Synthesis and characterization of some novel coumarin based 2-pyridone heterocycles with their broad spectrum antimicrobial potency

: The synthesis of a novel series of 6-((arylidene)amino)-2-oxo-1-((1-(2-oxo-2 H -chromen-3-yl)ethylidene)amino)-4-phenyl-1,2-dihydropyridine-3,5-dicarbonitriles 4a-o were synthesized and structures of compounds have been elucidated by IR, 1 H NMR, 13 C NMR and mass spectral data. Antimicrobial activity was measured against certain strains of bacteria and fungi by serial broth dilution. Evaluation of antimicrobial activity shown that the compounds ( 4g , 4j , 4k and 4o) were found to be most active against selected bacterial strains and compounds ( 4d , 4m and 4n) were found to be most active against selected fungal strains.


Introduction
Coumarin and 2-pyridone derivatives play a vital role in the theoretical development of heterocyclic chemistry along with being used extensively in organic synthesis. The action of bacterial infections still remains a significant therapeutic problem. It led to develop infectious diseases and the increasing number of multidrug-resistant microbial pathogens. Therefor since the past few decades much attention has been given to the design and synthesis of new types of pharmacologically diverse structural hybrid molecules [1]. The present work is dealing with the synthesis of novel series of 6-((arylidene)amino)-2-oxo-1-((1-(2-oxo-2H-chromen-3-yl)ethylidene)amino)-4-phenyl-1,2 dihydropyridine-3,5-dicarbonitriles 4a-o. It is well known that coumarin derivatives possess a wide range of medicinal indications, such as anthelmintic, anticoagulant, hypnotic and insecticidal properties [2]. The antimicrobial activity of coumarin derivatives is reported in the literature [3,4,5,6,7]. Potential of various natural and synthetic coumarin derivatives proved its importance as anti-inflammatory [8] antioxidant properties [9] anticancer [10,11] and monoamine oxidase inhibitors [12]. Novobiocin and chlorobiocin have reported as antimicrobials containing a coumarin skeleton [13]. Pyridone and their derivatives play a vital role in some biological processes and have significant chemical and pharmacological importance [14,15,16]. 2-Pyridones motifs found to possess useful pharmacological activities, such as analgesic [17], antimalarial [18], anti-HIV [19], phytotoxic [20], and antitumoral [21] properties. The versatility of 2-pyridone and its potential to yield derivatives with a wide range of biological activities has made it a useful structure for further molecular investigation. Moreover, 2-pyridones are a class of newly exposed potent antibacterial agents that are of specific concern due to their in vitro and in vivo antibacterial potencies against the bacterial type II DNA topoisomerases, which consist of two highly homologous enzymes-DNA gyrase and topoisomerase IV [22,23]. In accordance with our previous work [24,25,26] and medicinal importance of coumarin and 2-pyridone motifs we report herein the synthesis of a new class of 6-((arylidene)amino)-2-oxo-1-((1-(2-oxo-2H-chromen-3-yl)ethylidene)amino)-4-phenyl-1,2dihydropyridine-3,5-dicarbonitriles 4a-o as potential antimicrobial agents.

Characterization of compounds 4a-o
The structure of compound 4a-o was confirmed on the basis of spectral data. The IR spectrum of compound 4a-o showed strong absorption band at 1700-1730 cm -1 due to >C=O stretching, coumarin ring. Absorption band appeared at 2203-2221 cm -1 due to stretching vibrations corresponding to cyano group and absorption band at 1591-1599, 1551-1562 cm -1 corresponding to C=C, C=N stretching, aromatic ring. Moreover absorption bands appeared in compounds 4a-o at 1251-1268 cm -1 indicated the C-O-C linkage present in coumarin ring. In 1 H NMR spectra, the appearance of singlet peaks in compounds 4a-o at δ = 8.65-9.30 and 9.05-9.25 ppm was due to one proton of coumarin ring (C4-H) and Ar-CH=N-linkage. Three proton of Ar-C(CH 3 )=N-displayed singlet at δ = 2.03-2.10 ppm. Remaining all aromatic protons appeared multiplet in the region δ= 6.90-8.53 ppm. The 13 C NMR spectrum of compound 4a-o showed characteristic signal at δ = 159.2-159.9 ppm due to carbonyl carbon at coumarine motif as well as the appearance of signal around δ = 115.5 (2) ppm was assignable to cyano group of the 2-pyridone ring. Carbon of -CH=Nlinkage showed signal around δ= 163.2 ppm. The mass spectrum revealed a molecular ion peak in compound 4a-o at m/z = 509. 15-599.18 in mass spectra, molecular ion peak was in agreement with proposed molecular weight and elemental analysis.

Antibacterial activity
In preliminary screening, compounds 4a-o against the test microbes are listed in Table 1 along with MIC values of reference compounds ampicillin (for bacteria) and griseofulvin (for fungi). Although all coumarine base 2-pyridone derivatives 4a-o were found to show antimicrobial activity against different strains in these two assays, compounds 4g, 4j, 4k and 4o are clearly outstanding in their antibacterial properties. For several pathogens, these compounds were more active than the reference drugs.

Antifungal activity
Compounds 4a-o were tested for antifungal activity in six sets against C. albicans, A. niger and A. clavatus at various concentrations of 1000, 500, 200 and 100 μg/ml as shown in Table 1. Synthesized compounds are diluted at 1000 μg ml -1 concentration, as a stock solution. The data of antimicrobial evaluation of compounds 4a-o were collected in Table 1. Compound 4d selectively inhibits the growth of C. albicans and A. niger. Compound 4m, expressed excellent activity against A. niger and A. clavatus while compound 4n exhibited excellent activity against A.niger with a two to four fold higher (12.5-25 µg/ml) MIC value than the reference drug griseofulvin. Detailed activity results are summarized in Table 1.

Materials and methods
All reactions except those in aqueous media were carried out by standard techniques for the exclusion of moisture. 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. 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. 1 H NMR spectra were recorded on Varian Gemini 300 MHz and 13 C NMR spectra are recorded with a Varian Mercury-400 (100 MHz) NMR spectrometer, using tetramethylsilane as the internal reference, with dimethyl sulfoxide (DMSO-d 6 ) as solvent. Mass spectra were scanned on a Shimadzu LCMS 2010 spectrometer.

SAR studies
The structure-activity relationships (SAR) of compounds 4a-o were determined on the basis of results presented in Table 1. The substitution pattern on the coumarin based 2-pyridone ring system derivatives was carefully selected to confer different electronic environment of the molecules. From the activity data, compounds containing electron withdrawing group at para position lead to enhance bacterial activity. Compounds 4g (-4-NO 2 -C 6 H 4 ), 4j (-4-Cl-C 6 H 4 ) and 4k (-4-F-C 6 H 4 ) containing 4-NO 2, 4-Cl and 4-F group showed highest inhibition at MIC = 12.5-25 μg ml -1 against bacterial strain E. coli and P. aeruginosa. Same results observed in compounds 4j (-4-Cl-C 6 H 4 ) and 4k (-4-F-C 6 H 4 ) showed highest inhibition at MIC = 12.5-25 μg ml -1 against S. pyogenes and compound 4o -4-Br group showed highest inhibition at MIC =12.5 μg ml -1 against P. aeruginosa and S. aureus. On the basis of screening results, it has been observed that the compound containing electron releasing groups such as -OH, -OCH 3 lead to enhance fungal activity. Compounds 4d (-4-OH-C 6 H 4 ), 4m (-4-OCH 3 -C 6 H 4 ) and 4n (-3,4,5-(OCH 3 ) 3 -C 6 H 2 ) exhibited excellent inhibitory action against A.niger. While compounds 4d (-4-OH-C 6 H 4 ) and 4m (-4-OCH 3 -C 6 H 4 ) also showed reasonably excellent inhibition against C. albicans and A. clavatus respectively. SAR studies revealed that the presence of electron withdrawing and electron releasing group in title compound increase the antibacterial and antifungal activity respectively.