Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X Vol. 4(2), 56-59, February (2014) Res. J. Chem. Sci. International Science Congress Association 56 Synthesis and in vitro Antimicrobial Evaluation of 5’-Acetamido-2'-Hydroxy Chalcone derivatives Amita S. Rao, Lalitha Simon2*, K.K. Srinivasan, Sudheer Moorkoth, Zarah A. Haleem, Shivender S. Jadon, Ram M. Matsa2 and Ravivarma Sagiraju2 Department of Microbiology, Melaka Manipal Medical College (Manipal Campus), Manipal University, Manipal, Karnataka, INDIA Department of Chemistry, Kasturba Medical College International Center, Manipal University, Manipal, Karnataka, INDIA Department of Chemistry, Shri Madhwa Vadiraja Institute of Technology and Management, Bantakal, Udupi , Karnataka, INDIA Department of Pharmaceutical Quality Assurance, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, Karnataka, INDIA Available online at: www.isca.in, www.isca.me Received 8th January 2014, revised 23rd January 2014, accepted 10th February 2014Abstract A series of chalcones (3a-j) were synthesized by Claisen-Schmidt condensation of aromatic aldehydes with 5’-acetamido-2’-hydroxyacetophenone. Paracetamol was acetylated using acetic anhydride and sulphuric acid. The acetylated paracetamol underwent Fries rearrangement to form 5’-acetamido-2’-hydroxy acetophenone. The antibacterial and antifungal activity of the test compounds were evaluated using the agar well diffusion method. The test compounds displayed activity against Candida albicans. The fluorinated chalcone 3c exhibited maximum inhibition. However the test compounds failed to show antibacterial activity. Keywords: Chalcone, antibacterial, antifungal, agar well diffusion method. Introduction Chalcones (trans-1,3-diaryl-2-propen-1-ones) are precursors of open chain flavonoids and isoflavonoids, which are abundant in edible plants. Chalcones have been reported to be very active compounds and have displayed a broad spectrum of pharmacological activities. The pharmaceutical importance of these compounds lies in the fact that they can be effectively utilized as antibacterial, antifungal, anti-inflammatory, antiviral, antioxidant, anti-allergic, antimalarial, and anticancer agents1-6A number of chalcone derivatives have also been found to inhibit several important enzymes in cellular systems, including xanthine oxidase, aldose reductase, epoxide hydrolase, protein tyrosine Kinase, and quinone reductase. The presence of unsaturated ketone group in chalcones is responsible for their diverse activities. Resistance to antimicrobial drugs has become an increasingly important global problem8,9. Structural modification of antimicrobial drugs to which resistance has developed has proven to be an effective means of extending the lifespan of antifungal agents such as the azoles, the antiviral agents such as the non-nucleoside reverse transcriptase inhibitors, and various antibacterial agents including lactams and quinolones10. Broad empirical screening of chemical entities for antimicrobial activity represents a strategy for the development of novel drugs. The aim of the present study was to evaluate the antimicrobial activity of synthesized 2’-Hydroxy chalcones against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Candida albicans using the agar well diffusion method. The scheme for synthesis is shown in figure 1. Material and Methods Experimental: All purchased chemicals were of analytical grade and used without further purification. Melting points were determined by open capillary method and are uncorrected. Purity of synthesized compounds was checked by thin layer chromatography. values were recorded by using pre-coated silica gel aluminium backed plates Kieselgel 60 F254 Merck (Germany). Synthesis of N-(4-Acetylphenyl)acetamide (1): A mixture of paracetamol (0.1mol), acetic anhydride (0.01 mol) and concentrated sulphuric acid (0.001mol) was heated on a water bath at 50-60°C with occasional stirring for about 15min. After cooling the flask,100 ml water was added to it. The solid separated was filtered and crystallized from ethanol. Synthesis of 5’-acetamido-2’-Hydroxyacetophenone (2): N- (4-Acetylphenyl) acetamide (0.1 mol) was added to anhydrous AlCl (0.15mol) and nitromethane (0.4mol) and the mixture was refluxed at 130°C for 3hrs. To the cooled reaction mixture crushed ice was added slowly and the resulting solution was extracted with diethyl ether. The organic fraction was dried over anhydrous MgSO and the solvent was evaporated under reduced pressure to get 5’-acetamido-2’-Hydroxyacetophenone. Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(2), 56-59, February (2014) Res. J. Chem. Sci. International Science Congress Association 57 Synthesis of substituted chalcone derivatives (3a-j): A mixture of 5’-acetamido-2’-Hydroxy acetophenone (0.01mol) and aryl aldehyde (0.01 mol) was dissolved in ethanol (30ml).To this, aqueous potassium hydroxide solution (0.03 mol) was added slowly and stirred for 24 h, at room temperature. After completion of the reaction, the reaction mixture was poured into crushed ice and acidified with HCl. The solid separated was filtered and crystallized from ethanol. OH R COCH (CHCO) OH COCH + OHC R ' OH R O R ' ( i )(iii)( ii)3 a-jR - CHCONHReagents and conditions: (i) Con.HSO, reflux for 15 min, (ii) AlCl,130°C,3hrs (iii) NaOH/EtOH stir for 18-24 hrs Figure-1 Scheme for the synthesis of compounds (3a-j) Table-1 Physical data of compounds (3a-3j) Compound Mol Formula R’ Yield (%) MP (ºC) Rf* value 3a C 18 H 17 O 5 N 4-OH,3-OCH 3 72 158-161 0.64 3b C 17 H 14 O 3 NCl 4-Cl 90 148 0.71 3c C 17 H 14 O 3 NF 4-F 88 160-162 0.69 3d C 17 H 15 O 5 N Piperonaldehyde 93 188 0.60 3e C 19 H 19 O 4 N 4-OC 2 H 5 88 158-160 0.66 3f C 18 H 17 O 4 N 3-OCH 3 89 187 0.58 3g C 17 H 15 O 3 N H 90 163 0.64 3h C 18 H 17 O 5 N 3-OH,4-OCH 3 75 142 0.38 3i C 19 H 19 O 5 N 3,4-OCH 3 86 114 0.55 3j C 17 H 14 O 5 N 2 3-NO 2 79 138 0.60 *Solvent system Hexane: EtOAc 6:5 Table-2 Spectral data of a representative compound3aUV max (nm) IR data IR (KBr) cm-1Mass spectral data (ESI MS) 1 H NMR data values (DMSO, 400MHz) 240, 330 3244 (br., Ar-OH str), 1674 (C=O str. Side chain), 1626 (C=O str. Chalcone), 3342 (NH def), 3404 (NH str), 3036 (CH str Ar) 328 (M+1) Peak in the positive ionisation mode 2.38 (s, 3H, CH of acetamido), 3.58 (s, 3H, CH of methoxy), 6.77 (s, 1H, -C=CH), 10.07 (s, 1H, -NH), 9.78 (s, 1H, C-OH), 9.85 (s,1H,C-OH), 7.50-8.73 (m, 7H, Ar-H) Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(2), 56-59, February (2014) Res. J. Chem. Sci. International Science Congress Association 58 Antimicrobial Screening12-15: All the synthesized 2’-Hydroxy chalcone derivatives were screened for their antibacterial and antifungal activity. For antibacterial studies microorganisms employed were Gram-positive bacteria (Staphylococcus aureus ATCC 25923), Gram-negative bacteria (Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 and clinical isolates Klebsiella pneumonia. Candida albicans was used forthe antifungal activity study. The antimicrobial activity was determined using the agar well diffusion method. The selected bacterial and fungal strains were revived by plating on nutrient agar medium and Sabouraud dextrose agar medium respectively. After overnight incubation at 37°C, isolated colonies were selected and identities of the organisms were confirmed. Isolated bacterial colonies were then transferred to sterile Muller-Hinton Broth (MHB) and Candida albicans was transferred to Sabouraud Dextrose Broth (SDB) and incubated overnight. The growth concentration was adjusted to 10 organisms/ml by using 0.5 McFarland's turbidity standard. Determination of Antibacterial Activity: Petri dishes containing 20 ml of Muller-Hinton Agar (MHA) were used. The bacterial culture was spread over the surface of the MHA plate.4 mm diameter wells were punched into the agar and filled with 20l solution of 2'-Hydroxy chalcones of various concentrations (1000 µg/ml, 500 µg /ml, 250 µg /ml and 50 µg/ml).The plates were then incubated at 37°C for 18 hrs. Tests were done in triplicates and the average was taken. Determination of antifungal activity: The agar well diffusion method11 was modified. Sabouraud’s dextrose agar (SDA) was used for fungal cultures. Overnight cultures were grown at 37°C in Sabouraud’s dextrose Broth (SDB) and adjusted to contain 10 organisms/ml. Petri dishes containing 20 ml of Sabouraud’s dextrose agar (SDA) , were used. The fungal culture was spread over the surface of the SDA plate.4 mm diameter wells were punched into the agar and filled with 20l of the solution of 2'-Hydroxy chalcones of various concentrations (1000 µg/ml, 500 µg/ml, 250 µg/ml and 50 µg/ml). The plates were then incubated at 37°C for 24 hrs. Tests were done in triplicates and the average was taken. Results and Discussion A series of chalcones (3a-3j) was prepared by reacting acetylated 5’-acetamido-2’-hydroxyacetophenone with various aryl aldehydes in the presence of alkali (figure-1 and table-1).Formation of the substituted chalcones were confirmed by various spectral techniques. All the chalcones were obtained in good yield. Physical data such as melting point and Rf values also were determined and are presented in table 1. The UV, IR, mass and 1HNMR spectral data of a representative compound is shown in table 2. The effect of synthesized chalcones on bacterial and fungal strains are summarized in Table 3. The diameter of the clear zone of inhibition surrounding the well was measured in mm. The results indicate that 5’-Acetamido-2'-Hydroxy Chalcones displayed promising anti-fungal activity against Candida albicans. Among the chalcones, the fluorinated chalcone (3c) showed maximum activity against Candida albicans at all concentrations. However, the chalcones did not show any antibacterial activity. Table-3 Antimicrobial activity screening data Compound Code Zone of Inhibition of Different Concentrations of Compounds for Different Organisms in mm Antifungal Activity Antibacterial Activity Candida albicans Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia Concentration 1000 µg /ml 500µg /ml 250 µg /ml 50 µg /ml All four concentrations of compounds 3a-j did not show activity against the bacterial organisms:Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia 3a8.0 8.0 5.0 00 3b13.0 11.0 9.0 00 3c17.0 15.0 15.0 12.0 3d7.0 5.0 00 00 3e14.0 13.0 11.0 9.0 3f14.0 10.0 8.0 7.0 3g10.0 9.0 6.0 6.0 3h12.0 10.0 7.0 6.0 3i13.0 8.0 8.0 7.0 3j9.0 7.0 00 00 DMSO00 00 00 00 Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(2), 56-59, February (2014) Res. J. Chem. Sci. International Science Congress Association 59 ConclusionThe 5’-Acetamido-2’-Hydroxy chalcones displayed promising anti-fungal activity against Candida albicans. The chalcone containing fluorine (3c) showed maximum activity at all concentrations. In the antibacterial screening, none of the chalcones showed activity against the bacterial strains used. It can be concluded that 5’-Acetamido-2’-Hydroxy chalcones may be considered as an antifungal agent and can be used as a source of antimycotic substance for possible treatment of fungal infections. Acknowledgment Authors thank Manipal University and KMC International Center for providing the necessary laboratory facilities. 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