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[4, 5-b] quinoline 9a-d, which afforded the thiazolotriazolopyrimido [4, 5-b] ... Key words: Pyrazole, Triazole, Thiazolidinone, Pyrimido [4, 5-b] quinoline, Antitumor ...
Arch Pharm Res Vol 35, No 3, 437-445, 2012 DOI 10.1007/s12272-012-0306-5

Synthesis of New Pyrazole, Triazole, and Thiazolidine-Pyrimido [4, 5-b] quinoline Derivatives with Potential Antitumor Activity Ameen Ali Abu-Hashem1,2 and Ahmed S. Aly1 1

Photochemistry Department (Heterocyclic Unit), National Research Center, 12622, Dokki, Giza, Egypt and 2Chemistry Department, Faculty of Science, Jazan University, 2097 Jazan, Saudi Arabia (Received June 8, 2011/Revised August 8, 2011/Accepted August 16, 2011)

2-Hydrazinyltetrahydropyrimido [4, 5-b] quinolin-4(3H)-one (3) was prepared by desulfurization reaction of S- and N-dimethyl derivatives 2 with hydrazine hydrate. Reactions of (3) with malonitrile, carbondisulfide, potassium thiocyanate, phthalic anhydride and aromatic aldehydes afforded 3, 5-di aminopyrazolopyrimido [4, 5-b] quinoline (4), triazolotetrahydropyrimido [4, 5-b] quinoline (5), aminotriazolopyrimido [4, 5-b] quinoline (6), aminophthalimidopyrimido [4, 5-b] quinoline (7) and N-arylidene hydrazinepyrimido [4, 5-b] quinoline 8a-d, respectively. Furthermore, 8a-d reacted with mercaptoacetic acid gave the thiazolidinonepyrimido [4, 5-b] quinoline 9a-d, which afforded the thiazolotriazolopyrimido [4, 5-b] quinolinone 10a-d upon treatment with ethanolic potassium hydroxide. The newly synthesized compounds were characterized by elemental analyses, IR, 1H-NMR, 13C-NMR and mass spectrometer. The investigated compounds were screened for their cytotoxicity. Compounds 4, 6 and 5 exhibited potent antitumor activity. Key words: Pyrazole, Triazole, Thiazolidinone, Pyrimido [4, 5-b] quinoline, Antitumor activity

INTRODUCTION Cancer is a major worldwide health problem and previous researches have shown that pyrimidoquinoline derivatives are among the potential cures for cancer. Pyrimidoquinoline derivatives are of importance due to their biological activities; especially, Pyrimido [4,5-b]quinoline derivatives have received much attention over the years due to their interesting pharmacological properties, including antimicrobial (El-Sayed et al., 2004; Selvi et al., 2006; El-Gazzar et al., 2009c), anti-inflammatory (El-Gazzar et al., 2009a, 2009b, 2009c), anti-allergy (Althuis et al., 1979), antifolates (Perner et al., 2003), anti-HIV infections (Dorjsuren et al., 2006), antimalarial (Joshi et al., 2005; Joshi and Viswanathan, 2006), antitumor (Singh et al., 1998; Ali et al., 2007, 2008a, 2008b), antibacterial (Taguchi et al., 1992; Chen et al., 2001), antioxidant, anti-inflamCorrespondence to: Ameen Ali Abu-Hashem, National Research Center, Photochemistry Department, 12622, Dokki, Giza, Egypt Tel: 2-12-521-1700, 966-59-136-3915, Fax: 2-2-3337-0931, 966-7324-5212 E-mail: [email protected]

matory and analgesic (El-Gazzar et al., 2009a, 2009b) activities. Our research group has been interested in developing a synthetic strategy for polyfunctionalized heterocycles (El-Gazzar et al., 2009a, 2009b) and in developing products with antitumor activity (AbuHashem et al., 2010; Abu-Hashem et al., 2011; Gouda et al., 2011).

MATERIALS AND METHODS General All melting points are in oC and were determined on Gallenkamp electric melting point apparatus. The IR spectra were recorded potassium bromide (KBr) on a Perkin–Elmer 1430 spectrometer. The 1H-NMR and 13 C-NMR spectra (δ, ppm) were recorded on JEOLECA 500 and JEOL JNM-LA-400 FT NMR Spectrometers, and chemical shifts were expressed as δ values against TMS as an internal standard. Mass spectra were recorded on GCMS-QP 1000 EX (Shimadzu) (Gas Chromatography-Mass spectrometer). Microanalytical data were obtained at the Microanalytical Center at Cairo University and National Research Center, Egypt. The pharmacological evaluations were carried

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out in the pharmacological unit, Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Egypt.

(E)-9-(4-methoxybenzylidene)-5-(4-methoxyphenyl)-3-methyl-2-(methylthio)-6, 7, 8, 9-tetra hydropyrimido [4, 5-b] quinolin-4 (3H)-one (2) Ethanolic sodium ethoxide solution (10 mmol sodium metal in 40 mL absolute ethanol) was added each of 1 (4.56 g, 10 mmol) under boiling. The heating was continued for 30 min and the mixture was allowed to cool to room temperature, and methyl iodide (2.82 mL, 20 mmol) was added. The mixture was stirred under reflux for 5 h, then cooled to room temperature and poured into cold water (100 mL). The solid product that precipitated was filtered off, washed with water and crystallized from benzen to yield (2) golden crystals. Yield 85%; m.p. 175-177oC; IR (KBr): ν/cm−1 = 1695 (CO), 1620 (C=N). 1H-NMR (CDCl3): δ/ppm = 1.66-1.70 (m, 2H, CH2), 2.27 (t, 2H, J = 12.51 Hz, CH2), 2.65 (s, 3H, SCH3), 2.76 (t, 2H, J = 12.55 Hz, CH2), 3.81, 3.84 (2s, 6H, 2OCH3), 4.22 (s, 3H, N-CH3), 7.11-7.66 (m, 8H, Ar-H), 8.12 (s, 1H, methine-H). MS (70 ev, %) m/z 487 (M+ + 2, 10.4), 486 (M+ + 1, 35.7), 485 (M+, 90). Anal. calcd for C28H27N3O3S (485.6): C, 69.25; H, 5.60; N, 8.65%. Found: C, 69.10; H, 5.55; N, 8.50%. (E)-2-hydrazinyl-9-(4-methoxybenzylidene)-5-(4methoxyphenyl)-3-methyl-6, 7, 8, 9-tetrahydropyrimido [4, 5-b] quinolin-4 (3H)-one (3) A suspension of (2) (4.85 g, 10 mmol) in hydrazine hydrate (30 mL) was stirred under gentle reflux for 12 h and the reaction mixture was allowed to cool to room temperature. The separated solid was filtered, washed with ethanol, dried as yellow crystals and recrystallized from DMF. Yield 83%; m.p. 245-247oC; IR (KBr): ν/cm−1 = 3205 (NH), 1675 (CO), 1625 (C=N). 1 H-NMR (DMSO-d6): δ/ppm = 1.65-1.71 (m, 2H, CH2), 2.26 (t, 2H, J = 12.51 Hz, CH2), 2.74 (t, 2H, J = 12.56 Hz, CH2), 3.78, 3.82 (2s, 6H, 2OCH3), 4.20 (s, 3H, NCH3), 7.10-7.68 (m, 8H, Ar-H), 8.11 (s, 1H, methine-H), 9.10, 11.30 (2brs, 3H, NH2 and NH D2O exchangeable). 13 C-NMR (DMSO-d6): δ/ppm = 28.4, 29.7, 30.8, 33.9, 55.8 (3C, CH2; 1C, CH3; 2C, OCH3), 114.2, 114.8, 119.7, 129.6, 130.0, 130.2, 130.3, 130.6, 133.4, 134.00, 152.5, 152.9, 155.9, 156.1, 158.2, 159.8 (Ar-C), 163.5 (CO, amide). MS (70 ev, %) m/z 471 (M+ + 2, 14.5), 470 (M+ + 1, 45.5), 469 (M+, 96). Anal. calcd for C27H27N5O3 (469.5): C, 69.07; H, 5.80; N, 14.92%. Found: C, 69.10; H, 5.75; N, 14.82%.

A. A. Abu-Hashem and A. S. Aly

(E)-2-(3, 5-diamino-1H-pyrazol-1-yl)-9-(4-methoxybenzylidene)-5-(4-methoxy phenyl)-3-methyl-6, 7, 8, 9-tetrahydropyrimido [4, 5-b] quinolin-4 (3H)-one (4) A mixture of (3) (4.69 g, 10 mmol) and malononitrile (0.66 g, 10 mmol) in absolute ethanol (35 mL) containing a few drops of piperidine was refluxed for 7 h. The separated solid was filtered, dried and recrystallized from ethanol. Yield 80%; m.p. 270-272oC; IR (KBr): ν/cm−1 = 3350 (NH2), 1680 (CO), 1630 (C=N). 1 H-NMR (DMSO-d6): δ/ppm = 1.64-1.70 (m, 2H, CH2), 2.25 (t, 2H, J = 12.52 Hz, CH2), 2.75 (t, 2H, J = 12.55 Hz, CH2), 3.79, 3.81 (2s, 6H, 2OCH3), 4.23 (s, 3H, NCH3), 6.351 (s, 1H, pyrazol H-4), 6.10, 6.60 (2brs, 4H, 2NH2, D2O exchangeable), 7.12-7.70 (m, 8H, Ar-H), 8.10 (s, 1H, methine-H). MS (70 ev, %) m/z 537 (M+ + 2, 20.4), 536 (M+ + 1, 25.5), 535 (M+, 80), 505 (15), 469 (90), 454 (30), 439 (12), 425 (18), 319 (21), 201 (40), 133 (100). Anal. calcd for C30H29N7O3 (535.6): C, 67.27; H, 5.46; N, 18.31%. Found: C, 67.20; H, 5.40; N, 18.26%. 11-(4-methoxybenzylidene)-7-(4-methoxyphenyl)5-methyl-2-thioxo-8, 9, 10, 11-tetrahydro [1, 3,4] triazolo [2', 3'- 2, 3] pyrimido [4, 5-b] quinolin6-one (5) A mixture of (3) (4.69 g, 10 mmol) in ethanolic potassium hydroxide solution (10 mmol in 30 mL ethanol and 5 mL water) and CS2 (10 mmol) was refluxed for 8 h. The reaction mixture was cooled, diluted with cold water (30 mL) and acidified with acetic acid. The solid was filtered, dried and recrystallized from ethanol. Yield 80%; m.p. 210-212oC. IR (KBr): ν/cm−1 = 3320 (NH), 1684 (CO), 1635 (C=N). 1HNMR (DMSO-d6): δ/ppm = 1.65-1.71 (m, 2H, CH2), 2.25 (t, 2H, J = 12.50 Hz, CH2), 2.74 (t, 2H, J = 12.56 Hz, CH2), 3.80, 3.82 (2s, 6H, 2OCH3), 4.24 (s, 3H, NCH3), 7.11-7.72 (m, 8H, Ar-H), 8.12 (s, 1H, methineH), 9.20 (brs., NH, D2O exchangeable). MS (70 ev, %) m/z 513 (M+ + 2, 15.1), 512 (M+ + 1, 22.2), 511 (M+, 87). Anal. calcd for C28H25N5O3S (511.6): C, 65.74; H, 4.93; N, 13.69%. Found: C, 65.60; H, 4.85; N, 13.60%. 2-amino-11-(4-methoxybenzylidene)-7-(4-methoxyphenyl)-5-methyl-8, 9, 10, 11-tetrahydro [1, 3, 4] triazolo [2', 3' -2, 3] pyrimido [4, 5-b] quinolin-6-one (6) A mixture of (3) (4.69 g, 10 mmol) and potassium thiocyanate (0.97 g, 10 mmol) was heated under reflux in acetic acid for 10 h. The precipitate was collected by filtration, dried and crystallized from dioxane as yellow powder. Yield 83%; m.p. 310-212oC; IR (KBr): ν/cm−1 = 3355 (NH2), 1680 (CO), 1633 (C=N). 1H-NMR

Pyrimidoquinoline Derivatives & Antitumor Activities

(DMSO-d6): δ/ppm = 1.67-1.73 (m, 2H, CH2), 2.26 (t, 2H, J = 12.51 Hz, CH2), 2.75 (t, 2H, J = 12.55 Hz, CH2), 3.81, 3.84 (2s, 6H, 2OCH3), 4.25 (s, 3H, N-CH3), 6.60 (brs., 2H, NH2, D2O exchangeable), 7.12-7.74 (m, 8H, Ar-H), 8.11 (s, 1H, methine-H). 13C-NMR (DMSO-d6): δ/ppm = 23.9, 28.4, 30.2, 30.8, 55.8 (3C, CH2; 1C, CH3; 2C, OCH3), 114.2, 114.8, 128.1, 129.6, 130.1, 130.2, 130.3, 131.1, 137.1, 143.1, 149.2, 153.1, 155.2, 155.9, 157.8, 159.6 (Ar-C), 163.6 (CO, amide). MS (70 ev, %) m/z 496 (M+ + 2, 12.4), 495 (M+ + 1, 28.8), 494 (M+, 80.0). Anal. calcd for C28H26N6O3 (494.5): C, 68.00; H, 5.30; N, 16.99%. Found: C, 68.20; H, 5.38; N, 16.85%.

(E)-2-(9-(4-methoxybenzylidene)-5-(4-methoxyphenyl)-3-methyl-4-oxo-3, 4, 6, 7, 8, 9-hexahydropyrimido [4, 5-b] quinolin-2-ylamino) isoindoline-1, 3-Dione (7) A mixture of compound (3) (4.69 g, 10 mmol) and phthalic anhydride (10 mmol) in methanol (40 mL) was refluxed for 6 h. The solid obtained was filtered, dried and recrystallized from dioxane. Yield 80%; m.p. 285-287oC. IR (KBr): ν/cm−1 = 3310 (NH), 1790, 1735 (CO-N-CO), 1680 (CO), 1645 (C=N). 1H-NMR (DMSOd6): δ/ppm = 1.69-1.75 (m, 2H, CH2), 2.27 (t, 2H, J = 12.52 Hz, CH2), 2.77 (t, 2H, J = 12.56 Hz, CH2), 3.82, 3.85 (2s, 6H, 2OCH3), 4.24 (s, 3H, N-CH3), 7.12-7.88 (m, 12H, Ar-H), 8.11 (s, 1H, methine-H), 9.10 (brs., 1H, NH, D2O exchangeable). MS (70 ev, %) m/z 601 (M+ + 2, 1.9), 600 (M+ + 1, 2.5), 599 (M+, 95), 585 (14), 479 (16), 361 (26), 216 (35), 201 (29), 187 (27), 133 (100). Anal. calcd for C35H29N5O5 (599.6): C, 70.11; H, 4.87; N, 11.68%. Found: C, 70.23; H, 4.77; N, 11.60%. (9E)-2-(2-(p-sub)-benzylidenehydrazinyl)-9-(4methoxybenzylidene)-5-(4-methoxyphenyl)-3methyl-6, 7, 8, 9-tetrahydropyrimido [4, 5-b] quinolin-4 (3H)-one (8a-d) General procedure A mixture of compound (3) (4.69 g, 10 mmol) in absolute ethanol (45 mL) and the appropriate aromatic aldehyde (10 mmol) was refluxed for 6 h, concentrated to half the volume and left to cool. The precipitates so obtained were recrystallized to give 8a-d, respectively. (9E)-2-(2-benzylidenehydrazinyl)-9-(4-methoxybenzylidene)-5-(4-methoxy phenyl)-3-methyl-6, 7, 8, 9-tetrahydropyrimido [4, 5-b] quinolin-4 (3H)one (8a) Brown powder, yield 82%; m.p. 275-277oC (methanol); IR (KBr): ν/cm−1 = 3270 (NH), 1675 (CO), 1650 (C=N). 1 H-NMR (DMSO-d6): δ/ppm = 1.68-1.74 (m, 2H, CH2), 2.28 (t, 2H, J = 12.52 Hz, CH2), 2.78 (t, 2H, J = 12.57 Hz, CH2), 3.81, 3.84 (2s, 6H, 2OCH3), 4.23 (s, 3H, N-

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CH3), 7.11-7.85 (m, 13H, Ar-H), 8.10 (s, 1H, azomethineH), 8.25 (s, 1H, methine-H), 10.10 (brs., 1H, NH, D2O exchangeable). MS (70 ev, %) m/z 559 (M+ + 2, 6.9), 558 (M+ + 1, 4.5), 557 (M+, 100). Anal. calcd for C34H31N5O3 (557.6): C, 73.23; H, 5.6; N, 12.56%. Found: C, 73.15; H, 5.55; N, 12.60%. (9E)-9-(4-methoxybenzylidene)-2-(2-(4-methoxybenzylidene) hydrazinyl)-5-(4-methoxyphenyl)3-methyl-6, 7, 8, 9-tetrahydropyrimido [4, 5-b] quinolin-4 (3H)-one (8b) Yellow powder, yield 81%; m.p. 312-314oC (ethanol); IR (KBr): ν/cm−1 = 3275 (NH), 1677 (CO), 1652 (C=N). 1 H-NMR (DMSO-d6): δ/ppm = 1.69-1.75 (m, 2H, CH2), 2.30 (t, 2H, J = 12.51 Hz, CH2), 2.80 (t, 2H, J = 12.55 Hz, CH2), 3.82, 3.85, 3.90 (3s, 9H, 3OCH3), 4.24 (s, 3H, N-CH3), 7.10-7.87 (m, 12H, Ar-H), 8.12 (s, 1H, azomethine-H), 8.30 (s, 1H, methine-H), 10.30 (brs., 1H, NH, D2O exchangeable). MS (70 ev, %) m/z 589 (M+ + 2, 16.2), 588 (M+ + 1, 14.7), 587 (M+, 90). Anal. calcd for C35H33N5O4 (587.6): C, 71.53; H, 5.66; N, 11.92%. Found: C, 71.5; H, 5.55; N, 11.85%. (9E)-2-(2-(4-hydroxybenzylidene)hydrazinyl)-9-(4methoxybenzylidene)-5-(4-methoxyphenyl)-3methyl-6, 7, 8, 9-tetrahydropyrimido [4, 5-b] quinolin-4 (3H)-one (8c) White powder, yield 80%; m.p. 212-214oC (methanol); IR (KBr): ν/cm−1 = 3350-2650 (brs., OH, NH), 1677 (CO), 1652 (C=N). 1H-NMR (DMSO-d6): δ/ppm = 1.66-1.74 (m, 2H, CH2), 2.28 (t, 2H, J = 12.52 Hz, CH2), 2.78 (t, 2H, J = 12.57 Hz, CH2), 3.81, 3.84 (2s, 6H, 2OCH3), 4.23 (s, 3H, N-CH3), 7.12-7.87 (m, 12H, Ar-H), 8.14 (s, 1H, azomethine-H), 8.34 (s, 1H, methine-H), 9.30 (brs., 1H, OH, D2O exchangeable), 10.40 (brs., 1H, NH, D2O exchangeable). MS (70 ev, %) m/z 575 (M+ + 2, 9.8), 574 (M+ + 1, 18.5), 573 (M+, 80). Anal. calcd for C34H31N5O4 (573.6): C, 71.19; H, 5.45; N, 12.21%. Found: C, 71.12; H, 5.41; N, 12.14%. (9E)-2-(2-(4-chlorobenzylidene) hydrazinyl)-9-(4methoxybenzylidene)-5-(4-methoxyphenyl)-3methyl-6, 7, 8, 9-tetrahydropyrimido [4, 5-b] quinolin-4 (3H)-one (8d) Yellow powder, yield 82%; m.p. 292-294oC (ethanol); IR (KBr): ν/cm−1 = 3345 (NH), 1678 (CO), 1653 (C=N). 1 H-NMR (DMSO-d6): δ/ppm = 1.65-1.73 (m, 2H, CH2), 2.28 (t, 2H, J = 12.51 Hz, CH2), 2.79 (t, 2H, J = 12.55 Hz, CH2), 3.80, 3.83 (2s, 6H, 2OCH3), 4.25 (s, 3H, NCH3), 7.14-7.87 (m, 12H, Ar-H), 8.14 (s, 1H, azomethineH), 8.34 (s, 1H, methine-H), 10.31 (brs., NH, D2O exchangeable). MS (70 ev, %) m/z 594 (M+ + 2, 11.6), 593 (M+ + 1, 10.2), 592 (M+, 83). Anal. calcd for C34H30ClN5O3

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(592.1): C, 68.97; H, 5.11; N, 11.83%. Found: C, 68.9; H, 5.1; N, 11.76%.

9-(4-Methoxybenzylidene)-5-(4-methoxyphenyl)3-methyl-2-(4-oxo-2-(sub)-phenyl-thiazolidin-3ylamino) - 6, 7, 8, 9-tetrahydro -3H-pyrimido [4, 5-b] quinolin-4-one (9a-d) General procedure A mixture of each (8a-d) (10 mmol) and mercaptoacetic acid (0.015 mol) was refluxed in dry benzene (40 mL) using a dean stark water separator for 4-8 hours. Excess benzene was evaporated under reduced pressure and the resulting residue was triturated with saturated NaHCO3 solution. The solids so obtained were filtered, then washed with H2O, dried and recrystallized to give 9a-d, respectively. 9-(4-Methoxybenzylidene)-5-(4-methoxyphenyl)-3methyl-2-(4-oxo-2-phenylthiazolidin-3-ylamino)-6, 7, 8, 9-tetrahydro-3H-pyrimido [4, 5-b] quinolin4-one (9a) White powder, yield 82%; m.p. 137-139oC (dioxane); IR (KBr): ν/cm−1 = 3290 (NH), 1680, 1715 (2CO), 1651 (C=N). 1H-NMR (DMSO-d6): δ/ppm = 1.66-1.72 (m, 2H, CH2), 2.27 (t, 2H, J = 12.52 Hz, CH2), 2.75 (t, 2H, J = 12.56 Hz, CH2), 3.80, 3.82 (2s, 6H, 2OCH3), 3.853.95 (2d, 2H, thiazolidinone CH2), 4.24 (s, 3H, N-CH3), 6.10 (s, 1H, thiazolidinone H-2), 7.11-7.87 (m, 13H, Ar-H), 8.20 (s, 1H, methine-H), 10.20 (brs., 1H, NH, D2O exchangeable). MS (70 ev, %) m/z 633 (M+ + 2, 14.7), 632 (M+ + 1, 17.5), 631 (M+, 90). Anal. calcd for C36H33N5O4S (631.7): C, 68.44; H, 5.27; N, 11.09%. Found: C, 68.40; H, 5.20; N, 11.11%. 9-(4-Methoxy-benzylidene)-5-(4-methoxyphenyl)2-[2-(4-methoxy-phenyl)-4-oxo-thiazolidin-3-ylamino]-3-methyl-6, 7, 8, 9-tetrahydro-3H-pyrimido [4, 5-b] quinolin-4-one (9b) Brown powder, yield 70%; m.p. 190-192oC (methanol); IR (KBr): ν/cm−1 = 3291 (NH), 1681, 1716 (2CO), 1650 (C=N). 1H-NMR (DMSO-d6): δ/ppm = 1.65-1.71 (m, 2H, CH2), 2.28 (t, 2H, J = 12.50 Hz, CH2), 2.77 (t, 2H, J = 12.55 Hz, CH2), 3.8, 3.82, 3.84 (3s, 9H, 3OCH3), 3.86-3.96 (2d, 2H, thiazolidinone CH2), 4.25 (s, 3H, NCH3), 6.12 (s, 1H, thiazolidinone H-2), 7.12-7.86 (m, 12H, Ar-H), 8.21 (s, 1H, methine-H), 10.23 (brs, 1H, NH, D2O exchangeable). MS (70 ev, %) m/z 663 (M+ + 2, 4.6), 662 (M+ + 1, 3.5), 661 (M+, 82). Anal. calcd for C37H35N5O5S (661.7): C, 67.15; H, 5.33; N, 10.58%. Found: C, 67.10; H, 5.30; N, 10.50%.

A. A. Abu-Hashem and A. S. Aly

2-[2-(4-Hydroxyphenyl)-4-oxo-thiazolidin-3-ylamino]-9-(4-methoxybenzylidene)-5-(4-methoxyphenyl)-3-methyl-6, 7, 8, 9-tetrahydro-3H-pyrimido [4, 5-b] quinolin-4-one (9c) Yellow powder, yield 80%; m.p. 170-172oC (DMF); IR (KBr): ν/cm−1 = 3355-2640 (brs., OH, NH), 1679, 1720 (2CO), 1651 (C=N). 1H-NMR (DMSO-d6): δ/ppm = 1.641.70 (m, 2H, CH2), 2.27 (t, 2H, J = 12.52 Hz, CH2), 2.76 (t, 2H, J = 12.56 Hz, CH2), 3.80, 3.82 (2s, 6H, 2OCH3), 3.85-3.95 (2d, 2H, thiazolidinone CH2), 4.23 (s, 3H, N-CH3), 6.13 (s, 1H, thiazolidinone H-2), 7.117.88 (m, 12H, Ar-H), 8.20 (s, 1H, methine-H), 9.50 (brs., 1H, OH, D2O exchangeable), 10.50 (brs., 1H, NH, D2O exchangeable). MS (70 ev, %) m/z 649 (M+ + 2, 14.8), 648 (M+ + 1, 12.4), 647 (M+, 80). Anal. calcd for C36H33N5O5S (647.7): C, 66.75; H, 5.14; N, 10.81%. Found: C, 66.70; H, 5.10; N, 10.77%. 2-[2-(4-Chloro-phenyl)-4-oxo-thiazolidin-3-ylamino]9-(4-methoxybenzylidene)-5-(4-methoxyphenyl)-3methyl- 6, 7, 8, 9-tetrahydro-3H-pyrimido [4, 5-b] quinolin-4-one (9d) Yellow powder, yield 85%; m.p. 120-122oC (dioxane). IR (KBr): ν/cm−1 = 3292 (NH), 1685, 1718 (2CO), 1653 (C=N). 1H-NMR (DMSO-d6): δ/ppm = 1.66-1.72 (m, 2H, CH2), 2.30 (t, 2H, J = 12.50 Hz, CH2), 2.77 (t, 2H, J = 12.55 Hz, CH2), 3.80, 3.82 (2s, 6H, 2OCH3), 3.843.94 (2d, 2H, thiazolidinone CH2), 4.24 (s, 3H, N-CH3), 6.11 (s, 1H, thiazolidinone H-2), 7.11-7.89 (m, 12H, Ar-H), 8.24 (s, 1H, methine-H), 10.21 (brs, NH, D2O exchangeable). 13C-NMR (DMSO-d6): δ/ppm = 23.9, 24.9, 28.3, 30.7, 32.4, 55.9 (4C, CH2; 1C, CH3; 2C, OCH3), 69.2, 114.3, 114.8, 119.9, 122.6, 126.9, 127.1, 127.9, 128.9, 130.4, 131.5, 132.4, 133.7, 136.5, 142.1, 143.4, 150.2, 153.4, 155.9, 159.3 (Ar-C), 163.8, 171.6 (2CO, amide, thiazolidinone). MS (70 ev, %) m/z 668 (M+ + 2, 24.7), 667 (M+ + 1, 30.5), 666 (M+, 85), 555 (8), 454 (12), 439 (17), 425 (15), 319 (24), 201 (28), 187 (48), 133 (100). Anal. calcd for C36H32ClN5O4S (666.2): C, 64.90; H, 4.84; N, 10.51%. Found: C, 64.75; H, 4.80; N, 10.46%.

1-(p-sub-phenyl)-4-methyl-6-(4-methoxyphenyl)10-(4-methoxybenzylidene)-[1,3]-thiazolo [4',3' : 5'',1'']-[1,2,4]-triazolo[3'',4'' : 2,3]-7,8,9–trihydropyrimido[4,5 – b]quinolin-5-one (10a-d) General procedure A mixture of compound (9a-d) (10 mmol) in ethanolic potassium hydroxide (10 mmol in 40 mL ethanol) was refluxed for 6-9 h. The reaction mixture was cooled, diluted with cold water (35 mL) and acidified with hydrochloric acid. The solid that formed was filtered, dried and recrystallized from proper solvent to give 10a-d, respectively.

Pyrimidoquinoline Derivatives & Antitumor Activities

4-methyl-6-(4-methoxyphenyl)-10-(4-methoxybenzylidene)-1-phenyl-[1,3]-thiazolo[4',3' : 5'',1'']-[1,2,4]triazolo[3'',4'': 2,3]-7, 8, 9–trihydropyrimido[4,5 b]quinolin-5-one (10a) Yellowish powder, yield 79%; m.p. 220-222oC (dioxane). IR (KBr): ν/cm−1 = 1678 (CO), 1645 (C=N). 1H-NMR (DMSO-d6): δ/ppm = 1.65-1.71 (m, 2H, CH2), 2.27 (t, 2H, J = 12.52 Hz, CH2), 2.76 (t, 2H, J = 12.56 Hz, CH2), 3.82, 3.83 (2s, 6H, 2OCH3), 4.12 (s, 1H, thiazolidine H5), 4.25 (s, 3H, N-CH3), 4.98 (s, 1H, thiazolidine H-2), 7.12-7.88 (m, 13H, Ar-H), 8.22 (s, 1H, methine-H). MS (70 ev, %) m/z 615 (M+ + 2, 20.2), 614 (M+ + 1, 35.1), 613 (M+, 83). Anal. calcd for C36H31N5O3S (613.7): C, 70.45; H, 5.09; N, 11.41%. Found: C, 70.41; H, 5.14; N, 11.55%. 1-(4-methoxyphenyl)-4-methyl-4-(4-methoxyphenyl)-10-(4-methoxybenzylidene)-[1, 3]-thiazolo [4', 3': 5'', 1'']-[1, 2, 4]triazolo[3'', 4'': 2, 3]-7, 8, 9–trihydropyrimido [4, 5-b] quinolin-5-one (10b) Brown powder, yield 81%; m.p. 232-234oC (DMF); IR (KBr): ν/cm−1 = 1679 (CO), 1644 (C=N). 1H-NMR (DMSOd6): δ/ppm = 1.66-1.72 (m, 2H, CH2), 2.29 (t, 2H, J = 12.50 Hz, CH2), 2.76 (t, 2H, J = 12.55 Hz, CH2), 3.81, 3.82, 3.85 (3s, 9H, 3OCH3), 4.14 (s, 1H, thiazolidine H5), 4.26 (s, 3H, N-CH3), 4.96 (s, 1H, thiazolidine H-2), 7.14-7.90 (m, 12H, Ar-H), 8.24 (s, 1H, methine-H). MS (70 ev, %) m/z 645 (M+ + 2, 22.4), 644 (M+ + 1, 30.3), 643 (M+, 85). Anal. calcd for C37H33N5O4S (643.7): C, 69.03; H, 5.17; N, 10.88%. Found: C, 69.12; H, 5.21; N, 10.80%. 1-(4-hydroxyphenyl)-4-methyl-6-(4-methoxyphenyl)-10-(4-methoxybenzylidene)-[1, 3]-thiazolo[4',3' : 5'',1'']-[1, 2, 4]-triazolo [3'',4'' : 2,3]- 7, 8, 9–trihydropyrimido[ 4, 5-b]quinolin-5-one (10c) White powder, yield 80%; m.p. 262-264oC (dioxane); IR (KBr): ν/cm−1 = 3350-2655 (OH), 1678 (CO), 1645 (C=N). 1H-NMR (DMSO-d6): δ/ppm = 1.65-1.70 (m, 2H, CH2), 2.27 (t, 2H, J = 12.50 Hz, CH2), 2.76 (t, 2H, J = 12.55 Hz, CH2), 3.80, 3.81 (2s, 6H, 2OCH3), 4.12 (s, 1H, thiazolidine H-5), 4.25 (s, 3H, N-CH3), 4.95 (s, 1H, thiazolidine H-2), 7.12-7.91 (m, 12H, Ar-H), 8.23 (s, 1H, methine-H), 9.80 (brs., OH, D2O exchangeable). MS (70 ev, %) m/z 631 (M+ + 2, 25.5), 630 (M+ + 1, 40.2), 629 (M+, 80). Anal. calcd for C36H31N5O4S (629.7): C, 68.66; H, 4.96; N, 11.12%. Found: C, 68.55; H, 4.88; N, 11.77%. 1-(4-chlorophenyl)-4-methyl-6-(4-methoxyphenyl)10-(4-methoxybenzylidene)-[1, 3]-thiazolo [4', 3': 5'', 1'']-[1, 2, 4]-triazolo[3'',4'' : 2, 3]-7, 8, 9– trihydropyrimido[4, 5- b]quinolin-5-one (10d) Yellow powder, yield 82%, m.p. 182-184oC (methanol).

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IR (KBr): ν/cm−1 = 1680 (CO), 1640 (C=N). 1H-NMR (DMSO-d6): δ/ppm = 1.65-1.71 (m, 2H, CH2), 2.28 (t, 2H, J = 12.52 Hz, CH2), 2.76 (t, 2H, J = 12.56 Hz, CH2), 3.80, 3.81 (2s, 6H, 2OCH3), 4.12 (s, 1H, thiazolidine H5), 4.24 (s, 3H, N-CH3), 4.94 (s, 1H, thiazolidine H-2), 7.15-7.91 (m, 12H, Ar-H), 8.22 (s, 1H, methine-H).13CNMR (DMSO-d6): δ/ppm = 24.1, 24.9, 32.6, 35.7, 55.9 (3C, CH2; 1C, CH3; 2C, OCH3), 65.7, 79.3, 112.1, 114.1, 114.8, 122.7, 125.6, 126.8, 127.1, 128.1, 128.9, 130.1, 131.4, 132.3, 138.9, 142.5, 144.1, 148.7, 151.1, 153.6, 154.2, 155.9, 159.8 (Ar-C), 164.5 (CO, amide). MS (70 ev, %) m/z 650 (M+ + 2, 35.8), 649 (M+ + 1, 47.5), 648 (M+, 86), 537 (9), 523 (19), 442 (14), 321 (26), 203 (33), 187 (37), 133 (100). Anal. calcd for C36H30ClN5O3S (648.1): C, 66.71; H, 4.67; N, 10.80%. Found: C, 66.65; H, 4.55; N, 10.75%.

Pharmacological studies The human carcinoma cancer cell lines KB, CNE2, MGC-803 and MCF-7 supplemented from The National Cancer Institute, Cairo University, Egypt. The method used The in vitro cytotoxicity of the synthesized compounds against different cancer cell lines was assessed with the MTT assay as described previously (Mosmann, 1983; Skehan et al., 1990). The MTT assay is based on the reduction of soluble 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) into a bluepurple formazan product, mainly by mitochondrial reductase activity inside living cells. The cells used in the cytotoxicity assay were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum. Cells suspended in the medium (2Y' 104/mL) were plated in 96-well culture plates and incubated at 37oC in a 5% CO2 incubator. After 12 h, the test sample (2 µL) was added to the cells (2Y' 104) in 96-well plates and cultured at 37oC for 3 days. The cultured cells were mixed with 20 µL of MTT solution and incubated for 4 h at 37oC. The supernatant was carefully removed from each well and 100 µL of DMSO was added to each well to dissolve the formazan crystals that were formed by the cellular reduction of MTT. After mixing with a mechanical plate mixer, the absorbance of each well was measured by a microplate reader using a test wavelength of 570 nm. The results were expressed as the IC50, which is the concentration of a drug inducing 50% inhibition of cell growth of treated cells when compared to the growth of control cells. Each experiment was performed at least 3 times. There was good reproducibility between replicate wells with standard errors below 10%. The results are summarized in Table I.

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Table I. Cytotoxic activity of pyrimido [4, 5-b] quinoline derivatives against human tumor cells Compounds 1 2 3 4 5 6 7 8a 8b 8c 8d 9a 9b 9c 9d 10a 10b 10c 10d 5-Fluorouracil

In vitro cytotoxicity IC50 (µM) a

MCF-7

MGC-803a

CNE2a

KBa

>50 >50 >50 19.6 ± 2.5 23.2 ± 2.4 22.1 ± 2.1 >50 >50 >50 >50 >50 44.2 ± 2.4 31.4 ± 2.2 33.9 ± 2.6 29.2 ± 2.3 28.1 ± 2.4 25.7 ± 2.6 26.8 ± 2.5 24.1 ± 2.5 14.5 ± 2.2

>50 >50 >50 16.2 ± 1.2 20.1 ± 1.3 19.4 ± 1.4 38.4 ± 1.5 >50 43.4 ± 1.2 >50 41.5 ± 1.3 35.6 ± 1.4 27.8 ± 1.5 29.4 ± 1.6 25.6 ± 1.4 24.5 ± 1.1 20.8 ± 1.4 22.6 ± 1.5 18.9 ± 1.5 12.9 ± 1.2

>50 >50 46.8 ± 1.6 13.8 ± 1.7 16.5 ± 1.4 15.2 ± 1.5 32.1 ± 1.4 44.9 ± 1.4 39.1 ± 1.2 42.9 ± 1.3 36.2 ± 1.6 30.2 ± 1.5 24.3 ± 1.4 27.6 ± 1.8 22.5 ± 1.1 20.3 ± 1.3 17.1 ± 1.5 19.2 ± 1.2 15.8 ± 1.7 13.7 ± 1.6

>50 49.8 ± 1.1 47.5 ± 1.2 12.9 ± 1.4 15.3 ± 1.2 14.1 ± 1.1 35.2 ± 1.3 45.2 ± 1.1 41.8 ± 1.5 43.4 ± 1.2 38.5 ± 1.2 32.4 ± 1.4 27.1 ± 1.3 29.5 ± 1.2 25.4 ± 1.1 22.5 ± 1.6 19.3 ± 1.3 20.2 ± 1.5 17.1 ± 1.4 12.8 ± 1.3

a

KB cells are drug-sensitive human oral carcinoma cells, CNE2 cells are drug-sensitive human nasopharyngeal carcinoma cells, MGC-803 cells are drug-sensitive human gastric carcinoma cells and MCF-7 cells are drug-sensitive human breast adenocarcinoma cells.

RESULTS AND DISCUSSION The new rings of pyrimido [4, 5-b] quinolin-4-one derivatives seem to be effective as antitumor since the compounds [for examples, 2-(3, 5-diamino-1H-pyrazol1-yl)-9-(4-methoxybenzylidene)-5-(4-methoxy phenyl)3-methyl-tetrahydropyrimido [4, 5-b] quinolin-4(3H)one 4, 11-(4-methoxybenzylidene)-7-(4-methoxy phenyl)5-methyl-2-thioxo-tetrahydro [1, 3, 4] triazolopyrimido [4, 5-b] quinolin-6-one 5, 2-amino-11-(4-methoxybenzylidene)-7-(4-methoxyphenyl)-5-methyl-tetrahydro [1,3, 4] triazolo pyrimido [4, 5-b] quinolin-6-one 6 and 1-(4chloro phenyl)-4-methyl-6-(4-methoxyphenyl)-10-(4methoxybenzylidene)-[1, 3] thiazolo [1, 2, 4] triazolo-

trihydropyrimido [4, 5-b] quinolin-5-one 10d] showed the highest antitumor activity (potent cytotoxicity against cancer cell lines). Because these compounds (4, 5, 6 and 10d) contained different functional groups and new rings for example, two amino groups with pyrazole ring, thioxo with triazole ring, one amino with triazole ring and 4-Cl-phenyl with thiazol-triazole ring which may be potentiate the effects of these new compounds rather than the original ones.

Chemistry Alkylation of 9-arylidene-5-aryl-2-thioxo-2,3,6,7,8,9hexahydro-1H-pyrimido[4,5-b]quinolin-4-one 1 (ElGazzar et al., 2009a, 2009b) using methyl iodide in

Scheme 1. Synthesis of 2-hydrazinyl-tetrahydropyrimido[4, 5-b]quinolin-4-one derivatives. Reagents and conditions: compound 2 with hydrazine hydrate reflux for 12 h, 83% yield.

Pyrimidoquinoline Derivatives & Antitumor Activities

443

Scheme 2. Synthesis of 3, 5-diamino-pyrazole, triazole, amino-triazole, amino-phthalimido-pyrimido[4, 5-b]quinoline derivatives. Reagents and conditions: compound 3 with many reagents, for example, malononitrile, carbon disulfide, potassium thiocyanate, and phthalic anhydride, refluxed for 6-10 h, 80-83% yield.

ethanolic potassium hydroxide yielded S,N-dimethyl derivative 2. Desulfurization of compound 2 with hydrazine hydrate afforded 2-hydrazinyl derivative 3. The IR spectrum of compound 3 showed absorption bands at ν 3205 cm−1, corresponding to NH-NH2 (cf. experimental and Scheme 1). Moreover, compound 3 was treated with malonitrile to give (E)-2-(3,5-diamino-1H-pyrazol-1-yl)-9-(4-methoxybenzylidene)-5-(4-methoxyphenyl)-3-methyl-6,7,8,9tetrahydropyrimido[4,5-b] quinolin-4 (3H)-one 4. The IR spectrum of compound 4 revealed the absence of the NH-NH2 band and absorption bands at ν 3350 cm−1, corresponding to amino of the pyrazole ring, and in the 1H-NMR spectrum, a singlet appeared at δ 6.351 for pyrazole H-4. On the other hand, compound 3 reacted with carbon disulfide in the presence of ethanolic potassium hydroxide to yield the fused 2mercaptotriazole derivative 5. In the 1H-NMR spectrum for 5, a singlet appears at δ 9.20 for NH in triazole ring (D2O exchangeable). When compound 3 was reacted with potassium thiocyanate in acetic acid, fused 2-aminotriazole derivative 6 was obtained in

good yield. In the 1H-NMR spectrum of the latter compound, a broad peak appears at δ 6.60 for NH2 (D2O exchangeable). 4-Oxo-hexahydropyrimido [4, 5-b] quinolin-2-ylamino) isoindoline-1, 3-dione 7 was obtained by reaction of 3 with phthalic anhydride in absolute methanol. Mass spectra of 5, 6 and 7 showed molecular ion peaks at m/z 511 (87%), 494 (80%) and 599 (100%), respectively. The structures of the final products were established by physical and spectral methods (cf. experimental and Scheme 2). Treatment of 3 with aromatic aldehydes, namely, benzaldehyde, 4-methoxybenzaldehyde, 4-hydroxybenzaldehyde and 4-chlorobenzaldehyde, yielded the corresponding Schiff bases (9E)-2-benzylidenehydrazinyl)-9-(4-methoxybenzylidene)-5-(4-methoxy-phenyl)-3methyl-6, 7, 8, 9-tetra hydropyrimido[4, 5-b] quinolin4(3H)-one 8a-d. 1H-NMR spectrum showed a singlet at δ 8.10 for azomethine proton (N=CH), a singlet at δ 8.25 for methylenic-proton and a broad peak at 10.10 for NH (D2O exchangeable). Compounds 8a-d were reacted with mercaptoacetic acid in dry benzene to yield thiazolidinone derivatives 9a-d. The IR spectra

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Scheme 3. Synthesis of N-arylidenehydrazino, thiazolidinone, thiazolo-triazolo-pyrimido[4, 5-b] quinolinone derivatives. Reagents and conditions: aromatic aldehyde derivatives, mercaptoacetic acid and ethanolic potassium hydroxide refluxed for 4-9 h, 70-85% yield.

of compounds 9a-d showed absorption bands at ν 1680, 1715 cm−1 corresponding to amide carbonyl and carbonyl thiazolidinone ring. Compounds 9a-d were cyclized by refluxing in ethanolic potassium hydroxide, where the nitrogen atom attack on the carbonyl group was followed by elimination of water molecule, to afford fused 1-(p-sub-phenyl)-4-methyl-[1,3]-thiazolo [4',3' : 5'',1'']-[1,2,4]-triazolo[3'',4'' : 2,3]-7,8,9–trihydro pyrimido[4,5 – b]quinolin-5-one 10a-d derivatives. IR spectrum of 10a showed absorption bands at ν 1678 cm−1 corresponding to the amide carbonyl group and an absence of bands corresponding to carbonyl thiazolidine ring. The 1H-NMR spectrum showed a singlet at δ 4.12 corresponding to thiazolidine H-5 and a singlet at 4.98 corresponding to thiazolidine H-2. The 1H-, 13 C-NMR spectra and mass spectra supported the suggested structures (cf. experimental and Scheme 3).

Pharmacology evaluation In vitro cytotoxicity The results indicated that the pyrimidoquinoline group introduced to the structure improved the antitumor activity since the compounds without pyrimido [4, 5-b] quinolin-4-one group displayed very weak antitumor activity against the tumor cells. As seen in Table I, compounds 4, 6, 5 and 10d showed potent cytotoxicity against the KB cancer cell line (IC50 12.9, 14.1, 15.3, 17.1 µM) respectively; compound 4 showed potent cytotoxicity against KB and CNE2 cancer cell

lines (IC50 12.9, 13.8 µM); and compounds 4, 6, 5 and 10d showed moderate cytotoxicity against MGC-803 (IC50 16.2, 19.4, 20.1, 18.9 µM) and MCF-7a (IC50 19.6, 22.1, 23.2, 24.1 µM) cancer cell lines, respectively. However, the rest of the compounds showed weak cytotoxicity activity. Based on the structure activity relationship (SAR), the presence of 3, 5-diaminopyrazolo; 2-amino-1, 3, 4-triazolo; 1, 3, 4-triazolo and triazolothiazolidine moieties linked to pyrimido[4,5-b]quinoline enhanced the cytotoxicity activity as in compounds 4, 6, 5 and 10d.

ACKNOWLEDGEMENTS The author wishes to express his most sincere thanks to Prof. Dr. Farid Abd-Elraheem Badria (Department of Pharmacognosy, Faculty of Pharmacy and Mansoura University 35516, Egypt) for carrying out the biological activity tests. The present work was supported by the Department of Photochemistry (Heterocyclic unit); Chemical Industries Research Division, National Research Centre in Cairo, Egypt.

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