One-pot synthesis of some novel N-aryl-1,4-dihydropyridines derivatives bearing nitrogen mustard

The synthesis of a novel dihydropyridine, bearing carbethoxy groups at C(3) and C(5), respectively, has been achieved by applying three component Hantzsch-type condensation. The products were assayed for their in vitro biological assay antibacterial activity against with two Gram-positive bacteria Staphylococcus aureus MTCC-96, Streptococcus pyogenes MTCC 443, two Gram-negative bacteria Escherichia coli MTCC 442, Pseudomonas aeruginosa MTCC 441 and three fungal strains Candida albicans MTCC 227, Aspergillus Niger MTCC 282, Aspergillus clavatus MTCC 1323 taking ampicillin, chloramphenicol, ciprofloxacin, norfloxacin, nystatin, and griseofulvin as standard drugs.


INTRODUCTION
1,4-Dihydropyridine derivatives (1,4-DHPs) form a class of heterocyclic compounds with interesting pharmacological and biological properties [1]. It is well known that the 1,4-DHP nucleus serves as the scaffold of important cardiovascular drugs and it has been well established that the calcium modulator activity of this family of compounds is determined by structural requirements [2]. The systematic structural modification of the 1,4-DHP ring yields different compounds used in the treatment of TB and cancer [3]. Recently, studies showed that 3,5-dicarbamoyl derivatives of 1,4-dihydropyridine (DHP) with lipophilic groups have considerable antitubercular activity against M. tuberculosis H37Rv [4][5][6][7][8]. It was also observed that esters or substituted isosters of pyridine and pyrazine carboxylic acids (such as tetrazoles) have been more active than the parent acids especially against resistant strains. These esters are presumably activated by an esterase to parent acid [9][10][11][12]. Indeed, esters of pyrazinoic acids have been shown to possess activity against pyrazinamide-resistant isolates which has been attributed to a deficiency of nicotinamidase [9][10][11][12]. In addition, pyrazoles exhibited significant antitubercular activity [12]. Moreover, Nitrogen mustard is another important class bio-active scaffolds. In addition, nitrogen mustard and isosteric to pyridine and are associated with interesting biologic activities such as anti TB, antibacterial, anticancer, antiviral, cardiotonic etc. Over the years, molecular hybridization based drug design approach [13,14] has been exploited by many researchers in order to develop some promising new hybrid chemical entities (NHCEs), displaying significant therapeutic values. The combination of two pharmacophores into a single molecular skeleton is a well established approach for designing more potent drugs with significant increase in activity. A hybrid molecule acting on manifold targets is considered to be a better drug candidate than drug combinations, since administration of single drug will have more predictable pharmacokinetic and pharmacodynamic properties with improved patient compliance. Owing to their well appreciated antitubercular and anticancer properties, we envisaged to design hybrid structures having substituted N-aryl-1,4-dihydropyridines and nitrogen mustard motifs connected with a linker. However, to the best of our knowledge, biological activity of 1,4-dihydropyridines bearing N-aryl-substitution or C-3 and C-5 acetyl group is not much explored.
Therefore, in view of the above facts and in continuation of our search on biologically active hybrid molecules [15][16], herein we report the synthesis of novel N-aryl-1,4dihydropyridines hybrid analogs (1a-1k).

General Procedures
Melting points of all the compounds are uncorrected and have been recorded by open capillary method. Room temperature, wherever mentioned, normally corresponds to 280 -330C. Silica gel-G was used for preparing the TLC plates using different solvent systems. Infra red spectra of all the compounds were scanned on SHIMADZU-FOURIER TRANSFORM INFRA RED (FTIR) -8400 Spectrophotometer using KBr pellet method. PMR Spectra were recorded on BRUKER Spectrophotometer (300 MHz) using TMS as a internal standard and CDCI 3 as solvent. Electron Impact (EI) mass spectra were recorded on GCMSQP2010 Mass Spectrometer.

Antimicrobial evaluation
All of the synthesized compounds (1a to 1k) were tested for their antibacterial and antifungal activity (MIC) in vitro by broth dilution method [11][12] with two Gram-positive bacteria Staphylococcus aureus MTCC-96, Streptococcus pyogenes MTCC 443, two Gramnegative bacteria Escherichia coli MTCC 442, Pseudomonas aeruginosa MTCC 441 and three fungal strains Candida albicans MTCC 227, Aspergillus Niger MTCC 282, Aspergillus clavatus MTCC 1323 taking ampicillin, chloramphenicol, ciprofloxacin, norfloxacin, nystatin, and griseofulvin as standard drugs. The standard strains were procured from the Microbial Type Culture Collection (MTCC) and Gene Bank, Institute of Microbial Technology, Chandigarh, India.
The minimal inhibitory concentration (MIC) values for all the newly synthesized compounds, defined as the lowest concentration of the compound preventing the visible growth, were determined by using microdilution broth method according to NCCLS standards [11]. Serial dilutions of the test compounds and reference drugs were prepared in Muellere-Hinton agar. Drugs (10 mg) were dissolved in dimethylsulfoxide (DMSO, 1 mL). Further progressive dilutions with melted Muellere-Hinton agar were performed to obtain the required concentrations of 1.56, 3.12, 6.25, 10, 12.5, 25, 50, 62.5, 100, 125, 250, 500 and 1000 µg mL -1 . The tubes were inoculated with 10 8 cfu mL -1 (colony forming unit/mL) and incubated at 37 ºC for 24 h. The MIC was the lowest concentration of the tested compound that yields no visible growth (turbidity) on the plate. To ensure that the solvent had no effect on the bacterial growth, a control was performed with the test medium supplemented with DMSO at the same dilutions as used in the experiments and it was observed that DMSO had no effect on the microorganisms in the concentrations studied. The results obtained from antimicrobial susceptibility testing are depicted in Table 1. Compounds 1e and 1f are display broad spectrum antibacterial activities against both gram-positive and gram-negative bacteria as compared with ciprofloxacin. As observed with high antibacterial potency of 1e and 1f a may

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ILCPA Volume 48 be attributed to the presence of electron withdrawing substituents such as chloro and fluoro at 4 th position of phenyl ring of pyridine compounds. In comparison to the standard drug griseofulvin, the in vitro antifungal activity results indicated that compound 1f substituted with fluoro group at 4 th position of phenyl ring was found to be the most potent against A. niger, A. clavatus and C. albicans respectively. Table 1. Antibacterial and antifungal activity of synthesized compounds 1a to 1k.

CONCLUSIONS
In the present paper, we report the synthesis, spectral studies and antimicrobial activity of various pyridine derivatives. The high bioactivity of these compounds makes them suitable tools for additional in vitro and in vivo evaluations, in order to develop new class of antimicrobial drugs or prodrugs with potential antimicrobial activity in the treatment of several dieses. Further studies and modification in this area are in progress in our laboratory.

Acknowledgments
We acknowledge the support of UGC through Grant no. 41-198/ 2012(SR), New Delhi, India, for financial support and National Facility for Drug Discovery (NFDD) for providing instrumental support. I also wish to acknowledge Professor and Head, Department of Chemistry, Saurashtra University Rajkot for providing research facility. P. M. Singala is thankful to RGNF for meritorious Fellowship.