GREEN CHEMISTRY APPROACH FOR THE SYNTHESIS OF NOVEL TETRAZOLE DERIVATIVES AND EVALUATION OF ANTIFUNGAL ACTIVITY

New 2-substituted-4-(5-phenyl-1H-tetrazol-1-yl)-2,3,5a,9a-tetrahydro-1H-1,5-benzodiazepine derivatives were synthesized by conventional as well as microwave method. Benzonitrile and sodium azide in the presence of ammonium chloride and DMF produces 5-phenyltetrazole; this on reaction with acetic anhydride forms 5-phenyl-1-acetyl tetrazole which reacted with different aromatic aldehydes in the presence of the alkaline medium, to yield corresponding chalcones. Chalcones on further reaction with o-phenylenediamine yield 2-substituted-4-(5phenyl-1H-tetrazol-1-yl)-2,3,5a,9a-tetrahydro-1H-1,5-benzodiazepines (4a-4j). The structures of newly synthesized compounds were characterized by physical and spectral characteristics by FT-IR and H NMR spectroscopy. All synthesized compounds were evaluated for their antifungal activity by MIC (minimal inhibitory concentration, broth dilution method) against A. niger and C. albicans. All synthesized compounds show moderate to good antifungal activity.


Introduction
Tetrazoles have been attracted as an important class of heterocyclic compounds in the field of clinical research and medicinal chemistry. Tetrazoles have not been found in nature, but they are resistant to biological degradation. This property makes it possible to use tetrazoles as isosteric substituents of various functional groups in the development of biologically active substances. 1 Tetrazole and their derivatives have great importance in pharmaceutical chemistry due to their diverse biological activity such as antifungal, 2 antibacterial, 3 antiinflammatory, 4 antituberculous, 5 antihypertensive agents, 6 anticancer, 7 antibiotic 8 and anticonvulsant. 9 Development of the tetrazole chemistry has mainly been associated with the wide-scale application of these compounds in medicine, biochemistry and agriculture. The tetrazole functionality plays a vital role in medicinal chemistry, primarily due to its ability to serve as the bioequivalent (bioisoster) of the carboxylic acid group. In particular, 1-substituted tetrazoles and 5-thio-substituted tetrazoles have been used in the synthesis of pharmacologically active drugs. 3 The 1,5-benzodiazepines moiety is a privileged class of pharmacophore, as compounds bearing this structural unit possess a broad spectrum of biological activities, as antimicrobial, 10 anti-inflammatory 11 , anticancer 12 and anticonvulsant activities. 13 The synthesis of the 1,5benzodiazepines moiety involves the reaction of chalcones with o-phenylenediamine. 14 Tetrazoles clubbed with benzodiazepines will help to improve the antifungal properties of the pharmacophore leading to more potent compounds.
In recent years, organic reactions involving a green chemistry approach have received considerable attention in organic synthesis because of their ease handling, enhanced reaction rates, more excellent selectivity, simple workup and recoverability of the products. 15 The synthesis of novel tetrazole based benzodiazepines derivatives and investigation of their chemical and biological behavior has gained more importance in recent decades for biological and pharmaceutical reasons.
In continuation of research in the field of green chemistry, an attempt is made to synthesize tetrazole containing benzodiazepine and the compounds have been evaluated for antifungal activity, which has not been reported yet.

Experimental
Melting points were determined with open capillary and were uncorrected. FT-IR spectra were recorded on a 'JASCO FT-IR-4600' spectrophotometer, 1 H-NMR spectra were recorded in BRUKER AVANCE II400'NMR spectrometer at 400 MHz frequency in DMSO using TMS as an internal standard.

Synthesis of 5-phenyltetrazole (1)
A mixture of benzonitrile (3.3 g, 0.10 mol), sodium azide (0.65 g, 0.10 mol) dimethylformamide (10 mL) and ammonium chloride (5.3 g, 0.10 mol) was heated in an oil bath for 7 h at 125 °C. The solvent was removed under reduced pressure. The residue was dissolved in 100 mL of water and carefully acidified with concentrated hydrochloric acid to pH 2. The solution was cooled to 5 °C in an ice bath. Compound 1 has been recrystallized from aqueous methanol.

Synthesis of 5-phenyl-1-acetyltetrazole (2)
A solution of 5-phenyl tetrazole (12.8 g, 0.08 mol), acetic anhydride (0.08 mol) and 2-3 drops of concentrated sulphuric acid were heated for 15-20 min on a water bath, then cooled and poured into ice-cold water. The product was filtered and dried and recrystallized from ethanol.
General procedure for the preparation of chalcones (3a-3j): Method 1. Conventional synthesis A solution of 5-phenyl-1-acetyltetrazole (8.5g, 0.005 mol) and the aromatic aldehyde (0.005 mol) in ethanol (12 mL) was cooled to 5 to 10 o C in an ice bath. The cooled solution was treated with dropwise addition of aqueous potassium hydroxide (2.5 mL, 50 %). The reaction mixture was stirred for 30 min and then left overnight. The resulting dark solution was diluted with ice water and carefully acidified using diluted hydrochloric acid. The chalcone was collected by filtration and washed with aqueous sodium bicarbonate and water then recrystallized from ethanol.

Method 2. Microwave-assisted synthesis
A mixture of 0.01 mol 5-phenyl-1-acetyltetrazoles, 0.01 mol of aromatic aldehydes, ethanol (5 mL) and 2.5 mL of NaOH (6 M) was kept in a microwave oven at level 2 and time for 2 min. The removed mixture was cooled in an icebath and acidified with concd. HCl. The chalcone was collected by filtration.

Results and discussions
The tetrazole derivatives were synthesized using conventional as well as microwave-assisted synthesis methods according to Scheme. Spectral data confirmed the structure of all synthesized derivative. 5-Phenyltetrazole (compound 1) was prepared by the reaction of benzonitrile with sodium azide in the presence of ammonium chloride and DMF. 5-Phenyltetrazole (1) was converted to 5-phenyl-1-acetyltetrazole (2) by the reaction with acetic anhydride and sulphuric acid. Compounds 3a-3j were obtained by treatment of 2 with aromatic aldehydes in the presence of NaOH. Compounds 3a-3j on treatment with o-phenylenediamine in the presence of ethanol and NaOH yielded a compounds 4a-4j, respectively.
The 1 H-NMR spectra show the chemical shift at 6.9-7.8 due to aromatic protons, 3.2-5.6 due to (5H benzodiazepine part). The results of spectral data are in good agreement with the structure of synthesized compounds.  The results of antifungal activity are depicted in Table 2 and Fig. 1, revealing that all compounds show antifungal activity against Aspergillus niger and Candida albicans. The activities are comparable with control standard Fluconazole shows potent activity at MIC of 85 and 110 µg mL -1 . Compounds 4b and 4g (4-OH, 3-NO2) have shown good antifungal activity against A. niger while compounds, 4c, 4e and 4f, (4-Br, 4-NO2, 2-Cl) have shown good antifungal activity against C. Albicans. Compounds 4a and 4i (4-Cl, 4-N(CH3)2) have shown moderate while compounds 4d, 4h and 4j showed weak antifungal activity against A. niger and C. albicans.

Conclusions
Tetrazole derivatives were synthesized from 5-phenyltetrazole which was synthesized from benzonitrile and sodium azide in good yields. The compounds 4b and 4g possess 4-OH, 3-NO2 potent anti-inflammatory activity in comparison with control. The compounds 4c, 4e and 4f containing 4-Br, 4-NO2, 2-Cl substitution produce moderate anti-inflammatory activity. The compounds 4d, 4h and 4j have shown weak anti-fungal activity against A. niger and C. Albicans.