Synthesis and Antimicrobial Activity of a New Class of Sulfone Linked Bisheterocycles

A new and novel class of bis(heterocycles) viz., bis pyrroles, pyrrolyl pyrazoles and pyrrolyl isoxazoles are prepared from 1-aroyl-2-styrylsulfonylethenes by 1,3-dipolar cycloaddition of tosylmethyl isocyanide, diazomethane, nitrile imines and nitrile oxides. The lead compounds are screened for antimicrobial activity.


Antimicrobial Testing
The compounds 2, 3, 7-9 were tested for antimicrobial activity at two different concentrations 100 and 200 μg/mL.The antibacterial activity was screened against Staphylococcus aureus, Bacillus subtilis (Gram-positive bacteria) and Escherichia coli, Klebsiella pneumoniae (Gram-negative bacteria) on nutrient agar plates at 37˚C for 24 hr using chloramphenicol as reference drug.The compounds were also evaluated for their antifungal activity against Fusarium solani, Curvularia lunata and Aspergillus niger using ketoconazole as standard drug.Fungi cultures were grown on potato dextrose agar medium (PDA) at 25˚C for 3 days.The spore suspension was adjusted to 106 pores/mL at an mg/mL concentration by the Vincent and Vincent method [23].
The results of the compounds of preliminary antibacterial testing are shown in Table 3.The results revealed that the compounds 2 and 3 exhibited least activity against Gram-positive bacteria and almost no activity against Gram-negative bacteria.However, the other compounds showed higher inhibitory activity against Grampositive bacteria than that of Gram-negative bacteria.It was reported that good DNA binding properties are a prerequisite for antibacterial activity [8].This was evidenced by the fact that the compounds 7 showed good activity when compared with compounds 8.This may be due to the bulkier tetrasubstituted pyrazole destroys DNA binding and activity.In fact, the compounds having trisubstituted pyrazole (7) and disubstituted isoxazole (9) units exhibited good activity when compared with the compounds having terasubstituted pyrazole (8) unit.It was observed that the presence of chloro substituent enhances the activity.The compounds 7c and 9c displayed excellent activity against Gram-positive bacteria (inhibitory zone > 28 mm) and good activity against Gramnegative bacteria (inhibitory zone > 22 mm).All the test compounds showed moderate to high inhibitory effect towards tested fungi (Table 4).
The MIC values were determined as the lowest concentration that completely inhibited visible growth of the microorganisms (Table 5).The structure-antimicrobial activity relationship of the tested compounds revealed that disubstituted pyrazole and trisubstituted isoxazole in  combination with pyrrole displayed greater activity.The compounds having tetrasubstituted pyrazole with pyrrole exhibited least activity.The maximum activity wasobserved with the compounds 7c and 9c.

Experimental Section
Melting points were determined in open glass capillaries on a Mel-Temp apparatus and are uncorrected.The purity of the compounds was checked by TLC (silica gel H, BDH, ethyl acetate/hexane, 1:3).The IR spectra were recorded on a Thermo Nicolet IR 200 FT-IR spectrometer as KBr pellets and the wave numbers were given in cm -1 .The 1 H and 13 C NMR spectra were run in CDCl 3 / DMSO-d 6 on a Jeol JNM spectrometer operating at 400 and 100 MHz.All chemical shifts were reported in δ ppm using TMS as an internal standard.The elemental analyses were determined on a Perkin-Elmer 24˚C elemental analyzer.The starting material 1-aroyl-2-styrylsulfonylethene (1) was prepared by the literature procedure [24].under vacuum resulted in a solid which was purified by column chromatography (ethyl acetate/hexane, 1:4).General procedure for the synthesis of 4-aroyl-3-(4'phenyl-4',5'-dihydro-1'H-pyrazol-3'-ylsulfonyl)-1H-pyr role (4) To a cooled solution of 2 (5 mmol) in dichloromethane (20 mL), an ethereal solution of diazomethane (40 ml, 0.4 M) and triethylamine (0.12 g) were added.The reaction mixture was kept at -20 to -15 ˚C for 40 -48 hr.The solvent was removed under reduced pressure and the resultant solid was recrystallized from 2-pro-panol.

Scheme 1. Synthesis of bis heterocycles.
1H NMR spectrum of 2a showed two singlets at δ 7.01 and 8.02 ppm for C 2 -H, and C 5 -H of pyrrole ring protons.Two doublets are observed at 6.79 and 7.48 ppm corresponding to olefin protons in addition to the signals of the aromatic protons.
5 -H) ppm apart from signals due to aromatic protons (Table 2).The olefin moiety in 2 is used to develop different het-

Table 1 . Physical characterization data of compounds 2-9.
a Yield in Method-2; b Yield in Method-3