Hybrid Approach for Synthesis and Antimicrobial Activity of Heterocyclic Compounds

. We have synthesized novel series of N -(1-(2-(1-phenyl-3-( p -tolyl)-1 H -pyrazol-4-yl)-5-(pyridin-4-yl)-1,3,4-oxadiazol-3(2 H )-yl)ethylidene)arylaniline and their derivatives. The structures of synthesized compounds were well characterized by spectroscopic techniques. Antimicrobial activity of the newly synthesized derivatives was evaluated against gram positive ( S. aureus and S. pyogenes ), gram negative bacteria ( E. coli and P. aeruginosa ), and strains of fungi ( C. albicans , A. niger and A. clavatus ). Among the screened derivatives 5c , 5f , 5i , 5l and 5t demonstrated superior antimicrobial activity against microbial strains.


Introduction
Nitrogen-rich heterocycles, particularly pyrazole 1,3,4-oxadiazole and pyridine, represent an exclusive class of diversified frameworks exhibiting a broad spectrum of biological functions. Some microbial species are responsible for dangerous diseases such as food poisoning, typhoid and due to this; it is the global concern for public health. All these species are fully resisted against old fashionable antimicrobial drugs. For this reason new discovery of antimicrobials are important for the medicinal chemists. Heterocycles comprising nitrogen especially five member rings have captured huge interest due to their availability in many natural products and as vital part of synthetic bioactive molecules [1]. In this framework, pyridine, pyrazole and 1,3,4-oxadiazole templates featuring one, two nitrogen's and an oxygen atom [2] is recognized as a member of fortunate structural class particularly from medicinal point of view [3]. Commercially available drugs ( Fig. 1) celecoxibe, zibotentan and sulphapyridine sequentially contain pyrazole [4], 1,3,4-oxadiazole [5] and pyridine heterocycles. Among the important heterocyclic scaffolds of biological and pharmacological interest, the pyrazole ring is able with diverse medicinal activities [6][7][8][9][10][11][12].
Molecular hybridization is a significant tool for discovery of novel chemical entities. In the past some decades much attention has been given to the plan and synthesis of new types of pharmacologically diverse structural hybrid molecules [21]. Encouraged by the above-mentioned explanation, we have focused onto construct some novel antimicrobial derivatives containing pyrazole, 1,3,4-oxadiazole and pyridine scaffolds screened for antimicrobial activity. Previously our research group has worked on the synthesis of pyrazole and their derivatives as potential antimicrobials [22][23][24]. In furtherance to this, N-(1-(2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-5-(pyridin-4-yl)-1,3,4-oxadiazol-3(2H)-yl)ethylidine)arylanilines (5a-t) have been synthesized and screened for their antimicrobial activity against standard strains of bacteria and fungi [25]. All final compounds were categorised by different spectroscopic methods such as, infrared spectroscopy, 1 H and 13 C nuclear magnetic resonance and mass.

Chemistry
All reactions except those in aqueous media were carried out by standard techniques for the keeping out of wetness. Melting points were determined on an electro thermal melting point apparatus and were reported uncorrected. TLC on silica gel plates (Merck, 60, F 254 ) was used for purity checking and reaction monitoring. Column chromatography on silica gel (Merck, 70-230 mesh and 230-400 mesh ASTH for flash chromatography) was applied when necessary to isolate and purify the reaction products. Elemental analysis (% C, H, N) was carried out by a Perkin-Elmer 2400 CHN analyzer. IR spectra of all compounds were recorded on a Perkin-Elmer FT-IR spectrophotometers in KBr. 1 H NMR and 13 C NMR spectra were recorded on Varian Gemini 500 MHz in DMSO-d 6 as a solvent and tetramethylsilane (TMS) as an internal standard. Mass spectra were scanned on a Shimadzu LC-MS 2010 spectrometer. Anhydrous reactions were carried out in oven-dried glassware in nitrogen atmosphere.
Novel compounds (5a-t) were categorized by different spectroscopic methods such as, IR, 1 H NMR, 13 C NMR, and mass. In compound 5l, group >C=N-and >C=C< stretching vibrations appeared at 1597 and 1537 cm -1 . Nitro group of aniline showed stretching vibrations at 1504 cm -1 . C-CH 3 group which was attached to 1,3,4-oxadiazole ring showed stretching vibrations at 1355 cm -1 . The absorption band at 1292 and 1224 cm -1 is due to stretching vibration of >C=N-and C-O-C oxadiazole ring. In 1 H NMR characterization C 2 -H and C 6 -H protons of pyridine ring showed doublet at 8.82 ppm. In pyridine ring two protons C 3 -H and C 5 -H appeared at 8.12 ppm as doublet. Singlet signal of pyrazole ring was given by C 5 -H at 7.96 ppm. All other aromatic 13 protons showed multiple in range of 6.95-8.15 ppm. At 5.85 ppm singlet signals appeared by one

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ILCPA Volume 77 proton of oxadiazole ring. Remaining six protons showed two singlet signals at 2.39 and 2.28 ppm out of six three protons are of -CH 3 ring and another three from -CH 3 -C=N. Looking to the 13 C NMR spectra, the chemical shifts of the final compound 5l has carbons that varied from δ = 158.3-21.5 ppm. Signal of C 2 and C 5 in 1,3,4-oxadiazole ring appeared at 158.0 at and 78.3 ppm. In pyridine ring C 2 , C 3 and C 5 showed peak at 149.2, 138.2 and 124.7 ppm. In pyrazole ring system C 3 , C 4 and C 5 signals appeared at 149.5, 123.8 and 117.8 ppm. One carbon of -CH=N-displayed peak at 156.3 ppm. Furthermore, the mass spectrum of (5a-t) appeared a molecular ion peak corresponding to molecular formula (5a-t) onward of other fragment peaks, which supported the proposed structure of final compounds. Detail discussion of experimental data is given in supporting section.

3-chloro-4-fluoro-N-(1-(2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-5-(pyridin-4-yl)-1,3,4oxadiazol-3(2H)-yl)ethylidene)aniline: 5t
IR  Table 2 exposed that most of the tested compounds displayed variable inhibitory effects. These all titled compounds (5a-t) were shown screening at different concentration of 1000, 500, 200, 100, 50, and 25 μg/mL by broth dilution method [27][28][29]. In this method 2% DMSO used as a diluents and standard drugs to test upon standard microbial strains. At 1000 μg/ML concentration synthesized compounds were diluted. The control tube containing no antibiotic was immediately subcultured [before inoculation] by spreading a loopful evenly over quarter of a plate of medium suitable for the growth of test organisms. At 37ºC, for 24 h the culture tubes were then incubated and the development of bacteria was monitored visually and spectrophotometrically. 10 μg/mL suspensions were more inoculated on a suitable media, after 24 and 48 h development of bacterial strains was noted. The lowest concentration (the highest dilution) required to arrest the growth of bacteria was regarded as MIC. DMSO and sterilized distilled water were used as negative control, while chloramphenicol antibiotic (1 U strength) was used as positive control. The recently synthesized derivatives 5a-t was displayed for their antifungal activity against in six sets against C. albicans (MTCC-227), A. niger (MTCC-282) and A. clavatus (MTCC-1323) at various primary concentrations of 1000, 500 and 250 μg ml -1 . The primary screen active compounds were similarly diluted to obtain 200, 125, 100, 62.5, 50, 25 and 12.5 μg ml -1 concentrations for secondary screening to test in a second set of dilution against all microorganisms, For antifungal activity griseofulvin used as a standard drug, which showed 500, 100 and 100 μg ml -1 MIC against responsible fungal strains such as C. albicans, A. niger and A. clavatus. For development of fungi, in the present method, we have used Sabourauds dextrose broth at 28ºC in aerobic condition for 48 h. Sterilized distilled water and DMSO and used as negative controls while 'griseofulvin' (1 U strength) was used as a positive control".

Antimicrobial studies
For antibacterial activity ampicillin used as a standard drug and results displayed in Table 2. Compounds 5c and 5l having (C 6 H 4 -4-F), (C 6 H 4 -4-NO 2 ) displayed excellent activity against E. coli. In case of gram negative bacteria P. aeruginosa, compounds 5c (C 6 H 4 -4-F) and 5r (C 6 H 4 -4-NO 2 ) International Letters of Chemistry, Physics and Astronomy Vol. 77 exhibited very good and good activity at MIC = 25 and 50 µg ml -1 . Compound 5t (C 6 H 3 -3-Cl-4-F) had shown MIC = 12.5 µg ml -1 which observed as the highest inhibition against S. pyogenes. Other compounds showed moderate activity against bacterial strains as shown in Table 2. For antifungal activity MIC was carried out on C. albicans, A. niger and A. clavatus by conventional broth dilution method. Griseofulvin was used as a standard drug for antifungal activity at different concentration of 1000, 500, 250, 100, 50, 25 and 12.5 μg/mL. Excellent and good activity posseded against C. albicans and A. clavatus by derivatives 5i (C 6 H 4 -4-OCH 3 ) and 5f (C 6 H 4 -4-CH 3 ). Other derivatives showed moderate antifungal activity against responsible fungal strains. The results of the antifungal studies are reported in Table 2. Data given in table shown that the presence of functional group at para position amended antibacterial activity as compared to ortho and meta substituted derivatives. SAR studies SAR studies exposed that the antimicrobial activity in heterocyclic class of pyrazole, 1,3,4oxadiazole and pyridine scaffolds rely on the character of the peripheral substituent and their spatial relationship within this framework. Different electronic environment of the molecules was confirmed by the pattern of substitution. The electronic nature of the substituent escort to important variance in antimicrobial activity. The occurrence of chloro, fluoro and nitro substituents at para situation on aromatic ring arrangement has enlarged the antibacterial activity of compounds

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ILCPA Volume 77 compared to those of electron withdrawing groups. The presence of electron donating such as methyl and methoxy group on phenyl ring provides a positive effect on antifungal activity.

Conclusion
We have synthesized novel series of (5a-t) by conventional method with growth in product of reactions. The excellent properties of this new series of titled compounds of antimicrobial activity deserve further examination. The mode of action at molecular level explains the activity observed. The effectiveness and deeper insight in to titled compounds' SAR study committed to show additional physiochemical and biological parameters.