Synthesis and antimicrobial activity of metal complexes from 2-( 1 ’ / 2 ’-hydroxynaphthyl ) benzoxazoles

Synthesis and antimicrobial activity of new metal [Mg(II), Fe(II), Co(II), Ni(II), Zn(II) and Cd(II)] complexes from 2-(1’/2’-hydroxynaphthyl)benzoxazoles have been described. Some of the metal complexes show significant antifungal activity (MIC <3.12 μg/ml). Further, 2-(1’/2’hydroxynaphthyl)benzoxazoles exhibit excited-state intramolecular proton transfer mechanism that has been studied using absorbance and fluorescence spectroscopy.


Introduction
The excited-state intramolecular proton transfer (ESIPT) of 2-(2'-hydroxyphenyl)benzoxazoles (HBOs) has been studied under physiological conditions using absorbance and steady-state emission spectroscopy and inhibition of ESIPT via metal coordination showed a significant wavelength shift 1 .HBOs exhibit fluorescent and luminescent properties 2 and good thermal and photo stabilities 3 and are excellent materials for plastic scintillation applications 4 and some of their metal complexes are of interest for the organic light emitting diode (OLED) technology 5 .It has also been proposed that HBO behave as structural mimic of DNA base pair for which tautomerism may be initiated at a defined time and position within duplex DNA 6 .HBO moiety is also present in a number of synthetic metal ion chelators 7 .A natural product bis(benzoxazole) (UK-1) also having this moiety has been reported to possess anticancer activity and the metal ion binding studies of UK-1 indicates that it is capable of binding a variety of biologically important metal ions 8 .Recently, we have also demonstrated the metal-mediated DNA binding of UK-1 by ESI-MS that it forms complexes with a variety of metal ions 9 .The numerous applications of HBO promoted to undertake the synthesis of 2-(1'/2'-hydroxynaphthyl)benzoxazoles by replacing the 2'-hydroxyphenyl moiety in HBO with 1'/2'-hydroxynaphthyl moiety and to study

Results and Discussion
The reaction of 2-aminophenol (1) with 1-hydroxynaphthaldehyde (2) in presence of iodobenzene diacetate (IBD) resulted in 2-(1'-hydroxynaphthyl)benzoxazole (4) via the intermediacy of 3 (Scheme 1) in one pot and in 70% yield which was purified by column chromatography.All the physical and spectroscopic (UV, IR, NMR) data were in consonance with the structure.

Scheme 2
Further, the reaction of 2-aminophenol (1) with 2-hydroxynaphthaldehyde in presence of IBD afforded 2-(2'-hydroxynaphthyl)benzoxazole ( 6) which was purified by column chromatography.All the physical and spectroscopic (UV, IR, NMR) data were in consonance with the structure.The absorption and fluorescence peaks of 6 are at 373 and 443 nm, respectively.The large difference in absorption and emission spectra is again expected due to an excited-state intramolecular proton transfer.In an alternate method, 4 and 6 were also obtained by one step reaction of 1 and 1hydroxynaphthoic acid and 2-hydroxynaphthoic acid, respectively in presence of polyphosphoric acid in good yields.Furthermore, we examined the reactions of 4 and 6 with metal [Mg(II), Fe(II), Co(II), Ni(II), Zn(II) and Cd(II)] salts in 1:2 ratio that generated new metal complexes 5 and 7, respectively (Schemes 1 and 2).The structures of new metal complexes were established through elemental analysis, UV, IR and mass spectral studies.The absorption and fluorescence values of metal complexes (5 and 7) indicate the influence of metal cation binding on the proton transfer process (Table 1).The chelation of metal cations competes with protonation of the ligand donor atoms.The proton transfer is disrupted by coordination of the metal ion, but the red-shift tautomer emission did not disappear in favour of normal emission, instead, the complexation led to a change in both the absorption and emission spectra (Table 1).
The IR spectra of 5 and 7 are also in complete agreement with their structures.There was sharp modification between the IR spectra of the metal complexes and the ligands.Most of the bands changed their pattern in the region probably due to coordination of the oxygen atom of OH group and nitrogen atom of the ligand to metal ions (vide experimental).were employed for bacterial and fungal growth, respectively.Minimum Inhibitory Concentrations (MIC) were determined by means of standard serial dilution method 11 and are presented in Table 2.All the ligands/complexes exhibited appreciable in vitro activity against the tested strains.# The MIC of standard drugs for antibacterial activity (Tetracycline, Chloramphenicol, Kanamycin, Cefazoline sodium and Cefotaxime) and antifungal activity (Cycloheximide, Carbendazim and Fluconazole) were found to be <3.12µg/ml.Table 2 indicates that metal complex 5c, 5e and 7c-7e showed good antibacterial activity against Bacillus subtilis and Escherichia coli, respectively.The metal complex 5c also displayed good antibacterial activity against Staphylococcus aureus.Further, the antifungal activity of metal complex 7d was found to be significant against Aspergillus ficuum, while the ligand 6 and its metal complexes 7b-7f showed similar antifungal activity against Aspergillus parasiticus.Further, the antifungal activity of metal complex 5a was found to be significant against Candida albicans among others.Finally, the metal complex 5f was the only one to display better antifungal activity against Aspergillus niger.Most of the metal complexes showed better activity than their respective ligands ( 4 and 6).These results indicate that the increase in the size of the transition metal ions from iron to cadmium has no substantial effect on the antimicrobial activity except the nickel complex (7d) that possesses appreciable antifungal activity against Aspergillus ficuum.Interestingly the magnesium complex (5a) was found to exhibit significant antifungal activity against Candida albicans.It was also noticed that all the compounds, ligands and metal complexes were more active as antifungal than as antibacterial.
In conclusion, we have synthesized new ligands and their metal complexes and evaluated their antimicrobial activity.The results clearly showed that the magnesium complex (5a) and the nickel complex (7d) possess significant antifungal activity against Candida albicans and Aspergillus ficuum.Further, it guides us to design and synthesize the analogs of

Experimental Section
The melting points were determined in open capillaries and are uncorrected.The UV (methanol) and florescence spectra (solid) were recorded on Cary 5000 and Fluromax G (SPEX) spectrophotometer.The FTIR spectra were obtained in KBr on Perkin Elmer Spectrum RX1 instruments and are reported in cm -1 . 1 H and 13 C NMR spectra were recorded on Bruker Avance II 400 MHz and 100 MHz NMR spectrometer, respectively in CDCl 3 and are expressed as ppm with respect to TMS.Elemental analysis was carried out on Perkin Elmer 2400 instrument.The mass spectra were recorded on Voyager Elite MALDI-TOF instrument.Iodobenzene diacetate and metal salts [magnesium(II) nitrate, iron(II) nitrate, cobalt(II) nitrate, nickel(II) nitrate, zinc(II) acetate and cadmium(II) acetate] were purchased from Aldrich and were used without further purification.All the solvents were purified using standard procedures.The new ligands (4 and 6) have been made utilizing literature procures. 12,13
(100g) at 180°C for 6 h under nitrogen atmosphere.The reaction mixture was cooled to room temperature and poured in water to afford 4 and 6, in 67% and 69% yields, respectively which were purified by column chromatography using hexane as eluent.

Metal complexes
General procedure: To a solution of 4/6 (261 mg, 1 mmol) in methanol (15 ml) was added a few drops of 10% aq.sodium hydroxide and a solution of metal salt (0.5 mmol) in methanol (5 ml).The reaction mixture was refluxed for 24 h and cooled.The product thus separated was filtered, washed with water and dried to give 5a-5f and 7a-7f.

Biological studies
The in vitro antibacterial and antifungal activity of 2-(1'/2'-hydroxynaphthyl)benzoxazoles (4 and 6) and their metal complexes (5a-5f and 7a-7f) were carried out against Bacillus subtilis, Escherichia coli, Staphylococcus aureus Aspergillus ficuum, Aspergillus parasiticus, Candida albicans and Aspergillus niger using serial dilution technique in double strength nutrient broth-I.P. and Sabouraud dextrose broth-I.P. as a medium The ligands and metal complexes were dissolved in DMSO to give a concentration of 100 µg/ml (stock solution).

Antibacterial assay
A 24 h fresh cultures were obtained by inoculation of respective bacteria in double strength nutrient broth-I.P. followed by incubation at 37±1°C.The stock solution of ligands (4 and 6) and their metal complexes (5a-5f and 7a-7f) was serially diluted in tube containing 1 ml of sterile double strength nutrient broth-I.P. to get a concentration of 100 to 3.12 µg/ml and then inoculated with 100 µl of suspension of respective organisms in sterile saline (B.subtilis, E. coli and S. aureus).The inoculated tubes were incubated at 37±1°C for 24 h and minimum inhibitory concentrations (MIC) were determined.From the observed MIC values, the exact MIC values were determined by making suitable dilution of stock solution.

Antifungal assay
The antifungal activity of 4, 5a-5f, 6 and 7a-7f against the fungal species A. ficcum, A. parasiticus, C. albicans and A. niger was determined by serial dilution method similar to Antibacterial assay using Sabouraud dextrose broth-I.P. following the incubation condition of 25±1°C for a period of 7 days, except C. albicans (37±1°C for a period of 36 h).

Table 2 .
The in vitro antimicrobial activity of metal complexes (MIC in µg/ml)