Novel Coumarin Derivatives: Synthesis, Characterization and Antimicrobial Activity

The novel coumarin derivatives (2, 3, 4, 5, 6, 7) have been synthesized from the reaction of o-acetyloxy benzoic acid with thionyl chloride yielding 2-acetoxy benzoyl chloride, which on further treatment with ethylacetoacetate gave 4-hydroxycoumarin. Substituted pyrazolones and thiazoles reacted with 4-hydroxy coumarin to give pyrazolones and methyl thiazoles related coumarin derivatives. The newly synthesized products were characterized with IR, H and C NMR, mass spectroscopic techniques and elemental analysis. The synthesized compounds were screened for their antibacterial and antifungal activity. All the compounds were found to have significant activity against the tested microorganisms.


Introduction
In plants, coumarin derivatives are present in significant quantities and more than 1300 coumarins were identified from natural sources. 1 ThIS family of compounds serve as an important model for the advanced design and synthesis of more active analogous coumarins, since natural compounds possess potent antioxidants and radical-scavenging properties as reportedin various experimental models. 2 The synthesis of coumarin and its derivatives have attracted considerable attention from organic and medicinal chemists for many years, as large numbers of natural products contain this heterocyclic nucleus. 3 They are widely used as optical brighteners 4 and dispersed fluorescent and laser dyes, 5 additives in food, 6 perfumes, 7 cosmetics 8 and pharmaceuticals 9 so the synthesis of this heterocyclic nucleus is of much interest. The coumarin derivatives possess a broad spectrum of biological activities such as, antifungal, 10 antibacterial, 11 anti-inflammatory, 12 antiproliferative, 13 antitumor, 14 antiviral, 15 antioxidant, 16 anticoagulant, 17 anticancer 18 and anti-HIV 19 activities. The reaction of o-acetyloxy benzoic acid and thionyl chloride to give 2-acetoxy benzoyl chloride, which then reacts with ethylacetoacetate to yield hydroxycoumarin. Coumarin heterocycles have been found to be very useful compounds for different types of activities. 4-Hydroxy coumarin derivatives are useful starting materials for the synthesis of new Coumarin derivatives. [20][21][22][23] Coumarins have been synthesized by several routes including Pechmann, 24 Perkin, 25 Knoevenagel, 26 Reformatsky 27 and the Wittig reaction. 28 Bucumolol, 29 Chromonar, 30 Folescutol and 4-methyl umbelliferone 31 are coumarin derivatives and are clinically used as antiarrhythmic, 32 vasodilator, 30 capillary and antispectively agents. Warfarin 33 and acenocomarol, 34 which are coumarin derivatives, exhibit anticoagulant activity. [35][36][37] Coumarin derivatives are also used to synthesize fluorescent polymers 38 and fluorescent chemosensors for Mg +2 ion. 39 We report herein the design and synthesis of pyrazolone and methyl thiazole related coumarins derivatives with significant biological importance.

Methods and Materials
All the chemicals were procured from Sigma-Aldrich, and used without further purification. The 4-hydroxy coumarin was prepared by Knoevenagel's literature procedure. 21 Initially, the purity of synthesized compounds was confirmed using aluminium-coated TLC plates (E. Merck). Melting points were determined using a Stuart SMP10 MP apparatus and are uncorrected. The IR spectra (ν, cm -1 ) were recorded on a 8400 FT-IR-435 spectrometer using KBr pellets. Elemental analysis was performed on an ECS 4010 Elemental Combustion System. A Bruker-Avance 400 MHz spectrometer was used to record the 1 H-NMR and 13 C-NMR spectra using TMS as an internal standard. The chemical shifts were reported in parts per million (δ-LC, ppm). Mass spectra was carried out on Waters Micro mass Q-Tof Micro having range of 4000 amu in quadruple and 20 000 amu in ToF.

Synthesis of Intermediates Chromen-2-one Derivatives (2,
3) (Scheme 1) 4-Hydroxy coumarin 1 (1.0 mole), DMF (4 vol) and potassium carbonate (1.1 mole) was heated to 85°C. Dibromoalkane (2.3 mole) in DMF (1 vol) was added in to the reaction mixture. The reaction mass stirred for 5 h at 85°C. The completion of the reaction was checked on TLC (hexane/ethyl acetate 7:3, Rf = 0.85 for compound 2 and 0.57 for compound 1). After completion of the reaction, the reaction mass was filtered and washed with DMF. The collected filtrate was poured into water and stirred for 30 min. The obtained solid was filtered, washed with water and purified by recrystallization from acetone to give compound 2 and compound 3.
During the reaction, Impurity-A and Impurity-B formed, which are insoluble in DMF so they can easily be removed through filtration. The method of preparation for Impurity-A and Impurity-B is incorporated in the supplementary information. The purification of compounds 2 and 3 were carried out via recrystallization in acetone.

Scheme 1
Synthesis of compounds 2 and 3.

Scheme 2
Synthesis of compounds 4 and 5.

Antimicrobials and Antifungal Activities (MIC)
The antimicrobial and antifungal activities were performed by Agar diffusion method. 40 The standard microbial strains used for the antimicrobial activity were procured from the Institute of Microbial Technology, Chandigarh. The antibacterial activity of the synthesized compounds was tested against two Gram-positive bacteria [Staphylococcus aureus (MTCC 96), Bacillus subtilis (MTCC 441)] and two Gram-negative bacteria [Escherichia coli (MTCC 443) and Pseudomonas aeruginosa (MTCC 1688)] using nutrient agar medium. The antifungal activity of the compounds was tested against Candida albicans (MTCC 227) using Sabouraud dextrose agar medium. All of the tests were performed in triplicate. The sterilized medium was inoculated (1 mL 100 mL -1 of medium) with the suspension (10 5 cfu mL -1 ) of the microorganism (matched to McFarland barium sulphate standard) and poured into a Petri dish to give a depth of 3-4 mm. The paper impregnated with the test compounds (100 µg disc -1 ) was placed on the solidified medium. The plates were pre-incubated for 1 hour at room temperature and incubated at 37°C for 24 h for antibacterial and 48 h for antifungal activities. Ampicillin 43 (100 µg disc -1 ) and Flucanazole 41 (10 µg disc -1 ) were used as standard for anti-bacterial and anti-fungal activities respectively. The MIC values for standards are obtained under the identical experimental condition as the test compounds. The results are presented in Table 1.
Scheme 3 Synthesis of compounds 6 and 7. and 13 C NMR spectroscopic techniques, mass spectra and elemental analysis. The IR spectra of compounds (4, 5) showed a band in the region 2993-3076 cm -1 due to the -OH groups. 42 The C-H bending vibrations are observed as a sharp medium to strong band at 1329 cm -1 in all compounds. The C-H linkage of the six-member ring caused a weak and sharp absorption band at 800-850 cm -1 . The C=O group is observed as a strong and sharp band at 1650-1710 cm -1 in these compounds. Further, 1 H NMR spectra exhibited multiplet in the region at δ-LC 7.04-7.94 ppm for eight aromatic protons (four aromatic protons of coumarin and four aromatic protons of benzene) (4). Four protons present in -CH 2 of compounds (4) are found to resonate as multiplet at δ-LC 4.54-4.34 ppm (alkene). One proton present in -OH of compound (4) is found to resonate as singlet at δ-LC 5.88 ppm. Six protons present in -CH 3 of compounds (4) are found to resonate as triplets at δ-LC 1.35-1.39 ppm (thiazole) and singlet at δ-LC 2.71 ppm (thiazole). Two protons of the -CH 2 group are observed at δ-LC 2.71 ppm as a multiplet compound (4).The IR spectra of compounds (6-7) showed a broad band in the region 3084-3176 cm -1 due to the -OH groups. The C=O group is observed as a strong and sharp band at 1631 cm -1 in these compounds. The C-H linkage of the mono substituted six-member ring caused a weak and sharp absorption band at 730-780 cm -1 in all the compounds. The 1 H NMR spectra of compounds 6 and 7 show identical peaks for coumarin moiety but show significant differences for pyrazolone moiety. The mono substituted aromatic rings proton found in between δ-LC 7.54-8.03 ppm for compounds 6 and -NH proton observed as broad peat at δ-LC 8.50 ppm for compound 7.
The anti-bacterial activity of the synthesized compounds was tested against two Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis) and two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) using nutrient agar medium. The antifungal activity of the compounds was tested against Candida albicans using Sabouraud dextrose agar medium. The results given in Table 1 indicated that most of the compounds tested and compared to standard Ampicillin exhibited considerable in vitro antimicrobial activities against Staphylococcus aures, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. All the compounds having minimum inhibitory concentration (MIC) values ranging from 10 to16 µg mL -1 . Activity of compounds 4 and 5 was the best against Pseudomonas aeruginosa and Escherichia coli.  Table 2.

Conclusion
In summary, a series of novel bioactive coumarin derivatives have been synthesized, purified and characterized. The result of antibacterial activity with the Ampicillin as standard at MIC >100 µg mL -1 against Pseudomonas aeruginosa and Escherichia coli at 300 µg mL -1 . All compounds exhibited slightly reduced activities (than the positive controls), and the best activity was for compounds 4 and 5. Against Staphylococcus aureus, Bacillus subtilisat 300 µg all compound showed slightly reduces activities, and the best performance was shown by compounds 4 and 7.
The result of antifungal activities with the comparison of Flucanazole at MIC 10 µg against Candida albicans at 40 µg all compound activity showed less activity. Given the above result, these kind of compounds could be further studied and explored as good antibacterial agents.