8Eclética Química, 36 (3), 8-14, 2011. selective binding affinity at cholecystokinin CCK1 receptors (IC50=156.5±33.2 nM) [6,7]. Classical ben- zodiazepines show anxiolytic, sedative, and anticon- vulsant activities whereas, several 1,4-benzodiazepine derivatives such as diazepam, triazolam or midazolam, several others have demonstrated activity as antitu- mor antibiotics, anti-HIV agents, and antiarrhytmics [10,11,12].The synthesis of new indazolone and pyr- azolone derivatives starting from methyl anthranilate presents advantageous alternative for the synthesis of the target heterocycles, which implies the use of the environmentally friendly oxidizer PIFA [phenyliod- ine(III)bis-(trifluoroacetate)] [11] Keeping in view the importance of hydrazides [12-18] and their complexes with Copper, Nickel and Zinc [20,21] we here by re- ported the hydrazide derived from methyl anthranilate through the condensation with hydrazine, to get a new modified diammine adding to the previous ones we reported so far [22-24]. All these various studies forced us to make complexes of such compounds and to study the effect on their bioassay studies. Introduction Methyl anthranilate is an aromatic ester, and is widely used in the synthesis of biologically active alkaloids. These alkaloids include quinazoline, qui- naxaline, cinnoline, phthalazine and other three or four membered ring azaheterocycles such as, pteridine, al- loxazine, isoalloxazine [1]. It is also used as repellents for deterring wildlife entrance into an area. The pri- mary component of synthetic grape flavoring, methyl anthranilate, has been identified to be a powerful avian irritant [2]. It has been successfully tested as a topical repellent, to deter goose grazing on grass [3], to reduce bird damage to blueberries [4], and to repel birds at landfills and standing water on airports [5]. Methyl anthranilate is a substrate reacted with N- Boc protected amino aldehydes to get the product 5-oxo-1,2,3,4-tetrahydro-5H-1,4-benzodiaze- pine. The resulting product was further alkylated and acylated by reacting with phenylalanine and trypto- phan to get the derived product which show significant Abstract: The present report is concerned with the synthesis and coordination compounds of 2-amino-(N-am- inobezoyl)benzohydrazide[ABH]. The ligand was charcterized by proton NMR, mass spectrometry, elemental and infrared studies. The ligand has got –NH2 moeity which is capable of chelation. Therefore complexes of Ni, Cu, and Zn were prepared. These complexes were characterized by elemental, infrared, thermal, conductance, and magnetic susceptibility studies. Infrared spectra shows that the ligand form complexes through –NH2 moe- ity, while the elemental studies suggest M(ABH)X2 composition of the coordination compounds. Knowing about the importance of -N-N- linkage in the biologically active compounds, the synthesized complexes were studied for their biological activities against Gram Negative bacteria which include E. coli, Salmonella typhi, Entero- bacter aerogenes, Proteus vulgaris and Pseudomonas aeruginosa, selected Gram Positive bacterial strain is Staph aureus and Fungus like Candida albican. These activities show that the hydrazide containing metal complexes become more potentially resistive to the microbial activities as compared to the sole ligand. Keywords: aminobenzohydrazine, coordination compounds, bioassay studies. EFFECT ON THE INHIBITORY ACTIVITY OF POTENTIAL MICROBES ON THE COMPLEXATION OF METHYL ANTHRANILATE DERIVED HY- DRAZIDE WITH CU, NI AND ZN(II) METAL IONS Saeed-Ur-Rehman*1, Mazhar-Ul-Islam1, Muhammad Ikram1, Sadia Rehman1, Syed Muhammad Shah1, Kamran Mahdi 1 and Farhat Ullah2 1Institute of Chemical Sciences, University of Peshawar, Peshawar, Pakistan 2Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan *1Corresponding author. E-mail address: srachem@yahoo.com , Tele Phone: 0092 91-5641658/915843456 9Eclética Química, 36 (3), 9-14, 2011. drug gentamycin for bacterial strains and mystasin for fungal species. In order to clarify any participating role of DMSO in the biological screening, separate studies were carried out with the solutions alone of DMSO and they showed no activity against any bacterial strains. All these complexes were found to be potentially ac- tive against these bacterial strains. Preparation of Ligand According to the procedure adopted by Shadia A. Galal [8,9,28], 0.060 mole of methyl anthranilate was mixed with 0.022 mole of hydrazine monohydrate in a round bottom flask, the resulting reaction mix- ture was refluxed at 80-90 ºC for 1 hour with constant stirring. Needle type crystals of the prepared ligand appeared by keeping the reaction mixture for about two hours in a refrigerator. Crystals were washed with n-hexane and recrystallized with dry ethanol. The yield was 48%. 2.4 General Preparation of the Solid Com- plexes All complexes of 2-aminobenzohydrazide (ABH) were prepared using the same general proce- dure. The required amount of partially dehydrated salts were dissolved in a minimum amount of anhydrous ethanol or methanol. Dehydration of the metal salts was carried out by reacting with the calculated amount of 2,2-dimethoxy propane as dehydrating agent. The solution was stirred up for about half an hour in order to ensure complete dehydration. Dissolved ligand was added slowly to the metal salt solution with constant stirring. The solid complex was formed immediately on mixing the two solutions or in either case complex was obtained by reducing the volume of the solution on a rotary evaporator. The products were filtered through sintered glass crucible, washed several times with n-hexane or dried ethanol and dried under vacu- um. Results and discussions The ABH is characterized by elemental anal- yses, mass spectrum and NMR (proton and 13C). The mass spectrum of ABH shows a peak at m/Z 270. This is due to ABH+. Elemental analytical data of ABH and its com- plexes are very close to the theoretical values as shown in Table1. ABH Ligand Experimental Materials and Methods All chemicals and solvents used were of Analar grade. Salts of transition metals were obtained from Riedel- de-Haen, Germany and used without further purifica- tion. The partial dehydration of the salts was carried out by drying the hydrated salts in a vacuum oven for several hours at 80 – 100o C. Methyl anthranilate was obtained from Across Organics, USA and hydrazine monohydrate from PANREAC Quimica SA, Barcelo- na, Espania. Solvents were distilled twice before use. Instrumentation Infrared spectra were taken in the range of 4000-600 cm on PYE UNICAM Infrared Spectropho- tometer in KBr disc. The far IR spectra were examined in KBr discs in the region of 400- 200 cm-1(T- IR SHIMADZU) . The absorption spectra of solution of complex- es in the range of 200- 900 nm using different solvents were obtained on Jasco DEC-1 Spectrophotometer with 1 cm matched quartz –cells. Molar conductances of the solution of the metal complexes were deter- mined with a conductivity meter type HI 8333. All measurements were carried out at room temperature at 30 oC with freshly prepared solution. Magnetic sus- ceptibilities were measured by Gouy method at room temperature using Hg[Co[SCN]4 as a standard [25], magnetic moments were thus calculated. The cations and anions were estimated by using typical analytical procedure [26]. Bioactivities were investigated using agar-well diffusion method [27]. Two to eight hours old bacterial strains in column containing approxi- mately 104–106 colony forming units (CFU)/mL were used in these assays. The wells were dug in the media with the help of a sterile metallic borer with centers at least 24 mm. Recommended concentration (100 μl) of the test sample 1 mg/mL in DMSO was introduced in the respective wells. Other wells supplemented with DMSO and reference antibacterial drug, gentamycin served as negative and positive controls, respectively. The plates were incubated immediately at 37◦C for 20 hours. Activity was determined by measuring the diameter of zones showing complete inhibition (mm). Growth inhibition was compared with the standard 10Eclética Química, 36 (3), 10-14, 2011. The 1H-NMR show doublet of doublet for protons located at 3 and 19, 4 and 18, 5 and 17, 6 and 16, which all are observed at 6.7ppm to 7.5 ppm in a very complex form. While the protons located at positions 10 and 11 are observed around 9ppm [28]. While the protons at 7 and 20 positions give a singlet at around 6.3ppm. 13C-NMR show seven peaks for all the seven identical carbons viz., 12 and 8, 1 and 15, 2 and 14, 3 and 19, 4 and 18, 5 and 17, 6 and 16 respec- tively. *Calculated values are given in parenthesis Elemental analysis show that the metal to li- gand ratio is 1:1 and the composition of metal complex is M(ABH)X2 (Where X is Cl-, I-, and NO3-). Conductance and melting points of the com- plexes are given in Table.2, conductance data show that the metal complexes are non-electrolyte indicating the halide or nitrate ions are located inside the coordi- nation sphere. All the complexes are magnetic suscep- tible except Zn(II) complexes as shown in Table. 2. IR data which is shown in Table. 3 shows broadening of -NH2 peak, which suggest coordination through this site. While the carbonyl peak and of hydrazide –NH remain unaltered. Table 2. Conductance, melting points and magnetic moments data 11Eclética Química, 36 (3), 11-14, 2011. complex is non-electrolytic. Zinc complexes The conductance data of Zn(II) complexes indicate the non-ionic species. As the complexes of the cyclic diamines have not been reported so far, how- ever, they can be compared with cyclic amidines. In view of the well-known tendencies of Zn(II) to form tetrahedral complexes [6,29,30], same structure may be proposed for Zn(II) ABH complexes with ABH acting as bidentate ligand. Trzaskowski et al observed by density functional calculations that deprotonated serine and cystein, which are close in structural features to ABH, forms tetrahedral geometry around the zinc(II) ion with only four ligands around the metal ion [31]. These calculations are in agreement with the known experimental crystal structures of some zinc-contain- ing complexes. Dudev et al using Continuum Dielec- tric Calculations showed that zinc monochloride and dichloride tetrahedral complexes were calculated to be more stable than the respective octahedral com- plexes by -4.5 and -8.6 kcal/mol, respectively, at the B3LYP/6-31++G(2d,2p) level. Furthermore, stationary points for zinc octahedral complexes containing three or four Cl- could not be found since they isomerized Table 3. IR spectra for ABH and its complexes (cm-1) of selected region Nickel Complexes The visible absorption spectra of Ni(II) com- plexes (Table 3 and Fig.2) show broad peak around 17,800 cm-1 with shoulder around 16,600 cm-1, assigned to (F)®3T1(P) and 3T1(F)®3T2(F) transi- tion probably indicating tetrahedral geometry. The magnetic moment (3.06 B.M for [Ni(NO3)2 (C14H- 14N4O2)] and 3.09 B.M for [NiI2(C14H14N4O2)]) and non-electrolytic behavior of these complexes are consistent with distorted tetrahedral symmetry of [Ni(ABH)X2]. Copper Complexes The complexes of Cu (II) show an absorption band in the region 13000-15000cm-1(Table 3 and Fig.3). The envelopes of these bands are generally unsymmetrical, seeming to encompass several over- lapping transitions. This band is similar to absorption maximum at 16,000cm-1 observed in the case of [Cu(- daco-diac)2X2], a well known tetrahedral structure [15]. The magnetic moment is around 2.0 B.M; which is very close to the spin only value for the the unpaired electron. The conductance behaviour shows that the 12Eclética Química, 36 (3), 12-14, 2011. tially active against these bacterial strains. It is evident that the overall potency of the ligand was enhanced on coordination as shown in comparative graphs in Fig. 1-5. Fig.1 Comparative Graph for Activity shown by [Ni(NO3)2 (C14H14N4O2)] against Microbes into tetrahedral (4 + 2) complexes during optimization [32]. Roe et al also showed that tetrahedral geometry for the zinc+2 complexes are the most stable ones [30]. This geometry would also be consistent with the non-electrolytic behavior of the complexes [32]. Bio-Assay Investigations The complexes of ABH were investigated for their bioactivity against various available microorgan- isms. These microorganisms include gram positive and gram negative bacteria along with a selected species of fungi. Gram Negative bacteria include E. coli, Salmo- nella typhi, Enterobacter aerogenes, Proteus vulgaris and Pseudomonas aeruginosa, selected gram Positive bacterial strain is Staph aureus and fungus like Candi- da albican. These studies show that the metal complexes become more biologically active as compared to neat organic moiety. The results are reported in Table 6. Table 4. Biological activities of complexes against gram negative, gram positive and fungus Gram Negative: E.coli, Salmonella typhi, Enterobacter aerogenes, Proteus vulgaris and Pseudomonas aerugi- nosa, Gram Positive: Staph aureus, Fungus:Candida albican All these complexes were found to be poten- 13Eclética Química, 36 (3), 13-14, 2011. Fig.5 Comparative Graph for Activity shown by [ZnCl2(C14H14N4O2)] against Microbes 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 400 450 500 550 600 650 700 750 800 Wa veleng th, nm A bs or ba nc e, a u Fig. 6 Visible Spectrum of [Cu(NO3)2 (C14H14N4O2)] 0 0.02 0.04 0.06 0.08 0.1 450 500 550 600 650 700 750 800 Wavelength, nm Ab so rb an ce , a u Fig.7 Visible Spectra of Nickel Complexes (x x x x x for [Ni(NO3)2 (C14H14N4O2)] and □ □ □ □ □ for [NiI2 (C14H14N4O2)]) Conclusion The synthesized complexes of ABH ligand show tetrahedral geometries. Magnetic moment stud- ies prove the assigned geometries. The synthesized hydrazine derived ligand showed antibacterial/ antifun- gal properties. In comparison, the copper(II), nick- Fig.2 Comparative Graph for Activity shown by [NiI2 (C14H14N4O2)] against Microbes Fig.3 Comparative Graph for Activity shown by [Cu(NO3)2 (C14H14N4O2)] against Microbes Fig.4 Comparative Graph for Activity shown by [Zn(NO3)2 (C14H14N4O2)] against Microbes 14Eclética Química, 36 (3), 14-14, 2011. and Raul SanMartin, Tetrahedron 62 (48), 11100- 11105 (2006). 12. Hoose C., Eberhardt K., Hartmann W. and Wosniok W., Pneumologie, 44(1), 458 (1990). 13. Saliaev V.N., Titov N.S. and Ermolaev V.P., Far- maologiia I Toksikologiia, 43(4), 327 (1980). 14. Vidrio H., Fernandez G., Medina M., Alvarez E. and Orallo F., Vasc. Pharmacol., 40(1), 13 (2003). 15. Delaney J. and Timbrell J.A., Xenobiotica, 25(12), 1399 (1995). 16. MacRae W.D. and Stich H.F., Mutat. Res., 68(4), 351 (1979). 17. Strolin-Benedetti M. and Tipton K.F., J. Neural Transm. Suppl., 52, 149 (1998). 18. 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London, ELBS and Longmann, , London, 1978. 27.Atta-ur-Rahman, Choudhary M.I. and Thomsen W.J., Bioassay Techniques for Drug Development. Amsterdam, The Netherlands: Harwood Academic; 2001. WWW.ECLETICA.IQ.UNESP.BR Volume 36, número 3, 2011 el(II), and zinc(II) metal complexes of this compound showed more activity against one or more bacterial/ fungal strains, thus introducing a novel class of met- al-based bactericidal and fungicidal agents. Acknowledgment We are grateful to Higher Education Pakistan for providing the funding facilities, to HEJ Research Institute of Chemistry, University of Karachi, Paki- stan, for providing us the facility of Mass spectrum and we are also grateful to Chemistry Department of Quaid-i-Azam University, Islamabad for providing NMR facilities. Our gratitude also goes to the Depart- ment of Environmental Sciences, University of Pesha- war for providing FTIR facility. Recebido em : 29/02/2010 Aceito em: 13/06/2012 References 1. I.L. 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