Acta Pharm. 59 (2009) 431–440 Original research paper 10.2478/v10007-009-0040-9 Synthesis, anticancer and cytostatic activity of some 6H-indolo[2,3-b]quinoxalines SUBHAS S. KARKI1* RAHUL HAZARE1 SUJEET KUMAR1 VIVEK S. BHADAURIA1 JAN BALZARINI2 ERIK DE CLERCQ2 1Department of Pharmaceutical Chemistry KLE College of Pharmacy, 2nd Block Rajajinagar, Bangalore-560010, India 2Rega Institute for Medical Research Katholieke Universiteit Leuven B-3000 Leuven, Belgium Accepted October 22, 2009 Various 6-aralkyl-9-substituted-6H-indolo[2,3-b]quinoxalines were synthesized by reaction of 1,5-disubstituted 2,3-dioxo-2,3-dihydroindole and orthophenylene diamine. Appreciable anticancer activity of compounds 5b, 5d, 5g and 5l at various cell lines among 59 human tumor cell panels was observed. All the synthesized compounds were evaluated for cytostatic activity against human Molt 4/C8 and CEM T-lymphocytes as well as for murine L1210 leukemia cells. Compound 5h exhibited an IC50 of 71 mmol L–1 against Molt 4/C8 and 117 mmol L–1 against CEM compared to melphalan 3.2 mmol L–1 and 2.5 mmol L–1, respectively. The IC50 for compound 7i against L1210 was 7.2 mmol L–1 compared to melphalan 2.1 mmol L–1. Keywords: indolo[2,3-b]quinoxaline, cytostatic activity, anticancer activity Quinoxaline derivatives seem to have very interesting biological properties (1–3). The plant alkaloid ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) has DNA-intercalating and antitumor activity and is active against the herpes simplex virus (4, 5). Graslund et al. (6) have studied ellipticine and the derivatives 2,3-dimethyl-6-(2-dimethyl-aminoethyl)6H-indolo[2,3-b]quinoxaline and 6-(2-dimethylaminoethyl)6H-indolo[2,3-b] quinoxaline for their interaction with oligodeoxynucleotide duplexes. They reported that compounds were intercalated in a non-specific fashion and by an AT-specific interaction. Recently, Sauvain et al. (7) reported that 3-(4’-chloro)phenylquinoxaline-2-carbonitrile-1,4-di-N-oxide had potent antimalarial activity particularly against a chloroquine-resistant strain of Plasmodium falciparum. Moarbess et al. (8) examined imidazo[1,2-a]quinoxaline, imidazo[1,5-a]quinoxaline and pyrazolo[1,5-a]quinoxaline derivatives for their in vitro and in vivo anti-tumoral activities. Toshima et al. (9) have designed and evaluated quinoxaline-carbohydrate hybrids as novel and selective photo-induced DNA cleaving and cytotoxic agents. * Correspondence; e-mail: subhasskarki@gmail.com 431 S. S. Karki et al.: Synthesis, anticancer and cytostatic activity of some 6H-indolo[2,3-b]quinoxalines, Acta Pharm. 59 (2009) 431–440. H3 C N N N H N CH 3 N Ellipticine 5,11-dimethyl-6H-pyrido[4,3-b]carbazole CH 3 N N CH 3 CH 3 H3C 6(2-dimethylaminoethyl) 6H-indolo[2,3-b]quinoxalines N N N H3C CH 3 2,3-dimethyl-6(2-dimethylaminoethyl) 6H-indolo[2,3-b]quinoxaline Fig. 1. Structures of ellipticine and other indolo[2,3-b]quinoxalines. Some quinoxalin-2-ones have shown antifungal activity (10, 11) whereas quinoxalin-1-oxides have antibacterial activity (12). 6H-indolo[2,3-b]quinoxaline can be regarded as an aza analogue of ellipticine. It was therefore of interest to test 6,9-disubstituted-6H-indolo[2,3-b]quinoxalines for their anticancer and cytostatic activity. EXPERIMENTAL Melting points were determined in open capillaries and were uncorrected. Rf values were obtained using silica gel thin layer chromatography plates and a solvent system of chloroform/methanol (9:1). 2,3-Dioxy-2,3-dihydroindoles/5-substituted 2,3-dioxy-2,3-dihydroindoles and 1-(4-substituted)arylmethyl-2,3-dioxy-2,3-dihydroindoles were prepared according to literature (13, 14). The infrared spectra of all compounds were determined by a diffuse reflectance technique using potassium bromide powder on a Jasco 460 FTIR machine (Jasco, Japan). 13C NMR for 5i and 5j and 1H NMR spectra (400 MHz) of all compounds were generated in dimethylsulfoxide-d6/CDCl3 on a Bruker Ultraspec spectrophotometer (Germany). FAB mass spectra for 5c and 5j were obtained by using a Jeol SX-102 instrument (Jeol, Japan). CHN for all compounds were generated on an elemental analyzer Vario EL III (Germany). 432 S. S. Karki et al.: Synthesis, anticancer and cytostatic activity of some 6H-indolo[2,3-b]quinoxalines, Acta Pharm. 59 (2009) 431–440. General procedure for the synthesis of 6-aralkyl-9-substituted-6H-indolo[2,3-b]quinoxalines (5a-m) A mixture of 1-arylmethyl-2,3-dioxy-2,3-dihydroindole (0.005 mol), orthophenylene diamine (0.005 mol), glacial acetic acid (0.5–1.0 mL) and anhydrous ethanol (100 mL) was heated under reflux until the reaction was complete (4 h). Approximately half of the ethanol was removed in vacuo and the solution was left overnight at room temperature. The solid that precipitated was collected, washed with cold ethanol and recrystallized from suitable solvent. Products obtained were: 9-fluoro-6-(4-fluorobenzyl)-6H-indolo[2,3-b]quinoxaline (5a), 6-benzyl-9-fluoro-6H-indolo[2,3-b]quinoxaline (5b), 9-methyl-6-(4-methylbenzyl)-6H-indolo[2,3-b]quinoxaline (5c), 6-benzyl-9-methyl-6H-indolo[2,3-b]quinoxaline (5d), 9-chloro-6-(4-fluorobenzyl)-6H-indolo[2,3-b]quinoxaline (5e), 6-benzyl-9-chloro-6H-indolo[2,3-b]quinoxaline (5f), 6-(4-fluorobenzyl)-6H-indolo[2,3-b]quinoxaline (5g), 6-(4-methylbenzyl)-6H-indolo[2,3-b]quinoxaline (5h), 9-bromo-6-(4-methylbenzyl)-6H-indolo[2,3-b]quinoxaline (5i), 9-chloro-6-(4-methylbenzyl)-6H-indolo[2,3-b]quinoxaline (5j), Table I. Physicochemical data of synthesized compounds Compd. Yield (%) M.p. (°C) 5a 65 218–220 5b 68 220–222 5c 72 220–225 5d 75 215–218 5e 75 235–240 5f 68 198–201 5g 72 162–163 5h 80 185–190 5i 75 193–195 5j 80 192–194 5k 71 212–215 5l 71 177–180 5m 76 223–225 Mol. formula (Mr) C21H13F2N3 345.34 C21H14FN3 327.35 C23H19N3 337.42 C22H17N3 323.39 C21H13ClFN3 361.80 C21H14ClN3 343.81 C21H14FN3 327.35 C22H17N3 323.39 C22H16BrN3 402.29 C22H16ClN3 357.84 C21H13BrN3F 406.25 C22H16FN3 341.38 C22H16FN3 341.38 Rf value 0.76 0.52 0.48 0.55 0.40 0.55 0.70 0.76 0.60 0.56 0.31 0.80 0.60 Elemental analysis calcd./found (%) C H N 73.04 72.99 77.05 76.88 81.87 81.26 81.71 81.65 69.71 70.00 73.36 72.98 77.05 77.25 81.71 80.98 65.68 65.01 73.84 73.01 62.09 62.45 77.40 76.97 77.40 76.78 3.79 3.56 4.31 4.25 5.68 5.66 5.30 5.22 3.62 3.55 4.10 4.01 4.31 3.99 5.30 5.32 4.01 3.75 4.51 4.11 3.23 3.01 4.72 4.39 4.72 4.51 12.17 12.25 12.84 13.11 12.45 12.75 12.99 13.09 11.61 11.55 12.22 12.39 12.84 13.02 12.99 13.28 10.45 11.01 11.74 12.13 10.34 11.01 12.31 12.55 12.31 12.81 433 S. S. Karki et al.: Synthesis, anticancer and cytostatic activity of some 6H-indolo[2,3-b]quinoxalines, Acta Pharm. 59 (2009) 431–440. 9-bromo-6-(4-fluorobenzyl)-6H-indolo[2,3-b]quinoxaline (5k), 9-fluoro-6-(4-methylbenzyl)-6Hindolo[2,3-b]quinoxaline (5l), 6-(4-fluorobenzyl)-9-methyl-6H-indolo [2,3-b] quinoxaline (5m). Physico-chemical data for synthesized compounds 5a-m are reported in Table I. Anticancer activity Compounds 5b, 5d, 5g and 5l were submitted to in vitro disease-oriented antitumor screen (15). This assay involves determination of a test agent’s effect on growth parameters against a panel of approximately 60 human tumor cell lines, derived largely from solid tumors, including non-small cell lung, colon, central nervous system, melanoma, ovarian, prostate and breast cancers, plus a few leukemia and renal cell lines. Compounds were tested at a 1.0 ´ 10–5 mol L–1 using DMSO as solvent; a 48-h continuous drug exposure protocol was applied and a sulforhodamine B (SRB) protein assay was used to estimate cell viability or growth. The measured effect of the compound on a cell line was calculated according to one of the following two expressions: if (Atest – Atzero) ³ 0, then percentage growth = 100 (Atest – Atzero) / Actrl – Atzero) if (Atest – Atzero) < 0, then percentage growth = 100 (Atest – Atzero) / Atzero where, Atzero is the average of absorbance measurements of SRB-derived color just before exposure of cells to the test compound, Atest is the average absorbance measurements of SRB-derived color just after 48-h exposure of cells to the test compound, Actrl is the average of absorbance measurements of SRB-derived color just after 48 h with no exposure of cells to the test compound. Cytostatic activity The methodology for cytostatic activity assays in Molt 4/C8, CEM and L1210 assays has been published previously (16). In brief, varying concentrations of compounds were incubated at 37 °C with the cells for 72 h (human Molt 4/C8 or CEM T-lymphocytes) or 48 h (murine L1210 cells). After the incubation period cell number was counted by a coulter counter (Harpenden Herz, UK). RESULTS AND DISCUSSION We have synthesized a series of thirteen derivatives of 6H-indolo[2,3-b]quinoxalines (5a-m) containing an N-aralkyl group at the 6th position by reacting N-aralkylisatin with 434 S. S. Karki et al.: Synthesis, anticancer and cytostatic activity of some 6H-indolo[2,3-b]quinoxalines, Acta Pharm. 59 (2009) 431–440. NH 2 NH2 O HO NH HN R R O N NH R O NH 2 O N alcohol N CH 3COOH R' R' 1 R' 2 3 H2O H2 N N R N N R N O N –H 2O R' R' 5a-m 4 5 R R' 5 R R' a F F h H CH3 b F H i Br CH3 c CH3 CH3 j Cl CH3 d CH3 H k Br F e Cl F l F CH3 f Cl H m CH3 F g H F Scheme 1 orthophenylene diamine, as depicted in Scheme I. The reaction of isatin with orthophenylene diamine has been reported to give spirobenzimidazoline, isatin-3-imine and/or 6H-indolo[2,3-b]quinoxaline in various kinds of solvents. No spiro compound or Schiff’s base Table II. Spectral data of synthesized compounds Compd. 5a 5b 5c IR (n, cm–1) 3100–3030, 2917–2849, 1604, 1584, 1508, 1486 3066–3004, 2904–2849, 1618, 1582, 1511, 1489 3052–3019, 2920–2851, 1615, 1578, 1513, 1487 1H/ 13C NMR (d, ppm) (DMSO-d6) MS 5.73 (s, 2H, -CH2-), 7.02–7.06 (m, 2H, Ar-H), 7.40–7.43 (m, 2H, Ar-H), 7.52–7.57 (m, 1H, Ar-H), 7.62 (d, 1H, J = 8.0, Ar-H), 7.75–7.79 (m, 1H, Ar-H), 7.83–7.87 (m, 1H, Ar-H), 8.19–8.24 (m, 2H, Ar-H), 8.38 (d, 1H, J = 8.0, Ar–H) 5.77 (s, 2H, -CH2-), 7.28–7.31 (m, 4H, Ar-H), 7.32–7.39 (m, 2H, Ar-H), 7.42 (d, 1H, Ar-H), 7.74–7.79 (m, 1H, Ar-H), 7.82–7.86 (m, 1H, Ar-H), 8.19–8.24 (m, 2H, Ar-H), 8.38 (d, 1H, J = 8.0, Ar-H) 2.34 (s, 3H, CH3), 2.59 (s, 3H, CH3), 5.71 (s, 2H, -CH2-), 7.14 (d, 2H, J=7.6, Ar-H), 7.27 (d, 2H, J = 8.0, Ar-H), 7.30–7.32 (m, 2H, Ar-H), 7.49 (d, 1H, J = 8.4, Ar-H), 7.74 338 (100) (t, 1H, J = 14.8, Ar-H), 7.81 (t, 1H, J = 14.8, Ar-H), 8.20 (d, 1H, J = 8.0, Ar-H), 8.35–8.37 (m, 1H, Ar-H) 435 S. S. Karki et al.: Synthesis, anticancer and cytostatic activity of some 6H-indolo[2,3-b]quinoxalines, Acta Pharm. 59 (2009) 431–440. 5d 5e 5f 5g 3059–3024, 2966–2850, 1616, 1578, 1508, 1489 3066, 2917–2848, 1605, 1581, 1509, 1474 3062, 2918–2849, 1609, 1581, 1509, 1455 3045–3013, 2954–2849, 1610, 1583, 1513, 1469. 5h 3053–3022, 2964–2920, 1612, 1583, 1510, 1471 5i 3053–3021, 2918–2849, 1609, 579, 1516, 1453 5j 3054-3022, 2972-2849, 1613, 1581, 1516, 1467 5k 5l 5m 436 3066-3024, 2917-2848, 1605, 1578, 1508, 1475 3054-3029, 2917-2849, 1613, 585, 1515, 1485 3055-3026, 2919-2849, 1603, 579, 1506, 1488 2.59 (s, 3H, -CH3), 5.76 (s, 2H, -CH2-), 7.29–7.38 (m, 6H, Ar-H), 7.49 (d, 1H, J = 8.4, Ar-H), 7.72–7.76 (m, 1H, Ar-H), 7.79–7.83 (m, 1H, Ar-H), 8.18–8.20 (m, 1H, Ar-H), 8.36–8.38 (m, 2H, Ar-H) 5.73 (s, 2H, -CH2-), 7.01–7.37 (m, 4H, Ar-H), 7.61–7.64 (m, 2H, Ar-H), 7.76–7.80 (m, 1H, Ar-H), 7.84–7.88 (m, 1H, Ar-H), 8.22 (d, 1H, J = 8.0, Ar-H), 8.39 (d, 1H, J = 8.0, Ar-H), 8.52–8.53, (m, 1H, Ar-H) 5.77 (s, 2H, -CH2-), 7.28–7.35, (m, 6H, Ar-H), 7.57–7.62, (m, 1H, Ar-H), 7.75–7.87 (m, 2H, Ar-H), 8.19–8.22 (m, 1H, Ar-H), 8.36–8.38, (m, 1H, Ar-H), 8.53, (s, 1H, Ar-H) 5.69 (s, 2H, -CH2-), 7.00 (t, 2H, J = 16, Ar-H), 7.31–7.41 (m, 4H, Ar-H), 7.63 (t, 1H, J = 16.0, Ar-H), 7.69–7.73 (m, 1H, Ar-H), 7.76–7.80 (m, 1H, Ar-H), 8.16 (d, 1H, J = 8.8, Ar-H), 8.32–8.34 (m, 1H, Ar-H), 8.51 (d, 1H, J = 7.6, Ar-H) 2.34 (s, 3H, CH3), 5.74 (s, 2H, -CH2-), 7.15 (d, 2H, J = 8.0, Ar-H), 7.27–7.34 (m, 2H, Ar-H), 7.44 (d, 2H, J = 8.0, Ar-H), 7.68 (t, 1H, J = 15.6, Ar-H), 7.73–7.77 (m, 1H, Ar-H), 7.83 (t, 1H, J = 15.2, Ar-H), 8.22 (d, 1H, J = 8.4, Ar-H), 8.39 (d, 1H, J = 8.4, Ar-H), 8.55 (d, 1H, J = 7.6, Ar-H) 2.29 (s, 3H, CH3), 5.66 (s, 2H, -CH2-), 7.10 (d, 2H, J = 8.0, Ar-H), 7.20 (d, 2H, J = 7.6, Ar-H), 7.24 (s, 1H, Ar-H), 7.67–7.73 (m, 2H, Ar-H), 7.79 (t, 1H, J = 13.8, Ar-H), 8.16 (d, 1H, J = 8.0, Ar-H), 8.32 (d, 1H, J = 8.0, Ar-H), 8.61 (s, 1H, Ar-H). 21.17 (s), 29.79 (s), 44.90 (s), 111.72 (s), 114.02 (s), 121.31 (s), 125.40 (s), 126.48 (s), 127.21 (s), 128.01 (s), 129.32 (s), 129.58 (s), 133.09 (s), 133.48 (s), 137.64 (s), 138.71 (s), 139.69 (s), 140.92 (s), 142.73 (s) 2.34 (s, 3H, -CH3), 5.72 (s, 2H, -CH2-), 7.15 (d, 2H, J=8, Ar-H), 7.25 (d, 2H, J=8, Ar-H), 7.34-7.36 (m, 1H, Ar-H), 7.59-7.61 (m, 1H, Ar-H), 7.75-7.86 (m, 2H, Ar-H), 8.19-8.22 (m, 1H, Ar-H), 8.35-8.38 (m, 1H, Ar-H), 8.51 (d, 1H, Ar-H). 357 (100) 21.18 (s), 29.80 (s), 44.90 (s), 111.27 (s), 120.80 (s), 122.39 (s), 126.44 (s), 126.80 (s), 127.22 (s), 128.01 (s), 129.29 (s), 129.52 (s), 129.01 (s), 130.81 (s), 133.15 (s), 137.62 (s), 138.84 (s), 139.65 (s), 140.90 (s), 142.35 (s), 145.83 (s). 5.67 (s, 2H, -CH2-), 7.00 (t, 2H, J=16, Ar-H), 7.23-7.31 (m, 4H, Ar-H), 7.69-7.75 (m, 2H, Ar-H), 7.78-7.82 (m, 1H, Ar-H), 8.16 (d, 1H, J=8.8, Ar-H), 8.33 (d, 1H, J=8, Ar-H). 2.30 (s, 3H, CH3), 5.68 (s, 2H, -CH2-), 7.12 (d, 2H, Ar-H), 7.22 (d, 2H, Ar-H), 7.29-7.37 (m, 3H, Ar-H), 7.69-7.73 (m, 1H, Ar-H), 7.77-7.81 (m, 1H, Ar-H), 8.15-8.18 (m, 1H, Ar-H), 8.33 (d, 1H, J=8.4, Ar-H). 2.55 (s, 3H, CH3), 5.66, (s, 2H, -CH2-), 6.95-6.98 (m, 2H, Ar-H), 7.23-7.32 (m, 3H, Ar-H), 7.45 (d, 1H, J=8.4, Ar-H), 7.68-7.71 (m, 1H, Ar-H), 7.75-7.79 (m, 1H, Ar-H), 8.14 (d, 1H, J=8.8, Ar-H), 8.32 (d, 2H, J=6.8, Ar-H). S. S. Karki et al.: Synthesis, anticancer and cytostatic activity of some 6H-indolo[2,3-b]quinoxalines, Acta Pharm. 59 (2009) 431–440. were detected. Compounds 5a-m showed absorption bands ranging from 3052–3000 cm–1 for C-H aromatic stretching, 2960–2900 cm–1 for C-H aliphatic stretching; there were also some bands for C=C and C=N at 1580–1575 cm–1 and 1505–1480 cm–1, respectively (Table II). In 1H NMR spectra, the presence of a singlet between d 5.8–5.6 ppm was observed for the methylene group and multiplets were observed between d 8.55–7.00 ppm for aromatic protons. In the 13C NMR spectra, signals from d 142.73 to 111.72 ppm were observed for aromatic carbon and d 44.90 to 21.12 ppm for alkyl carbons. Fast atomic bombardment (FAB) mass spectra showed accurate molecular ion peaks at m/z 338 and 357 for 5c and 5j, respectively (Table II). The anti-cancer activity of compounds 5b, 5d, 5g and 5l at concentrations 1.0 ´ 10–5 mol L–1 was tested against 59 human tumor cell lines representing leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate and kidney. The data for anticancer activity, in terms of the percent growth of treated cells, is given in Table III. Table III. Growth of cancer cells (%) when treated with compounds 5b, 5d, 5g and 5la Panel/cell line Non-small cell lung cancer HOP-92 Colon cancer HCC-2998 HCT-116 Breast cancer HS 578T Ovarian cancer IGROV1 Leukemia HL-60(TB) K-562 MOLT-4 RPMI-8226 SR Renal cancer UO-31 Melanoma SK-MEL-2 SK-MEL-5 Prostate cancer PC-3 CNS cancer SF-539 SNB-75 a Percentage of growth (%) 5b 5d 5g 5l 34.69 47.75 – 57.14 55.67 69.24 – – – – – 69.57 – – 54.68 68.94 69.90 – 44.75 55.85 – – 7.31 50.03 – – 32.40 45.51 58.90 – – – – – – 30.11 69.85 – – 18.70 – – 66.05 – – – – – 37.12 59.20 – – – – – 58.63 – – – – – – 66.09 60.96 c = 1.0 ´ 10–1 mol L–1 437 S. S. Karki et al.: Synthesis, anticancer and cytostatic activity of some 6H-indolo[2,3-b]quinoxalines, Acta Pharm. 59 (2009) 431–440. Table IV. Cytostatic activity: IC50 values Compd. L1210a Molt4/C8a CEMa 5a 289 ± 14 381 ± 136 303 ± 191 5b 91 ± 45 > 610 189 ± 52 5c 285 ± 24 272 ± 24 246 ± 59 5d 260 ± 78 389 ± 161 290 ± 22 > 552 ³ 552 5e 85 ± 2.8 5f 75 ± 17 513 ± 23 119 ± 55 5g 199 ± 27c 170 ± 18 c 188 ± 40c 5h 117 ± 74 71 ± 31 117 ± 19 5i 7.2 ± 5.3 222 ± 25 c 247 ± 28c 5j 5k 5l 32 ± 25 50 ± 27b 164 ± 2.9c 5m 32 ± 16 Melphalan 2.1 ± 0.02 284 ± 8 c 279 ± 7c > 500 400 ± 142c ³ 1465 764 ± 179c 424 ± 81c 3.2 ± 0.6 > 500 2.5 ± 0.2 DMSO – negative control. IC50 – 50 % inhibitory concentration (mmol L–1) required to inhibit tumor cell proliferation by 50 %. a Values are mean ± SEM, n = 2 to 3. Significant difference vs. melphalan: b p < 0.05, c p < 0.01. Synthesized compounds and melphalan as a standard were also evaluated for their in vitro cytostatic activity against human Molt 4/C8 and CEM T-lymphocytes as well as murine leukemia L1210 cells. The results of cytostatic activity are presented in Table IV. Only melphalan showed IC50 values consistently lower than 10 mmol L–1 (IC50 range 2.1–3.2 mmol L–1). The test compounds had IC50 values ranging between 23 and ³ 1465 mmol L–1 (except for 5i that had and IC50 of 7.2 mmol L–1 for L1210 cell proliferation). Compound 5h emerged as the only compound that consistently inhibited cell proliferation of all three tumor cell lines at an IC50 ranging between 71 and 117 mmol L–1. It is unclear why 5i is markedly more cytostatic to L1210 cells than to the human lymphocyte cell lines. Also, for 5e, 5j, 5k, 5l and 5m, there was a clear trend of higher cytostatic activity against the murine L1210 than against the human lymphocyte cell lines. Structure activity relation Quinoxaline derivatives with substituents like F and CH3 on benzyl at 4th position and F, CH3, Cl, Br at 9th position exhibited anticancer activity against various cell lines of HOP-92, HCT-116, IGROV1, K-562 Molt-4 and RPMI-8226. The order of potency was found to be 5l > 5b > 5d ³ 5g as it is evident from the overall percentage of growth inhibition. In addition, compound 5l is selective against prostate cancer. Quinoxaline derivatives with substituents like F and CH3 on 4-aralkyl at 6th position and Cl, F, Br and CH3 at 9th position were studied for cytostatic activity against human 438 S. S. Karki et al.: Synthesis, anticancer and cytostatic activity of some 6H-indolo[2,3-b]quinoxalines, Acta Pharm. 59 (2009) 431–440. Molt 4/C8 and CEM T-lymphocytes as well as murine L1210 leukemia cells. In most cases, there was a more pronounced cytostatic activity against the murine leukemia L1210 cells than against the human lymphocyte cells. Among the tested compounds 5b, 5e, 5f, 5h, 5i, 5j and 5k were moderately cytostatic for the leukemia cell line L1210, as it is evident from Table IV (IC50 < 100 mmol L–1). The order of cytostatic activity for L1210 was 5i > 5f ³ 5b, therefore it suggests that by placing a halogen like bromine on the ninth position and a methyl on the 4th position of aralkyl on the 6th position of quinoxaline produced compounds which were less cytostatic than the standard melphalan. By keeping the electron donating groups like methyl on 6 and 9th position of aralkyl, there was not any improvement of cytostatic activity (5c). CONCLUSIONS The reaction of 5-substituted isatin with orthophenylene diamine provided the corresponding quinoxalines 5a-m in good yields. Investigations of cytostatic activities revealed better cytostatic activity of several compounds (5b, 5e, 5f, 5i, 5j, 5k, 5m) against the leukemia cell line L1210 than the human lymphocytic cell lines (the lowest IC50 of 7.2 mmol L–1 was noted for 5i) compared to melphalan (IC50 2.1 mmol L–1). Acknowledgements. – The authors thank B. G. Desai of the KLE College of Pharmacy, Bangalore, India, for providing the necessary facilities. The authors thank V. L. Narayanan and his group at NIH, Bethesda, USA, for anti-cancer screening at the National Cancer Institute. Appreciation is extended to the Belgian Fonds voor Wetenschappelijk Onderzoek (Vlaanderen) which supported the cytostatic activity assays and to L. van Berckelaer for excellent technical assistance. REFERENCES 1. D. J. Brown (Ed.), Quinoxalines, Wiley, New York 2004. 2. G. W. H. Cheeseman and R. F. Cookson (Eds.), Condensed Pyrazines, Wiley, New York 1979. 3. A. Carta, P. Corona and M. 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Spojevi 5b, 5d, 5g i 5l pokazali su zna~ajno antitumorsko djelovanje na 59 humanih tumorskih stanica. Svi sintetizirani spojevi ispitani su na citostatsko djelovanje na stani~ne linije Molt 4/C8 i CEM T-limfocite, te na murin L1210 stanice leukemije. IC50 za spoj 5h je 71 mmol L–1 na stani~nu liniju Molt 4/C8 i 117 mmol L–1 na CEM/0, dok su vrijednosti za melfalan 3,2, odnosno 2,5 mmol L–1. IC50 spoja 7i na stanice L1210/0 je 7,2, dok je za melfalan 2,1 mmol L–1. Klju~ne rije~i: indolo[2,3-b]kinoksalin, citostatsko djelovanje, antitumorsko djelovanje Department of Pharmaceutical Chemistry, KLE College of Pharmacy, 2nd Block Rajajinagar Bangalore-560010, India Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium 440