Scholarly article on topic 'Synthesis and antitumor activity of benzo[6″,7″]cyclohepta[1″,2″:4′,5′]pyrido[2′,3′-d][1,2,4]triazolo[4,3-a]pyrimidin-5-ones'

Synthesis and antitumor activity of benzo[6″,7″]cyclohepta[1″,2″:4′,5′]pyrido[2′,3′-d][1,2,4]triazolo[4,3-a]pyrimidin-5-ones Academic research paper on "Chemical sciences"

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{Hydrazones / "Penta-heterocyclic compounds" / "Oxidative cyclization" / "HEPG-2 “liver” cancer cell line" / "MCF-7 “breast” cancer cell line"}

Abstract of research paper on Chemical sciences, author of scientific article — Mastoura M. Edrees, Thoraya A. Farghaly

Abstract 2-Thioxo-3,9,10,11-tetrahydro-1-phenyl-benzo[6′,7′]cyclohepta[1′,2′:4,5]pyrido[2,3-d]pyrimidin-4-one (4) was prepared by the reaction of 2-dimethylaminomethylenebenzosuberone (1) with 6-amino-1-phenyl-2-thioxo-2,3-dihydro-pyrimidin-4-one (2). 2-Hydrazino-9,10,11-trihydro-1-phenyl-benzo-[6′,7′]cyclohepta[1′,2′:4,5]pyrido[2,3-d]pyrimidin-4-one (5) was prepared in good yield by reaction of 4 with hydrazine hydrate under reflux. Reaction of compound 5 with different aldehydes in acetic acid gave the corresponding hydrazono derivatives 6. Cyclization of the latter compounds with bromine in acetic acid afforded a series of novel pentaheterocyclic compounds namely, 7,8,9-trihydro-4-phenyl-benzo[6″,7″]cyclohepta[1″,2″:4′,5′]pyrido[2′,3′-d][1,2,4]triazolo[4,3-a]pyrimidin-14-ones (7a–g). Some of the newly synthesized compounds showed potent antitumor activity against HEPG-2 “liver” cancer cell line and MCF-7 “breast” cancer cell line.

Academic research paper on topic "Synthesis and antitumor activity of benzo[6″,7″]cyclohepta[1″,2″:4′,5′]pyrido[2′,3′-d][1,2,4]triazolo[4,3-a]pyrimidin-5-ones"

Arabian Journal of Chemistry (2013) xxx, xxx-xxx

King Saud University Arabian Journal of Chemistry

www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE

Synthesis and antitumor activity

of benzo[6,,,7,,]cyclohepta[1,,,2,,:4,,5,]pyrido[2,,3-d|

[1,2,4]triazolo[4,3-a]pyrimidin-5-ones

Mastoura M. Edrees ab, Thoraya A. Farghaly C *

a Department of Organic Chemistry, National Organization for Drug Control and Research (NODCAR), Giza 12311, Egypt b Department of Chemistry, Faculty of Science, KiING KHALID University, P.O. Box 9004, ABHA 61413, Saudi Arabia C Department of Chemistry, Faculty of Science, University of Cairo, Giza, Egypt

Received 11 January 2013; accepted 4 June 2013

KEYWORDS

Hydrazones; Penta-heterocyclic compounds;

Oxidative cyclization; HEPG-2 "liver'' cancer cell line;

MCF-7 "breast'' cancer cell line

Abstract 2-Thioxo-3,9,10,11-tetrahydro-1-phenyl-benzo[6',7']cyclohepta[1',2':4,5]pyrido[2,3-d] pyrimidin-4-one (4) was prepared by the reaction of 2-dimethylaminomethylenebenzosuberone

(1) with 6-amino-1-phenyl-2-thioxo-2,3-dihydro-pyrimidin-4-one (2). 2-Hydrazino-9,10,11-tri-hydro-1-phenyl-benzo-[6',7']cyclohepta[1',2':4,5]pyrido[2,3-d]pyrimidin-4-one (5) was prepared in good yield by reaction of 4 with hydrazine hydrate under reflux. Reaction of compound 5 with different aldehydes in acetic acid gave the corresponding hydrazono derivatives 6. Cyclization of the latter compounds with bromine in acetic acid afforded a series of novel pentaheterocyclic compounds namely, 7,8,9-trihydro-4-phenyl-benzo[6",7"]cyclohepta[1",2":4',5']pyrido[2',3'-d][1,2,4]triazolo[4,3-a]pyrimidin-14-ones (7a-g). Some of the newly synthesized compounds showed potent antitumor activity against HEPG-2 "liver" cancer cell line and MCF-7 "breast" cancer cell line.

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1. Introduction

The search for anticancer drugs led to the discovery of several hydrazones having anticancer activity. A report on aryl-idenehydrazinopyrimidines deals with the activity of these

compounds as anticancer and immunomodulating agents (Dlugosz and Machon, 1995). Easmon et al. (2001) reported on the cytotoxic effect of N-heteroaryl hydrazones that are particularly selective against colon carcinoma and that exhibit a novel mechanism of action. Hwu et al. have reported that photolytic cleavage of the nitrogen-nitrogen single bond in benzaldehyde phenylhydrazones produced aminyl (R2N.) and iminyl (R2C=N.) radicals. This photochemical property was utilized in the development of hydrazones as photoinduced DNA cleaving agents (Hwu et al., 2004). Many derivatives of fused benzocycloheptanone are potent antitumoral agents (Afonso et al., 2001; Benoit et al., 2000) against L1210 murine leukemia and HT29 cell lines. Also, the inhibition of the

* Corresponding author. Tel.: +20 235676608. E-mail address: thoraya-f@hotmail.com (T.A. Farghaly). Peer review under responsibility of King Saud University.

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farnesyl protein transferase, a novel approach to antitumor therapy is based on compounds such as substituted cyclohep-tapyridines (Ganguly et al., 2001). In continuation of our research work concerned with the synthesis of bioactive heterocyclic compounds (Abdel Hafez et al., 2010; Riyadh et al., 2010a,b; Farghaly and Abdalla, 2009; Farghaly et al., 2012; Edrees et al., 2010), we report herein the synthesis of a new series of 2-[(substitutedmethylene)hydrazono]-9,10,11-trihydro-1-phenyl-benzo[6',7']cyclohepta[1',2':4,5]pyrido[2,3-d] pyrimidin-4-one and benzo[6'',7'']cyclohepta[1'',2'':4',5']pyr-ido[2',3'-d][1,2,4]triazolo[4,3-a]pyrimidin-14-ones to evaluate their anti-tumor properties against two human tumor cell lines (MCF7 "breast" and HEPG2 "liver" cancers).

2. Experimental

2.1. Chemistry

Melting points were determined on a Gallenkamp apparatus and are uncorrected. IR spectra were recorded in a Pye-Unicam SP300 instrument in potassium bromide disks. NMR and 13C NMR spectra were recorded in a Varian Mercury VXR-300 spectrometer (300 MHz for and 75 MHz for 13C) in DMSO-d6 and the chemical shifts were related to that of the solvent. Mass spectra were recorded in a GCMS-QP 1000 EX Shimadzu Spectrometer, the ionizing voltage was 70 eV. Elemental analyses were carried out at the Microanalyt-ical Laboratory of Cairo University, Giza, Egypt. The anti-tumor activities were evaluated at the Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt.

2.1.1. Synthesis of 2-thioxo-3,9,10,11-tetrahydro-1-phenyl-benzo [6',7']cyclohepta[1',2':4, 5]pyrido[2, 3-d]pyrimidin-4-one (4)

A mixture of enaminone 1 (Farghaly et al., 2010) (2.15 g, 10mmol) and 6-amino-1-phenyl-2-thioxo-3H-pyrimidin-4-one (2) (2.19 g, 10mmol) in glacial acetic acid (30 mL) was heated under reflux for 5 h. After cooling, the reaction mixture was poured into ice/HCl mixture and the solid product was collected and recrystallized from dioxane as white crystals, yield (3.15 g, 85%) mp > 300 0C. *H NMR (DMSO-d6) d ppm: 2.16-2.54 (m, 6H, 3CH2), 7.09-7.52 (m, 9H, ArH), 8.35 (s, 1H, pyridine-H), 12.89 (s, 1H, NH, D2O exchangeable). IR v cm"1: 3107 (NH), 1704 (C=O), 1291 (C=S) cm"1. Ms m/z (%) 373 (M++2, 4), 372 (M+ + 1, 19), 371 (M + , 67), 370 (100), 312 (3), 180 (6), 156 (1), 142 (2), 77 (23). Anal. Calcd. for C22H17N3OS (371.45) C, 71.14; H, 4.61; N, 11.31. Found: C, 71.03; H, 4.36; N, 11.51%.

2.1.2. Synthesis of 2-hydrazino-9,10,11-trihydro-1-phenyl-benzo [6' ,7']cyclohepta[1' ,2':4,5]pyrido[2,3-d]pyrimidin-4-one (5) To thione 4 (1.86 g, 5 mmol) in ethanol (20 mL) was added hydrazine hydrate (10 mL, 80%). The reaction mixture was re-fluxed till all H2S (30 h) ceased to evolve, and then cooled. The solid that precipitated was filtered off and crystallized from ethanol to give compound 5 (1.44 g, 78%) as yellow solid. m.p. 260-262 oc. NMR (DMSO-d6) d ppm: 2.16-2.50 (m, 6H, 3CH2), 3.37 (br, 2H, NH2, D2O exchangeable), 6.92-8.01 (m, 9H, ArH), 8.18 (s, 1H, pyridine-H), 10.81 (s,

1H, NH, D2O exchangeable). 13C NMR (DMSO-d6) d ppm: 28.71, 30.63, 32.56, 118.68, 120.67, 121.00, 123.96, 126.34, 128.58, 128.88, 129.78, 135.39, 137.15, 138.04, 139.36, 140.66, 151.54, 158.02, 160.32, 166.68. IR v cm"1: 3350, 3290, 3188 (NH, NH2), 1644 (C=O) cm"1. Ms m/z (%) 369 (M + , 12), 353 (28), 339 (15), 312 (14), 207 (7), 166 (13), 153 (11), 127 (16), 113 (10), 91 (8), 90 (6), 89 (13), 77 (100). Anal. Calcd. for C22H19N5O (369.42) C, 71.53; H, 5.18; N, 18.96. Found: C, 71.25; H, 5.25; N, 18.74%.

2.1.3. Preparation of hydrazones 6a-g

A mixture of hydrazine 5 (0.92 g, 2.5 mmol) and the appropriate aldehyde 6 (2.5 mmol) in acetic acid (20 mL) and few drops of conc. hydrochloric acid (1 mL) was heated under reflux for 3 h. The reaction mixture was then cooled and diluted with water. The so-formed solid product was then collected by filtration, dried and crystallized from the proper solvent to afford the corresponding hydrazones 6a-g.

2.1.3.1. 2-[(Phenylmethylene)hydrazono]-9,10,11-trihydro-1-phenyl-benzo[6', 7' ]cyclohepta[1', 2' :4,5]pyrido[2, 3-d]pyrimi-din-4-one (6a). Pale yellow crystals (0.71 g, 75%) m.p. 268270 0C (ethanol) 1H NMR (DMSO-d6) d ppm: 1.91-2.35 (m, 6H, 3CH2), 7.31-7.41 and 7.95-7.97 (m, 14H, ArH), 7.68 (s, 1H, N=CH), 8.19 (s, 1H, pyridine-H), 11.60 (br s, 1H, NH, D2O exchangeable). IR v cm"1: 3173 (NH), 1680 (C=O) cm"1. Ms m/z (%) 458 (M + +1, 10), 457 (M + , 40), 192 (40), 165 (12), 103 (34), 92 (85), 77 (100). Anal. Calcd. for C29H23N5O (457.53) C, 76.13; H, 5.07; N, 15.31. Found: C, 76.01; H, 4.99; N, 15.19%.

2.1.3.2. 2-[(4-Methylphenylmethylene)hydrazono]-9,10,11-tri-hydro-1-phenyl-benzo[6 7 ]cyclohepta[1 2 :4 5]pyrido[2 3-d]pyrimidin-4-one (6b). Yellow crystals (0.94 g, 80%) m.p. 218-220 0C (ethanol) 1H NMR (DMSO-d6) d ppm: 2.17-2.59 (m, 6H, 3CH2), 2.45 (s, 3H, CH3), 7.17-8.04 (m, 13H, ArH), 8.25 (s, 1H, N=CH), 8.65 (s, 1H, pyridine-H), 10.97 (s, 1H, NH, D2O exchangeable). 13C NMR(DMSO-d6) d ppm: 20.83, 28.58, 30.30, 32.12, 108.85, 120.53, 126.20, 126.60, 127.69, 128.32, 128.46, 128.54, 128.85, 129.03, 129.14, 129.31, 129.69, 132.09, 136.50, 137.05, 138.10, 139.35, 139.44, 150.13, 158.69, 162.58. IR v cm"1: 3342 (NH), 1696 (C=O) cm"1. Ms m/z (%) 472 (M+ 1, 20), 471 (M + , 25), 380 (33), 221 (8), 193 (24), 144 (18), 120 (21), 77 (49). Anal. Calcd. for C30H25N5O (471.55) C, 76.41; H, 5.34; N, 14.85. Found: C, 76.25; H, 5.20; N, 14.70%.

2.1.3.3. 2-[(4-Methoxyphenylmethylene)hydrazono]-9,10,11-trihydro-1-phenyl-benzo[6' ,7' ]cyclohepta[1' ,2' :4,5]pyrido[2,3-d]pyrimidin-4-one (6c). Yellow solid (0.99 g, 82%) m.p. 300302 0C (ethanol) 1H NMR (DMSO-d6) d ppm: 2.15-2.80 (m, 6H, 3CH2), 3.28 (s, 3H, OCH3), 6.93-8.03 (m, 13H, ArH), 8.04 (s, 1H, N=CH), 8.25 (s, 1H, pyridine-H), 10.93 (s, 1H, NH, D2O exchangeable). IR v cm"1: 3205 (NH), 1687 (C=O) cm"1. Ms m/z (%) 488 (M++1, 21), 487 (M + , 73), 380 (54), 271 (6), 207 (15), 122 (6), 120 (12), 107 (30), 91 (49), 77 (100). Anal. Calcd. for C30H25N5O2 (487.55) C, 73.90; H, 5.17; N, 14.36. Found: C, 73.68; H, 5.01; N, 14.22%.

2.1.3.4. 2-[(4-Chlorophenylmethylene)hydrazono]-9,10,11-tri-hydro-1-phenyl-benzo[6' ,7' ]cyclohepta[1' ,2' :4,5]pyrido[2,3-d]pyrimidin-4-one (6d). Yellow crystals (0.92 g, 75%) m.p. 240-242 0C (ethanol) NMR (DMSO-d6) d ppm: 2.18-2.50 (m, 6H, 3CH2), 7.18-8.04 (m, 13H, ArH), 8.08 (s, 1H, N=CH), 8.27 (s, 1H, pyridine-H), 11.25 (s, 1H, NH, D2O exchangeable). IR v cm"1: 3434 (NH), 1709 (C=O) cm"1. Ms mjz (%) 493 (M + +2, 20), 492 (M+ + 1, 28), 491 (M + , 41), 380 (100), 353 (29), 339 (3), 312 (4), 205 (8), 165 (19), 153 (18), 124 (14), 111 (34), 91 (7), 90 (13), 89 (85), 77 (82). Anal. Calcd. for C29H22ClN5O (491.97) C, 70.80; H, 4.51; N, 14.24. Found: C, 70.66; H, 4.28; N, 14.51%.

2.1.3.5. 2-[(4-Nitrophenylmethylene)hydrazono]-9,10,11-trihy-dro-1-phenyl-benzo[6' ,7' ]cyclohepta[1', 2' :4,5]pyrido[2,3-d]pyrimidin-4-one (6e). Dark orange solid (0.90 g, 72%) m.p. 250-252 0C (ethanol) 1H NMR (DMSO-d6) d ppm: 2.21-2.60 (m, 6H, 3CH2), 7.27-8.32 (m, 14H, ArH, NH D2O exchangeable), 8.35 (s, 1H, N=CH), 8.69 (s, 1H, pyridine-H). IR v cm"1: 3421(NH), 1716 (C=O) cm"1. Ms mjz (%) 503 (M + , 2), 392 (24), 367 (8), 353 (5), 339 (2), 295 (100), 225 (2), 193(7), 131 (7), 111 (15), 91 (36), 89 (4), 77(4). Anal. Calcd. for C29H22N6O3 (502.52) C, 69.31; H, 4.41; N, 16.72. Found: C, 69.20; H, 4.21; N, 16.65%.

2.1.3.6. 2-[(4-Dimethylaminophenylmethylene)hydrazono]-9,10,11-trihydro-1-phenyl-benzo[6' ,7' ]cyclohepta[1', 2':4, 5]pyr-ido[2,3-d]pyrimidin-4-one (6f). Yellow solid (65%) m.p. >300 0C (ethanol/dioxane) 1H NMR (DMSO-d6) d ppm: 2.20-2.62 (m, 6H, 3CH2), 3.55 (s, 6H, 2CH3), 6.87-7.69 (m, 14H, ArH, NH D2O exchangeable), 7.89 (s, 1H, N=CH), 8.60 (s, 1H, pyridine-H). IR v cm"1: 3315 (NH), 1690 (C=O) cm"1. Ms mjz (%) 500 (M + , 1), 499 (1), 336 (14), 281 (35), 254 (27), 251 (12), 207 (11), 191 (20), 76 (100). Anal. Calcd. for C31H28N6O (500.59) C, 74.38; H, 5.64; N, 16.79. Found: C, 74.27; H, 5.39; N, 16.52%.

2.1.3.7. 2-[(2-Furfurylmethylene)hydrazono]-9,10,11-trihydro-1-phenylbenzo[6' ,7' ]cyclohepta[1' ,2' :4,5]pyrido[2,3-d]pyrimi-din-4-one (6g). Yellow crystals (0.76 g, 68%) m.p. 270-272 0C (ethanol) 1H NMR (DMSO-d6) d ppm: 1.91-2.63 (m, 6H, 3CH2), 6.66-7.64 (m, 13H, ArH, NH, D2O exchangeable), 7.89 (s, 1H, N=CH), 8.67 (s, 1H, pyridine-H). IR v cm"1: 3422 (NH), 1718 (C=O) cm"1. Ms mjz (%) 447 (M + , 2), 380 (3), 368 (6), 353 (3), 339 (3), 221 (3), 195 (11), 142 (5), 76 (14). Anal. Calcd. for C27H21N5O2 (447.49) C, 72.47; H, 4.73; N, 15.65. Found: C, 72.32; H, 4.60; N, 15.42%.

2.1.4. Synthesis of 7,8,9-trihydro-4-phenyl-benzo[6'',7"]cyclo-hepta[1 ,2 :4 ,5 ]pyrido[2 ,3 -d][1,2,4]triazolo[4,3-a]pyrimidin-14-ones (7a-g)

Bromine (0.052 g, 1 mmol) in acetic acid (5 mL) was added dropwise to a stirred solution of the appropriate hydrazone 6a-g (1 mmol of each) in acetic acid (10 mL). The reaction mixture was then poured into ice cold water, and the solid that precipitated was filtered off, washed with sodium bicarbonate solution and then with water, dried and crystallized from the appropriate solvent to give the respective compounds 7a-g.

2.1.4.1. 1,4-Diphenyl-7,8,9-trihydro-benzo[6'' ,7" ]cyclohepta[1'', 2 :4 ,5 ]pyrido[2 ,3 -d][1,2,4]triazolo[4,3-a]pyrimidin-14-one

(7a). Yellow solid (0.28 g, 62%) m.p. 246-248 0C (ethanol). 1H NMR (DMSO-d6) d ppm: 2.22-2.62 (m, 6H, 3CH2), 7.34-8.25 (m, 14H, ArH), 8.69 (s, 1H, pyridine-H). IR v cm"1: 1717 (C=O) cm"1. Ms mjz (%) 457 (M+ +2, 5), 456 (M+ + 1, 22), 455 (M + , 54), 454 (16), 295 (49), 192 (29), 178 (10), 165 (19), 115 (14), 103 (29), 77 (100). Anal. Calcd. for C29H21N5O (455.51) C, 76.47; H, 4.65; N, 15.37. Found: C, 76.62; H, 4.41; N, 15.06%.

2.1.4.2. 1-( 4-Methylphenyl)-7 ,8 ,9-trihydro-4-phenyl-benzo [6'',7'']cyclohepta[1'',2'':4',5']pyrido[2',3'-d][1,2,4]triazolo [4,3-a]pyrimidin-14-one (7b). Pale yellow (0.32 g, 68%) m.p. 258-260 0C (ethanol) 1H NMR (DMSO-d6) d ppm: 2.34-2.61(m, 6H, 3CH2), 2.63 (s, 3H, CH3), 7.31-7.99 (m, 13H, ArH), 8.48 (s, 1H, pyridine-H). IR v cm"1: 1695 (C=O) cm"1. Ms mjz (%) 471 (M+ +2, 11), 469 (M + , 35), 296 (20), 295 (37), 192 (32), 165 (30), 118 (20), 115 (33), 91 (44), 89 (22), 77 (100). Anal. Calcd. for C30H23N5O (469.54) C, 76.74; H, 4.94; N, 14.92. Found: C, 76.52; H, 4.63; N, 14.66%.

2.1.4.3. 1-( 4-Methoxyphenyl)-7,8,9-trihydro-4-phenyl-benzo [6'',7'']cyclohepta[r,2'':4',5']pyrido[2',3'-d][1,2,4]triazol-o[4,3-a]pyrimidin-14-one (7c). Orange crystals (0.33 g, 69%) m.p. 230-232 0C (ethanol) 1H NMR (DMSO-d6) d ppm: 2.19-2.55 (m, 6H, 3CH2), 4.58 (s, 3H, OCH3), 7.31-7.99 (m, 13H, ArH), 8.55 (s, 1H, pyridine-H). IR v cm"1: 1705 (C=O) cm"1. Ms mjz (%) 486 (M+ + 1, 21), 485 (M + , 18), 297 (18), 296 (36), 191 (29), 165 (32), 164 (32), 103 (18), 89 (21), 77 (100). Anal. Calcd. for C30H23N5O2 (485.54) C, 74.21; H, 4.77; N, 14.42. Found: C, 74.10; H, 4.60; N, 14.20%.

2.1.4.4. 1-(4-Chlorophenyl)-7,8,9-trihydro-4-phenyl-benzo-[6'',7'']cyclohepta[r',2'':4',5']pyrido[2',3'-d][1,2,4]triazolo-[4,3-a]pyrimidin-14-one (7d). Yellow solid (0.30 g, 62%) m.p. 290-292 0C (ethanol) 1H NMR (DMSO-d6) d ppm: 2.06-2.62 (m, 6H, 3CH2), 7.16-8.05 (m, 13H, ArH), 8.62 (s, 1H, pyri-dine-H). IR v cm"1 1698 (C=O) cm"1. Ms mjz (%) 491 (M++2, 6), 490 (M++1, 18), 489 (M + , 55), 297 (48), 233 (15), 111 (83), 89 (67), 77 (100). Anal. Calcd. for C29H20ClN5O (489.95) C, 71.09; H, 4.11; N, 14.29. Found: C, 71.24; H, 4.28; N, 14.08%.

2.1.4.5. 1-( 4-Nitrophenyl)-7,8,9-trihydro-4-phenyl-benzo[ 6'',7" ] cyclohepta[1'' ,2" :4' ,5' ]pyrido[2',3'-d][1,2,4]triazolo[4,3-a] pyrimidin-14-one (7e). Orange solid (64%) m.p. >300 0C(diox-ane) 1H NMR (DMSO-d6) d ppm: 1.90-2.41 (m, 6H, 3CH2), 7.03-7.80 (m, 13H, ArH), 8.35 (s, 1H, pyridine-H). IR v cm"1 1690 (C=O) cm"1. Ms mjz (%) 501 (M+ + 1, 10), 500 (M + , 25), 89 (60), 77 (100). Anal. Calcd. for C29H20N6O3 (500.51) C, 69.59; H, 4.03; N,16.79. Found: C, 69.31; H, 4.10; N, 16.63%.

2.1.4.6. 1-(4-Dimethylaminophenyl)-7,8,9-trihydro-4-phenyl-benzo[6'',7'']cyclohepta[r',2":4',5']pyrido[2',3'-d][1,2,4]triaz-olo[4,3-a]pyrimidin-14-one (7f). Orange solid (62%) m.p. >300 0C (dioxane) 1H NMR (DMSO-d6) d ppm: 2.01-2.48 (m, 6H, 3CH2), 3.62 (s, 6H, 2CH3), 6.97-8.02 (m, 13H, ArH), 8.58 (s, 1H, pyridine-H). IR v: 1685 (C=O) cm"1. Ms mjz (%) 499 (M+ + 1, 2), 498 (M + , 24), 124 (45), 114 (85), 89 (58), 77 (100). Anal. Calcd. for C31H26N6O (498.58) C,74.68; H, 5.26; N, 16.86. Found: C, 74.42; H, 5.09; N, 16.80%.

-N(Me)2

N N "S

AcOH / reflux -/"

NH2NH2 / ethanol reflux

ArCHO / AcOH / HCl reflux

^ ^N N

« Ph H 6

Jk /^Ar

Br2/AcOH

stirring

Ar : C6H4X

X: a, H; b, 4-CH3; c, 4-OCH3; d, 4-Cl; e, 4-NO2; f, 4-N(Me)2

Ar ■ g, -Q

Scheme 1 Synthesis of compounds 6a-g and 7a-g.

2.1.4.7. 1-(2-Furfuryl)-7,8,9-trihydro-4-phenyl-benzo[6'' ,7'' ] cyclohepta[1'',2'':4',5']pyrido[2',3'-d][1,2,4]triazolo[4,3-a] pyrimidin-14-one (7g). Yellow solid (0.30 g, 60%) m.p. 286288 oc (ethanol) NMR (DMSO-d6) d ppm: 1.91-2.40 (m, 6H, 3CH2), 7.35-7.89 (m, 12H, ArH), 8.47 (s, 1H, pyridine-H). IR v cm"1 1678 (C=O) cm"1. Ms m/z (%) 445 (M + , 100), 367 (15), 114 (18), 89 (25), 77 (95). Anal. Calcd. for C27H19N5O2 (445.47): C, 72.80; H, 4.30; N, 15.72. Found: C, 72.65; H, 4.22; N, 15.57%.

2.2. Pharmacology

2.2.1. Anticancer activity

The anticancer activity of the synthesized compounds 5, 6 and 7 was determined against the human breast cell line (MCF-7) and the liver carcinoma cell line (HEPG-2), using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and doxorubicin was used as a reference drug.

Data generated were used to plot a dose response curve of which the concentration of test compounds required to kill 50% of cell population (IC50) was determined. Cytotoxic activity was expressed as the mean IC50 of three independent experiments (Table 1). The method applied is similar to that reported by Vijayan et al. (2004) using Crystal violet stain (1%). Cells were seeded in 96-well plate at a cell concentration 1 x 104 cells per well in 100 il of growth medium. Fresh medium containing different concentrations of the test sample was added after 24 h of seeding. Serial twofold dilutions of the tested chemical compound were added to confluent cell mono-layers, flat-bottomed microtiter plates using a multichannel pipette. The microtiter plates were incubated at 37 oc in a humidified incubator with 5% CO2 for 48 h. Three wells were used for each concentration of the test sample. Control cells were incubated without test sample with DMSO. After incubation of the cells for 24 h at 37 oC, various concentrations of sample (50, 25, 12.5, 6.25, 3.125, and 1.56 ig) were added,

and the incubation was continued for 48 h and the viable cells' yield was determined by a colorimetric method (Carmichael et al., 1987). After the end of the incubation period, media were aspirated and the crystal violet solution was added to each well for at least 30 min. The stain was removed and the plates were rinsed using tap water until all excess stain is removed. Acetic acid (30%) was then added to all wells and mixed thoroughly, and then the absorbance of the plates was measured after gently shaking on Microplate reader, using a test wavelength of 490 nm. All results were corrected for background absorbance detected in wells without added stain. Treated samples were compared with the cell control in the absence of the tested compounds. All experiments were carried out in triplicate. The cell cytotoxic effect of each tested compound was calculated (Table 1).

3. Results and discussion

3.1. Chemistry

Reaction of 2-dimethylaminomethylenebenzosuberone (1) with 6-amino-1-phenyl-2-thioxopyrimidin-4-one (2) in glacial acetic acid under reflux gave novel tetra-heterocyclic ring system, namely, 2-thioxo-3,9,10,11-tetrahydro-1-phenyl-benzo-[6',7']cyclohepta[1',2':4,5]pyrido[2,3-d]pyrimidin-4-one (4) (Scheme 1). Mass, IR spectra and elemental analysis data of the isolated product were consistent with each of the isomeric structures 3 and 4 (Scheme 1), while, *H NMR spectra revealed singlet signal at d 8.35 ppm assigned for pyridine-2H proton not pyridine-4H proton (Quiroga et al., 2002; Ahmed and Farghaly, 2009) which was consistent with the isomeric structure 4 not the isomer structure 3.

Our target 2-hydrazino-9,10,11-trihydro-1-phenyl-ben-zo[6 ,7 ]cyclo-hepta[1 ,2 :4,5]pyrido[2,3-d]pyrimidin-4-one (5) was prepared by treating compound 4 with hydrazine hydrate in absolute ethanol under reflux till all H2S ceased to evolve about 30 h (Scheme 1). The structure of compound 5 was elucidated on the basis of spectral data and microanalysis. For example, the IR spectrum revealed absorption bands at 3350, 3290, 3188, 1644 cm"1 assignable to NH2, NH, and C=O, respectively. The 1H NMR spectrum showed characteristic broad signal at d = 3.37 ppm assigned for the protons of the NH2 group. In addition, the 13C NMR revealed 19 carbon at d 28.71, 30.63, 32.56, 118.68, 120.67, 121.00, 123.96, 126.34, 128.58, 128.88, 129.78, 135.39, 137.15, 139.36, 140.66, 151.54, 158.02, 160.32, 166.68 ppm.

Condensation of 2-hydrazino-9,10,11-trihydro-1-phenyl-benzo[6 ,7 ]cyclohepta[1 ,2 :4,5]pyrido[2,3-d]pyrimidin-4-one (5) with different aldehydes in acetic acid containing few drops of conc. hydrochloric acid gave the corresponding hydrazone derivatives 6a-g (Scheme 1). The mass spectra of the isolated products 6a-g showed the molecular ion peaks at the expected m/z values. Their IR spectra showed the disappearance of the NH2 group, and revealed in each case a carbonyl band in the region 1717-1695 cm" 1. Also, 1H NMR spectra showed, in each case, the presence of the azomethine at d = 7.688.35 ppm.

Treatment of each of the hydrazones 6 with bromine in acetic acid in the presence of sodium acetate with stirring gave, in each case, a single product as evidenced by TLC analysis. Elemental analyses and mass spectra revealed that each of such isolated

Table 1 IC50 of the selected tested compounds against HEPG-2 ''liver'' cancer cell line and MCF-7 ''breast'' cancer cell line.

Compound No. IC50 (lM) HEPG-2 cell line IC50 (iM) MCF-7 cell line

5 16.38 1.98

6a 22.35 21.56

6b 6.58 12.34

6c 0.18 0.26

6d 10.24 21.01

6f 0.49 0.88

6g 45.21 35.24

7a >50 >50

7b 3.65 0.85

7c 1.05 5.14

7e 3.94 >50

7f 2.68 14.07

7g >50 >50

Doxorubicin 0.469 0.426

products has two hydrogens less than the respective hydrazone. This finding was confirmed by *H NMR spectra which indicated the absence of the -N=CH- and hydrazone -NH-N=C protons. On the basis of this finding, the isolated products were assigned the structure of 7,8,9-trihydro-4-phenyl-benzo [6'',7'']cyclohepta[1'',2'':4',5']pyrido[2',3'-d][1,2,4]triazolo[4,3-a] pyrimidin-14-ones (7a-g) (Scheme 1).

3.2. Pharmacology 3.2.1. Anticancer activity

The in vitro anti-tumor activity of the tested compounds 5, 6a-d,f,g and 7a-c,e-g was evaluated at the Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt, against HEPG-2 "liver" cancer cell line and MCF-7 "breast" cancer cell line. Doxorubicin was used as a reference and showed IC50 = 0.469 and 0.426 ig/mL against (HEPG-2) and (MCF-7) cell lines, respectively. Some of the tested compounds showed effects against both cancer cell lines as shown in Table 1.

3-Hydrazino-2,7,8,9-tetrahydro-1-phenyl-benzo[6 ,7 ]cyclo-hepta[1',2':4,5]pyrido[2,3-d] pyrimidin-4-one (5) has activity against MCF-7 "breast" cancer cell line with IC50 = 1.98 ig/mL. Regarding the activities of the hydrazone derivatives 6a-d,f,g in Table 1, compounds 6c showed reactivity on the two tumoral cell lines more than the reference drug. Also, compound 6f revealed activity near to the reference doxorubicin against the two cell lines used. While the other derivatives showed moderate to low activity.

For compounds 7a-c,e-g derivatives 7c has activity against HEPG-2 "liver" cancer cell line with IC50 = 1.05 ig/mL. In addition, compound 7b showed activity against MCF-7 "breast" cancer cell line (IC50 = 0.85 ig/mL) near to the reference drug. From these data we can conclude that the most reactive compounds are the hydrazone derivatives 6c and 6f which carry electron donating groups (OCH3 and N(CH3)2, respectively at the phenyl methylenehydrazono group at position 3 of compound 6.

4. Conclusion

In summary, we report herein the synthesis of a new series of 7,8,9-trihydro-4-phenyl-benzo[6'',7'']cyclohepta[1'',2'':4',5']pyr-ido[2',3'-d][1,2,4]triazolo[4,3-a]pyrimidin-14-ones (7a-g) from oxidative cyclization of 2-[(substitutedmethylene)hydrazono]-9,10,11-trihydro-1-phenyl-benzo[6',7']cyclohepta[1',2':4,5]pyr-ido[2,3-d]pyrimidin-4-one (6a-g). Compound 6c exhibited high anti-tumor activity against HEPG-2 "liver" cancer cell line and MCF-7 "breast" cancer cell line which is more potent than the reference standard doxorubicin. The other synthesized compounds revealed good to moderate anti-tumor properties against HEPG-2 "liver" cancer cell line and MCF-7 "breast" cancer cell line.

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