Synthesis and In-vitro Antifungal Evaluation of 5-Pyrazolones

Experimental: All chemicals and solvents, reagents used in the present study were of analytical grade and solvents were used after distillation. All the melting points of the synthesized compounds were determined by open capillary and are uncorrected. The purity of the compounds was checked using precoated TLC plates (MERCK) using chloroform: ethyl acetate (8:2) solvent system. The developed chromatographic plates were visualized under UV at 254nm. IR spectra were recorded using KBr on Perkin Elmer spectrophotometer. HNMR and CNMR spectra in CDCl3 on a BRUKER FT-NMR instrument using TMS as internal standard and chemical shift values were expressed in ppm. Elemental analysis (CHN) was performed on Elementarvario MICRO cube CHN analyser.


INTRODUCTION
In recent years, fungal infections spreaded worldwide infection to life threatening systemic ailments involving the internal organs ranging from minor superficial skin and mucous membrane.The search for new, effective and safe nuclei leads to important modifications in the existing drugs by increasing their efficacy as well as formulating new bioactive agents by molecular modifications.The role of medicinal chemistry is essential and sustainable for previous and current generation.In the practice of medicinal chemistry developed from an empirical organic synthesis of new compound based on the modification of structure and identifies their biological activity [1,2].Moreover, various heterocyclic and biologically active compounds have five-member nitrogen, sulphar, oxygen containing heterocyclic ring [3].Pyrazolone is a five membered lactum ring, containing two nitrogen and one ketonic group in its structure.
Pyrazolones have acquired versatile importance as drug substances in pharmaceutical industry of their biological importance.For instance, various pyrazolones drugs, viz.phenazone, propyphenazone, ampyrone and metamizole are useful antipyretic and analgesic drugs [4].Therefore, pyrazolones possess antimicrobial, antifungal [5], antimycobacterial [6,7], antibacterial [8], anti-inflammatory [9], antitumor [10], gastric secretion stimulatory [11], antidepressant [12] and antifilarial activities [13].Many attempts have been made to synthesize, characterize and to study biological activity of pyrazolones [14].Interest in the chemistry of a new organic photochromic compound containing pyrazolone-ring was synthesized and characterized by Liu et al.They evaluated their photocromic properties were related to the photoisomerization and time-dependent UV-vis spectra, and these compounds exhibited good antibacterial activities [15].Atudosie et al. [16] have been reported the synthesis of new 5-substituted-2-[2-(2-substituted-10Hphenothiazin-10-yl)-2-oxoethyl]-2,4-dihydro-3H-pyrazol-3 one containing phenothiazine unit by reaction of Nchloroacetyl compound, ethyl acetoacetate with hydrazine hydrate and their were evaluated antiproliferative activity.In the recent years, the chemistry and antibacterial activity of pyrazolone have been investigated and synthesized to be novel pyrazolones from easily available starting materials and their broad range of antimicrobial and anti-inflammatory activity were evaluated [17 -23].The study was aimed at exploring our synthesis of some new biologically active pyrazolone derivatives by the reaction of thiosemicarbazide and ethyl-2-chloro acetoacetate.

MATERIALS AND METHODS
All materials were of commercial reagent grade and purchased from Sigma-Aldrich.All reaction were monitored by column chromatography with E-Merck silica precoated plates and visualization was executed by ultraviolet and iodine chamber.Melting points were measured by open glass capillary and are uncorrected. 1H and 13 C NMR spectra in CDCl 3 were taped on a BRUKER AVANCE II 129 400 MHz FT-NMR spectrometer (Bruker Bio Spin, Switzerland) at 400 and 100 MHz, respectively.IR spectra were taped using Perkin Elmer FTIR spectrophotometer.Elemental analysis was performed on Elementarvario MICRO cube CHN analyser.All yields refer to isolated products.

Disc Diffusion Assay
It is done by using Kirby-Bauer method to determine the antibacterial susceptibility at a fixed concentration [24].For this, few colonies of organism were inoculated in 2-5 ml broth and grown for 2.5 hours.Before inoculation, dried agar plates to prevent flow of inoculated material during incubation.A sterile cotton swab is dipped into the bacterial suspension and used to evenly spread the diluted culture on the agar surface.After the inoculation dried, impregnated discs were placed on the agar surface with flamed forceps and gently pressed down to ensure contact [25].The sterile (6 mm diameter) discs impregnated with fixed doses 600µg/ml were assassible on the pre-inoculated accede.The seeded petri-dishes were incubated within 30 minutes at 37 o C for 48 hours.Similar plates were prepared for the standard drug, the reference antifungal drug fluconazole was used.Dimethylformamide was used as control solvent in the assay.The zone of inhibition is directly proportional to the degree of sensitivity of fungal strain and the concentration of compound.The antifungal activity of the compounds was calculated by using the formula as given below:

Determination of Minimum Inhibitory Concentration (MIC) Value
The antibacterial screening was determined using disc diffusion method by measuring zone of inhibition in mm.

Biological Assay
The antifungal activity of all synthesized compounds (3a-g) were tested against three fungal strains, viz; A. niger, C. albicans and Curvularia, using disc diffusion technique.Compounds (3a-g) were liquefy in dimethylformamide at different concentrations and the discs were dipped into the respective compounds and put on the petri dishes of specified organisms against a control of dimethylformamide.After 48 hours, the zone of inhibition was mascurated in mm. and the details of antifungal activity were furnished in Table 2.

RESULTS AND DISCUSSION
Synthetic route of the target compounds (3a-g) is depicted in Scheme 1.Here, we reported newly synthesized biologically active pyrazolone derivatives by the reaction of thiosemicarbazide and ethyl-2-chloro acetoacetate with DMF as a solvent.Dimethylformamide is a polar (hydrophilic) aprotic solvent with a high boiling point as compared to ethanol.It can easily react with NHNH 2 SCN reactant compound and makes it possible to react with ethyl-2-choloro acetoacetate and leads to the formation of target compounds (3a-g).
Scheme. 1. Synthesis of substituted pyrazolone derivatives.The structures of the synthesized compounds (3a-g) were determined on the basis of their FT-IR, 1 H-NMR, 13 C-NMR and elemental analysis.The IR spectra of target compounds (3a-g) showed the presence of these groups N-H, C=O, C-N, C-Cl and C=S absorptions band at 3,200-3,430 cm -1 , 1,600 cm -1 , 750 cm -1 , 1400-1490 cm -1 and 1,150 cm, -1 respectively.In 1 H-NMR spectra, all the compounds were characterized due to the presence of aromatic protons (Ar-CH) expected multiplet near δ 7.22-7.67ppm.Another N-H protons also exhibited singlets at δ 2.20-2.30ppm, respectively whereas, the pyrazolone methyl protons seemed high as a singlet near δ 1.32 ppm. 13C NMR spectra recorded signals correspond to thiosemicarbazide moiety and other aromatic corbons.The mass spectrum of pyrazolone (3a) sustained molecular ion peak at m/z= 267.02 (M) + , with the molecular formula C 11 H 10 ClN 3 OS.And all compounds gave satisfactory elemental analysis.

Antifungal Activity
The antifungal activities were found of compounds (3a-g) against Candida albicans, Aspergillus niger and Curvularia fungal strain.Table 2 shows result of in-vitro antifungal bioassay.The reference antifungal drug was Fluconazole.Compounds 3d and 3g were assigned to inhibit the growth of Aspergillus niger and Candida albicans, respectively, compounds 3c and 3f exhibited remarkable inhibition on Curvularia, respectively.Here we can conclude that different methyl and methoxy group on aromatic ring increases the antifungal activity of different derivatives.

CONCLUSION
In the present work, substituted pyrazolones have been successfully synthesized by using thiosemicarbazide and ethyl-2-chloro acetoacetate as a starting materials.The synthesized compounds were deduced by spectral analysis ( 1 H -NMR, 13 C -NMR, IR, Mass) and elemental analysis.The result of screening clearly indicated the nature of substitution in newly synthesized compounds affected in-vitro antifungal activity.The presence of electron-withdrawing group on the aromatic ring of thiosemicarbazide increases the antifungal activity of tested compounds.Here, electron-donating group were also shows moderate activity against tested pathogens such as Aspergillus niger, Candida albicans and Curvularia.All the synthesized compounds exposed better antifungal activities against a wide range of microorganisms.