synthesis , characterization and pharmacological evaluation of mixed ligand-metal complexes containing omeprazole and 8-hydroxyquinoline

Purpose: To synthesize a series of mixed ligand-metal complexes and to evaluate their alkaline phosphatase inhibitory capacities, antioxidant potential and antimicrobial activities. Method: Mixed ligand-metal complexes of Zn (II), Ni (II), Co (II), Cu (II), omeprazole and 8hydroxyquinoline were synthesized. The ligand-metal complexes were characterized by various physicochemical techniques, including elemental analysis, magnetic susceptibility, scanning electron microscope (SEM), mass spectrometry (EI-MS), ultraviolet-visible (UV-Vis) spectrophotometry, Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (H-NMR) and conductance studies. The new compounds were also investigated for their alkaline phosphatase (ALPs) inhibition, 2,2’-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging and antimicrobial activities. Results: Spectroscopic studies revealed the chemical composition of synthesized compounds as well as the bidentate bonding behavior of the coordinating ligands with metal ions. Conductance measurement suggested that the metal complexes were non-electrolytes. Ni(II) complex exhibited antioxidant activity (30.48 ± 0.32 M) higher than those of BHT (standard) and other complexes. Stronger inhibition of ALPs by Ni (II) mixed ligand complex compared to the other complexes was evident. The synthesized compounds showed moderate to very good antimicrobial activity against bacterial strains, i.e., Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Actinomyces viscosus, Staphylococcus aureus, Bacillus subtilis; as well as against the fungal strains, Candida albicans, Aspergillus flavus and Aspergillus niger. Conclusion: All the mixed ligand complexes demonstrate higher antioxidant, ALPs-inhibitory and antimicrobial activities than their corresponding ligands. This indicates their therapeutic potential as future drug candidates for the concerned diseases.


INTRODUCTION
Omeprazole belongs to a class of substituted benzimidazole and has some clinical applications in treatment of peptic-acid diseases like gastric and esophageal ulceration and prevention of their relapse [1].Acid inhibition strategies such as histamine H 2 -receptor antagonist and use of anti-cholinergic agents have been employed to treat peptic ulcer disease.Omeprazole is a proton pump inhibitor which promotes reduction of parietal cells by a series of prototropic reactions.The long duration of action of parietal cell H + -K + -ATPase is due to prolonged binding of the active form of the drug [2][3][4].Omeprazole has a privileged structure in pharmacophore and medicine chemistry.The importance of this compound is due to its bicyclic structure which is a fusion of benzene and imidazole rings.The plasma elimination half-life (t 1/2β ) of omeprazole is usually shorter than one hour, although a few individuals exhibit slower elimination (t 1/2β ≈ 2h).These individuals have recently been shown to be poor hydroxylators of S-mephenytoin, which suggests that a major part of omeprazole metabolism is mediated by S-mephenytoin hydroxylase, an enzyme within the cytochrome P450 system [5,6].
Omeprazole is completely metabolized and its average plasma clearance is 30 L/h.The drug also shows good antimicrobial activity against Helicobacter pylori [7-9].8-hydroxyquinoline is well-known for its antimicrobial activity.It has been comprehensively used for analytical determination of metal ions [10].Cheatum et al [11] studied the dynamic excited state of HQL dimmers.
In the present study, an attempt has been made to synthesize Zn (II), Co(II), Ni(II) and Cu(II) mixed ligand complexes of omeprazole and 8hydroxyquinoline.All the complexes were characterized by spectroscopic techniques and their biological activities were studied.

EXPERIMENTAL
All chemicals and solvents of analytical grade were purchased from Merck and Sigma-Aldrich.UV-visible spectra were recorded from 200 -800 nm on Jasco UV visible spectrophotometer model V-770 UV-VIS.FT-IR spectra were recorded on Nicolet IS50, FT-IR spectrophotometer from 400-4000 cm -1 .Elemental analyses were performed on a CHNS/O-2400 Series II elemental analyzer.Magnetic susceptibility was determined using Holmarc's Magnetic Susceptibility (model HO-ED-EM-07).Mass spectral measurement was performed on Finnigan MAT LCQ TM mass spectrometer.The molar conductance of the synthesized complexes in DMSO was measured using a Conductivity Meter, Model WD-35604-00 at 25 °C temperature.SEM images were recorded in a Hitachi SEM analyzer. 1H-NMR spectra were recorded in DMSO-d 6 at room temperature using TMS as internal standard on a Bruker 400 MHz spectrophotometer.

Synthesis of mixed ligand metal complexes
Mixed ligand complexes were synthesized by mixing water-ethanol solutions (1:1, v/v) of metal salts (1 mmol), ethanolic solution of primary ligand (L) omeprazole (0.345 g, 1mmol) and secondary ligands 8-hydroxyquinoline (L2, 0.145 g, 1 mmol) in molar ratio of 1:1:1 (M:L:L2).The resultant mixture was refluxed for 3-4 h and colored precipitates were obtained in each case.The solids precipitated were filtered and washed with diethyl ether-ethanol (1:1 v/v).Stability constants were also calculated. Figure 1 shows the structural formula of the mixed ligand metal complexes.

Assay of inhibitory effects of metal complexes on ALPs
The assay of inhibition of ALPs was done spectrophotometrically as described earlier [12].Working substrate was prepared by mixing four parts of diethanolamine (pH = 9.8, 2 mol/dm 3 ) and MgCl 2 (0.5 mmol/dm 3 ), and one part of p-NPP (50 mmol/dm 3 ).This substrate was incubated at 37 o C for 5 min.The ALP assay of OMZ, HQL and metals complexes were determined by addition of 2 mL of working substrate to 40 µL of human serum.ALP activity was assayed with 25 mM stock solutions of OMZ, HQL and the synthesized complexes.After the incubation period, absorbance was recorded at 405 nm [13].All measurements were done in triplicate and the mean of these values was used to calculate the percentage inhibition of ALPs activity.The principle of this assay is that colorless p-Nitrophenyl phosphate (substrate) in alkaline medium changes to a yellow-colored due to liberation of p-Nitrophenol by ALPs.

Determination of DPPH radical scavenging activity
DPPH free radical scavenging activity is a rapid technique for assay of antioxidant activities of compounds.Using the method as described by Khan et al [14] the DPPH free radical scavenging potential of the mixed ligand complexes were determined relative to standard i.e. butylated hydroxytoluene (BHT).In the assays, OMZ, HQL and synthesized mixed ligand metals complexes at various concentrations (500, 250, 125 and 60 μg/mL) were incubated with 3 ml of 0.1 mM DPPH in methanol for 60 min at room temperature.Absorbance was measured at 517 nm against methanol.The absorbance was decreased by conversion of DPPH to the more stable DPPH 2 molecules, which serves as an indication of the capacity of antioxidant compounds to donate hydrogen ion [15].All measurements were taken in triplicates.DPPH radical scavenging activity was calculated using the formula: DPPH scavenging (%) = {(Ac -As)/Ac}100… (1) where Ac is the absorbance of control and As is the absorbance of tested compounds.

Antimicrobial assay
The antimicrobial properties of OMZ, HQL and synthesized complexes were determined using the agar-well diffusion method [16].Six bacterial strains including three Gram-negative strains, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, and three Grampositive strains, Actinomyces viscosus, Staphylococcus aureus, and Bacillus subtilis were used.Antifungal activity was determined against Candida albicans, Aspergillus nigar and Aspergillus flavus.The culture media was prepared by dissolving 28 g of nutrient agar in 1L of distilled water (for bacterial strains), and 38 g of potato dextrose agar in 1L of distilled water for fungi.The media were autoclaved at 121°C and 15 LD/sq inch for 15 min.The stock solution (100 μL in each dish) was put into Petri dishes and then allowed to attain room temperature.The microorganisms were cultured and incubated for 24 h at 37 °C (for bacterial cultures) and for 48 h at 28 °C (for fungal cultures) [17,18].Zones of inhibition in specimen Petri dishes were measured in mm with venire caliper in mm.The anti-fungal drug fluconazole and antibacterial drug ampicillin (100 μg/mL) were used as standard controls for comparison with the tested specimens under similar conditions.These assays were performed in triplicate, and the mean was taken as the final reading.

Statistical analysis
Analysis of data was carried out using Microsoft Excel-2016 software.All computations were executed in triplicate and the results were expressed as mean ± SEM (n = 3).Antimicrobial, DPPH and ALP assays were computed with suitable dilutions for each sample and different statistical techniques such as Duncan multiple range method, one-way analysis of variance were used for analysis of data obtained from different samples.P < 0.05 was taken as indicative of statistical significance.

RESULTS
The synthesized mixture of ligand complexes showed yields that ranged from good to excellent yield (up to 89 %).The purity of the complexes was checked by melting point determination.Sharp melting points were obtained with fairly pure complexes.Low values of conductance measurements suggested the non-electrolyte nature of synthesized mixed ligand metal complexes.The chemical structures of all the synthesized compounds were deduced with the aid of physical and spectroscopic methods.Physical data and elemental (CHNS/O) analysis of these complexes were in good agreement with the proposed formulae.The general reaction equation ( 2) for the synthesis of the complexes is shown in Eq 2.

Magnetic susceptibility
The mixed ligand metal complexes had magnetic moments in the range of

FT-IR studies
The two main regions are of interest in mixed ligand complexes because they give valuable information about the coordination behavior of ligands to metals.These are C=N and S=O (sulphonic acid group).In the IR spectra of OMZ, a peak appeared at 3560 cm -1 . This peak was due to the stretching vibration of v(N-H) in benzimidazole.The stretching vibration of C=N occurs at 1586 cm -1 while that of S=O occurs at 1010 cm -1 .In the case of 8-hydroxyquinoline (HQL) molecules, the stretching vibration of v(CO) appears in medium peak at 1712 cm -1 whereas v(C=N) and v(OH) stretching vibrations occur at 1643 cm -1 and 3453 cm -1 , respectively.
In the FTIR spectra of the mixed ligand metal complexes, the stretching vibration of v(N-H) of the benzimidazole appeared at ~3060 cm -1 , indicating its non-involvement in coordination with metal ions.The peaks for v(S=O) (sulphonic acid group) appeared between 1023 and 1032 cm -1 , with a shift of 13-22 cm -1 to the higher wave number relative to OMZ, indicating the involvement of S=O group in the coordination of the complexes.The region between 1559-1534 cm -1 was allocated to the v(C=N) bnz vibrations of all the complexes with shift of 27-52 cm -1 to the lower wave number, than C=N bond become weak due to dπ-pπ (metal to ligand) back donation and it shifts to lower wave number which indicating its involvement in coordination with metal ions.In the case of stretching vibrations, v(C=N) HQL appeared at 1623-1637 cm -1 but it also shift to lower wave number of 6-20 cm -1 .
The stretching vibration of v(CO) (HQL) appeared between 1723-1733 cm -1 , with shift of 11-21 cm -1 to the higher wave number, confirming attachment to metal ions.The shifting of peaks of the C=N, S=O and CO on complexation gives valuable information about the point of attachment of metal ions to OMZ and 8-HQL; this also confirms that OMZ and HQL behave as bidentate ligands.In the case of HQL there is a considerable shift in the frequencies of O and C=N group and also acting as a bidentate ligand due to coordination with metal in all the complexes.The broad-stretching vibration bands found within the range 3600-2700 cm -1 was assigned to the coordination of water molecules in the Cu(II) and Ni(II) complexes.New absorption bonds of M-O stretching mode of vibration were assigned 479-488 cm -1 of the synthesized complexes, while M-N stretching vibration mode was assigned within the range of 435-449 cm -1 .

Electronic absorption spectra
The absorption spectra of 2.5 x 10 -3 M solutions of OMZ, HQL and synthesized complexes in DMSO were recorded from 200 to 800 nm at room temperature.UV spectra showed high absorption intensity in ultraviolet (UV) region due to presence of benzimidazole chromosphere and these complexes are inactive in the visible region.Two strong bands were observed for OMZ at 257 and 300 nm, and for 8hydroxyquinoline at 263 and 293 nm.These bands were attributed to n→π* and π→π* electronic transition in the framework of the synthesized complexes.In all the complexes, bands that commonly appeared in-between 289-343 nm were attributed to intra ligand transitions consistent with electronic transition of C=N group.Upon complexation, the absorption bands of the complexes were slightly shifted to higher wavelength (red shift) compared to those of the free ligand omeprazole as well as 8hydroxyquinoline.These modifications of the shifts and intensities of the absorption bands indicate the coordination of the ligand to the metal ions.A square-planar arrangement around Zn (II) complex is supported due to occurrence of intermolecular charge transfer and ligand-tometal charge transfer (LMCT) at 289 and 229 nm, respectively.However in Co (II), Ni (II) and Cu (II) complexes, the structural geometry is octahedral due to attachment of water molecule.This was confirmed by FT-IR spectra of these complexes.

Morphological and structural properties of synthesized metal ligand complexes
The morphological and structural properties of the mixed ligand metal complexes were evaluated using scanning electron microscopy (SEM).The SEM micrographs are shown in Figure 2. Zn (II) and Co (II) complexes showed particles with sizes in the nanometer range, and their structures are homogenous.Some Ni (II) and Cu (II) complexes were irregularly-shaped and well-separated from each other, while most are present in agglomerated forms.Thus, these SEM results confirmed the nano-structured behavior of the synthesized metal complexes.

H NMR Spectra
The 1 H NMR spectra of the synthesized mixed ligand complexes was obtained in deuterated DMSO-d 6 .In 1 H NMR spectrum, the signal that appeared around the δ 3.42-3.51ppm was assigned to O-CH 3 , while methylene (-CH 2) protons were responsible for the doublet peak at δ 4.64-4.69ppm in all the synthesized complexes.The NMR signals for CH group were observed downfield in comparison to normal position because of the electron-withdrawing effect of neighboring atoms like nitrogen and oxygen in five-membered rings which make it more de-shielded.
There was a weak signal at 2.16 ppm which may be assigned to the methyl (-CH 3 ) protons but its integration showed otherwise.It may be that it was unusually merged with the DMSO peak.Due to non-involvement of NH proton in coordination, the singlet peak was observed at 11.23 ppm in all the synthesized complexes.The aromatic hydrogens were observed at δ 6.916-8.446ppm. Figure 3 shows the 1 H NMR spectra of Zn (II) mixed ligand complex.

Mass spectra
The mass spectra were recorded for synthesized mixed ligand complexes at 300 °C and 70 eV.All the spectra exhibited parent peaks due to molecular ions.The proposed molecular formulae of these complexes were confirmed by comparing their molecular formula weights with m/z values.Molecular ion peaks were observed in complexes of Zn (II), Ni (II), Cu (II) and Co (II) at m/z 554, m/z 584, m/z 589, m/z 584, respectively.This was in line with the composition of synthesized complexes.Mass spectra of Zn (II) and Ni (II) complexes are shown in Figure 4 and Figure 5.

Antibacterial activities of synthesized metal ligand complexes
The antibacterial activity of OMZ, HQL and synthesized complexes were investigated against three gram-negative strains, E. coli, P. aeruginosa, and K. pneumoniae, as well as three gram-positive strains, A. viscosus, S. aureus and B. subtilis.Cu(II) complex had the largest zones of inhibition against E. coli, S. aureus and A. viscosus.Ni(II) complex produced the largest antibacterial activity against P. aeruginosa and E. coli.Zn (II) complex higher inhibition zones for K. pneumoiae than any of the other complexes or Ampicillin.The order of antibacterial activity of the compounds was Cu(II) > Ni(II) > OMZ > Zn(II) > HQL > Ampicillin.The antibacterial effects of OMZ, HQL and synthesized complexes against both gram-negative and gram-positive microscopic organisms are shown in Table 1.

Antifungal activity of synthesized complexes
The antifungal activities of OMZ, HQL and synthesized complexes were determined against three parasitic strains: C. albicans, A. flavus, and A. niger.The complexes showed highest antifungal activity against fungal strains due to presence of S=O and C=N groups in their structures.Zn (II) complex showed the highest zones of inhibition against C. albicans and A. niger, while Co (II) complex had the highest activity against C. albicans and A. flavus.Cu (II) complex was more active against A. niger but lower in activity than OMZ.The order of antifungal activity of the compounds was Zn(II) >Co(II) >Ni(II) >Cu(II) >OMZ > HQL > Fluconazole.The antifungal activity of OMZ, HQL and synthesized complexes are shown in Table 2.