Synthesis and Antibacterial Evaluation of Novel 3,6-Disubstituted Coumarin Derivatives

Abstract A novel series of 3,6-disubstituted coumarin derivatives were synthesized by the reaction of ethyl-2-(3-acetyl-2-oxo-2H-chromen-6-yl)-4-methylthiazole-5-carboxylate with thiosemicarbazide and various phenacyl bromides / 3-(2-bromoacetyl)-2H-chromen-2-ones / 2-(2-bromoacetyl)-3H-benzo[f]chromen-3-one in ethanol having catalytic amount of acetic acid under reflux conditions with good yields. All the synthesized compounds were fully characterized by spectral studies and evaluated for their in vitro antibacterial activity against Pseudomonas aeruginosa, Bacillus subtilis (Gram positive), Escherichia coli, and Azatobacter (Gram negative) bacterial strains. Activity results revealed that the compound 6h against Escherichia coli and compound 6i against Pseudomonas aeruginosa and Escherichia coli have shown maximum zones of inhibition. Remaining compounds showed moderate to good activity against all the tested bacterial strains compared with the standard drug cefotaxime. GRAPHICAL ABSTRACT


INTRODUCTION
The natural and synthetic coumarin derivatives played an important role in medicinal chemistry because of their biological and pharmacological properties. Hence the synthesis of various coumarin derivatives occupied an important place in organic synthesis. Coumarins and their derivatives displayed antimicrobial, [1,2] antiviral, [3] anticancer, [4] anti-inflammatory, [5] antioxidant, [6] anti-HIV, [7] and antiasthmatic [8] activities. These also act as acetylcholinesterase [9] and monoamine oxidase [10] inhibitors for depression and Alzheimer's disease. In particular, 3,6-disubstituted comarins act as mechanism-based inhibitors of thrombin and factor Xa. [11] Similarly, numerous biologically active molecules have thiazole rings as an integral part of their structure. This nucleus constitutes an integral part of all the available penicillins [12] for controlling bacterial diseases. The literature survey revealed that thiazoles act as antimicrobial, [13,14] antitumor, [15] anti-inflammatory, [16] anticonvulsant, [17] and anticoagulant [18] agents. In view of the therapeutic properties of these moieties (coumarins and thiazoles), as well as in continuation of our earlier studies on coumarin derivatives, [19][20][21] we report the design and synthesis of a novel series of 3,6-disubstituted coumarin derivatives under conventional method. [22,23]

Antibacterial Activity
All the newly synthesized compounds (6a-l) were assessed for their in vitro antibacterial activity against Pseudomonas aeruginosa, Bacillus subtilis (Gram positive), Escherichia coli, and Azatobacter (Gram negative) bacterial strains with compared to standard antibiotic drug cefotaxime. Zone of inhibition (mm) values for analogues and positive control drug cefotaxime at were determined by agar disc diffusion method. [24] The bacterial strains were grown and maintained on nutrient agar plates. All the compounds and standard were dissolved in dimethylsulfoxide (DMSO; 100 µg/ mL and 30 µg/mL respectively) and transferred to each disc with the help of micropipette. After 24 h incubation at 37°C, the resulting zone of inhibition was measured.
The antibacterial data revealed that almost all the compounds 6a-l have shown moderate to good antibacterial activity ( Table 2). Compounds 6f, 6h-k, and 6l have shown good activity whereas compounds 6a, 6b, 6d, 6g have shown moderate activity against both Gram-positive and Gram-negative strains. Compond 6c is inactive towards Bacillus subtilis and Escherichia coli, whereas compound 6e is inactive only on Bacillus subtilis. In the overview of antibacterial data, compound 6h against Escherichia coli and compound 6i against Pseudomonas aeruginosa and Escherichia coli have shown maximum zones of inhibition (11 mm) when compared with the standard drug cefotaxime. The graphical representation of antibacterial activity of the compounds as well as standard drug cefotaxime are shown in Fig. 1.

EXPERIMENTAL
All the reagents and solvents were purchased from Aldrich/Merck and used without further purifications. Melting points were determined in open capillaries using Stuart SMP30 apparatus and are uncorrected. The progress of the reactions as well as purity of compounds were monitored by thin-layer chromatography (TLC) with F 254 silica-gel precoated sheets using hexane / ethyl acetate 8/2 as eluent; ultraviolet light and iodine vapors were used for detection. IR spectra were recorded on a Perkin-Elmer 100S spectrometer utilizing KBr pellets. 1 H NMR and 13 C NMR spectra were obtained at 400 MHz and 100 MHz respectively on Bruker using dimethylsulfoxide (DMSO-d 6 ) as solvent and tetramethylsilane (TMS) as internal standard. Elemental analyses were performed on a Carlo-Erba model EA1108 analytical unit and the values are AE0.4% of theoretical values. Mass spectra were recorded on a Jeol JMSD-300 spectrometer.

CONCLUSION
In conclusion, we have synthesized a novel series of 3,6-disubstituted coumarin derivatives by using conventional methods. All synthesized compounds were determined from their spectral (IR, 1 H NMR, 13 C NMR) and mass data and assessed for their in vitro antibacterial activity.

ACKNOWLEDGMENT
We thank the director of the National Institute of Technology, Warangal, for providing facilities.

SUPPORTING INFORMATION
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