Synthesis, Characterization and Antimicrobial Evaluation of Novel (E)-N’-(4-(1-((3,4-dimethoxypyridin-2-yl)methyl)-1H- 1,2,3-triazol-4-yl)benzylidene)benzohydrazide Derivatives

The synthesis of novel 1,2,3-triazole-hydrazone derivatives embedded with 3,4-dimethoxy pyridine ring nucleus is described. These derivatives were prepared utilizing, 2-(chloromethyl)-3,4dimethoxypyridine 1, 4-ethynylbenzaldehye 5 and various benzohydrazides7a-7j. The structures of the newly synthesized 1,2,3-triazole-hydrazones 8a-j was established on the basis of the spectroscopic techniques like 1H NMR, mass and IR data. They were evaluated against a panel of bacterial and fungal pathogens such as Staphylococcus. pyogens, Staphylococcus. Aureus (Gram positive bacteria), Escherichia.coli, Pseudomonas. aeruginosa (Gram negative bacteria) and Aspergillus niger and Candida albicans (Fungal stains). Compounds 8b, 8c, 8d, 8e and 8f with R = 4-OH, 4-OMe, 4-SO2Me, 3,45,-OMe and 3-NO2 respectively showed moderate antibacterial activity while compounds 8b, 8d, 8i and 8j with R = 4-OH, 4-SO2Me, 3,5-dichloro and 2,5-difluoro substitution exhibited very good fungal activity.


INTRODUCTION
The growth of 1,2,3-triazoles for drug discovery and industrial use has been shown to be very resourceful.The interest in the 1,2,3-triazole is due to it being non-toxic, benign and stable.Triazoles are predominantly attractive for medicinal use because they are more expected to be water soluble than normal aromatic compounds, and are stable in biological systems 1 .These triazole products readily connect with biological targets, through hydrogen bonding and dipole interactions 2 .Some of the pharmaceutically prominent triazole antifungal drugs are fluconazole 3 , isavuconazole 4 , itraconazole 5 , voriconazole 6 , pramiconazole 7 , and posaconazole 8 .Also, the examples of triazole crop protection fungicides includesepoxiconazole 9 , triadimenol 10 , propiconazole 11 , metconazole 12 , cyproconazole 13 , tebuconazole 14 , flusilazole 15 and paclobutrazol 16 .Derivatives of 1,2,3-triazole have been found to have anti-HIV 17 , anti-allergenic 18 , cytostatic 19 , virostatic 20 , anti-inflammatory 21 activities, furthermore, these triazoles are also being studied for the treatment of obesity 22 and anti-oxidant activity 23 .
The frequency of bacterial and fungal infections is an important contemporary problem due to the emerging new infectious diseases and increasing multi-drug resistance of microbial pathogens 30 .The widespread use of antibiotics has contributed to the growing infection rate since fungal infections occur after antibiotic therapy, which has the effect of killing the beneficial bacteria that normally suppress fungi.The development of new effective antifungal and antibacterial agents is strongly needed.Herein, we report the synthesis and antimicrobial evaluation of new 1,2,3 triazole derivatives linked with pyridine and hydrazone fragments.The structures of the synthesized compounds were determined by 1H NMR, mass and IR spectroscopy.
The str uctures of the synthesized compounds were confirmed by 1 H NMR, Mass and IR data.As a representative example, the 1 H NMR spectra of (E)-N'-(4-(1-((3,4-dimethoxypyridin-2yl)methyl)-1H-1,2,3-triazol-4-yl)benzylidene)-4methoxybenzohydrazide is as follows, the protons resonating at 11.78 ppm and 8.48 ppm as broad singlet corresponds to the characteristic -CONH and -CH=N-respectively, while the protons resonating at 8.72 ppm (pyridine ring proton) and 7.23 ppm (overlap of triazole and pyridine ring proton signal), is assigned to triazole ring and pyridine ring protons.The multiplet at 7.94-7.91ppm (four proton integration), a broad singlet at 7.81 ppm (two proton integration) and a doublet at 7.07 ppm (two proton integration is assigned to the protons of the 4-methoxy phenyl ring and the phenyl ring flanked to triazole ring.The aliphatic protons are observed in the expected region.The mass and IR spectral data of the hydrazone compounds are in agreement with the desired structures.
In case of fungal screening evaluation, hydrazone derivatives 8b, 8d, 8i and 8j with R = 4-OH, 4-SO 2 Me, 3,5-dichloro and 2,5-difluoro substitution exhibited very good fungal activity and the remaining hydrazone derivatives in the series showed moderate antifungal activity against the tested fungal strains with reference to Greseofluvin as the standard drug.

EXPERIMENTAL
The solvents were purified according to standard procedures prior to use, and all commercial chemicals were used as received.For thin-layer chromatography (TLC) analysis, Merck pre-coated plates (silica gel 60 F254) were used and spots were visualized with UV light.Merck silica gel 60 (230-400 mesh) was used for flash column chromatography and the eluting solvents are indicated in the procedures.Melting point (m.p.) determinations were performed by using Mel-temp apparatus and are uncorrected. 1H NMR spectra were recorded in Varian MR-400 MHz instrument.Chemical shifts are reported in d ppm (parts per million) downfield from tetramethylsilane (TMS) with reference to internal standard and the signals were reported as s (singlet), d (doublet), dd (doblet of doblet), t (triplet), q (quartet), m (multiplet) and coupling constants are in Hz.The mass spectra were recorded on Agilent ion trap MS.Infrared (IR) spectra were recorded on a Perkin Elmer FT-IR spectrometer.

2-(azidomethyl)-3,4-dimethoxypyridine 2
Sodium azide (0.365 g, 5.60 mmol) was added to a solution of compound 1(1g, 5.33 mmol) in DMF (5 mL) at room temperature.The reaction mixture was heated to 100°C for 1h.After completion of the reaction (by T.L.C), the reaction contents were cooled to room temperature and diluted with ethylacetate (15 mL) and water (5 mL).The organic layer was separated and washed with brine solution (2 X 5 mL), separated, dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to obtain the crude compound 2. The crude compound was utilized in the next step without further purification.

Experimental procedure for synthesis of benzohydrazides7a-j [34-35]
Benzoic acids a-j (8.12 mmol) was dissolved in ethanol (15 mL) and added catalytic qty of conc.H 2 SO 4 and heated to reflux for 10 h.Ethanol was evaporated and the obtained residue was diluted with ethylacetate (25 mL).The organic layer was washed with aqueous saturated NaHCO 3 (3 X 15 mL) followed by water (2 X 15 mL) and brine solution (20 mL).The organic layer was separated, dried over sodium sulphate, filtered and evaporated to obtain respective ethyl benzoates.
To the above prepared respective ethyl benzoates (6.65 mmol) in ethanol was added hydrazine-hydrate (40.0 mmol) and refluxed for 8 h.Ethanol was evaporated from the reaction mixture and the precipitated solids were slurred with petether (5 times) and filtered at the pump and dried to obtain benzohydrazides7a-7j.

General procedure for the synthesis of hydrazone derivatives 8a-8j
To a solution of compound 6 (100 mg, 0.308 mmol) in ethanol was added corresponding benzohydrazides 7a-7j (0.308 mmol) and heated to reflux for 30 min.The precipitated solids were filtered at the pump and dried to afford hydrazone derivatives 8a-8j in quantitative yields.