Title of Invention

"NOVEL SUBSTITUTED 1H-BENZ[DE]ISOQUINOLINE-1,3-DIONES"

Abstract

Novel substituted 1 H-benz[de]isoquinoline-1,3-diones : The present invention provides novel substituted 1H-benz[de]isoquinoline-1,3-diones of formula 1 where R is H, halogen, nitro, X = N(CH2)nNH2; N(CH2)nNHCONHCH2CH2CI; N(CH2)nNHCONHCH2CH3; N(CH2)nNHCON(NO)CH2CH2CI; N(CH2)nNHCON(NO)CH2CH3 where n = 2,3 . The present invention also provides a process for preparation of novel substituted 1H-benz[de]isoquinoline-1,3-diones by reacting naphthalic anhydride / substituted naphthalic anhydrides with diaminoalkanes .

Full Text

This invention relates to novel substituted 1H-benz[de]isoquinoline-1,3-diones. The present invention also relates to a process for preparation of substituted 1H-benz[de]isoquinoline-1,3-diones. The present invention particularly relates to the 'Synthesis of substituted 1H-benz[de]isoquinoline-1,3-diones as a novel group of anticancer agents'. The main utility of this invention is to prepare hitherto unknown groups of compounds which may be useful in the treatment of human cancers. Further objects and advantages of this invention will be more apparent from the ensuing description. Despite the advent of other types of chemotherapeutic agents, a number of alkylating agents still retain their key role in several human cancers. Among the alkylating agents, the subclass of nitrosoureas containing the established anti-tumour highly cytotoxic functionality as R - NH - CO - N(NO) - CH2CH2CI best represented by BCNU, CCNU, Me-CCNU, chlorozotocin and also less cytotoxic R - NH - CO - N(NO) -CH2CH3 as streptozotocin which are useful in various human cancers including brain-tumours since these have the capacity to cross the blood-brain barrier. Several structural patterns have been used as the carrier molecule for the NU groups. However, literature survey reveals that there are no reports, to our knowledge, in which the substituted naphthalimides have been used as their carrier which was particularly chosen since it has been found that some substituted naphthalimides containing N-{2-dimethylaminoethyl) chain best represented by amonafide and mitonafide have exhibited substantial cytotoxicities in various animal and human tumours and are under clinical trial. Hence the synthesized compounds described in this invention may have synergistic activity since the molecules contain the DNA binder naphthalimide group as well as the highly cytotoxic 2-chloroethylnitrosourea or comparatively less cytotoxic ethylnitrosourea functionalities embedded in it. The main object of the present invention is to provide novel substituted 1H-benz[de]isoquinoline-1,3-diones of formula 1. Another object of the present invention is to provide a process for preparation of substituted 1 H-benz[de]isoquinoline-1,3-diones useful as anticancer agents. Yet another object of the present invention is to provide novel groups of compounds of formula 1d-1e having low toxicity. A further object of this invention is to prepare amino compounds of substituted naphthalimides of formula 1 which may have application in chemical industry Accordingly the present invention provides novel substituted 1H-benz[de]isoquinoline-1,3-diones of formula 1 where R is H, halogen, nitro, X = N(CH2)nNH2 ; N(CH2)nNHCONHCH2CH2CI ; N(CH2)nNHCONHCH2CH3 ; N(CH2)nNHCON(NO)CH2CH2CI ; N(CH2)nNHCON(NO)CH2CH3 where n = 2, 3. (Formula Removed) Accordingly the present invention provides novel substituted 1H-benz[de]isoquinoline-1,3-diones, Structural formula for 1a is given below : (Formula Removed) where n = 2,3 R = H, 6-Br, 6-CI, 5-NO 2,6-NO2 Accordingly the present invention provides novel substituted 1H-benz[de]isoquinoline-1(Formula Removed) ,3-diones, Structural formula for 1 b is given below : where n = 2,3 R = H, 6-Br, 6-CI, 5-NO 2,6-NO2 Accordingly the present invention provides novel substituted 1H-benz[de]isoquinoline-1,3-diones. Structural formula for 1c is given below : (Formula Removed) where n = 2,3 R = H, 6-Br, 6-CI, 5-NO 2,6-NO2 Accordingly the present invention provides novel substituted 1H-benz[cte]isoquinoline-1,3-diones, Structural formula of 1d is given below : (Formula Removed) where n = 2,3 R = H, 6-Br, 6-CI, 5-NO 2,6-NOa Accordingly the present invention provides a process for preparation of novel substituted 1 H-benz[de]isoquinoline-1,3-diones of formula 1 where R is H, halogen, nitro, X = N(CH2)nNH2 ; N(CH2)nNHCONHCH2CH2CI N(CH2)nNHCONHCH2CH3 ; N(CH2)nNHCON(NO)CH2CH2CI N(CH2)nNHCON(NO)CH2CH3 where n = 2, 3. (Formula Removed) which comprises (I) reacting naphthalic anhydride / substituted naphthalic anhydrides with diaminoalkanes in a polar solvent at a temperature in the range of 0 to 80RC for a period in the range of 1 to 24 hr, isolating the amino alkyl derivative of compounds of formula 1 wherein R is H, halogen, nitro, X = N(CH2)nNH2 and n = 2, 3 by conventional methods (ii) reacting the above amino alkyl compound • obtained in step (I) with isocyanate compound in an organic solvent at an ambient temperature for a period in the range of 1 to 12 hr, isolating the ureido alkyl derivative compound of formula wherein R is H, halogen, nitro, X = N(CH2)nNHCONHCH2CH2CI, N(CH2)nNHCONHCH2CH3 where n = 2 , 3 from reaction mixtures by conventional methods, (iii) reacting the above ureido alkyl derivative compounds obtained in step (ii) with carboxylic acid and sodium nitrite in water at a temperature of in the range of -10 to ORC for a period in the range of 8 to 12 hr isolating the nitroso derivative of formula 1 where R is H, halogen, nitro, X = N(CH2)nNHCON(NO)CH2CH2CI, N(CH2)nNHCON(NO)CH2CH3 where n = 2,3 from reaction mixture by conventional methods, In an embodiment of the present invention the substitution in naphthalic anhydrides used may be selected from halogen, nitro group. In another embodiment of the present invention the diaminoalkane used may be selected from 1,2-diaminoethane, 1,3-diaminopropane. In yet another embodiment of the present invention the polar solvent used in step (I) is selected from water, dimethylformamide. In still another embodiment of the invention the organic solvent used in step (ii) may be selected from ethanol, chloroform. In yet still another embodiment of the invention the acid used in step (iii) may be carboxylic acid such as formic acid. In further still another embodiment of the invention the isocyanate used in step (iii) may be 2-chloroethyl isocyanate or ethyl isocyanate. The compounds of formula 1a-e may be isolated from the reaction mixtures by filtration of the precipitate obtained after completion of reaction and work up. According to this invention, the known three step process was followed for preparing 2-chloroethyl nitrosourea or ethyl nitrosourea derivatives of naphthalimides / substituted naphthalimides of formula 1d and 1e respectively. Thus in the first step, amino compounds of formula 1a were obtained from the corresponding naphthalic anhydride / substituted 1,8-naphthalic anhydride derivatives by treating with 1,2-diaminoethane or 1,3-diaminopropane under various reaction conditions. Next compounds of formula 1b or 1c were obtained from the compounds of formula 1a by treating the latter with 2-chloroethyl isocyanate or ethyl isocyanate preferably at room temperature in good yields. Finally compounds of formula 1d or 1e were obtained in good yields from the ureido compounds of formula 1b or 1c by nitrosation with sodium nitrite in the presence of formic acid at a temperature of ORC. By following the above synthetic process, nine new title compound of 1d and eight new title compound of 1e ( n = 2, 3) have been obtained (Table 4 and 5). The following examples are given by way of illustration and should not construed to the limit of the scope of the invention. Synthesis of the title compounds 1 New compounds (Table 1-5) were characterised by PMR spectra measured in a Brucker 300-DPX spectrometer with the solvents as indicated and chemical shifts were expressed in 5 units (ppm) using tetramethyl silane as internal standard. IR and UV spectra were recorded in spectrophotometers as Perkin-Elmer FT-IR model no RX-1 and Shimadzu model no. UV-160A respectively. Melting points were determined on a Thomas-Hoover Unimelt capillary melting point apparatus and were uncorrected. Wherever possible, purity of compounds were checked with Waters HPLC system at ambient temperature (µ-bondapak C18 steel column, 30 cm x 3.9 mm; isocratic mobile phase acetonitrile-water in varying proportions (up to 50 : 50) at a flow rate of 1.0 ml/min; uv detection at 250 nm). TLC plates coated with silica gel were used. Example 1 : Synthesis of 1d (n = 2, R = 6-Bromo). 6-Bromo -2-(2-aminoethyl)-1 H-benzfdelisoauinoline-1,3-dione da. n = 2. R = 6-Bromo) To a stirred solution of 1,2-diaminoethane (2.2 ml) in water (12.6 ml) at 75°C was added portion wise 4-bromonaphthalic anhydride (1.38 gm) suspended in water (4.9 ml) over 10 minute. Soon a fine yellow precipitate began to appear. The solution was kept at this temperature for 30 minute and filtered. The precipitate was collected, washed with water and dried over phosphorus pentoxide in vacuo. Wt of the crude yellow solid was 1.71 gm. This was continuously extracted with chloroform under boiling condition, cooled, filtered from insoluble residues and the filtrate was concentrated in vacuo to furnish a yellow solid (1.03 gm ). m.pt. 150 -151°C ; TLC RF 0.35 (CHCI3: MeOH, 90 : 10) ; UMAX (KBr) 3373, 2924, 2855, 1706, 1662, 1574, 1532,1480, 1434, 1367, 1345, 1236, 1175, 1077, 781 cm'1; 1HNMR (CDCI3) 2.18 (m, 2H, NH2), 3.07 (t, 2H, C/^NH2), 4.27 [m, 2H, CH2N(CO)2)], 7.84 (m, 1H, arom. H), 8.04 (d, 1H, arom. H), 8.42 (d, 1H, arom. H), 8.55 (m,1H, arom. H), 8.65 (m,1H, arom. H). 6-Bromo-2-[2[(2-chloroethvnureido)ethyl]-1H-benzfdelisoauinoline-1.3-dione (1b. n = 2. R = 6-Bromo) To a clear solution of the above compound 1a (1 gm) in chloroform was added 2-chloroethylisocyanate (0.28 ml) at a time at room temperature. A yellow precipitate began to appear after 1 hr. It was stirred further for 3 hr. The solid was filtered and washed with chloroform to furnish the desired product 1 b as yellow powder (860 mg) ; m.pt. 220-222°C; TLC RF 0.72 (CHCI3: MeOH, 90 : 10); UMAX (KBr) 3397, 3276, 2924, 1658, 1577, 1512, 1436, 1372, 1?40, 1182, 1025, 849, 701 cm'1; 1HNMR(d6-DMSO) 3.16 (m, 2H, CH2CI), 3.35 (m, 4H, 2 x CH2NH) , 4.11 [t, 2H, CH2N(CO)2], 6.08 and 6.18 (t, 2H, 2 x NH), 8.04 (m,1H, arom. H), 8.25 (m,1H, arom H), 8.35 (m,1H, arom H), 8.58 (m,2H, arom H). 6-Bromo-2-[2{(2-chloroethyl)-3-nitrosoureido)ethyll-1H-benz[de]isoauinoline-1.3-dione (1d. n = 2.R = 6-Bromo) To a cooled (0°C) solution of the above compound 1b (240 mg) in formic acid (3.5 ml) was added sodium nitrite solution (43 mg) in water (0.4 ml) drop wise. A solid precipitate began to appear after an hour. It was stirred further for 2 hr. The yellow solid was filtered, washed with water and dried; Crude weight 180 mg. Column chromatography over silica gel (4 gm) followed by repeated crystallisation from chloroform furnished a light yellow coloured solid (140 mg); m.p. 144 - 145°C ; TLC RF 0.82 (CHCI3: MeOH, 90 : 10) ; UMAX (KBr) 3359, 2938, 1737, 1697, 1653, 1589, 1524, 1476, 1368, 1345, 1240, 1151,1075, 879, 785 cm'1; 1HNMR(CDCI3) 3.53 (m, 2H, CH2CI), 3.65 (m, 2H, CH2NH) , 4.30 [t, 4H, CH2N(CO)2 and NCH2], 7.20 (m,2H, 2 x NH), 7.84 (t,1H, arom. H), 8.04 (t,1H, arom. H), 8.40 (d,1H, arom H) & 8.62 (m, 2H, arom H). Example 2 : Synthesis of 1d (n = 2, R = 5-nitro). 5-Nitro -2-(2-aminoethyl)-1H-benzrdelisoauinoline-1.3-dione (1a,n = 2,R = 5-Nitro) To a stirred solution of 1,2-diaminoethane (0.66 ml) in dimethylformamide (DMF) (5.0 ml) at 0-2°C was added drop wise a solution of 3-nitronaphthalic anhydride (1.22 gm) in DMF (37.5 ml) over 30 minute. The solution was stirred at this temperature for 2 hour and left overnight at room temp, when a brownish solid precipitate appeared. It was filtered, the filtrate was diluted with water and the solid was collected by filtration, dried over phosphorus pentoxide in vacuo. Wt of the crude brown solid was 1.25 gm. m.pt. 198-200°C [vide M.F.Brana et al Eur. J.Med.Chem. 16 (1981) pp 207-212 ] ; TLC RF 0.47 (CHCI3: MeOH, 90 : 10) ; UMAX (KBr) 3371, 3079, 1664, 1592, 1538, 1435, 1345, 1245, 1099, 953, 917, 832, 785, 756, 537 cm'1; 1HNMR (d6-DMSO) 2.82 (t, 2H, NH2), 3.31 (m, 2H, CH2N), 4.09 [m, 2H, CH2N(CO)2)], 8.04 (t, 1H, arom. H), 8.51 (d, 1H, arom. H), 8.67 (d, 1H, arom. H), 8.94 (app. s, 1H, arom. H), 9.47 (m,1H, arom. H). 5-Nitro-2-[2{[2-chloroethynureido)ethtl)-1H-benzrde]isoauinoline-1.3-dione (1b. n = 2. R = 5-Nitro) To the above compound 1a (288 mg) was added dry ethanol (20 ml) and the mixture was refluxed for 1 hour, cooled and filtered to remove undissolved compound. To the clear filtrate was added 2-chloroethylisocyanate (0.085 ml) at a time at room temperature. Next the mixture was stirred at 75°C for 4 hour. After evaporation of the solvent to dryness, a solid residue was obtained which was filtered and washed with ethanol followed by with acetone to furnish the desired product 1b as yellow powder (114 mg); m.pt. 220-222°C; TLC RF 0.73 (CHCI3: MeOH, 90 : 10) ; UMAX (KBr) 3361, 3073, 2926, 1707, 1667, 1630, 1574, 1532, 1434, 1371, 1334, 1246, 1180, 1085, 790, 757 cm-1; 1HNMR(d6-DMSO) 3.13 (m, 2H, CH2CI), 3.32 (m, 4H, 2 x CH2N) , 4.19 [m, 2H, CH2N(CO)2], 6.08 and 6.18 (t, 2H, 2 x NH), 8.04 (m,1H, arom. H), 8.67 (d,1H, arom H), 8.77 (d,1H, arom H), 8.95 (s,1H, arom H), 9.48 (s,1H, arom H). 5-Nitro-2-[2{(2-chloroethyl)3-nitrosoureido)ethyl1-1H-benzfdelisoauinoline-1.3-dione (1d. n = 2.R = 5-Nitro) To a cooled (0°C) solution of the above compound 1b (200 mg) in formic acid (2.0 ml) was added sodium nitrite solution (40 mg) in water (0.4 ml) drop wise. A solid precipitate began to appear after an hour. It was stirred further for 4 hr. The yellow solid was filtered, washed with water and dried; Crude weight 140 mg. Crystallisation from chloroform furnished a light yellow coloured solid (100 mg); m.p. 110 -112°C ; TLC RF 0.80 (CHCI3: MeOH, 90 : 10) ; UMAX (KBr) 3384, 3080, 2961,1708, 1668, 1598, 1538, 1434, 1334, 1245, 1091, 920, 788, 758 cm'1; 1HNMR(de-DMSO) 3.37 - 3.67 (m, 4H, CH2CI and CH2N), 4.06 (m, 2H, CH2N), 4.33 [m, 2H, CH2N(CO)2], 7.26 (m,1H, NH), 7.94 (t,1H, arom. H), 8.43 (t,1H, arom H), 8.77 (m,1H, arom H), 9.16 (t,1H, arom H), 9.31 (q,1H, aromH). Example 3 : Synthesis of 1d (n = 3, R = 5-nitro). 5-Nitro -2-[3-aminopropyl-1H-benz[delisoauinoline-1.3-dione (1a. n = 3.R = 5-Nitro) To a stirred solution of 1,3-diaminopropane (0.83 ml) in dimethylformamide (DMF) (5.0 ml) at 0-2°C was added drop wise a solution of 3-nitronaphthalic anhydride (1.22 gm) in DMF (37.5 ml) over 30 minute. The solution was stirred at this temperature for 2 hour and left ovenight at room temp. The solvent was removed in vacuo to furnish a solid residue. It was washed repeatedly with water and the solid was collected by filtration, dried over phosphorus pentoxide in vacuo. Wt of the crude brown solid was 1.25 gm. m.pt. 138 -140°C [ lit 138-140°C vide D.M.LGoodgame et al Transition Met. Chem.(London) 16 (1991) pp 223-229.] ; TLC RF 0.21 (CHCI3: MeOH, 90 : 10) ; UMAX (KBr) 3405, 3071, 2947, 1704, 1662, 1596, 1536, 1438, 1332, 1245, 1175, 1088, 917, 787, 756 cm'1; 1HNMR (CDCI3) 1.98 (m, 2H, CH2), 2.79 (t, 2H, CH2N), 4.28 - 4.37 [m, 4H, NH2 and CH2N(CO)2)], 7.96 (m, 1H, arom. H), 8.44 (d, 1H, arom. H), 8.77 (d, 1H, arom. H), 9.13 - 9.30 (m, 2H, arom. H). 5-Nitro-2-[3[(2-chloroethynureido)propyl-1H-benz[delisoauinoline-1.3-dione (1b. n = 3. R = 5-Nitro) To a clear solution of the above compound 1a (0.38 gm) in chloroform (15 ml) was added 2-chloroethylisocyanate (0.15 ml) at a time at room temperature. A orange turbidity appeared immediately. It was stirred further for 12 hr. It was filtered and filtrate evaporated to dryness. The solid residue was washed with chloroform to furnish the desired product 1b as orange powder (280 mg); m.pt. 182-184°C; TLC RF 0.59 (CHCI3: MeOH, 90 : 10) ; UMAX (KBr) 3346, 3070, 2972, 1707, 1658, 1627, 1596, 1538, 1439, 1422, 1334, 1248, 1178, 1088, 788, 757 crrf1; 1HNMR(CDCI3) 1.98 (m,2H, CH2),3.26 (m, 2H, CH2CI), 3.48 - 3.65 (m, 4H, 2 x CH2N) , 4.31 [t, 2H, CH2N(CO)2], 4.82 and 5.19 (t, 2H, 2 x NH), 7.96 (t,1H, arom. H), 8.45 (d,1H, arom. H), 8.78 (t,1H, arom H), 9.15 (d,1H,aromH), 9.29 (m,1H, arom H). 5-Nitro-2-[3[(2-chloroethyl)-3-nitrosoureido)DroDvll-1H-benz[delisoauinoline-1.3-dione (1d. n = 3,R = 5-Nitro) To a cooled (0°C) solution of the above compound 1b (140 mg) in formic acid (2.0 ml) was added sodium nitrite solution (30 mg) in water (0.3 ml) drop wise. A solid precipitate began to appear after an hour. It was stirred further for 4 hr. The yellow solid was filtered, washed with water and dried; Crude weight 110 mg. Crystallisation from chloroform furnished a light yellow coloured solid (90 mg); m.p. 124 - 125°C ; TLC RF 0.70 (CHCI3: MeOH, 90 :10) ; UMAX (KBr) 3345, 3038, 1726, 1697, 1660, 1598, 1531, 1478, 1424,1345, 1243, 1176, 1135, 1080, 1004, 860, 791, 759 cm'1; 1HNMR(CDCI3) 1.91 (m, 2H, CH2), 3.77 (m, 4H, CH2CI and CH2N), 3.97 (m, 2H, CH2N), 4.17 [m, 2H, CH2N(CO)2)], 7.28 (m, 1H, NH), 7.94 (t, 1H, arom. H), 8.43 (d, 1H, arom. H), 8.76 (d, 1H, arom. H), 9.13 (m,1H, arom. H), 9.30 (m,1H, arom. H). Example 4 : Synthesis of 1e (n = 2, R = 6-Chloro). 6-Chloro -2-[2-aminoethyl)-1H-benz[de]isoauinoline-1.3-dione da. n = 2.R = 6-Chloro) To a stirred solution of 1,2-diaminoethane (2.2 ml) in water (12.6 ml) at 75°C was added portion wise 4-chloronaphthalic anhydride (1.16 gm) suspended in water (4.9 ml) over 10 minute. Soon a fine yellow precipitate began to appear. The solution was kept at this temperature for 30 minute and filtered. The precipitate was collected, washed with water and dried over phosphorus pentoxide in vacuo. Wt of the crude yellow solid was 1.43 gm. This was continuously extracted with chloroform under boiling condition, cooled, filtered from insoluble residues and the filtrate was concentrated in vacuo to furnish a yellow solid (1.10 gm ). m.pt. 150 -152°C ; TLC RF 0.38 (CHCI3: MeOH, 90 : 10) ; UMAX (KBr) 3366, 2952, 2918, 2850, 1700, 1663, 1588, 1573,1461, 1434, 1370, 1345, 1234, 1171, 1080, 780,750 cm'1; 1HNMR (CDCI3) 2.17 (m, 2H, NH2), 3.07 (t, 2H, CA£NH2), 4.27 [m, 2H, CH2N(CO)2)], 7.85 (m, 2H, arom. H), 8.50 (d, 1H, arom. H), 8.65 (m,2H, arom. H). 6-Chloro-2-[2-[3-ethylureido)ethyl]-1H-benzfdelisoauinoline-1.3-dione (1c. n = 2. R = 6-Chloro} To a clear solution of the above compound 1a (1.5 gm) in chloroform was added ethyl isocyanate (0.4 ml) at a time at room temperature. A yellow precipitate began to appear after 1 hr. It was stirred further for 3 hr. The solid was filtered, washed with chloroform and re-crystallised from methanol to furnish the desired product 1c as yellow powder (600 mg); m.pt. 228 -230°C; TLC RP 0.63 (CHCI3: MeOH, 90 : 10); UMAX (KBr) 3340, 2973, 2864, 1696, 1623, 1585, 1454, 1349, 1237, 1184, 1053, 785 cm'1; 1HNMR(d6-DMSO) 0.83 (t, 3H, CH3), 2.85 (m, 2H, NCH2), 3.3 (m, 2H, NCH2) , 4.11 [t, 2H, CH2N(CO)2], 5.71 and 5.90 (t, 2H, 2 x NH), 8.02 (m,2H, arom. H), 8.43 (m,1H, arom H), 8.58 (m,2H, arom H). 6-Chloro-2-[2-[3-ethyl-3-nitrosoureido]ethyl]-1H-benz[de]isoauinoline-1.3-dione (1e. n = 2.R = 6-Chloro) To a cooled (0°C) solution of the above compound 1c (250 mg) in formic acid (5 ml) was added sodium nitrite solution (190 mg) in water (2 ml) drop wise. A solid precipitate began to appear after an hour. It was stirred further for 2 hr. The yellow solid was filtered, washed with water and dried; Crude weight 100 mg. Crystallisation from chloroform furnished a light yellow coloured solid (80 mg); m.p. 143 - 145°C ; TLC RF 0.82 (CHCI3: MeOH, 90 : 10) ; UMAX (KBr) 3386, 3072, 2985, 1702, 1654, 1591, 1530, 1472, 1368, 1349, 1240, 1171,1040, 888, 785 cm'1; 1HNMR(CDCI3) 0.91 (t, 3H, CH3), 3.8 (m, 2H, NCH2), 3.85 (m, 2H, NCH2) , 4.5 [t, 2H, CH2N(CO)2], 7.42 (m,1H, NH), 7.84 (t,2H, arom. H), 8.46 (m,1H, arom H), 8.61 (m,2H, arom H). Example 5 : Synthesis of 1e (n = 3, R = H). 2-[3-Aminopropyll)-1H-benz[de]isoauinoline-1.3-dione da. n = 3.R = H) To a stirred solution of 1 ,3-diaminopropane (2.78 ml) in water (12.5 ml) at 75°C was added drop wise naphthalic anhydride (1 gm) suspended in water (5 ml) over 10 minute. Soon a fine yellow precipitate began to appear. The solution was kept at this temperature for 30 minute and filtered. The precipitate was collected, washed with water and dried over phosphorus pentoxide in vacuo. Wt of the crude yellow solid was 0.7 gm. m.pt. 135 -138°C [ lit 138-140°C vide D.M.LGoodgame et al Transition Met. Chem.(London) 16 (1991) pp 223-229.] ; TLC RF 0.2 (CHCI3 : MeOH, 90 : 10) ; UMAX (KBr) 3516, 3358, 2952, 1695, 1659, 1589, 1441, 1348, 1235, 1178, 1056, 850, 785 cm'1; 1HNMR (CDCI3) 1.88 (m, 2H, CH2), 2.77 (t, 2H, CH2N), 4.28 [m, 2H, CH2N(CO)2)], 7.76 (m, 2H, arom. H), 8.20 (d, 2H, arom. H), 8.60 (d, 2H, arom. H). 2-[3-[3-Ethylureido)propyl]-1H-benzfdelisoauinoline-1.3-dione (1c. n = 3. R = H) To a clear solution of the above compound 1a (0.38 gm) in chloroform (15 ml) was added ethyl isocyanate (0.12 ml) at a time at room temperature. It was stirred for 12 hr whereupon a solid precipitate was obtained which was filtered. The solid residue was washed with chloroform and crystallised from methanol to furnish the desired product 1c as white amorphous powder (175 mg) ; m.pt. 240 -242°C; TLC RF 0.73 (CHCI3: MeOH, 90 : 10) ; UMAX (KBr) 3323, 2962, 2873, 1699, 1665, 1620, 1589, 1441, 1346, 1237, 1182, 1061, 901, 779, 655 cm'1; 1HNMR(d6-DMSO) 0.97 (t, 3H, CH3), 1.73 (m,2H, CH2), 3.02 (m, 4H, CH2N), 4.1 [t, 2H, CH2N(CO)2], 5.87 (m, 2H, 2 x NH), 7.87 (t,2H, arom. H), 8.47 (m,4H, arom. H). 2-[3-[3-Ethyl-3-nitrosoureido)DroDvl1-1H-benz[delisoauinoline-1.3-dione (1,e. n = 3.R = H) To a cooled (0°C) solution of the above compound 1c (150 mg) in formic acid (5.0 ml) was added sodium nitrite solution (120 mg) in water (1 ml) drop wise. A solid precipitate began to appear after an hour. It was stirred further for 4 hr. The yellow solid was filtered, washed with water and dried; Crude weight 80 mg. Crystallisation from chloroform furnished a light yellow coloured solid (60 mg); m.p. 118 - 120°C ; TLC RF 0.90 (CHCI3: MeOH, 90 :10) ; UMAX (KBr) 3363, 2973, 1737, 1697, 1651, 1592, 1529, 1471, 1386, 1346, 1233, 1180, 1046, 1011, 867, 811, 779 cnY1; 1HNMR (CDCI3) 1.03 (t, 3H, CH3), 2.1 (m, 2H, CH2), 3.52 (m, 2H, CH2N), 3.88 (m, 2H, CH2N), 4.33 [m, 2H, CH2N(CO)2)], 7.70 (m, 3H, NH and arom. H), 8.20 (m, 2H, arom. H), 8.6 (d, 2H, arom. H). In a similar fashion other compounds 1a - 1e having different substituents as indicated (Table 1-5) were prepared by following essentially the same procedure. The structural formula 1a-e depicted in the tables 1-5 are described below: (Formula Removed) where n = 2,3 R = H, 6-Br, 6-CI, 5-NO 2,6-NO2 Table 1. Physicochemical data of the Compounds la [ X = N(CH2)nNH2 ] (Table Removed) *k = known unk = unknown till date, to our knowledge Table 2. Physicochemical data of Compounds Ib [ X = N(CH2 )nNHCONHCH2CH2Cl ] (Table Removed) *unk = unknown Table 3. Physicochemical data of Compounds Ic [ X = N(CH2 )nNHCONHCH2CH3 ] (Table Removed) *unk = unknown Table 5. Physicochemical data of Compounds le [ X = N(CH2 )nNHCON(NO)CH2CH3] (Table Removed) *unk = unknown Studies with such new compounds 1d have demonstrated that they have displayed excellent reproducible activity in two different murine tumours having 'curative effects' and also having comparable results with two standard clinical drugs namely 5-fluorouracil (5-FU) and cyclophosphamide (endoxan). Anti - Tumour Screening assay with the compounds Mice Closed colony bred Swiss albino male mice of about 6 weeks of age weighing 24 ± 2 g were used, maintained and divided in two groups as control (untreated) and treated . At least 6 animals were used for a particular group in an experiment. Vehicle Physiological saline containing 2% polyoxyethylenesorbitan monooleate (Tween 80, Sigma Inc., USA) was used for drug administration by intraperinoteal (i.p.) route. The drug solutions were administered to the experimental mice through injection. The drug solutions were prepared daily just prior to the injection. Tumour Ehrlich ascites carcinoma (EAC) and Sarcoma - 180 (S-180) cells obtained from National Centre for Cell Sciences, Pune, were used. Tumour cell suspensions in physiological saline were prepared to final concentrations of 5 x 106 cells/ml as described . Mice were inoculated with 1.0 x 106 viable cells/mouse at an injection volume of 0.2 ml on day 0 for in vivo study. Schedule In in vivo screening increase in life span (ILS) study against EAC & S-180, groups of mice (6 animals each for treated and control groups) were inoculated. Compounds 1d in respective doses was administered to each mouse in treated groups as per table 6. The control groups received equal volumes of vehicle (0.2 ml) on those days. The testing was evaluated by calculating the median survival time (M.S.T.) of drug-treated (T) tumor-bearing animals compared to untreated control (C) tumour bearing animals and expressed as percent T/C value. A T/C percentage value greater than 125 was considered as significant. Two standard clinical drugs as cyclophosphamide (endoxan) and 5-fluorouracil (5-FU) were used as positive controls for comparison in these tumour systems. Results and Discussions - Antitumoural screening The anticancer efficacy of compounds 1d have been assessed in S-180 and EAC by measuring three parameters as the ILS value, ascites cell and ascites fluid. It was found that the highly significant (P Compounds 1d have displayed "curative" effects in different schedules since 1-5 /6 tumour bearing animals treated with these compounds survived more than 60 days and got cured while untreated tumour bearing animals have got median survival time of 22 - 26 days (Table 6). In order to simulate a clinical situation where cancer chemotherapeutic agent is often used at an advanced stage of disease, the efficacy of compounds 1d were also evaluated in different doses in mice bearing EAC and S-180 for 5 days and highly advanced 10 days before the drug challenge. It is interesting to note significant increases in the median survival times in the treated groups with '1-2 'cure's/6 animals having survival rates over 60 days in EAC & S-180 (Table 6). These have also displayed much greater activity than cyclophosphamide (endoxan) in these two tumour systems when all of them are administered i.p. This may be due to the reason that cyclophosphamide differ from other types of alkylating agents in that it requires a multistep activation process before functioning as an anti-tumour agent. In liver cells the microsomal oxidase system converts it into active metabolites and for cyclophosphamide an oral or intraveneous route is preferred. It is also worthy to note that compounds 1d have displayed comparable ILS values with 5-FU which also displayed 'curative effects' having long term survivors in EAC and S-180. Table 6. In vivo anti-tumour screening data (Table Removed) % T/C value > 125 is considered as significant *advanced tumour Reference may be made to the inherent toxicities associated with anticancer drugs in general. Some of the newly invented active compounds have been subjected to the toxicological assay in vivo in normal and tumour-bearing mice at their optimum doses by measuring sequentially drug-induced changes in hematological parameters, femoral bone marrow cellularity and splenic cellularity. Drug-induced hepatotoxicity and nephrotoxicity were also sequentially evaluated sequentially in normal and tumour-bearing mice but no such toxicities were detected. Toxic symptoms were not observed externally in animals, in terms of their general appearance, in respect of their skin and hair texture and in their behavioural pattern in respect of food and water intake and activity. The internal organs like heart, spleen, liver and kidney all retained their usual distinctive colours and appearances in the treated groups on different days compared with those of normal mice. Example 1. In vivo Toxiocological assay of 1d ( n = 2, R = 6 - Bromo) in mouse. The toxicological assay results with the representative compound 1d ( n = 2, R = 6 - Bromo) are described below. 1) Hematoloaical studies Blood samples were collected from tail veins or by cardiac puncture from just sacrificed animals under exposure to ether for counting erythrocytes, thrombocytes or leukocytes in an improved Neubauer bright field counting chamber by standard procedures using freshly prepared RBC and WBC counting fluids. Differential counts of leucocytes were performed by staining the blood smears with Leishman's stain (pH 6.8) and slides were observed under light microscope. Hemoglobin concentrations were measured by the standard method. Sera were obtained from blood samples collected as above. Standard methods and reagents were used to measure the serum alkaline phosphatase (SAKP), glutamic oxaloacetic transaminase (SGOT), glutamic pyruvic transaminase (SGPT) and blood urea nitrogen (BUN) levels. 2) Femoral bone marrow cellularity Mice were sacrificed under exposure to ether and femoral marrow cells were obtained as per standard procedure. Ends of the femur bone were snipped open immediately thereafter with scissors and the marrow plug was flushed out by forcefully injecting cold HBSS (Ca++ and Mg++ free) through the bone cavity by inserting a 26 gauze needle. The marrow plug, was dissociated into single cell suspension by repeatedly passing this suspension through 22 gauze needle and the total volume was measured. Total number of nucleated cells per femur were counted in a hemocytometer after treating the cells with 2% glacial acetic acid. 3) Splenic cellularity Whole spleen was removed from mice sacrificed under exposure to ether and processed essentially as per the published procedure. The spleen was minced in cold HBSS (Ca++ and Mg++ free) and the resultant mixture was passed sequentially through 22 gauze needles to make a single cell suspension. The total number of nucleated cells in the spleen was counted in a hemocytometer similarly as in the case of femoral marrow after treatment with 2% glacial acetic acid. Results - Toxicoloalcal screening To study the hematological changes associated with the application of the representative compound 1d (n = 2, R = 6 - Bromo) , four parameters - (a) hemoglobin level (b) erythrocyte count (c) leukocyte count (total and differential) and (d) platelet count were determined in the peripheral blood of normal treated (NT) as well as in S-180 tumour bearing mice (treated groups ST ; control groups SC respectively) on days 9, 15 and 21 sequentially following drug administration from day 1 to 7 (dose - 50 mg/kg). The following cellularities namely (e) femoral bone marrow and (f) splenic were also determined in all these groups The detailed data obtained for each parameter in NT, ST and SC groups were expressed in Fig. 1 and 2 as the percent control of the respective values obtained for normal control (NC) mice [Respective values for normal control mice are Hemoglobin - 14.5 gm/dl ; RBC - 7.7 x 106 /ul ; WBC - 8200 /ul ; Platelet - 8.9 x 105 /ul; Femoral bone marrow cell count - 12.2 x 106 /ul ; Splenic cell count-15.2 x107/ul]. From Fig. 1, it is noted that initially there were about 10% decrease in the hemoglobin levels in the NT and ST groups observed on day 9 which gradually tends to reach the NC value at a later stage. In contrast, the hemoglobin concentrations in the SC group was found to decrease gradually with the progression of tumour, a common observation noted earlier in several tumor systems by others. Erythrocyte counts in NT and ST groups fully corroborated the above observation as erythrocyte count fell by 17-20% on day 9 reaching NC value on day 21. Like the hemoglobin content, the erythrocyte counts in the SC group was also found to decrease gradually with the progression of tumours as expected supporting the above and earlier observations. A marked increase in circulating leukocytes was observed to be associated with the growth of different animal tumors including S-180. Similar observations and trends were noted in SC mice. Results from WBC total count reflected that there was no significant decrease or increase in any of the treated groups up to the final day of observation i.e. day 21 (Fig. 1). Differential counts of leucocytes in peripheral blood smears of all these tumour bearing control mice showed a shift from lymphocytosis, noted in normal mice to a neutrophilia of approximately 70-75% in accordance with earlier observations. Thus we have found the reversal of lymphoid-myeloid ratio from approximately 3:1 for normal mice to approximately 1:3 in SC control group of mice. It was found that in the treated groups the lymphoid-myeloid ratio was in the range of (56-61 % : 33-40 %) which was close to that of NC ratio (71% : 23%). Platelet counts showed that neither thrombocytosis nor thrombocytopenia occured in the treated groups except slight decrease (10%) from that of NC value was observed initially in ST group on day 9. It is evident that thrombocytosis had occurred in SC group during the progression of tumour. As revealed from the Fig. 2, it was found that there was slight decrease (13%) in the femoral marrow cell count occurred particularly in the ST group which persisted till day 15. Recovery from the bone marrow suppression was noted within 10-12 days post drug therapy in ST group. During this period, gradual decrease in this cellularity was observed in the SC group. Hyposplenic cellularity more prounced in the ST group (decrease by 35%) was noted on day 9. After withdrawal of the drug gradual increase in the splenic cellularities were observed in these groups and the normal count was soon reached within day 15-21 (Figure 2). The average spleen weights recorded in the NT and ST groups, however, did not exhibit any such change or decrase. During tumorigenesis under the stress of tumour burden, splenomegaly have been observed in S-180 control group. This trend is also in full agreement with the earlier observations made by others. In order to evaluate the drug-induced hepatotoxicity and nephrotoxicity, the levels of SAKP, SGPT, SGOT and BUN were biochemically measured in normal and S-180 bearing mice sequentially after 48 hr (day 9) for noting immediate effects, after 192 hr (day 15) for intermediate effects and after 336 hr ( day 21) for late effects, if any, following drug therapy. For normal control mice the respective values recorded are 16.0 IU/I, 6.5 IU/I, 6.6.KA unit and 12.0 mg/dl respectively which are within the range of reported values (Figure 3). It is worth noting that all the above levels remained near the normal range in the treated groups of mice. Discussion - Toxicological screening Literature survey reveals that tumorigenesis and its progression has been accompanied with the following changes compared with normalcy : (1) gradual decrease in hemoglobin content, erythrocyte count and bone marrow cellularity (2) gradual increase in leucocytes and thrombocytes and splenic cellularity and (3) reversal of lymphoid-myeloid ratio in the differential WBC count. As noted in Figure 1, it was observed that there was negligible decreases (10-15%) in the hemoglobin level and erythrocyte count in the treated groups and these levels were elevated within 14 days of drug therapy with 1d(n = 2, R = 6- Bromo). The initial decrease (13.0%) in femoral marrow cellularity observed in the ST group on day 9 (Figure 2) appears to be due to the combined effects of tumorigenesis and the compound 1d. However, the normal value was quickly reached as tumour bearing animals gradually became normal following treatment. It was also evident that neither leucocytosis and thrombocytosis nor leucopenia and thrombocytopenia occured in these groups. However, initial hyposplenic activity was observed in ST group which reached normal value on day 21 (Figure 2). Thus the results indicate that compound 1d (n = 2, R = 6 - Bromo) does not adversely affect hematopoiesis at its optimum dose. It is well known that there are significant elevations in the levels of SAKP and SGPT in liver diseases and disorders and in hepatocellular damages caused by a number of agents. An increase in the SGOT level is observed in patients with cardiac damage due to myocardial infarction and with liver disorders. Biochemical measurements of these parameters showed that those values remained within the normal range in treated groups (Figure 3). An increase in the BUN level is noted in cases of renal diseases and damage. Since the BUN values in all the treated groups remained within the normal range (Figure 3), the said compound 1d has not displayed nephrotoxicity (BUN level > 30.0 mg/dl is considered significant for toxicity). Essentially similar toxicological screening assay results were obtained for other 1d compounds (n = 2;R = H,6- Chloro, 5 - NO2) at their optimum doses. We Claim: (1) novel substituted 1 H-benz[de]isoquinoline-1,3-diones of formula 1 where R is H, halogen, nitro, X = N(CH2)nNH2 ; N(CH2)nNHCONHCH2CH2CI N(CH2)nNHCONHCH2CH3 ; N(CH2)nNHCON(NO)CH2CH2CI N(CH2)nNHCON(NO)CH2CH3 where n = 2, 3. (Formula Removed) (2) Novel substituted 1 H-benz[de]isoquinoline-1,3-diones as claimed in claim 1, Structural formula for 1a is given below: (Formula Removed) where n = 2,3 R = H, 6-Br, 6-CI, 5-NO 2,6-NO2 (3) Novel substituted 1 H-benz[cte]isoquinoline-1,3-diones as claimed in claim 1, Structural formula(Formula Removed) for 1b is given below where n = 2,3 R = H, 6-Br, 6-CI, 5-NO 2,6-NO2 (4) Novel substituted 1 H-benz[de]isoquinoline-1,3-diones as claimed in claim 1, Structural formula of 1c is given below : (Formula Removed) where n = 2,3 R = H, 6-Br, 6-CI, 5-NO 2,6-NO2 (5) Novel substituted 1H-benz[de]isoquinoline-1,3-diones as claimed in claim 1, Structural formula of 1d is given below : (Formula Removed) where n = 2,3 R = H, 6-Br, 6-CI, 5-NO 2,6-NO2 (6) Novel substituted 1 H-benz[de]isoquinoline-1,3-diones as claimed in claim 1, Structural formula of 1e is given below : (Formula Removed) where n = 2,3 R = H, 6-Br, 6-CI, 5-NO 2, 6-NO2 (7) A process for preparation of novel substituted 1H-benz[de]isoquinoline-1,3- diones of formula 1 where R is H, halogen, nitro, X = N(CH2)nNH2 N(CH2)nNHCONHCH2CH2CI ; N(CH2)nNHCONHCH2CH3 N(CH2)nNHCON(NO)CH2CH2CI ; N(CH2)nN/HCON(NO)CH2CH3 where n = 2, 3. (Formula Removed) which comprises (I) reacting naphthalic anhydride / substituted naphthalic anhydrides with diaminoalkanes in a polar solvent at a temperature in the range of 0 to 80RC for a period in the range of 1 to 24 hr, isolating the amino alkyl derivative of compounds of formula 1 wherein R is H, halogen, nitro, X = N(CH2)nNH2 and n = 2, 3 by conventional methods (ii) reacting the above amino alkyl compounds obtained in step (I) with isocyanate compound in an organic solvent at an ambient temperature for a period in the range of 1 to 12 hr, isolating the ureido alkyl derivative compound of formula wherein R is H, halogen, nitro, X = N(CH2)nNHCONHCH2CH2CI, N(CH2)nNHCONHCH2CH3 where n = 2 , 3 from reaction mixtures by conventional methods, (iii) reacting the above ureido alkyl derivative compound obtained in step (ii) with carboxylic acid and sodium nitrite in water at a temperature in the range of -10 to 0°C for a period in the range of 8 to 12 hr isolating the nitroso derivative of formula 1 where R is H, halogen, nitro, X = N(CH2)nNHCON(NO)CH2CH2CI, N(CH2)nNHCON(NO)CH2CH3 where n = 2,3 from reaction mixture by conventional methods, (8) A process as claimed in claim 7 where the substitution in naphthalic anhydride is selected from hydrogen, halogen, nitro group. (9) A process as claimed in claims 7-8 where the diaminoalkane used is selected from 1,2-diaminoethane, 1,3-diaminopropane. (10) A process as claimed in claims 7-9 where the polar solvent used in step (i) is selected from water, dimethylformamide. (11) A process as claimed in claims 7-10 where the compounds of formula 1 are isolated from the reaction mixture by filtration of the precipitate obtained after completion of reaction and work up. (12) A process as claimed in claims 7-11 where the isocyanate used is selected from 2-chloroethyl isocyanate or ethyl isocyanate. (13) A process as claimed in claims 7-12 where the solvent used is selected from chloroform, ethanol. (14) A process as claimed in claims 7-13 where the acid used in step (iii) is selected from lower carboxylic acid such as formic acid. (15) Novel substituted 1 H-benz[de]isoquinoline-1,3-diones as claimed in claims 1-14. (16) A process for preparation of novel substituted 1 H-benz[de]isoquinoline-1,3- diones substantially as wherein described with reference to the examples.

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1163-del-2000-abstract.pdf

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