Synthesis and Antimicrobial Activity of Some New 3,5-Disubstituted Pyrazoles and Isoxazoles

A new class of 3,5-disubstituted pyrazoles and isoxazoles were prepared from the Michael acceptors 1-furanyl / thiophenyl / pyridinyl-3-indole-prop-2-en-1-ones under ultrasonication and evaluated for antimicrobial activity. Amongst all the tested compounds fluoro substituted thiophene linked compounds 12b and 18b displayed promising antibacterial activity particularly against Bacillus subtilis and antifungal activity against Aspergillus niger. Furthermore, compounds with more number of electron withdrawing groups showed higher antimicrobial activity. This result indicates that compounds 12b and 18b can be used as lead compounds in the future studies.


Experimental Protocols
All the chemicals were purchased from commercial sources and used without further purification. Melting points were determined in open capillaries on a Mel-Temp apparatus and are uncorrected. The homogeneity of the compounds was checked by TLC (silica gel H, BDH, hexane/ethyl acetate, 3:1). 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 NMR spectra were recorded in CDCl 3 /DMSO-d 6 on a Jeol JNM λ-400 MHz spectrometer. The 13 C NMR spectra were recorded in CDCl 3 /DMSO-d 6 on a Jeol JNM spectrometer operating at λ-100 MHz. High-resolution mass spectra were recorded on Micromass Q-TOF micromass spectrometer using electrospray ionization. All chemical shifts were reported in δ (ppm) using TMS as an internal standard. The microanalyses were performed on a Perkin-Elmer 240C elemental analyzer. The temperature was measured by flexible probe throughout the reaction. Ultrasonication was performed in a Bandelin Sonorex RK 102H ultrasonic bath operating at frequency of 35 KHz.

Antimicrobial Testing
The compounds 8-19 were dissolved in DMSO at different concentrations of 50 and 100 µg/well.

Antibacterial and antifungal assays
The in vitro antimicrobial studies were carried out by agar well diffusion method against test organisms [35,36]. Nutrient broth (NB) plates were swabbed with 24 h old broth culture (100 µl) of test bacteria. Using the sterile cork borer, wells (6 mm) were made into each petriplate. Various concentrations of DMSO dissolved compounds (50, 100 µg/ well) were added into the wells by using sterile pipettes. Simultaneously the standard antibiotics, Chloramphenicol for antibacterial activity and Ketoconazole for antifungal activity (as positive control) were tested against the pathogens. The samples were dissolved in DMSO which showed no zone of inhibition acts as negative control. The plates were incubated at 37°C for 24 h for bacteria and at 28°C for 48 h for fungi. After appropriate incubation, the diameter of zone of inhibition of each well was measured. Duplicates were maintained and the average values were calculated for eventual antibacterial activity.
Broth dilution test was used to determine minimum inhibitory concentration (MIC) of the above mentioned samples [37,38]. Freshly prepared nutrient broth was used as diluents.  [39] and minimum fungicidal concentration (MFC) [40] for each set of test tubes in the MIC determination, a loopful of broth was collected from those tubes which did not show any growth and inoculated on sterile nutrient broth (for bacteria) and PDA (for fungi) by streaking. Plates inoculated with bacteria and fungi were incubated at 37°C for 24 h and at 28°C for 48 h, respectively. After incubation, the lowest concentration was noted as MBC (for bacteria) or MFC (for fungi) at which no visible growth was observed.

Biological evaluation
Antimicrobial activity: The compounds 8-19 were evaluated for antimicrobial activity at two concentrations (50 and 100 µg/well) by agar well diffusion and broth dilution methods. The results regarding the antibacterial activity presented in Table 1 and Figure 1 indicated that Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) were more susceptible against the tested compounds than Gramnegative bacteria (Pseudomonas aureginosa and Klebsiella pneumoniae). The compounds 8b, 9a, 10a and 14b displayed low activity whereas the compounds 8a and 14a showed no activity. Amongst all the compounds 12b (39 µg/well) and 18b (41 µg/well) displayed higher antibacterial activity than the standard Chloramphenicol (38 µg/well) particularly against B. subtilis. However, the remaining compounds showed moderate to good activity. The compounds having pyrazole (11)(12)(13) and isoxazole units (17)(18)(19) displayed higher antibacterial activity than pyrazoline (8)(9)(10) and isoxazoline rings (14)(15)(16). The presence of electron withdrawing groups on the aromatic ring enhanced the activity.
All the tested compounds 8-19 inhibited the spore germination against the fungi Aspergillus niger and Penicillium chrysogenum except 8a and 14a (Table 2 and Figure 2). In general, all the compounds showed comparatively higher antifungal activity against A. niger than P. chrysogenum. Amongst all the tested compounds 12b (37 µg/well) and 18b (39 µg/well) displayed higher antifungal activity than the standard drug (36 µg/well). It was observed that thiophene linked compounds (9, 12, 15 and 18) inhibited the spore germination against the tested fungi when compared with the other compounds. The MIC, MBC and MFC values of the tested compounds are shown in Table 3. The compounds 12b and 18b exhibited low MIC values. The MBC value of compounds 12b and 18b is 2 × MIC in case of B. subtilis and MFC value of 12b and 18b is 2 × MIC in case of A. niger.
The structure-activity relationship of the tested compounds revealed that thiophene linked compounds showed greater activity than furan and pyridine moieties. Further, it was observed that aromatized heterocycles 11-13 and 17-19 showed greater antimicrobial       activity than non-aromatized compounds 8-10 and 14-16. Moreover aromatized heterocyclic compounds having isoxazole unit displayed slightly higher activity than pyrazole unit. This is may be due to the presence of electron withdrawing oxygen atom. It was also noticed that compounds having electron withdrawing fluoro substituent on aromatic ring enhanced the activity when compared with the unsubstituted ones. Besides, the compounds having more number of electron withdrawing groups showed increased antimicrobial activity. Amongst all the compounds 12b and 18b were found to be potential antimicrobial agents particularly against Bacillus subtilis and Aspergillus niger. This result indicates that compounds 12b and 18b can be used as lead compounds in the future studies.

Conclusion
A new class of 3,5-disubstituted pyrazoles and isoxazoles were prepared from the Michael acceptors 1-furanyl / thiophenyl / pyridinyl-3-indole-prop-2-en-1-ones under ultrasonication and evaluated for antimicrobial activity. In fact, the target compounds were obtained in shorter reaction times with high yield under ultrasonication when compared with conventional method. Amongst all the tested compounds 12b and 18b displayed promising antimicrobial activity particularly against Bacillus subtilis and Aspergillus niger. The presence of electron withdrawing fluoro substituent on the aromatic ring enhanced the activity than the unsubstituted ones. Furthermore, compounds with more number of electron withdrawing groups showed higher antimicrobial activity. This result indicates that compounds 12b and 18b can be used as lead compounds in the future studies.