untitled European Journal of Chemistry 2 (4) (2011) 455‐462 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2011 EURJCHEM DOI:10.5155/eurjchem.2.4.455‐462.171 European Journal of Chemistry Journal homepage: www.eurjchem.com Novel 4(3H)‐quinazolinones containing biologically active thiazole, pyridinone and chromene of expected antitumor and antifungal activities Khairy Abdel Hameed Mohsen El‐Bayoukia, Wahid Mohamed Basyounia, Yahia Abdel Fatah Mohamedb, Mohsen Mohamed Alyb and Samir Youssef Abbasa,* a Organometallic and Organometalloid Chemistry Department, National Research Centre, Cairo, 12311, Egypt b Department of Chemistry, Faculty of Science, Al‐Azhar University, Cairo, 11884, Egypt *Corresponding author at: Organometallic and Organometalloid Chemistry Department, National Research Centre, Cairo, 12311, Egypt. Tel.: +202.33335929; fax: +202.33370931. E‐mail address: samiryoussef98@yahoo.com (S.Y. Abbas). ARTICLE INFORMATION ABSTRACT Received: 16 June 2010 Received in revised form: 19 January 2011 Accepted: 03 February 2011 Online: 31 December 2011 KEYWORDS Novel 4(3H)‐quinazolinone derivatives with biologically active moieties were synthesized. Reactions of 2‐cyano‐N‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐yl) acetamide with carbon disulfide, isothiocyanates followed by cycloalkylation afforded acrylamide, 1,3‐dithiazole, 1,3‐ dithiane, thiazole and pyrazole derivatives. The 2‐pyridone derivatives were obtained via reaction of cyanoacetamide with some acetylacetone or arylidenes. Cyclocondensation reaction of cyanoacetamide with o‐hydroxy aldehydes furnished chromene derivatives. Screening for some selected compounds was carried for their potential antitumor and antifungal activities. 2‐Cyano‐N‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐yl)‐2‐(4‐methyl‐3‐ phenyl‐thiazol‐2(3H)ylidene)‐acetamide with 3‐side chain incorporated with substituted thiazole moiety was found to be of high to moderate activity towards cells. Also, the latter product showed high activity against Aspergillus ochraceus Wilhelm with inhibition zone (18 mm) compared with (20 mm) Nystatin inhibition zone. 4(3H)‐Quinazolinone Thiazole Pyridinone Chromene Antitumor Antifungal activity 1. Introduction The chemistry of 4(3H)‐quinazolinone system has received an increasing interest because of its biological significance. Many derivatives of this system showed antifungal [1], antibacterial [2], antitumor [3], antiinflammatory [4], anticonvulsant [5‐7], analgesic [8,9] and anti‐tuberculosis [10,11] activities. Cyanoacetamides are highly reactive compounds. The carbonyl and the cyano functions of these compounds are suitably situated to enable reactions with common reagents to form a variety of heterocyclic compounds. Also, the active methylene of cyanoacetamide can take part in a variety of condensation and substitution reactions. Moreover, cyanoacetamides and their related heterocyclic derivatives have generated great attention due to their interesting biological, therapeutic value and pharmaceutical activities e.g. as herbicidal [12] plant growth regulators [13] anti‐ inflammatory [14] anti‐tumor [15] and analgesic properties [16]. Therefore, it was aimed in the present investigation to synthesize and characterize newer 4(3H)‐quinazolinone derivatives for their expected antitumor and antimicrobial activities. 2. Experimental 2.1. Instrumentation All melting points are uncorrected. Microanalyses were carried out by the Microanalytical Laboratory, National Research Centre, Cairo, Egypt and the Microanalytical Center, Cairo University. Infrared spectra (KBr disc) were recorded using a Jasco FT/IR‐300E spectrophotometer and FTIR 5300 spectrometer (ν, cm‐1). 1H NMR spectra were recorded using Varian mercury 300 MHz or Varian Gemini 200 MHz with chemical shift in δ from Me4Si and Jeol 270, 500 MHz. Mass spectra were recorded on GC/MS Finnigan SSQ 7000 spectrophotometer &or GC Ms‐QP 1000 EX mass spectrometer at 70 eV. 2.2. Synthesis 2.2.1. 2‐Cyano‐N‐(6‐iodo‐2‐methyl‐4(3H)‐quinazolinon‐3‐yl) acetamide (2) 3‐Amino‐quinazolinone, 1, (0.01 mol) was fused with excess ethyl cyanoacetate at ~210 °C in an oil‐bath for 40 min. Excess ethyl cyanoacetate was evaporated under vacuum. The solid product remained was triturated with ethanol (20 mL) then filtered. The ethanolic filtrate was poured onto crushed ice. The solid product obtained was filtered and crystallized from toluene:ethyl acetate (1:1) to give 2 as yellow brownish crystals (Scheme 1). Yield: 80%. M.p.: 215‐217 °C. FT‐IR (ν, cm‐1): 3210 (NH), 2264 (C≡N), 1690 (C=O). 1H NMR (DMSO‐d6, δ, ppm): 2.39 (s, 3H, CH3), 4.10 (m, 2H, CH2), 7.43 (d, 1H, J = 8.40 Hz, ArH at C8‐H), 8.15 (d, 1H, J = 8.40 Hz, ArH at C7‐H), 8.37 (s, 1H, ArH at C5‐H), 11.57 (s, 1H, NH, D2O‐exchangeable). MS (m/z, %): 368 (M+; 100). Anal. calcd for C12H9IN4O2: C, 39.15; H, 2.46; N, 15.22. Found: C, 39.10; H, 2.50; N, 15.20%. 2.2.2. General procedure for the syntheses of 2‐(6‐iodo‐2‐ methyl‐3(4H)‐quinazolinonylamino)‐2‐oxo‐N'‐p‐arylaceto‐ hydrazonoylcyanide (3a,b) 456 El‐Bayouki et al. / European Journal of Chemistry 2 (4) (2011) 455‐462 Scheme 1 To aromatic amine (0.01 mole) concentrated HCl (3 mL) was added and cooled to ~0‐5 oC in ice bath then cooled sodium nitrite solution (1.0 g in 10 mL of water) was added to the mixture drop wise during 10 minutes. The reaction mixture was then stirred for 10 minutes. A cold mixture of the cyanoacetamide derivative 2 (0.01 mole) and sodium acetate (4.10 g; 0.05 mole) in ethanol (50 mL), was then added drop wise to the reaction mixture with stirring. The stirring was continued for 30 minutes and the reaction mixture was left for 1 hour at room temperature. The solid product obtained was collected by filtration and crystallized from ethanol to give the corresponding hydrazono derivatives, 3a,b, as yellow crystals (Scheme 1). 2‐((6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐yl)amino)‐2‐oxo‐ N'‐phenylacetohydrazonoyl cyanide (3a): Yield: 80%. M.p.: 234‐ 236 °C. FT‐IR (ν, cm‐1): 3228 (NH), 2216 (C≡N), 1686 (C=O). MS (m/z, %): 472 (M+; 8.4), 301 (100), 473 (3.2%), 301 (18.5), 245 (9.7), 172 (4.0), 111 (5.4). Anal. calcd. for C18H13IN6O2: C, 45.78; H, 2.77; N, 17.80. Found: C, 45.80; H, 2.70; N, 17.80%. 2‐(6‐Iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐ylamino)‐2‐oxo‐ N'‐p‐tolylacetohydrazonoyl cyanide (3b): Yield: 90%. M.p.: >300 °C. FT‐IR (ν, cm‐1): 3230 (NH), 2218 (C≡N), 1689 (C=O). 1H NMR (DMSO‐d6, δ, ppm): 2.31 (s, 3H, CH3), 2.40 (s, 3H, CH3), 7.15 (d, 2H, J = 8.08 Hz, AB system), 7.50 (d, 1H, J = 8.57 Hz, ArH at C8‐H), 7.61 (d, 2H, J = 8.08 Hz, AB system), 8.10 (d, 1H, J = 8.57 Hz, ArH at C7‐H), 8.40 (s, 1H, ArH at C5‐H), 11.15 (b, 1H, NH), 12.30 (b, 1H, NH). Anal. calcd. for C19H15IN6O2: C, 46.93; H, 3.11; N, 17.28. Found: C, 46.90; H, 3.10; N, 17.30%. 2.2.3. General procedure for synthesizing 2‐cyano‐2‐ (substituted)‐N‐(6‐iodo‐2‐methyl‐4(3H)quinazolinonyl) acetamides (4,5 and acrylamide 6) To a stirred suspension of finely powdered potassium hydroxide (0.02 mole) in dry DMF (10 mL) cyanoacetamide 2 (0.01 mole) was added, the resulted mixture was cooled at 10 oC in an ice bath, then carbon disulfide (0.01 mol) was added slowly over the course of 10 min (Scheme 1). After complete addition, stirring of the reaction mixture was continued for additional 2 h. Then dibromoethane, dibromopropane or dimethylsulfate (0.01 mol) was added to the mixture while cooling (~15 oC) and stirring for 1 h. then poured into crushed ice, the resulting precipitate was filtrated off, dried and crystallized from the proper solvent to give: 2‐Cyano‐2‐(1,3‐dithiolan‐2‐ylidene)‐N‐(6‐iodo‐2‐methyl‐4‐ oxoquinazolin‐3(4H)‐yl)acetamide (4): As yellow brownish crystals (benzene:ethanol; 1:1). Yield: 70%. M.p.: 243‐245 °C. FT‐IR (ν, cm‐1): 3266 (NH), 2971 (CHaliph), 2204 (C≡N), 1696, 1666 (C=O). 1H NMR (CDCl3, δ, ppm): 2.44 (s, 3H, CH3), 3.75 (s, 4H, 2CH2), 7.50 (d, 1H, J = 8.10 Hz, ArH at C8‐H), 8.20 (d, 1H, J = 8.10 Hz, ArH at C7‐H), 8.42 (s, 1H, ArH at C5‐H), 11.20 (s, 1H, NH, D2O‐exchangeable). MS (m/z, %): 470 (M+, 22.2), 170 (100), 471 (2.8), 172 (17.2), 75 (16.5). Anal. calcd. for C15H11IN4O2S2: C, 38.31; H, 2.36; N, 11.91. Found: C, 38.30; H, 2.40; N, 11.90%. 2‐Cyano‐2‐(1,3‐dithian‐2‐ylidene)‐N‐(6‐iodo‐2‐methyl‐4‐ oxoquinazolin‐3(4H)‐yl)acetamide (5): As yellow brownish crystals (benzene:ethanol; 1:1). Yield: 75%. M.p.: 238‐240 °C. FT‐IR (ν, cm‐1): 3242 (NH), 2926 (CHaliph), 2200 (C≡N), 1703, 1665 (C=O). 1H NMR (CDCl3, δ, ppm): 2.20 (p, 2H, J = 6.80 Hz, CH2), 2.50 (s, 3H, CH3), 3.05 (t, 2H, J = 6.60 Hz, CH2), 3.21 (t, 2H, J = 6.60 Hz, CH2), 7.42 (d, 1H, J = 8.50 Hz, ArH at C8‐H), 8.14 (d, J = 8.50 Hz, 1H, ArH at C7‐H), 8.35 (s, 1H, ArH at C5‐H), 11.06 (s, 1H, NH, D2O‐exchangeable). Anal. calcd. for C16H13IN4O2S2: C, 39.68; H, 2.71; N, 11.57. Found: C, 39.70; H, 2.70; N, 11.60%. 2‐Cyano‐N‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐yl‐3,3‐ bis(methylthio)acrylamide (6): As yellow brownish crystals (methanol). Yield: 60%. M.p.: 173‐175 °C. FT‐IR (ν, cm‐1): 3212 (NH), 2964, 2926 (CHaliph), 2200 (C≡N), 1688 (C=O). MS (m/z, %): 472 (M+, 22.2), 368 (30.9), 328 (37.8), 271 (49.0), 216 (16.9), 172 (14.8), 116 (18.6), 75 (43.8). Anal. calcd. for C15H13IN4O2S2: C, 38.14; H, 2.77; N, 11.86. Found: C38.10, H, 2.80; N, 11.90%. El‐Bayouki et al. / European Journal of Chemistry 2 (4) (2011) 455‐462 457 N N I CH3 O H N CN O NS Ph CH3 2 8 N N I CH3 O H N CN O KS NHPh Cl CH2COCH3 N N I CH3 O H N O S H2N NHPh 7 COCH3 KOH PhNCS Cl CH2COOC2H5 N N I CH3 O H N O N N NHPh N H3C CH3 12 O O N2H4 N N I CH3 O H N O N H NH2N NHPh 11 N N I CH3 O H N CN O H3CS NHPh 10 (CH3)2SO4 N N I CH3 O H N CN O NS Ph 9 O Scheme 2 2.2.4. 2‐Cyano‐N‐(6‐iodo‐2‐methyl‐4(3H)‐quinazolinon‐3‐yl)‐ 2‐(substituted) acetamide (8,9) To a stirred suspension of finely powdered potassium hydroxide (0.01 mole) in dry DMF (10 mL) cyanoacetamide 2 (0.01 mole) was added, and then phenyl isothiocyanate (0.01 mol) was added slowly (Scheme 2). After complete addition, stirring of the reaction mixture was continued for additional 5 h. Then chloroacetone or ethyl chloroacetate (0.01 mole) was added to the mixture and the reaction mixture was stirred for 2 h. The reaction mixture was poured into crushed ice. The resulting precipitate was filtrated off, dried and crystallized to give: 2‐Cyano‐N‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐yl‐2‐(4‐ methyl‐3‐phenylthiazol‐2(3H)ylidene)acetamide (8): As yellow brownish crystals (acetic acid). Yield: 75%. M.p.: 248‐250 °C. FT‐IR (ν, cm‐1): 2973, 2926 (CHaliph), 2181 (C≡N), 1708 (C=O). 1H NMR (DMSO‐d6, δ, ppm): 1.93 (s, 3H, CH3 [thiazole]), 2.40 (s, 3H, CH3 [quinazoline]), 7.03 (s, 1H, CH [thiazole]), 7.52 (d, 1H, J = 8.10 Hz, ArH at C8‐H), 7.66 (m, 5H, ArH), 8.17 (d, 1H, J = 8.10 Hz, ArH at C7‐H), 8.40 (s, 1H, ArH at C5‐H), 9.93 (s, 1H, NH, D2O‐ exchangeable). MS (m/z, %): 541(M+, 1.8), 214 (100), 327 (48.2), 243 (45.7), 187 (8.2), 116 (11.0), 75 (17.5). Anal. calcd. for C22H16IN5O2S: C,48.81; H, 2.98; N, 12.94. Found: C, 48.80; H, 3.00; N, 12.90%. 2‐Cyano‐N‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐yl)‐2‐(4‐ oxo‐3‐phenylthiazolidin‐2‐ylidene)acetamide (9): As yellow brownish crystals (ethanol:dioxane; 2:1). Yield: 70%. M.p.: 193‐ 195 °C. FT‐IR (ν, cm‐1): 3188 (NH), 2924 (CHaliph), 2220 (C≡N), 1688 (C=O). 1H NMR (DMSO‐d6, δ, ppm): 2.35 (s, 3H, CH3), 4.00 (s, 2H, CH2[thiazole]), 7.30‐7.70 (m, 6H, [Ph‐H , ArH at C8‐H]), 8.10 (d, 1H, J = 8.10 Hz, ArH at C7‐H), 8.35 (s, 1H, ArH at C5‐H), 10.60 (s, 1H, NH, D2O‐exchangeable). Anal. calcd. for C21H14IN5O2S: C, 46.42; H, 2.60; N, 12.89. Found: C, 46.40; H, 2.60; N, 12.90%. 2.2.5. 2‐Cyano‐N‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐ yl)‐3‐(methylthio)‐3‐(phenylamino)acrylamide (10) To suspension of potassium hydroxide (0.01 mole) in dry DMF (10 mL) cyanoacetamide 2 (0.01 mole) was added during stirring, phenyl isothiocyanate (0.01 mol) was dropped slowly to the reaction mixture. After complete addition, stirring of the reaction was continued for 5 h. and dimethyl sulfate (0.01 mol) was added. The reaction mixture was stirred for 2 h. then, poured into crushed ice. The resulting precipitate was filtrated off, dried and crystallized from benzene to give 10 as yellow brownish crystals (Scheme 2). Yield: 70%. M.p.: 144‐145 °C. FT‐ IR (ν, cm‐1): 3244 (NH), 2194 (C≡N), 1658 (C=O). 1H NMR (DMSO‐d6, δ, ppm): 2.30 (s, 3H, SCH3), 2.45 (s, 3H, CH3), 7.35 (d, 1H, J = 8.50 Hz, ArH at C8‐H), 7.46 (m, 5H, ArH), 8.20 (d, 1H, J = 8.50 Hz, ArH at C7‐H), 8.41 (s, 1H, ArH at C5‐H), 10.81 (s, 1H, NH, NHPh D2O‐exchangeable), 11.78 (s, 1H, NH, CONH, D2O‐ exchangeable). MS (m/z, %): 517 (M+), 127 (100). Anal. calcd. for C20H16IN5O2S: C, 46.43; H, 3.12; N, 13.54. Found: C, 46.40; H, 3.10; N, 13.50%. 2.2.6. 5‐amino‐N‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐ yl)‐3‐(phenylamino)‐1H‐pyrazole‐4‐carboxamide (11) A mixture of 10 (0.01 mol) and hydrazine hydrate (0.012 mol) in ethanol (30 mL) was heated under reflux for 3 hrs and allowed to cool. The solid product obtained was filtrated and crystallized from acetic acid to give 11 as yellow crystals (Scheme 2) Yield: 65%. M.p.: 300 °C. FT‐IR (ν, cm‐1): 3311, 3199, 3159 (NH, NH2), 1684 (C=O). 1H NMR (DMSO‐d6, δ, ppm): 2.6 (s, 3H, CH3), 5.8 (s, 2H, NH2), 6.8‐8.4 (m, 8H, ArH), 9.1 (s, 1H, NH), 9.2 (s, 1H, NH), 10.0 (s, 1H, NH). Anal. calcd. for C19H16IN7O2: C, 45.52; H, 3.22; N, 19.56. Found: C, 45.50; H, 3.20; N, 19.60%. 2.2.7. N‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐yl)‐5,7‐ dimethyl‐2)phenylamino)pyrazolo[1,5‐a]pyrimidine‐3‐ carboxamide (12) A mixture of compound 11 (0.01 mol) and acetyl acetone (0.01 mol) was heated under reflux in glacial acetic acid (20 mL) for 3 hrs then left to cool. The obtained solid product was filtrated and crystallized from dioxane to give 12 as yellow brownish crystals (Scheme 2). Yield: 65%. M.p.: >300 °C. FT‐IR (ν, cm‐1): 3253 (NH), 1705, 1656 (C=O). 458 El‐Bayouki et al. / European Journal of Chemistry 2 (4) (2011) 455‐462 Scheme 3 1H NMR (DMSO‐d6, δ, ppm): 2.50 (s, 6H, 2CH3), 2.63 (s, 3H, CH3), 6.90 (t, 1H, J = 7.55 Hz, ArH at C4 of phenyl ring), 7.11 (s, 1H, CH‐pyrimidine), 7.35 (t, 2H, J = 7.55 Hz, ArH at C3,5 of phenyl ring), 7.48 (d, 1H, J = 8.40 Hz, ArH at C8‐H of quinazoline), 7.74 (d, 2H, J = 8.40 Hz, ArH at C2,6 of phenyl ring), 8.15 (d, 1H, J = 8.40 Hz, ArH at C7 of quinazoline), 8.38 (s, 1H, ArH at C5 of quinazoline), 9.11 (b, 1H, NH), 10.37 (b, 1H, NH). MS (m/z, %): 565 (28.0), 265 (100), 566 (6.9), 238 (9.6), 174 (2.6), 134(7.8). Anal. calcd. for C24H20IN7O2: C, 50.99; H, 3.57; N, 17.34. Found: C, 51.00; H, 3.60; N, 17.30%. 2.2.8. 1‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐yl)‐4,6‐ dimethyl‐2‐oxo‐1,2‐dihydro‐pyridine3‐carbonitrile (13) A mixture of cyanoacetamide 2 (0.01 mol), acetyl acetone (0.012 mol) and pipridine (few drops) was placed in a conical flask and fused for 15 min. then allowed to cool. The mixture was triturated with ethanol (20 mL) and the solid obtained was collected by filtration and crystallized from dioxane to give 13 as pale grey crystals (Scheme 3). Yield: 85%. M.p.: >300 °C. FT‐ IR (ν, cm‐1): 2958 (CHaliph), 2218 (C≡N), 1680 (C=O). 1H NMR (DMSO‐d6, δ, ppm): 2.25 (m, 6H, 2CH3), 2.48 (s, 3H, CH3), 6.67 (s, 1H, Ar‐H), 7.54 (d, 1H, J = 8.40 Hz, ArH at C8‐H), 8.25 (d, 1H, J = 8.40 Hz, ArH at C7‐H), 8.41 (s, 1H, ArH at C5‐H). MS (m/z, %): 432 (M+), 416 (100), 433 (38.3), 434 (6.7), 290 (7.8), 257 (6.6), 216 (19.8), 116(15.0), 75 (36.1). Anal. calcd. for C17H13IN4O2: C, 47.24; H, 3.03; N, 12.96. Found: C, 47.20; H, 3.00; N, 13.00%. 2.2.9. General procedure for the syntheses of 6‐amino‐1‐(6‐ iodo‐2‐methyl‐4(3H)‐quinazolin‐3‐yl)‐4‐alkyl‐2‐oxo‐1,2‐ dihydro‐Pyridine‐3,5‐dicarbonitrile (14a,b) To a mixture of 2‐cyanoacetamide 2 (0.01 mol), formaledehyde or acetaldehyde (0.01 mol) and malononitrile (0.01 mol) in ethanol (30 mL), few drop of pipridine was added. The reaction mixture was heated under reflux for 3 hrs. The solid product which formed while hot was collected by filtration and crystallized from dioxane to give 14a,b as yellow crystals (Scheme 3). 6‐Amino‐1‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐yl)‐2‐ oxo‐1,2‐dihydropyridine‐3,5‐dicarbonitrile (14a): M.p.: 197‐195 °C. Yield: 70%. FT‐IR (ν, cm‐1): 3353, 3204 (NH2), 2191 (C≡N), 1694 (C=O). MS (m/z, %): 444 (M+, 1.5), 286 (100). Anal. calcd. for C16H9IN6O2: C, 43.26; H, 2.04; N, 18.92. Found: C, 43.30; H, 2.00; N, 18.90%. 6‐Amino‐1‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐yl)‐4‐ methyl‐2‐oxo‐1,2‐dihydropyridine‐3,5‐dicarbonitrile (14b): M.p.: 277‐280°C. Yield: 75%. FT‐IR (ν, cm‐1): 3302, 3143 (NH2), 2218 (C≡N), 1696 (C=O). 1H NMR (DMSO‐d6, δ, ppm): 2.20 (s, 3H, CH3‐quinazoline), 2.50 (s, 3H, CH3‐ pyridine), 7.53 (d, 1H, J = 8.40 Hz, ArH at C8‐H), 8.15 (d, 1H, J = 8.70 Hz, ArH at C7‐H), 8.40 (s, 1H, ArH at C5‐H), 9.22 (b, 2H, NH2). Anal. calcd. for C17H11IN6O2: C, 44.56; H, 2.42; N, 18.34. Found: C, 44.60; H, 2.40; N, 18.40%. 2.2.10. General procedure for the syntheses of 6‐amino‐1‐(6‐ iodo‐2‐methyl‐4(3H)‐quinazolinonyl)‐4‐(aryl)‐2‐oxo‐1,2‐ dihydro‐pyridine‐3,5‐dicarbonitriles (15a,b) To equimolar amounts of 2‐cyanoacetamide 2 and 2‐(4‐ (chloro or methoxy) benzylidene) malononitrile, (0.01 mol) in ethanol (30 mL) it was added few drop of pyridine. The reaction mixture was heated under reflux for 3 hrs then allowed to cool and poured into cold diluted HCl solution. The obtained product was collected by filtration and crystallized from the proper solvent to give pyridinone derivatives 15a,b (Scheme 3). 6‐Amino‐1‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐yl)‐4‐(4‐ methoxyphenyl)‐2‐oxo‐1,2‐dihydropyridine‐3,5‐dicarbonitrile (15a): M.p.: 178‐180 °C. Yield: 55%. FT‐IR (ν, cm‐1): 3300, 3150 (NH2), 2214 (C≡N), 1690 (C=O). MS (m/z, %): 550 (M+, 94.7), 508 (100), 551 (28.2), 552 (6.6). Anal. calcd. for C23H15IN6O3: C, 50.20; H, 2.75; N, 15.27. Found: C, 50.20; H, 2.75; N, 15.30%. 6‐Amino‐4‐(4‐chlorophenyl)‐1‐(6‐iodo‐2‐methyl‐4‐oxo‐ quinazolin‐3(4H)‐yl)‐2‐oxo‐1,2‐dihydropyridine‐3,5‐dicarbo‐ nitrile (15b): M.p.: >300 °C. Yield: 65%. FT‐IR (ν, cm‐1): 3285, 3200 (NH2), 2219 (C≡N), 1695 (C=O). 1H NMR (DMSO‐d6, δ, ppm): 2.40 (s, 3H, CH3), 7.53 (d, 1H, J = 8.10 Hz, ArH at C8‐ quinazoline), 7.70 (m, 4H, ArH), 8.20 (d, 1H, J = 8.10 Hz, ArH at C7‐ quinazoline), 8.43 (s, 1H, ArH at C5‐quinazoline), 9.40 (b, 2H, NH2, D2O‐exchangeable). Anal. calcd. for C22H12ClIN6O2: C, 47.63; H, 2.18; N, 15.15. Found: C, 47.60; H, 2.20; N, 15.10%. El‐Bayouki et al. / European Journal of Chemistry 2 (4) (2011) 455‐462 459 Scheme 4 2.2.11. 6‐amino‐4‐(4‐(2,2‐dicyanovinyl)phenyl)‐1‐(6‐iodo‐2‐ methyl‐4‐oxoquinazolin‐3(4H)‐yl‐2‐oxo‐1,2dihydropyridine‐ 3,5‐dicarbonitrile (16) To a mixture of 2‐cyanoacetamide 2 (0.01 mol), ter‐ phthaledehyde (0.01 mol) and malononitrile (0.02 mol) in ethanol (30 ml), few drop of pyridine was added. The reaction mixture was heated under reflux for 3 hrs. The solid product which formed while hot was collected by filtration and crystallized from dioxane to give 16 as red crystals (Scheme 3). Yield: 85%. M.p.: 258‐261 °C. FT‐IR (ν, cm‐1): 3444, 3320, 3158 (NH2), 2212 (C≡N), 1644 (C=O). MS (m/z, %): 596 (M+, 1.6), 341 (100), 286 (77.8), 246 (34.3), 114 (35.0), 90 (17.7) Anal. calcd. for C26H13IN8O2: C52.37; H, 2.20; N, 18.79. Found: C, 52.40; H, 2.20; N, 18.80%. 2.2.12. General procedure for the syntheses of chromene‐3‐ carboxamide derivatives (17‐19) A mixture cyanoacetamide 2 (0.01 mol) and salicylaldehyde, β‐naphthaldehyde or 7‐hydroxy‐5‐methoxy‐2‐ methyl‐4‐oxo‐4H‐chromone‐6‐carboxaldehyde (0.01 mol) in ethanol (30 mL) containing ammonium acetate (0.3 g) was heated under reflux for 0.5 hrs (Scheme 4). The obtained solid product which formed while hot was collected by filtration and crystallized from ethanol:dioxane (2:1) to give: 2‐Imino‐N‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐yl)‐2H‐ chromene‐3carboxamide (17): As greenish crystals. Yield: 80%. M.p.: 219‐221 °C. FT‐IR (ν, cm‐1): 3242 (NH), 1682 (C=O). 1H NMR (DMSO‐d6, δ, ppm): 2.50 (s, 3H, CH3), 6.90‐7.50 (m, 5H, [Ar‐H + NH]), 7.90 (d, 1H, J = 8.10 Hz, ArH at C8‐H), 8.10 (d, 1H, J = 8.10 Hz, ArH at C7‐H), 8.40 (s, 1H, ArH at C5‐H), 9.10 (s, 1H, CH‐chromene), 10.60 (b, 1H, NH, D2O‐exchangeable). MS (m/z, %): 472 (M+), 301 (100), 474 (2.3), 396 (2.4), 368 (5.0), 245 (19.6), 137 (20.5), 145 (12.6), 75 (11.7). Anal. calcd. for C19H13 IN4O3: C48.32; H, 2.77; N, 11.86. Found: C, 48.30; H, 3.80; N, 11.90%. 2‐Imino‐N‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐yl)‐2H‐ benzo[h]chromene‐3carboxamide (18): As greenish crystals (ethanol:dioxane; 2:1). Yield: 85%. M.p.: 254‐256 °C. FT‐IR (ν, cm‐1): 3284 (NH), 1680 (C=O). 1H NMR (DMSO‐d6, δ, ppm): 2.50 (s, 3H, CH3), 6.90‐7.50 (m, 5H, [Ar‐H + NH]), 7.90 (d, 1H, J = 8.10 Hz, ArH at C8‐H), 8.10 (d, 1H, J = 8.10 Hz, ArH at C7‐H), 8.40 (s, 1H, ArH at C5‐H), 9.10 (s, 1H, CH‐chromene), 10.60 (b, 1H, NH, D2O‐exchangeable). MS (m/z, %): 522 (M+), 301 (100), 523 (M+1; 24.5), 494 (90.3), 477 (38.7), 222 (78.7), 193 (55.3), 139 (37.6), 111 (20.6), 75 (46.6). Anal. calcd. for C23H15 IN4O3: C, 52.89; H, 2.89; N, 10.73. Found: C, 52.90; H, 2.90; N, 10.70%. 2‐Imino‐N‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐yl)‐5‐ methoxy‐8‐methyl‐6‐oxo‐2,6‐dihydropyrano[3,2‐g]chromene‐ 3carboxamide (19): As yellow crystals (dioxane). Yield: 80%. M.p.: 254‐356 °C. FT‐IR (ν, cm‐1): 3186 (NH), 1718, 1690 (C=O). MS (m/z, %): 584 (M+), 325 (100), 555 (31.0), 490 (13.6), 286 (75.9), 245 (33.0), 145 (3.5), 90 (7.1). Anal. calcd. for C24H17 IN4O6: C, 49.33; H, 2.93; N, 9.60. Found: C, 49.30; H, 2.90; N, 9.59%. 2.3. Antitumor activity against Ehrlich Ascites Carcinoma cells (EAC) [17] In vitro short term cytotoxic activity of the compounds was determined using Ehrlich Ascites Carcinoma (EAC) cells. The EAC cells that were collected from the animal peritoneum by aspiration were washed repeatedly with phosphate buffered saline (PBS) solution to free it from blood. The viability of the cells was checked in a haemocytometer. The cells (2.5 x 105 tumor cells/mL PBS) were incubated in clean sterile tubes with the test compounds (25, 50, 100 µg/mL in dimethylsulfoxide (DMSO)) for 2h at 37 °C, keeping the final volume at 0.9 mL. The volume of DMSO was pegged below 0.1% of the total volume. The control tube had 10 µL of solvent. The final volume was made up to 0.9 mL with PBS. To each tube100 µL of Tryphan blue solution was added. The live (without stain) and dead (with blue stain) cells were counted using haemocytometer and percent cell death was calculated. Exclusion test was carried out to calculate the percentage of nonviable cells. % Cytotoxicity = (Tdead- Cdead) Ttotal 100X (1) where Tdead is the number of dead cells in the treated group, Cdead is that in the control group and Ttotal is the total number of dead and live cells in the test compound treated group. Cis‐ platin was used as the standard. 2.4. Antifungal activity of some selected synthesized compounds Two test organisms were used: Aspergillus ochraceus Wilhelm and Fusarium oxysporium (local strainidentified in Regional Center for fungi and its applications in Al‐Azher University). Sterilized discs method [18] was used. Fresh stock solutions (1 mg/mL) of the synthesized compounds were prepared in redistilled dimethylsulfoxide according to the required concentrations. The discs were impregnated in DMSO. 460 El‐Bayouki et al. / European Journal of Chemistry 2 (4) (2011) 455‐462 Each disc was impregnated with 0.1 mL DMSO, so total amount of compound contained in each disk was 100 µg. Media: Dox agar was prepared and sterilized by autoclaving for 15 min at 121 °C, then the medium was cooled to 50 °C inoculated with the previously prepared fungi suspension and poured in sterilize petri dishes. The medium was allowed to solidify after a simple circular movement of the plates. Paper discs previously impregnated with the solution of compounds to be tested were placed on the plates. The plates were then incubated for 72 h at 28 °C. On each plate an appropriate reference Nystatin disc was applied. To ensure that the solvent had no effect on fungi growth; a control test was performed with test medium supplemented with DMSO as the same procedures as used in the experiments. DMSO showed no inhibition zones. Dilution method: To determine the MIC for highly active compounds dilution method [19] was used. The compounds were dissolved and then diluted using DMSO, two‐fold serial concentrations of the compounds were employed to determine the MIC ranging from 100 to 6 µg/mL. The MIC‐value was determined as the lowest concentration of the compound that completely inhibited macroscopic growth of microorganism. 3. Results and discussion The solvent‐free reaction of aryl amines with ethyl cyanoacetate is well known to constitute one of the most widely used synthetic methods. Thermal fusion of 3‐amino‐ 4(3H)‐quinazolinone [20], 1, (above its melting point) with ethyl 2‐cyanoacetate afforded 2‐cyano‐N‐(6‐iodo‐2‐methyl‐4‐ oxoquinazolin‐3(4H)‐yl) acetamide, 2. In order to probe the reactivity of the latter cyanoacetamide having active methylene moiety, its diazotization reaction was tried. Thus, upon its reaction with the desired diazonium chloride (obtained in situ by diazotization of the desired aromatic amine using a mixture of sodium nitrite and HCl) in the presence of sodium acetate; the corresponding quinazolinone hydrazono derivatives, 3a,b, were obtained. Structures of the acetamide 2 and the hydrazono derivatives 3a,b were inferred from correct analytical analyses and spectral determinations. IR spectrum of product 2 showed absorption bands at 3210 (NH), 2264 (C≡N) and 1690 cm‐1 (C=O). Its 1H NMR spectrum revealed the following signals at δ: 2.39 (s, 3H, CH3), 4.10 (m, 2H, CH2), 7.43 (d, 1H, J = 8.40 Hz, ArH at C8‐H), 8.15 (d, 1H, J = 8.40 Hz, ArH at C7‐H), 8.37 (s, 1H, ArH at C5‐H), 11.57 (s, 1H, NH, D2O‐ exchangeable). Its mass spectrum exhibited a molecular ion peak at m/z = 368 (base peak). 1H NMR spectrum of 3b showed two singlet at 11.15 and 12.30 ppm for 2 NH. Mass spectrum of 3a revealed a molecular ion peak at m/z = 472 (8.4%) with a base peak at m/z = 301. Upon stirring the cyanoacetamide 2 with carbon disulfide in the presence of potassium hydroxide in N,N‐ dimethylformamide followed by cycloalkylation with 1,2‐ dibromoethane afforded 1,3‐dithiolane derivative, 4. Also, stirring of 2 under the same reaction conditions with 1,3‐ dibromopropane yielded 2‐cyano‐2‐(1,3‐dithian‐2‐ylidene)‐N‐ (6‐iodo‐2‐methyl‐4‐oxo‐quinazolin‐3(4H)‐yl) acetamide, 5, in good yield. Furthermore, reaction of 2 with CS2 in the presence of KOH, followed by addition of dimethyl sulfate while stirring and cooling, afforded 2‐cyano‐N‐(6‐iodo‐2‐methyl‐4‐ oxoquinazolin‐3(4H)‐yl)‐3,3‐bis(methylthio)acrylamide, 6 (Scheme 1). Elemental analyses and spectroscopic data accorded well the proposed structures of the acetamide derivatives, 4, 5 and 6. IR spectra of compounds 4, 5 and 6 showed characteristic bands for NH, CH‐aliphatic, C≡N and C=O groups. 1H NMR spectrum of the compound 4 revealed signal at δ 3.75 ppm (s, 4H, 2CH2‐dithiolane), mass spectrum of compound 4 showed a molecular ion peak at m/z = 470 (22.2%) with a base peak at m/z = 170 (100%). 1H NMR spectrum of compound 5 showed signals for dithiene moiety at δ 2.20 ppm (p, 2H, J = 6.80 Hz, CH2), 3.05 (t, 2H, J = 6.60 Hz, CH2), 3.21 (t, 2H, J = 6.60 Hz, CH2). Mass spectrum of 6 showed a molecular ion peak at m/z = 472 with a base peak at m/z = 301 (100%). The reactivity of 3(4H)‐quinazolinone cyanoacetamide, 2, toward isothiocyanates was investigated. Thus, when 2 was left to react with phenyl isothiocyanate in the presence of solution of potassium hydroxide at room temperature and then chloroacetone was added; the corresponding 4‐methylthiazole derivative, 8, was obtained, as a clean cut product, in good yield without affording the expected thiophene structure of type 7. Probably the reaction mechanism is assumed to proceed via initial alkylation followed by in situ heterocyclization through nucleophilic addition of secondary amino group to carbonyl group of chloroacetone to yield the cyclic product: 2‐cyano‐N‐ (6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐yl)‐2‐(4‐methyl‐3‐ phenylthiazol‐2(3H)‐ylidene)acetamide, 8. Similarly, the novel 2‐cyano‐N‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐yl)‐2‐(4‐ oxo‐3‐phenylthiazolidin‐2‐ylidene)‐acetamide, 9, was synt‐ hesized from reaction of 2 with phenyl isothiocyanate in the presence of potassium hydroxide followed by in situ heterocyclization of the resulting adduct with ethyl chloroacetate. Also, when 2‐cyano‐N‐(6‐iodo‐2‐methyl‐4‐ oxoquinazolin‐3(4H)‐yl) acetamide, 2, was left to react with the same isothiocyanate in the presence of (CH3)2SO4 in alkaline medium, the corresponding 2‐cyano‐N‐(6‐iodo‐2‐methyl‐4‐ oxoquinazolin‐3(4H)‐yl)‐3‐(methyl‐thio)‐3‐(phenylamino)‐ acrylamide, 10, was afforded. Cyclocondensation of the acryl amide, 10, with hydrazine hydrate in refluxing ethanol furnished 5‐amino‐N‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐ yl)‐3‐(phenylamino)‐1H‐pyrazole‐4‐carboxamide, 11. Presumably, formation of the aminopyrazole 11 is assumed to proceed via Michael addition of the hydrazino amino group to the ethylenic bond side chain in 10 with elimination of SCH3 group followed by intramolecular cyclization at the cyano group. When product 11 was heated under reflux with acetylacetone in glacial acetic acid; the pyrazolo[1,5‐ a]pyrimidine derivative, 12, was afforded (Scheme 2). 1H NMR spectrum (DMSO‐d6) of 8 displayed the following signals at δ 1.93 (s, 3H, CH3 [thiazole]), 2.40 (s, 3H, CH3 [quinazoline]), 7.03 (s, 1H, CH [thiazole]), 9.93 ppm (s, 1H, NH, D2O‐exchangeable). Its mass spectrum revealed a molecular ion peak at m/z = 541 with a base peak at m/z = 214 (100%). 1H NMR spectrum of 9 revealed signals at δ = 4.00 (s, 2H, CH2[thiazole]), 10.60 ppm (s, 1H, NH (D2O‐exchangeable)). 1H NMR spectrum of 10 displayed the following signals at δ = 2.30 (s, 3H, SCH3), 2.45 (s, 3H, CH3), 10.81 ppm (s, 1H, NH, NHPh (D2O‐ exchangeable)), 11.78 (s, 1H, NH, CONH‐(D2O‐exchangeable)). Mass spectrum of 10 revealed a molecular ion peak at m/z = 517 with a base peak at m/z = 127. IR spectrum of 11 showed bands at 3311, 3199, 3159 (NH, NH2) and 1684 (C=O) cm‐1. Its 1H NMR spectrum showed signals at δ = 2.6 (s, 3H, CH3), 5.8 (s, 2H, NH2), 6.8‐8.4 (m, 8H, ArH), 9.1 (s, 1H, NH), 9.2 (s, 1H, NH), 10.0 ppm (s, 1H, NH). 1H NMR spectrum (DMSO‐d6) of 12 displayed signals a δ = 2.50 (s, 6H, 2CH3), 2.63 (s, 3H, CH3), 6.90 (t, 1H, J = 7.55 Hz, ArH at C4 of phenyl ring), 7.11 (s, 1H, CH‐pyrimidine), 7.35 (t, 2H, J = 7.55 Hz, ArH at C3,5 of phenyl ring), 7.48 (d, 1H, J = 8.40 Hz, ArH at C8 of quinazoline), 7.74 (d, 2H, J = 8.40 Hz, ArH at C2,6 of phenyl ring), 8.15 (d, 1H, J = 8.40 Hz, ArH at C7 of quinazoline), 8.38 (s, 1H, ArH at C5 of quinazoline), 9.11 (b, 1H, NH), 10.37 (b, 1H, NH). Its Mass spectrum revealed a molecular ion peak at m/z = 566 with a base peak at m/z = 265. It is well known that many 2‐pyridone derivatives exhibited diverse biological activities e.g. as cardiotonic agents, potential HIV‐1 specific reverse transcriptase inhibitors [21,22] and elastase inhibitors [23]. The present study was continued to report the reactivity of cyanoacetamide derivative 2 towards certain nucleophilic reagents. Thus, when 2 was thermally fused with acetylacetone in the presence of a catalytic amount of piperidine cyclocondensation reaction occurred and the 4,6‐ dimethyl‐2‐pyridine derivative 13 was smoothly afforded. It El‐Bayouki et al. / European Journal of Chemistry 2 (4) (2011) 455‐462 461 can be postulated that the reaction initially proceeds via a nucleophilic attack to form Michael adduct (13a) which in turn cyclized to the adduct (13b) then lost two water molecules affording 1‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐yl)‐4,6‐ dimethyl‐2‐oxo‐1,2‐dihydro‐pyridine‐3‐carbonitrile, 13, (Scheme 3). IR spectrum of the pyridine, 13, exhibited bands at 2958 (CH‐aliphatic), 2218 (C≡N) and 1680 (C=O) cm‐1. Its 1H NMR spectrum displayed the following signals 2.25 (m, 6H, 2CH3), 2.48 (s, 3H, CH3), 6.67 (s, 1H, Ar‐H), 7.54 (d, 1H, J = 8.40 Hz, ArH at C8‐H), 8.25 (d, 1H, J = 8.40 Hz, ArH at C7‐H), 8.41 (s, 1H, ArH at C5‐H). Mass spectrum revealed a molecular ion peak at m/z = 432 corresponding to a molecular formula C17H13IN4O2 together with a base peak at m/z = 416 (100%). One‐pot reactions of the cyanoacetamide derivative, 2, with malononitrile and (formaldehyde or acetaldehyde) (1:1:1 molar ratio) at reflux temperature in ethanol in the presence of piperidine afforded the 2‐pyridinone derivatives, 14a,b, respectively. On the other hand, the 2‐pyridinone derivatives 15a,b and 16 were obtained via reaction of cyanoacetamide, 2, with 2‐(4‐chlorobenzylidene) malononitrile or 2‐(4‐methoxy‐ benzylidene)malononitrile (1:1:1 molar ratio) or with ter‐ phthaldehyde and malononitrile (1:1:2 molar ratio) upon heating under reflux in the presence of a catalyst. Structural assignment of the pyridinones 14a,b, 15a,b and 16 was confirmed on the basis of correct elemental analyses and spectral determination. IR spectrum of 14b showed bands at 3353, 3204 cm‐1 for NH2 group. 1H NMR spectrum of 14b revealed signal at 2.20 (s, 3H, CH3‐quniazoline), 2.50 (s, 3H, CH3‐pyridine), 7.53 (d, 1H, J = 8.40 Hz, ArH at C8‐H), 8.15 (d, 1H, J = 8.70 Hz, ArH at C7‐H), 8.40 (s, 1H, ArH at C5‐H), 9.22 (b, 2H, NH2) ppm. Mass spectrum of 15a showed a molecular ion peak at m/z = 550 (94.70%) with base beaks at m/z = 508. Also, one pot reaction of cyanoacetamide derivative 2 with ter‐ phthaldehyde and malononitrile (1:1:2 molar ratio) at reflux temperature in ethanol in the presence of piperidine (few drops) afforded the 2‐pyridinone derivative, 16, (Scheme 3). Mass spectrum of 16 showed a molecular ion peak at m/z = 596 (1.6%) corresponding to a molecular formula, C26H13IN8O2, with a base peak at m/z = 341 (100 %). Chromene derivatives are widely used for production of highly effective fluorescent dyes for synthetic fibers and daylight fluorescent pigments [24,25]. Some derivatives play also a vital role in electro photographic and electroluminescent devices [26] Moreover, many other derivatives are well known for their considerable biological and medicinal activities [27]. Cyclocondensation reaction of 2 with salicylaldehyde or 2‐ hydroxynaphthaldehyde or 7‐hydroxy‐5‐methoxy‐2‐methyl‐4‐ oxo‐4H‐chromone‐6‐carboxaldehyde in ethanol containing ammonium acetate furnished smoothly 2‐imino‐N‐(6‐iodo‐2‐ methyl‐4‐oxoquinazolin‐3(4H)‐yl)‐2H‐chromene‐3‐carbox‐ amide, 17, 2‐imino‐N‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐ 3(4H)‐yl)‐2H‐benzo[H]chromene‐3‐carbox amide, 18, and 2‐ imino‐N‐(6‐iodo‐2‐methyl‐4‐oxoquinazolin‐3(4H)‐yl)‐5‐met‐ hoxy‐8‐methyl‐6‐oxo‐2,6‐dihydropyrano[3,2‐g]‐ chromene‐3‐ carboxamide, 19, respectively (Scheme 4). 1H NMR spectrum of 17 showed characteristic signals at 9.11 for CH‐chromene and at 10.60 ppm for NH. Mass spectrum of 17 exhibited a molecular ion peak at m/z = 472. Mass spectrum of compound 18 exhibited a molecular ion peak at m/z = 522. IR spectrum of the pyrano‐chromene 19 showed absorption bands at 3186 and 1718 cm‐1 for NH and C=O, respectively. Its mass spectrum revealed molecular ion peak at m/z = 584. Some selected examples from the synthesized products were tested using the short term in vitro cytotoxicity towards Ehrlich ascites carcinoma cells (EAC) as a preliminary screening technique of tryphan blue exclusion method (cell viability test) for their potential cytotoxicity activity using 100, 50 and 25 µg/mL concentrations. Structure activity relationship (SAR) indicated that the thio compounds 4, 8 and 10 showed that the quinazolinone derivative 4 with C‐3 side chain having dithiolane moiety was found of no activity at the used concentrations. Product 8 with 3‐side chain incorporated with substituted thiazole moiety was found to be of high to moderate activity towards cells at 100 (50%) and 50 (20%) µg/mL, respectively. Presumably the activity showed by compound 8 was due to the presence of the thiazole ring in its structure. On the other hand, product 10 with methyl mercapto group attached in its 3‐position was of low activity (10%) only at 100 µg/mL (Table 1). Table 1. Antitumor activity of the screened compounds. Compound No. % Cell death at different concentrations (µg/mL) after 2 h 100 50 25 4 0 0 0 8 50 20 0 10 10 0 0 The preliminary in vitro antifungal activity screening for some selected examples of the synthesized compounds was carried out using paper disc method against Aspergillus ochraceus Wilhelm and Fusarium oxysporium fungi. Fresh stock solutions (1 mg/mL) of the tested compounds were prepared in redistilled DMSO according to the required concentrations. Serial concentrations of the compounds were employed to determine the (MIC) ranging from 100 to 6.25 μg/mL. The incubation for impregnated discs was 72 h at 28 °C. The antifungal Nystatin and the DMSO solvent were used as positive and negative controls, respectively. The results showed compound 8 incorporating a thiazole moiety and the chromene product 19 were only of high activity against Fusarium oxysporium with inhibition zones (18 mm) and (16 mm), respectively, compared with (20 mm) Nystatin inhibition zone. MIC of compounds 8, 19 was 12.5 μg/mL. All the other tested compounds showed no activity towards the used fungi (Table 2). Table 2. Antimicrobial activities data of some synthesized compounds. Compound No. Fungi Asperg. Sp. Fuzarium sp. 8 0 18 (12.5) 13 0 0 15a 0 0 19 0 16 (12.5) Nystatin 12 20 4. Conclusion Novel 4(3H)‐quinazolinone derivatives bearing thiazole, pyrazole, 1,3‐dithiazole, pyridine, pyrazolopyrimidine, chromene and pyranochromene moieties were synthesized and characterized. 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