INTRODUCTION:

The azines are a class of hydrazides having nitrogen donar atoms. Heterocyclic azines exhibit wide applicability; viz. inhibit marine tumor growth¹, acts on fluorescent brightening agents, photosensitizes², azines are used as an ion selective electrodes and optical sensors³. There has been a lot of interest^4-7 in reactions of transition metal ions with hydrazine derivative because of a variety of resultant products. The complexes of azines have potential applications for the fixation of phosphoric nitrogen via transition metal dinitrogen complexes, antitubercular and antifungul agents. Salicyladehyde derivates are also known to possess wide biological activities. The hydrazones are known to exhibit bacteriostatic and tuberculostatic activities and are useful therapy^8-12. Earlier studies^13-17 showed that, open chain diazine ligands present several possible mononucleating and dinucleating coordination modes due to the flexibility of the ligand around N- N- single bond. The azines derived from two heterocyclic aldehydes are versatile ligands and can coordinate to metal ions in different ways.

Metal ions play a vital role in a vast number of widely different biological processes. The attraction of these ions with biologically active ligands, for ex; in drugs, is a subject of considerable interest. Hence, in present investigation we have synthesized the Co(II), Ni(II) and Cu(II) metal complexes with newly synthesized NO donor asymmetric aldazines derived from substituted salicylaldehyde and benzophenone hydrazone, which are characterized by spectral (IR, Uv-Vis.,) magnetic and molar conductivities. The azines and their metal complexes have been studied for their biological activity against various pathogenic bacterial strains.

EXPERIMENTAL:

Analysis and Physical measurements

Carbon, hydrogen and nitrogen were estimated by using Elemental Analyzer Carlo Erba EA1108 analyzer. The IR spectra of the azines and their Co(II), Ni(II) and Cu(II) complexes were recorded on a Shimadzu 1000 FTIR spectrometer in the range of 400 - 4000 cm^-1 in KBr disc. The electronic spectra of the complexes were recorded in HPLC grade chloroform solvent on a Shimadzu electronic spectrophotometer in the region of 200-1100 nm. Molar conductivity measurements were recorded on ELICO-CM-82 T Conductivity Bridge with a cell having cell constant 0.829 cm^-1. Magnetic susceptibility of paramagnetic solid substance was measured at room temperature in Gouy method.

Synthesis

Synthesis of ligand aldazines L^Iand L^II

The asymmetric aldazines L^I and L^II were synthesized by refluxing the reaction mixture of hot ethanol solution (30 mL) of benzophenone hydrazone (0.01mol) and hot ethanol solution (30 mL) of substituted salicylaldehyde (0.01mol) for 4-5 h with addition of a 2-3 drops of hydrochloric acid. The precipitate formed during refluxion was filtered, washed with cold EtOH, and recrystallized from hot EtOH. yield 84%.

Synthesis of Co(II), Ni(II) and Cu(II) Complexes [1-6]

An alcoholic solution of aldazines (L^Iand L^II; 0.02mol) (30 mL) was refluxed with 0.01mol of CoCl₂.6H₂O/ NiCl₂.6H₂O/CuCl₂.2H₂O in ethanol (30 mL) on water bath for 1h. Then, to the reaction mixture 1 mmol of sodium acetate was added and reflux was continued for 3h. The separated complex was filtered, washed thoroughly with water, Ethanol, Ether and finally dried in vacuum over fused CaCl₂.

In vitro antibacterial and antifungal assay

The biological activities of synthesized asymmetric aldazines and their Co(II), Ni(II) and Cu(II) complexes have been studied for their antibacterial and antifungal activities by agar and potato dextrose agar diffusion method^{18, 19} respectively. The antibacterial activities were done against Pseudomonas aeruginosa and Klebsiella. Aspergillus niger and Aspergillus flavous were used for antifungal activities at 10 mgmL^-1 concentrations in DMSO used as control. The bacteria were subcultured in agar medium. These bacterial strains were incubated for 24h at 37ºC and fungal strains were incubated for 48h. at 37ºC. Standard antibacterial (Gentamycine) and antifungal drug (Niyastatin) was used for comparison under similar conditions.

RESULTS AND DISCUSSION:

All the Co(II), Ni(II) and Cu(II) complexes are colored, (Table 1) stable non-hygroscopic in nature. These complexes are less soluble in common organic solvents but soluble in chloroform, nitrobenzene, DMF and DMSO. The elemental analyses show that, the Co(II), Ni(II) and Cu(II) complexes have 1:2 stoichiometry of the type ML₂.2H₂O, where L stands for a deprotonated ligand (Scheme). The molar conductance values are too low to account for any dissociation of the complexes in nitrobenzene, indicating the non-electrolytic nature of the complexes (Table 1). Several attempts made to develop the singal crystals were failed due to poor solubility of the complexes in common organic solvents.

Table 1. Elemental analysis of Co(II), Ni(II) and Cu(II) complexes of ligands along with magnetic moment data.

Ligand	Complex			Percentage Composition
				Metal			C			H			N
				Found		Calc.	Found		Calc.	Found	Calc.		Found		Calc.		µ_effB.M
L^I	Co(II) (1)			7.54		7.53	61.44		61.42	3.84	3.82		7.16		7.19		4.09
	Ni(II) (2)			7.63		7.60	62.4		62.39	4.14	4.12		7.28		7.26		3.1
	Cu(II) (3)			8.13		8.28	62.08		62.00	3.88	3.82		7.25		7.21		1.73
L^II		Co(II) (4)	7.54		7.53		61.44	61.42		3.84	3.82	7.16		7.19		4.09
		Ni(II) (5)	7.63		7.60		62.4	62.39		4.14	4.12	7.28		7.26		3.1
		Cu(II) (6)	8.13		8.28		62.08	62.00		3.88	3.82	7.25		7.21		1.73

Table 2. The important infrared frequencies (in cm^-1) of asymmetric azines and their metal complexes.

Compound	Co-ordinated water ν (OH)	ν (CH=N)	H-bonded –OH Stretching	Phenolic ν (C-O)	ν (M-N)	ν (M-O)
L^I	-	1625	2782	1298	-	-
L^II	-	1622	2860	1319	-	-
1	3200	1607	-	1298	458	557
2	3215	1605	-	1307	438	514
3	3210	1606	-	1307	438	565
4	3198	1607	-	1298	458	557
5	3205	1605	-	1307	455	514
6	3210	1618	-	1317	450	557

In order to establish whether the water molecules present in the complexes are coordinated to the metal ion, the weighed complex was heated for about 2 h at 105⁰C; then, cooled in desiccator and weighed again; no loss in weight of the complex was observed²⁰. These observations suggest that, the water molecules present in the complexes are coordinated to the metal ion.

Infrared spectral studies

The infrared spectral data of asymmetric azines and their Co(II), Ni(II) and Cu(II) complexes are presented in Table 2. The IR spectra of the azines show a characteristic high intensity band at 1625-1622 cm^-1 which is attributed to the ν(C=N) vibration²¹. A set of medium to strong intensity bands at 1618, 1519 – 1585, 1487 and a high intensity band at 1319-1298 cm^-1 in the IR spectra of the azines are assigned to aromatic (C=C) vibrations and phenolic ν(C-O) vibrations respectively. The free hydroxy group is expected to occur at 3400cm^-1, which shifts to lower frequency with inter and intra molecular hydrogen bonding. However the intra molecular hydrogen bonding being stronger, further shifts the band to lower frequency²². Hence, in the present study the broad band around 2860-2782 cm^-1, is attributed to intramolecularly H-bonded –OH stretching vibrations of the asymmetric azines.

In comparison with the spectra of the azines, all the complexes exhibited downward shift of ν(C=N) around 1618-1605 cm^-1 indicating that, the nitrogen is coordinated to the metal ion²³. The band due to H-bonded –OH stretching around 2860-2782 cm^-1in the azines disappeared in the spectra of complexes. The high intensity band due to phenolic ν(C-O) at 1298 cm^-1 in the ligands appeared as a medium to high intensity band in the region 1317-1298 cm^-1 in the complexes. The higher side shift of ν(C-O) in the metal complexes is may be due to the expected high mesomeric interaction in the complex that is probably activated by the presence of the metal ion²⁴. Rupini et. al., and El-sharief et. al., have reported the similar features of ν(C=N) and phenolic OH groups^{23, 24}. The presence of coordinated water molecules in the complexes are confirmed by a broad band around 3200 cm^-1 and two weak bands in the region 750-800 and 700-720 cm^-1 due to ν (-OH) rocking and wagging modes of vibrations, respectively^{23, 25}. The new bands in the region of 550-490 and 450 cm^-1 in all the complexes are assigned to stretching frequencies of (M-O) and (M-N) bonds respectively^{26, 27}.

Thus the IR spectral results provide strong evidences for the complexation of the asymmetric azines in bidentate mode.

Electronic spectral studies

The electronic spectra of octahedral Co(II) complexes exhibited absorption bands in the region 8000-10000 cm^-1 and 18000-20000 cm^-1 corresponding to ν₁ and ν₃transitions respectively, which are attributed to the transitions ⁴T_1g (F) → ⁴T_2g (F) (ν₁); ⁴T_1g (F) → ⁴T_1g (P) (ν₃). In the present investigation, brown Co(II) complexes show the absorption bands in the region 9782-9734 and 19886-19844 cm^-1 corresponding to ν₁ and ν₃ transitions respectively. These bands are characteristic of high spin octahedral Co(II) complex^{25, 28}. However, ν₂ band is not observed because of its proximity to strong ν₃ transition.

The yellowish green Ni(II) complex exhibited three bands around 10586, 16372 and 26106 cm^-1 attributed to the ³A_2g →³T_2g (ν₁); ³A_2g → ³T_1g (F) (ν₂) and ³A_2g → ³T_1g (P) (ν₃) transitions respectively, which indicate octahedral geometry around Ni(II) ion.

The Electronic spectra of Cu(II) complexes display two prominent bands. A low intensity broad band around 14486 cm^-1 is assignable to ²T_2g ← ²E_g transition. Another high intensity band at 25426 cm^-1 is due to symmetry forbidden ligand → metal charge transfer. This band shows hypsochromic shift with distortion. Further, Cu(II) is a d⁹ system which is highly vulnerable to John – Tellar distortion effect. On the basis of electronic spectra distorted octahedral geometry around Cu(II) ion is suggested²⁹.

Magnetic studies

The magnetic moments obtained at room temperature are listed in Table 1. The magnetic measurement for Co(II) complexes showed magnetic moment value around 4.2 BM which is just below the 4.3BM for octahedral Co(II) complexes. The observed low values may be due to the ligand contribution. Nickel (II) complexes show magnetic moments in the range of 3.1-3.6 BM agreeable to two unpaired electrons. The Ni (II) is a d⁸ ion and its spin only value is 2.8 BM. In octahedral state, it shifts to higher value depending on the spin orbital contribution. Hence, the observed magnetic moments indicate that the Nickel (II) is in octahedral environment^{30, 31}. The Cu(II) complexes showed magnetic moment 1.73-2.1 BM, is slightly higher than the spin-only value 1.73 BM expected for one unpaired electron. The observed values suggest that the Cu(II) in these complexes is devoid of any spin interaction which offers possibility of an octahedral geometry³².

Table 3. Antimicrobial results of asymmetric aldazines and their metal complexes.

Comp-ound	% Inhibition against Bacteria		% Inhibition against Fungi
Comp-ound	*Klebsiella*	*P. aeruginosa*	*A Niger*	*A Flavus*
L^I	4.32	10.12	7.8	2.9
L^II	5.7	9.2	11.02	5.61
1	16.80	12.20	15.34	3.46
2	5.32	10.36	10.34	6.91
3	13.00	15.20	9.66	7.66
4	-	13.22	16.44	5.66
5	13.27	14.33	10.66	7.00
6	16.66	12.33	11.66	8.44

<10 mm: Inactive; 10-12: Weakly active; 13-15: Moderately active; >16: Highly active

Biological Studies

The antimicrobial results are systematized in Table 3. In case of antibacterial studies it was observed that, the azines were found to be active against P. aeruginosa. All the Co(II) and Cu(II) metal complexes shown high potential activity against P. aeruginosa and Ni(II) complexes of L^II exhibited good activity against both the tested organisms. In case of antifungal activity, the azines shown moderate activity against A. niger. The Co(II), Ni(II) and Cu(II) metal complexes showed good antifungal activity against A. niger, particularly Co(II) complexes found to exhibit potential activity. All the metal salts exhibited negligible activity towards all bacterial and fungi species.

It is evident from the results that, the biological activity of the metal complexes is higher than the ligands. This enhancement in the activity of the metal complexes can be explained on the basis of chelation theory³³. Chelating tends to make the Schiff bases act as more powerful and potent bactereostatic agents, thus inhibiting the growth of bacteria and fungi more than the parent Schiff bases. It is suspected that, factors such as solubility, conductivity, dipole moment and cell permeability mechanism (influenced by the presence of metal ions) may be the possible reasons for the increase in activity. It has been reported in past that the metal complexes with Schiff bases possess high antimicrobial activity^34,
35.

CONCLUSION:

The newly synthesized asymmetric aldazines act as bidentate ligands. The metal ion was coordinated through the azomethine nitrogen, phenolic oxygen. The bonding of ligand to metal ion was confirmed by the analytical, spectral and magnetic studies. Biological study reveals that, the azines and some metal complexes were found to be highly active against P. aeruginosa. In case of antifungal studies Co(II) complexes are found to be highly active against A. niger.

All these observations put together lead us to propose the following structure (Figure 1) in which, the complexes having the stoichiometry of the type ML₂.2H₂O (M=Co(II), Ni(II) and Cu(II)).

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Received on 31.12.2013 Modified on 15.01.2014

Asian J. Research Chem. 7(2): February 2014; Page 200-203

Comples No	R	Complex
1	Cl	L^I– Co(II)
2	Cl	L^I– Ni(II)
3	Cl	L^I– Cu(II)
4	CH₃	L^II– Co(II)
5	CH₃	L^II– Ni(II)
6	CH₃	L^II– Cu(II)