One-pot synthesis of carbazole based 3-hydroxy-4H- chromen-4-ones by a modified Algar–Flynn–Oyamada reaction and their antimicrobial activity J. Serb. Chem. Soc. 80 (11) 1361–1366 (2015) UDC 547.759.32+546.76–36:542.913: JSCS–4802 542.96:615.28–188 Original scientific paper 1361 One-pot synthesis of carbazole based 3-hydroxy-4H- chromen-4-ones by a modified Algar–Flynn–Oyamada reaction and their antimicrobial activity DONGAMANTI ASHOK*, SIDDA RAVI, BOMMIDI VIJAYA LAKSHMI and ARRAM GANESH Department of Chemistry, Osmania University, Hyderabad - 500 007, India (Received 3 December 2014, revised 30 March, accepted 10 June 2015) Abstract: A new series of 2-(9-ethyl-9H-carbazol-3-yl)-3-hydroxy-4H-chro- men-4-ones were synthesized from substituted 2-hydroxyacetophenones and 9-ethyl-9H-carbazole-3-carbaldehyde using NaOH and H2O2 by a modified Algar–Flynn–Oyamada reaction. In this method, the flavonols were synthe- sized in good yields (70–82 %) without isolating chalcones. The structures of the compounds were established based on 1H-NMR, 13C-NMR, FT-IR and mass spectral and analytical data. All the compounds were evaluated for their antimicrobial activity against bacteria, such as Staphylococus aureus, Bacillus subtilis, Escherichia coli and Klebsiella pneumoniae, as well as fungi, such as Aspergillus flavus and Fusarium oxysporum. Keywords: 2-(9-ethyl-9H-carbazol-3-yl)-3-hydroxy-4H-chromen-4-ones; modi- fied Algar–Flynn–Oyamada reaction; antimicrobial activity. INTRODUCTION Flavonoids constitute an important class of secondary metabolites that are widely distributed in plants. Their widespread distribution in nature, their struc- tural variability, relatively low toxicity and antioxidant activities have increased interest in flavonoids. Furthermore, flavonoids possess multimodal biological activities, such as anticancer,1 antihypertensive,2 anti-inflammatory,3 antibac- terial4 and antifungal5 activities. Chromones are interesting structural scaffolds and have been assigned as privileged structures for drug discovery. Substituted chromones were reported to show potential anticancer,6 antihistamine7 and anta- gonistic8 activities against leukotriene D4. On the other hand, carbazole derivat- ives are an important class of heterocyclic compounds that are known to possess important biological properties, such as antibacterial, antifungal,9 antitumor,10 antioxidant11 and antidiabetic12 properties. * Corresponding author. E-mail: ashokdou@gmail.com doi: 10.2298/JSC141203051A 1362 ASHOK et al. In the light of biological importance of the chromone and carbazole scaffolds and in continuation of the ongoing search for biologically active heterocyclic molecules,13 herein, the one pot synthesis of 2-(9-ethyl-9H-carbazol-3-yl)-3-hyd- roxy-4H-chromen-4-ones using a modified Algar–Flynn–Oyamada reaction (Scheme 1) and the antimicrobial properties of the obtained derivatives are reported. Scheme 1. Synthetic route for the preparation of carbazole based 3-hydroxy-4H-chromen-4-ones (3a–i). RESULTS AND DISCUSSION The original Algar–Flynn–Oyamada reaction14 is a two step process for the synthesis of 3-hydroxy chromenones. In the first step, 2-hydroxy chalcones are formed, which on subsequent cyclisation in the second step in the presence of alkaline hydrogen peroxide yields the corresponding flavonols, whereas the modified Algar–Flynn–Oyamada reaction15 is a one step process for the syn- thesis of flavonols from 2-hydroxyacetophenone and aromatic aldehydes in the presence of alkaline hydrogen peroxide (Table I). In this modified version there is no need to isolate the intermediate chalcones. As a model case, 2-hydroxy- acetophenone 1a was condensed with 9-ethyl-9H-carbazole-3-carbaldehyde 2 at room temperature using alkali and subsequently treated with alkaline hydrogen peroxide at room temperature to yield the flavonol derivative. It was identified as 2-(9-ethyl-9H-carbazol-3-yl)-3-hydroxy-4H-chromen-4-one 3a by IR, 1H-NMR, 13C-NMR and mass spectral data, which ruled out the formation of corres- ponding aurones and some other benzofuran derivatives, which were reported to form as by-products in Algar–Flynn–Oyamada reaction. In the 1H-NMR spectra of 3a, the OH proton appeared at δ 7.08 ppm as broad singlet and H4′ proton appeared as a doublet at δ 9.03 ppm. In the 13C-NMR spectra of 3a, the carbonyl carbon appeared at δ 173.0 ppm and the N–CH2 carbon resonated at δ 37.7 ppm. The ESI mass spectra of 3a showed a molecular ion peak at m/z = 356 [M+H]+. SYNTHESIS OF CARBAZOLE BASED 3-HYDROXY CHROMENONES AND ANTIMICROBIAL ACTIVITY 1363 TABLE I. Physical data of the synthesized compounds 3a–i Compd. M.p., °C R1 R2 R3 Time, h Yield, %a 3a 134–136 H H H 6 72 3b 128–130 F H H 7 78 3c 178–180 Cl H H 5 82 3d 190–192 Br H H 8 76 3e 140–142 CH3 H H 6 80 3f 206–208 Cl CH3 H 6 82 3g 212–214 Cl H Cl 7 80 3h 148–150 H OCH3 H 7 76 3i 172–174 H OC2H5 H 8 70 aIsolated yield Antibacterial activity The newly synthesized compounds 3a–i were screened in vitro for their antibacterial activity against two Gram-positiveve bacterial strains (Staphylo- coccus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633)) and two Gram- -negative bacterial strains (Escherichia coli (ATCC 25922), Klebsiella pneu- moniae (ATCC 13883)) at two different concentrations 20 and 40 µg mL–1. The zone of inhibition was measured in mm and ciprofloxacin was used as a standard antibacterial substance, under similar conditions for comparison. All the syn- thesized compounds showed good activity against the tested microorganisms (Table II). Among all, compounds 3a, 3h and 3i showed maximal zones of inhi- bition against the tested bacterial strains. It could be concluded that 3-hydroxy chromenones with electron releasing groups, such as methoxy, ethoxy and unsub- stituted compounds, showed the maximum activity. Furthermore, the antibac- terial results observed for other substitutions on the phenyl ring were very similar TABLE II. Antimicrobial activities (zones of inhibition in mm) of the synthesized compounds 3a–i Concentration, µg mL-1 Compd. S. aureus B. subtilis E. coli K. pneumoniae A. flavus F. oxysporum 20 40 20 40 20 40 20 40 50 50 3a 16 28 14 26 14 30 13 26 10 12 3b 11 22 6 14 05 12 7 15 5 10 3c 12 24 9 18 08 17 8 15 6 5 3d 11 20 9 17 10 17 9 19 6 4 3e 10 22 7 16 7 7 5 10 9 8 3f 11 21 9 18 9 10 7 15 11 4 3g 13 23 10 21 7 15 9 19 8 12 3h 16 29 16 29 17 32 24 35 13 16 3i 18 30 17 30 19 33 25 36 15 19 Ciprofloxacin 15 28 16 30 18 35 23 35 – – Amphotericin-B – – – – – – – – 12 15 1364 ASHOK et al. to each other. It was also concluded that changing the halogen substituent from F to Cl and Br does not provide any significant changes in antibacterial activity. Antifungal activity The antifungal activities of the synthesized compounds 3a–i were tested against two pathogenic fungi, Aspergillus flavus (ATCC-9643) and Fusarium oxysporum (ATCC-48112), at a concentration of 50 µg mL–1 and the results were compared with those of the standard, amphotericin-B. All the compounds showed good activity against the tested fungal strains (Table II). Among all the compounds, 3a, 3h and 3i showed maximal zones of inhibition against the tested fungal strains. Thus, electron releasing groups on 3-hydroxy chromenone, i.e., methoxy and ethoxy substitutions and the unsubstituted compound showed the highest antifungal activities, followed by the halogen substituted compounds. EXPERIMENTAL Materials All the employed materials were obtained commercially, mostly from Sigma–Aldrich, and used without further purification. Equipment The melting points were determined in open capillaries and are uncorrected. The purity of the compounds was checked by TLC on silica gel 60 F254 (Merck). The 1H-NMR and 13C-NMR spectra were recorded on a Bruker Avance II 400 spectrometer using TMS as an internal standard. The IR spectra were recorded in KBr on a Shimadzu FTIR 8400S spec- trophotometer. The mass spectra were recorded on a Shimadzu LCMS 2020 mass spec- trometer. The elemental microanalysis was realised on a Perkin Elmer CHN-2400 analyzer. The physical, analytical and spectral data of compounds 3a–i are given in the Supple- mentary material to this paper. General procedure for the synthesis of 2-(9-ethyl-9H-carbazol-3-yl)-3-hydroxy-4H-chromen- -4-ones To a well stirred solution of 2-hydroxyacetophenone 1a–i (1 mmol) and 9-ethyl-9H- -carbazole-3-carbaldehyde 2 (1 mmol) in EtOH (20 mL) was added NaOH (4 mmol in 10 mL of EtOH) at room temperature. The reaction mixture was further stirred for 4–5 h. After consumption of reactants (as indicated by TLC), the reaction mixture was dissolved in aque- ous NaOH (5 mmol in 5 mL), 3 mL of 30 % H2O2 was added dropwise and the stirring was continued for 2–3 h. After completion of reaction (monitored by TLC), the resulting light yellow reaction mixture was poured onto crushed ice and neutralized with dilute HCl. The thus-obtained light yellow solid was filtered, washed with water and dried. The crude product was purified by column chromatography on silica gel using hexane:ethyl acetate (7:3) as eluent to afford the desired products 3a–i. The respective yields are given in Table I. Biological assay Synthesized compounds were screened for their antibacterial activities against patho- genic bacteria, i.e., S. aureus, B. subtilis, E. coli and K. pneumoniae and their antifungal acti- vity against A. flavus and F. oxysporum. SYNTHESIS OF CARBAZOLE BASED 3-HYDROXY CHROMENONES AND ANTIMICROBIAL ACTIVITY 1365 The test organisms were cultured on agar slants, incubated for 24 h at 37±0.5 °C and 24– –48 h at 27±0.2 °C for the bacteria and fungi, respectively, to obtain freshly prepared cultures. The synthesized compounds were evaluated for antibacterial activity and antifungal activity against these freshly prepared strains of test organisms by the agar diffusion method and the poison plate technique, respectively. Muller–Hinton agar (MHA) and potato dextrose agar (PDA) were used as nutrient media for bacterial and fungal strains, respectively. The broth cultures were diluted with sterilized saline to bring the final size of the inoculum to approx- imately 105–106 CFU mL-1. The compounds were diluted in acetone, dimethyl sulphoxide (DMSO) and diethyl ether for biological assays. Of the three solvents, diethyl ether is found to be the best. The bacterial cultures were placed on the media and incubated at 37 °C for 24 h along with the diluted compounds introduced through discs (diameter 5 mm) dipped and placed over the nutrient media. The discs of ciprofloxacin (20–40 µg) and amphotericin-B (50 µg) were also incorporated into the medium for comparison. The same procedure was employed for determining the antifungal activity except that the culture strains of fungi were maintained on PDA and spores were transferred into PDA medium and the plates were incubated at 27±0.2 °C for 24–48 h. Inhibition of growth of the test organisms (bacterial and fungal) in presence of the test material and the standards was measured with the help of a standard scale. The values of the inhibition zones are reported in Table II. CONCLUSIONS In conclusion, an easy, facile and one-pot route for the synthesis of 2-(9- -ethyl-9H-carbazol-3-yl)-3-hydroxy-4H-chromen-4-ones in good yields is rep- orted. In this method, there is no need to isolate the intermediate chalcones, which tremendously reduces the man power, time and cost and also improves the overall yields. The antimicrobial assay of these compounds revealed that com- pounds 3a, 3h and 3i showed maximal zones of inhibition against the tested mic- roorganisms compared with the standards. SUPPLEMENTARY MATERIAL Physical, analytical and spectral data of compounds 3a–i are available electronically from http://www.shd.org.rs/JSCS/, or from the corresponding author on request. Acknowledgements. The authors are thankful to the Head of Department of Chemistry for providing the laboratory facilities and the Director of Central Facilities for Research and Development (CFRD), Osmania University, India, for providing the IR and NMR spectral analysis. Financial support for SR from CSIR, New Delhi, is gratefully acknowledged. И З В О Д СИНТЕЗА КАРБАЗОЛСКИХ ДЕРИВАТА 3-ХИДРОКСИ-4H-ХРОМЕН-4-ОНА МОДИФИКОВАНОМ АЛГАР–ФЛИН–ОЈИМАДИНОМ РЕАКЦИЈОМ И ИСПИТИВАЊЕ ЊИХОВЕ АНТИМИКРОБНЕ АКТИВНОСТИ DONGAMANTI ASHOK, SIDDA RAVI, BOMMIDI VIJAYA LAKSHMI и ARRAM GANESH Department of Chemistry, Osmania University, Hyderabad - 500 007, India Синтетисана је серија деривата 2-(9-етил-9H-карбазол-3-ил)-3-хидрокси-4H-хро- мен-4-она полазећи од супституисаних 2-хидрокси-ацетофенона и 9-етил-9H-карбазол- -3-карбалдехида помоћу NaOH и H2O2 у модификованој Алгар–Флин–Ојамадином реакцији. Овим поступком синтетисани су флавоноли, без изоловања халкона, у добром 1366 ASHOK et al. приносу (70–82 %). Структуре једињења утврђене су 1H-NMR и 13C-NMR техникама, FT- IR спектроскопијом, масеном спектрометријом и елементалном анализом. Испитана је антимикробна активност добијених једињења према бактеријама Staphylococus aureus, Bacillus subtilis, Escherichia coli и Klebsiella pneumoniae као и према гљивицама Aspergillus flavus и Fusarium oxysporum. (Примљено 3. децембра 2014, ревидирано 30. марта, прихваћено 10. јуна 2015) REFERENCES 1. E. Genoux, E. Nicolle, A. Boumendjel, Curr. Org. Chem. 15 (2011) 2608 2. Z. P. Xiao, Z. Y. Peng, M. J. Peng, W. B. Yan, Y. Z. Ouyang, H. L. Zhu, Mini-Rev. Med. Chem. 11 (2011) 169 3. H. P. Kim, K. H. Son, H. Chang, S. S. Kang, Nat. Prod. Sci. 2 (1996) 1 4. A. Mishra, A. K. Sharma, S. Kumar, A. K. Saxena, A. K. Pandey, BioMed Res. Int. 10 (2013) 915 5. M. Friedman, J. Agric. Food Chem. 62 (2014) 6025 6. M. Grazul, A. Kufelnicki, M. Wozniczka, I. P. Lorenz, P. Mayer, A. Jozwiak, M. Czyz, E. Budzisz, Polyhedron 31 (2012) 150 7. E. O. Meltzer, Allergy Asthma Proc. 26 (2005) 445 8. M. Q. Zhang, Y. Wada, F. Sato, H. Timmerman, J. Med. Chem. 38 (1995) 2472 9. R. Velmurugan, A. V. Vijayasankar, M. Sekar, Indian J. Chem., B 52 (2013) 414 10. T. Indumathi, A. Muthusankar, P. Shanmughavel, K. Prasad, J. Rajendra, Med. Chem. Commun. 4 (2013) 450 11. B. P. Bandgar, L. K. Adsul, S. V. Lonikar, H. V. Chavan, S. 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