An efficient synthesis and antimicrobial evaluation of some new pyrazoline, pyrimidine and benzodiazepine derivatives bearing 1,3,5- triazine core

In our present investigation a new class of diverse sets of acetyl pyrazolines (6a-e), amino pyrimidines (7a-e) and 1,5-benzodiazepines (8a-e) bearing 1,3,5-triazine core were synthesised from chalcones (5a-e). Treatment of chalcone with hydrazine hydrate, guanidine hydrochloride and ophenylenediamine afforded the corresponding acetyl pyrazoline, amino pyrimidine and 1,5-benzodiazepine derivatives respectively. The structures of all the newly synthesised compounds were assigned on the basis of FTIR, 1 H NMR, 13 C NMR, mass spectral data as well as elemental analysis. In vitro antimicrobial proficiency of the title compounds were assessed against selected pathogens S. aureus MTCC 96, S. pyogeneus MTCC 442, E. coli MTCC 443 and P. aeruginosa MTCC 1688 bacteria for antibacterial activities as well as antifungal activities against C. albicans MTCC 227, A. niger MTCC 282 and A. clavatus MTCC 1323 were used. The minimum inhibitory concentration (MIC) was determined by broth dilution method and recorded at the lowest concentration inhibiting growth of the organism. Among the synthesised compounds 6b, 6c, 7b, 8b, 8d and 8e exhibited excellent antimicrobial activity and said to be the most proficient members of the series.


INTRODUCTION
Varieties of infectious diseases caused by microbes are increasing rapid in recent year and still a major threat to public health in the world [1], even though incredible development in medicinal chemistry. Owing to non-availability of requisite medicines and emergence of prevalent drug resistance, the impact is more delicate in developing countries. Scientists and doctors are still struggling to find solutions with various forms of medications. Thus the demand for designing of new antimicrobial agents having excellent potential with high beneficial index with novel structural classes and mechanisms is necessary. 1,3,5-Triazine has received an enormous attention among the researchers and chemists as a consequence of essential role in novel drug discovery owing to threefold symmetry, allows for versatile modifications, uncomplicated by region chemical concerns and has proven a useful biological target with diverse pharmacological properties of the molecules [2][3]. And therefore, it is an ongoing effort to synthesise novel antimicrobial agents bearing 1,3,5triazine core. Chalcones are compounds of chalconoid group chemically known as 1,3-diarylprop-2en -1-one and key precursors in the synthesis of a large array of biologically important heterocycles [4][5][6][7][8]. It is therefore, not surprising that many synthetic methods have been developed for the preparation of heterocycles starting from chalcones precursors. Pyrazoline is an important five member heterocyclic compound containing nitrogen as a hetero atom and various methods have been worked out for their synthesis. Due to the interesting activity of variously substituted pyrazolines as biological agents considerable attention has been focused on this class of compounds. They are used as anti-inflammatory [9], antitumor [10], anticancer [11], antimicrobial [12] etc… . Among the existing various pyrazoline type derivatives, 1-acetyl pyrazolines have been identified as one of the most promising derivatives. In view of the importance of these derivatives, the title compound (6a-e) is achieved. Pyrimidine is a six member heterocyclic compound

1. Material
All the chemicals and solvents used for reaction were of analytical reagent (AR) grade. All the melting points were resolute in open capillary method and are uncorrected. IR spectra were recorded on Shimadzu FTIR 8401 spectrophotometer using potassium bromide pellets. 1 H NMR and 13 C NMR spectra were recorded on a Bruker Avance 400 F (MHz) spectrometer (Bruker Scientific Corporation Ltd., Switzerland) using CDCl 3 , DMSO as a solvent and TMS as an internal standard at 400 MHz. Chemical shifts are reported in parts per million (ppm) and coupling constant (J) are reported in Hertz. Elemental analysis was carried out by Perkin-Elmer 2400 series-II elemental analyser (Perkin-Elmer, USA). Mass spectra were scanned on a Shimadzu LCMS 2010 spectrometer (Shimadzu, Tokyo, Japan). Purity of the compounds were checked by thin layer chromatography using TLC aluminum sheets Silica Gel 60 F-254 (Merck) plates of 0.25 mm thickness and detection of the components were made by exposure to UV light or keeping the plates in iodine chamber.

2 A. General procedure for the compounds (A), (B), (C) and chalcones (5a-e)
The starting precursors A, B and C were prepared by the reported procedure [20]. The chalcones (5a-e) were prepared by the reported method [12] in good yields by a base catalysed Claisen-Schmidt condensation of appropriately substituted benzaldehydes and substituted ketone (C).

1. Chemistry
The aim of the present study was to develop an efficient protocol with good to excellent yields in a short span of time without formation of any side product. The formation of designed compounds was confirmed by their IR, 1 H NMR and 13 C NMR spectral and elemental analysis. The IR spectrum of compound 6a exhibited a strong band at 1610 cm -1 for C=O stretching of acetyl group attached at N 1 position in pyrazoline ring. The C-H stretching was observed at 2900 cm -1 . Characteristic band of C=N stretching of pyrazoline moiety appeared at 1583 cm -1 . A strong absorption band was observed at 1365 cm -1 due to the presence of the CH 3 group. The aromatic C-H bending vibrations for 1,4 disubstituted benzene ring and C=N stretching of 1,3,5-triazine core were observed at 833 and 800 cm -1 respectively. The 1 H NMR spectrum of compound 6a showed a singlet at δ 2.4 ppm for the COCH 3 protons. The pro-chiral methylene protons C 4 ''-H of pyrazoline appeared as two distinct doublets of a doublet at δ 3.0 ppm (J = 9.7 & 15.4 Hz) and at δ 3.6 ppm (J = 9.7 & 12.4 Hz) for the CH x -CH and CH y -CH protons, thereby indicating that both the protons are magnetically non-equivalent and diastereotopic. The chiral C 5 ''-H proton of pyrazoline appeared as a doublets of a doublet at δ 5.6 ppm (J = 4.0 & 11.5 Hz) due to CH-CH 2 -Ar proton. The other remaining twelve aromatic protons appeared as a multiplet signal at δ 6.7-7.0 ppm. In the 13 C NMR spectrum of compound 6a, the signal appeared at δ 23.4 ppm was assigned to the methyl carbon and the most deshielded signal that appeared at δ 168.5 ppm was assigned to the carbonyl carbon of the acetyl group attached with the pyrazoline unit. The signals for aromatic carbons appeared between δ 20 ILCPA Volume 57 111.4-146.2 ppm in the 13 C spectrum. The IR spectrum of compound 7a showed a strong characteristic band at 1672 cm -1 and 3307 cm -1 due to the C=N and NH 2 group of pyrimidine ring. The aromatic C=C stretching and C-H bending vibrations for 1,4 disubstituted benzene ring were appeared at 1510 and 831 cm -1 respectively. The 1 H NMR spectrum of compound 7a showed a singlet at δ 5.18 ppm due to NH 2 proton on pyrimidine nucleus which confirmed the cyclisation of pyrimidine moiety. The other remaining thirteen aromatic protons resonate as a multiplet signal at δ 6.9-8.0 ppm. 13 C NMR spectrum of compound 7a showed a signal at δ 163.8 ppm assigned to the C=N carbon of pyrimidine ring which assigned the pyrimidine unit. The signals for aromatic carbons appeared between δ 114.8-142.6 ppm in the 13

2. Antimicrobial evaluation
The antibacterial and antifungal activity of newly synthesised compounds (6a-e), (7a-e) and (8a-e) was carried out by broth dilution method [21] according to National Committee for Clinical Laboratory Standards (NCCLS, 2002). It is one of the non automated in vitro bacterial / fungal susceptibility tests. This classic method yields a quantitative result for the amount of antimicrobial agents that is needed to inhibit growth of specific microorganisms which is carried out in tubes. Upon reviewing antimicrobial data (  6c, 6d, 7a, 7b, 7d, 8a, 8c and 8d (MIC = 500 µg/mL) were found to have equipotent activity to Greseofulvin (MIC = 500 µg/mL) against Candida albicans (MTCC 227). None of the compound showed promising antifungal activity against Aspergillus niger (MTCC 282) and Aspergillus clavatus (MTCC 1323).

CONCLUSION
In summary, the synthesis of biologically important three elegent protocols (acetyl pyrazolines, amino pyrimidines and 1,5-benzodiazepines) has been developed using the potential of chalcones with the hope of generating new pharmacologically important molecules that could be useful as potent antimicrobial agents. The method reported in this investigation is effective in giving excellent conversion to the product, less -energy consuming and apparently substituent insensitive. There is no necessary to do any extra or special work during isolation of product. The results indicated that all the derivatives of new chemical entities exhibited appreciable activities against multidrug-resistant bacteria. Among the fifteen newly synthesised compounds, analogs 6b, 6c, 7b, 8b, 8d and 8e were found to be the most proficient members of the series. It is interesting to note that compounds bearing the electron withdrawing chloro group in the aryl moiety 6b, 7b and 8b showed the highest antibacterial and antifungal activities. Further in depth SAR study of the active scaffolds and the mechanism research are also required. These result suggest that chalcones and their derivatives have an opportunity to behave as generation of newer antimicrobial agents and have exellent scope for further development as commercial antimicrobial agents.