A convergent approach for the synthesis of new pyrazolyl bipyridinyl substituted coumarin derivatives as antimicrobials

Some new 3-[3-(1-phenyl-3-aryl-1 H -pyrazol-4-yl)acryloyl]coumarins 3a-f were synthesized (coumarin chalcones) by the condensation of various 3-acetyl coumarins 1 and appropriate 1-phenyl-3-aryl-1 H -pyrazole-4-carbaldehyde 2 . These coumarin chalcones 3a-f were then employed for the synthesis of pyrazolyl bipyridinyl substituted coumarins 7a-f , 8a-f , and 9a-f under Krohnke’s reaction condition. The characterization of all the synthesized compounds was carried out by elemental analysis, IR, 1 H-NMR, 13 C-NMR, DEPT-135 and mass spectral analysis. In addition to that, in vitro antimicrobial competency of the title compounds was assessed against selected pathogens. Compounds 3b , 3e , 7b , 8b , 8c and 9b exhibited excellent antimicrobial activity and said to be the most proficient members of the series.


INTRODUCTION
During past few decades, there has been an alarming increase in bacterial resistance to antibiotic drugs. The evolution and spread of these multidrug resistant bacteria have become a major threat to global health care [1].
Consequently, the discovery and design of new efficient antimicrobial agents is of crucial need to counteract the resistant pathogens. Therefore numerous research groups have directed their efforts towards designing the new anti-bacterial drugs with new targets, distinctive modes of action, low toxicity and low probability of inducing resistance to multi resistant bacteria [2][3][4].
Coumarin is extensively investigated as class of naturally occurring compounds and its derivatives have tremendous contribution in therapeutic field. Many coumarin derivatives used as effective drug in marketplace such as Warfarin, Novbiocin, Dicoumarol, Imperatorin, Calophyllolide and Neo-tarnshinlactone.
Such striking information from literature survey directed us to detail study of various heterocyclic substituted coumarin derivatives.
During our literature survey we came across some bipyridines derivatives which have been reported to possess wide applications in the field of bioinorganic chemistry [12], supramolecular chemistry [13] and polymeric material [14]. Bipyridine derivatives exhibit wide range of physiological activities such as anticancer [15], cardiotonic [16], DNA binding properties [17] and antibacterial properties [18]. In addition to that, we came across some pyrazolyl substituted pyridines which have been reported to possess insecticidal [19], antiviral [20] and cardiotinic activities [21].
More efficacious antibacterial compounds can be designed by joining two or more biologically active heterocyclic systems together in a single molecular framework [22]. Such structural hybridizations show synergistic influence on the anticipated activity, hoping to discover new entities that would have astonishing antimicrobial activity.
In view of this background and medicinal significance of pyridyl substituted coumarins, bipyridines and pyrazolyl substituted pyridines encouraged us to hybrid these three bioactive moieties in single scaffold.
Hence, in continuation our efforts to synthesize such biologically active pyridyl substituted coumarin derivatives [10,11], we herein, report the synthesis and antimicrobial activity of pyrazolyl bipyridinyl coumarin derivatives7a-f, 8a-f, and 9a-f.
The plausible mechanism for the synthesis of target compounds 7a-f, 8a-f and 9a-f is demonstrated in Scheme 2.
The corresponding intermediate underwent cyclization in presence of ammonium acetate which upon subsequent loss of two water molecules afforded the target compounds in good yields.
A general observation was made from the activity data ( Table 1) that compounds 7a-c, 8a-c and 9a-c having no substitution in coumarin ring were more potent than their analogous having methoxyl substitution in most of the cases against all the bacterial strains. Compounds 9a-f bearing 4-pyridyl ring showed poor activity compared to standard drugs against gram negative bacterial strains. Compounds 7b and 8b, with R 1 = CH 3 showed better activity than their analogous against all the bacterial strains while 9b with R 1 = CH 3 showed better activity against gram positive bacterial strains only.

1. Chemistry
All reactions were performed with commercially available reagents and they were used without further purification. Organic solvents were purified by standard methods and stored over molecular sieves. All reactions were monitored by thin-layer chromatography (TLC, on aluminium plates coated with silica gel 60 F 254 , 0.25 mm thickness, Merck) and detection of the components was made by exposure to UV light. Melting points were determined in open capillaries and are uncorrected. Infrared spectra were recorded on Shimadzu FTIR 8401 spectrophotometer using potassium bromide pellets in the range 4,000-400 cm -1 and frequencies of only characteristic peaks are expressed in cm -1 . 1 H and 13 C NMR spectra were recorded in CDCl 3 on a Bruker Avance 400 (MHz) spectrometer (Bruker Scientific Corporation Ltd., Switzerland) using TMS signal as an internal standard at 400 MHz and 100 MHz respectively. Chemical shifts are reported in parts per million (ppm). The coupling constants (J) are given in Hertz (Hz). Mass spectrum of one representative compound was scanned on a Shimadzu QP 2010 spectrometer (Shimadzu, Tokyo, Japan). The compounds were purified by column chromatography using silica gel (60-120 mesh). Reference drugs ampicillin, griseofulvin, nystatin were of commercial grade.

General procedure for the synthesis of bipyridinyl substituted coumarins (7a-f, 8a-f, and 9a-f).
In a 100 mL round bottom flask equipped with a condenser, guard tube and magnetic needle, an appropriate pyridoyl methyl pyridinium iodide salt 4, 5 and 6 ,0.003 mol (4,5and 6: 1g) in glacial acetic acid (15mL) was taken. To this ammonium acetate, 0.03 mol (2.36g) was added with stirring at room temperature. Then a solution of an appropriate 3-[3-(1,3-diaryl-1H-pyrazol-4-yl)acryloyl]coumarin 3a-f, 0.003 mol (3a:1.33g, 3b:1.37g, 3c:1.42g, 3d:1.42g, 3e:1.46g, 3f:1.5g) in glacial acetic acid (15 mL) was added with stirring at room temperature and the reaction mixture was further stirred for 1 hour at room temperature and then refluxed for 8 hours at 140°C. It was then allowed to come to room temperature and was poured into ice-cold water (75 mL). A crude solid obtained was extracted with chloroform (3 х 30 mL). The organic layer was washed with 5% sodium bicarbonate solution (3 х 20 mL), water (2 х 20 mL) and dried over anhydrous sodium sulfate.        tube) were sub cultured and incubated overnight at 37°C. The amount of growth from the control tube before incubation (which represents the original inoculum) was compared. Subcultures might show (i) similar number of colonies indicating bacteriostatic (ii) a reduced number of colonies indicating a partial or slow bactericidal activity (iii) no growth if the whole inoculum has been killed. The test must include a second set of the same dilutions inoculated with an organism of known sensitivity. Each synthesized compound was diluted obtaining 2000 µg/mL concentration as a stock solution. In primary screening 500, 250 and 200 µg/mL concentrations of the synthesized compounds were taken. The compounds which found active in this primary screening were further tested in a second set of dilution using 100, 62.5, 50 and 25 µg/mL concentrations against all microorganisms. The highest dilution showing at least 99% inhibition is taken as MIC.

CONCLUSION
Present study described successful hybridization strategy of three bioactive moieties, pyridyl substituted coumarin, bipyridine and pyrazolyl substituted pyridine in single scaffold. The target compounds were synthesized in good yield by adopting Krohnke's protocol. Majority of the compounds were found to be active against Staphylococcus aureus and Bacillus subtilis. In antifungal activity, majority of the compounds showed excellent activity against Candida albican as compared to griseofulvin. Antimicrobial screening results revealed that compounds 3b, 3e, 7b, 8b, 8c and 9b were found to be the most proficient members of the series. Reviewing the antimicrobial data, it is worth mentioning here that coumarins bearing bipyridine and pyrazole entities as substitution serve as promising lead scaffolds for further generation of new antimicrobial agents.