untitled European Journal of Chemistry 2 (1) (2011) 65‐69 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2011 EURJCHEM DOI:10.5155/eurjchem.2.1.65‐69.303 European Journal of Chemistry Journal homepage: www.eurjchem.com Synthesis and antifungal activity of some new pyrido[2,3‐d]pyrimidines Fatin Ismail Hanafy Department of Chemistry, Faculty of Education, Ain Shams University, Roxy, Cairo, EG‐11711, Egypt Corresponding author at: Department of Chemistry, Faculty of Education, Ain Shams University, Roxy, Cairo, EG‐11711, Egypt. Tel.: +2025088247; fax: +2025088247. E‐mail address: hsseleem@yahoo.com (F.I. Hanafy). ARTICLE INFORMATION ABSTRACT Received: 03 March 2010 Received in revised form: 21 July 2010 Accepted: 25 July 2010 Online: 31 March 2011 KEYWORDS Some new pyrido[2,3‐d]pyrimidine derivatives (3a‐c) were synthesized from 2‐amino‐5‐ cyano‐6‐methoxy‐4‐(4‐methoxyphenyl)pyridine‐3‐carboxamide. 7‐methoxy‐5‐(4‐methoxy phenyl)‐4‐oxo‐2‐phenyl‐3,4‐dihydropyrido[2,3‐d]pyrimidine‐6‐carbonitrile (3b) and 7‐met‐ hoxy‐5‐(4‐methoxyphenyl)‐4‐oxo‐3,4‐dihydropyrido[2,3‐d]pyrimidine‐6‐carbonitrile (3c) are used in synthesizing 7a,b, then 8a,b. 7‐methoxy‐5‐(4‐methoxyphenyl)‐2‐methyl‐4‐oxo‐3,4‐ dihydropyrido[2,3‐d]pyrimidine‐6‐carbonitrile (3a) and 4‐hydrazinyl‐7‐methoxy‐5‐(4‐ methoxyphenyl)pyrido[2,3‐d]pyrimidine‐6‐carbonitrile (8b) were condensed with different carbonyl compounds to produce compounds 4, 5, 6 and 9, 10, 11, 12. 3‐Methoxy‐1‐(4‐ methoxyphenyl)‐6‐phenyl‐7‐hydropyridino[2,3‐d]1,2,3,4‐tetrazolo[1,5‐e]pyrimidine‐2‐carbo‐ nitrile (13) was synthesized from 8a or 7a. Condensation of 8b with acetophenone to yield 14, which on further reaction gave 15 then 16. 4‐Hydrazinyl‐7‐methoxy‐5‐(4‐ methoxyphenyl)‐2‐phenylpyrido[2,3‐d]pyrimidine‐6‐carbonitrile (8a) also condensed with 4‐ amino antipyrine giving 17 then 18. Structures of these compounds have been deduced upon the basis of elemental analysis and spectral data. Significant antifungal activities were observed for some of the synthesized compounds. Pyrido[2,3‐d]pyrimidine Fusion Condensation Active methyl group Oxidative cyclization Antifungal activity 1. Introduction Pyrido[2,3‐d]pyrimidine and few of its derivatives display potentially useful biological activities [1,2]. They show dihydrofolate reductase inhibition and antitumor [3‐5] as well as diuretic properties [6]. Moreover, some of these compounds possess antimicrobial [7,8], antibacterial [9,10], and cytotoxic activities [11,12]. Therefore, a few new pyrido[2,3‐ d]pyrimidine derivatives condensed with 1,2,4‐triazole, pyrazole, tetrazole and 1,2,4‐triazine rings were synthesized. These new prepared compounds were tested for their antifungal activities. 2. Experimental 2.1. Instrumentation Melting points were determined on a digital Stuart SMP3 and are uncorrected. Infrared spectra were measured on Perkin‐Elmer 293 spectrophotometer (γ in cm‐1), using KBr disks. 1H NMR spectra were measured on Gemini‐200 spectrometer (200 MHz), using DMSO‐d6 as a solvent and TMS (δ, 0.0 ppm) as internal standard. The mass spectra were recorded on gas chromatographic GCMSqp 1000‐ex Shimadzu instrument or HP‐MS 5988 mass spectrometer by direct inlet operating at 70 eV. Elemental microanalyses were performed on Perkin Elmer CHN‐2400 analyzer or C, H, N manual in micro‐analysis center at Cairo University. 2.2. 2‐Amino‐5‐cyano‐6‐methoxy‐4‐(4‐methoxyphenyl) pyridine‐3‐carboxamide (2) To 100 mL of an alcoholic solution of KOH (5%), 0.01 mole of compound 1 was added and the reaction mixture was refluxed for 30 min. After cooling, the reaction mixture was diluted with water and the formed solid was filtered off, washed with water and recrystallized from DMF to give 2 as yellow crystals (Scheme 1). Yield: 82%. M.p.: 250 oC. IR (KBr, max, cm‐1): 3380 (amide NH2), 1720 (C=O amide), 1604 (C=C aromatic), 2230 (CN). 1H NMR (DMSO, δ ppm): 2.80‐3.02 (6H, s, ‐OCH3); 6.88 (4H, d, Ar‐H); 10.03 (4H, br, NH2). MS (m/z, %): 298 (M+, 72.22), 191 (88.89), 147 (100), 121 (61.11), 107(61.11), 76 (55.56), 44 (83.33), 32 (55.56), 26 (61.11). Anal. Calcd. for C15H14N4O3 (298): C, 60.40; H, 4.98; N, 18.79. Found: C, 60.32; H, 4.90; N, 18.53.   Scheme 1 66 Hanafy / European Journal of Chemistry 2 (1) (2011) 65‐69 2.3. 7‐Methoxy‐5‐(4‐methoxyphenyl)‐2‐substituted‐4‐oxo‐3‐ hydrpyridino[2,3‐d] pyrimidine‐6‐carbonitrile (3a‐c) A mixture of 2 (0.01 mole) and acetic anhydride, benzoyl chloride or triethyl orthoformate (0.01 mole) was fused at 150 oC for 2 hours. After cooling, the formed solid was washed with ethanol and recrystallized from the proper solvent; 3a from ethanol and 3b,c from DMF (Scheme 1). 7‐methoxy‐5‐(4‐methoxyphenyl)‐2‐methyl‐4‐oxo‐3,4‐dihydro pyrido[2,3‐d]pyrimidine‐6‐carbonitrile (3a): Reddish brown crystals. Yield: 73%. M.p.: 270 oC. IR (KBr, max, cm‐1): 3050 (C‐H aromatic), 2980 (C‐H aliphatic), 1650 (C=O), 1604 (C=C aromatic), 2240 (CN). 1H NMR (DMSO, δ ppm): 1.8 (3H, s, CH3), 2.73‐3.01 (6H, s, OCH3), 6.72 (4H, d, Ar‐H), 11.50 (1H, br, NH). Anal. Calcd. for C17H14N4O3 (322): C, 63.35; H, 4.35; N,17.39. Found: C, 63.05; H, 4.29; N, 17.41. 7‐methoxy‐5‐(4‐methoxyphenyl)‐4‐oxo‐2‐phenyl‐3,4‐dihydro pyrido[2,3‐d]pyrimidine‐6‐carbonitrile (3b): Yellow crystals. Yield: 58%. M.p.: 285 oC. IR (KBr, max, cm‐1): 3020 (C‐H aromatic), 2920 (C‐H aliphatic), 1690 (C=O), 1590 (C=C aromatic), 2230 (CN). 1H NMR (DMSO, δ ppm): 2.90‐3.03 (6H, s, OCH3), 6.93‐7.85 (9H, m, Ar‐H), 11.20 (1H, br, NH). Anal. Calcd. for C22H16N4O3 (384): C, 68.74; H, 4.17; N, 14.58. Found: C, 68.69; H, 4.02; N, 14.53. 7‐methoxy‐5‐(4‐methoxyphenyl)‐4‐oxo‐3,4‐dihydropyrido[2,3‐d] pyrimidine‐6‐carbonitrile (3c): Yellow crystals. Yield: 65%. M.p.: 292 oC. IR (KBr, max, cm‐1): 3040 (C‐H aromatic), 2950 (C‐H aliphatic), 1650 (C=O), 1600 (C=C aromatic), 2210 (CN). 1H NMR (DMSO, δ ppm): 2.75‐3.05 (6H, s, OCH3), 7.05‐7.73 (5H, m, Ar‐H), 10.80 (1H, br, NH). MS (m/z, %): 322 (M+, 46.0), 215 (66.5), 107 (100), 108 (39.3), 80 (62.3), 76 (76.5), 65 (26.2), 32 (20.8), 15 (35.1). Anal. Calcd. for C16H12N4O3 (308): C, 62.34; H, 3.90; N, 18.18. Found: C, 62.25; H, 3.82; N, 18.09. 2.4. 2‐((1E)‐2‐phenylvinyl)‐7‐methoxy‐5‐(4‐methoxyphenyl)‐ 4‐oxo‐3‐hydro pyridino[2,3‐d]pyrimidine‐6‐carbonitrile (4) A mixture of 3a (0.01 mole) and benzaldehyde (0.012 mole) in ethanol (30 mL) was refluxed for one hour. After cooling, the solid obtained was filtered off and recrystallized from ethanol to give 4 as yellow crystals (Scheme 2). Yield: 55%. M.p.: 230 oC. IR (KBr, max, cm‐1): 3044 (C‐H aromatic), 2978 (C‐H aliphatic), 1690 (C=O), 1632 (C=C), 2240 (CN). 1H NMR (DMSO, δ ppm): 2.73‐3.10 (6H, s, OCH3), 3.5 (2H, d, CH=CH‐Ph), 7.2 (9H, m, Ar‐H), 12.05 (1H, br, NH). Anal. Calcd. for C24H18N4O3 (410): C, 70.24; H, 4.39; N,13.66. Found: C, 70.19; H, 4.32; N, 13.58. Scheme 2 2.5. 2‐Methoxy‐4‐(4‐methoxyphenyl)‐7,9‐dimethyl‐5‐oxo‐6‐ hydropyridino[2,3‐d] pyridino[1,2‐a]pyrimidine‐3‐ carbonitrile (5) A mixture of 3a (0.01 mole) and the corresponding β‐ dicarbonyl derivative; acetyl acetone, (0.012 mole) in ethanol (30 mL) was refluxed for one hour. After cooling, the solid obtained was filtered off and recrystallized from ethanol to give 5 as yellow crystals (Scheme 2). Yield: 65%. M.p.: 290 oC. IR (KBr, max, cm‐1): 3035 (C‐H aromatic), 2950 (C‐H aliphatic), 1710 (C=O), 1645 (C=C), 2237 (CN). 1H NMR (DMSO, δ ppm): 2.03 (6H, s, ‐CH3), 2.7‐3.0 (6H, s, ‐OCH3), 6.8 (6H, m, Ar‐H, H‐8, H‐10). MS (m/z, %): 386 (M+, 11.8), 278 (30.5), 250 (42.5), 171 (100), 115 (32.6), 107 (30.9), 79 (13.8), 30 (17.1), 26 (15.8). Anal. Calcd. for C22H18N4O3 (386): C, 68.39; H, 4.66; N, 14.51. Found: C, 68.41; H, 4.52; N, 14.60. 2.6. 2‐Methoxy‐4‐(4‐methoxyphenyl)‐8‐methyl‐5,7‐dioxo‐6‐ hydropyridino[2,3‐d]3‐pyridino[1,2‐a]pyrimidine‐3‐ carbonitrile (6) A mixture of 3a (0.01 mole) and ethyl pyruvate (0.012 mole) in ethanol (30 mL) was refluxed for one hour. After cooling, the solid obtained was filtered off and recrystallized from ethanol to give 6 as pale brown crystals (Scheme 2). Yield: 65%. M.p.: 275 oC. IR (KBr, max, cm‐1): 2980 (aliphatic C‐H), 1720 (C=O), 1604 (C=C aromatic), 2230 (CN). 1H NMR (DMSO, δ ppm): 2.03‐3.20 (9H, d, ‐CH3, ‐OCH3), 7.3 (5H, m, Ar‐H, H‐9). Anal. Calcd for C20H14N4O4 (374): C, 64.17; H, 3.74; N, 14.97. Found: C, 64.09; H, 3.69; N, 15.03. 2.7. 4‐Chloro‐7‐methoxy‐5‐(4‐methoxyphenyl)‐2‐substituted pyridino[2,3‐d]pyrimidine‐6‐carbonitrile (7a,b) A mixture of 3b or 3c (5 g) and POCl3 (25 mL) was refluxed for 3 hours. The reaction mixture was cooled and poured on 200 g ice water. The formed solid was filtered off, washed with water and recrystallized from ethanol to give 7a and 7b as yellow crystals (Scheme 3). 4‐chloro‐7‐methoxy‐5‐(4‐methoxyphenyl)‐2‐phenylpyrido [2,3‐d]pyrimidine‐6‐carbonitrile (7a): Yield: 70%. M.p.: 185 oC. IR (KBr, max, cm‐1): 3020 (aromatic C‐H), 2980 (aliphatic C‐H), 1604 (C=C aromatic), 2230 (CN). 1H NMR (DMSO, δ ppm): 2.3‐3.1 (6H, s, ‐OCH3), 6.80‐7.20 (9H, m, Ar‐H). Anal. Calcd. for C22H15N4O2Cl (402.5): C, 65.59; H, 3.73; N,13.91; Cl, 8.82. Found: C, 65.62; H, 3.70; N, 13.89; Cl, 8.70. 4‐chloro‐7‐methoxy‐5‐(4‐methoxyphenyl)pyrido[2,3‐d] pyrimidine‐6‐carbonitrile (7b): Yield: 75%. M.p.: 172 oC. IR (KBr, max, cm‐1): 3040 (aromatic C‐H), 2950 (aliphatic C‐H), 1600 (C=C aromatic), 2210 (CN). 1H NMR (DMSO, δ ppm): 2.8‐3.5 (6H, s, ‐OCH3), 7.20‐7.77 (5H, m, Ar‐H). Anal. Calcd. for C16H11N4O2Cl (326.5): C, 58.81; H, 3.37; N, 17.15; Cl, 10.87. Found: C, 59.03; H, 3.40; N, 17.18; Cl, 10.90. Scheme 3 2.8. 4‐Hydrazino‐7‐methoxy‐5‐(4‐methoxyphenyl)‐2‐ substituted pyridino[2,3‐d] pyrimidine‐6‐carbonitrile (8a,b) A mixture of 7a or 7b (0.01 mole) and hydrazine hydrate (98%; 0.05 mole) in 50 mL ethanol was refluxed for 3 hours. After cooling, the formed solid was filtered off and recrystallized from the proper solvent to give 8a (ethanol) and 8b (DMF) as colorless crystals (Scheme 3). 4‐Hydrazinyl‐7‐methoxy‐5‐(4‐methoxyphenyl)‐2‐phenylpyrido [2,3‐d]pyrimidine‐6‐carbonitrile (8a): Yield: 55%. M.p.: 220 oC. IR (KBr, max, cm‐1): 3020 (aromatic C‐H), 2980 (aliphatic C‐H), 1604 (C=C aromatic), 2230 (CN). 1H NMR (DMSO, δ ppm): 2.3‐2.9 (6H, s, ‐OCH3), 7.3 (9H, m, Ar‐H), 11.9 (3H, br, NH‐NH2). Anal. Calcd. for C22H18N6O2 (398): C, 66.33; H, 4.52; N, 21.11. Found: C, 66.36; H, 4.50; N, 21.20. 4‐Hydrazinyl‐7‐methoxy‐5‐(4‐methoxyphenyl)pyrido[2,3‐d] pyrimidine‐6‐carbonitrile (8b): Yield: 63%. M.p.: 235 oC. IR (KBr, Hanafy / European Journal of Chemistry 2 (1) (2011) 65‐69 67 Scheme 4 max, cm‐1): 3030 (aromatic C‐H), 2910 (aliphatic C‐H), 1580 (C=C aromatic), 2230 (CN). 1H NMR (DMSO, δ ppm): 2.7‐3.1 (6H, s, ‐OCH3), 7.05‐7.63 (5H, m, Ar‐H), 11.7 (3H, br, NH‐NH2). Anal. Calcd. for C16H14N6O2 (322): C, 59.63; H, 4.35; N, 26.09, Found: C, 59.59; H, 4.33; N, 26.12. 2.9. 4‐[((1E)‐1‐aza‐2‐phenylvinyl)amino]‐7‐methoxy‐5‐(4‐ methoxyphenyl)‐2‐phenyl pyridino[2,3‐d]pyrimidine‐6‐ carbonitrile (9) A mixture of 8a (0.01 mole) and the benzaldehyde (0.012 mole) in acetic acid (20%; 30 mL) was refluxed for one hour. The solid formed was filtered off and recrystallized from the ethanol to give 9 as pale yellow crystals (Scheme 4). Yield: 85%. M.p.: 235 oC. IR (KBr, max, cm‐1): 3480(NH), 3020 (aromatic C‐H), 2980 (aliphatic C‐H), 2230 (CN), 1660 (δ NH), 1610‐1590 (C=C and C=N). 1H NMR (DMSO, δ ppm): 2.3‐ 2.8 (6H, s, ‐OCH3), 6.8‐8.0 (15H, m, Ar‐H, CH=N), 11.9 (1H, br, NH). Anal. Calcd. for C29H22N6O2 (486): C, 71.60; H, 4.53; N, 17.28. Found: C, 71.62; H, 4.60; N, 17.33. 2.10. 3‐methoxy‐1‐(4‐Methoxyphenyl)‐6,8‐diphenyl‐7‐ hydropyridino[2,3‐d]1,2,4‐triazolo[4,5e] pyrimidine‐2‐ carbonitrile (10) Compound 9, was cyclized by oxidation with FeCl3 on refluxing in ethanol for 6 hours. The formed solid was filtered off and recrystallized from ethanol to give 10 as colorless crystals (Scheme 4). Yield: 65%. M.p.: 290 oC. IR (KBr, max, cm‐ 1): 3030 (aromatic C‐H), 2970 (aliphatic C‐H), 1600 (C=C and C=N), 2220 (CN). 1H NMR (DMSO, δ ppm): 2.3‐2.9 (6H, s, ‐ OCH3), 6.8‐8.1 (14H, m, Ar‐H). MS (m/z, %): 484 (M+, 54.51), 377 (33.4), 270 (23.26), 116 (75.97), 107 (100), 77 (52.27), 32 (64.43). Anal. Calcd. for C29H20N6O2 (486): C, 71.90; H, 4.13; N, 17.36. Found: C, 71.87; H, 4.20; N, 17.40. 2.11. 4‐(3,5‐dimethylpyrazolyl)‐7‐methoxy‐5‐(4‐methoxy phenyl)‐2‐phenylpyridino [2,3d] pyrimidine‐6‐carbonitrile (11) A mixture of 8a (0.01 mole) and β‐dicarbonyl derivative; acetyl acetone (0.012 mole) in ethanol (30 mL) was refluxed for 5 hours. After cooling, the reaction mixture was poured onto cold water and the solid obtained was filtered off and recrystallized from ethanol to give 11 as colorless crystals (Scheme 4). Yield: 55%. M.p.: 190 oC. IR (KBr, max, cm‐1): 3030 (aromatic C‐H), 2970 (aliphatic C‐H), 1595 (C=C and C=N), 2220 (CN). 1H NMR (DMSO, δ ppm): 2.3 (6H, s, ‐CH3), 3.02 (6H, s, ‐OCH3), 7.8‐8.01 (10H, m, Ar‐H, H‐4[pyrzolo‐]). MS (m/z, %): 462 (M+, 62.50), 376 (68.75), 238 (75.00), 131 (62.50), 129 (62.50), 107 (62.50), 95 (52.30), 76 (100), 32 (75.00). Anal. Calcd. for C27H22N6O2 (462): C, 70.13; H, 4.76; N, 18.18, Found: C, 70.10; H, 4.52; N, 18.60. 2.12. Ethyl 1‐[7‐amino‐6‐cyano‐5‐(4‐methoxyphenyl)‐2‐ phenylpyridino[3,2‐e] pyrimidin‐4‐yl]‐3‐methyl‐5‐oxo‐2‐ pyrazoline‐4‐carboxylate (12) A mixture of 8a (0.01 mole) and ethyl cyanoacetate (0.01 mole) in acetic anhydride (20 mL) was refluxed for 3 hours. After cooling, the reaction mixture was poured onto cold water and the solid obtained was filtered off and recrystallized from ethanol to give 12 as a colorless crystals (Scheme 4). Yield: 55%. M.p.: 240 oC. IR (KBr, max, cm‐1): 2980 (aliphatic C‐H), 1740 (C=O), 1604 (C=C and C=N), 2230 (CN). 1H NMR (DMSO, δ ppm): 2.1 (3H, s, ‐CH3), 2.7 (3H, t, ‐CH3 of the ester), 3.8 (6H, s, ‐OCH3), 4.2 (2H, q, ‐CH2 of the ester), 7.4‐8.0 (9H, m, Ar‐H). MS (m/z, %): 536 (M+, 45.83), 367 (41.67), 238 (45.83), 168 (41.67), 140 (41.67), 131 (62.50), 129 (45.83), 107 (87.52), 95 (100), 76 (45.83), 45 (70.83), 28 (87.50). Anal. Calcd for C29H24N6O5 (536): C, 64.93; H, 4.78; N, 15.67. Found: C, 65.02; H, 4.80; N, 15.70. 2.13. 3‐Methoxy‐1‐(4‐methoxyphenyl)‐6‐phenyl‐7‐hydro pyridino[2,3‐d]1,2,3,4‐tetrazolo[1,5‐e]pyrimidine‐2‐ carbonitrile (13) A solution of 8a (0.01 mole) in acetic acid (5 mL) and water (1 mL) was treated with a solution of NaNO2 (0.015 mole) in water (2 mL) at 0‐5 oC. The solid obtained upon diluting the reaction mixture with 10 mL of water was filtered off and recrystallized from ethanol to give 13 as yellow crystals (Scheme 4). Yield: 65%. M.p.: 235 oC. This compound was also synthesized by refluxing of a mixture of 7a (0.01 mole) in DMF (30 mL) and sodium azide (0.01 mole) in water (2 mL) for 3 hours, then the reaction mixture was cooled, poured onto cold water and the resulting solid was filtered off (Scheme 4). IR (KBr, max, cm‐1): 2960 (aliphatic C‐H), 1600 (C=C and C=N), 2230 (CN). 1H NMR (DMSO, δ ppm): 3.19 (6H, s, ‐OCH3), 6.9‐ 7.5 (9H, m, Ar‐H). Anal. Calcd for C22H15N7O2 (409): C, 64.55; H, 3.67; N, 23.96. Found: C, 64.49; H, 3.71; N, 23.89. 2.14. 4‐[((1E)‐1‐Aza‐2‐phenyl prop‐1‐enyl)amino]‐7‐ methoxy‐5‐(4‐methoxy phenyl) pyridino[2,3‐d]pyrimidine‐6‐ carbonitrile (14) An equimolar mixture of 8b and acetophenone in ethanol was refluxed for 15 minutes. After cooling, the resulting solid was recrystallized from ethanol to give 14 as colorless crystals (Scheme 5). Yield: 70%. M.p.: 210 oC. IR (KBr, max, cm‐1): 3260‐ 3120 (NH), 3050 (aromatic C‐H), 2990 (aliphatic C‐H), 1630‐ 1600 (C=C and C=N), 2220 (CN). 1H NMR (DMSO, δ ppm): 68 Hanafy / European Journal of Chemistry 2 (1) (2011) 65‐69 2.6‐3.0 (9H, d, ‐CH3, ‐OCH3), 7.1‐8.0 (10H, m, Ar‐H, H‐2), 12.09 (1H, br, NH). Anal. Calcd. for C24H20N6O2 (424): C, 67.92; H, 4.72; N, 19.18, Found: C, 67.90; H, 4.30; N, 19.72. R = C6H4-OCH3-p R N NC MeO N N NHNH2 (8b) R N NC MeO N N HN N CH3 Ph (14) R N NC MeO N N HN H N CH3 CN Ph Conc. HCl R N NC MeO N H N N N CH3 O Ph (16) (15) N NCH3 CH3 NH2 O Ph N N CH3 CH3H2N Ph NR N NC MeO N N HN (17) N N CH3 CH3 Ph N N N CH3 R N NC MeO N N (18) PhCOCH3 KCN AcOH AcONa AcOH Scheme 5 2.15. 4‐[2‐(1‐cyano‐1‐phenylethyl)hydrazino]‐7‐methoxy‐5‐ (4‐methoxyphenyl) pyridino [2,3‐d]pyrimidine‐6‐ carbonitrile (15) A mixture of 14 (0.01 mole) and KCN (0.01 mole, in 10 mL water) in glacial acetic acid (50 mL) and ethanol (10 mL) was refluxed for 2 hours. After cooling and diluting with cold water, the resulting solid was filtered off and recrystallized from ethanol to give 15 as colorless crystals (Scheme 5). Yield: 50%. M.p.: 221 oC. IR (KBr, max, cm‐1): 3260‐3130 (NH), 3010 (aromatic C‐H), 2950 (aliphatic C‐H), 1630‐1580 (C=C and C=N), 2260 (CN). 1H NMR (DMSO, δ ppm): 2.9 (3H, s, ‐CH3), 3.1 (6H, s, ‐OCH3), 7.3‐7.9 (10H, m, Ar‐H, H‐2), 11.9 (2H, br, NH‐ NH). Anal. Calcd. for C25H21N7O2 (451): C, 66.52; H, 4.56; N, 21.73. Found: C, 66.55; H, 4.61; N, 21.70. 2.16. 3‐Methoxy‐1‐(4‐methoxyphenyl)‐9‐methyl‐8‐oxo‐9‐ phenyl‐7‐hydro‐10H‐pyridino[2,3‐d]1,2,4‐triazino[4,3‐ e]pyrimidine‐2‐carbonitrile (16) Compound 15 (2 g) was refluxed in concentrated HCl (50%; 5 mL) for 4 hours. After cooling and diluting with water, the resulting solid was filtered off washed with water and recrystallized from ethanol to give 16 as colorless crystals (Scheme 5). Yield: 30%. M.p.: >300 oC. IR (KBr, max, cm‐1): 3250‐3100 (NH), 3010 (aromatic C‐H), 2950 (aliphatic C‐H), 1640‐1600 (C=C and C=N), 1700 (C=O) 2240 (CN). 1H NMR (DMSO, δ ppm): 1.3 (3H, s, ‐CH3), 2.9 (6H, m, ‐OCH3), 7.8 (10H, m, Ar‐H, H‐6), 10.3 (1H, s, NH). Anal. Calcd. for C25H20N6O3 (452): C, 66.37; H, 4.42; N, 18.58. Found: C, 66.42; H, 4.30; N, 19.06. 2.17. 4‐{[(4‐Amino‐2,3‐dimethyl‐1‐phenyl](3‐pyrazolin‐5‐ ylidene))azamethyl]amino)‐7‐methoxy‐5‐(4‐methoxy phenyl)pyridino[2,3‐d]pyrimidine‐6‐carbonitrile (17) A mixture of 8b (0.01 mole) and 4‐aminoantipyrine (0.01 mole) in absolute ethanol (50 mL) and few drops of acetic acid was refluxed for one hour. The reaction mixture was then cooled, filtered off and recrystallized from ethanol to give 17 as pale yellow crystals (Scheme 5). Yield: 40%. M.p.: 135 oC. IR (KBr, max, cm‐1): 3400‐3200 (NH), 3020 (aromatic C‐H), 2910 (aliphatic C‐H), 1640‐1600 (C=C and C=N), 1700 (C=O), 2220 (CN). 1H NMR (DMSO, δ ppm): 1.8‐2.9 (12H, br, ‐CH3, ‐OCH3), 7.3 (10H, m, Ar‐H, H‐2), 10.3‐11.02 (3H, br, NH, NH2). Anal. Calcd. for C27H25N9O2 (507): C, 63.91; H, 4.93; N, 24.85. Found: C, 64.09; H, 5.07; N, 24.78. 2.18. 7‐Methoxy‐5‐(4‐methoxyphenyl)‐4‐(2,3,5‐trimethyl‐1‐ phenyl(3‐pyrzolino[4,5‐e]1,2,4‐triazin‐6‐yl))pyridino[2,3‐ d]pyrimidine‐6‐carbonitrile (18) Compound 17 (0.01 mole) in glacial acetic acid (50 mL) and fused sodium acetate (10 g), was refluxed for 6 hours. After cooling and diluting with water, the formed solid was filtered off and recrystallized from ethanol to give 18 as yellow crystals (Scheme 5). Yield: 50%. M.p.: 260 oC. IR (KBr, max, cm‐1): 3030 (aromatic C‐H), 2960 (aliphatic C‐H), 1650‐1630 (C=C and C=N), 2220 (CN). 1H NMR (DMSO, δ ppm): 1.8‐2.1 (9H, s, ‐CH3), 3.02 (6H, s, ‐OCH3), 6.7‐7.2 (10H, m, Ar‐H, H‐2). MS (m/z, %): 531 (M+, 30.30), 291 (33.33), 240 (84.85), 184 (100), 158 (63.64), 127 (30.30), 107 (51.52), 76 (57.58), 31 (48.48), 26 (72.73). Anal. Calcd. for C29H25N9O2 (531): C, 65.54; H, 4.71; N, 23.73. Found: C, 65.50; H, 4.69; N, 23.67. 3. Results and discussion 3.1. Synthesis The synthesis of heterocyclic systems containing pyrido[2,3‐d]pyrimidine moiety has gained much attention due to high biological activity possessed by these compunds [1‐12]. 2‐amino‐6‐methoxy‐4‐(4‐methoxyphenyl)pyridine‐3,5‐dicar‐ bonitrile (1) was prepared [13]. Hydrolysis of 1 using alcoholic solution of KOH (5%) gave the amide derivative 2, which on condensation with acetic anhydride, benzoyl chloride or triethyl orthoformate [14] yielded the pyrido[2,3‐d]pyrimidine derivatives 3a‐c, respectively (Scheme 1). Due to the high reactivity of the methyl group in position‐2, compound 3a underwent condensation reactions with aromatic aldehydes such as benzaldehyde, to give 2‐((1E)‐2‐ phenylvinyl)‐7‐methoxy‐5‐(4‐methoxyphenyl)‐4‐oxo‐3‐hydro pyridino[2,3‐d]pyrimidine‐6‐carbonitrile (4). Also, a conden‐ sation reaction with β‐diketones, as acetyl acetone, gave 2‐ methoxy‐4‐(4‐methoxyphenyl)‐7,9‐dimethyl‐5‐oxo‐6‐hydro pyridino[2,3‐d]pyridino [1,2‐a]pyrimidine‐3‐carbonitrile (5). Also, 3a was condensed with ethyl pyruvate to give 2‐methoxy‐ 4‐(4‐methoxyphenyl)‐8‐methyl‐5,7‐dioxo‐6‐hydropyridino [2,3‐d]3‐pyridino[1,2‐a]pyrimidine‐3‐carbonitrile (6), (Scheme 2). 7‐Methoxy‐5‐(4‐methoxyphenyl)‐2‐phenyl‐4‐oxo‐3‐hydro pyridino[2,3‐d] pyrimidine‐6‐carbonitrile (3b) and 7‐methoxy‐ 5‐(4‐methoxyphenyl)‐4‐oxo‐3‐hydrpyridino[2,3‐d] pyrimidine‐ 6‐carbonitrile (3c), were refluxed with POCl3 to give 4‐chloro‐ 7‐methoxy‐5‐(4‐methoxyphenyl)‐2‐substituted pyridino[2,3‐ d]pyrimidine‐6‐carbonitrile (7a,b), respectively, which on reaction with hydrazine hydrate yielded the hydrazino‐ derivatives 8a,b, respectively (Scheme 3). Treatment of 4‐hydrazino‐7‐methoxy‐5‐(4‐methoxy phenyl)‐2‐phenylpyridino[2,3‐d] pyrimidne‐6‐carbonitrile (8a) with benzaldehyde, gave 4‐[((1E)‐1‐aza‐2‐phenylvinyl)amino]‐ 7‐methoxy‐5‐(4‐methoxyphenyl)‐2‐phenylpyridino[2,3‐d] pyri‐ midine‐6‐carbonitrile (9), which on oxidative cyclization using FeCl3, yielded the fused pyridino[2,3‐d]1,2,4‐triazolo[4,5e] pyrimidine, 10. β‐Dicarbonyl reagents such as acetyl acetone, reacted with 8a to give 4‐(3,5‐dimethyl pyrazolyl)‐7‐methoxy‐ 5‐(4‐methoxyphenyl)‐2‐phenyl pyridine [2,3‐d]pyrimidine‐6‐ carbonitrile (11), [15]. Moreover, 8a on refluxing with a mixture of acetic anhydride and ethyl cyanoacetate afforded ethyl‐1‐[7‐amino‐6‐cyano‐5‐(4‐methoxyphenyl)‐2‐phenylpyri‐ Hanafy / European Journal of Chemistry 2 (1) (2011) 65‐69 69 dino[3,2‐e]pyrimidin‐4‐yl]‐3‐methyl‐5‐oxo‐2‐pyra‐zoline‐4‐ carboxylate (12) [15]. Reaction of 8a with nitrous acid and the reaction of 7a with sodium azide in DMF yielded 3‐methoxy‐1‐ (4‐methoxyphenyl)‐6‐phenyl‐7‐hydro‐pyridino[2,3‐d]1,2,3,4‐ tetrazolo[1,5‐e]pyrimidine‐2‐carbonitrile (13) [16,17] (Scheme 4). Compound 8b was condensed with acetophenone in glacial acetic acid to give the hydrazone 14 which underwent addition of HCN in acetic acid‐ethanol mixture, giving the cyano hydrazone 15. Further, acidic hydrolysis of 15 by refluxing in concentrated HCl, led to the formation of 3‐methoxy‐1‐(4‐ methoxyphenyl)‐9‐methyl‐8‐oxo‐9‐phenyl‐7‐hydro‐10H‐pyri‐ dino[2,3‐d]1,2,4‐triazino[4,3‐e]pyrimidine‐2‐carbonitrile (16) [18]. Also, 8b, was condensed with 4‐aminoantipyrine in absolute ethanol in presence of few drops of acetic acid to give 4‐{[(4‐amino‐2,3‐dimethyl‐1‐phenyl](3‐pyrazolin‐5‐ylidene)) azamethyl]amino)‐7‐methoxy‐5‐(4‐methoxyphenyl)pyridine [2,3‐d]pyrimidine‐6‐carbonitrile (17), which on refluxing with glacial acetic acid‐fused sodium acetate, via acylation followed by cyclo‐condensation, produced 7‐methoxy‐5‐(4‐methoxy phenyl)‐4‐(2,3,5‐trimethyl‐1‐phenyl(3‐pyrazolino[4,5‐e]1,2,4‐ triazin‐6‐yl))pyridino[2,3‐d]pyrimidine‐6‐carbonitrile (18), (Scheme 5). 3.2. Biological screening A few newly synthesized compounds were screened for their antifungal activities against three types of fungi, Alternaria alternata, Aspergillus niger, and Aspergillus flavipes, using the disk diffusion method [19‐21]. The tested compounds were dissolved in DMF, which was used as a control to get 1 mg/mL solution. The inhibition zones of microbial growth surrounding the filter paper disc (2.5 mm) were measured in millimeters at the end of an incubation period at 30 oC for 3 days. Activity of each compound was compared with that of flucanazole as the standard. The investigation of fungicidal screening data revealed that all the tested compounds showed variable activities towards the investigated fungi used, indicating that the compounds are biologically active due to the presence of different heterocycles and functional groups. Compounds 3a, 13 and 18 showed very high activities, whereas compounds 6, 10 and 17 showed high activity against them. On the other hand, compounds 3b and 7a showed moderate activities against Alternaria alternata and Aspergillus niger, while compound 11 showed low activity against Alternaria alternata and Aspergillus niger and moderate activity against Aspergillus flavipes (Table 1). Table 1. Antifungal activities data of some of the prepared compounds. Compound Diameter of inhibition zone (mm)* Alternaria alternata Aspergillus niger Aspergillus flavipes 3a ++++ ++++ ++++ 3b ++ ++ + 5 + ++ ++ 6 +++ +++ +++ 7a ++ ++ ++ 10 +++ +++ +++ 11 + + ++ 13 ++++ ++++ ++++ 16 +++ ++ ++ 17 +++ +++ +++ 18 ++++ ++++ ++++ Fluconazole ++++ ++++ ++++ *Very high activity = ++++ (inhibition zone > 30 mm), High activity = +++ (inhibition zone 21‐30 mm), Moderate activity = ++ (inhibition zone 11‐20 mm), Low activity = + (inhibition zone 1‐10 mm). References [1]. Burova, O. A.; Bystrykova, I. D.; Smirnova, N. M.; Safonova, T. S. Chem. Heterocyc. Comp. 1991, 27, 394‐398. [2]. Rahman, L. K. A.; Chhabra, S. R. Med. Res. Rev. 1988, 8, 95‐155. [3]. Gangjee, A.; Vasudevan, A.; Queener, F.; Kisliuk, R. J. Med. Chem. 1996, 39, 1438‐1446. [4]. Zink, M.; Lanig, H.; Troschutz, R. Eur. J. Med. Chem. 2004, 39, 1079‐ 1088. [5]. Cordeu, L.; Cubedo, E.; Bandres, E.; Rebollo, A.; Saenz, X.; Chozas, H.; Dominguez, M.; Echeverria, M.; Mendivil, B.; Sanmartin, C.; Palop, J.; Font, M.; Foncillas, J. Bioorg. Med. Chem. 2007, 15, 1659‐1669. [6]. Monge, A.; Martinez, V.; San Martin, C.; Simon, M. A. Spanish Patent ES 1994 2,056,742. [Chem. Abstr. 1995, 122, 105912q]. [7]. Parmar, K.; Suthar, B.; Suthar, A.; Maheta, A. J. Heterocycl. Chem. 2009, 46, 975‐679. [8]. Koeckkritz, P.; Ruhmann, C.; Fieblinger, D.; Schroeder, C.; Joksch, B.; Heider, B.; Weiher, B. Liebscher J. Ger. Offen. DE 4,117,802 [Chem. Abstr. 1993, 118, 191550s]. [9]. Narayana, B.; Ram Rao, A.; Shanthan Rao, P. Eur. J. Med. Chem. 2009, 44, 1369‐1376. [10]. Kanth, S.; Reddy, G.; Kishore, K.; Shanthan Rao, P.; Narsaiah, B.; Murthy, U. Eur. J. Med. Chem. 2006, 41, 1011‐1016. [11]. El‐Subbagh, H. I.; Abuzaid, S. M.; Mahran, M. A.; Badria, F. A.; Al‐Obaid, A. M. J. Med. Chem. 2000, 43, 2915‐2921. [12]. Ashalatha, B. V.; Narayana, B.; Vijaya Raj, K. K.; Suchetha Kumari, N. Eur. J. Med. Chem. 2007, 42, 719‐728. [13]. Seada, M.; El‐Behairy, M. A.; Jahine, H.; Hanafy, F. Orient. J. Chem. 1989, 5, 273‐280. [14]. Chan, J.; Gustin, D.; Divirgilio, E. S.; Guram, A.; Faul, M. M. Synthesis 2007, 23, 3678‐3682. [15]. Seada, M.; Abdel‐Rahman, R. M.; El‐Behairy, M.; Hanafy, F. Asian J. Chem. 1992, 4, 604‐614. [16]. Okafor, C. O. J. Org. Chem. 1973, 38, 4386‐4390. [17]. Dehuri, S. N.; Pradhan, P. C.; Nayak, A. J. Indian Chem. Soc. 1983, 60, 475‐481. [18]. Seada, M.; Abdel‐Rahman, R. M.; Hanafy, F. J. Indian Chem. Soc. 1992, 69, 882‐884. [19]. Khan, K. M.; Saify, Z. S.; Zeesha, A. K.; Ahmed, M.; Saeed, M.; Schick, M.; Bkohlbau, H. J.; Voelter, W. Arzneim‐Forsch. 2000, 50, 915‐922. [20]. Rahman, A. U.; Choudhary, M. I.; Thomsen, W. J. “Bioassay Techniques for drug development, 16 the Netherlands”: Harwood Academic Publishers 2001. [21]. Jorgensen, J. H.; Crawford, S. A.; Mc Elmeel, M. L.; Fiebelkorn, K. R. J. Clin. Microbiol. 2004, 42, 1800‐1802.