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Heterocyclic compounds based on 3-(4-bromophenyl) azo-5-phenyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity / Heterociklički derivati 3-(4-bromfenil) azo-5-fenil-2(3H)-furanona: Djelovanje na virus ptičje gripe (H5N1)

Heterocyclic compounds based on 3-(4-bromophenyl) azo-5-phenyl-2(3H)-furanone: Anti-avian... Acta m. 62 (2012) 593­606 DOI: 10.2478/v10007-012-0037-7 Short communication Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity EMAN M. FLEFEL1,* RANDA E. ABDEL-MAGEID1 WALED A. TANTAWY1 MOHAMED A. ALI2 ABD EL-GALIL E. AMR3,4 1 Deptment of otochemistry National Resech Centre, Cairo, Egypt Virology Laboratory, Environmental Resech Division, National Resech Center, Cairo, Egypt Drug Exploration & Development Chair (DEDC), College of macy, King Saud University, Riyadh 11451, Saudi abia Applied Organic Chemistry Deptment National Resech Center, Dokki 12622 Cairo, Egypt 3-[2-(4-omenyl)hydrazono]-5-enyl-furan-2(3H)-one (1) was used for prepation of some novel pyrazole, pyridazinone, oxadiazole, triazole, thiazolidine and thioxopyrimidine derivatives. Some of the preped products were tested for anti-avian influenza virus activity and revealed promising antiviral activity against H5N1 virus [A/Chicken/Egypt/1/20 % (H5N1)] by determination of both EC50 and LD50 and confirmed by plaque reduction assay on Madin-Dby canine kidney cells. Compounds 3-[2-(4-omoenyl)hydrazono]-5-enylfuran-2(3H)-one (1), 1-(4-omoenyl)-N-hydroxy-5-enyl-1H-pyrazole-3-cboxamide (5) and 1-(4-omoenyl)-N-{2,3-dihydro-4-hydroxy-3-enyl-6-oxo-2-thioxopyrimidin-1(6H)-yl}-5-enyl-1H-pyrazole-3-cboxamide (12a) showed the highest effects. Detailed synthesis, spectroscopic data, and antiviral activity of the synthesized compounds e reported. Keywords: furanone, pyrazole, pyridazinone, oxadiazole, triazole, anti-avian influenza virus (H5N1) Accepted September 10, 2012 2(3H)-Furanones e a type of five-membered heterocyclic compounds of synthetic and biological importance. The importance of this type is due to the facile opening of the lactone ring to give acyclic products, which undergo ring closure to give other synthetically and biologically important heterocyclic compounds (1, 2). The chemistry of pyrazole-containing compounds is pticully interesting for antiviral (1), anti-angiogenic activity (3), and as apoptosis-inducing agents (4), potent peroxisome proliferator-activated receptor gamma [PPg] ptial agonists (5) and antibiotics (6). Moreover, pyridazinones have promising biological activities as antiplatelet (7), antihypertensive agents (8). Biochemical studies revealed that oxadiazole caused activation on glutamic oxalo- * Correspondence; e-mail: emanflefel@yahoo.com E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. acetic transaminase (GOT) and glutamate pyruvate transaminase (GPT) enzymes, inhibition of gamma-glutamyl transpeptidase (g-GT) enzyme activity (9) and potent inhibition of tyrosine (10). On the other hand, substituted 1,3,4-oxadiazoles exhibit fungicidal (11), antimicrobial and antitubercul activity (12). Simple 1,2,4-triazoles also display some biological activities as antioxidant, urease inhibitors (13) and antiviral (14) agents. These diverse macological activities prompted us to convert furanone into other heterocycles being pyrazolyl moiety, and evaluate them for anti-viral activity. On the other hand, influenza viruses e respiratory pathogens that affect humans and e responsible for substantial morbidity, mortality and decreased productivity worldwide (15). Several synthetic compounds have already been utilized as potential inhibitors of the avian virus (H5N1) and some of them demonstrated the ability to inhibit viral replication at a level compable to the approved anti-influenza drugs zanamivir and oseltamivir (16). EXPERIMENTAL Melting points were measured using an Electrothermal 9100 digital melting point appatus (Büchi, Switzerland) and e uncorrected. IR spectra were recorded on a Perkin-Elmer 1600 FTIR (Perkin-Elmer, USA) in K discs. 1H NMR spectra were measured on a Jeol 270 MHz spectrometer (Jeol, Japan) and a uker Avance spectrometer (300 MHz) (uker, Germany) in DMSO-d6, and chemical shifts were recorded in d ppm relative to the internal standd TMS. Mass spectra were run at 70 eV with a Finnigan SSQ 7000 spectrometer (Thermo electron corporation, USA) using EI; m/z values e indicated in Dalton. Elemental analyses were performed on a Perkin-Elmer 2400 analyzer (Perkin-Elmer) and were found within ±0.4 % of the theoretical values (Table I). Follow-up of the reactions and checking of the purity of compounds were done by TLC on silica gel-precoted aluminum sheets (type 60 F254, Merck, Germany). All solvents and reagents were purchased from Aldrich (Germany). ysicochemical and spectral data for the synthesized compounds e given in Tables I and II, respectively. Syntheses 3-[2-(4-omoenyl)hydrazono]-5-enylfuran-2(3H)-one (1). ­ To a solution of omoaniline (6.88 g, 0.04 mol) in glacial acetic acid (15 mL), conc. HCl (8 mL) was added, then while the mixture was being cooled in ice under stirring, sodium nitrite (4.96 g, 0.068 mol) was added dropwise. To the diazonium mixture, freshly fused sodium acetate (6.0 g) was added. oyl propionic acid (6.0 g, 0.04 mol) was added to acetic anhydride (40 mL), the mixture was refluxed for 3 h, then allowed to cool at room temperature. This solution was added dropwise, with stirring and cooling in ice, to the diazo- nium mixture. The reaction mixture was allowed to stand overnight and the precipitate was collected, washed with water several times and recrystallized from acetic acid to give high yield (2). E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. 2-[2-(4-omoenyl)hydrazono]-4-oxo-4-enylbutane hydrazide (2). ­ A mixture of furanone 1 (3.42 g, 0.01 mol) and hydrazine hydrate (0.64 g, 0.02 mol, 98 %) in absolute ethanol (20 mL), was stirred at room temperature for 0.5 h. The solvent was evaporated under vacuum and crystals were obtained. 4-[2-(4-omoenyl)hydrazinyl]-6-enylpyridazin-3(2H) one (3). ­ A mixture of furanone 1 (3.42 g, 0.01 mol) and hydrazine hydrate 98 % (0.64 g, 0.02 mol) in absolute ethanol (25 mL) was refluxed for 2 h. The solvent was evaporated under vacuum and the solid residue was recrystallized from benzene. 4-[2-(4-omoenyl)hydrazono]-1,2-dihydro-1,6-dienylpyridazin-3(4H)-one (4). ­ A mixture of furanone 1 (3.42 g, 0.01 mol) and enylhydrazine (1.3 g, 0.012 mol) in absolute ethanol (25 mL) was refluxed for 3 h. The solvent was evaporated under vacuum and the solid residue was recrystallized from benzene/petroleum ether (60­80 °C, 1:1). 1-(4-omoenyl)-N-hydroxy-5-enyl-1H-pyrazole-3-cboxamide (5). ­ A mixture of furanone 1 (3.42 g, 0.01 mol), hydroxylamine hydrochloride (0.7 g, 0.01 mol) and sodium acetate (1 g) in ethanol (25 mL) was refluxed for 4 h. The solvent was evaporated under vacuum and the solid residue was recrystallized from benzene/petroleum ether (60­80 °C, 1:1). 5-[1-(4-omoenyl)-5-enyl-1H-pyrazole-3-yl]-1,3,4-oxadiazole-2(3H)-thione (6). ­ A mixture of hydrazide 2 (1.13 g, 0.003 mol), cbon disulfide (5 mL) and sodium hydroxide (4 %, 3 mL) in ethanol (30 mL) was stirred at room temperature for 6 h. The solvent was evaporated under vacuum, dissolved in hot water, and then the filtrate was neutralized with diluted hydrochloric acid. The yellow precipitate was collected after washing with water several times. The product obtained was recrystallized from benzene/petroleum ether (60­80 °C, 1:1). 2-{[1-(4-omoenyl)-5-yl-1H-pyrazol-3-yl]cbonyl}-N-enyl-hydrazinecbo-thioamides (7a,b). ­ A mixture of hydrazides 2 (3.75 g, 0.01 mol) and enyl or p-methoxyenyl isothiocyanate (0.01 mol) in ethanol (30 mL), was refluxed for 2 h. The mixture was cooled, and then filtered off. The solid so obtained was recrystallized from benzene/ethanol (1:2.5). 1-(4-omoenyl)-5-enyl-N'-(enylcbonyl)-1H-pyrazole-3-cbohydrazide (8). ­ To a suspension of hydrazide 2 (1.13 g, 0.003 mol) in dry benzene (15 mL), benzoyl chloride (0.42 g, 0.003 mol) was added at 25 °C and the mixture was heated under reflux at 75 °C for 3 h. The solvent was evaporated under vacuum, and the solid residue was recrystallized from benzene. 2-{1-(4-omoenyl)-5-enyl-1H-pyrazole-3-yl}-5-enyl-1,3,4-oxadiazole (9). ­ A mixture of dioylhydrazine 8 (0.92 g, 0.002 mol) and osorus oxychloride (10 mL) was refluxed for 1 h. The mixture was cooled, poured onto crushed ice and then neutralized with 4 % NaOH. The white precipitate was collected by filtration, washed with water and recrystallized from benzene. 4-(4-omoenyl)-5-(1-yl-5-enyl-1H-pyrazol-3-yl)-2H-1,2,4-triazole-3(4H)-thiones (10a,b). ­ A mixture of thiosemicbazides 7a,b (0.001 mol) and NaOH (4 %, 20 mL) was E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. refluxed for 2 h. The mixture was cooled, poured onto crushed ice and then neutralized with diluted hydrochloric acid. The white precipitate was collected by filtration and washed with water. The solid obtained was recrystallized from ethanol/water (5:1). 1-(4-omoenyl)-N-(4-oxo-3-ylthiazolidine-2-ylidene)-5-enyl-1H-pyrazole-3-cbohydrazides (11a,b). ­ A mixture of thiosemicbazide 7a or b (0.5 g, 0.001 mol), ethylchloroacetate (0.12 g, 0.001 mol) and NaOH pellets (0.4 g) in absolute ethanol (30 mL) was reTable I. ysical and analytical data of new compounds Analysis (%) (calcd./found) C 56.00 56.20 51.22 51.32 53.80 53.72 60.98 60.79 53.65 53.69 51.14 51.23 56.10 56.00 55.18 55.20 59.88 59.64 62.32 62.12 58.23 58.00 57.15 56.98 56.40 56.38 55.52 55.49 55.72 55.61 54.92 54.69 H 3.23 3.20 4.03 4.38 3.67 3.68 3.95 3.78 3.38 3.28 2.78 2.64 3.68 3.61 3.86 3.66 3.71 3.50 3.41 3.21 3.40 3.19 3.60 3.40 3.41 3.43 3.52 3.50 3.24 3.13 3.41 3.20 23.28 23.21 21.30 21.16 22.37 22.10 18.44 18.10 22.31 22.06 20.01 19.89 16.23 16.13 15.30 15.09 17.32 17.12 18.03 17.87 16.84 16.62 15.84 15.62 15.01 14.84 14.21 14.11 14.26 14.00 13.53 13.19 N 8.16 8.10 14.93 14.73 15.68 15.00 12.93 12.60 11.73 11.60 14.03 13.88 14.22 14.00 13.41 13.21 12.15 12.00 12.64 12.48 14.76 14.63 13.88 13.48 13.15 13.00 12.45 12.39 12.50 12.22 11.86 11.66 6.76 6.49 6.36 6.34 6.02 5.91 5.70 5.68 5.72 5.70 5.43 5.39 8.03 8.00 6.51 6.58 6.12 6.00 S Compd. Formula (Mr) C16H11N2O2 (342.80) C16H15N4O2 (375.22) C16H13N4O (357.22) C22H17N4O (433.30) C16H12N3O2 (358.19) C17H11N4OS (399.26) C23H18N5OS (492.39) C24H20N5O2S (522.42) C23H17N4O2 (461.31) C23H15N4O (443.30) C23H16N5S (474.38) C24H18N5OS (504.40) C25H18N5O2S (532.41) C26H20N5O3S (562.44) C26H18N5O3S (560.42) C27H20N5O4S (590.45) M.p. (°C) 295­296 89­90 160­161 84­85 103­104 179­180 199­200 147­148 120­121 167­168 189­190 225­226 191­192 139­140 186­187 124­125 Yield (%) 65 80 86 81 78 79 76 74 77 80 77 81 72 76 70 72 1 2 3 4 5 6 7a 7b 8 9 10a 10b 11a 11b 12a 12b E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. fluxed for 2 h. The solvent was evaporated under reduced pressure; the reaction mixture was poured onto water. The obtained precipitate was filtered off, washed with water, dried and recrystallized from ethanol. 1-(4-omoenyl)-N-{2,3-dihydro-4-hydroxy-3-yl-6-oxo-2-thioxopyrimidin-1(6H)-yl}-5-enyl-1H-pyrazole-3-cboxamide (12a,b). ­ A solution of 7a or b (0.001 mol) in sodium ethoxide (0.023 g sodium metal in 30 mL ethanol) was heated for 30 min at 80 °C. The reaction mixture was cooled, and then diethylmalonate (0.016 g, 0.001 mol) was poured into water, with stirring at 70 °C for 3 h. The precipitated solid was filtered off, washed with water, dried and recrystallized from methanol to give compounds 12a,b. Table II. Spectral data of the new compounds Compd. IR (nmax, cm­1) 1735 (C=O), 3335 (NH) 1652, 1690 (2 C=O), 3270­3335 (NH, NH2) 1H 1H and 13C NMR (d, ppm) MS (m/z, %) 342 (M+, 100) NMR: 6.32 (s, 1H, =CH), 3.42 (s, 1H, NH, D2O exchangeable), 7.80 (s, 1H, NH, D2O exchangeable), 7.30­7.45 (m, 9H, -H) 1H NMR: 2.60­2.85 (m, 2H, ­CH2), 3.42 (s, 2H, NH2, D2O exchangeable), 7.00 (s, 1H, NHCO­, D2O exchangeable), 7.30­7.45 (m, 9H, ­H), 8.65 (s, 1H, NH, D2O exchangeable) NMR: 5.60 (s, 1H, H pyridazinone), 6.55 (d, 2H, J = 8Hz, -H), 7.25 (d, 2H, J = 8Hz, -H), 7.40 (m, 3H, -H), 7.65 (m, 2H, -H), 7.90 (s, 1H, NH, pyridizinone, D2O exchangeable), 8.70 (s, 1H, NH, D2O exchangeable), 11.80 (s, 1H, NH-, D2O exchangeable) 374 (M+, 100) 1H 1648 (C=O), 3275­3315 (NH) 357 (M+, 100) 1655 (C=O), 3235 (NH) 1645 (C=O), 3130 (NH), 3340 (OH) 1250 (C=S), 3100 (NH) 1252 (C=S), 1646 (C=O), 3200­3290 (NH) 1252 (C=S), 1650 (C=O), 3196­3292 (NH) NMR: 5.9 (s, 1H, H pyridazinone), 6.35­7.4 (m, 14H, -H), 8.2 (., 1H, NHCO­, D2O exchangeable), 11.1 (s, 1H, NH exchangeable) 1H 1H 332 (M+, 100) 357 (M+, 100) 398 (M+, 100) NMR: 7.00 (s, 1H, pyrazole H), 7.20­7.48 (m, 9H, -H + -H), 9.20 (s, 1H, OH, D2O exchangeable), 11.10 (s, 1H, NH, D2O exchangeable) NMR: 7.08 (s, 1H, pyrazole H), 7.20­7.40 (m, 9H, -H + -H), 14.35 (, 1H, NH, D2O exchangeable) 1H 7a NMR: 7.14 (s, 1H, pyrazole H), 7.30­7.66 (m, 14H, 3 -H), 9.70 (, 2H, NH, D2O exchangeable), 10.30 (s, 1H, NH, D2O exchangeable) 13C NMR: 108.31, 120.56­138.70, 143.30 (-20C), 146.38 (C=N), 161.05 (C=O), 198.40 (C=S) NMR: 3.7 (s, 3H, OCH3), 7.11 (s, 1H, pyrazole H), 7.31­7.62 (m, 13H, 3 -H), 9.90 (, 2H, NH, D2O exchangeable), 10.60 (s, 1H, NH, D2O exchangeable) 1H 1H 491 (M+, 100) 7b 523 (M+, 100) E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. Table II. cont. 1H 1632 (C=N), 1675 (C=O), 3202 (NH) NMR: 7.10 (s, 1H, pyrazole H), 7.20­7.90 (m, 14H -H), 10.20 (s, 1H, NH, D2O exchangeable), 10.60 (s, 1H, NH exchangeable) 13C NMR: 108.86, 121.91, 128.05­132.70, 139.02, 144.87 (-20C), 146.41 (C=N), 161.05, 166.19 (2C=O) 1H 460 (M+, 100) 9 10a 1600 (C=N) 1251 (C=S), 3080 (NH) 1252 (C=S), 3085 (NH) NMR: 7.00 (s, 1H, pyrazole), 7.2­7.78 (m, 14H, -H) NMR: 6.70 (s, 1H, pyrazole H), 6.9­7.56 (m, 14H, 3-H), 14.16 (, 1H, NH, D2O exchangeable) 443 (M+,100) 475 (M+,100) 504 (M+,100) 1H 10b NMR: 3.40 (s, 3H, OCH3), 6.70 (s, 1H, pyrazole H), 6.90­7.66 (m, 13H, 3-H), 14.20 (, 1H, NH, D2O exchangeable) 1H 1H 11a 3085 (NH), 1680 (C=O) NMR: 4.86 (s, 2H, CH2), 6.70 (s, 1H, pyrazole H), 6.90­7.66 (m, 14H, 3-H), 10.60 (s, 1H, NH, D2O exchangeable); 13C NMR: d 48.1 (CH2), 106.3 (pyrazole C-4), 122.12­159.90 (18 -C, pyrazole C-3,5 and thiazole C-2,5), 170.36, 172.52 (2C=O) 13C NMR: 40.1 (CH ), 106.3, 116.12­142.90 (-20C), 2 152.10, 155.25 (2 C=N), 170.36, 172.52 (2 C=O) 1H 531 (M+, 100) 11b 3088 (NH), 1681 (C=O) NMR: 3.60 (s, 1H, OCH3), 4.82 (s, 2H, CH2), 6.70 (s, 1H, pyrazole H), 6.92­7.62 (m, 13H, 3-H), 10.2 (s, 1H, NH, D2O exchangeable) 13C NMR: 39.70 (CH ), 55.32 (CH ), 107.10, 114.42, 2 3 118.65, 121.37­132.29, 138.27, 144.81 (-20C), 153.08, 154.57 (2C=N), 160.10, 168.64 (2C=O) 1H 563 (M+, 100) 12a (C=S), (C=O), (C=O), (NH) (C=S), (C=O), (C=O), (NH) NMR: 6.70 (s, 1H, pyrazole H), 6.9­7.66 (m, 14H, 3-H), 14 (,1H, NH, D2O exchangeable) 13C NMR: 39.84 (CH ), 108.69, 121.46, 127.47­132.29, 2 138.50, 139.34, 144.26 (-20C), 145.96 (C=N), 166.65, 168.17, 168.49 (3C=O), 181.16 (C=S) 1H 559 (M+, 100) 12b NMR: 3.4 (s, 3H, OCH3),5.4 (s, 1H, ethylene), 6.7 (s, 1H, pyrazole H), 7.1­7.66 (m, 13H, 3-H), 9.2 (s, 1H, NH, D2O exchangeable), 15.2 (, 1H, OH, D2O exchangeable) 589 (M+, 100) Virus and cells. ­ Avian influenza-A virus (H5N1) [isolated from cloacal swabs from chicken, in Qalubiya governorate, Egypt, 2006 (A/chicken/Egypt/1/2006 (H5N1), accession no. FJ472343], was used to prepe low pathogenic rH5N1 by plasmid-based reverse genetics. The preped H5N1 vaccine strain was used in this study to evaluate antiviral activity of some synthetized compounds. Madin-Dby canine kidney (MDCK) cells kindly provided by Dr. Richd Webby (St. Jude Children's Resech Hospital, Memis, TN, USA) were used for virus propagation. The MDCK cells were routinely E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. passaged in Dulbecco's modified Eagle medium (DMEM) containing 10 % fetal bovine serum (FBS) and 1 % antibiotic-antimycotic mixture (penicillin-streptomycin-amotericin B). Compound prepation for biological assays. ­ Preped compounds were dissolved in 10 % dimethyl suloxide (DMSO) in doubly destilled water to a concentration of 10 mg mL­1 stock solutions used for further dilutions according to the assay applied. Cytotoxicity assay (MTT assay). ­ Cytotoxic activity of the extracts was tested in MDCK cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dienyltetrazolium omide (MTT) method (17) with minor modification. iefly, the cells were seeded in 96 well-plates (100 µL per well containing 3 × 105 cells mL­1) and treated with 5, 10, 20, 40, 80 and 120 µg of the sample per well. At 24 h, cells were washed with sterile osate buffer (PBS) three times and the supernatant was discded. MTT solution (20 µL of 5 mg mL­1) was added to each well and incubated at 37 °C for 4 h. Then the medium was aspirated. In each well, the formed formazan crystals were dissolved with 200 µL of acidified isopropanol (0.04 mol L­1 HCl in absolute isopropanol). Absorbance of formazan was detected with a dual wavelength UV spectrometer at 540 nm with 620 nm reference wavelength. The percentage of cytotoxicity comped to the untreated cells was determined. The 50 %-cytotoxicity (LD50) for each compound was calculated from standd curve of percentage of cytotoxicity vs. sample concentration. Antiviral activity and therapeutic index. ­ Antiviral activity of the compounds was determined using the cytopathogenicity (CPE) assay against low pathogenic reassortant avian influenza virus (rH5N1). Cells were seeded in 96-well cell culture plates (100 µL per well at a density of 3 × 105 cells mL­1) and grown to confluency. Cells were then infected with 100 µL of stock virus. After the virus adsorption period on cells of 2 hours at 37 °C, the virus was removed and serial dilutions (10, 20, 40 µg µL­1) of the tested compounds were added, then incubated with infected cells using maintenance DMEM with 2 % FBS (100 µl per well) at 37 °C for 3 days of monitoring until complete CPE was observed in the infected and untreated virus control. Plaque reduction assay. ­ In a six-well cell culture plate, confluent MDCK cells were infected with a pre-incubated mixture of 100 µL of avian influenza H5N1 virus (80­100 plaques per well) and 100 µL of DMEM [containing 2 % antibiotics and 1 mg mL­1 of L-1-tosyl-amido-2-enylethyl chloromethyl ketene and a different concentration of each compound (5, 10, 20, and 40 mg mL­1)]. The plates were incubated for 1 h at 37 °C in 5 % CO2 to allow virus adsorption. After adsorption, 2 mL of agose overlayer (2 % agose in DMEM 2 x containing 1 % FBS) was added to each well and mixed. The cultures were incubated at 37 °C in 5 % CO2 for 3­4 days of plaque formation monitoring. Plaques were fixed with 10 % formalin in osate-buffered saline for 2 h followed by removal of the ag overlayer and staining with 0.1 % crystal violet in distilled water. Plaques were counted manually from triplicate wells based on the plaque number but not plaque size. Viral counts and percentage of virus reduction were calculated according to Hayden et al. (18). E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. RESULTS AND DISCUSSION Chemistry 3-[2-(4-omoenyl)hydrazono]-5-enylfuran-2(3H)-one (1) was preped by coupling the diazotized aniline with 5-enyl-2(3H)-furanone according to the reported procedures (2). Furanone 1 was reacted with hydrazine hydrate in absolute ethanol at room temperature to give the corresponding acid hydrazide 2, which was refluxed in ethanol to give pyridazinone derivative 3. Also, compound 3 was preped by refluxing compound 1 with hydrazine hydrate in absolute ethanol. The reaction of furanone 1 with enylhydrazine was also investigated. When the reaction was cried out under reflux conditions, pyridazinone derivative 4 was formed. Moreover, furanone 1 was reacted with hydroxylamine hydrochloride in ethanol under reflux to give pyrazole derivative 5 (Scheme 1). H N H N O 2 O N NH2NH2 EtOH, r.t. NH2 EtOH, reflux H N N H N N O 1 O NHNH2 EtOH, reflux N N H 4 O O NH2OH EtOH, reflux N N 5 N H H 3 N N H O N N H NH2NH2 EtOH, reflux = Scheme 1 Treatment of 2 with cbon disulide in alcoholic sodium hydroxide gave oxadiazolthione derivative 6. Hydrazide 2 was reacted with enyl or p-methoxyenyl isothiocyanate to give the corresponding thiosemicbazide derivatives 7a,b, respectively. Also, hydrazide 2 was reacted with benzoyl chloride in dry benzene to give the corresponding pyrazole derivative 8, which was treated with osorus oxychloride to afford the corresponding 1,3,4-oxadiazole derivative 9 (Scheme 2). E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. S N O CS2, NaOH EtOH, r.t. N 6 N O N H N H N S ' H N H N ' N=C=S EtOH, r.t. N N 7a,b O COCl dry benzene N H O 2 O NH2 H N N O N N 8 ' O N POCl3 reflux N N OMe Scheme 2 Ring closure of thiosemicbazides 7a,b using sodium hydroxide solution, led to the formation of the corresponding triazolthiones 10a,b. Also, the reaction of thiosemicbazides 7a,b with ethyl chloroacetate in the presence of alcoholic potassium hydroxide afforded thiazolidine derivatives 11a,b, respectively. Moreover, when thiosemicbazides 7a,b were reacted with diethylmalonate in the presence of sodium ethoxide, the corresponding thioxopyrimidin derivatives 12a,b were formed (Scheme 3). Antiviral bioassays Cytotoxicity assay (MTT) was cried out on all preped compounds and according to the results obtained, only compounds 1, 5, 7a, 8, 9, 10a, 11a, and 12a showed notable toxic effects on the biology of cells used in assays. The LD50, EC50, and therapeutic index (TI) e presented in Fig. 1. Calculated LD50 value indicated that only 1, 5, 12a passed the antiviral activity bioassay. It was obvious that at concentrations of 10, 20 and 40 mg mL­1 compounds 1, 5 and 12a showed higher therapeutic indices than the other tested compounds comped to the anti-influenza drug zanamivir. Plaque reduction assay confirmed the antiviral activity of the three compounds as shown in Fig. 2. E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. S N NaOH reflux ' N N N N 10a,b O N H N S ' N N N N N 12a,b H O O ' O N N N 7a,b H H N S H N ClCH2COOEt, ' NaOH, EtOH, r.t. COOEt N N O O 11a,b S a: ' COOEt NaOH, reflux b: OMe ' O S N N N N H O N OH Scheme 3 Structure-activity relationship Structure modifications on the lead compounds 1 and 2 afforded derivatives with a viety of anti-avian influenza virus (H5N1) activity. For example, pyrazole-3-cboxyamide derivative 5 demonstrated remkable activity due to the chromoor in the skeleton of this compound and displayed a significant EC50 value in regd to the pent compound 1 (Fig. 1). However, product 2 does not appe to be advantageous in terms of anti-avian influenza virus with respect to the pent 1 because of its open chain structure and higher hydroilicity. Furthermore, chemical transformation/structure conversion of 2 into 7 and 8 influenced positively the activity profiles against influenza virus comped to their pent. On the contry, a dramatic drop of activity was observed for compound 9 because of the building of the oxazole ring. This behavior confirmed that such functionalization hindered the interaction with the host cell receptor for virus entry and decreased the activity. Transformation of 7a,b into 10a,b and 12a,b was accomplished with significant changes in activity. Increase of activity here can be attributed to the E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. Fig. 1. The 50 % effective concentration (EC50) and the concentration that exhibited 50 % cytotoxicity (LD50) of the tested compounds, comped to the anti-influenza drug zanamivir. Data presented at mean ± SE (n = 5). triazole and pyrimidine ring formation. In pticul, 10a and 12a showed remkable activity to the pent 7a whereas a dramatic drop of activity was noted in case of 11a comped to 7a which was probably due to the thiazolidine ring formation. In conclusion, it follows that pyrazole nucleus is essential for anti-avian influenza virus (H5N1) activity; increased number of nitrogen atoms and amide functions shply increases the activity; open chains containing amide C=O conjugated with pyrazole ring e more active than cyclized ones. Fig. 2. Antiviral activity of compounds 1, 5, 12a at different concentrations. CONCLUSIONS A series of novel pyrazole, pyridazinone, oxadiazole, triazole, thiazolidine and thioxopyrimidine derivatives were preped and assayed in a viety of biological tests for antiviral activity. Also, some of the preped products were tested for anti-avian influenza virus activity and revealed promising antiviral activity against H5N1 virus by determination of both EC50 and LD50 confirmed by the plaque reduction assay on MDCK cells. Compounds 3-[2-(4-omoenyl)hydrazono]-5-enylfuran-2(3H)-one (1), 1-(4-omoenyl)-N-hydroxy-5-enyl-1H-pyrazole-3-cboxamide (5) and 1-(4-omo- E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. enyl)-N-{2,3-dihydro-4-hydroxy-3-enyl-6-oxo-2-thioxopyrimidin-1(6H)-yl}-5-enyl-1H-pyrazole-3-cboxamide (12a) showed the highest anti-H5N1 activity of virus reduction vying from 75 % obtained by 10 mg per well to 84 % by 40 mg per well. Acknowledgement. ­ The authors extend their appreciation to the Deanship of Scientific Resech at King Saud University for funding their work through the resech group project No. RGP-VPP-172. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Pharmaceutica de Gruyter

Heterocyclic compounds based on 3-(4-bromophenyl) azo-5-phenyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity / Heterociklički derivati 3-(4-bromfenil) azo-5-fenil-2(3H)-furanona: Djelovanje na virus ptičje gripe (H5N1)

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Abstract

Acta m. 62 (2012) 593­606 DOI: 10.2478/v10007-012-0037-7 Short communication Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity EMAN M. FLEFEL1,* RANDA E. ABDEL-MAGEID1 WALED A. TANTAWY1 MOHAMED A. ALI2 ABD EL-GALIL E. AMR3,4 1 Deptment of otochemistry National Resech Centre, Cairo, Egypt Virology Laboratory, Environmental Resech Division, National Resech Center, Cairo, Egypt Drug Exploration & Development Chair (DEDC), College of macy, King Saud University, Riyadh 11451, Saudi abia Applied Organic Chemistry Deptment National Resech Center, Dokki 12622 Cairo, Egypt 3-[2-(4-omenyl)hydrazono]-5-enyl-furan-2(3H)-one (1) was used for prepation of some novel pyrazole, pyridazinone, oxadiazole, triazole, thiazolidine and thioxopyrimidine derivatives. Some of the preped products were tested for anti-avian influenza virus activity and revealed promising antiviral activity against H5N1 virus [A/Chicken/Egypt/1/20 % (H5N1)] by determination of both EC50 and LD50 and confirmed by plaque reduction assay on Madin-Dby canine kidney cells. Compounds 3-[2-(4-omoenyl)hydrazono]-5-enylfuran-2(3H)-one (1), 1-(4-omoenyl)-N-hydroxy-5-enyl-1H-pyrazole-3-cboxamide (5) and 1-(4-omoenyl)-N-{2,3-dihydro-4-hydroxy-3-enyl-6-oxo-2-thioxopyrimidin-1(6H)-yl}-5-enyl-1H-pyrazole-3-cboxamide (12a) showed the highest effects. Detailed synthesis, spectroscopic data, and antiviral activity of the synthesized compounds e reported. Keywords: furanone, pyrazole, pyridazinone, oxadiazole, triazole, anti-avian influenza virus (H5N1) Accepted September 10, 2012 2(3H)-Furanones e a type of five-membered heterocyclic compounds of synthetic and biological importance. The importance of this type is due to the facile opening of the lactone ring to give acyclic products, which undergo ring closure to give other synthetically and biologically important heterocyclic compounds (1, 2). The chemistry of pyrazole-containing compounds is pticully interesting for antiviral (1), anti-angiogenic activity (3), and as apoptosis-inducing agents (4), potent peroxisome proliferator-activated receptor gamma [PPg] ptial agonists (5) and antibiotics (6). Moreover, pyridazinones have promising biological activities as antiplatelet (7), antihypertensive agents (8). Biochemical studies revealed that oxadiazole caused activation on glutamic oxalo- * Correspondence; e-mail: emanflefel@yahoo.com E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. acetic transaminase (GOT) and glutamate pyruvate transaminase (GPT) enzymes, inhibition of gamma-glutamyl transpeptidase (g-GT) enzyme activity (9) and potent inhibition of tyrosine (10). On the other hand, substituted 1,3,4-oxadiazoles exhibit fungicidal (11), antimicrobial and antitubercul activity (12). Simple 1,2,4-triazoles also display some biological activities as antioxidant, urease inhibitors (13) and antiviral (14) agents. These diverse macological activities prompted us to convert furanone into other heterocycles being pyrazolyl moiety, and evaluate them for anti-viral activity. On the other hand, influenza viruses e respiratory pathogens that affect humans and e responsible for substantial morbidity, mortality and decreased productivity worldwide (15). Several synthetic compounds have already been utilized as potential inhibitors of the avian virus (H5N1) and some of them demonstrated the ability to inhibit viral replication at a level compable to the approved anti-influenza drugs zanamivir and oseltamivir (16). EXPERIMENTAL Melting points were measured using an Electrothermal 9100 digital melting point appatus (Büchi, Switzerland) and e uncorrected. IR spectra were recorded on a Perkin-Elmer 1600 FTIR (Perkin-Elmer, USA) in K discs. 1H NMR spectra were measured on a Jeol 270 MHz spectrometer (Jeol, Japan) and a uker Avance spectrometer (300 MHz) (uker, Germany) in DMSO-d6, and chemical shifts were recorded in d ppm relative to the internal standd TMS. Mass spectra were run at 70 eV with a Finnigan SSQ 7000 spectrometer (Thermo electron corporation, USA) using EI; m/z values e indicated in Dalton. Elemental analyses were performed on a Perkin-Elmer 2400 analyzer (Perkin-Elmer) and were found within ±0.4 % of the theoretical values (Table I). Follow-up of the reactions and checking of the purity of compounds were done by TLC on silica gel-precoted aluminum sheets (type 60 F254, Merck, Germany). All solvents and reagents were purchased from Aldrich (Germany). ysicochemical and spectral data for the synthesized compounds e given in Tables I and II, respectively. Syntheses 3-[2-(4-omoenyl)hydrazono]-5-enylfuran-2(3H)-one (1). ­ To a solution of omoaniline (6.88 g, 0.04 mol) in glacial acetic acid (15 mL), conc. HCl (8 mL) was added, then while the mixture was being cooled in ice under stirring, sodium nitrite (4.96 g, 0.068 mol) was added dropwise. To the diazonium mixture, freshly fused sodium acetate (6.0 g) was added. oyl propionic acid (6.0 g, 0.04 mol) was added to acetic anhydride (40 mL), the mixture was refluxed for 3 h, then allowed to cool at room temperature. This solution was added dropwise, with stirring and cooling in ice, to the diazo- nium mixture. The reaction mixture was allowed to stand overnight and the precipitate was collected, washed with water several times and recrystallized from acetic acid to give high yield (2). E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. 2-[2-(4-omoenyl)hydrazono]-4-oxo-4-enylbutane hydrazide (2). ­ A mixture of furanone 1 (3.42 g, 0.01 mol) and hydrazine hydrate (0.64 g, 0.02 mol, 98 %) in absolute ethanol (20 mL), was stirred at room temperature for 0.5 h. The solvent was evaporated under vacuum and crystals were obtained. 4-[2-(4-omoenyl)hydrazinyl]-6-enylpyridazin-3(2H) one (3). ­ A mixture of furanone 1 (3.42 g, 0.01 mol) and hydrazine hydrate 98 % (0.64 g, 0.02 mol) in absolute ethanol (25 mL) was refluxed for 2 h. The solvent was evaporated under vacuum and the solid residue was recrystallized from benzene. 4-[2-(4-omoenyl)hydrazono]-1,2-dihydro-1,6-dienylpyridazin-3(4H)-one (4). ­ A mixture of furanone 1 (3.42 g, 0.01 mol) and enylhydrazine (1.3 g, 0.012 mol) in absolute ethanol (25 mL) was refluxed for 3 h. The solvent was evaporated under vacuum and the solid residue was recrystallized from benzene/petroleum ether (60­80 °C, 1:1). 1-(4-omoenyl)-N-hydroxy-5-enyl-1H-pyrazole-3-cboxamide (5). ­ A mixture of furanone 1 (3.42 g, 0.01 mol), hydroxylamine hydrochloride (0.7 g, 0.01 mol) and sodium acetate (1 g) in ethanol (25 mL) was refluxed for 4 h. The solvent was evaporated under vacuum and the solid residue was recrystallized from benzene/petroleum ether (60­80 °C, 1:1). 5-[1-(4-omoenyl)-5-enyl-1H-pyrazole-3-yl]-1,3,4-oxadiazole-2(3H)-thione (6). ­ A mixture of hydrazide 2 (1.13 g, 0.003 mol), cbon disulfide (5 mL) and sodium hydroxide (4 %, 3 mL) in ethanol (30 mL) was stirred at room temperature for 6 h. The solvent was evaporated under vacuum, dissolved in hot water, and then the filtrate was neutralized with diluted hydrochloric acid. The yellow precipitate was collected after washing with water several times. The product obtained was recrystallized from benzene/petroleum ether (60­80 °C, 1:1). 2-{[1-(4-omoenyl)-5-yl-1H-pyrazol-3-yl]cbonyl}-N-enyl-hydrazinecbo-thioamides (7a,b). ­ A mixture of hydrazides 2 (3.75 g, 0.01 mol) and enyl or p-methoxyenyl isothiocyanate (0.01 mol) in ethanol (30 mL), was refluxed for 2 h. The mixture was cooled, and then filtered off. The solid so obtained was recrystallized from benzene/ethanol (1:2.5). 1-(4-omoenyl)-5-enyl-N'-(enylcbonyl)-1H-pyrazole-3-cbohydrazide (8). ­ To a suspension of hydrazide 2 (1.13 g, 0.003 mol) in dry benzene (15 mL), benzoyl chloride (0.42 g, 0.003 mol) was added at 25 °C and the mixture was heated under reflux at 75 °C for 3 h. The solvent was evaporated under vacuum, and the solid residue was recrystallized from benzene. 2-{1-(4-omoenyl)-5-enyl-1H-pyrazole-3-yl}-5-enyl-1,3,4-oxadiazole (9). ­ A mixture of dioylhydrazine 8 (0.92 g, 0.002 mol) and osorus oxychloride (10 mL) was refluxed for 1 h. The mixture was cooled, poured onto crushed ice and then neutralized with 4 % NaOH. The white precipitate was collected by filtration, washed with water and recrystallized from benzene. 4-(4-omoenyl)-5-(1-yl-5-enyl-1H-pyrazol-3-yl)-2H-1,2,4-triazole-3(4H)-thiones (10a,b). ­ A mixture of thiosemicbazides 7a,b (0.001 mol) and NaOH (4 %, 20 mL) was E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. refluxed for 2 h. The mixture was cooled, poured onto crushed ice and then neutralized with diluted hydrochloric acid. The white precipitate was collected by filtration and washed with water. The solid obtained was recrystallized from ethanol/water (5:1). 1-(4-omoenyl)-N-(4-oxo-3-ylthiazolidine-2-ylidene)-5-enyl-1H-pyrazole-3-cbohydrazides (11a,b). ­ A mixture of thiosemicbazide 7a or b (0.5 g, 0.001 mol), ethylchloroacetate (0.12 g, 0.001 mol) and NaOH pellets (0.4 g) in absolute ethanol (30 mL) was reTable I. ysical and analytical data of new compounds Analysis (%) (calcd./found) C 56.00 56.20 51.22 51.32 53.80 53.72 60.98 60.79 53.65 53.69 51.14 51.23 56.10 56.00 55.18 55.20 59.88 59.64 62.32 62.12 58.23 58.00 57.15 56.98 56.40 56.38 55.52 55.49 55.72 55.61 54.92 54.69 H 3.23 3.20 4.03 4.38 3.67 3.68 3.95 3.78 3.38 3.28 2.78 2.64 3.68 3.61 3.86 3.66 3.71 3.50 3.41 3.21 3.40 3.19 3.60 3.40 3.41 3.43 3.52 3.50 3.24 3.13 3.41 3.20 23.28 23.21 21.30 21.16 22.37 22.10 18.44 18.10 22.31 22.06 20.01 19.89 16.23 16.13 15.30 15.09 17.32 17.12 18.03 17.87 16.84 16.62 15.84 15.62 15.01 14.84 14.21 14.11 14.26 14.00 13.53 13.19 N 8.16 8.10 14.93 14.73 15.68 15.00 12.93 12.60 11.73 11.60 14.03 13.88 14.22 14.00 13.41 13.21 12.15 12.00 12.64 12.48 14.76 14.63 13.88 13.48 13.15 13.00 12.45 12.39 12.50 12.22 11.86 11.66 6.76 6.49 6.36 6.34 6.02 5.91 5.70 5.68 5.72 5.70 5.43 5.39 8.03 8.00 6.51 6.58 6.12 6.00 S Compd. Formula (Mr) C16H11N2O2 (342.80) C16H15N4O2 (375.22) C16H13N4O (357.22) C22H17N4O (433.30) C16H12N3O2 (358.19) C17H11N4OS (399.26) C23H18N5OS (492.39) C24H20N5O2S (522.42) C23H17N4O2 (461.31) C23H15N4O (443.30) C23H16N5S (474.38) C24H18N5OS (504.40) C25H18N5O2S (532.41) C26H20N5O3S (562.44) C26H18N5O3S (560.42) C27H20N5O4S (590.45) M.p. (°C) 295­296 89­90 160­161 84­85 103­104 179­180 199­200 147­148 120­121 167­168 189­190 225­226 191­192 139­140 186­187 124­125 Yield (%) 65 80 86 81 78 79 76 74 77 80 77 81 72 76 70 72 1 2 3 4 5 6 7a 7b 8 9 10a 10b 11a 11b 12a 12b E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. fluxed for 2 h. The solvent was evaporated under reduced pressure; the reaction mixture was poured onto water. The obtained precipitate was filtered off, washed with water, dried and recrystallized from ethanol. 1-(4-omoenyl)-N-{2,3-dihydro-4-hydroxy-3-yl-6-oxo-2-thioxopyrimidin-1(6H)-yl}-5-enyl-1H-pyrazole-3-cboxamide (12a,b). ­ A solution of 7a or b (0.001 mol) in sodium ethoxide (0.023 g sodium metal in 30 mL ethanol) was heated for 30 min at 80 °C. The reaction mixture was cooled, and then diethylmalonate (0.016 g, 0.001 mol) was poured into water, with stirring at 70 °C for 3 h. The precipitated solid was filtered off, washed with water, dried and recrystallized from methanol to give compounds 12a,b. Table II. Spectral data of the new compounds Compd. IR (nmax, cm­1) 1735 (C=O), 3335 (NH) 1652, 1690 (2 C=O), 3270­3335 (NH, NH2) 1H 1H and 13C NMR (d, ppm) MS (m/z, %) 342 (M+, 100) NMR: 6.32 (s, 1H, =CH), 3.42 (s, 1H, NH, D2O exchangeable), 7.80 (s, 1H, NH, D2O exchangeable), 7.30­7.45 (m, 9H, -H) 1H NMR: 2.60­2.85 (m, 2H, ­CH2), 3.42 (s, 2H, NH2, D2O exchangeable), 7.00 (s, 1H, NHCO­, D2O exchangeable), 7.30­7.45 (m, 9H, ­H), 8.65 (s, 1H, NH, D2O exchangeable) NMR: 5.60 (s, 1H, H pyridazinone), 6.55 (d, 2H, J = 8Hz, -H), 7.25 (d, 2H, J = 8Hz, -H), 7.40 (m, 3H, -H), 7.65 (m, 2H, -H), 7.90 (s, 1H, NH, pyridizinone, D2O exchangeable), 8.70 (s, 1H, NH, D2O exchangeable), 11.80 (s, 1H, NH-, D2O exchangeable) 374 (M+, 100) 1H 1648 (C=O), 3275­3315 (NH) 357 (M+, 100) 1655 (C=O), 3235 (NH) 1645 (C=O), 3130 (NH), 3340 (OH) 1250 (C=S), 3100 (NH) 1252 (C=S), 1646 (C=O), 3200­3290 (NH) 1252 (C=S), 1650 (C=O), 3196­3292 (NH) NMR: 5.9 (s, 1H, H pyridazinone), 6.35­7.4 (m, 14H, -H), 8.2 (., 1H, NHCO­, D2O exchangeable), 11.1 (s, 1H, NH exchangeable) 1H 1H 332 (M+, 100) 357 (M+, 100) 398 (M+, 100) NMR: 7.00 (s, 1H, pyrazole H), 7.20­7.48 (m, 9H, -H + -H), 9.20 (s, 1H, OH, D2O exchangeable), 11.10 (s, 1H, NH, D2O exchangeable) NMR: 7.08 (s, 1H, pyrazole H), 7.20­7.40 (m, 9H, -H + -H), 14.35 (, 1H, NH, D2O exchangeable) 1H 7a NMR: 7.14 (s, 1H, pyrazole H), 7.30­7.66 (m, 14H, 3 -H), 9.70 (, 2H, NH, D2O exchangeable), 10.30 (s, 1H, NH, D2O exchangeable) 13C NMR: 108.31, 120.56­138.70, 143.30 (-20C), 146.38 (C=N), 161.05 (C=O), 198.40 (C=S) NMR: 3.7 (s, 3H, OCH3), 7.11 (s, 1H, pyrazole H), 7.31­7.62 (m, 13H, 3 -H), 9.90 (, 2H, NH, D2O exchangeable), 10.60 (s, 1H, NH, D2O exchangeable) 1H 1H 491 (M+, 100) 7b 523 (M+, 100) E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. Table II. cont. 1H 1632 (C=N), 1675 (C=O), 3202 (NH) NMR: 7.10 (s, 1H, pyrazole H), 7.20­7.90 (m, 14H -H), 10.20 (s, 1H, NH, D2O exchangeable), 10.60 (s, 1H, NH exchangeable) 13C NMR: 108.86, 121.91, 128.05­132.70, 139.02, 144.87 (-20C), 146.41 (C=N), 161.05, 166.19 (2C=O) 1H 460 (M+, 100) 9 10a 1600 (C=N) 1251 (C=S), 3080 (NH) 1252 (C=S), 3085 (NH) NMR: 7.00 (s, 1H, pyrazole), 7.2­7.78 (m, 14H, -H) NMR: 6.70 (s, 1H, pyrazole H), 6.9­7.56 (m, 14H, 3-H), 14.16 (, 1H, NH, D2O exchangeable) 443 (M+,100) 475 (M+,100) 504 (M+,100) 1H 10b NMR: 3.40 (s, 3H, OCH3), 6.70 (s, 1H, pyrazole H), 6.90­7.66 (m, 13H, 3-H), 14.20 (, 1H, NH, D2O exchangeable) 1H 1H 11a 3085 (NH), 1680 (C=O) NMR: 4.86 (s, 2H, CH2), 6.70 (s, 1H, pyrazole H), 6.90­7.66 (m, 14H, 3-H), 10.60 (s, 1H, NH, D2O exchangeable); 13C NMR: d 48.1 (CH2), 106.3 (pyrazole C-4), 122.12­159.90 (18 -C, pyrazole C-3,5 and thiazole C-2,5), 170.36, 172.52 (2C=O) 13C NMR: 40.1 (CH ), 106.3, 116.12­142.90 (-20C), 2 152.10, 155.25 (2 C=N), 170.36, 172.52 (2 C=O) 1H 531 (M+, 100) 11b 3088 (NH), 1681 (C=O) NMR: 3.60 (s, 1H, OCH3), 4.82 (s, 2H, CH2), 6.70 (s, 1H, pyrazole H), 6.92­7.62 (m, 13H, 3-H), 10.2 (s, 1H, NH, D2O exchangeable) 13C NMR: 39.70 (CH ), 55.32 (CH ), 107.10, 114.42, 2 3 118.65, 121.37­132.29, 138.27, 144.81 (-20C), 153.08, 154.57 (2C=N), 160.10, 168.64 (2C=O) 1H 563 (M+, 100) 12a (C=S), (C=O), (C=O), (NH) (C=S), (C=O), (C=O), (NH) NMR: 6.70 (s, 1H, pyrazole H), 6.9­7.66 (m, 14H, 3-H), 14 (,1H, NH, D2O exchangeable) 13C NMR: 39.84 (CH ), 108.69, 121.46, 127.47­132.29, 2 138.50, 139.34, 144.26 (-20C), 145.96 (C=N), 166.65, 168.17, 168.49 (3C=O), 181.16 (C=S) 1H 559 (M+, 100) 12b NMR: 3.4 (s, 3H, OCH3),5.4 (s, 1H, ethylene), 6.7 (s, 1H, pyrazole H), 7.1­7.66 (m, 13H, 3-H), 9.2 (s, 1H, NH, D2O exchangeable), 15.2 (, 1H, OH, D2O exchangeable) 589 (M+, 100) Virus and cells. ­ Avian influenza-A virus (H5N1) [isolated from cloacal swabs from chicken, in Qalubiya governorate, Egypt, 2006 (A/chicken/Egypt/1/2006 (H5N1), accession no. FJ472343], was used to prepe low pathogenic rH5N1 by plasmid-based reverse genetics. The preped H5N1 vaccine strain was used in this study to evaluate antiviral activity of some synthetized compounds. Madin-Dby canine kidney (MDCK) cells kindly provided by Dr. Richd Webby (St. Jude Children's Resech Hospital, Memis, TN, USA) were used for virus propagation. The MDCK cells were routinely E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. passaged in Dulbecco's modified Eagle medium (DMEM) containing 10 % fetal bovine serum (FBS) and 1 % antibiotic-antimycotic mixture (penicillin-streptomycin-amotericin B). Compound prepation for biological assays. ­ Preped compounds were dissolved in 10 % dimethyl suloxide (DMSO) in doubly destilled water to a concentration of 10 mg mL­1 stock solutions used for further dilutions according to the assay applied. Cytotoxicity assay (MTT assay). ­ Cytotoxic activity of the extracts was tested in MDCK cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dienyltetrazolium omide (MTT) method (17) with minor modification. iefly, the cells were seeded in 96 well-plates (100 µL per well containing 3 × 105 cells mL­1) and treated with 5, 10, 20, 40, 80 and 120 µg of the sample per well. At 24 h, cells were washed with sterile osate buffer (PBS) three times and the supernatant was discded. MTT solution (20 µL of 5 mg mL­1) was added to each well and incubated at 37 °C for 4 h. Then the medium was aspirated. In each well, the formed formazan crystals were dissolved with 200 µL of acidified isopropanol (0.04 mol L­1 HCl in absolute isopropanol). Absorbance of formazan was detected with a dual wavelength UV spectrometer at 540 nm with 620 nm reference wavelength. The percentage of cytotoxicity comped to the untreated cells was determined. The 50 %-cytotoxicity (LD50) for each compound was calculated from standd curve of percentage of cytotoxicity vs. sample concentration. Antiviral activity and therapeutic index. ­ Antiviral activity of the compounds was determined using the cytopathogenicity (CPE) assay against low pathogenic reassortant avian influenza virus (rH5N1). Cells were seeded in 96-well cell culture plates (100 µL per well at a density of 3 × 105 cells mL­1) and grown to confluency. Cells were then infected with 100 µL of stock virus. After the virus adsorption period on cells of 2 hours at 37 °C, the virus was removed and serial dilutions (10, 20, 40 µg µL­1) of the tested compounds were added, then incubated with infected cells using maintenance DMEM with 2 % FBS (100 µl per well) at 37 °C for 3 days of monitoring until complete CPE was observed in the infected and untreated virus control. Plaque reduction assay. ­ In a six-well cell culture plate, confluent MDCK cells were infected with a pre-incubated mixture of 100 µL of avian influenza H5N1 virus (80­100 plaques per well) and 100 µL of DMEM [containing 2 % antibiotics and 1 mg mL­1 of L-1-tosyl-amido-2-enylethyl chloromethyl ketene and a different concentration of each compound (5, 10, 20, and 40 mg mL­1)]. The plates were incubated for 1 h at 37 °C in 5 % CO2 to allow virus adsorption. After adsorption, 2 mL of agose overlayer (2 % agose in DMEM 2 x containing 1 % FBS) was added to each well and mixed. The cultures were incubated at 37 °C in 5 % CO2 for 3­4 days of plaque formation monitoring. Plaques were fixed with 10 % formalin in osate-buffered saline for 2 h followed by removal of the ag overlayer and staining with 0.1 % crystal violet in distilled water. Plaques were counted manually from triplicate wells based on the plaque number but not plaque size. Viral counts and percentage of virus reduction were calculated according to Hayden et al. (18). E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. RESULTS AND DISCUSSION Chemistry 3-[2-(4-omoenyl)hydrazono]-5-enylfuran-2(3H)-one (1) was preped by coupling the diazotized aniline with 5-enyl-2(3H)-furanone according to the reported procedures (2). Furanone 1 was reacted with hydrazine hydrate in absolute ethanol at room temperature to give the corresponding acid hydrazide 2, which was refluxed in ethanol to give pyridazinone derivative 3. Also, compound 3 was preped by refluxing compound 1 with hydrazine hydrate in absolute ethanol. The reaction of furanone 1 with enylhydrazine was also investigated. When the reaction was cried out under reflux conditions, pyridazinone derivative 4 was formed. Moreover, furanone 1 was reacted with hydroxylamine hydrochloride in ethanol under reflux to give pyrazole derivative 5 (Scheme 1). H N H N O 2 O N NH2NH2 EtOH, r.t. NH2 EtOH, reflux H N N H N N O 1 O NHNH2 EtOH, reflux N N H 4 O O NH2OH EtOH, reflux N N 5 N H H 3 N N H O N N H NH2NH2 EtOH, reflux = Scheme 1 Treatment of 2 with cbon disulide in alcoholic sodium hydroxide gave oxadiazolthione derivative 6. Hydrazide 2 was reacted with enyl or p-methoxyenyl isothiocyanate to give the corresponding thiosemicbazide derivatives 7a,b, respectively. Also, hydrazide 2 was reacted with benzoyl chloride in dry benzene to give the corresponding pyrazole derivative 8, which was treated with osorus oxychloride to afford the corresponding 1,3,4-oxadiazole derivative 9 (Scheme 2). E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. S N O CS2, NaOH EtOH, r.t. N 6 N O N H N H N S ' H N H N ' N=C=S EtOH, r.t. N N 7a,b O COCl dry benzene N H O 2 O NH2 H N N O N N 8 ' O N POCl3 reflux N N OMe Scheme 2 Ring closure of thiosemicbazides 7a,b using sodium hydroxide solution, led to the formation of the corresponding triazolthiones 10a,b. Also, the reaction of thiosemicbazides 7a,b with ethyl chloroacetate in the presence of alcoholic potassium hydroxide afforded thiazolidine derivatives 11a,b, respectively. Moreover, when thiosemicbazides 7a,b were reacted with diethylmalonate in the presence of sodium ethoxide, the corresponding thioxopyrimidin derivatives 12a,b were formed (Scheme 3). Antiviral bioassays Cytotoxicity assay (MTT) was cried out on all preped compounds and according to the results obtained, only compounds 1, 5, 7a, 8, 9, 10a, 11a, and 12a showed notable toxic effects on the biology of cells used in assays. The LD50, EC50, and therapeutic index (TI) e presented in Fig. 1. Calculated LD50 value indicated that only 1, 5, 12a passed the antiviral activity bioassay. It was obvious that at concentrations of 10, 20 and 40 mg mL­1 compounds 1, 5 and 12a showed higher therapeutic indices than the other tested compounds comped to the anti-influenza drug zanamivir. Plaque reduction assay confirmed the antiviral activity of the three compounds as shown in Fig. 2. E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. S N NaOH reflux ' N N N N 10a,b O N H N S ' N N N N N 12a,b H O O ' O N N N 7a,b H H N S H N ClCH2COOEt, ' NaOH, EtOH, r.t. COOEt N N O O 11a,b S a: ' COOEt NaOH, reflux b: OMe ' O S N N N N H O N OH Scheme 3 Structure-activity relationship Structure modifications on the lead compounds 1 and 2 afforded derivatives with a viety of anti-avian influenza virus (H5N1) activity. For example, pyrazole-3-cboxyamide derivative 5 demonstrated remkable activity due to the chromoor in the skeleton of this compound and displayed a significant EC50 value in regd to the pent compound 1 (Fig. 1). However, product 2 does not appe to be advantageous in terms of anti-avian influenza virus with respect to the pent 1 because of its open chain structure and higher hydroilicity. Furthermore, chemical transformation/structure conversion of 2 into 7 and 8 influenced positively the activity profiles against influenza virus comped to their pent. On the contry, a dramatic drop of activity was observed for compound 9 because of the building of the oxazole ring. This behavior confirmed that such functionalization hindered the interaction with the host cell receptor for virus entry and decreased the activity. Transformation of 7a,b into 10a,b and 12a,b was accomplished with significant changes in activity. Increase of activity here can be attributed to the E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. Fig. 1. The 50 % effective concentration (EC50) and the concentration that exhibited 50 % cytotoxicity (LD50) of the tested compounds, comped to the anti-influenza drug zanamivir. Data presented at mean ± SE (n = 5). triazole and pyrimidine ring formation. In pticul, 10a and 12a showed remkable activity to the pent 7a whereas a dramatic drop of activity was noted in case of 11a comped to 7a which was probably due to the thiazolidine ring formation. In conclusion, it follows that pyrazole nucleus is essential for anti-avian influenza virus (H5N1) activity; increased number of nitrogen atoms and amide functions shply increases the activity; open chains containing amide C=O conjugated with pyrazole ring e more active than cyclized ones. Fig. 2. Antiviral activity of compounds 1, 5, 12a at different concentrations. CONCLUSIONS A series of novel pyrazole, pyridazinone, oxadiazole, triazole, thiazolidine and thioxopyrimidine derivatives were preped and assayed in a viety of biological tests for antiviral activity. Also, some of the preped products were tested for anti-avian influenza virus activity and revealed promising antiviral activity against H5N1 virus by determination of both EC50 and LD50 confirmed by the plaque reduction assay on MDCK cells. Compounds 3-[2-(4-omoenyl)hydrazono]-5-enylfuran-2(3H)-one (1), 1-(4-omoenyl)-N-hydroxy-5-enyl-1H-pyrazole-3-cboxamide (5) and 1-(4-omo- E. M. Flefel et al.: Heterocyclic compounds based on 3-(4-omoenyl) azo-5-enyl-2(3H)-furanone: Anti-avian influenza virus (H5N1) activity, Acta m. 62 (2012) 593­606. enyl)-N-{2,3-dihydro-4-hydroxy-3-enyl-6-oxo-2-thioxopyrimidin-1(6H)-yl}-5-enyl-1H-pyrazole-3-cboxamide (12a) showed the highest anti-H5N1 activity of virus reduction vying from 75 % obtained by 10 mg per well to 84 % by 40 mg per well. Acknowledgement. ­ The authors extend their appreciation to the Deanship of Scientific Resech at King Saud University for funding their work through the resech group project No. RGP-VPP-172.

Journal

Acta Pharmaceutica – de Gruyter

Published: Dec 1, 2012

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