{Microwave assisted synthesis of substituted 4-chloro-8-methyl-2-(1,3-diphenyl-1h-pyrazol-4-yl)-1,5-dioxa-2h-phenanthren-6-ones and their antimicrobial activity} J. Serb. Chem. Soc. 82 (2) 117–125 (2017) UDC 547.814.1+547.587.51+547.772.2: JSCS–4952 537.5–962:615.281–188 Original scientific paper 117 Microwave assisted synthesis of substituted 4-chloro-8-methyl-2- -(1,3-diphenyl-1H-pyrazol-4-yl)-1,5-dioxa-2H-phenanthren-6- -ones and their antimicrobial activity DONGAMANTI ASHOK1*, BACHI REDDY VANAJA1, MDDERLA SARASIJA2 and B. VIJAYA LAKSHMI1 1Green and Medicinal Chemistry Laboratory, Department of Chemistry, Osmania University, 500007 Hyderabad, India and 2Department of Chemistry, Satavahana University, Karimnagar, 505001 Telangana, India (Received 9 February, revised 5 December, accepted 14 December 2016) Abstract: Due to the potential antimicrobial activity of pyranochromenones and pyrazolines moieties, hybrid compounds containing both substituted 4-chloro- -8-methyl-2-(1,3-diphenyl-1H-pyrazol-4-yl)-1,5-dioxa-2H-phenanthren-6-ones (4a–g), were synthesized from substituted (E)-1-(7-hydroxy-4-methyl-8- -coumarinyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-2-propen-1-ones (3a–g) in good yield using the Vilsmeier reaction, by the microwave-assisted method. The structures of all the compounds were established based on their analytical and spectral data. All the synthesized compounds were tested in vitro for their antibacterial and antifungal activities. Some of the compounds showed very good activity compared to standard drugs against all the tested pathogenic bacteria and fungi. Keywords: antimicrobial activity; chromene; coumarin; microwave irradiation; pyrazolines; Vilsmeier reagent. INTRODUCTION The coumarin and chromene core moieties are important six-membered oxygen heterocyclic motifs embedded in several natural products and drugs. These systems are widely distributed in nature, and their derivatives have been shown to exhibit significant pharmacological activities.1 Chromenes and fused chromenes are biologically important compounds due to their antibacterial,2 anti- fungal,3 antitumor4 and antiviral5 activities. Coumarin derivatives were reported to exhibit anti-inflammatory,6,7 antimicrobial,8 antioxidant,9 anticancer10 and chemoprophylactic11 activities. Hybrid compounds containing both coumarin and chromene moieties, called pyranochromenone, due to the combined effect, * Corresponding author. E-mail: ashokdou@gmail.com doi: 10.2298/JSC160209001A 118 ASHOK et al. may exhibit better biological activity. The compounds embedded with pyrano- chromenone (Fig. 1), soulattrolide, inophyllum G-1, cordatolide A and oblon- gulide were reported by Patil et al. to have potential application for the treatment of HIV.12 Fig. 1.Representative examples of pyranochromenones that exhibit anti-HIV activity. Compounds with the backbone of chalcones were reported to possess vari- ous biological activities, such as antimicrobial, anti-inflammatory, analgesic, antiplatelet, anti-ulcerative, antimalarial, anticancer,13 antiviral, antileishmanial, antioxidant,14 antitubercular,15 antihyperglycemic, immunomodulatory, inhi- biters of chemical mediators release,16 inhibitors of leukotriene B4,17 inhibiters of tyrosinase18 and inhibitors of aldose reductase,19 estrogenic activities.20 Pyr- azolines were found to possess antimicrobial,21 antibacterial,22 anti-amoebic,23–24 antidepressant,25 anticonvulsant,26 anti–inflammatory27–28 and antitumor act- ivities. The recent literature is enriched with progressive findings concerning the synthesis and pharmacological properties of pyrazolines.21–28 Microwave irradiation has gained popularity in the past decade as a powerful tool for rapid and efficient synthesis of a variety of compounds because of sel- ective absorption of microwave energy by polar molecules.29 The application of microwave irradiation provides enhanced reaction rates and improved products in the field organic synthesis and is quite successful in the formation of a variety of carbon–heteroatom bonds. Our research group has been making considerable efforts in the design and realization of innovative synthetic protocols in organic synthesis adopting a more eco-sustainable approach.30–32 RESULTS AND DISCUSSION The synthetic route to compounds 4a–g is shown in Scheme 1. Compounds 3a–g were synthesized according to a previous work.33 The condensation of 8-acetyl-7-hydroxy-4-methyl coumarin (1) with 1-aryl-3-phenyl-1H-pyrazole-4- -carbaldehydes (2a–g) in the presence of piperdine under microwave irradiation gave (E)-1-(7-hydroxy-4-methyl-8-coumarinyl)-3-(1,3-diphenyl-1H-pyrazol-4- -yl)-2-propen-1-ones (3a–g). Subsequently these chalcones 3a–g on reaction with Vilsmeier reagent (DMF/POCl3) yielded substituted 4-chloro-8-methyl-2-(1,3- -diphenyl-1H-pyrazol-4-yl)-1,5-dioxa-2H-phenanthren-6-ones (4a–g). However, SYNTHESIS AND MICROBIAL ACTIVITY OF PYRANOCHROMENONE–PYRAZOLINE HYBRIDS 119 as the Vilsmeier reagent (DMF/POCl3) serves for formylation of electron-rich aromatic rings, it was highly efficient for intramolecular cyclization of 2′-hyd- roxychalcones to 4-chloro-2H-chromenes.34-36 Initially, the reaction was per- formed at room temperature but no product was formed. Optimum results were obtained when the temperature was maintained at 90–100 °C by taking 6 equi- valents of POCl3. In case of the microwave irradiation method, optimum results were obtained by irradiating at 160 W for 4–5 min. The crude products were purified using column chromatography to afford the pure products. Scheme 1. Synthetic route to substituted 4-chloro-8-methyl-2-(1,3-diphenyl-1H-pyrazol-4-yl)- -1,5-dioxa-2H-phenanthren-6-ones; a, R1=H, R2=H; b, R1=OCH3, R2=H; c, R1=OCH3, R2= OCH3; d, R1=CH3, R2=H; e, R1=F, R2=H; f, R1=Cl, R2=H; g, R1=Br, R2=H. It was observed that the yields were better when the microwave irradiation method was used rather than the conventional heating method, Table I. It is known that microwave irradiation is used for a variety of organic reactions due to TABLE I. Comparisons of the yields of the synthesized compounds 4a–g Compd. Conventional method MWI t / h Yield, % t / min Yield, % 4a 5 54 4 81 4b 5 61 4.5 89 4c 6 64 4 87 4d 6 57 5 88 4e 5.5 58 4.5 88 4f 5.5 61 4 90 4g 3 54 2 86 120 ASHOK et al. short reaction times, cleaner reactions, easier work-up and good yield. All the newly synthesized compounds were characterized using spectral analysis. The results are given in the Supplementary material to this paper. The IR spectra of intermediate compounds 3a–g showed characteristic peaks of functional group C=N stretching between 1576–1598 cm–1, C=O stretching (of chalcone) between 1633–1654 cm–1 and OH stretching between 3436–3448 cm–1. For the final derivatives 4a–g, characteristic bands were present in each spectra in wave number (cm–1) ranges: 703–721, 1078–1086, 1654–1665 and 1725–1736, which correspond to C–Cl, C–O–C, C=N and C=O stretching, res- pectively. The 1H-NMR spectra of chalcones 3a–g showed characteristic signals in the δ (ppm) ranges: 8.03–8.05, 8.15–8.18 and 8.60–8.64, corresponding to Hα, Hβ and the triazole H, respectively. In case of compounds 4a–g, the protons, H2, H7, H3 and H10 appeared as doublets in the δ ranges 5.96–5.96, 6.11–6.20, 6.18–6.21 and 6.83–6.89, respectively. In the 13C-NMR spectra, the carbonyl carbon of the chalcones 3a–g appeared in the δ 192.4–196.1. In the case of the final compounds 4a–g, C2 appeared in δ range 75.5–78.0. The rest of the carbons appeared in their usual regions, which supports the formation of compounds 3a–g and 4a–g. In the mass spectra of 3a–g and 4a–g, the molecular ion peaks, observed as [M+1] peaks, confirmed the molecular weights of the compounds. Antibacterial activity All the compounds were screened for their antibacterial activity against Sta- phylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli using ampicillin as the standard drug (Table II). The activity was determined using the cup plate agar diffusion method by measuring the zone of inhibition in mm. The compounds were screened at a concentration of 50 μg mL–1 in DMSO. From the screening studies, it was evident that the synthesized compounds 4b and 4c showed good antibacterial activity against all the tested organisms. It was further observed that the electron rich compound 4c, with both aryl rings pos- sessing a methoxy substituent showed the best activity, closely followed by 4b, TABLE II. Antibacterial activity (zone of inhibition, mm) of compounds 4a–g Compd. Gram-positive bacteria Gram-negative bacteria S aureus B. subtilis P. aeruginosa E. coli 4a 20 7 5 22 4b 30 15 12 30 4c 32 16 13 33 4d 18 10 6 18 4e 22 11 8 25 4f 10 8 2 23 4g 28 11 9 28 Ampicillin 30 12 10 30 SYNTHESIS AND MICROBIAL ACTIVITY OF PYRANOCHROMENONE–PYRAZOLINE HYBRIDS 121 which has only one methoxy substituent. This led to the conclusion that electron rich chalcones showed higher activity. Furthermore, changing in the halogens from F to Cl or Br did not provide any significant change in the levels of activity against the bacteria. Antifungal activity All the compounds were screened for their antifungal activity against Asper- gillus nigerzeae, Penicillium italicum and Fusarium oxysporum using grieseo- fulvin as the standard drug (Table III). The activity was determined using the cup plate agar diffusion method by measuring the zone of inhibition in mm. The compounds were screened at a concentration of 50 μg mL–1 in DMSO. From the screening studies, it is evident that the synthesized compounds 4a, 4c and 4g showed good antifungal activity against all the tested organisms. The unsubstitiuted compound 4a showed the highest activity against the fungi. The highly electron rich compound 4c showed activity which was comparable to that of 4a, but in general, substituents on the pyrazole were detrimental to the observed activity. Among the halogen derivates, the bromo substituent (4g) showed significantly higher activity than the chloro (4f) and fluoro (4e) substituted compounds. TABLE III. Antifungal activity (zone of inhibition, mm) of compounds 4a–g Cmpd. Fungi A. nigerzeae P. italicum F. oxysporum 4a 17 24 26 4b 8 14 19 4c 14 20 26 4d 7 15 18 4e 10 12 12 4f 8 11 12 4g 13 21 24 Grieseofulvin 12 20 25 EXPERIMENTAL Materials All the used materials were obtained commercially, mostly from Sigma–Aldrich, and were used without further purification. Equipment 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 spectrophoto- meter. The high-resolution electron spray ionization mass spectra (ESI-HRMS) were recorded 122 ASHOK et al. on a Micromass Q-Tof (ESI-HR-MS) mass spectrometer. The microwave reactions were performed in the milestone multiSYNTH microwave system. General procedure for the synthesis of substituted of (E)-1-(7-hydroxy-4-methyl-8-coum- arinyl)-3-(1-phenyl-3-phenyl-1H-pyrazol-4-yl)-2-propen-1-one analogues 3a–g A mixture of 8-acetyl-7-hydroxy-4-methyl coumarin 1 (1 mmol), substituted 1-(phenyl)- -3-phenyl-1H-pyrazole-4-carbaldehyde (2a–g, 1 mmol) in ethanol (2 ml) and few drops of piperidine was taken in a glass vial equipped with a cap and then subjected to microwave irradiation at 100 watts, by maintaining 80 °C for 10 to 15 min. The progress of the reaction was monitored by TLC. After completion of the reaction, the mixture was diluted with cold water and acidified with dil. HCl. The precipitate that formed was filtered, dried and recrys- tallized from ethanol to afford pure chalcone. General procedure for the synthesis of substituted 4-chloro-8-methyl-2-(1,3-diphenyl-1H-pyr- azol-4-yl)-1,5-dioxa-2H-phenanthren-6-ones 4a–g a) Conventional method A. DMF (5 mL) was taken in a round-bottomed flask and cooled to 0–5 °C. POCl3 (0.006 mol) was added drop wise under stirring. The mixture was stirred at 0–5 °C for 15 min and then a substituted (E)-1-(7-hydroxy-4-methyl-8-coumarinyl)-3-(1,3- -diphenyl-1H-pyrazol-4-yl)-2-propen-1-one (3a–g, 0.001 mol) solution in 3 mL of DMF was added at 0–5 °C and the temperature was maintained at 0–5 °C for 30 min. The reaction mixture was then heated in a water bath at 90 °C for 6–8 h. After completion of the reaction (monitored by TLC, EtOAc:hexane in 1:4 volume ratio), the reaction mixture was poured into ice–water and neutralized with 10 % NaOH solution and extracted with chloroform (2×20 mL). The combined organic layer was washed with 10 mL water and dried over anhydrous magnesium sulfate. The solvent was evaporated and the residue was purified by silica gel column chromatography to afford pure product 4a–g. b) Microwave method B. DMF (5 mL) was taken in a round-bottomed flask and cooled to 0–5 °C. POCl3 (0.006 mol) was added drop wise under stirring. The stirring at 0–5 °C was continued for 15 min and then a substituted (E)-1-(7-hydroxy-4-methyl-8-coumarinyl)-3-(1,3- diphenyl-1H-pyrazol-4-yl)-2-propen-1-one (3a–g, 0.001 mol) solution in 3 mL of DMF was added at 0–5 °C. The temperature was maintained at 0–5 °C for 30 min. The reaction mixture was placed in a microwave oven and subjected to microwave irradiation at 160 W by maintaining 90 °C for 4–5 min. The progress of reaction was monitored by TLC (EtOAc:hex- ane in 1:4 volume ratio). After completion of the reaction, the mixture was poured into ice water, neutralized with 10 % NaOH solution and extracted with chloroform (2×20 mL). The combined organic layer was washed with 10 mL water and dried over anhydrous magnesium sulfate. The solvent was evaporated and the residue was purified by silica gel column chromatography to afford pure product 4a–g. Biological assay Antibacterial activity. The synthesized novel compounds 3a–g and 4a–g were screened for their antibacterial activity against different types of bacterial strains, i.e., the Gram-neg- ative bacterial strains P. aeruginosa and E. coli and the Gram-positive bacterial strains B. subtilis and S. aureus at a concentration of 50 µ mL-1. The cultures were diluted with 5 % autoclaved saline and the final volume was made with a concentration of approximately 105–106 CFU mL-1. The synthesized compounds were diluted in acetone for antibacterial biological assays. For the agar disc diffusion method,37 the liquid form of the test compound was soaked on to the disc and then allowed to air dry, so that SYNTHESIS AND MICROBIAL ACTIVITY OF PYRANOCHROMENONE–PYRAZOLINE HYBRIDS 123 the disc became completely saturated with test compound. The saturated chemical discs were introduced onto the upper layer of the medium evenly loaded with the bacteria. The discs dipped in different chemical samples were placed over the evenly spread bac- terial nutrient media and incubated at 37 °C for 24 to 48 h for better inhibition of bacteria. The zones of inhibition were measured after 24 to 48 h. All the experiments were carried out in triplicates and the results were expressed as zone of Inhibition in mm. The zones of inhibition of synthesized compounds 4a–g were compared with the zone of inhibition of standard antibiotic concentration of Ampicillin (50 µg/mL). The Antibacterial activity was evaluated and the results are presented in Table I. Antifungal activity. The antifungal activity of synthesized compounds 4a–g was tested against three pathogenic fungi, namely Fusarium oxysporum, Aspergillus niger, and Penicil- lium italicum, by the poison plate technique at a concentration of 50 µg mL-1. The three kinds of fungi were incubated in potato dextrose agar (PDA) at 25±1 °C for 5 days to obtain new mycelium for the antifungal assay, then a mycelia as disks of approximately 0.45 cm diameter cut from the culture medium were picked up with a sterilized inoculation needle and ino- culated in the center of the PDA plate. Test compounds were dissolved in acetone (10 mL) then added to the medium PDA (90 mL). The final concentration of compounds in the medium was adjusted to 50 µg mL-1. The inoculated plates were incubated at 25±1 °C for 5 days. Acetone was diluted with sterilized distilled water and used as control, while grieseo- fulvin (50 µg mL-1) was used as standard control for each treatment. Three replicates of the experiments were performed. The radial growth of the fungal colonies was measured on the sixth day. CONCLUSIONS A new series of compounds (4a–g) were synthesized under conventional and microwave irradiation conditions. Using the microwave irradiation method, the reactions were completed in a shorter time with better yields as compared to the conventional method. All the new compounds were screened for their antimic- robial activity. It was observed that compounds 4b and 4c exhibited a broad spectrum of antibacterial activity, and compounds 4a, 4c and 4g showed a broader spectrum of antifungal activity against all the tested strains as compared to the standard drugs with their respective concentrations. SUPPLEMENTARY MATERIAL Analytical and spectral data of the compounds are available electronically at the pages of journal website: http://www.shd.org.rs/JSCS/, or from the corresponding author on request. Acknowledgments. We are thankful to The Head of the Department of Chemistry, Osma- nia University, Hyderabad, India, for providing the Laboratory facilities and The Director, CFRD, Osmania University, Hyderabad, for providing spectral analysis facilities. 124 ASHOK et al. И З В О Д СИНТЕЗА ОЗРАЧИВАЊЕМ МИКРОТАЛАСИМА СУПСТИТУИСАНИХ 4-ХЛОР-8-МЕТИЛ-2-(1,3-ДИФЕНИЛ-1H-ПИРАЗОЛ-4-ИЛ)-1,5-ДИОКСА-2H- -ФЕНАНТРЕН-6-ОНА И ЊИХОВА АНТИМИКРОБНА АКТИВНОСТ DONGAMANTI ASHOK1, BACHI REDDY VANAJA1, MDDERLA SARASIJA2 и B. VIJAYA LAKSHMI1 1 Green and Medicinal Chemistry Laboratory, Department of Chemistry, Osmania University, 500007 Hyderabad, India и 2Department of Chemistry, Satavahana University, Karimnagar, 505001 Telangana, India Због потенијалне антимикробне активности пиранохроменонских и пиразолинских једињења, Вилсмајеровом реакцијом су синтетисани хибридни деривати супституисаних 4-хлор-8-метил-2-(1,3-дифенил-1H-пиразол-4-ил)-1,5-диокса-2H-фенантрен-6-она (4a– –g), полазећи од супституисаних (E)-1-(7-хидрокси-4-метил-8-кумаринил)-3-(1,3- дифенил-1H-пиразол-4-ил)-2-пропен-1-она (3a–g), у добром приносу, применом микро- таласа. Структура свих једињења утврђена је на основу аналитичких и спектралних података. Одређена је in vitro антибактеријска и антифунгална активност свих синте- тисаних једињења. Поједини деривати показују веома добру активност према свим испитиваним бактеријама и гљивицама, у поређењу са стандардним лековима. 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