Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X Vol. 1(8), 6-11, Nov. (2011) Res.J.Chem.Sci. International Science Congress Association 6 Potentiation of the Antimicrobial Activity of 4-Benzylimino-2, 3-Dimethyl-1-Phenylpyrazal-5-One by Metal ChelationElemike E.E., Oviawe A.P.1 and Otuokere I.E.2 Department of Chemistry, University of Benin, NIGERIA Department of Chemistry, Michael Okpara University of Agriculture, NIGERIA Available online at: www.isca.in (Received 25th July 2011, revised 19th August 2011, accepted 08th September 2011)Abstract A Schiff base ligand, 4-benzylimino-2-3-dimethyl-l-phenylpyrazal-5-one have been synthesized by the condensation of Benzaldehyde and 4-aminoantipyrine. Its divalent metal complexes of Fe, Co, Ni, Cu and Zn were also synthesized. The ligand and the complexes were characterized by FTIR, UV/visible, H-NMR, 13C-NMR, and GCMS. The ligand behaved as a bidentate donor by using its carbonyl and azomethine N as binding sites for the metals. Tetrahedral structures were proposed for the all complexes excepting the Cu(II) complex. The ligand showed low activity against some microbes but the complexes were remarkably active against the bacteria and fungi species. Keywords: 4-benzylimino-2-3-dimethyl-l-phenylpyrazal-5-one, benzaldehyde, 4-aminioantipyrine, antimicrobial. Introduction Schiff base are derived by condensation reaction of aldehydes or ketones and primary amines. They are compounds containing –N=CHR group. Many Schiff base ligands have been synthesized from heterocyclic compounds. Schiff base having oxygen, nitrogen, and sulphur donor atoms have been reported by several scientists. In this work we have synthesized a Schiff base ligand by condensation reaction between benzaldehyde and 4-aminoantipyrine (4-amino-2, 3-dimethyl-1-phenylpyrazal-5-one). Benzaldehyde is a typical aromatic aldehyde. It is a colourless oily liquid and a component of complex compound in the seeds of bitter almonds, peach and cherry seeds. It has been applied in food flavouring, synthetic perfumes, manufacture of cinnamic and benzoic acids and equally as a dye intermediate. Antipyrine is very much used in medicine5 and it is believed that its amino derivative would equally be of much use in medicine possibly as intermediates in antipyretic and analgesic drugs. Material and Methods All reagents used in this analysis are of analytical grade and obtained from Sigma-Aldrich Chemical Ltd and BDH chemicals. The reagents include: Benzaldehyde, 4-aminoantipyrine, ethanol, dimethyl sulphoxide (DMSO), dimethyl formamide (DMF), NiCl.6HO, CuCl.2HO, CoCl.6HO, ZnCl and FeCl2.. 4HO Instrumental analysis: The melting point was detected using the melting point apparatus, electronic spectra were determined using UNICAM UV 2120 spectrophotometer. IR spectra were also determined using FTIR-8400S spectrophotometer. H-NMR and 13C-NMR were recorded in -DMSO on a Shimadzu FTNMR spectrometer. The GCMS was performed using GCMS – QP2010 plus Schimadzu . Synthesis of 4-benzylimino-2-3-dimethyl-l-phenylpyrazal-5-one: The Schiff base derived from benzaldehyde and 4-aminioantipyrine was prepared by adding an ethanol solution (25ml) of 4-aminoantipyrine (2.03g, 0.01mol) to 1.01ml benzaldehyde (0.01mol). The mixture was stirred and refluxed for two hours. This was then filtered and left for 2 days to crystallize. The crystal formed was recrystallized with hot ethanol and dried in a desiccator over CaClvaccum. The yield was recorded. Preparation of the complexes: The complexes were prepared by the reaction of the ligand(0.01mol) with the respective metal (II) salts in ethanol medium (0.01mol NiCl. 6HO (2.37g); 0.01mol CuCl.2HO (1.72g); 0.01mol CoCl.6HO (2.78g); 0.01mol ZnCl (1.37g) and 0.01mol FeCl .4HO (1.98g)). The various 0.01mol of the metal salts were each refluxed with 0.01mol of the ligand in ethanol medium for 2 hours. They were all filtered and washed several times with ethanol after which they were left for 2-4 days to recrystallize. The resulting crystals were then dried. The yield was recorded. Sensitivity test: The sensitivity tests on the samples were carried out using agar well diffusion method. The nutrient agar was prepared according to the manufacturer’s recommendation and was poured into Petri dishes to set. The Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 1(8), 6-11, Nov. (2011) Res.J.Chem.SciInternational Science Congress Association 7 test organisms (bacteria)-Klebsiella pneumoniae, Staphyloccocus aureus, Escherichia coli, Enterococcus feacalis, (fungi)- Candida albicans and Microsporum audoni were cultured.The overnight bored cultures of the test organisms were properly diluted to the turbidity of Mac-farland’s standards and were inoculated on the surface of the agar . The inoculated agar was left for 20 minutes and holes were bored into it using cork borer.The prepared ligand and complexes were dissolved in DMSO and were then introduced into the agar using sterile swob stick. The innoculated plates were then incubated at 37C for 18 hours thereafter the resultant zones of inhibition were measured using meter rule and results obtained in centimeters were recorded. Ciprofloxacin and fluconazole which are antibacterial and antifungal agents respectively were used as control drugs.Results and Discussion The synthesis of 4-benzylimino-2-3-dimethyl-l-phenylpyrazal-5-one ligand is shown in scheme-1. The proposed structures of the complexes are shown in figure-1.NH OH +CH3CH3NOH EthanolReflux Scheme-1 Synthesis of 4-benzylimino-2-3-dimethyl-l-phenylpyrazal-5-one ligand NiCl CuCl ZnClCl FeClCl CoCl Figure - 1 Proposed structures of the metal complexes Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 1(8), 6-11, Nov. (2011) Res.J.Chem.SciInternational Science Congress Association 8 All the complexes are air stable, colored solids and non-hygroscopic. The physical properties of the compounds are presented in table-1. Infra-red spectra: The infra-red spectra of the ligand and complexes are presented in table-2 The infra-red spectra of cobalt and nickel complexe exhibited a broad band at 3425 and 3245cm-1 respectively. This is due to the presence of water molecules. The sharp bands between 1570 – 1592cm-1are due to C=N azomethine vibrations. The free ligand has the C=N vibration at 1569cm-1 so the shifting of the band to higher frequencies in the complexes indicates complexation 10. The bands that appeared below 650cm-1 are assigned to the metal-nitrogen (M-N),metal-oxygen(M-O) and metal chlorine (M-Cl) bonds. UV/Visible electronic spectra: The electronic spectra of the ligand and its complexes were recorded and their assignment given in table-3.The ligand’s spectra data displayed two bands at 20498cm-1 and 24739cm-1 which results from intra-ligand charge transfer (ILCT), phenyl ring and n* (HC=N) transitions 11. Two bands were observed in the spectrum of FeL which are 20728cm-1 and 25274cm-1. These bands have been assigned MLCT and transition and a tetrahedral geometry is therefore proposed 12. For the complex (CoL), three absorption bands are observed 15175cm-1, 16536cm-1 and 24636cm-1 These bands are as a result of metal ligand charge transfer (MLCT), dd transfer and  ligand transfer. The intensity of the band suggested an tetrahedral geometry and their assignments thus 1g2g, 1g1g and 1g2g. Ni(II) complex exhibited three bands at 25199cm-1, 28150cm-1 and 30827cm-1. A square planar geometry is suggested for this complex. Copper (II) complex (CuL) has two bands 19396cm-1 and 25785cm-1 which represents E transitions and the intensity of the bands suggest trigonal geometry. ZnL complex have two bands at 20551cm-1 and 24569cm-1. The absorptions are as a result of intra-ligand charge transfer and a tetrahedral geometry is proposed. Table – 1 Physical properties of the ligand and complexes Compound Colour Melting point o C Molecular weight Yield % L Yellow 160 291.01 44.0 FeL Dark red 146-147 416.12 49.0 CoL Yellowish brown 134-136 401.77 75.6 NiL Light green 186-188 401.53 73.7 CuL Brown 268 388.36 63.9 ZnL Yellow 279 425.68 70.3 L= 4-benzylimino-2-3-dimethyl-l-phenylpyrazal-5-one Table-2 Infrared data of the ligand and complexes (cm-1) Compounds OH, H 2 O  C - H aromatic  C=O  C=N  C=C aromatic  M - N  M - Cl L - 3042 1649 1569 1481 - - FeL - 3044 1650 1571 1483 585 441 CoL 3425 3043 1649 1570 1483 530 442 NiL 3245 3050 1639 1577 1481 591 447 CuL - 3171 1623 1620 - 497 387 ZnL - 3051 1819 1592 - 618 421 L= 4-benzylimino-2-3-dimethyl-l-phenylpyrazal-5-one Table-3 UV/Visible electronic spectra data and possible assignments Compounds 1 cm - 1  2 cm - 1 Assignment L 20498 24739  * FeL 20728 25274 5 E 5 T 2 CoL 15175 16536 4 T 1g  4 T 2g , 4 T 1g  4 T 1g , 4 T 1g  4 T 2g NiL 25199 28150  * CuL 19396 25785 2 T 2  2 E ZnL 20551 24569  * L=4-benzylimino-2-3-dimethyl-l-phenylpyrazal-5-one Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 1(8), 6-11, Nov. (2011) Res.J.Chem.SciInternational Science Congress Association 9 HNMR spectra data: HNMR spectra of the ligand and its complexes have four protons environments. The different chemical shifts are shown in Table 4. In the ligand, the first chemical shift appeared at 2.00ppm indicates the CH(alkyl). The azomethine hydrogen (HC=N) appeared at 2.53ppm for the ligand but shifted downfield for the complexes showing complexation through the azomethine linkage13. CH-N band is shifted lower field compared to the CH-C due to the presence of nitrogen group that deshields the electrons. Within the region of 7.12 – 7.87ppm, there are multiples of peaks which indicated the presence of aromatic protons14. 13CNMR: 13CNMR spectra data is presented in table-5. In the ligand, the C=0 carbon resonates at 190ppm but in the complexes, C=0 band shifted downfield due to complexation. The azomethine carbons C=N appearred between 145-152ppm in both the ligands and metal complexes though there was a downfield shift for the complexes 15. There is a prominent peak that appeared in all the spectra at 40 ppm, this resulted from the solvent d-DMSO used in the analysis. There are a total of 14 peaks which confirmed the structure of the ligand. There is also a concentration of peaks between 102 – 152 ppm which indicated sp hybridized carbons and they consists of azomethine carbons, C=C carbons, benzylic carbons and aromatic carbons. Table – 4 H-NMRChemical shifts of the different compounds (all values in ppm) Compound HC=N Aromatic H CH-N` CH-C L 2.53 7.12-7.87 2.49 2.00 FeL 2.73 6.68-7.08 2.44 1.80 CoL 2.78 7.02-7.28 2.54 1.60 NiL 3.23 7.78-8.08 2.64 2.00 CuL 3.97 7.48-7.78 2.23 2.00 ZnL 2.33 7.18-7.48 2.29 1.80 L= 4-benzylimino-2-3-dimethyl-l-phenylpyrazal-5-one Table – 5 13C-NMR data for the ligand and complexes in ppm Compound C=0 C=N C=C Aromatic carbons CH-N CH3 L 190 151 146,149 112-142 32 18 FeL 204 148 142,145 112-138 28 16 CoL 213 149 141,146 103-135 27 13 NiL 214 149 142,146 111-138 25 16 CuL 211 149 144,146 107-142 32 18 ZnL 218 152 146,149 118-144 22 12 L= 4-benzylimino-2-3-dimethyl-l-phenylpyrazal-5-one NH +CH-C-N-CH M/Z = 56 (base peak)M/Z = 77M/z = 104 Scheme-2GCMS major fragmentations Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 1(8), 6-11, Nov. (2011) Res.J.Chem.SciInternational Science Congress Association 10 Table – 6 Antimicrobial test results (cm) Compounds Escheriachia coli Enterococus feacalis Klebsiella pneumoniae Staphylococcus aureus Candida albicans(fungus) Monosporum audonii (fungus) L 18 10 17 - 15 10 FeL 16 15 16 15 17 14 CoL 23 20 18 17 21 32 NiL 19 15 11 18 18 30 CuL 19 18 20 27 26 23 ZnL - - - 18 30 18 Control 16 11 17 - 15 10 L= 4-benzylimino-2-3-dimethyl-l-phenylpyrazal-5-oneGCMS spectra: In the GCMS analysis of ligand and complexes, there were many peaks. The molecular ion peak of the ligand had mass/charge (m/z) ratio of 291 for the ligand which corresponded to the molecular mass of the compound. There was a small peak at( m/z) 292, this is because of the natural abundance of 13C . The base peak had m/z ratio at 56. Other fragments occurred at 199, 188, 171, 157, 146, 130, 121, 103, 91, 77, 54 and 39.The major fragmentations are represented in scheme-2. The results in table-6 are zones of inhibition. From the results, we can see that most of the complexes proved potent against some bacteria and fungi. Complexation improved the antimicrobial activities of the ligand. CuL inhibited the growth of Staphyloccucus aureus more than other complexes. The standard drug used (ciprofloxacin) did not show any inhibition against Staphyloccucus aureus. CoL, CuL and NiL have shown great antibacterial and antifugal activities than the ligand and other complexes used in this work. The ZnL is not potent against bacteria but showed great activity against fungi. Conclusion The potentiation of the antimicrobial activity of 4benzylimino-2-3-dimethyl-l-phenylpyrazal-5-one by metal chelation has been studied. A Schiff base ligand, 4benzylimino-2-3-dimethyl-l-phenylpyrazal-5-one have been synthesized by the condensation of Benzaldehyde and 4-aminoantipyrine. Its divalent metal complexes of Fe, Co, Ni, Cu and Zn were also synthesized. The ligand and the complexes have been characterized by FTIR, UV/visible, H-NMR, 13C-NMR, and GCMS. The ligand behaved as a bidentate donor by using its carbonyl and azomethine N as binding sites for the metals. Tetrahedral structures were proposed for the all complexes excepting the Cu(II) complex. The ligand showed low activity against some microbes but the complexes were remarkably active against the bacteria and fungi species. We hereby suggest that this ligand and its metal complexes be used as metal based drugs. Acknowledgement We acknowledge Mr Tobias Egemba of City university, London. He helped us in NMR spectra characterization. References 1. Bahl B.S. and Bahl A., Elementary Organic Chemistry, Schand and Company Ltd, New Delhi, 60 (1993) 2. Kovala-Demertzi D., Platinum(II) and palladium(II) Schiff base complexes of pyridine-2-carbaldehyde thiosemicarbazone as alternative antiherpes simplex virus agents., Bio. Chem. App., (1), 1-6 (2007) 3. Silverstein R.M., Bassler G.C. and Morril T.C., Spectrometric identification of Organic Compounds, John Wiley and Sons, New York ,50-56 (1974) 4. Finar I.L. Organic Chemistry, Vol 2, 5th Ed., Dorling Kindersley Publishers Ltd, India, 627(2007) 5. Agarwal R.K., Singh L. and Sharma D.K. ,Synthesis, spectral, and biological properties of copper(II) complexes of thiosemicarbazones of Schiff bases derived from 4-aminoantipyrine and aromatic aldehydes, Bio.Chem. Appl.,.2006, 1-10 (2006) 6. Scovill J.P., Klayman D.L. and Franchino C.F., 2-acetylpyridine thiosemicarbazones complexes with transition metals as antimalarial and antileukemic agents, J. of Med.Chem., 25(10),1261–1264 (2009) 7. Costa R.F.F., Rebolledo A.P. and Matencio T., Metal complexes of 2-benzoylpyridine-derived thiosemicarbazones: structural, electrochemical and biological studies, J. of Coord Chem., 58(15), 1307–1319 (2005) 8. Hugo, W.B. and Russel, A.D. Pharmaceutical Microbiology, Black Well Science, Ltd, U.S.A. 182 (2002) Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 1(8), 6-11, Nov. (2011) Res.J.Chem.SciInternational Science Congress Association 11 9. Sulekh C. and Amit K. S., Antitingal and Spectra Studies of Cr(III) and Mn(II) compexes derived from 3, 3- Thiodropropionic Acid derivative, Res. Lett. Inorg. Chem., 89 (2009) 10. Quiroga A.G., P´erez J.M. and L´opez-Solera I., Novel tetranuclear orthometalated complexes of Pd(II) and Pt(II) derived from p-isopropylbenzaldehyde thiosemicarbazone with cytotoxic activity in cis-DDP resistant tumor cell lines Interaction of these complexes with DNA, J. of Med. Chem., 41(9), 1399–1408 (1988) 11. Kovala-Demertzi D., Boccarelli A. and Coluccia M., In vitro antitumor activity of 2-acetyl pyridine 4N-ethyl thiosemicarbazone and its platinum(II) and palladium(II) complexes, Chemotherapy. 53(2), 148–152 (2007) 12. Pandey O.P., Synthesis, spectral and antibacterial studies of binuclear titanium (IV) / zirconium(IV) complexes of piperazine dithiosemicarbazones, Bio. Chem Appl., 1(1), 35–44. (2003) 13. Shipman J. Thiosemicarbazones of 2-acetylpyridine, 2-acetylquinoline, 1-acetylisoquinoline, and related compounds as inhibitors of herpes simplex virus in vitro and in a cutaneous herpes guinea pig model, Avr. Res.6(4), 197–222 (1986) 14. Beraldo H. Semicarbazones and thiosemicarbazones: their wide pharmacological profile and clinical applications. Quim. Nova, 27(3),461–471(2004) 15. Quiroga A.G., P´erez J.M. and L´opez-Solera I. Binuclear chloro-bridged palladated and platinated complexes derived from p-isopropylbenzaldehyde thiosemicarbazone with cytotoxicity against cisplatin resistant tumor cell lines, J. of Inorg. Biochem., 69(4), 275–281(1988)