Synthesis, Characterization and Biological Evaluation of Co(II), Cu(II), Ni(II) and Zn(II) Complexes With Cephradine

Some Co(II), Cu(II), Ni(II) and Zn(II) complexes of antibacterial drug cephradine have been prepared and characterized by their physical, spectral and analytical data. Cephradine acts as bidentate and the complexes have compositions, [M(L)2X2] where [M = Co(II), Ni(II) and Zn(II), L = cephradine and X = Cl2] showing octahedral geometry, and [M(L)2] where [M = Cu(II), L = cephradine] showing square planar geometry. In order to evaluate the effect of metal ions upon chelation, eephradine and its complexes have been screened for their antibacterial activity against bacterial strains, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.


INTRODUCTION
Evidences supporting the introduction of metallic elements in several biological processes are rapidly accumulatingr6. Schubert 7 and Kirschner 8 have investigated the antibacterial, antiviral and anticancer activities of more than 25 inorganic compounds, which included the metal atom as potentially significant part of the molecule. They suggested 8'9 that the transfer of metal ion from the ligand to the cancer-associated viruses was a mechanism for releasing the anticancer drug in the locality of the tumor. Due to the significant nature of the metallic ions, their metal complexes are now, being included in the search for ideal anticancer drugs. Such significant examples of which are, palladium and platinum complexes of 6-mercaptopurine that destroy 1 adenocarcinomas and similarly, the complexes of dialkyldithiophosphate which reduce some tumors1. A wide range of activities of even the simplest complexes of metallic elements has been reviewed by Rosenberg 2 who noticed that the viral-induced cancers all respond to the treatment with even the simplest metal-amine halide complexes. It has also been demonstrated that ehelation/complexation tend to make inactive substances/ligands active and active compounds/drugs become more active and less toxic316. All these evidences however, highlight the need to study and evaluate more the biological applications of metallic elements for therapeutic potentials. Cephradine is a first-generation eephalosporin class of antibiotic7, actively used against Gram-positive cocci, and Gram-negative bacilli. It has similar structural and antibacterial relationship to that of its closely related analogue cephalexin. It contains the (NH), (COOH), (NH) and (C=O) functional groups and its molecular model reveals that its structure is suitable for chelation/complexation. A detailed biological evaluation of the copper(II) and Zn(II) complexes of cephalexin has been reported . In order to evaluate the biological comparison of both the analogues, we, therefore, report in this paper the preparation, characterization and biological properties of cobalt(II), copper(II), niekel(II) and Zn(II) complexes of eephradine which may be an addition to reveal the significance of the strong relationship between metals, or their complexes, and bactericidal activity.

Material and Methods
All chemicals and solvents used were of Analar grade. All metal(II) salts were used as chlorides. Sodium salt of cephradine was obtained by reacting equimolar amount of sodium hydroxide with cephradine. IR spectra were recorded on a Philips Analytical PU 9800 FTIR spectrophotometer as KBr discs. UV-Visible spectra were obtained in DMF on a Hitachi U-2000 double-beam spectrophotometer. C, H and N analyses was carried out by Butterworth Laboratories Ltd. Conductance of the metal complexes was determined in DMF on a Hitachi YSI-32 model conductometer. Magnetic measurements were made on solid complexes using the Gouy method. Melting points were recorded on a Gallenkamp apparatus and are uncorrected.

Preparation of cobalt(ll)-cephrafline Complex
An ethanol solution (20 mL) of the cobalt chloride hexahydrate (0.24 g, 0.001 M) was added to a magnetically stirred sodium salt of cephradine (0.8 g, 0.002 M) in distilled water (25 mL). The mixture was refluxed for h and cooled to room temperature. On cooling, pink precipitates were formed, filtered and washed with ethanol, acetone and ether, and dried by suction. Crystallization in aqueous ethanol (70 30) gave the desired metal complex. All other metal complexes were formed following the same method.

Antibacterial Studies
The synthesized metal complexes, in comparison to the uncomplexed cephradine were screened for their antibacterial activity against pathogenic bacterial species, Escherichia coli, Staphylococcus aureus and of Co(II), Cu(lI), Ni(ll) and Zn(II) Complexes with Cephradine Pseudomonas aeruginosa. The paper disc diffusion method was adopted for the determination of antibacterial activity"r4"16.

RESULTS AND DISCUSSION
All the complexes are intensely colored and stable amorphous solids, which did not have sharp melting points and decompose above 225C. The complexes are only soluble in DMF and DMSO and insoluble in all other common organic solvents. Their melting behavior, solubility and crystalline nature suggest that they are monomers. Molar conductance values (14-18 ohmlcmZmol"l) of the complexes in DMF solution indicated 19 that all ofthe complexes are non-electrolytes. The IR spectra of the complexes in comparison to the uncomplexed cephradine are listed in Table 2  carboxyl group is coordinated to the metal ion. Also, the band due to v(NH2) at 3345 cm " was found shifted to lower wave number (15-20 cm"l) in the spectra of its metal complexes. And a new band at 415-420 cm -l assigned to v(M-N) was evolved in the spectra of the complexes indicating 2 involvement of the v(NH) group via nitrogen in the coordination. Moreover, In the far infrared region a new band at 335 cm 1 was found in the spectra of the Co(II), Ni(lI) and Zn(II) complexes and not observed in the spectrum of the Cu(II) complex assigned to v(M-CI) modes respectively. It in turn, indicated that two chloride atoms are also coordinated to the metal atom. These evidences however, indicated that the Co(II) Ni(II) and Zn(II) complexes possess an octahedral geometry ( Fig 1A) and Cu(II) complex to show square planar geometry H and lC NMR spectra of the uncomplexed cephradine and its metal complexes have been recorded in DMSO-d6 with TMS as internal reference and are summerized in Table 2 with some tentative assignments.
The spectra of cephradine exhibited peaks as expectedza. Signals for the NH and COOH protons in the spectra of uncomplexed Cephradine at 1.1 and 10.8 ppm disappeared in the complexes. This indicated that Cephradine is coordinated to the metal ions by deprotonation. In the complexes the aromatic proton signals 24 25 appeared downfield due to increased conjugation during coordination UV-Visible spectral bands of the complexes are recorded in Table 2. The cobalt(II) complex shows magnetic moment 4.7 B.M. at room temperature (Table. 1). These high value of the magnetic moment and the stoichiometry suggest a coordination number of six for the central cobalt(II) ion and an octahedral geometry. The electronic spectrum of the complex is consistent with its octahedral environment around the cobalt(II) ion (Fig.lA). The spectrum displays two bands at 20,715 and 28,580 cm "1 attributed to 4Tlg 4Tlg and 4Tlg 4 26 27 T2g transitions, respectively, in a high-spin octahedral geometry   C-NMR (DMSO-d6) (ppm) 13  of Co(ll), Cu(ll), Ni(ll) and Zn(ll) Complexes with Cephradine configuration and may be assigned to 2Big -2Alg and 2Big --2Eg transitions respectively2s'29. The strong high-energy band is assigned to metal --ligand charge transfer. Also, the magnetic moment value (1.4 B.M) for the Cu(II) complex is found to be consistent with the proposed square planar geometry (Fig 1B) for the Cu(II) complex.
The electronic spectra of the nickel(II) complex exhibited absorption bands at 16,570 and 27,375 cm , attributed to 3A2g 3Tlg (F) and 3AEg 3Tlg (P) transitions, respectively, in an octahedral geometry3'3. The calculated values of the ligand field parameter lie in the range reported for an octahedral structure. Also, the value ofthe magnetic moment (3.3 B.M.) may be taken as an additional evidence for its octahedral structure. The diamagnetic zinc(II) complex did not show any d-d bands and its spectrum is dominated only by_ the charge transfer band. This charge transfer band at 27,555 cm was assigned to the transition 2Eg ---, 2T2g, possibly in an octahedral environment3.
On the basis of the above observations, it is tentatively suggested that the Co(II), Ni(II) and Zn(II) complexes show an octahedral geometry (Fig. 1A) in which the two cephradine molecules act as bidentate and at axial positions two chlorides are coordinated to the metal atom. In the Cu(II) complex the two cephradine molecules respectively, acting as bidentate show a square planar geometry of the complex (Fig. 1B) by possibly accommodating themselves around the metal atom in such a way that a stable chelate ring is formed.

Antibacterial Properties
Cephradine in comparison to its metal(II) chelates was evaluated for antibacterial activity against the standard bacterial strains, Escherichia coli (a),), Staphylococcus aureus (b) and Pseudomonas aeruginosa (c) The compounds were tested at a concentration of 30 lag/0.01 mL in DMF solution using the paper disc diffusion method reported 33'34 as earlier. The susceptibility zones were measured in diameter (mm) and the results are reproduced in Table 3. The susceptibility zones were the clear zones around the discs. Cephradine and its complexes individually were found to be biologically active showing various degrees of inhibitory effects on the growth of the tested bacterial species. The antibacterial results evidently show that the complexation improved the antibacterial activity. These observations however, suggest that the metallic elements should be seriously considered in <drug designing>. Their use could provide an easy way of improving the bactericidal activity of many drugs/antibacterial agents.