untitled European Journal of Chemistry 2 (3) (2011) 365‐371 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2011 EURJCHEM DOI:10.5155/eurjchem.2.3.365‐371.289 European Journal of Chemistry Journal homepage: www.eurjchem.com Synthesis of novel 3,4‐dihydroquinoxalin‐2(1H)‐one derivatives Mohammed Shabaan, Azza Taher Taher* and Eman Omar Osman Department of Organic Chemistry, Faculty of Pharmacy, Cairo University, Cairo, 11562, Egypt *Corresponding author at: Department of Organic Chemistry, Faculty of Pharmacy, Cairo University, Cairo, 11562, Egypt. Tel.: +2.0101470792; fax: +2.023639307. E‐mail address: azzataher2005@yahoo.com (A.T. Taher). ARTICLE INFORMATION ABSTRACT Received: 27 October 2010 Received in revised form: 22 December 2010 Accepted: 24 December 2010 Online: 30 September 2011 KEYWORDS New derivatives of 3,4‐dihydroquinoxaline‐2(1H)‐one were synthesized and characterized. Representative examples were evaluated for their antimicrobial and antifungal activities using Tetracycline and Nystatin as reference compound. One of the tested compounds 10a was found to exhibit slight activity against Staphylococcus aureus. Compounds 10b, 11b and 14b showed slight activity against Escherichia coli. Moreover, nineteen compounds were screened for their inhibition effect on CDK5, CK1, and GSK‐3β. None of the tested compounds showed an inhibition activity below 10 µM concentration. Quinoxaline 3,4‐dihydroquinoxalin Antimicrobial activity CK1 inhibitors CDK5 inhibitors GSK‐3β inhibitors 1. Introduction Quinoxaline ring system represents the building block of many biologically active compounds that possess anti‐ inflammatory [1,2], antibacterial [3‐5], antifungal [6], anticancer [7,8], antimalarial [9], CNS depressant [10] and hypoglycemic [11] activities. Cyclin‐dependent kinase 5 (CDK5) is a protein kinase believed to play a critical role in the early development of the central nervous system [12,13]. It is shown to be involved in cellular processes like neuronal differentiation [14], cell adhesion [15], and axonal guidance [16]. Recently, a large body of evidence suggests that deregulation of CDK5 is implicated in the pathology of a number of neurodegenerative disorders. As a consequence, CDK5 inhibitors are of potential therapeutic uses for diseases such as Alzheimer's Disease [17], Parkinson's disease [18], amyotrophic lateral sclerosis [19] and ischemic stroke [20]. From high throughput screening efforts, we identified the structure activity relationship of potent CDK2 and CDK5 inhibitors based on five member ring attached to quinoline‐ 2(1H)‐one, or 3,4‐dihyro‐1H‐quinazoline‐2‐ones [21,22]. In our efforts to discover more potent inhibitors and an extension of these studies we introduced some modification, firstly the designed compounds included fusion of quinoxaline ring either with six member pyridinone ring compound 4 or five member furan ring compounds 9a‐c. Secondly, quinoxaline‐2(1H)‐ones were linked either to 3‐aryl‐six member ring compounds 5, 6a‐ c or 3‐aryl‐five member ring via methylene spacer compounds 10a, b (Scheme 1). In Scheme 2, quinoxaline‐2(1H)‐ones ring is linked directly either to phenylpyrazole compound 12, or aryloxazine compounds 14a, b. The derivatives prepared in this study were evaluated for their ability to inhibit purified human CDK and the IC50 values were reported in Table 1. Unfortunately, none of the tested compounds showed any inhibitory activity. On the other hand, some quinoxaline derivatives were found to have good antibacterial activity against Staphylococcus aureus and Bacillus subtilis which was more potent than tetracycline. Therefore, it was interesting to test the antibacterial and antifungal activities of new quinoxalines. 2. Experimental 2.1. Chemistry Melting points were determined on a Griffin apparatus and were uncorrected. IR spectra were determined as KBr discs on Shimadzu IR 435 spectrophotometer and values were represented in cm‐1. 1H NMR was carried out on Varian Gemini 200 MHz and 300 MHz spectrophotometer, Microanalytical center, Cairo University, Egypt, using TMS as an internal standard and chemical shifts were recorded in ppm on δ scale. Mass spectra were run on a Hewlett Packard 5988 spectrometer, Microanalytical center, Cairo University; Elemental analyses were carried out at the Microanalytical center, Cairo University. Progress of the reactions was monitored by thin‐layer chromatography (TLC) using TLC sheets pre‐coated with UV fluorescent silica gel Merck 60 F254 that were visualized using UV lamp. 2.2. Synthesis 2.2.1. Ethyl acetylpyruvate sodium salt 1,3‐Acetonylquino‐ xalin‐2(1H)‐one 2,3‐(2‐oxo‐4‐phenylbut‐3‐en‐1‐yl) quinoxalin‐2(1H)‐ones (3a) and 3‐(2‐oxo‐4‐(4‐chloro phenyl)‐but‐3‐en‐1‐yl)quinoxalin‐2(1H)‐ones (3b) Ethyl acetylpyruvate sodium salt 1,3‐acetonylquinoxalin‐ 2(1H)‐one 2,3‐(2‐oxo‐4‐phenylbut‐3‐en‐1‐yl)quinoxalin‐2(1H)‐ 366 Shabaan et al. / European Journal of Chemistry 2 (3) (2011) 365‐371 N H N O O N H N O O Ar N H N O O N ArCHO N H N O O Paraformaldehyde NaOH ArN H N HN O NH CN PhCHO / Acetic acid / Reflux H N N O O 8 Ethyl cyanoacetate Ammonium acetate ArCHO Malononitrile Ammonium acetate ArCHOAcetic acid or PhCHO / Fusion Br2 / Acetic acid / Sodium acetate or reflux in ethylene glycol N H N HN O O CN Ar N N O Ar N H N NHN Ar H2SO4 NH2NH2 Ethanol O NH2 NH2 O O O 1 Ethanol Ethanol Acetic anhydride + 2 3 4 5a,b 6a-c 7 10a,b 9a,b Ar = C6H5 p-ClC6H4 m-NO2C6H4 p-FC6H4 Acetic acid Scheme 1 ones (3a), 3‐(2‐oxo‐4‐(4‐chlorophenyl)‐but‐3‐en‐1‐yl) quino‐ xalin‐2(1H)‐ones (3b) were prepared as reported [23‐25] (Scheme 1). 2.2.2. 3‐(2‐oxo‐4‐(3‐nitrophenyl)‐but‐3‐en‐1‐yl)quinoxalin‐ 2(1H)‐ones (3c) and 3‐(2‐oxo‐4‐(4‐flourophenyl)‐but‐3‐en‐1‐ yl)quinoxalin‐2(1H)‐ones (3d) To a suspension of 3‐acetonylquinoxalin‐2(1H)‐one(2) (0.5 g, 0.0025 mol) in water (10 mL) and 10% sodium hydroxide (2 mL), the appropriate aldehyde (0.0025 mol) was sequentially added with continuous stirring. The formed solid product was filtered, dried and crystallized from acetone (Scheme 1). 3‐(2‐oxo‐4‐(3‐nitrophenyl)‐but‐3‐en‐1‐yl)quinoxalin‐2(1H)‐ ones (3c) : Yield: 65%. M.p.: 210‐212 οC. IR (KBr, cm‐1): 3450, 3400 (NH), 1685, 1680 (C=O), 1620(C=N). 1H NMR (DMSO‐d6, δ, ppm): 6.35 (s, 1H, vinylic CH), 7.10‐7.78 (m, 10H), 13.88‐ 14.00 (br, 2H, NH, OH, exchanged with D2O). MS (m/z (%)): 213 (100%), 335 (M+., 26.96%), 336(M+1┐+., 5.83%). anal. Calcd.of C18H13N3O4 (335): C, 64.48; H, 3.88; N, 12.54. Found: C, 64.71; H, 3.97; N, 12.68%. 3‐(2‐oxo‐4‐(4‐flourophenyl)‐but‐3‐en‐1‐yl)quinoxalin‐2(1H)‐ ones (3d) : Yield: 72%. M.p.: 213‐214 οC. IR (KBr, cm‐1): 3400, 3350 (NH), 1690, 1680 (C=O), 1630(C=N). 1H NMR (DMSO‐d6, δ, ppm): 6.33 (s, 1H, vinylic CH), 7.05‐7.83 (m, 10H), 12.01, 13.82 (2s, 2H, NH, OH, exchanged with D2O). Anal. Calcd. for C18H13FN2O2 (308): C, 70.12; H, 4.23; N, 9.09. Found: C, 70.33; H, 4.22; N, 8.79%. 2.2.3. 10‐Phenyl‐5H‐pyrido[1,2‐a]quinoxaline‐6,8‐dione (4) Method A: Benzaldehyde (0.53 g, 0.005 mol) was added to a solution of 2 (1.01 g, 0.005 mol) in acetic acid (25 mL). The mixture was heated under reflux for 5 h. The product formed was filtered and crystallized from ethanol (Scheme 1). Yield: 75%. M.p.: >300 °C. Method B: A mixture of 2 (1.01 g, 0.005 mol) and benzaldehyde (0.53 g, 0.005 mol) was heated at 180‐190 °C for 2 h. The solid was then washed with ethanol and crystallized from ethanol. Yield: 70%. M.p.: >300 °C [26]. Method C: To a stirred solution of 3a (1.45 g, 0.005 mol) and sodium acetate (0.82 g, 0.01 mol) in acetic acid (10 mL), bromine (0.8 g, 0.005 mol) in acetic acid (2.3 mL) was added dropwise. The reaction mixture was heated under reflux for 7 h. Water (20 mL) was then added and the solid product separated was filtered and crystallized from ethanol. Yield: 60%. M.p.: >300 °C. Shabaan et al. / European Journal of Chemistry 2 (3) (2011) 365‐371 367 Method D: A solution of 3a (1.45 g, 0.005 mol) in ethylene glycol (5 mL) was heated under reflux for 6 h. The mixture was cooled to room temperature and then poured onto an ice‐water mixture. The product was filtered and crystallized from ethanol. Yield: 70%. M.p.: >300 °C. IR (KBr, cm‐1): 3249 (NH), 1660, 1620 (C=O). 1H NMR (CHCl3/TFA, (5:1), δ, ppm): 6.94‐ 7.58 (m, 11H). MS (m/z (%)): 288 (M+., 100%), 289 (M+1┐+., 27.01%). Anal. Calcd. for C18H12N2O2 (288): C, 75.00; H, 4.17; N, 9.72. Found: C, 74.95; H, 3.80; N, 9.92%. 2.2.4. 3[(4‐Aryl‐5‐cyano‐6‐oxo‐1,6‐dihydropyridin‐2‐yl) methyl]quinoxalin‐2(1H)‐ones (5a,b) Method A: A mixture of 2 (1.01 g, 0.005 mol), the appropriate aldehyde (0.005 mol), ethyl cyanoacetate (0.56 g, 0.005 mol) and ammonium acetate (3.08 g, 0.04 mol) in absolute ethanol (10 mL) or n‐butanol (10 mL) was heated under reflux for 6 h. The solvent was evaporated under reduced pressure and the residue was triturated with ethanol, filtered, washed with ethanol and crystallized from acetone (Scheme 1). Method B: A mixture of the chalcone analogue 3a (1.45 g, 0.005 mol), ethyl cyanoacetate (0.56 g, 0.005 mol) and ammonium acetate (3.08 g, 0.04 mol) in n‐butanol (10 mL) was heated under reflux for 10 h. The solvent was evaporated under reduced pressure and the residue was triturated with ethanol, filtered, washed with ethanol and crystallized from acetone (Scheme 1). 3[(4‐Phenyl‐5‐cyano‐6‐oxo‐1,6‐dihydropyridin‐2‐yl)methyl] quinoxalin‐2(1H)‐ones (5a): Yield: 68% (Method A), 77% (Method B). M.p.: 276‐278 οC. IR (KBr, cm‐1): 3450, 3200 (NH), 1690, 1660 (C=O), 2200 (CN). 1H NMR (DMSO‐d6, δ, ppm): 3.42 (s, 2H, CH2), 7.18‐7.48 (m, 10H, aromatic protons), 12.14 (br, 2H, 2NH, exchanged with D2O). MS (m/z (%)): 251 (100%), 354 (M+., 0.28%). Anal. Calcd. for C21H14N4O2 (354): C, 71.18; H, 3.95; N, 15.81. Found: C, 70.80; H, 4.20; N, 15.72%. 3[(4‐(3‐nitrophenyl)‐5‐cyano‐6‐oxo‐1,6‐dihydropyridin‐2‐ yl)methyl]quinoxalin‐2(1H)‐ones (5b): Yield: 51% (Method A). M.p.: 292‐294 οC. IR (KBr, cm‐1): 3450, 3350 (NH), 1690, 1670 (C=O), 2200 (CN). 1H NMR (DMSO‐d6, δ, ppm): 3.31 (s, 2H, CH2), 7.21‐7.42 (m, 9H, aromatic protons), 12.11 (br, 2H, 2NH, exchanged with D2O). Anal. Calcd. for C21H13N5O4 (399): C, 63.16; H, 3.25; N, 17.54. Found: C, 63.42; H, 3.57; N, 17.25%. 2.2.5. 3[(4‐Aryl‐5‐cyano‐6‐amino‐1‐pyridin‐2‐yl)methyl] quinoxlin‐2(1H)‐ones (6a‐c) A mixture of 2 (1.01 g, 0.005 mol), the appropriate aldehyde (0.005 mol), malononitrile (0.33 g, 0.005 mol) and ammonium acetate (3.08 g, 0.04 mol) in absolute ethanol (10 mL) was heated under reflux for 6 h. The solvent was evaporated under reduced pressure and the residue was triturated with ethanol, filtered, washed with ethanol and crystallized from acetone (Scheme 1). 3[(4‐phenyl‐5‐cyano‐6‐amino‐1‐pyridin‐2‐yl)methyl]quin‐ oxlin‐2(1H)‐one (6a): Yield: 55%. M.p.: >300 οC. IR (KBr, cm‐1): 3350, 3200 (NH), 1685 (C=O), 2200 (CN). 1H NMR (DMSO‐d6, δ, ppm): 3.42 (s, 2H, CH2), 7.15‐8.15 (m, 10H, aromatic protons), 7.74, 7.80 and 11.80 (s, 3H, NH exchanged with D2O). MS (m/z (%)): 83 (100%), 353 (M+., 7.26%). Anal. Calcd. for C21H15N5O (353): C, 71.38; H, 4.24; N, 19.85. Found: C, 71.10; H, 4.28; N, 19.79%. 3[(4‐(4‐Chlorophenyl)‐5‐cyano‐6‐amino‐1‐pyridin‐2‐yl) methyl]quinoxlin‐2(1H)‐one (6b): Yield: 60%. M.p.: >300 οC. IR (KBr, cm‐1): 3450, 3400 (NH2), 1690 (C=O), 2200 (CN). 1H NMR (DMSO‐d6, δ, ppm): 3.47 (s, 2H, CH2), 7.06‐8.18 (m, 9H, aromatic protons), 11.00, 11.10 and 11.76 (s, 3H, NH exchanged with D2O). Anal. Calcd. for C21H14ClN5O (387.5): C, 65.03; H, 3.61; N, 18.06. Found: C, 65.33; H, 3.62; N, 17.99%. 3[(4‐(3‐nitrophenyl)‐5‐cyano‐6‐amino‐1‐pyridin‐2‐yl) methyl]quinoxlin‐2(1H)‐one (6c): Yield: 50%. M.p.: 238‐240 οC. IR (KBr, cm‐1): 3300, 3250 (NH2), 1690 (C=O), 2200 (CN). 1H NMR (DMSO‐d6, δ, ppm): 3.90 (s, 2H, CH2), 7.05‐8.63 (m, 9H, aromatic protons), 9.05, 9.15 and 10.95 (s, 3H, NH exchanged with D2O). Anal. Calcd. for C21H14N6O3 (398): C, 63.31; H, 3.51; N, 21.10. Found: C, 63.57; H, 3.84; N, 20.86%. 2.2.6. 3‐(1‐methylidene‐2‐oxopropyl)quinoxalin‐2(1H)‐one (7) A solution of 3‐acetonylquinoxalin‐2(1H)‐one 2 (1.01 g, 0.005 mol), paraformaldehyde (0.15 g, 0.005 mol) in acetic acid (1 mL) and ethanol (10 mL) was heated under reflux for 3 h, cooled and the separated solid was filtered and crystallized from acetic acid (Scheme 1). Yield: 85%. M.p.: 277‐278 οC. IR (KBr, cm‐1): 3400 (NH), 1660, 1620 (C=O), 1375 (CH3 bending). 1H NMR (CHCl3/TFA, (5:1), δ, ppm): 2.24 (s, 3H, CH3), 4.98, 5.28 (2s, 2H, CH2), 7.50‐7.97 (m, 4H, aromatic protons). MS (m/z (%)): 53 (100%), 214 (M+., 18%). Anal. Calcd. for C12H10N2O2 (214): C, 67.28; H, 4.67; N, 13.08. Found: C, 67.40; H, 4.90; N, 13.06%. 2.2.7. 3‐[2‐Oxo‐1‐(piperidin‐1‐ylmethyl)propyl]quinoxalin‐ 2(1H)‐one (8) A solution of 2 (1.01 g, 0.005 mol), paraformaldehyde (0.15 g, 0.005 mol) and piperidine (0.005 mol) in acetic acid (1 mL) and ethanol (10 mL) was heated under reflux for 3 h, cooled and the separated solid was filtered and crystallized from acetic acid (Scheme 1). Yield: 75%. M.p.: >300 οC. IR (KBr, cm‐1): 3400 (NH), 1680, 1650 (C=O), 1380 (CH3 bending). 1H NMR (CHCl3/TFA, (5:1), δ, ppm): 1.59‐1.94 (br., 6H, piperidine), 2.38 (s, 3H, CH3), 3.03 (br., 4H, piperidine), 3.37 (s, 1H, CH‐CH2), 3.90 (d, 2H, CH2N), 7.47‐8.06 (m, 4H, aromatic protons). MS (m/z (%)): 52 (100%), 300 (M+1┐+., 28.5%). Anal. Calcd. for C17H21N3O2 (299): C, 68.22; H, 7.02; N, 14.04. Found: C, 68.58; H, 7.34; N, 14.18%. 2.2.8. 2‐(2‐Arylethenyl)furo[2,3‐b]quinoxalines (9a,b) A mixture of 3a,b (0.003 mol), acetic anhydride (10 mL) and sulphuric acid (few drops) was heated under reflux for 5 h. The reaction mixture was cooled and poured on ice (10 mL) and stirred. The formed product was filtered, dried and crystallized from ethanol (Scheme 1). 2‐(2‐phenylethenyl)furo[2,3‐b]quinoxalines (9a): Yield: 50%. M.p.: >300 οC. IR (KBr, cm‐1): 1660 (C=N). MS (m/z (%)): 69 (100%), 272 (M+., 26.69%), 273 (M+1┐+., 5.13%). Anal. Calcd. for C18H12N2O (272): C, 79.41; H, 4.41; N, 10.29. Found: C, 79.34; H, 4.45; N, 10.65%. 2‐[2‐(4‐ Chlorophenyl)ethenyl]furo[2,3‐b]quinoxalines (9b): Yield: 55%. M.p.: >300 οC. IR (KBr, cm‐1): 1650(C=N). 1H NMR (DMSO‐d6, δ, ppm): 7.33‐7.85 (m, 11H, aromatic protons). MS (m/z (%)): 306 (M+., 100%), 307 (M+1┐+., 34.08%), 308(M+2┐+., 32.11%). Anal. Calcd. for C18H11ClN2O (306.5): C, 70.47; H, 3.59; N, 9.14. Found: C, 70.72; H, 3.33; N, 9.46. 2.2.9. 3‐[(5‐Aryl‐1H‐pyrazol‐3‐yl)methyl]quinoxalin‐2(1H)‐ ones (10a,b) A solution of 3c,d (0.003 mol) and 99% hydrazine hydrate (0.75 g, 0.015 mol) in absolute ethanol (15 mL) containing few drops of glacial acetic acid was heated under reflux for 5 h. The reaction mixture was cooled, and the formed solid was filtered, dried and crystallized from acetone (Scheme 1). 3‐{[5‐(3‐nitrophenyl)‐1H‐pyrazol‐3‐yl]methyl}quinoxalin‐ 2(1H)‐ones (10a): Yield: 50%. M.p.: 232‐234 οC. IR (KBr, cm‐1): 3300‐3250 (NH), 1685 (C=O). 368 Shabaan et al. / European Journal of Chemistry 2 (3) (2011) 365‐371 Scheme 2 1H NMR (DMSO‐d6, δ, ppm): 4.27 (s, 2H, CH2), 6.77‐8.60 (m, 9H, aromatic protons), 12.51, 13.06 (2s, 2H, 2NH, exchanged with D2O). MS (m/z (%)): 347 (M+., 100%), 348 (M+1┐+., 23.17%). Anal. Calcd. for C18H13N5O3 (347): C, 62.25; H, 3.74; N, 20.17. Found: C, 62.20; H, 3.85; N, 19.91%. 3‐{[5‐(4‐fluorophenyl)‐1H‐pyrazol‐3‐yl]methyl}quinoxalin‐ 2(1H)‐ones (10b): Yield: 61%. M.p.: 228‐230 οC. IR (KBr, cm‐1): 3400 (NH), 1670 (C=O). 1H NMR (DMSO‐d6, δ, ppm): 4.17 (s, 2H, CH2), 6.51‐7.76 (m, 9H, aromatic protons), 12.45, 12.77 (2s, 2H, 2NH, exchanged with D2O). Anal. Calcd. for C18H13FN4O (320): C, 67.50; H, 4.06; N, 17.50. Found: C, 67.50; H, 4.11; N, 17.37%. 2.2.10. 3‐[1‐(2‐Arylhydrazinylidene)‐2‐oxopropyl] quinoxalin‐2(1H)‐ones (11a,b) A mixture of 2 (2.02 g, 0.01 mol), sodium hydroxide (1.6 g, 0.04 mol) and water (25 mL) was stirred for 10 min until a clear solution was formed, then chilled at ‐5 °C. To this solution, an ice cooled solution of aryldiazonium salt [prepared from the appropriate aromatic amine (0.01 mol), concentrated hydrochloric acid (3 mL) and sodium nitrite (0.69 g, 0.01 mol) in water (15 mL)] was added. The reaction mixture was maintained at ‐5 °C for 30 min then acidified with glacial acetic acid till pH 5‐5.5. The formed solid was filtered, washed with water and crystallized from ethyl acetate (Scheme 2). 3‐[1‐(2‐phenylhydrazinylidene)‐2‐oxopropyl]quinoxalin‐ 2(1H)‐ones (11a): Yield: 60%. M.p.: 140‐142 οC. IR (KBr, cm‐1): 3500‐3450 (NH), 1690, 1675 (C=O), 1370 (CH3 bending). 1H NMR (DMSO‐d6, δ, ppm): 2.63 (s, 3H, CH3), 7.02‐7.78 (m, 9H, aromatic protons), 11.69, 13.38 (2s, 2H, 2NH exchanged with D2O). MS (m/z (%)): 77 (100%), 306 (M+., 41.77%), 307 (M+1┐+., 9.06%). Anal. Calcd. for C17H14N4O2 (306): C, 66.67; H, 4.58; N, 18.30. Found: C, 66.36; H, 4.30; N, 18.10%. 3‐[1‐(2‐(4‐Chlorophenyl)hydrazinylidene)‐2‐oxopropyl] quinoxalin‐2(1H)‐ones (11b): Yield: 80%. M.p.: 224‐226 οC. IR (KBr, cm‐1): 3450‐3400 (NH), 1680, 1660 (C=O), 1360 (CH3 bending). 1H NMR (CDCl3/TFA, (5:1), δ, ppm): 2.89 (s, 3H, CH3), 7.55‐7.95 (m, 8H, aromatic protons). MS (m/z (%)): 340 (M+., 100%), 341(M+1┐+., 24.1%), 342 (M+2┐+., 38.4%). Anal. Calcd. for C17H13ClN4O2 (340.5): C, 59.90; H, 3.82; N, 16.45. Found: C, 60.10; H, 4.05; N, 16.34%. 2.2.11. 3‐(4‐Hydroxy‐1‐phenyl‐1H‐pyrazol‐3‐yl)quinoxalin‐ 2(1H)‐one (12) To a stirred solution of 11a (1.53 g, 0.005 mol) and sodium acetate (0.82 g, 0.01 mol) in acetic acid (10 mL), bromine (0.8 g, 0.005 mol) in acetic acid (2.3 mL) was added dropwise. The reaction mixture was stirred overnight. Water (20 mL) was then added and the solid product was filtered and crystallized from methanol (Scheme 2). Yield: 55%. M.p.: 244‐246 °C. IR (KBr, cm‐1): 3500‐3350 (NH, OH stretching), 1660 (C=O stretching). 1H NMR (DMSO‐d6, δ, ppm): 7.33‐8.27 (m, 10H, aromatic protons), 11.04, 13.27 (2s, 2H, NH, OH, exchanged with D2O). MS (m/z (%)): 143 (100%), 304 (M+., 15.25%), 305 (M+1┐+., 9.83%). Anal. Calcd. for C17H12N4O2 (304): C, 67.10; H, 3.94; N, 18.42. Found: C, 66.76; H, 4.00; N, 18.17%. 2.2.12. 3‐(2‐Oxo‐1‐oximinopropyl)quinoxalin‐2(1H)‐one (13) A solution of 2 (1.01 g, 0.005 mol) in glacial acetic acid (25 mL) was stirred at room temperature for 5 min; then cooled to ‐5 °C. To this solution solid sodium nitrite (0.48 g, 0.007 mol) was added portionwise over a period of 30 min. After stirring for an additional 30 min at room temperature, the reaction mixture was diluted with water, and then filtered. The obtained solid was washed with water and crystallized from ethanol (Scheme 3). Yield: 90%. M.p.: 236‐238 °C. IR (KBr, cm‐1): 3200‐ 3150 (NH, OH stretching), 1690, 1660 (C=O stretching), 1370 (CH3 bending). 1H NMR (DMSO‐d6, δ, ppm): 2.49 (s, CH3, 3H), 7.31‐7.80 (m, 4H, aromatic protons), 12.61, 12.87 (2s, 2H, NH, OH exchanged with D2O). MS (m/z (%)): 172 (100%), 231 (M+., 29.28%), 232(M+1┐+., 4.22%). Anal. Cald. for C11H9N3O3 (231): C, 57.14; H, 3.89; N 18.18. Found: C, 57.40; H, 3.90; N, 18.06%. Shabaan et al. / European Journal of Chemistry 2 (3) (2011) 365‐371 369 N H N O O N H N O O N OH N H N OH O HN O Ar Sodium nitrite Acetic acid ArCHO NaOH 14a,b 2 13 Ar = C6H5, p-BrC6H4 Scheme 3 2.2.13. 3‐(6‐Aryl‐4‐hydroxy‐2H‐1,2‐oxazin‐3‐yl)quinoxalin‐ 2(1H)‐ones (14a,b) To a suspension of 13 (0.46 g, 0.002 mol) in water (10 mL), 10% aqueous sodium hydroxide (2 mL) solution and the appropriate aldehyde (0.002 mol) were sequentially added. The reaction mixture was heated under reflux for 5 h. The formed solid was filtered, washed with ethanol and crystallized from methanol (Scheme 3). 3‐(6‐phenyl‐4‐hydroxy‐2H‐1,2‐oxazin‐3‐yl)quinoxalin‐2(1H)‐ ones (14a): Yield: 60%. M.p.: 285‐287 °C. IR (KBr, cm‐1): 3600‐ 3400 (NH, OH stretching), 1660 (C=O stretching) . 1H NMR (DMSO‐d6, δ, ppm): 6.37 (s, 1H, CH of oxazine), 7.35‐7.78 (m, 9H, aromatic protons), 7.91, 10.66, 12.03 (3s, 3H, NH, OH, exchanged with D2O). MS (m/z (%)): 303 (100%), 319 (M+., 0.4%). Anal. Calcd. for C18H13N3O3 (319): C, 67.71; H, 4.07; N, 13.16. Found: C, 67.58; H, 3.91; N, 13.33%. 3‐(6‐(4‐bromophenyl)‐4‐hydroxy‐2H‐1,2‐oxazin‐3‐yl) quinoxalin‐2(1H)‐ones (14b): Yield: 43%. M.p.: 179‐181 °C. IR (KBr, cm‐1): 3400‐3300 (NH, OH stretching), 1670 (C=O stretching). 1H NMR (DMSO‐d6, δ, ppm): 6.00 (s, 1H, CH of oxazine), 6.88‐7.69 (m, 8H, aromatic protons). Anal. Calcd. for C18H12BrN3O3 (398): C, 54.27; H, 3.01; N, 10.55. Found: C, 54.29; H, 2.92; N, 10.90%. 2.3. Microbiology Test 2.3.1. Test organisms and culture media Twelve compounds were tested for their antimicrobial activity using: Staphylococcus aureus and Bacillus subtilis (Gram positive bacteria), Escherichia coli and Pseudomonas aeruginosa (Gram negative bacteria) and Candida albicans (fungus). Culture media: Nutrient broth, Sabouraud’s broth and agar. 2.3.2. Method Agar plate diffusion technique: Agar plates containing 15 mL of agar medium [nutrient agar for bacteria and Sabouraud’s broth for fungi] were seeded with 0.2 mL of broth culture of each organism and cultered for 18 h. Sterile filter paper discs (6 mm in diameter) were impregnated each with 10 µL of a 1% solution of the test compound in DMF and allowed to air dry. The discs were then placed onto the surface of agar plates and incubated at 37 oC for 24 h. Control discs impregnated with DMF were used alone to determine the solvent activity. The antibacterial reference tetracycline and the antifungal reference nystatin discs were tested concurrently as standards. The diameter of the inhibition zone around each disc was measured (mm). 2.4. Biological tests Kinase preparations and assays were performed according to the reported procedures [27‐29]. Kinase activities were assayed in Buffer A (10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 25 mM Tris‐HCl (pH = 7.5), 50 µg heparin/mL.) or C (60 mM ß‐ glycerophosphate, 15 mM p‐nitrophenylphosphate, 25 mM Mops (pH = 7.2), 5 mM EGTA, 15 mM MgCl2, 1 mM DTT, 1 mM sodium vanadate, 1 mM phenylphosphate) at 30 °C, at a final ATP concentration of 15 µM. Blank values were subtracted and activities expressed in % of the maximal activity, i.e. in the absence of inhibitors. Controls were performed with appropriate dilutions of DMSO. CDK1/cyclin B (M phase starfish oocytes, native), CDK2/cyclin A, CDK2/cyclin E, CDK5/p25 and CDK7/cyclin H (human, recombinant) were prepared as previously described. Their kinase activity was assayed in buffer C, with 1 mg histone H1/mL, in the presence of 15 µM [‐33P] ATP (3,000 Ci/mmol; 10 mCi/mL) in a final volume of 30 µL. After 30 min incubation at 30 °C, 25 µL aliquots of supernatant were spotted onto 2.5 x 3 cm pieces of Whatman P81 phosphocellulose paper, and, 20 sec later, the filters were washed five times (for at least 5 min each time) in a solution of 10 mL phosphoric acid/liter of water. The wet filters were counted in the presence of 1 mL ACS (Amersham) scintillation fluid (Table 1). GSK‐3 (porcine brain, native) was assayed, as described for CDK1 but in Buffer A and using a GSK‐3 specific substrate (GS‐1: YRRAAVPPSPSLSRHSSPHQSpEDEEE) (pS stands for phosphorylated serine). GS‐1 was synthesized by Millegen (Labege, France). CK1 (porcine brain, native) was assayed as described for CDK1 but using the CK1‐specific peptide substrate RRKHAAIGpSAYSITA, obtained from Millegen (Labege, France). 3. Results and discussion 3.1. Chemistry The target compounds were prepared according to Schemes 1‐3. Compound 2 can exist in two tautomeric forms: the imine and the enamine which is the more predominant tautomer [30]. In this study, our target was to synthesize novel quinoxalines through the application of aliphatic electrophilic substitution reactions on the methyl and methylene of the side 370 Shabaan et al. / European Journal of Chemistry 2 (3) (2011) 365‐371 chain of 3‐acetonylquinoxalin‐2(1H)‐one 2, followed by cyclization reactions. Table 1. Inhibition effect (µM) on CK1, CDK5 and GSK3β of selected compounds. Compound CK1 CDK5 GSK‐3β Compound 3a > 10 > 10 > 10 Compound 3b > 10 > 10 > 10 Compound 3c > 10 > 10 > 10 Compound 5a > 10 > 10 > 10 Compound 5b > 10 > 10 > 10 Compound 6a > 10 > 10 > 10 Compound 6b > 10 > 10 > 10 Compound 6c > 10 > 10 > 10 Compound 7 > 10 > 10 > 10 Compound 8 > 10 > 10 > 10 Compound 9a > 10 > 10 > 10 Compound 9b > 10 > 10 > 10 Compound 10a > 10 > 10 > 10 Compound 10b > 10 > 10 > 10 Compound 11a > 10 > 10 > 10 Compound 11b > 10 > 10 > 10 Compound 12 > 10 > 10 > 10 Compound 13 > 10 > 10 > 10 Compound 14a > 10 > 10 > 10 In Scheme 1, the formation of chalcone product was catalyzed by a basic or acidic catalyst [31]. In alcoholic alkali medium the electrophilic substitution reaction occurred on the terminal methyl group. 1H NMR spectrum of compound 3 revealed the disappearance of the singlet signal at δ 2.17 ppm corresponding to the terminal methyl protons and the presence of a singlet signal at δ 6.38 ppm corresponding to the vinylic protons. On the other hand, reaction in acetic acid afforded compound 4 and its 1H NMR spectrum showed a multiplet signal at δ 6.94‐7.58 ppm corresponding to the aromatic protons and the disappearance of both aliphatic protons at δ 2.17 ppm and the vinylic proton at δ 6.01 ppm. Mass spectrum of the product showed a molecular ion peak at m/z 288. Compound 4 was prepared by two different ways, firstly by refluxing of compound 2 with benzaldehyde in glacial acetic acid or fusing the starting compound 2 with benzaldehyde at 180‐190 oC. Furthermore, refluxing of compound 3a in bromine/acetic acid afforded compound 4. Secondly, Hantzsch reaction was applied on 2 using ethyl cyanoacetate in ethanol or n‐butanol resulted in the same product 5a in yields of 68% and 77%, respectively. The reaction mechanism as guided by Katritzky [32] may proceed via the formation of the enaminone intermediate. The second step involved Micheal addition of this intermediate to the α,β‐ unsaturated ester, initially formed from the reaction of the appropriate aldehyde with ethyl cyanoacetate, followed by cyclization and aromatization to yield the target compounds 5a,b. On the other hand, the reaction of compound 2 with aromatic aldehydes, malononitrile and ammonium acetate in ethanol gave the products 6a‐c. Moreover, heating compound 2 with paraformaldehyde in ethanol containing acetic acid yielded product 7 [33]. 1H NMR spectrum of compound 7 showed a singlet signal at δ 2.24 ppm corresponding to the terminal methyl protons and the absence of the vinylic proton. Aminomethylation of compound 2 was carried out using paraformaldehyde and piperidine in ethanol containing few drops of acetic acid gave product 8. The structure assignment depends on the position at which the electrophilic substitution reaction took place. IR spectra showed bands at 1375 and 1380 cm‐1, respectively, corresponding to the bending vibration of the terminal methyl group [34] in addition to bands at 3450 and 3400 cm‐1, respectively, due to NH group and bands at 1690‐1650 cm‐1 due to C=O groups. 1H NMR spectrum showed a singlet signal at δ 2.38 ppm corresponding to the terminal methyl group protons and the absence of the vinylic proton. Dehydration of compounds 3a,b in acetic anhydride/sulphuric acid mixture gave the required compounds 9a,b . IR showed the absence of bands corresponding to NH and C=O groups. 1H NMR spectrum revealed the absence of the singlet signal of the vinylic proton and the absence of the exchangeable singlet signals corresponding to NH protons. Refluxing compounds 3a,c with hydrazine hydrate in ethanol containing few drops of glacial acetic acid afforded compounds 10a,b [35]. Mass spectrum of the compound 10a showed molecular ion peak at m/z 302. 1H NMR spectrum of compound 10b showed a singlet signal at δ 4.27 ppm corresponding to methylene protons, a multiplet signal at δ 6.77‐8.60 ppm corresponding to the aromatic protons and two exchangeable singlet signals at δ 12.51 and 13.06 ppm corresponding to 2 NH protons. Reaction of compound 3a,c with hydrazine hydrate afforded 3‐[(5‐aryl‐ 1H‐pyrazol‐3‐yl)methyl]‐quinoxalin‐2(1H)‐ones (10a,b). The proposed mechanism for this reaction may involve either the intermediate formation of hydrazones or the initial Micheal addition of the hydrazine on the chalcone. Coupling the alkaline solution of 3‐acetonylquinoxalin‐ 2(1H)‐one (2) with freshly prepared diazonium salts at pH= 5.0‐5.5 yielded products 11a,b. IR spectrum showed a band at 1380‐1370 cm‐1 due to the bending vibration of the terminal methyl group. 1H NMR spectra of compounds 11a showed a singlet signal at δ 2.63 ppm corresponding to the terminal methyl group and the absence of the singlet signal corresponding to the vinylic proton. Reaction of 11a with bromine in acetic acid containing sodium acetate yielded 12. Mass spectrum showed a molecular ion peak at m/z 304 and 1H NMR spectrum which revealed a multiplet signal at δ 7.33‐8.27 ppm corresponding to the aromatic protons, in addition to two exchangeable singlet signals at δ 11.04 and 13.27 ppm corresponding to NH and OH protons, respectively [36]. Treating a solution of compound 2 in acetic acid with solid sodium nitrite, following the directions of Shawali and Fahmi [37] yielded 13 whose structure depends on the position at which the electrophilic substitution took place. 1H NMR spectrum showed a singlet signal at δ 2.49 ppm corresponding to the methyl protons and the absence of the singlet signal at δ 6.01 ppm due to the vinylic proton. It was concluded that the weak non‐bulky electrophile attacked the more activated position 1 at the side chain. Furthermore, reaction of oxime 13 with benzaldehyde in aqueous sodium hydroxide gave product 14, which showed the expected molecular weight at m/z 319. 1H NMR spectrum showed a singlet signal at δ 6.37 ppm. This suggested an internal Micheal addition of the OH group of the chalcone analogue on the C=C to afford an oxazine ring. One of the tested compounds 10a was found to exhibit slight activity against staphylococcus aureus. 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