The alarming rate of emerging and reemerging microbial threat of bacterial resistance has heightened the urgency to discover and develop effective agents with novel mechanisms of action and enhanced activity. Despite the development of several new antibacterial agents,their clinical value is limited in treating an increasing array of life threatening systemic infections. Thus,the development of potent and effective antimicrobial agents is most important to overcome the emerging multi-drug resistance strains of bacteria and fungi.

2-Pyridones represent a unique class of pharmacophores,which are observed in various antibiotics and therapeutic agents ^[1]. In recent years,2-pyridones have been found to exhibit several biological activities,such as analgesic ^[2],anti-HIV ^[3] and antitumoral ^[4] properties. Moreover,2-pyridones are a class of recently discovered potent antibacterial agents that are of particular interest due to their in vitro and in vivo antibacterial potency against the bacterial type II DNA topoisomerases,which include two highly homologous enzymes-DNA gyrase and topoisomerase IV ^[5]. In addition,benzimidazoles are considered as a promising class of bioactive heterocyclic compounds surrounding a diverse range of biological activities such as antihypertensive ^[6],anticoagulant ^[7],anti-inflammatory ^[8] and antimicrobial ^[9]. The azole group of heterocyclic compounds possessed favorable pharmacokinetic properties and lipophilicity that influence the ability of drug to reach the target by transmembrane diffusion and showed promising activity against resistant TB by inhibiting the biosynthesis of lipids ^[10].

Molecular hybridization is an important tool for discovery of new chemical entities. In the past several decades much attention has been given to the design and synthesis of new types of pharmacologically diverse structural hybrid molecules ^[11]. One shining example is vilazodone,which combined serotonin reuptake inhibitor (SSRI) and 5-HT1A receptor partial agonist and was marketed under the tread name Viibryd ^[12]. Motivated by the above findings and our previous work ^{[13, 14]},it was thought worthwhile to synthesize new benzimidazole bearing 2-pyridone derivatives 5a-k that contain the aforementioned moieties in a single molecular framework in order to investigate their in vitro antibacterial and antifungal activity. 2. Experimental

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 spectrophotometer in KBr. 1H NMR spectra were recorded on a Varian Gemini 300 MHz and 13C NMR spectra on a Varian Mercury-400,100 MHz in DMSO-d6 as a solvent and tetramethylsilane (TMS) as an internal standard. Mass spectra were scanned on a Shimadzu LC-MS 2010 spectrometer.

General procedure for the synthesis of title compounds 5a-k: Compound 4 (0.01 mol),different substituted aromatic aldehydes (0.01 mol) and ethanol (50 mL) were taken in a round bottom flask and refluxed for 5 h. After 5 h,the reaction mass was poured onto crushed ice and separated solid was filtered,dried and recrystallized from DMSO. The detail characterization data of intermediate compounds 2,4 and 5a-k were given in Supporting information. 3. Results and discussion

The synthetic strategies adopted for the synthesis of target benzimidazole bearing 2-oxopyridine derivatives 5a-k are depicted in Scheme 1. In Scheme 1,the synthesis of 1-(1Hbenzo^{[ d]}imidazol-2-yl)ethanone 1 with equimolar quantity of cyanoacetic acid hydrazide in refluxing 1,4-dioxane afforded a intermediate 2. The presence of the reactive methylene group in the hydrazide 2 makes it a versatile precursor for the Michael type condensation with Knoevenagel product 3 in the presence of a catalytic amount of piperidine in ethanol (95%) as a solvent giving 2-oxopyridine compound 4. Condensation of 2-oxopyridine derivative 4 with appropriate aromatic aldehydes in boiling ethanol produced the targeted benzimidazole bearing 2-pyridones 5a-k. The structures of the final compounds 5a-k were established by their spectral analysis. Using compound 5i as a representative example,its IR spectrum showed the disappearance of -NH₂ band of derivative 4. A strong absorption band appeared at 1682 cm^-1 and was assigned to >C=O group. Its 1H NMR spectrum revealed a singlet of emine proton at 9.48, alongside the vanishing of the primary amine singlet. The 13CNMR spectrumof compound 5e displayed,besides the expected methyl and aromatic signals,three characteristic signals at 115.9,160.1 and 163.8 due to the carbons of CN,C=O and CH=N,respectively. The mass spectrum of 5e showed a molecular ion peak at m/z 560.12 (M + 1),which is in agreementwith its proposed structure ^[15].

Scheme 1

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Scheme 1.Synthetic route for the preparation of title compounds 5a-k.

All the newly synthesized compoundswere evaluated for their in vitro antimicrobial activity against different bacterial and fungal strains by the conventional broth-dilution method ^[16] using standard drugs. The results of antimicrobial studies are presented in Table 1. Intermediates 2 and 4 showed poor antimicrobial activity against all tested bacterial and fungal strains as compared to final derivatives 5a-k. In general, compounds 5a-k showed improved antibacterial activity compared to antifungal activity. Compounds 5b,5c,5j and 5k were found to be highly active against all the bacterial strains,showing inhibition in the range of 12.5-100 μg/mL. Among them, compounds 5b and 5j emerged as the most effective antibacterial agents with a 2 to 4-fold higher MIC (12.5-25 μg/mL) than the reference drug chloramphenicol. Compounds 5c and 5k exhibited comparable antibacterial activity with MIC values of 25-100 μg/mL. From these results,it can be observed that the antibacterial activity was considerably affected by the substitution pattern on the phenyl ring and the most active compounds contain an inductively electron withdrawing substituent at the meta or para position of the phenyl ring (m > p). In contrast, the presence of electron donating groups on the phenyl ring resulted in a substantial decrease in antimicrobial activity for compounds 5f-5i. Compounds 5b and 5j,substituted with inductively electron withdrawing fluoro and nitro groups, respectively,at the meta position showed the highest antibacterial activity (F > NO₂). The presence of lipophilic substituent at the meta position of phenyl ring provided a positive influence on the antibacterial activity. Further,the results of the antifungal activity indicated that compound 5d endowed with chlorine emerged as the most effective antifungal agent and showed an MIC in range of 25-62.5 μg/mL against three fungal strains using ketoconazole as a positive control.

Table 1

Table 1 Antimicrobial screening results of compounds 2,4 and 5a-k.

Table 1 Antimicrobial screening results of compounds 2,4 and 5a-k.

The cytotoxic potential of compounds 5b,5c,5j,5k and 5d was also determined in human cancer cell lines such as A549,HL-60, and HepG2 according protocols ^[17]. None of the tested compounds exhibited significant cytotoxic activity (IC₅₀ > 100 μg/mL) at the highest does used,indicating good selectivity. They possessed potent antibacterial and antifungal activity without the cytotoxicity in mammalian cells ^[18]. 4. Conclusion

In summary,we have accomplished the synthesis of new derivatives of benzimidazole bearing 2-pyridones 5a-k having two cyano groups with the hope of generating new bioactive molecules that could be useful as potent antimicrobial agents. Among the eleven newly synthesized compounds,analogs 5b,5c,5j and 5k possessing electron withdrawing atom/group such as fluoro and nitro at the meta or para position were identified as the most potent antibacterial agents and compound 5d was found to be the most effective antifungal agent with relatively low cytotoxicity. The results described here merit further investigations in our laboratory using a forward chemical genetic approach in finding lead molecules as antimicrobial agents. Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2013.11.026.