HETEROCYCLES

Paper | Regular issue | Vol. 85, No. 10, 2012, pp. 2421-2436
Received, 8th June, 2012, Accepted, 25th July, 2012, Published online, 16th August, 2012.
DOI: 10.3987/COM-12-12523

■ Novel Heterocyclic Derivatives of 2-Quinolinone Associated with Antibacterial and Antitumor Potencies

Hany M. Hassanin* and Somaya M. El-Edfawy

Department of Chemistry, Faculty of Education, Ain Shams University, Roxy, Cairo 11711, Egypt

Abstract

3-(2,2-Dichloroacetyl)-4-hydroxy-1-methyl-quinolin-2(1H)-one (2) was utilized to obtain several new quinolinones bearing imidazole, quinoxaline, 1,2,4-triazine, pyridotriazine and triazolotriazine heterocycles as substituents at position 3. Condensation of compound 2 with aminotriazine 12 afforded the iminone 14 which was subjected to annellation reactions leading to some new quinolinones fused at face [c]; viz. pyrazole, pyrimidine and benzodiazepine. Structure of the new compounds was confirmed by spectral and elemental analyses. The synthesized compounds were screened in vitro for their antimicrobial and antitumor activities.

INTRODUCTION
The chemistry of quinolinone derivatives has been of increasing interest since many of these compounds have found useful chemotherapeutic agents. For example, quinolinones revealed potential hepatitis C polymerase inhibitors.1,2 Also, many quinolinone derivatives were found active as microsomal prostaglandin E2 synthase-13 and selective iNOS.4 Many quinolin­one derivatives have been found active as antibacterial,5,6 anti-hepatitis B virus (HBV) activities,7,8 anti-HIV-1 agents.9 Combination of pyrazole nucleus with quinoline moiety, in one molecular framework, was reported in literature to possess biological activity.10-12 Also, pyrimido[5,4-c]quinolin-5(1H)-ones showed antioxidant and toxicological activities.13 Many pyrimidoquinolines have antiviral activity at nanomolar concentrations against HIV-1,14 and exhibited good antibacterial and antifungal activity.15 In addition, imidazole derivatives display efficacy in human tumor xenograft models.16 On the other hand, 1,2,4-triazine derivatives have been shown to have significant antitumor activities with simultaneous non-toxicity to human normal cell line and HSF cells.17 Furthermore, 1,2,4-triazine derivatives exhibited comparable antibacterial potencies in vitro as that of ampicillin,18 and significant broad cytotoxic activity against cancer cell in low micromolar range.19 Based on these findings, it was of interest to introduce a new series of quinoline and its fused systems incorporated with the above biologically active moieties, especially 1,2,4-triazine, for the evaluation of their antimicrobial and antitumor properties.

RESULTS AND DISCUSSION
The dichloroacetyl derivative 2 was prepared by heating pyranoquinolinedione 1 with sulfuryl chloride in dioxane (Scheme 1).20,21

Reaction of compound 2 with some 1,3-binucleophilic reagents, namely; guanidine hydrochloride, cyanoguanidine, and thiourea, was carried out under reflux in DMF, to afford the corresponding 3-imidazolylquinolinones 3a–c, respectively (Scheme 2). The 1H-NMR spectrum of compound 3a revealed the presence of broad singlet at 9.74 ppm (NH). Moreover, its mass spectrum recorded the molecular ion peak at m/z 254 which agree well with the molecular formula and supports the identity of the structure. Also, the IR spectrum of compound 3b showed a characteristic absorption band due to the nitrile function at 2213 cm-1. While the IR spectrum of compound 3c showed a characteristic absorption band due to thioxo group at 1219 cm-1.

Condensation of compound 2 with 1,4-N,N-binucleophiles such as o-phenylenediamine and 1,6-diaminopyridine derivative 5,22 under reflux in DMF, afforded the quinoxalinylquinolinone 4 and the pyridotriazine derivative 6, respectively (Scheme 3). The 1H-NMR spectrum of compound 4 showed characteristic singlet signal at 8.30 ppm due the CHquinoxalin, in addition to the presence of aromatic protons. While in the 1H-NMR spectrum of compound 6 the integral count of protons appeared in the aromatic region revealed the presence of nine aromatic protons due to benzo protons of quinoline, 1,4-disubstituted benzene protons, and C3-H of pyridotriazine. IR spectrum of the compound 6 exhibits absorption bands at 2219, 2291 cm-1 due to two nitrile groups and 1686, 1660 cm–1 due to C=Opyridone and C=Oquinolinone, respectively.

Reaction of compound 2 with aminoguanidine and thiosemicarbazide as 1,4-binucleophiles, under reflux in DMF, yielded the corresponding 1,2,4-triazine derivatives 7a,b, respectively (Scheme 4). IR spectra of compounds 7a and 7b showed characteristic absorption bands due to the NH groups at 3191 and 3126 cm-1, respectively. The 1H-NMR spectra of compounds 7a and 7b showed characteristic singlets at 8.16 ppm and 8.24 ppm, respectively, which were assigned to the proton of 1,2,4-triazine. In addition to the deuterium exchangeable proton signal due to the N2-H, which appeared at 10.51 ppm and 12.67 ppm of 7a and 7b, respectively. Also, the mass spectrum of compound 7a revealed the molecular ion peak at m/z 269 and the base peak at m/z 242 which was in agreement with its molecular mass after loss of one molecule of HCN. In addition, the mass spectrum of 7b revealed a molecular ion peak at m/z 286 [M+] as the base peak, which is in good accordance with the molecular formula. Hydrazinolysis of the 1,2,4-triazine derivative 7b in DMF produced the hydrazinotriazine 8 (Scheme 4). The elemental analysis of the later compound showed absence of sulfur in the product revealing the replacement of the thiol group. IR spectrum exhibited stretching vibrational bands at 3411, 3335 cm-1 due to NH2 and at 3191, 3100 cm-1 due to OH and NH groups. Also, 1H-NMR spectrum displayed three characteristic signals due to exchangeable protons 8.43 ppm (NH2), 9.99 ppm (NH) and 13.36 ppm (OH). Moreover, the mass spectrum revealed the molecular ion peak at m/z 284 [M+] as the base peak. Thermal cyclocondensation of the hydrazinopyrimidine 8 with triethyl orthoformate, under fusion condition, was carried out to get the triazolotriazine derivative 9 (Scheme 4). The 1H-NMR spectrum of the product 9 revealed the disappearance of the NH2 signal, in addition to the appearance of singlet at 8.24 ppm assigned to the characteristic CHtriazole. Reaction of the hydrazinotriazine 8 with [bis(methylthio)methylene]malononitrile, in DMF under reflux, gave the compound 10. The IR spectrum of the reaction product 10 showed characteristic absorption bands at 3323, 3183 (NH2), 2214 (C≡N), 1671(C=O). 1H-NMR spectrum of the product 10 showed a new singlet signal at 2.88 ppm due to three protons of SCH3 group, in addition to a broad signal at 5.22 ppm due to two protons of NH2 group.

Reaction of the dichloroacetyl derivative 2 with 4-aminotriazine 1123 was carried out in boiling pyridine. Interestingly, the only product that obtained revealed absence of chlorine in its molecular formula according to findings of elemental and mass spectral analysis. In addition, IR spectrum showed three different strong absorption vibrations at 1709, 1667, and 1632 cm-1 indicating the presence of acetyl C=O. Moreover, 1H-NMR spectrum revealed an azomethine C-H singlet signal at 5.93 ppm. These results indicate that a nucleophilic replacement took place with leaving of a chlorine atom which is followed by elimination of hydrogen chloride leading to the α-iminone 12 (Scheme 5).

α-Iminone 12, as interesting starting material, was allowed to react with some binucleophilic reagents to prepare some new quinolinones fused at face [c] with pyrazole, pyrimidine and benzodiazepine heterocycles. Therefore, treatment of α-iminone 12 with hydrazine hydrate, guanidine and o-phenylenediamine, in DMF under reflux, afforded the pyrazoloquinolinone 13, pyrimidoquinolinone 14 and benzodiazepinoquinolinone 15, respectively (Scheme 5). The IR spectra of the three later products did not reveal the absorption bands due to C4-OH and acetyl C=O vibrational frequencies that supports the cyclization process. Moreover, the 1H-NMR spectrum of compound 13 showed signals due to two exchangeable protons characteristic for NHpyrazole and NHtriazine at 10.53 and 13.83 ppm. Mass spectrum of compound 13 exhibited a molecular ion peak at m/z 367, corresponding to its calculated molecular formula. The 1H- NMR spectrum of compound 14 revealed the appearance of three exchangeable protons due to three different N-H signals at 8.78, 11.37 and 12.23 ppm. Mass spectrum of compound 14 revealed a peak at 394 assigned to molecular ion peak. While, the 1H-NMR spectrum of compound 15 showed eight aromatic protons, in addition to two characteristic exchangeable singlet signals at 9.95 and 12.79 ppm due the 2 NH protons.

Biological activity
Antimicrobial Activity
The results depicted in Table 1 showed various activities against all species of microorganisms which suggest that the variations in the structures affect on the growth of the microorganisms. Thus, we can conclude from these results:
1) Most of the prepared compounds showed a low to high antimicrobial activity towards Gram positive bacteria, Gram negative bacteria and the fungal strain (Table 1).
2) Compounds 3c, 7b, 9 and 12 showed higher significant activity than the reference antibiotic Doxymycin against Staphylococcus aureus as Gram positive bacteria.
3) Compounds 3c and 7b showed equal activity to the standard antibiotic Doxymycin against Proteus vulgaris and Klebsiella as Gram negative bacteria, while compound 12 showed higher activity.
4) Compounds 3c, 9 and 10 showed higher activity than the standard Fluconazole as fungus strain.
5) The microbial activity of compounds 3c and 12 is due to the presence of two bioactive heterocycles as 1,2,4-triazine and quinolinone. Compounds 9 and 10 are good antimicrobial due to presence of triazolotriazine and pyrazolyltriazine, respectively.

The minimum inhibitory concentration (MIC)
The minimum inhibitory concentration (MIC, μg/mL) of the most active compounds 3c, 7b, 9, 10, and 12 against the selected bacterial and fungal strains were determined (Table 2). The minimum inhibitory concentration (MIC) for these compounds show that these compounds may be considered promising for the development of new antimicrobial agents.

Antitumor activity
The antitumor activity of some of the newly synthesized compounds was investigated against Ehrlich ascites carcinoma cells (EAC). The Antitumor efficacy of the compounds against EAC cell lines was demonstrated compared to doxorubicin. The obtained results revealed that compounds exhibited IC50 values less than the used standard drug (doxorubicin). The triazinylquinolinones 7b, 12, 13, and 14 displayed higher activity (less IC50 values). The reason for the higher reactivity of the triazinylquinolinones can be explained by the presence of two bioactive heterocycles, quinolinone and 1,2,4-triazinone, in one molecular frame, especially 6-methyl-5-oxo-3-thioxo-1,2,4-triazinone moiety in compounds 12-14 which is known with its activity towards tumor cells.24 It has been reported that quinolones are known as potent antitumor agents because they target topoisomerase II enzyme and are considered as therapeutic promise.25 In addition, 1,2,4-triazines show promising cytotoxic effect against different several tumor cell lines.26 Also, El-Naggar et al.,27 found that 1,2,4-triazines significantly reduce tumor growth in EAC bearing mice. The most potent compound in this series is compound 12 due to presence of pyrazole ring fused with quinolone. Xia et al.28 deduced that pyrazoloquinolones have the highest cytotoxic activity against tumor cells in comparison with other qiunolone derivatives due to highest affinity towards topoisomerase II enzyme.

EXPERIMENTAL
Melting points were determined on a digital Stuart SMP3 apparatus. Infrared spectra were measured on Perkin-Elmer 293 spectrophotometer (cm-1), using KBr disks. 1H-NMR spectra were measured on Gemini-300BB spectrometer 300 MHz (at 75 MHz for 13C), or Jeol Eca-500 MHz (at 125 MHz for 13C) using DMSO-d6 or CDCl3 as a solvent and TMS (δ) as the internal standard. Mass spectra were obtained using GC-2010 Shimadzu GC-Mass spectrometer (70 eV). Elemental microanalyses were performed on a Perkin–Elmer CHN-2400 analyzer. Compounds 1, 5 and 12 are obtained according to literature methods20,22,23
3-(2,2-Dichloroacetyl)-4-hydroxy-1-methylquinolin-2(1H)-one (2)
To a suspension of compound 1 (2.43 gm, 10 mmol) in dioxane (20 mL) sulfuryl chloride (3.3 mL, 40 mmole) was added portionwise, while the temperature was not allowed to rise above 50 °C, where it was then kept for additional 10 min. The mixture was quickly heated to the boil and poured into ice-water (100 mL). The solid obtained was filtered and crystallized from toluene to give 2 as yellow crystals, mp 188-190 °C (3.22 g, 51%).21
General procedure for the preparation of compounds 3a-c
The 1,3-binucleophile (10 mmol) was added in one portion to a solution of compound 2 (2.85 g, 10 mmol) in DMF (25 mL) and the mixture was heated under reflux for 4 h. The solid obtained, after cooling was filtered off and then dried and recrystallized.
1-Methyl-4-hydroxy-3-(2-imino-2H-imidazol-4-yl)quinolin-2(1H)-one (3a)
Obtained from guanidine hydrochloride (0.94 g), crystallized from acetic acid as yellow crystals, mp 273-275 °C (1.93 g, 76%). IR (KBr, cm-1): 3366 (OH), 3206 (NH), 3030 (CHarom.), 2917 (CHaliphatic), 1668 (C=Oquinolinone), 1622 (C=N), 1586 (C=C). 1H-NMR (500 MHz, DMSO-d6), δ: 3.49 (s, 3H, NCH3), 7.21 (t, 1H, J = 7.2 Hz, H-6), 7.59–7.67 (m, 2H, H-8 and H-7), 7.91(s, 1H, CHimidazole), 8.06 (d, 1H, J = 8.0 Hz, H-5), 9.74 (s, 1H, NH exchangeable with D2O), 12.59 (s, 1H, OH exchangeable with D2O). M/z (relative intensity): 255 [M+ +1, 6], 254 [M+, 33], 228 (20), 221 (11), 200 (18), 188 (25), 175 (100), 172 (21), 147 (24), 146 (20), 134 (23), 115 (14), 106 (14), 104 (24), 77 (34). Anal. Calcd for C13H10N4O2 (254.24): C, 61.41; H, 3.96; N, 22.04%. Found: C, 61.44; H, 3.83; N, 22.01%.
1-Methyl-4-hydroxy-3-(2-cyanoiminoimidazol-4-yl)quinolin-2(1H)-one (3b)
Obtained from cyanoguanidine (0.84 g), crystallized from DMF as yellow crystals, mp 262-264 °C (1.81 g, 65%). IR (KBr, cm-1): 3400 (OH), 3080 (CHarom.), 2924, 2855 (CHaliphatic), 2213 (C≡N), 1629 (C=Oquinolinone), 1611 (C=N). 1H-NMR (500 MHz, DMSO-d6), δ: 3.48 (s, 3H, CH3), 7.32 (t, 1H, J = 7.4 Hz, H-6), 7.64 (d, 1H, J = 8.0 Hz, H-8), 7.89 (t, 1H, J = 7.2 Hz, H-7), 8.01 (d, 1H, J = 8.2 Hz, H-5), 8.15 (s, 1H, CHimidazole), 14.25 (s, 1H, OH exchangeable with D2O). 13C-NMR (125 MHz, DMSO-d6), δ: 30.2, 86.3, 111.1, 117.7, 118.5, 121.9, 123.2, 125.1, 133.9, 135.7, 140.5, 149.1, 152.5, 152.6. M/z (relative intensity): 279 [M+, 42], 266 (95), 253 (42), 225 (59), 199 (17), 188 (50), 185 (76), 146 (11), 140 (44), 132 (14), 115 (25), 105 (28), 94 (100). Anal. Calcd for C14H9N5O2 (279.26): C, 60.21; H, 3.25; N, 25.08%. Found: C, 60.14; H, 3.22; N, 25.04%.
1-Methyl-4-hydroxy-3-(2-thioxo-1,2-dihydroimidazol-4-yl)quinolin-2(1H)-one (3c)
Obtained from thiourea (0.76 g), crystallized from acetic acid as yellow crystals, mp 255-257 °C (1.81 g, 67%). IR (KBr, cm-1): 3393 (OH), 3074 (CHarom.), 2977, 2833 (CHaliphatic), 1666 (C=Oquinolinone), 1624 (C=N), 1219 (C=S). 1H-NMR (300 MHz, DMSO-d6), δ: 3.63 (s, 3H, NCH3), 7.23 (t, 1H, J = 7.0 Hz, H-6), 7.67 (d, 1H, J = 8.2 Hz, H-8), 7.89 (t, 1H, J = 7.2 Hz, H-7), 8.05 (d, 1H, J = 8.2 Hz, H-5), 8.2 (s, 1H, CHimidazole), 13.85 (s, 1H, OH exchangeable with D2O). Anal. Calcd for C13H9N3O2S (271.26): C, 57.55; H, 3.34; N, 15.49; S, 11.80%. Found: C, 57.21; H, 3.12; N, 15.04%.

1-Methyl-4-hydroxy-3-(quinoxalin-2-yl)quinolin-2(1H)-one (4)
A mixture of compound 2 (2.85 g, 10 mmol) and o-phenylenediamine (1.1 g, 10 mmol), in DMF (25 mL), was heated under reflux for 3 h. The solid obtained during heating was filtered and crystallized from acetic acid to give 4 as brown crystals, mp 276–279 °C (2.03 g, 67%). IR (KBr, cm-1): 3411 (OH), 3070 (CHarom.), 2979, 2937 (CHaliphatic), 1654 (C=Oquinoline), 1600, 1567 (C=N and C=C). 1H-NMR (500 MHz, DMSO-d6), δ: 3.63 (s, 3H, NCH3), 7.24 (t, 1H, J = 7.2 Hz, H-6), 7.28 -7.83 (m, 6H, Ar-H), 8.06 (d, 1H, J = 8.0 Hz, H-5), 8.30 (s, 1H, CHquinoxaline), 13.85 (s, 1H, OH exchangeable with D2O). 13C-NMR (125 MHz, DMSO-d6), δ: 28.2, 98.7, 111.8, 114.25, 115.8, 119.3, 121.6, 126.0, 128.4, 129.3, 130.0, 132.8, 134.7, 137.8, 144.0, 144.5, 146.2, 153.2. M/z (relative intensity): 304 [M++1, 5], 303 [M+, 81], 293 (13), 288 (14), 285 (37), 281 (36), 280 (20), 253 (38), 252 (30), 250 (100), 249 (17), 220 (34), 218 (36), 214 (31), 202 (20), 185 (25), 136 (38), 134 (50), 128 (29), 118 (25), 97 (48), 77 (65). Anal. Calcd for C18H13N3O2 (303.32): C, 71.28; H, 4.32; N, 13.85%. Found: C, 71.24; H, 4.23; N, 13.74%.
1-Methyl-4-hydroxy3-(8-(4-chlorophenyl)-6-oxo-6H-7,9-dicarbonitrilepyrido[1,2-b][1,2,4]triazin-2-yl)quinolin-2(1H)-one (6)
A mixture of compound 2 (2.85 g, 10 mmol) and 1,6-diamino-4-(4-chlorophenyl)-2-oxo-1,2- dihydropyridine-3,5-dicarbonitrile (5) (2.85 g, 10 mmol) in DMF (25 mL), was heated under reflux for 3 h. The solid obtained during heating was filtered and crystallized from DMF to give 6 as white crystals, mp 291-293 °C (3.22 g, 67%). IR (KBr, cm-1): 3357 (OH), 2973, 2930 (CHaliphatic), 2291, 2219 (2C≡N), 1686 (C=Opyridone), 1660 (C=Oquinolinone), 1612 (C=N), 1583 (C=C). 1H-NMR (300 MHz, DMSO-d6), δ: 3.61 (s, 3H, NCH3), 7.34 (t, 1H, J = 6.0 Hz, H-6), 7.44 (d, 2H, Ar-H), 7.56 (d, 1H, J = 6.4 Hz, Ar-H), 7.82 (m, 3H, Ar-H), 8.13 (d, 1H, J = 8.4 Hz, H-5), 8.18 (s, 1H, CHpyridotriazine), 12.81 (bs, 1H, OH exchangeable with D2O). M/z (relative intensity): 482 [M++2, 7], 481 [M++1, 35], 480 [M+, 100], 479 [M+-1, 10], 453 (27), 452 (27), 424 (15), 396 (3), 382 (2), 265 (37), 266 (12), 240 (5), 225 (2), 200 (22), 146 (10), 132 (14), 120 (8), 105 (5), 104 (6), 77(15). Anal. Calcd for C25H13ClN6O3 (480.87): C, 62.44; H, 2.72; Cl 7.37; N, 17.48%. Found: C, 62.44; H, 2.73; N, 17.44%.
General procedure for the preparation of compounds 7a and 7b
The 1,4-binucleophile (10 mmol) was added in one portion to a solution of compound 2 (2.85 g, 10 mmol) in DMF (25 mL) and the reaction mixture was heated under reflux for 4 h. The solid obtained, after cooling was filtered off and then dried and recrystallized.
1-Methyl-4-hydroxy-3-(3-imino-2,3-dihydro-1,2,4-triazin-5-yl)quinolin-2(1H)-one (7a)
Obtained from aminoguanidine nitrate (1.37 g), crystallized from DMF as pale yellow crystals, mp 292-294 °C (1.96 g, 73%). IR (KBr, cm-1): 3411 (OH), 3191 (NH), 3070 (CHarom.), 2979, 2937 (CHaliphatic), 1654 (C=Oquinoline), 1600, 1567 (C=N). 1H-NMR (300 MHz, DMSO-d6), δ: 3.61 (s, 3H, NCH3), 7.30 (t, 1H, J = 6.8 Hz, H-6), 7.51 (d, 1H, J = 8.0 Hz, H-8), 7.86 (t, 1H, J = 7.0 Hz, H-7), 8.07 (d, 1H, J = 8.0 Hz, H-5), 8.16 (s, 1H, Htriazine), 9.02 (s, 1H, =NH exchangeable with D2O), 10.51 (s, 1H, NH exchangeable with D2O), 13.93 (s, 1H, OH exchangeable with D2O). M/z (relative intensity): 269 [M+, 8], 242 (M+-HCN, 100), 241 (91), 228 (15), 214 (14), 200 (25), 175 (85), 172 (22), 159 (24), 147 (31), 146 (27), 134 (50), 132 (35), 115 (26), 106 (30), 105 (37), 104 (63), 77(16). Anal. Calcd for C13H11N5O2 (269.26): C, 57.99; H, 4.12; N, 26.01%. Found: C, 57.44; H, 4.09; N, 26.04%.
1-Methyl-4-hydroxy-3-(3-thioxo-2,3-dihydro-1,2,4-triazin-5-yl)quinolin-2(1H)-one (7b)
Obtained from thiosemicarbazide (0.91 g), crystallized from DMF as pale brown crystals, mp 282-284 °C (2.20 g, 77%). IR (KBr, cm-1): 3400 (OH), 3126 (NH), 3030 (CHarom.), 2917 (CHaliphatic), 1668 (C=Oquinolinone), 1623 (C=N). 1H-NMR (500 MHz, DMSO-d6), δ: 3.62 (s, 3H, NCH3), 7.24 (t, 1H, J = 7.2 Hz, H-6), 7.45 (d, 1H, J = 8.0 Hz, H-8), 7.67 (t, 1H, J = 7.2 Hz, H-7), 8.01 (d, 1H, J = 8.0 Hz, H-5), 8.24 (s, 1H, CHtriazine), 12.67 (s, 1H, NH exchangeable with D2O), 14.36 (s, 1H, OH exchangeable with D2O). 13C-NMR (125 MHz, DMSO-d6), δ: 28.6, 102.3, 113.5, 115.3, 122.6, 124.9, 131.3, 136.0, 141.8, 154.1, 159.6, 165.0, 171.5. M/z (relative intensity): 287 [M++1, 19], 286 [M+, 100], 273 (31), 258 (21), 257 (62), 256 (21), 240 (13), 225 (12), 200 (22), 175 (15), 146 (9), 134 (19), 132 (16), 115 (12), 105 (28), 104 (32), 77(50). Anal. Calcd for C13H10N4O2S (286.31): C, 54.54; H, 3.52; N, 19.57, S, 11.20%. Found: C, 54.44; H, 3.53; N, 19.16%.
1-Methyl-4-hydroxy-3-(3-hydrazino-2,3-dihydro-1,2,4-triazin-5-yl)quinolin-2(1H)-one (8)
A mixture of compound 7b (2.86 g, 10 mmol) and hydrazine hydrate (0.58 mL, 12 mmol), in DMF (30 mL), was heated under reflux for 4 h. The solid deposited after cooling was filtered and crystallized from DMF to give compound 8 as white crystals, mp above 300 °C (1.70 g, 60%). IR (KBr, cm-1): 3411, 3335, (NH2), 3191, 3100 (OH, NH), 3070 (CHarom.), 2979, 2937 (CHaliphatic), 1654 (C=Oquinoline), 1600, 1567 (C=N and C=C). 1H-NMR (300 MHz, DMSO-d6), δ: 3.44 (s, 3H, NCH3), 7.26 (t, 1H, J = 7.2 Hz, H-6), 7.53 (d, 1H, J = 8.0 Hz, H-8), 7.71 (t, 1H, J = 7.2 Hz, H-7), 7.93 (d, 1H, J = 8.0 Hz, H-5), 8.25 (s, 1H, CHtriazine), 8.43 (s, 2H, NH2 exchangeable with D2O), 9.99 (s, 1H, NH exchangeable with D2O), 13.36 (s, 1H, OH exchangeable with D2O). M/z (relative intensity): 285 [M++1, 17], 284 [M+, 100], 256 (25), 255 (6), 254 (11), 216 (96), 215 (19), 214 (69), 172 (51), 146 (66), 132 (57), 115 (29), 105 (14), 104 (22), 77(67). Anal. Calcd for C13H12N6O2 (284.28): C, 54.93; H, 4.25; N, 29.56%. Found: C, 54.84; H, 4.23; N, 29.54%.
1-Methyl-4-hydroxy-3-[1,2,4-triazolo[4,3-b][1,2,4]triazin-7-yl]quinolin-2(1H)-one (9)
A solution of compound 8 (2.84 g, 10 mmol) in triethyl orthoformate (8.16 mL, 48 mmol) was heated under fusion for 2 h. The precipitate so formed on hot was filtered and crystallized from acetic acid to give compound 9 as pale yellow crystals, mp above 300 °C (1.85 g, 63%). IR (KBr, cm-1): 3446 (OH), 3075 (CHarom.), 2977, 2934 (CHaliphatic), 1673 (C=Oquinolinone), 1634 (C=N), 1598 (C=C). 1H-NMR (300 MHz, DMSO-d6), δ: 3.47 (s, 3H, NCH3), 7.24 (t, 1H, J = 6.0 Hz, H-6), 7.47 (d, 1H, J = 8.0 Hz, H-8), 7.67 (t, 1H, J = 6.4 Hz, H-7), 7.99 (d, 1H, J = 8.4 Hz, H-5), 8.01, 8.24 (2s, 2H, Htriazine and Htriazole), 14.36 (bs, 1H, OH exchangeable with D2O). 13C-NMR (75 MHz, DMSO-d6), δ: 28.2, 99.3, 113.3, 117.0, 118.9, 124.7, 125.4, 139.0, 141.5, 154.1, 156.4, 159.4, 163.0, 166.4. Anal. Calcd for C14H10N6O2 (294.27): C, 57.14; H, 3.43; N, 28.56%. Found: C, 57.12; H, 3.33; N, 28.54%.
5-Amino-1-[5-(4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-yl)-[1,2,4]triazine-3-yl]-3-methyl- sulfanyl-1H-pyrazole-4-carbonitrile (10)
A mixture of compound 8 (2.84 g, 10 mmol) and [bis(methylthio)methylene]malononitrile (1.70 gm, 10 mmol), in DMF (25 mL) was heated under reflux for 4 h. The reaction solution was left to cool at room temperature and the solid so formed was filtered and crystallized from dioxane to give compound 10 as pale yellow crystals, mp above 300 °C (2.48 g, 61%). IR (KBr, cm-1): 3400 (OH), 3323, 3183 (NH2), 3080 (CHarom.), 2924, 2855 (CHaliphatic), 2214 (C≡N), 1671(C=Oquinolinone), 1629 (C=N), 1611 (C=C). 1H-NMR (300 MHz, DMSO-d6), δ: 2.88 (s, 3H, SCH3), 3.61 (s, 3H, NCH3), 5.22 (bs, 2H, NH2 exchangeable with D2O), 7.22 (t, 1H, J = 6.2 Hz, H-6), 7.49 (d, 1H, J = 8.2 Hz, H-8), 7.77 (t, 1H, J = 6.4 Hz, H-7), 8.13 (d, 1H, J = 8.0 Hz, H-5), 8.23 (s, 1H, CHtriazine), 12.28 (bs, 1H, OH exchangeable with D2O). Anal. Calcd for C18H14N8O2S (406.43): C, 53.20; H, 3.47; N, 27.57; S, 7.89%. Found: C, 53.12; H, 3.43; N, 27.44; S, 7.85%.

1-Methyl-4-hydroxy-3-[2-(6-methyl-5-oxo-3-thioxo-2,3dihydro-1,2,4-triazin-4(5H)-ylimino)acetyl]-　quinolin-2(1H)-one (12)
A mixture of compound 2 (2.85 g, 10 mmol) and aminotriazine 11 (1.58 g, 10 mmol), in pyridine (30 mL), was heated under reflux for 4 h. Then, the solution was filtered and the clear solution was acidified by dilute HCl. The precipitate so formed was filtered, washed several times with water, air dried and crystallized from ethanol to give 12 as yellow crystals, mp 236-237 °C (2.22 g, 60%). IR (KBr, cm-1): 3419 (OH), 2981, 2934 (CHaliphatic), 1709 (C=O), 1667, 1632 (C=Otriazinone and C=Oquinolinone), 1599, 1575 (C=N and C=C). 1H-NMR (300 MHz, DMSO-d6), δ: 3.23 (s, 3H, CH3 triazine), 3.68 (s, 3H, NCH3), 5.93 (s, 1H, CH=N), 7.20 (t, 1H, J = 6.2 Hz, H-6), 7.44 (d, 1H, J = 8.0 Hz, H-8), 7.61 (t, 1H, J = 6.4 Hz, H-7), 7.89 (d, 1H, J = 8.4 Hz, H-5), 11.39 (bs, 1H, NH exchangeable with D2O), 13.23 (bs, 1H, OH exchangeable with D2O). 13C-NMR (75 MHz, DMSO-d6), δ: 13.6, 28.6, 61.8, 102.6, 113.8, 115.5, 117.0, 122.8, 125.2, 136.2, 142.0, 153.3, 160.1, 163.0, 171.8, 190.4. M/z (relative intensity): 371 [M+, 16], 343 (17), 326 (23), 292 (16), 278 (16), 275 (27), 269 (60), 241 (14), 223 (32), 197 (30), 175 (20), 174 (22), 158 (7), 146 (43), 136 (16), 132 (79), 114 (13), 105 (27), 104 (33), 77(79), 63 (100). Anal. Calcd for C16H13N5O4S (371.38): C, 51.75; H, 3.53; N, 18.86, S, 8.63%. Found: C, 51.74; H, 3.53; N, 18.74; S, 8.53%.
General procedure for the preparation of compounds 13, 14 and 15
Hydrazine hydrate (12 mmol), guanidine hydrochloride (10 mmol) and/or o-phenylenediamine (10 mmol) was added to a solution of compound 12 (3.71 g, 10 mmol) in DMF (25 mL) and the reaction mixture was refluxed for 4 h. The solid obtained after cooling was filtered off and then dried and recrystallized to give compounds 13, 14 and 15 respectively.
5-Methyl-3-[(6-methyl-5-oxo-3-thioxo-2,5-dihydro-3H-1,2,4-triazin-4-ylimino)methyl]-1,5-dihydro-pyrazolo[4,3-c]quinolin-4-one (13)
Obtained from hydrazine hydrate (0.58 mL, 12 mmol), crystallized from DMF as yellow crystals, mp above 300 °C (2.27 g, 62%). IR (KBr, cm-1): 3292 (NH), 2978, 2870 (CHaliphatic), 1662, 1630 (C=Otriazinone and C=Oquinolinone), 1603, 1572 (C=N and C=C). 1H-NMR (300 MHz, DMSO-d6), δ: 2.88 (s, 3H, CH3 triazine), 3.63 (s, 3H, NCH3), 5.57 (s, 1H, CH=N), 7.28 (t, 1H, J = 6.2 Hz, H-8), 7.32 (d, 1H, J = 8.0 Hz, H-6), 7.77 (t, 1H, J = 6.4 Hz, H-7), 8.13 (d, 1H, J = 8.4 Hz, H-9), 10.53 (bs, 1H, NH exchangeable with D2O), 13.83 (bs, 1H, NH exchangeable with D2O). 13C-NMR (75 MHz, DMSO-d6), δ: 16.2, 28.9, 62.9, 98.2, 114.5, 118.2, 119.9, 120.7, 122.2, 124.2, 128.5, 135.5, 145.4, 153.7, 155.6, 176.7. M/z (relative intensity): 367 [M+, 25], 366 [M+ -1, 20], 326 (27), 303 (25), 278 (26), 258 (20), 255(26), 249 (21), 201 (21), 175 (21), 148 (31), 134 (22), 115 (26), 105 (22), 104 (27), 86(100), 77 (36). Anal. Calcd for C16H13N7O2S (367.39): C, 52.31; H, 3.57; N, 26.69, S, 8.73%. Found: C, 52.21; H, 3.53; N, 26.64; S, 8.63%.
2-Imino-6-methyl-4-[(6-methyl-5-oxo-3-thioxo-2,5-dihydro-3H-1,2,4-triazin-4-ylimino)methyl]-2,6-dihydro-1H-pyrimido[5,4-c]quinolin-5-one (14)
Obtained from guanidine hydrochloride (0.94 g, 10 mmol), crystallized from acetic acid as pale yellow crystals, mp above 300 °C (2.40 g, 61%). IR (KBr, cm-1): 3253 (NH), 3072 (CHarom.), 2977, 2932 (CHaliphatic), 1674, 1634 (C=Otriazinone and C=Oquinolinone), 1599, 1569 (C=N and C=C). 1H-NMR (300 MHz, DMSO-d6), δ: 2.88 (s, 3H, CH3 triazine), 3.61 (s, 3H, NCH3), 5.86 (s, 1H, CH=N), 7.22 (t, 1H, J = 6.4 Hz, H-9), 7.43 (d, 1H, J = 8.4 Hz, H-7), 7.67 (t, 1H, J = 6.2 Hz, H-8), 8.10 (d, 1H, J = 8.0 Hz, H-10), 8.78 (bs, 1H, NH exchangeable with D2O), 11.37 (bs, 1H, NH exchangeable with D2O), 12.23 (bs, 1H, NH exchangeable with D2O). M/z (relative intensity): 394 [M+, 11], 393 [M+-1, 13], 379 (11), 365 (12), 331 (14), 322 (100), 321 (60), 294 (65), 268 (46), 266 (22), 257 (11), 240 (13), 189 (11), 175 (10), 146 (23), 132 (33), 132 (16), 115 (14), 108 (25), 105 (12), 93 (22), 77 (42). Anal. Calcd for C17H14N8O2S (394.41): C, 51.77; H, 3.58; N, 28.41; S, 8.13%. Found: C, 51.74; H, 3.53; N, 28.14; S, 8.09%.
8-Methyl-6-[(6-methyl-5-oxo-3-thioxo-2,5-dihydro-3H-1,2,4-triazin-4-ylimino)methyl]-13H-benzo- [1,5]diazepino[6,5-c]quinolin-7(8H)-one (15)
Obtained from o-phenylenediamine (1.1 g, 10 mmol), crystallized from acetic acid as pale yellow crystals, mp above 300 °C (2.91 g, 66%). IR (KBr, cm-1): 3294, 3252 (NH), 2980, 2934 (CHaliphatic), 1670, 1632 (C=Otriazinone and C=Oquinolinone), 1603, 1583 (C=N and C=C). 1H-NMR (300 MHz, DMSO-d6), δ: 3.15 (s, 3H, CH3 triazine), 3.63 (s, 3H, NCH3), 5.87 (s, 1H, CH=N), 7.23 (t, 1H, J = 6.4 Hz, Ar-H), 7.36 (d, 1H, J = 8.2 Hz, Ar-H), 7.42 (t, 1H, J = 6.4 Hz, Ar-H), 7.86-8.13 (m, 5H, Ar-H), 9.95 (s, 1H, NH exchangeable with D2O). 12.79 (bs, 1H, NH exchangeable with D2O). 13C-NMR (75 MHz, DMSO-d6), δ: 16.2, 29.1, 62.1, 92.2, 113.6, 114.3, 115.7, 117.0, 119.6, 127.2, 128.1, 129.2, 129.7, 130.4, 134.5, 138.5, 146.2, 152.2, 152.6, 157.3, 165.2, 174.9. Anal. Calcd for C22H17N7O2S (443.49): C, 59.58; H, 3.86; N, 22.11, S, 7.23%. Found: C, 59.54; H, 3.83; N, 22.02.
Antimicrobial Activity
The standardized disc agar diffusion method29 was followed to determine the activity of the synthesized compounds against the sensitive organisms Staphylococcus aureus as a Gram positive bacterium, Proteus vulgaris and Klebsiella as Gram negative bacteria and Candida albicans as a fungus strain. The antibiotic Doxymycin and Fluconazole were used in concentration 100 µg mL-1, as references for antibacterial and antifungal agents. The compounds were dissolved in DMSO which has no inhibition activity to get concentration of 100 µg mL-1. The test was performed on medium potato dextrose agars (PDA) which contain infusion of 200 g potatoes, 6 g dextrose and 15 g agar.30 Uniform size filter paper disks (3 disks per compound) were impregnated by equal volume (10 µL) from the specific concentration of dissolved tested compounds and carefully placed on inoculated agar surface. After incubation for 36 h at 27 °C in the case of bacteria and for 48 h at 24 °C in the case of fungi, inhibition of the organisms was measured and used to calculate mean of inhibition zones.
Antitumor activity
EAC cells were maintained by weekly intraperitoneal transplantation of 2.5 Х 105 cells in mice. The tumor is characterized by a moderately rapid growth, which leads to the death of the mice in about 20 days due to the distal metastasis. Ascites was withdrawn under aseptic conditions from the peritoneal cavity of tumor bearing mice by needle aspiration after 7 days of EAC cells inoculation. To adjust the number of EAC cells/mL, tumor cells obtained were diluted several times with normal saline. EAC viable cells were counted by trypan blue exclusion method where 10 µl trypan blue (0.05%) was mixed with 10 µl of the cell suspension. Within 5 min, the mixture was spread onto haemocytometer, covered with a cover slip and then the cells were examined under microscope. Dead cells are blue stained viable cells are not.31 Cell suspension was adjusted to contain 2.5 X 105 viable cells/ml. EAC cells, RPMI medium drugs, and DMSO were added in sterile test tubes according to trypan blue exclusion method.31 The cells were incubated for two hours at 37 °C under a constant over lay of 5% CO2. EAC viable cells were counted by trypan blue exclusion using haemocytometer as mentioned above. The cell surviving fraction was calculated from the relation T/C; where T and C represent the number of viable cells in a unit volume and the number of total (viable + dead) cells in the same unit volume, respectively.

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