ChemistryAll melting points were measured on a Griffin and George melting-point apparatus (Griffin & Georgy Ltd., Wembley, Middlesex, UK) and are uncorrected. IR spectra were recorded on the Pye Unicam SP1200 spectrophotometer (Pye Unicam Ltd., Cambridge, UK) by using the KBr wafer technique. 1H-NMR spectra were determined on a Varian Gemini 300 MHz on Bruker Avance III using tetramethylsilane as an internal standard (chemical shifts in δ scale), while 13C NMR spectra were run at 75 MHz. EI-MS was measured on a Shimadzu GC–MS (Columbia, MD) operating at 70 eV. Elemental analyses were carried out at the Microanalytical Unit, Faculty of Science, Ain Shams University, using a Perkin-Elmer 2400 CHN elemental analyzer (Waltham, MA), and satisfactory analytical data (± 0.4) were obtained for all compounds. The homogeneity of the synthesized compounds was controlled by thin layer chromatography (TLC), using aluminum sheet silica gel F254 (Merck).2-Amino-4-(2-chlorophenyl)-4H-benzo[h]chromene-3-carbonitrile 1a and 2-amino-4-(4-chlorophenyl)-4H-benzo[h]chromene-3-carbonitrile 1bTypical reaction procedure for the synthesis of naphthopyrane (multi-component step): 1-naphthol (5 g, 3.4 mmol) was mixed with malononitrile (1.88 mL, 3.4 mmol), o-chlorobenzaldehyde and/or p-chlorobenzaldehyde (3.8 mL, 3.4 mmol) in (50 mL) absolute ethanol containing piperidine (2 mL). The mixture was allowed to reflux for about 7 h. The obtained solid was filtered off and washed with ethanol then recrystallized from dioxane to give 1a and 1b, respectively.1a: brown crystals, yield: 91%, m.p. 239–240 0C (Lit. m.p. 238–240)39.1b: pale yellow crystals, yield: 86%, m.p. 234–235 0C (Lit. m.p. 230–232)39,40.7-(2-Chlorophenyl)-10-methyl-7,9-dihydro-8H-benzo-[7,8]chromeno[2,3-d]pyrimidin-8-one 2A solution of enaminonitrile 1a (1 g, 3 mmol) in acetic anhydride (20 mL) was heated under reflux for 12 h. The formed solid while reflux was filtered off, dried, and then crystallized from dioxane to give 2 as buff crystals; yield: 55%, m.p. > 300 oC. IR (KBr, cm-−1): 3156 (NH), 1655 (C=O). 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 2.34 (s, 3H, CH3), 5.70 (s, 1H, C4-pyran), 7.17–7.65 (m, 8H, Ar–H), 7.86 (d, 1H, Ar–H, J = 8.1 Hz), 8.23 (d, 1H, Ar–H, J = 8.1 Hz), 12.50 (s, 1H, NH, exchangeable with D2O). 13C-NMR (DMSO-d6) δ (ppm): 20.97, 35.85, 98.81, 117.78, 120.75, 123.07, 124.41, 125.63, 126.87, 126.93, 127.65, 127.71, 128.29, 129.51, 130.96, 131.88, 132.81, 142.66, 143.40, 159.01, 161.55, 162.18. MS (m/z, %): 376 (M+. + 2, 29.7%), 374 (M+., 25.71%), 217 (100%), 110 (36.12%), 72 (83.97%) Anal. Calc. for C22H15ClN2O2 (374.84): C, 70.50; H, 4.03; Cl, 9.46; N, 7.47. Found: C, 70.58; H, 4.13; Cl, 9.26; N, 7.56.10-(Chloromethyl)-7-(4-chlorophenyl)-7,9-dihydro-8H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-one 3A solution of enaminonitrile 1b (1 g, 3 mmol) and chloroacetyl chloride (0.23 mL, 3 mmol) in dioxane (15 mL) was heated under reflux for 6 h. The reaction mixture was cooled and then poured into ice-cold water. The obtained solid was filtrated off and crystallized from benzene to give 3 as pale-yellow crystals; yield: 74%, m.p: 294–295 oC. IR (KBr, cm-1): 3320 (br, NH), 2959, 2851(CH alph.), 1657 (C=O), 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 4.54 (s, 2H, CH2), 5.32 (s, 1H, C4-pyran), 7.25–7.67 (m, 8H, Ar–H), 7.90 (d, 1H, Ar–H, J = 7.8 Hz), 8.26 (d, 1H, Ar–H, J = 8.1 Hz), 13.02 (br s, 1H, NH, exchangeable with D2O). 13C-NMR (300 MHz, DMSO-d6) δ (ppm): 38.02, 42.13, 101.69, 118.23, 120.68, 123.05, 124.65, 126.51, 126.90, 126.98, 127.76, 128.38, 128.80, 129.42, 129.89, 131.28, 132.83, 143.54, 143.97, 156.61, 161.05, 162.09. MS (m/z, %): 411 (M+. + 2, 15.44%), 409 (M+., 18.22%), 36 (35.09%), 295 (100%), 161 (74.94%), 149 (48.47%), 106 (73.54%), 75 (43.53%). Anal. Calc. for C22H14Cl2N2O2 (409.27): C, 64.56; H, 3.45; Cl, 17.32; N, 6.84. Found: C, 64.65; H, 3.49; Cl, 17.22; N, 6.90.7-(2-Chlorophenyl)-10-methyl-7H,8H-benzo[7,8]chromeno[2,3-d] [1,3]oxazin-8-one 4A solution of enaminonitrile 1a (1 g, 3 mmol) in acetyl chloride (20 mL) was refluxed for 18 h on a water bath. The excess solvent was evaporated and the solid formed was crystallized from benzene to give 4 as brown crystals; yield: 53%, m.p. 225–228 0C. IR (KBr, cm−1): 1732 (C = O), 1656 (C=N). 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 2.59 (s, 3H, CH3), 5.92 (s, 1H, C4-pyran), 7.18–7.89 (m, 9H, Ar–H), 8.26 (d, 1H, Ar–H, J = 8.4 Hz). 13C-NMR (DMSO-d6) δ (ppm): 24.97, 38.65, 111.38, 117.11, 120.68, 122.85, 124.90, 125.25, 127.25, 127.79, 128.10, 128.30, 129.17, 129.88, 131.89, 132.97, 140.97, 160.26, 167.08. MS (m/z, %): 377 (M+. + 2, 7.87%), 375 (M+., 12.56%), 261(100%), 218 (56.87), 179 (47.61%), 143 (90.03%), 133 (50.17%). Anal. Calc. for C22H14ClN2O3 (375.81): C, 70.31; H, 3.75; Cl, 9.43; N, 3.73. Found: C, 70.23; H, 3.91; Cl, 9.54; N, 3.81.Ethyl N-[4-(2-chlorophenyl)-3-cyano-4H-benzo[h]chromen-2-yl] formimidate 5a and Ethyl N-[4-(4-chlorophenyl)-3-cyano-4H-benzo[h]chromen-2-yl] formimidate 5bA solution of enaminonitrile 1a or 1b (1 g, 3 mmol) in triethylorthoformate (20 mL) was heated under reflux for 8 h. The reaction solution was evaporated till dryness. The obtained solid was filtrated off and crystallized from ethanol to give 5a and 5b, respectively.5a: yellow crystals; yield: 79%, m.p. 177–179 0C. IR (KBr, cm-1): 2207 (C≡N), 1656 (C=N). 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 1.21 (Syn), 1.36 (Anti) (2t, 3H, CH2CH3, J = 6.9 Hz), 4.13 (Syn), 4.38 (Anti) (2q, 2H, CH2CH3, J = 6.9 Hz), 5.63 (s, 1H, C4-pyran), 6.97 (d, 1H, Ar–H, J = 8.7 Hz), 7.30–7.65 (m, 7H, Ar–H), 7.88 (d, 1H, Ar–H, J = 9 Hz), 8.36 (d, 1H, Ar–H, J = 7.2 Hz), 8.18 (Syn), 8.94 (Anti) (2 s, 1H, N = CH). 13C-NMR (300 MHz, DMSO-d6) δ (ppm): 13.92, 18.57, 54.01, 78.75, 115.37, 117.63, 121.32, 122.83, 124.93, 125.24, 127.04, 127.15, 127.65, 128.15, 129.50, 130.01, 131.82, 132.28, 133.01, 140.50, 143.12, 157.72, 161.92. MS (m/z, %): 390 (M+. + 2, 16.04%), 388 (M+., 23.09%), 375 (72%), 349 (52.68%), 254 (48.50%), 247 (100%), 245 (61.71%), 232 (63.75%), 204 (92.47%), 161 (43.96%), 134 (30.74%) Anal. Calc. for C23H17ClN2O2 (388.85): C, 71.04; H, 4.41; Cl, 9.12; N, 7.20. Found: C, 71.20; H, 4.50; Cl, 9.26; N, 7.11.5b: pale brown crystals; yield: 73%, m.p: 170–171 0C. IR (KBr, cm−1): 2205 (C≡N), 1654 (C=N). 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 1.36 (t, 3H, CH2CH3, J = 6.9 Hz), 4.42 (q, 2H, CH2CH3, J = 6.9 Hz), 5.22 (s, 1H, C4-pyran), 7.07 (d, 1H, Ar–H, J = 8.4 Hz), 7.34–7.67 (m, 7H, Ar–H), 7.89 (d, 1H, Ar–H, J = 8.4 Hz), 8.36 (d, 1H, Ar–H, J = 7.5 Hz), 8.94 (s, 1H, N=CH). 13C-NMR (300 MHz, DMSO-d6) δ (ppm): 13.92, 41.23, 63.99, 79.80, 116.06, 117.89, 121.31, 122.92, 124.84, 125.94, 127.05, 127.13, 127.66, 128.92, 130.13, 132.17, 132.95, 142.86, 142.99, 157.43, 162.01. MS (m/z, %): 390 (M+. + 2, 7.51%), 388 (M+., 10.95%), 380 (53.96%), 328 (62.27%), 286 (62.44%), 273 (61.03%), 147 (100%). Anal. Calc. for C23H17ClN2O2 (388.85): C, 71.04; H, 4.41; Cl, 9.12; N, 7.20. Found: C, 71.13; H, 4.21; Cl, 9.18; N, 7.04.N’-{4-(2-Chlorophenyl)-3-cyano-4H-benzo[h]chromen-2-yl}-N-(furan-2-ylmethyl) formimidamide 6A solution of 5a (1 g, 2.5 mmol) in dioxane (15 mL) and 2-furanylmethanamine (0.22 mL, 2.5 mmol) was refluxed for 12 h. The reaction mixture was cooled and then poured onto ice-cold water. The obtained solid was filtrated off and crystallized from benzene to give 6 as yellow crystals, yield: 82%, m.p. 138–139 0C. IR (KBr, cm-1): 3432 (NH), 2191 (C≡N), 1657 (C=N). 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 5.40 (s, 1H, C4-pyran), 5.83 (s, 2H, CH2), 6.13 (s, 1H, NH exchangeable with D2O), 6.29 (d, 1H, furan ring), 6.97–8.31 (m, 12H, 10Ar-H + 2H furan ring), 8.33 (s, 1H, N=CH). 13C-NMR (300 MHz, DMSO-d6) δ (ppm): 36.69, 37.93, 95.36, 106.08, 110.31, 116.7, 120.73, 122.64, 123.02, 124.01, 125.4, 126.73, 126.88, 126.94, 127.23, 127.71, 127.92, 128.17, 128.84, 129.29, 129.78, 130.14, 130.37, 131.19, 131.76, 131.95,132.81, 132.93, 140.09, 142.06, 142.18, 143.84, 152, 156.69, 160.36, 160.48, 162.08 . MS (m/z, %): 441 (M+. + 2, 24.71%), 439 (M+., 18.37%), 330 (100%), 234 (60.20%), 295 (90.28%), 165(32.71%), 122 (49.68%), 65 (34.37%) Anal. Calc. for C26H18ClN3O2 (439.90): C, 70.99; H, 4.12; Cl, 8.06; N, 9.55. Found: C, 70.85; H, 4.30; Cl, 8.26; N, 9.44.N’-{4-(2-Chlorophenyl)-3-cyano-4H-benzo[h]chromen-2-yl}-N-(pyridin-2-yl) formamidine 7A solution of 5a (1 g, 2.5 mmol) in dioxane (15 mL) and 2-aminopyridine (0.24 mL, 2.5 mmol) was refluxed for 24 h. The reaction mixture was cooled and then poured onto ice-cold water. The obtained solid was filtrated off and crystallized from ethanol to give 7 as pale-yellow crystals, yield: 65%, m.p. 222–224 0C. IR (KBr, cm−1): 3397 (NH), 2191 (C≡N), 1644 (C=N). 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 6.27 (s, 1H, C4-pyran), 7.00–7.91 (m, 11H, 8H Ar–H + 3H pyridine), 8.09–8.12 (m, 2H, C6-pyridine moiety + NH exchangeable with D2O), 8.26–8.33 (m, 2H, Ar–H), 8.57 (s, 1H, N = CH). 13C-NMR (300 MHz, DMSO-d6) δ (ppm): 36.39, 98.74, 113.66, 116.92, 118.70, 120.78, 122.97, 124.39, 125.56, 127.07, 127.78, 127.96, 128.29, 129.55, 130.39, 131.57, 131.79,132.97, 137.99, 140.41, 143.43, 148.00, 151.90, 156.59, 157.93, 162.85. MS (m/z, %): 439 (M+. + 2, 1.54%), 437 (M+., 10.89%), 404 (42.40), 394 (38.26%), 360 (30.35%), 254 (35.13%), 105 (100%), 94 (38.17%). Anal. Calc. for C26H17ClN4O (436.90): C, 71.48; H, 3.92; Cl, 8.11; N, 12.82. Found: C, 71.60; H, 3.82; Cl, 8.29; N, 12.88.7-(2-Chlorophenyl)-8-imino-7H-benzo[7,8]chromeno[2,3-d]-pyrimidin-9(8H)-amine 8A solution of 5a (1 g, 2.5 mmol) in excess hydrazine hydrate (3 mL) was stirred at room temperature for 10 h. The formed solid was filtered off, dried, and crystallized from toluene to give 8 as pale brown crystals, yield: 70%, m.p: 202–205 0C. IR (KBr, cm−1): 3336, 3319, 3284, 3180 (NH, NH2), 1652 (C=N). 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 5.74 (s, 2H, NH2 exchangeable with D2O), 5.79 (s, 1H, C4-pyran), 6.80 (br.s, 1H, NH exchangeable with D2O), 7.18–7.67 (m, 8H, Ar–H), 7.88 (d, 1H, Ar–H, J = 8.1 Hz), 8.23 (s, H, Ar–H), 8.25 (s, 1H, pyrimidine-H). 13C-NMR (300 MHz, DMSO-d6) δ (ppm): 37.21, 97.38, 116.91, 120.77, 122.98, 124.39, 125.50, 126.91, 127.73, 127.97, 128.83, 129.78, 131.10, 131.98, 132.85, 141.42, 143.29, 151.22, 155.04, 156.74. MS (m/z, %): 376 (M+. + 2, 27.86%), 374 (M+., 28.40%), 353 (95.12%), 347 (82.94%), 342 (100%), 287 (47.04%), 207 (61.97%). Anal. Calc. for C21H15ClN4O (374.83): C, 67.29; H, 4.03; Cl, 9.46; N, 14.95. Found: C, 67.14; H, 4.23; Cl, 9.52; N, 15.07.1-[{4-(2-Chlorophenyl)-3-cyano-4H-benzo[h]chromen-2-ylimino} methyl]thiosemicarbazide 9A mixture of iminoether derivative 5a (1 g, 2.5 mmol) and thiosemicarbazide (0.23 g, 2.5 mmol) in dioxane (20 mL) was heated under reflux for 15 h. The formed solid while reflux was filtered off, dried, and then crystallized from ethanol to give 9 as brown crystals, yield: 73%, m.p. 256–259 °C. IR (KBr, cm−1): 3467, 3308, 3155 (NH2, 2NH), 2191 (CN), 1645 (C = N), 1261 (C=S). 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 5.76 (s, 1H, C4-pyran), 6.35 (s, 1H, NH exchangeable with D2O), 6.98–7.41 (m, 7H, 5Ar-H + NH2 exchangeable with D2O), 7.54–7.74 (m, 3H, 3Ar-H + NH exchangeable with D2O), 7.87 (d, 1H, Ar–H, J = 8.1 Hz), 8.23 (s, 1H, N = CH), 8.29 (d, 1H, Ar–H, J = 8.1 Hz). 13C-NMR (300 MHz, DMSO-d6) δ (ppm): 37.04, 94.48, 116.95, 120.85, 123.06, 123.94, 125.74, 126.85, 126.92, 127.71, 127.94, 129.23, 130.34, 131.76, 131.95, 132.92, 140.26, 143.95, 156.90, 162.41, 162.72. MS (m/z, %): 435 (M+. + 2, 20.23%), 433 (M+., 35.18%), 412 (63.51), 399 (54.78%), 383 (50.44%), 334 (66.04%), 320 (70.40%), 303 (88.43%), 242 (56.11%), 198 (63.63%), 191 (74.85%), 168 (74.94%), 164 (100%), 120 (48.24%), 118 (82.67%), 112 (89.93%), 110 (84.68%), 92 (56.39%), 87 (42.86%), 72 (54.82%). Anal. Calc. for C22H16ClN5OS (433.91): C, 60.90; H, 3.72; Cl, 8.17; N, 16.14; S, 7.39. Found: C, 60.98; H, 3.57; Cl, 8.07; N, 16.32; S, 7.31.N’-{[(4-(2-Chlorophenyl)-3-cyano-4H-benzo[h]chromen-2-ylimino]methyl}-2-hydroxybenzo hydrazide 10A mixture of imnioether 5a (1 g, 2.5 mmol) and salicylhydrazide (0.39 g, 2.5 mmol) in dioxane (15 mL) was heated under reflux for 20 h. The reaction mixture was cooled and then poured onto ice-cold water. The obtained solid was filtrated off then boiled with petroleum ether and then crystallized from benzene to give 10 as pale brown crystals, yield: 75%, m.p. 253–256 °C. IR (KBr, cm-1): br. 3184 (NH, OH), 2192 (C≡N), 1660 (C=O). 1H-NMR (DMSO-d6) (δ ppm): 6.27 (s, 2H, C4-pyran + NH exchangeable with D2O), 6.94–8.34 (m, 15H, 14Ar-H + NH exchangeable with D2O), 9.74 (s, 1H, N = CH), 10.92 (s, 1H, OH, exchangeable with D2O). 13C-NMR (300 MHz, DMSO-d6) δ (ppm): 37.13, 38.21, 54.85, 114.96, 116.61, 117.30, 119.30, 119.31,119.77, 120.22, 120.28, 120.78, 121.63, 122.68, 124.14, 125.49, 126.82, 126.97, 127.29, 128.03,128.86, 128.94, 129.29, 129.43, 129.56, 129.69, 129.86, 131.26, 132.03, 132.86, 133.12, 133.52, 134.15, 136.57, 140.91, 142.22, 157.67, 158.54, 160.41, 165.80. MS (m/z, %): 496 (M+. + 2, 8.20%), 494 (M+., 10.73%), 314 (19.84%), 289 (100%), 252 (48.07%), 194 (34.89%), 139 (41.68%). Anal. Calc. for C28H19ClN4O3 (494.94): C, 67.95; H, 3.87; Cl, 7.16; N, 11.32. Found: C, 67.75; H, 3.95; Cl, 7.20; N, 11.18.N-{7-(2-Chlorophenyl)-8-imino-7H-benzo[7,8]chromeno[2,3-d]-pyrimidin-9(8H)-yl}-4-methylbenzenesulfonamide 11a and N-[7-(4-Chlorophenyl)-8-imino-7H-benzo[7,8]chromeno[2,3-d]pyrimidin-9(8H)-yl]-4-methylbenzenesulfonamide 11bA mixture of 5a or 5b (1 g, 2.5 mmol) and p-toluene sulphonohydrazide (0.48 g, 2.5 mmol) in dioxane (20 mL) was heated under reflux for 4 h. The formed solid while refluxing in each case was filtered off, dried, and crystallized from dioxane to give 11a and 11b, respectively.11a: white crystals, yield: 60%, m.p. 279–280 °C. IR (KBr, cm−1): 3444, 3233 (2NH), 1648 (C=N). 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 2.13 (s, 3H, CH3), 5.64 (s, 1H, C4-pyran), 6.85–6.87 (m, 2H, Ar–H), 7.00–7.03 (m, 1H, Ar–H), 7.20–7.48 (m, 6H, Ar–H), 7.59–7.61 (m, 3H, Ar–H), 7.76–7.86 (m, 3H, 1 Ar–H + 2NH exchangeable with D2O), 8.21–8.23 (m, 1H, Ar–H), 8.63 (s, 1H, pyrimidine-H). 13C-NMR (300 MHz, DMSO-d6) δ (ppm): 20.91, 38.38, 95.06, 114.79, 120.60, 122.54, 125.12, 125.43, 125.68, 127.35, 127.40, 127.86, 128.07, 128.89, 129.65, 131.34, 132.44, 132.69, 133.00, 137.34, 140.16, 140.48, 143.22, 153.15, 154.88, 159.38. MS (m/z, %): 531 (M + . + 2, 8.71%), 529 (M + ., 26.58%), 432 (50.86%), 312 (30.76%), 192 (69.75%), 82 (52.39%), 76 (100%), 65 (44.12%). Anal. Calc. for C28H21ClN4O3S (529.01): C, 63.57; H, 4.00; Cl, 6.70; N, 10.59; S, 6.06. Found: C, 63.37; H, 3.92; Cl, 6.84; N, 10.50; S, 6.18.11b: white crystals, yield: 83%, m.p. 280–282 °C. IR (KBr, cm-1): 3443 (NH), 1651(C=N). 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 2.20 (s, 3H, CH3), 5.41 (s, 1H, C4-pyran), 6.89 (d, 1H, Ar–H, J = 8.1 Hz), 7.21–7.35 (m, 8H, Ar–H), 7.57–7.70 (m, 3H, Ar–H), 7.91 (d, 1H, Ar–H, J = 7.8 Hz), 8.06 (br.s, 2H, 2NH exchangeable with D2O), 8.25 (d, 1H, Ar–H, J = 7.8 Hz), 8.62 (s, 1H, pyrimidine-H). 13C-NMR (300 MHz, DMSO-d6) δ (ppm): 20.76, 37.65, 96.66, 117.63, 120.46, 122.89, 125.46, 125.82, 127.17, 127.35, 127.82, 128.72, 128.80, 129.39, 131.98, 132.82, 140.13, 140.77, 141.25, 142.68, 152.99, 154.91, 158.90. MS (m/z, %): 531 (M + . + 2, 13.14%), 529 (M + ., 57.35%), 510 (87.64%), 417 (100%), 376 (63.78%), 237 (56.82%), 131 (48.89%). Anal. Calc. for C28H21ClN4O3S (529.01): C, 63.57; H, 4.00; Cl, 6.70; N, 10.59; S, 6.06. Found: C, 63.44; H, 4.05; Cl, 6.79; N, 10.51; S, 6.20.14-(4-Chlorophenyl)-2-phenyl-14H-benzo[7,8]chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine 12A mixture of imnioether 5b (1 g, 2.5 mmol) and benzoylhydrazide (0.34 g, 2.5 mmol) in dioxane (15 mL) was heated under reflux for 15 h. The reaction mixture was cooled and then poured onto ice-cold water. The obtained solid was filtrated off and crystallized from dioxane to give 12 as pale-yellow crystals; yield: 65%, m.p: 296–297 °C. IR (KBr, cm−1): 3046 (CH-Ar), 1619 (C = N). 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 5.98 (s, 1H, C4-pyran), 7.30–7.74 (m, 12H, Ar–H), 7.94 (d, 1H, Ar–H, J = 7.5 Hz), 8.16–8.17 (m, 1H, Ar–H), 8.36 (d, 1H, Ar–H, J = 8.1 Hz), 9.69 (s, 1H, pyrimidine-H). MS (m/z, %): 462 (M+. + 2, 6.48%), 460 (M+., 10.95%), 361 (58.62%), 321 (52.46%), 298 (86.61%), 40 (100%). Anal. Calc. for C28H17ClN4O (460.92): C, 72.96; H, 3.72; Cl, 7.69; N, 12.16. Found: C, 73.00; H, 3.82; Cl, 7.60; N, 12.21.2-{14-(2-Chlorophenyl)-14H-benzo[7,8]chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-2-yl}acetonitrile 13A mixture of imnioether derivative 5a (1 g, 2.5 mmol) and cyanoacetohydrazide (0.25 g, 2.5 mmol) in dioxane (20 mL) was heated at reflux for 20 h. The reaction mixture was concentrated and allowed to cool. The residue was poured onto ice-cold water and the formed solid was filtered off, dried, and then crystallized from ethanol to give 13 as yellow crystals, yield: 62%, m.p. 156–159 °C. IR (KBr, cm−1): 3340 (NH), 2261, 2192 (C≡N), 1658 (C=N). 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 3.58 (s, 2H, CH2CN), 4.39 (s, 1H, NH, exchangeable with D2O), 5.40 (s, 1H, C4-pyran), 6.27 (s, 1H, = CH), 6.98–8.23 (m, 10H, Ar–H), 9.73 (s, 1H, pyrimidine-H). MS (m/z, %): 425 (M+. + 2, 15.85%), 423 (M+., 4.17%), 413 (63.26%), 375 (41.30%), 303 (48.44%), 179 (70.39%), 75 (30.43%), 62 (100%). Anal. Calc. for C24H14ClN5O (423.86): C, 68.01; H, 3.33; Cl, 8.36; N, 16.52. Found: C, 68.09; H, 3.37; Cl, 8.26; N, 16.48.2-{14-(2-Chlorophenyl)-14H-benzo[7,8]chromeno[3,2-e][1,2,4]-triazolo[1,5-c]pyrimidin-2-y}-3-(4-methoxyphenyl) acrylonitrile 14A mixture of 13 (1 g, 2.3 mmol) and p-methoxybenzaldehyde (0.28 mL, 2.3 mmol) in dioxane (15 mL) containing drops of piperidine was refluxed for 9 h. The formed solid while heating was filtered off, dried, and then crystallized from dioxane/DMF (2:1) to give 14 as yellow crystals, yield: 52%, m.p. > 300 °C. IR (KBr, cm−1): 2221 (C≡N), 1626 (C=N). 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 3.83 (s, 3H, OCH3), 6.24 (s, 1H, C4-pyran), 7.08–7.93 (m, 13H, Ar–H), 8.15 (s, 1H, C = CH), 8.30 (d, 1H, Ar–H, J = 7.8 Hz), 9.69 (s, 1H, pyrimidine-H). MS (m/z, %): 544 (M+. + 2, 17.55%), 542 (M+., 27.25%), 514 (40.57%), 497 (30.49%), 464 (34.08%), 441 (100%), 397 (31.98%), 252 (54.39%), 141 (40.41%), 134 (74.54%), 107 (65.92%). Anal. Calc. for C32H20ClN5O2 (542): C, 70.91; H, 3.72; Cl, 6.54; N, 12.92. Found: C, 70.77; H, 3.76; Cl, 6.45; N, 12.97.3-[14-(2-Chlorophenyl)-14H-benzo[7,8]chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-2-yl]-2H-chromen-2-one 15A mixture of 13 (1 g, 2.3 mmol) and salicylaldehyde (0.24 mL, 2.3 mmol) in dioxane (20 mL) containing drops of piperidine was refluxed for 5 h. The formed solid while heating was filtered off, dried, and then crystallized from dioxane/DMF (2:1) to give 15 as pale-yellow crystals, yield: 53%, m.p. > 300 °C. IR (KBr, cm-−1): 1747 (CO), 1644 (C=N). 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 6.32 (s, 1H, C4-pyran), 7.01–8.34 (m, 14H, Ar–H), 8.31 (d, 1H, Ar–H), 8.63 (s, 1H, chromenone ring), 9.77 (s, 1H, pyrimidine-H). MS (m/z, %): 530 (M+. + 2, 18.60%), 528 (M+., 31.89%), 496 (40.38%), 386 (48.11%), 257 (37.17%), 96 (90.02%), 129 (46.64%), 59 (100%). Anal. Calc. for C31H17ClN4O3 (528.95): C, 70.39; H, 3.24; Cl, 6.70; N, 10.59. Found: C, 70.54; H, 3.31; Cl, 6.81; N, 10.55.14-(2-Chlorophenyl)-N-(2-hydroxyphenyl)-14H-benzo[7,8]chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine-2-carbohydrazonoyl cyanide 16To o-amino phenol (1.1 g, 10 mmol), concentrated HCl (3 mL) was added and cooled to ~ 0–5 °C in an ice bath then cooled sodium nitrite solution (1.0 g in 10 mL of water) was added to the mixture dropwise during 10 min. The reaction mixture was then stirred for 10 min. A cold mixture of the acetonitrile derivative 13 (4.35 g, 10 mmol) and sodium acetate (4.10 g, 50 mmol) in ethanol (50 mL), was then added dropwise to the reaction mixture with stirring. The stirring was continued for 30 min, and the reaction mixture was left for 1 h at room temperature. The solid product obtained was collected by filtration and crystallized from ethanol to give 16 as reddish-brown crystals, yield: 61%, m.p: 182–183 oC. IR (KBr, cm-1): br. 3427 (OH, NH), 2258 (C≡N), 1625 (C = N). 1H- NMR (300 MHz, DMSO-d6) δ (ppm): 3.42 (br s, 2H, NH + OH, exchangeable with D2O), 6.11(s, 1H, C4-pyran), 7.14–7.64 (m, 12H, Ar–H), 7.83 (d, 1H, Ar–H, J = 7.8 Hz), 8.26 (d, 1H, Ar–H, J = 8.1 Hz), 9.67 (s, 1H, pyrimidine-H). 13C-NMR (300 MHz, DMSO-d6) δ (ppm): 37.18, 116.05, 116.38, 120.55, 122.86, 124.67, 125.20, 126.99, 127.63, 127.78, 128.95, 129.80, 131.66, 131.90, 132.85, 140.53, 141.02, 143.34, 152.81, 154.34, 160.90. MS (m/z, %): 545 (M+. + 2, 14.13%), 543 (M+., 18.93%), 422 (58.61%), 360 (73.98%), 335 (76.45%), 258 (50.47%), 264 (55.33%), 234 (100%), 215 (47.95%), 155 (68.82%), 122 (34.45%), 68 (42.01%), 41 (78.09%). Anal. Calc. for C30H18ClN7O2 (543.97): C, 66.24; H, 3.34; Cl, 6.52; N, 18.02. Found: C, 66.32; H, 3.46; Cl, 6.43; N, 18.16.Biological evaluationMosquito larval colonyThe laboratory strain of C. pipiens was raised and maintained for roughly 24 generations in an insectary at the Entomology Department of the Faculty of Science, Ain Shams University, using the recommended techniques, under controlled conditions at 27 ± 2 °C and RH 75%, and photoperiod 12:12 light: dark hours57. TetraMin was administered to the newly hatched larvae. The pupae were placed within the (25 × 30 × 25) cm wooden cages before being moved. Adults received a 10% sucrose solution every day. The females were permitted to consume a meal of blood provided by a pigeon host58.Biological assayThe World Health Organization’s recommended larval bioassay test protocol was followed for conducting the experiments in a lab setting59. Twenty third-instar C. pipiens larvae were given a variety of insecticidal concentrations of the investigated chemicals ranging from 100 mg/L to 1000 mg/L. Before being diluted with water, the investigated substances were solubilized in dimethylformamide (DMF). Three replicates were used for each concentration, while DMF with water served as the control. The finding of larval mortality was noted after 12 h. Larvae were assumed to be dead if they did not react to touching53. A conventional insecticide “Chlorpyrifos” was tested also as a reference larvicidal compound under the same conditions used for testing our synthesized compounds.Statistical analysisThe data on larval mortality were examined using the LDP line program’s statistical capabilities. The lethal concentrations (LC25, LC50, LC90) were calculated using a 95% confidence interval (C.I.). Additionally, the Abbott formula was employed to account for control mortality, along with the Finney formula, the Chi-square test, and the goodness of fit test (r2)60,61. The toxicity index (T.I) of the investigated chemicals against mosquito larvae is then evaluated using the Sun equation62 which is applied as follows:$$\text{Toxicity index} = \frac{{\text{LC}}\text{50 }\text{of most effective compound}}{{\text{LC}}\text{50 }\text{of the compound used}} \, \times {100}$$Neural receptors 3D structure preparationsThe amino acid sequences for the target receptors of the house mosquito (C. pipiens): acetylcholinesterase (AChE) (Accession Number: Q86GC8), nicotinic acetylcholine receptor (nAChR) (Accession Number: A0A8D8NUM7), and voltage-gated sodium channel alpha subunit (VGSC α subunit) (Accession Number: A0A8D8AMN4), were obtained from the UniProt Knowledgebase (UniProtKB) (https://www.uniprot.org/).Due to the potential limitations of using pre-existing structures, homology modeling was employed to generate 3D models for the AChE enzyme, the nAChR, and the VGSC α subunit binding proteins. SWISS-MODEL (https://swissmodel.expasy.org/), a web-based server for protein structure prediction, was used for this purpose48,63. This server utilizes a combination of BLASTp and HHBlits algorithms to identify suitable template structures within the Protein Data Bank (PDB) and SWISS-MODEL Template Library (SMTL) databases for each receptor48. The identified templates are then used to build a reliable model for the target protein sequence.The quality of the generated homology models was evaluated using the Z-scoring functions, General Model Quality Estimate (GMQE), and Qualitative Model Energy Analysis (QMEAN), which are specifically designed for SWISS-MODEL outputs28,48,63. These scores provide an objective assessment of the model’s accuracy and reliability.Molecular docking assessmentA molecular docking simulation was performed to understand the potential mode of action underlying the larvicidal activity of the tested compounds. The 2D structures of the fifteen compounds were drawn using ChemDraw 20.0 (CambridgeSoft). The Molecular Operating Environment (MOE V. 2014.02 software; https://www.chemcomp.com/en/index.htm) was then employed for 3D structure generation of the compounds, protonation state assignment, partial charge calculation, and energy minimization. Geometry optimization and energy minimization of the 3D structures were further performed using Wave Function Spartan v 14.0 (Wavefunction Inc., Irvine, CA, USA) to improve docking accuracy26.The 3D structures of the target receptors (AChE,nAChRs, and VGSC α subunit) were prepared and used as receptors28. The MOE-Site-Finder function was used to define the active site for docking with alpha spheres. A non-bonded cut-off value of 8–10 Å was applied to the Lennard–Jones terms. The energy of the complex was minimized using the MMFF94x force field until the root-mean-square (RMS) gradient reached 0.1 kcal/mol/Å. For each compound-receptor pair, 100 docking poses were generated. The ten poses with the lowest docking energies for each molecule were selected for further analysis. The London ΔG energy scoring function was employed to rank and evaluate the binding affinity of each ligand-receptor complex.MOE software was used for cross-docking of three reference insecticides: Chlorpyrifos (AChE inhibitor), Nitenpyram (nAChRs agonist), and Indoxacarb (VGSC α subunit blocker) at the alpha pockets of these receptors. The fifteen synthesized compounds were also docked against the same three receptor pockets. Docking scores of the synthesized compounds were compared to the corresponding reference insecticide for each receptor.Ethics declarationThis study was approved by the Research Ethics Committee at Ain Shams University (Approval code: ASU-SCI/ENTO/2024/1/4) and was performed in accordance with the guidelines of the National Institute of Health (NIH). All methods are reported in accordance with ARRIVE guidelines.
