Abeer A El-Habeeb

Department of Chemistry,College of Science,Princess Nourah bint Abdulrahman University,Riyadh 11671,Saudi Arabia

Abstract Pipemidic acid is one of an efficient quinolone antibacterial drug. Thecomplexitybetween pipemidic acid “Hpipc” withgallium(Ⅲ),germanium(Ⅳ) and silicon(Ⅳ) afforded three molecular formulas of [Ga(pipc)2(H2O)(Cl)]·4H2O,1,[Ge(pipc)2(Cl)2]·4H2O,2 and[Si(pipc)2(Cl)2]·4H2O,3 complexes. These three new complexes were characterized based on elemental analysis,conductance,FTIR,UV-Vis,1HNMR and XRD spectroscopy. The pipc chelate exhibits O,O coordinated through the carbonyl and carboxylato (COO) of both oxygen atoms. Six coordinate geometry was proposed regarding complexes of 2 and 3,so the axial position was occupied by two coordinated chlorideatoms. In vitro,the antibacterial,antifungal,and anti-cancer assessments concerning the synthesized pipc complexes were scanned. These complexes have an excellent anti-microbial activity.

Keywords Pipemidic; Metal ions; Chelation; FTIR; XRD; Biological activity

Introduction

The chemical nomenclature of pipemidic acid (pipc) is “5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic acid” with substituted in two positions 2 and 8 by piperazinyl and ethyl moietiesas mentioned in Figure 1. The therapeutic agent of pipemidic acid as an antibiotic of quinolone derivatives is focused on urinary tract infections[1-2]. Literature survey revealed that the Ca2+,Sr2+,Ba2+,Sn4+complexes of the protonated pipc acid were synthesized and discussed[3],also,some of the lanthanides (La3+,Ce3+,Pr3+,Nd3+,Sm3+,Tb3+,Dy3+and Y3+) metal ions[4]as well as copper(Ⅱ) and managanese(Ⅱ) pentacoordinate complexes were prepared and well characterized[5-6].

In particular,the complexity of Ga(Ⅲ) by multi-chelators is expected to prevent hydrolysis processes,while improving the bioavailability and permeability of the cell membrane of the compounds[7-8].

Fig.1 Chemical structure of pipemidic acid (pipc)

The chemistry of coordinate silicon compounds has attracted considerable interest from many points of view such as reactivity,biological activity and structural features[9-10]. In 1991,it was suggested that silicon play a vital role allowing the interaction between one of the many extracellular matrix proteins detected and cell surface receptors[11]. Such a role would be a mechanism by which silicon could have beneficial measures that go beyond mere connective tissue,including cardiovascular health[12],wounding[13]and the immune function,that affected by pro-inflammatory cytokines.

Taking in view the biological and medicinal importance ofpipc and the efficiency role of metal ions in biology section,herein the article is reported the synthesis of GeⅣ,GaⅢand SiⅣcomplexes ofpipc ligandin situmethanol solvent at neutralized pH. In spite of the importance of Ga(Ⅲ) complexes as anticancer drugs,the single work reported for Ga-PPA complex[15]didn’t discuss this effect,sothis work will give more light about this point. Complexes of pipc drug can form a new class of mineral-based bioactive compounds,moreover,they may have a tendency to reduce resistance to bacterial strains. Therefore,these complexes were investigated in vitro antibacterial activity against Gram-positive/negative bacterial strains as well as human cellular carcinoma cells (HepG-2).

1 Experimental details

1.1 Materials and instrumentation

The pure chemicals and reagents used in this study were purchased from “Sigma Aldrich Chemical Corporation,St. Louis,Mo,USA”. These chemicals are summarized as: GaCl3; GeCl4; SiCl4and pipemidic acid. The microanalytical,physical and spectroscopic techniques as well as their models which were used in the interpretations of the solid synthesized complexes can be listed as follows:

Type of analysisModelsElemental analysesPerkin Elmer CHN 2400ConductanceJenway 4010 conductivity meterFTIR spectraBruker FTIR SpectrophotometerElectronic spectraUV2 Unicam UV/Vis SpectrophotometerMagnetic susceptibilityMagnetic Susceptibility Balance,1H-NMR spectraVarian Mercury VX-300 NMR spectrometer, 300 MHzXRDX 'Pert PRO PANanalytical, with copper target

1.2 Synthesis

The solid pipc complexes [Ga(pipc)2(H2O)(Cl)]·4H2O(1),Ge(pipc)2(Cl)2]·4H2O (2) and [Si(pipc)2(Cl)2]·4H2O (3) with yellow and white colors,respectively were synthesized by the same procedure: A mixtures of 2.0 mmol of GaCl3,GeCl4or SiCl4and 4.0 mmol of pipcwere stirred in 25 mL of CH3OH solvent. The mixtures were neutralized within pH=7～8 using ammonia solution,then refluxed for about three hours. The solutions of these mixtures were reduced by left them for one week. The solid yields of pipc are located within 60%～66% with higher melting point >300 ℃. The micro analytical analyses of the calculated (Calc. ) and experimental data (Found) of the three synthesized complexes are collected in compacted as follows:

ComplexElementCalc.Found1%C42.0441.87%H5.295.20%N17.5116.75%Cl4.434.392%C41.0039.83%H4.924.36%N17.0817.03%Cl8.648.583%C43.3643.31%H5.204.99%N18.0617.37%Cl9.148.98

1.3 Biological activities

Kirby-Bauer disc diffusion modified method was employed to assessment of theantimicrobial activity of the metal complexity of pipc samples[16]. In vitro,the cytotoxicity assay of the gallium(Ⅲ) pipc complex was performed on the human Hepatocellular carcinomacell lines(HepG-2)[17-18]. The HepG-2 cell line was received from VACSERA Tissue Culture Unit.

2 Result and discussions

2.1 Stoichiometry and molar conductance

The molar ratios and conductancebehaviors of gallium(Ⅲ),germanium(Ⅳ) and silicon(Ⅳ) complexes of pipc drug have been studied. The elemental analyses are in well agreement with 1∶2 stoichiometry (metal-to-ligand) as refereed in experimental section. All the synthesizedpipc complexes are stable at room temperature,with higher melting points. These complexes are insoluble in polar solventsbut soluble in commonly organic solvents (DMF & DMSO) with gently heating. Gallium(Ⅲ),germanium(Ⅳ) and silicon(Ⅳ) pipc solutions which were dissolved in DMSO showed slightly low conductance (Λm=20～29 ohm-1·cm2·mol-1) that confirmed the non-electrolyte nature of these respected complexes[19].

2.2 FTIR spectroscopy

An important information concerning the coordination mode of pipc ligand towards metal ions can be obtained in a first approach by comparative FTIR spectral analysis of the pipcchelate and itsgallium(Ⅲ),germanium(Ⅳ) and silicon(Ⅳ) complexes (Fig.2) and the data assignments are listed in (Table 1).

Fig.2 FT-IR spectra of pipcfree ligand and its complexes

Table 1 Infrared frequencies (cm-1) bands ofpipcligand and its complexes

2.3 Electronic UV-Vis spectra

2.4 1HNMR spectra

The spectral data of1H-NMR analysis concerningpipc free chelator and GaⅢand SiⅣcomplexes are assigned in (Table 2). By comparison between the pipcligand,[Ga(pipc)2(H2O)(Cl)]·4H2O and [Ge(pipc)2(Cl)2]·4H2O complexes,it was found a significant unrest on the chemical shift in the region of aromatic protons (H7 and H4) which are nearing the coordination sites[30]. The signal characteristic for piperazine proton (H-2) is exhibited atδ=8.85～8.95 ppm (unchanged),this meaning that nitrogen of piperazine ring doesn’t sharing in the coordination process. So,the pipc ligand coordinated towards respected metal ions through both oxygen atoms of pyridone and carboxylate rings.

Table 2 1H-NMR proton signals of the pipcligand and its complexes

Gallium(Ⅲ),germanium(Ⅳ) and silicon(Ⅳ) pipc synthesized complexes have been assigned using micro analytical analysis,1H-NMR,FT-IR and UV-Vis spectral studies as well as thermo gravimetric analyses. Molar ratio of prepared complexes were 1∶2 of (metal∶ligand). The complexes which have the general formula [Ga(pipc)2(H2O)(Cl)]·4H2O,[Ge(pipc)2(Cl)2]·4H2O and [Si(pipc)2(Cl)2]·4H2O (Fig.3).

Fig.3 Suggested structures of pipc complexesfor the Ga(Ⅲ),Ge(Ⅳ) and Si(Ⅳ)

2.5 Powder XRDspectroscopic analysis

The average size of the synthesized pipc complexes for the gallium(Ⅲ),germanium(Ⅳ) and silicon(Ⅳ) were determined from the Full width at half maximum of the respected XRD peaks (Fig.4) by Scherrer equation[31].

The data obtained from X-ray diffraction patterns refer to crystalline structures of the synthesized solid pipc metal complexes. Scherrer’s formula (DRX=0.9λ/Δcosθ) calculation is declared to the crystal size. Where 0.9 is a term constant of the equipment,λis the wavelength of radiation dueto the Cu Kα,peak andθis the Bragg’s angle. The grain size of the synthesized pipc complexes for the Ga(Ⅲ),Ge(Ⅳ) and Si(Ⅳ) is <100 nanometer.

2.6 Assessment of biological results

Invitro,the antibacterial and antifungal evaluations of the pipc complexes for the gallium(Ⅲ),germanium(Ⅳ) and silicon(Ⅳ) were assessed againstStaphylococcusAureus,Bacillussubtilis(G(+) bacteria) andEscherichiaColi,Pseudomonasaeruginosa(G(-) bacteria) and (CandidaAlbicansandAspergillusflavus) fungi (Table 3). The results indicate that the gallium(Ⅲ),germanium(Ⅳ) and silicon(Ⅳ) complexes have a significant active but less than the standard drugs Tetracycline and Amphotericin B. It is worthy mentioned that Si(Ⅳ) complex has an significant efficient against (22 mm·mg-1) Candida Albicans fungi species rather than standard Amphotericin B drug (19 mm·mg-1). The higher activity of silicon(Ⅳ) pipc complex against fungirevealed tothe nanosized form of this complex to make penetration during lipid membranes and blocking the sites of enzymes inthe microorganisms[32].

Fig.4 XRD pattern of pipc complexes forthe GaⅢ,GeⅣ and SiⅣ

The cytotoxicity (IC50) of the gallium(Ⅲ) pipc complex was assessed against human hepatocellular carcinoma and tabulated in (Table 4). The experimental of IC50 data of the gallium(Ⅲ) complex in vitro is equal to 123 μg·mL-1,which can be modified in future to become a promising anticancer agent.

Table 3 Antimicrobial assessments of GaⅢ,GeⅣ and SiⅣ pipc complexes

Table 4 The inhibitory activityof gallium(Ⅲ) pipc complex against HepG-2 cell line

Acknowledgement

This research was funded by the Deanship of Scientific Research at Princess Nourah bint Abdulrahman University through the Fast-track Research Funding Program.