Studies on Antimicrobial Evaluation of Some 1-((1-(1H-Benzo[d]imidazol-2-yl)ethylidene)amino)-6-((arylidene)amino)-2-oxo-4-phenyl-1, 2-dihydropyridine-3,5-dicarbonitriles

Abstract A new series of 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-((arylidene)amino)-2-oxo-4-phenyl-1,2-dihydropyridine-3,5-dicarbonitriles (4a–o) have been synthesized for the development of antimicrobial agents. Newly synthesized compounds were evaluated for their in vitro antibacterial activity against Gram-positive bacteria (Pseudomonas aeruginosa, Streptococcus pyogenes), Gram-negative bacteria (Escherichia coli, Staphylococcus aureus), and antifungal activity (Candida albicans, Aspergillus niger, Aspergillus clavatus). These compounds were characterized by infrared, 1H NMR, 13C NMR, and mass spectra. The synthesized compounds 4b, 4e, 4 h, and 4k showed potent antimicrobial activity against tested microorganisms. GRAPHICAL ABSTRACT

2-Pyridone derivatives have also attracted considerable attention because this skeleton is present in many compounds that have been isolated from natural substances, [22,23] having various biological activities. [24] The development of efficient synthesis of 2-pyridone is an important target in current organic synthesis. [25] Some derivatives containing a 2-oxopyridine ring system have been shown to possess useful pharmacological activities, such as milrinone (Primacor), a phosphodiesterase III inhibitor; olprinone, a cardiotonic agent; [26] and camptothecin (CPT), the DNA enzyme topoisomerase I (topo I) inhibitor. Two CPT analogs, topotecan and irinotecan, [27] have been approved and are used in cancer chemotherapy today. Pyridonebased drugs are well known as chemotherapy agents. Nothapodytine B (Fig. 1, Formula 2) is an excellent antiviral drug having 2-pyridones as core moiety. In addition, the predominance of nitrile-containing drugs has changed the treatment of several diseases because of the biocompatibility of the nitrile functionality. At present, more than 30 nitrile-containing drugs are prescribed for a diverse range of medicinal conditions and more than 20 additional nitrile-containing molecules are in clinical trials. Some shining examples of nitrile-based drugs are letrozole, etravirine, riplivirine, and lersivirine. [28] In the present program, our aim is to synthesize new molecules containing multiple heterocyclic systems and to study their characterization as well as their activities. In continuation to our previous work [29][30][31] and because of the medicinal importance of benzimidazole and 2-pyridone as a core moiety, we report herein the synthesis of a new class of 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-((arylidene)amino)-2-oxo-4-phenyl-1,2-dihydropyridine-3,5-dicarbonitriles (4a-o) and try to develop potential antimicrobials. The structures of newly synthesized compounds were elucidated on the basis of infrared (IR), 1 H NMR, 13 C NMR, and mass spectral analysis. These compounds were

Spectral Characterization
Characterization of newly synthesized compounds of the series is carried out by IR, 1 H NMR, 13 C NMR, and mass spectra and detailed discussions are given in the Experimental section. IR spectrum of compound 4a ( Fig. 2; molecular formula C 29 H 19 N 7 O, molecular weight 481.17 g=mol) has given stretching vibration at 3333 cm À1 , indicating the presence of N-H stretching of secondary amine. Absorption band at 3053 cm À1 indicates the presence of C-H stretching of aromatic hydrogen.

Biological Evaluation
Antibacterial screening. All the newly synthesized compounds (4a-o) were evaluated for their in vitro antibacterial activity against Gram-positive bacteria Staphylococcus aureus and Streptococcus pyogenes and Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa by conventional broth microdilution method using Ampicillin as a standard drug for antibacterial activity at different concentrations of 1000, 500, 200, 100, 50, 25, and 12.5 µg ml À1 as shown in Table 1. Among the synthesized compounds (4a-o), many of them had proven their antimicrobial potency varies from moderate to excellent. Compound 4e (-2-NO 2 -C 6 H 4 ) had excellent activity against E.coli and S. aureus. It is noteworthy that compound 4e (-2-NO 2 -C 6 H 4 ) showed the greatest inhibition at MIC ¼ 12.5 µg ml À1 , while compound 4 h (-2-Cl-C 6 H 4 ) showed inhibition at MIC ¼ 12.5 µg ml À1 against P. aeruginosa and S. pyogenes. These data revealed that compound 4 h (-2-Cl-C 6 H 4 ) was highly active against both organisms. Compounds 4e (-2-NO 2 -C 6 H 4 ) and 4 h (-2-Cl-C 6 H 4 ) showed very good activity at MIC ¼ 50 µg ml À1 . Compound Antifungal screening. Minimum inhibitory concentration (MIC) values of antifungal activity were observed against Candida albicans, Aspergillus niger, and Aspergillus clavatus by conventional broth microdilution method. Antifungal activity showed that compound 4k (-2-CH 3 -C 6 H 4 ) exhibited very good activity against A. clavatus at 50 µg ml À1 MIC. When we replaced hydrogen by group like (-4-OCH 3 -C 6 H 4 ) in compound 4n, the activity was slightly decreased against A. niger. The same results exhibited in compound 4 l (-3-CH 3 -C 6 H 4 ) possessed good activity against C. albicans and A. clavatus respectively. When hydrogen was replaced by (-2-OH-C 6 H 4 ) in compounds 4b (-2-CH 3 -C 6 H 4 ) and 4k (-2-CH 3 -C 6 H 4 ), both displayed excellent activity against C. albicans and A. niger with twofold greater MIC (12.5-25 µg ml À1 ) than the reference drug. The remaining compounds of the series showed feeble antifungal activity. Thus we have discussed and compared antifungal activity based on the standard drug griseofulvin shown in Table 1.

SAR Studies
The substitution patterns of the derivatives are carefully selected to confer different electronic environments of the molecules. The electronic nature of the substituent groups leads to significant variation in antimicrobial activity. Furthermore, considering the relationship between the structure of final compounds (4a-o) and antimicrobial property, the identity of different substituents proved to be a significant parameter for influencing the activity of reported compounds. The presence of chloro and nitro substituents present at ortho position on the aromatic ring has increased the antibacterial activity of compounds compared to those of electron-donating substituents. When we have changed the substitution position to para, the compound tends to lose its potency. Incorporation of electron-donating groups such as methyl, methoxy, and hydroxy diminished the antibacterial property. The presence of hydrophilic substituents on the phenyl ring provides a positive influence on antifungal activity. In agreement with these results, electron-donating groups on the orthosubstituted position showed optimum activity. It may be observed that position of substituent on the phenyl ring clearly affected the activity (e.g., same functional groups on meta and para positions are not found to be active as the standard drug). Eventually, it can be inferred from Table 1 that a compound without any substitution does not exhibit antimicrobial activity against a panel of microorganisms.

Materials and Methods
The required chemicals were purchased from E. Merck. Melting points were recorded on Gallenkamp apparatus and were left uncorrected. The completion of reaction and the purity of all compounds was checked on aluminum-coated thin-layer chromatography (TLC) plates 60 F 245 (E. Merck) using various solvent systems as mobile phase and visualized under ultraviolet (UV) light or iodine vapor. Elemental analysis (% C, H, N) was carried out by a Perkin-Elmer 2400 CHN analyzer. Infrared (IR) spectra were also recorded on a Perkin-Elmer FT-IR spectrophotometer. 1 H NMR and spectra were recorded on a Varian Gemini 400-MHz instrument and 13 C NMR spectra on a Varian Mercury 400 100-MHz instrument in dimethylsulfoxide (DMSO-d 6 ) as a solvent and tetramethylsilane (TMS) as an internal standard. Chemical shifts are reported in parts per million (d ppm). Mass spectra were scanned on a Shimadzu LCMS 2010 spectrometer. In the conventional method, compounds were synthesized by using a random synthesizer. Bookie Rota vapor was used for distillation.

CONCLUSION
The synthesized compounds were screened for their in vitro antibacterial and antifungal activity. It may be concluded from Table 1 that structural and electronic diversity of these products affected their biological activities. We have found compounds 4b, 4e, 4 h, and 4k to be the most distinctive derivatives identified in the present study because of their remarkable in vitro antimicrobial potency. SAR studies revealed that when the ortho position is substituted by electron-withdrawing groups such as nitro and chloro, compounds exhibited antibacterial activity. Likewise when the ortho position is substituted by electron-donating groups such as hydroxy and methyl, compounds displayed antifungal activity. It may be considered a promising lead for further design and development of new lead molecules. highly thankful to the UGC, New Delhi for providing a UGC-BSR one-time grant (2012).

SUPPLEMENTAL MATERIAL
Full experimental, antibacterial and antifungal assay, IR, 1 H NMR, and mass spectral details for this article can be accessed on the publisher's website.