NO180302B - 6,9-bis (substituted-amino) benzo [gÅisokinolin-5,10-diones - Google Patents

Description

This invention is directed to 6,9-bis(substituted-amino)-benzo[g]isoquinoline-5,10,diones, more specifically to 6,9-substituents which are (aminoalkyl)amino-substituents. These compounds have been shown to have antitumor activity in vitro and in vivo.

Background

Certain 1,4-bis[(aminoalkyl)amino]anthracene-9,10-diones have been reported, and they show antitumor activity in clinical trials. Of particular interest have been ametantrone, 1,4-bis-{[2-(2-hydroxyethylamino)ethyl]amino}anthracene-9,10-dione and mitoxantrone, 5,8-dihydroxy-1,4-bis{[2 -(2-Hydroxyethyl-amino)ethyl]amino}anthracene-9,10-dione. (Zee-Cheng et al, J. Med. Chem. (1978), 21, 291-4; Cheng et al, "Progress in Medicinal Chemistry", G.P. Ellis and G.B. West, eds; Else-vier: Amsterdam, 1983; pp. 20, 83 and literature references therein). Mitoxantrone is a broad-spectrum incolytic agent, and its activity is similar to that of the anthracycline antibiotic doxorubicin. Clinical trials have shown that mitoxantrone has particularly promising activity in the treatment of advanced breast cancer, acute leukemia and lymphoma (Legha, Drugs of Today, (1984), 20, 629). Although animal studies have shown reduced cardiotoxicity compared to doxorubicin, some clinical cardiotoxicity has also been observed with mitoxantrone, mostly in patients previously treated with doxorubicin (R. Stuart Harris et al, Lancet, (1984), 219, and literature references indicated therein.

Ametantrone is reported to be approximately 10 times less potent and cardiotoxic in animals than mitoxantrone. Because a delayed toxicity has been observed only with mitoxantrone after administration of the two drugs by i.p. administration to non-tumor-bearing rats at equivalently effective antitumor dosages, it has been suggested that the presence of the 5,8-dihydroxy subst in the tusion of mitoxantrone may be instrumental in the delayed deaths (Corbett et al, Cancer Chemother. Pharmacol., (1981), 6, 161).

In addition, both mitoxantrone and ametantrone have a remarkable myelodepressant toxicity, and both compounds show cross-resistance against cell histotypes and develop resistance to dexorubicin produced by overexpression of glycoprotein P. Such resistance, called multidrug resistance, implicates a number of antitumor antibiotics , such as amsacrine and podo-phyllotoxic derivatives, and it is one of the main reasons for therapeutic failure when treating solid tumors with the aforementioned antibiotics.

Therefore, it is necessary to search for new anthracenedione antitumor agents that have a higher therapeutic index than mitoxantrone and are effective both when it comes to inhibiting or delaying the growth of the solid tumors that are resistant to chemotherapeutic treatment (such as lung, breast and colon tumors) and against tumor histotypes that develop multi-drug resistance.

In the search for safer, active analogues of anthracene-diones, compounds having hydroxy substituents and/or (aminoalkyl)amino side chains in various positions on the anthracene-9,10-dione nucleus have been investigated (Cheng et al, Drugs of the Future, (1983), 8, 229) without noticeable improvement.

Aza- and diaza-anthracene-9,10-diones, such as 6,9-bis(ethoxycarbonylamino)benzo[g]quinoline-5,10-dione (1), 6,9-bis(ethoxycarbonylamino)benzo [g]isoquinoline-5,10-dione (2) 6,9-bis(ethoxycarbonylamino)benzo[g]quinazoline-5,10-dione (3), was described by Potts et al, (Synthesis, 1983, 31) . These compounds were reported to be related to antitumor - 1,4-bis-[(aminoalkyl)amino]anthracene-9,10-diones; but - no antitumor activity data were reported for any of the above compounds. The 1-[(aminoalkyl)amino]derivatives (4) of 6,9-dihydroxybenzo-[g]isoquinoline-5,10-dione related to mitoxantrone have been described as DNA intercalators, but they were completely devoid of any "in vitro " or "in vivo" antitumor activity (Croisy-Delcey et al, Eur. J. Med. Chem., (1988), 23, 101-106).

The 6,9-bis[(aminoalkyl)amino]benzo[g]quinoline-5,10-diones of formulas 5a-d (see Table I) were described in J. Med. Chem.

(1985), 28, 1124-26, where they were designated as 5,8-bis-[(aminoalkyl)amino]-1-aza-anthracenediones.

As reported in Table II below, the previously known compounds 5 are clearly less active than the corresponding carbocyclic analogues (6).

As reported in Table II, compounds 5a and 5b are indeed less cytotoxic than analogues 6a and 6b. Furthermore, compound 5a, which is slightly cytotoxic in vitro, is inactive in vivo, and it is both less active and less potent than the carbocyclic analogue 6a.

Although the introduction of a heteroatom is a common method in medicinal chemistry, its effect must be evaluated on a case-by-case basis.

In this particular case of aza-analogs of anthracene-diones, prior art clearly shows that the introduction of a nitrogen atom into the atracenedione structure is detrimental to antitumor activity. In fact, the aza-substituted anthracenediones are less cytotoxic than the corresponding anthracenediones and inactive "in vivo".

Thus, a person skilled in the art would not have considered introducing heteroatoms into the anthracenedione structure as a possible way to obtain more effective antitumor agents.

It is well known that the investigations to discover the antitumor agent mitoxantrone (J. Med. Chem. (1978), 21, 291-4) among a number of analogues have been carried out using the experimental model of mouse leukemia P388: this model is then predictive of antitumor activity in humans, at least for this class of antitumor antibiotics. Many other clinically active antitumor antibiotics are active on the murine leukemias P388 and L1210, such as m-amsacrine and doxorubicin.

Furthermore, mitoxantrone has shown good activity in other significant experimental tumors, such as Lewis lung carcinoma in mice and MX1 breast carcinoma in humans.

It has now been shown that the compounds according to the invention 6,9-bis[(aminoalkyl)amino]benzo[g]isoquinoline-5,10-diones are active as antitumor agents: they are highly effective against the murine leukemias L1210 and P388 and against Lewis lung carcinoma and MX1human breast carcinoma.

Summary of the invention

The compounds according to the invention have the formula (I)

The present invention also relates to the tautomeric forms, the individual enantiomers and diastereoisomers of the compounds of formula (I), as well as mixtures thereof.

where R is C2-C10 alkyl having a substituent selected from the group consisting of OR and -NRjP^, where ^ is selected from the group consisting of hydrogen, C1-C6 alkyl, -S(O2)R5 and C2-C6 alkyl , optionally substituted with OH;

R 2 and R 3 may be the same or different and are selected from the group consisting of hydrogen, C 1 -C 10 -alkyl, C 1 -C 3 -alkyl substituted with an OH group, C 1 -C 1 -alkoxy, -COR 1 , -COOR 5 , or R 1 and R 3 together with the nitrogen atom to which they are attached form an aziridine, morpholine or pyrrolidine ring; R 5 is C 1 -C 4 alkyl, such as free bases and their salts with pharmaceutically acceptable acids.

The present invention also relates to the non-toxic salts of the compounds of formula (I) with acids which are acceptable for pharmaceutical and veterinary medicinal use, such as salts obtained by the addition of inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, pyrophosphoric acid and/or organic acids, such as acetic acid, propionic acid, citric acid, benzoic acid, lactic acid, maleic acid, fumaric acid, succinic acid, tartaric acid, glutamic acid, aspartic acid, gluconic acid, ascorbic acid and the like.

Detailed description of the invention

Preferred examples of C 1 -C 4 alkyl groups are methyl, ethyl, n-propyl, sec-propyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl.

Particularly preferred are compounds of formula (I) where R is a C 1 -C 4 alkyl selected from the group consisting of: - a residue of formula (I) -(CH 2 )P~NH 2 where p is 2, 3 or 4; - a residue of formula -(CHjJp-NRjRjhwhere p is as defined above, and Rj and Rj are a C^Cj alkyl, or they form together with the nitrogen atom a heterocyclic ring selected from the group consisting of 1-aziridine, 1-pyrrolidine, 4-morpholine, - a residue of formula -(CHjJp-NRjRjhwhere p is as defined above, and Rj is hydrogen and R3 is Cj-C6-alkyl, - a residue of formula -(CH^p-NH-CCHj^-OH where p and q are independently of each other an integer selected from the group consisting of 2, 3, 4, - a residue of formula -(CH2)p-OH where p is as defined above, - a residue of formula -(CHjJp-O -fCHjJq-OH where p and q are as

above defined.

Specific examples of the preferred compounds in this invention are as follows: 6,9-bis{[2-(amino)ethyl]amino}benzo[g]isoquinoline-5,10-dione; 6,9-bis{[2-(4'-morpholine)ethyl]amino}benzo[g]isoquinoline-5,10-dione;

6,9-bis{[2-(dimethylamino)ethyl]amino}benzo[g]isoquinoline-5,10-dione;

6,9-bis{[2-(diethylamino)ethyl]amino}benzo[g]isoquinoline-5,10-dione;

6,9-bis{[2-[di(sec-propyl)amino]ethyl]amino}benzo[g]isoquinoline-5,10-dione;

6,9-bis{[2-(1'-pyrrolidine)ethyl]amino}benzo[g]isoquinoline-5,10-dione;

6,9-bis{[2-(1'-aziridine)ethyl]amino}benzo[g]isoquinoline-5,10-dione;

6,9-bis{[2-(methanesulfonyloxy)ethyl]amino}benzo[g]isoquinoline-5,10-dione;

6,9-bis{[4-(amino)butyl]amino}benzo[g]isoquinoline-5,10-dione; 6,9-bis{ [ 3 - (amino) pr opy 1] amino } benzo [ g ] isoquinoline-5,10-dione;

6,9-bis{[2-[(2-hydroxyethyl)amino]ethyl]amino}benzo[g]isoquinoline-5,10-dione;

6,9-bis{[3-(dimethylamino)propyl]amino}benzo[g]isoquinoline-5,10-dione;

6,9-bis{[(2-hydroxy)ethyl]amino}benzo[g]isoquinoline-5,10-dione;

6,9-bis{ [ (2-(2-hydroxy et oxy) ethyl] amino } benzo [g] isoquinoline-5, 10-dione;

6,9-bis{[2-(methylamino)ethyl]amino}benzo[g]isoquinoline-5,10-dione;

6,9-bis{[2-(ethylamino)ethyl]amino}benzo[g]isoquinoline-5,10-dione;

6,9-bis{[2-(n-propylamino)ethyl]amino}benzo[g]isoquinoline-5,10-dione;

6,9-bis{[2-(sec-propylamino)ethyl]amino}benzo[g]isoquinoline-

5,10-dione;

6,9-bis{[2-(guanidine)ethyl]amino}benzo[g]isoquinoline-5,10-dione;

6,9-Bis{[(2-amino-2,2-dimethyl)ethyl]amino}benzo[g]isoquinoline-5, 10-dione.

The compounds according to this invention can be prepared by reacting 6,9-difluorobenzo[g]isoquinoline-5,10-dione with formula (II)

with a compound of formula (III)

R'-NH2(III)

where R' has the same meanings as defined in formula (I) for R, or it is a group which can be converted to R, to give a compound of formula (Ia):

and then optionally performing one or more of the following steps: a) when R' is different from R, converting R' to R to obtain a compound of formula (I); b) when R' is one of the groups defined above for R in the compounds of formula (I), and in which case the compounds of formula (Ia) are the same as the compounds of formula (I), R' can optionally is converted to another R group to give another compound of formula (I);

c) optional salt formation and/or solvate formation of the obtained compounds of formula (I) or separation of

the isomers thereof.

The reaction of the compound of formula (II) with a compound of formula (III) is usually carried out in the presence of a stoichiometric amount or a small molar excess of a compound of formula (III) in a solvent, such as methylene chloride, chloroform, 1, 1,1-trichloroethane, dimethoxyethane, tetrahydrofuran, dimethylsulfoxide, dimethylformamide, pyridine, picoline, and mixtures thereof, or, if desired, to use compounds (III) in themselves as solvents, optionally in the presence of a inorganic base, such as an alkali or alkaline earth carbonate or hydrogen carbonate, or an organic base, such as trialkylamine, at a temperature from 0°C to the reflux temperature of the solvent.

Preferably, the reaction is carried out in a solvent, such as pyridine, chloroform or dimethylsulfoxide, using from 2-10 equivalents of compound (III) for 1 equivalent of compound (II) and working at a temperature varying from room temperature to 50°C .

If desired, the compounds of formula (I) wherein R is a hydroxyalkylaminoalkyl group of formula

-(CH2)p-NH-(CH2)q-OH, in which p and q are as defined above, is obtained by reacting a compound of formula (I), in which R is a group of formula -(CH2)p-OS (02)R5, with a compound of formula (IV)

H2N-(CH2)q-0-E (IV)

where E is a hydroxy protecting group, such as trialkylsilane, (dialkyl)arylsilane, formyl, acetyl, and this reaction can optionally be followed by removal of the protecting group E.

If desired, the compounds of formula (I), in which R is a group of formula -(CH^p-NRjRj, in which one of Rj or R3 is hydrogen and the other is hydrogen or a C 1 -C 6 alkyl group, can be obtained by turnover of the compound (II)

with a monoprotected diamine of formula (V):

wherein Rg is hydrogen or a C1-C8 alkyl group, and p and Rj are as defined in formula (I), to give a compound of formula (Ia), wherein R' is a group of formula -(CH2)p -NÉCOOR^Rg, and this reaction can be followed by removal of the protecting group COOR5.

Useful information for the removal of the above protecting groups can be found in T.W. Green, P.G.M. Wuts, "Protective Groups in Organic Synthesis", 2nd edition, John Wiley and sons, (1991).

The 6,9-Difluorobenzo[g] isoquinoline-5,10-dione compound of formula (II) can be prepared by a multi-step process involving the Friedel-Crafts acylation of 1,4-difluorobenzene with pyridine-3,4-dicarboxylic anhydride, and this results teres in compounds with the structure according to the formulas (Vila) and (Vllb):

Compounds of formulas (Vila) and (Vllb) can then be subjected to a cyclization reaction in 30% fuming sulfuric acid at about 140°C to give the compound of formula

(II).

The compounds of formula (III), (IV) and (V) are known and commercially available, or they can be prepared according to known methods.

For example, the compounds of formula (V) can be prepared according to the methods described in Synth. Comm., (1990), 20, 2559 and in J. Med. Chem. (1990), 33, 97.

The assessment of the biological activity of the compounds according to this invention was carried out "in vitro" and "in vivo" according to the protocols developed by the US National Cancer Institute.

The investigation of the "in vitro" cytotoxic activity of the compounds according to the invention was carried out with a human colon adenocarcinoma cell line (Lovo) isolated from a metastatic nodulus and a subline with acquired resistance to a number of antitumor agents, including doxorubicin, VP-16 and vincristine. This subline (named Lovo/DX) shows reduced accumulation of doxorubicin and overexpression of a protein (M. Grandi, C. Geroni, F.C. Giuliani, British, J. Cancer, (1986), 54, 515). The compounds were examined according to the MTT assay (T. Mosman, "Rapid Colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay", J. Immunol. Methods, (1983), 65, 55-63; L.M. Green , "Rapid colorimetric assay for cell viability; application to the quantitation of cytotoxic and growth inhibitory lymphokines", J. Immunol. Methods, (1984), 70, 257-268) in comparison with mitoxantrone, ametantrone and doxorubicin. The data are set out in Table VI.

In general, representative compounds of this invention were as cytotoxic as ametantrone and mitoxantrone in the Lovo cell line, and the highest cytotoxicity (superior to mitoxantrone) was shown by the compound 6,9-bis{[2-(dimethylamino)ethyl]amino}benzo [g] isoquinoline-5,10-dione (10a) described in Example 4 in the experimental section. When ametantrone and mitoxantrone were examined in the Lovo/DX cell line, a resistance index R.I. (defined as the ratio of the ICjo value of the resistant cell line to the IC^ value of the sensitive cell line) as high as 101.2 and 29.0 respectively, showing that this subline does not have an acquired resistance to mitoxantrone and ametanthrone . On the other hand, when representative compounds of this invention were examined in the same resistant subline, no cross-resistance occurred with mitoxantrone and ametantrone, which can be observed for compounds 10a, 12c, 10b, 10e and 10d described in Examples 4 , 22, 5, 8 and 7 in the experimental part, respectively. These compounds generally showed in this resistant subline the same IC^ values as shown by ametantrone in the sensitive Lovo cell line. In particular, compound 10a was more active than mitoxantrone in the Lovo/DX cell line.

These "in vitro" data suggest that representative compounds of this invention may be useful in overcoming the established multidrug resistance mechanism against tumors.

The "in vitro" cytotoxicity assessment was also performed on L 1210 murine leukemia cells using the above cells maintained in suspension cultures (McCoy's 5A medium supplemented with 10% horse serum, glutamine, penicillin and streptomycin) which were grown in a humid environment of 10% carbon dioxide and 100% air at 37°C. The compounds were dissolved with dimethylsulfoxide (DMSO) and added to the suspended cells in suitable concentrations. After 72 hours of continuous treatment, the cell concentration was counted using a Coulter counter, and weight inhibition was calculated using the formula:

The IC 50 value was calculated from the growth inhibition data and is given in Table VII compared to the previously known compound 5a (see Table I for the structure of 5a).

Studies of the biological activity "in vivo" of representative compounds according to the invention were carried out using P 388 and L 1210 murine leukemia models.

P 388 murine leukemia cells were injected intraperitoneally (ip) or intravenously (iv) into CD2Fl mice. The treatment was started approximately 24 hours after the tumor transplantation, and the dosages of the drug were administered ip (P 388 ip/ip) or iv (P 388 iv/iv) according to previously established standard methods, usually at 3-day (P 388 iv /iv) or 4-day (P 388 ip/ip) intervals. The studies were done over a 60-day period, and the dates of death for each animal were recorded. The T/C value in % was determined using the mean survival time (MST) for each group according to the formula

Fek showed two representative compounds according to this invention, namely 6,9-bis{[2-(amino)ethyl)amino}-benzo[g]isoquinoline-5,10-dione 10i as the dimaleate salt (10i maleate) described in Example 12 and 6,9-bis{[(2-dimethylamino)ethyl]amino}benzo[g]isoquinoline-5,10-dione 10a described in Example 4, showed an activity superior to mitoxantrone in P 388 iv/ the iv model. Compound 10i according to the present invention, both as hydrochloride (1Oi.HCl) and the dimaleate salt (10i.maleate) described in Example 12, was superior to mitoxantrone also in P

388 ip/ip model. Furthermore, the above-mentioned representative compounds according to the invention showed antileukemic activity in a white range of well-tolerated dosages, and in particular they were active at dosages that were lower than the maximum

tolerated dose, indicating a more favorable therapeutic index, compared to mitoxantrone. The results are shown in Table VIII and Table IX.

The antitumor activity of representative compounds according to this invention was also evaluated in the L 1210 mouse leukemia model.

L 1210 leukemia cells were injected intraperitoneally (ip) into CDF1 mice, and the treatment was started approximately 24 hours after tumor transplantation. Doses of the medication were administered ip according to previously established protocols, usually at 4-day intervals. The studies were done over a 60 day period, and the date of death for each animal was recorded. The T/C value in percent was determined using the mean survival time (MST) for each group according to the formula

The results are shown in Tables X and XI.

Table X shows the surprising, superior antileukemic activity of representative compounds according to the invention (namely compound 10a from example 4, compound 10i.HCl from example 12 and 6,9-bis{[2-(2-hydroxyethyl-amino)- ethyl]amino}benzo[g]isoquinoline-5,10-dione 101 prepared in Example 17 (dimaleate salt)) compared to the previously known compound 5a.

Table XI shows a clearly favorable comparison again between the excellent antileukemic activity of compound 10.HCl according to the invention and mitoxantrone.

Activity against solid tumors of the compounds of the invention was demonstrated using the murine Lewis lung carcinoma model and the human mammary carcinoma MX-1 model. These tumor models were incorporated into the panel of eight transplanted tumors used by US-NC1 as a screening panel for the selection of clinically useful antitumor agents (R.K.Y. Zee Cheng and CC. Cheng, "Screening and evaluation of anticancer agents", Meth. and Find . Expl. Clin. Pharmacol.,

(1988), 10(2), 67-101). As an example, Table XII shows activity data against these two solid tumors of one representative compound of this invention, 6,9-bis{[(2-amino)ethyl]-amino}benzo[g]isoquinoline-5,10 -dionedimaleate (1Oi-maleate; example 12).

Since representative compounds of this invention show good results against "in vivo" models of murine P 388 and L 1210 leukemias, murine Lewis lung carcinoma and human mammary carcinoma MX-1, which are predictive of good results in humans, as indicated above, it is expected the compounds described herein to be active against human leukemias and solid tumors.

The compounds according to the present invention can therefore be used as active ingredients in therapeutic compositions to induce regression and/or relief of cancer in mammals, when administered in amounts varying from about 1 mg - 0.4 g/kg body weight. A preferred dosage course would be from about 1 mg - about 50 mg/kg body weight/day. Unit dosages can be used so that from about 70 mg - about 3.5 g of the active compound for a patient of about 70 kg body weight is administered in a 24 hour period. The dosages can be adjusted to be compatible with other treatment regimens, such as radiation therapy.

The pharmaceutical composition can be in the form of tablets, capsules, gel capsules, suppositories, lyophilized powders and solutions for intravenous administration. The invention is illustrated by the following non-limiting examples, and variations which are obvious to those skilled in the art. The relevant formulas are indicated in tables I, III, IV and V.

Example 1

Pyridine-3,4-dicarboxylic acid anhydride (7)

A mixture of pyridine-3,4-dicarboxylic acid (15.0 g, 0.09 mol) and acetic anhydride (30 mL) was heated under reflux for 2 hours. The excess acetic anhydride was removed by distillation, and the anhydride was collected and purified by sublimation (123°C at 3 mm Hg) to give 7 as a white solid (10.1 g, 76%), m.p. 74-76°C. 1 H NMR (CDCl 3 ) 9.39 (S, 1H), 9.24 (d, 1H), 7.94 (d, 1H).

Example 2

4- (2', 5'- difluorobenzoyl) nicotinic acid ( 8a) and 3-( 2', 5'- difluorobenzoyl) isonicotinic acid ( 8b)

A mixture of 7 (5.0 g, 0.033 mol) and aluminum chloride (17.5 g, 0.131 mol) in 1,4-difluorobenzene was heated in an oil bath at 110 °C for 22 h. The excess 1,4-difluorobenzene was recovered by distillation. The residue was cooled in an ice bath, and the reaction was quenched with ice water (75 ml) and concentrated hydrochloric acid (6.3 ml). The precipitated solid was filtered and dried to give a white powder (7.7 g, 87%). This material could be crystallized from acetonitrile and water; m.p. 214-217°C; <*>H NMR (DMSO-d J , 9.15 (s), 8.90 (d), 8.80 (d), 7.90 (d), 7.5 (m) , 7.4 (m). Calcd for C13H7F2N03: C, 59.32; H, 2.69; N, 5.32. Found: C, 59.03; H, 2.55; N, 5.18.

Example 3

6. 9-difluorobenzofglycoquinoline-5, 10-dione (9)

The keto acid mixture of 8a and 8b (3.0 g, 0.011 mol) in fuming sulfuric acid (7.5 mL, 30% SO 3 ) was heated in an oil bath at 135-140°C for 3 h. After cooling to room temperature, the mixture was poured into ice (200 ml) and neutralized with sodium bicarbonate. Extraction with methylene chloride gave 9 as a yellow solid (2.0 g, 72%); mp 199-200°C; melt NMR (CDCl 3 ) 9.54

(S, 1H) , 9.12 (d, 1H) , 8.03 (d, 1H) , 7.57 (m, 2H) . Calcd for C13H5F2N02: C, 63.67; H, 2.04; N, 5.71. Found: C, 63.86; H, 1.66; N, 5.39.

Example 4

6. 9- bis- f f 2- (dimethylamino) ethyl] amino>benzofg] isoquinoline-5. 10-dione (10a)

A mixture of 6,9-difluorobenzof]isoquinoline-5,10-dione, 9 (20 mg, 0.08 mmol) and N,N-dimethylethylenediamine (0.8 mmol, 0.8 mL of a 1 M solution in pyridine) was stirred at room temperature for 48 hours. Most of the pyridine was allowed to evaporate, ether was added and the product 10a was removed by filtration (24 mg, 79%), mp 167-168°C; <t>ø NMR (CDCl 3 ) 11.06 (br t, 1H ), 10.97 (br t, 1H) , 9.63 (s, 1H) , 8.92 (d, 1H) , 8.13 (d, 1H), 7.32 (m, 2H), 3, 54 (q, 4H), 2.7 (t, 4H), 2.38 (s, 12H); mass spectrum m/z (relative intensity) 381 (2.0, M + ), 59 (5.4), 59 (100); UV 2 max (2-methoxyethanol) 580 (Sh, 6200), 614 (10.600), 661 (12.700).

Calculated for C2lKri}! 1502: C, 66.12; H, 7.13; N, 18.36. Found: C, 66.22; H, 7.10, N, 18.20.

Example 5

6. 9- bis- f f 2- fdiethylamino) etvllaminolbenzoralisoquinoline- 5. 10-dione ( 10b )

A solution of N,N-diethylethylenediamine (684 mg, 5.9 mmol) in pyridine (2 mL) was added to 9 (202 mg, 0.82 mmol) in pyridine (2 mL). The purple mixture was stirred at room temperature for 48 hours (TLC analysis at this point showed a blue component: silica gel, 5% MeOH/CHCl 3 ). The excess base and pyridine were removed by evaporation with a slow stream of nitrogen gas. The untreated material was then placed under vacuum overnight. Upon addition of ice water to the residue, the solid was collected by filtration and dried (330 mg, 84%); mp 142-144°C.<t>ø NMR (CDCl 3 ) 11.10 (t, 1H) , 11.00 (t, 1H) , 9.60 (s, 1H) , 8.92

(d, 1H) , 8.10 (d, 1H) , 7.25 (m, 3H) , 3.50 (m, 1H) , 2.82 (m, 1H), 2.35 (m, 3H) , 1.10 (m, 12H).

Calculated for CjsHjsNjOj: C, 68.62; H, 16.00; N, 7.31. Found: C, 67.95; H, 16.05; N, 7.28.

Example 6

6. 9- bis- f r2- (di f sec- propyl) amino) ethyl] amino>benzo[ g] isoquinoline- 5 . 10-dione f10c)

N,N-diisopropylethylenediamine (588 mg, 4.1 mmol) was added to compound 9 (100 mg, 0.41 mmol) in methanol (1 mL) and water (1 mL). The mixture was allowed to stand under stirring at room temperature for 88 hours, and then it was poured into ice water. The blue precipitate was recovered by filtration. The solid was purified by column chromatography over silica gel. The first eluent was chloroform followed by 2% and 20% methanol in chloroform. Concentration of the latter eluants led to 163 mg (81%) of product 10c. Mp 134-136°C; <X>H NMR (CDCl1) : 10.95-11.20 (m, 2H) , 9.63 (s, 1H) , 8.92 (d, 1H), 8.13 (d, 1H), 7.33 (m, 2H), 3.45 (2q, 4H), 3.10 (h, 4H), 2.82 (t, 4H), 1.08 (d, 24H) ,

Calculated for C^ B^^ ; C, 70.55; H, 8.78; N, 14,19. Found: C, 69.23; H, 8.71; N, 13.43.

Example 7

6. 9-bis-f f 2-(1'-pyrrolidine) ethyl] amino > benzo f er] isoquinoline-5. 10- dione flOd)

A solution of 1-(2-aminoethyl)pyrrolidine (690 mg, 6.0 mmol) in pyridine (2 mL) was added to 9 (202 mg, 0.82 mmol) in pyridine (2 mL). The mixture was stirred at room temperature for 49 hours. The excess base and pyridine were removed using a slow stream of nitrogen. The residue was placed under vacuum overnight. Cold water was added to the residue, and a sticky solid separated. The product was extracted with chloroform (3 x 35 mL), the extracts were dried over sodium sulfate, and the chloroform was removed by rotary evaporation to give the title compound (260 mg, 73%); TLC, silica gel, 5% MeOH/CHCl 3 , one blue spot; m.p. 141-142°C. 1 H NMR (CDCl 3 ) 11.15 (t, 1H), 11.00 (t, 1H), 9.65 (S, 1H), 8.90 (d, 1H), 8.05 (d, 1H) , 7.35 (m, 2H), 3.65 (m, 4H), 2.90 (m, 4H), 2.70 (m, 8H), 1.85 (m, 8H).

Calculated for C 2 H 2 N 2 O 2 : C, 69.26; H, 7.21; Sun, 16.15. Found: C, 69.02; H, 7.25; Thu, 16.00.

Maleate salt. Under a nitrogen atmosphere, a solution of maleic acid (271 mg; 2.34 mmol) in ethanol (5 mL) was added to a stirred suspension of compound 10d (450 mg; 1.04 mmol) in ethanol (20 mL). After stirring for 2 h, diethyl ether (25 mL) was slowly added, and the precipitate was filtered and dried under vacuum at 40°C to give the hygroscopic dimaleate salt of 10d (580 mg); m.p. 164-166°C.

Calculated for C 3 H 39 N 5 O 10 • H 2 O: C, 57.96; H, 6.04; N, 10.24. Found: C, 57.85, H, 5.99; N, 10,14.

Example 8

6. 9- bis- f r 2- f 4'- morph olin) etvll amino I benzo f q] isoquinoline- 5 . 10-dione (lOe)

A solution of 4-(2-aminoethyl)morpholine (220 mg, 1.7 mmol) in pyridine (2 mL) was added to 9 (102 mg, 0.42 mmol) in

pyridine (1 ml). The purple colored mixture was stirred at room temperature for 120 hours. The excess amine and pyridine were evaporated using a slow stream of nitrogen and the residue placed under vacuum overnight. The mixture was taken up in chloroform and then filtered. Removal of the chloroform left a blue solid as on TLC

(silica gel, 25% MeOH/75% CHCl 3 /a few drops of aqueous ammonium hydroxide) showed a blue spot (140 mg, 72%); <*>H NMR (CDCl 3 ) 11.015 (m, 1H) , 10.90 (m, 1H) , 9.62 (d, 1H) , 8.90 (d, 1H), 8.10 (d, 1H), 7.25 (m, 2H), 3.80 (m , 8H), 3.55 (m, 4H), 2.75 (t, 4H), 2.50 (m, 8H).

Calcd for C 25 H 31 N 5 O 4 : C, 64.22; H, 6.68; N, 11.98. Found: C, 63.95; H, 6.78; N, 11.89.

Example 9

6. 9- bis { r 2- f1'-aziridine) ethyl] amino>benzo Tg] isoquinoline- 5. 10-dione ( lOf)

The procedure in example 4 was repeated by reacting compound 9 with N-(2-aminoethyl)aziridine, and the residue obtained after evaporation of pyridine was purified by silica gel column chromatography.

A major blue band eluted with 10% CH 3 OH in CHCl 3 was collected. Crystallization from a mixture of methylene chloride and ligroin gave a dark blue solid, m.p. 100-103°C; <X>H NMR (CDCl 3 ) 11.23 (br, 1H) , 11.15 (br, 1H) , 9.73 (s, 1H) , 8.91 (d, 1H) , 8.12 (d, 1H) , 7.40 (d, 1H) , 7.38 (d, 1H) , 3.69 (q, 4H) , 2.60 (t, 4H), 1.85 (m , 4H), 1.24 (m, 4H); mass spectrum m/z (relative intensity) 377 (100, M<+>), 278 (72.7), 56 (12.6).

Calculated for Q^ A^ Afi^ ;. c, 66.82; H, 6.14; N, 18.55. Found: C, 66.50; H, 6.00; Sun, 18.20.

Example 10

6. 9- bis - f r 2 - f N- ( t- butoxsvcarbonyl) amino "| ethyl 1 amino> benzo f q 1 - isoquinoline- 5. 10- dione flOq)

A solution of 9 (0.10 g, 0.41 mmol) and N-(t-butoxycarbonyl)ethylenediamine (Synth. Comm., (1990), 20, 2559; 0.65 g, 4.10 mmol) in pyridine (4.0 ml) was stirred at room temperature for 24 hours. Addition of water (20 ml) gave 10g which was filtered, washed with water and dried (0.17g, 80%), m.p. 213-216°C. Crystallization from a CHC13:CCT4 mixture gave a dark blue solid, m.p. 215-216°C; <1>H NMR (CDCl 3 ) 11.07 (br, 1H) , 10.96 (br, 1H) , 9.49 (S, 1H) , 8.90 (d, 1H) , 7.99 (d, 1H), 7.32 (s, 2H), 5.30 (br, 2H), 3.58 (m, 4H), 3.47 (m, 4H), 1.56 (s , 18H); mass spectrum m/z (relative intensity) 525 (100, M<+>) 339 (51.9), 57 (87.7).

Calculated for C 2 H 2 N 2 O 2 : C, 61.70; H, 6.73; N, 13.32. Found: C, 61.18; H, 6.29; N, 12.88.

Example 11

6. 9- bis- f r2- (N- t- butoxxcarbonvl- N- methylamino) ethyl] aminol-benzofqlisoquinoline- 5. 10- dione f10h)

6,9-Difluorobenzo[g]isoquinoline-5,10-dione (0.29 g, 1.18 mmol) was added to a stirred solution of N-(t-butoxycarbonyl)-N-methylethylenediamine (0.824 g, 4, 73 mmol) (J. Med. Chem. 1990, 33, 97) in dry pyridine (5 ml). The reaction mixture was stirred in a nitrogen atmosphere for 24 hours at room temperature, and then a further 8 hours at 50°C. The solvent was removed under reduced pressure and the blue residue obtained was taken up with CRfili (50 ml), washed with a 5% NaHCO 3 solution (2 x 30 ml) and with water (30 ml). The combined organic phases were dried over Na 2 SO 4 , and the solvent was evaporated under reduced pressure.

The blue residue was purified by column chromatography (silica gel 230-400 mesh, 50 g, eluent CH 2 Cl 2 : AcOEt : MeOH 93:5:2) yielding 400 mg (61% blue crystals, mp 161.5 - 162.5° C after recrystallization from CHjClj: hexane.

1 H NMR (CDCl 3 ) 1.49 (s, 18H); 2.94 (p, 6H); 3.45-3.75 (m, 8H); 7.25-7.55 (m, 2H); 8.12 (d, 1H); 8.95 (d, 1H); 9.62 (s, 1H); 10.95-11.23 (m, 2H, exchangeable with D 2 O).

Example 12

6. 9- bis- f [ 2 -( amino) etvllamino> benzo[ q] isoquinoline- 5. 10- dione flOi)

From difluoride 9: Under a nitrogen atmosphere, 1,2-di-aminoethane (8.72 mL; 130.5 mmol) was added to a stirred solution of compound 9 (4.00 g, 16.3 mmol) in pyridine ( 80 ml). A slightly exothermic reaction followed, which was moderated by cooling in a water bath at 20°C. The reaction mixture was stirred at room temperature for 34 hours, then it was cooled to -5°C for 30 min, the solid was separated by suction under a stream of nitrogen and dried overnight under vacuum at 30°C. The crude material obtained (6.07 g) was recrystallized by dissolving in ethanol (20 ml) at 40°C and reprecipitated with methylene chloride (100 ml) and n-hexane (300 ml), which gave 5.12 g of 10i as a blue solid.

1 H NMR (CDCl 1 ) : 11.10-11.30 (2m, 2H), 9.53 (s, 1H), 8.93 (d, 1H), 8.13 (d, 1H), 7.35 (m, 2H), 3.55 (q, 4H), 3.10 (t, 4H).

HPLC-Lichrospher 100 RP18, 5/xm; eluent: sodium heptanesulfonate 2 g/l in H 2 O: CH 3 CN: dioxane 75:20:5, pH 3 with H 3 PO 4 ; n = 245 nm; flow 1 ml/min; RT = 8.80 min.

Maleate salt: Under a nitrogen atmosphere, the free base 10i (3.50 g; 10.8 mmol) was dissolved in a mixture of ethanol (170 mL) and methanol (30 mL) by heating at 50 °C for 10 min, where- Then a solution of maleic acid (2.87 g, 24.7 mmol) in ethanol (30 mL) was added. After heating for a further 5 min at 50°C, the mixture was allowed to cool to room temperature, and then allowed to stand at 4°C for 2 hours. The precipitate was separated by suction under a stream of nitrogen and dried at 40°C under vacuum, yielding 5.75 g of the crude dimaleate.

To a stirred suspension of this raw material (5.71 g) in water (90 ml) at 50°C, ethanol was added dropwise until a complete solution was obtained (about 300 ml), then more ethanol was added (400 ml), and the mixture was allowed to cool to room temperature. After 12 h, the precipitate was separated by suction, washed with ethanol (15 mL) and diethyl ether (10 mL) and then dried under vacuum at 30°C for 5 h to give 4.22 g of the dimaleate salt of 10i; m.p. 176-178°C shrinking, does not melt until 245°C;

Calculated for C17H19N502• 2C4H4O415 H20:

C, 51.37; H, 5.17; N, 11.98.

Found: C, 51.31; H, 4.99; N, 11.93.

<1>H NMR (DMSO-d6) 10.92 (br, 1H) , 10.87 (br, 1H) , 9.45 (s, 1H) , 9.01 (m, 1H) , 8.08 ( m, 1H), 7.59 (s, 2H), 6.03 (s, 4H), 3.76 (br, 4H), 3.08 (m, 4H). U.V., 1.528 mg in water (50 ml), nm (E 1%): 641 (271), 597 (245), 313 (98), 273 (257), 246 (479).

Hydrochloride salt:

a) From amine 10i: Hydrogen chloride gas was bubbled through a solution of the untreated amine in chloroform until the solution was no longer blue. The solid was filtered and dried to give a dark blue, hygroscopic solid (0.174 g, 26%), m.p. 209-212°C; <1>H NMR (DMSO-d J 10.91 (br, 2H) , 9.44 (S, 1H), 9.01 (d, 1H), 8.23 (br, 4H) , 8.09 (d, 1H), 7.74 (s, 2H), 3.84 (m, 4H), 3.02 (m, 4H).

Calculated for C17H19N502• 2HC1 • 41^0;

C, 44.94; H, 6.43; N, 15.41

Found: C, 44.52; H, 5.29; N, 14.53.

The free amine could be regenerated by adding solid potassium carbonate to the NMR sample. A thaw NMR analysis indicated the presence of free amine.

b) From deblocking<g>of t- Boc analogue lQq: Hydrogen chloride was bubbled through a solution of 10g (0.160g, 0.30mmol) in

dry chloroform (10 ml) for 30 min. A dark blue solid was filtered and dried (0.115 g, 95%); m.p. 213-215°C whose structure was confirmed as the hydrochloride salt of 10i by<*>H-NMR analyses.

Example 13

6. 9-bis- f r2- facetvlamino)etvllamino>benzora1 isoquinoline- 5. 10-dione flOi)

The procedure of Example 12 using compound 9 and 1,2-diaminoethane was repeated and the crude reaction mixture was applied to a silica gel column. Acetic anhydride (30 ml) was added to the column and it was allowed to stand for 15 min. A major blue fraction 10j was eluted with 1:4 CH 3 OH:CHCl 3 , and it was crystallized from a CHC 13 :CH 3 OH mixture to give a blue solid (0.240 g, 35%) m.p. 155-156°C; 1 H NMR (DMSO-dg) 11.12 (t, 1H), 11.02 (t, 1H), 9.42 (S, 1H), 8.95 (d, 1H), 8.15 (br, 2H), 8.03 (d, 1H), 7.62 (s, 2H), 3.57 (m, 8H), 1.83 (s, 6H); mass spectrum m/z (relative intensity) 409 (2.5, M<+>), 337 (6.4), 86 (100). Calculated for C₂R₂N₂C₂: C, 61.60; H, 5.67; N, 17.10. Found: C, 61.37; H, 5.42; N, 16.89.

Example 14

6. 9- bis- f r2- ( methylamino) ethylaminolbenzophal isoquinoline- 5. 10-dione trihydrochloride ( lOk)

Ethanolic HCl (6.7 N, 2.0 mL) was added to a solution of 6,9-bis-f. [2-(N-t-Butoxycarbonyl-N-methylamino)ethyl]amino}-benzo[g]isoquinoline-5,10-dione (10h) (440 mg, 0.795 mmol) in CHCl 3 (40 mL). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 24 hours, then the dark green crystals formed were collected by filtration, washed with absolute ethanol and dried at 40°C in vacuo to give 945 mg (94% yield) of pure product, m.p. 188°C decomposition (DSC assessment) .

Elemental analysis calculated for C19H26C13NS02■ 2H20:

C, 46.51; H, 6.03; N, 14.14; Cl, 23.60.

Found: C, 45.75; H, 6.06; N, 14.04, Cl, 21.32.

HPLC (Lichrospher 100 RP18, 5 jum; eluent KjHPC^25 mM, n-C7H15SO3Na 2 mg/ml in H2O: CH3CN: dioxane 80:15:5, pH 2.7 with HC104; n 275 nm, flow 1 ml/min ., Rx 20.11 min.

U.V., 1.07 mg in 20.0 ml water: nm (E 1%): 641 (290); 597

(267); 313 (106); 273 (278); 247 (486).

1 H NMR (D 2 O) 2.75 (s, 6H), 3.30-3.45 (m, 4H), 3.80-3.95 (m, 4H), 7.38 (α, 2H); 8.17 (d, 1H), 8.85 (d, 1H), 9.27 (S, 1H).

Example 15

6, 9- bisf(2-hydroxyethyl) amino] benzothalisoquinoline- 5, 10-dione

(Haha)

Difluoride 9 (1.0 g, 4.1 mmol) and 2-aminoethanol (2.5 g, 40.8 mmol) in pyridine (7 mL) were stirred at room temperature for 18 h. The mixture was poured into water (50 mL), and the product 11a was filtered and dried (1.26 g, 94%), m.p. 236-239°C. Recrystallization from methanol gave dark blue needles m.p. 237-239°C; 1 H NMR (DMSO-d J 11.28 (t, 1H) , 11.19 (t, 1H) , 9.43 (S, 1H), 8.94 (d, 1H), 8.03 (d, 1H ), 7.56 (s, 2H), 5.01 (t, 2H), 3.69 (m, 4H), 3.54 (m, 4H).

Calcd for C 17 H 17 N 3 O 4 : C, 62.38; H, 5.25; N, 12.83. Found: C, 62.21; H, 5.12; N, 12.50.

Example 16

6. 9- bis r ( 2- methanesulfonyloxyethyl) amino] benzo Tal isoquinoline-5. 10-dione (llh)

A solution of lia (0.30 g, 0.92 mmol) in dry pyridine (4 mL) was stirred at room temperature under nitrogen for 10 min. Methanesulfonyl chloride (0.24 g, 2.07 mmol) was added and the mixture was stirred for 20 min. The reaction was quenched with ice water (20 mL) and the solid was filtered. Crystallization from a chloroform-ligroin mixture gave a blue solid (0.294 g, 66%); m.p. 116-118°C; <X>H NMR (CDCl 3 ) 10.98 (br, 2H), 9.59 (S, 1H), 8.97 (d, 1H), 8.09 (d, 1H), 7.38 (d, 1H), 7.34 (d, 1H), 4.50 (t, 4H), 3.83 (q, 4H), 3.09 (s, 6H).

Calculated for C19H21N30gS2: C, 47.20; H, 4.39; N, 8.69.

Found: C, 46.80; H, 4.31; N, 8.48.

Example 17

6. 9- bis- f f 2- f ( 2- hydroxyethyl) aminol ethyl! amino > benzo f q] isoquinoline- 5 , 10-dione ( 101 )

a) From mesylate ( 11b)

A solution of (11b) (0.40 g, 0.83 mmol) and 2-trimethylsilyl-oxy)ethylamine (2.21 g, 16.5 mmol) in pyridine (5.0 mL) was stirred at room temperature under a nitrogen atmosphere for 48 hours. The pyridine was removed under a stream of nitrogen, and the residue was taken up in methylene chloride. This solution was washed with saturated sodium bicarbonate and dried over sodium sulfate. Removal of the solvent left a blue oil which was dried under vacuum overnight. Chromatography on a silica gel column resulted in cleavage of the Si-O bond to give 101. This product was eluted with a 1:5:5 Et 3 N:CH 3 OH:CHCl 3 mixture. Concentration gave 101 as an oil: 1 H NMR (CDCl 3 ) 11.19 (br, 1H), 11.06 (br, 1H), 9.46 (s, 1H), 8.83 (d, 1H), 7 .98 (d, 1H), 6.98 (s, 2H), 3.82 (m, 4H), 3.52 (m, 4H), 3.07 (m, 4H), 2.96 (m, 4H); UV (2-Methoxyethanol): 570 (sh, e = 7200), 612 (e = 13,200), 658 (e = 14,000).

b) From difluoride ( 9)

Under a nitrogen atmosphere, 2-[(2-hydroxyethyl)amino]-ethylamine, (6.19 mL; 61.2 mmol) was added to a stirred suspension of compound 9 (1.00 g; 4.08 mmol) in pyridine ( 30 ml) at 0°C.

After one hour, the reaction mixture was allowed to reach room temperature,

and it was allowed to stand at this temperature for 3 hours. The reaction mixture was applied to a chromatographic silica gel column (100 g) and eluted first with CHCl 3 :CH 3 OH 90:10, then with CHCl 3 :CH 3 OH 85:15 and finally with CHCl 3 :CH 3 OH:NH 4 OH 85:15:1. The fractions containing the product were combined, the solvents were removed, and the residue was subjected to a second purification by silica gel column chromatography (95 g), eluting with CHCl3:CH3OH:NH4OH concentrated from 95:5:0 - 80:20 :2. A final purification over grade I, basic alumina (25 g) eluting with CH 2 Cl 2 : EtOH 96:4 gave a residue of 500 mg which was crystallized from a mixture of ethanol (0.5 ml) and methylene chloride (10 ml). which gave 101 (142 mg).

Maleate salt; The free amine 101 (138 mg; 0.334 mmol) was dissolved in ethanol (6 mL) under a nitrogen atmosphere, and a solution of maleic acid (87 mg; 0.75 mmol) in ethanol (3 mL) was added at room temperature. After cooling for 3 h at 4°C, the obtained blue crystals were separated by suction under a stream of nitrogen, washed with ethanol (1 mL) and diethyl ether (2 mL) and dried under vacuum at 40°C to give the dimaleate salt of 101 (160 mg), m.p. 132-133°C; <1>H NMR (DMS0-d6) 10.94 (br, 1H) , 10.88 (br, 1H) , 9.47 (s, 1H) , 9.02 (d, 1H ), 8.60 (br, 2H), 8.09 (d, 1H), 7.63 (s, 2H), 6.01 (s, 4H), 5.32 (br, 2H), 3.89 (m, 4H), 3.71 (m, 4H), 3.26 (m, 4H), 3.12 (m, 4H). UV 1.355 mg in H20 (25 ml): nm (E 1%) : 641

(220), 598 (208), 312 (85), 272 (221), 246 (406).

Calculated for C^H^NjC^• 2C4H404■ H20:

C, 52.52; H, 5.57; N, 10.64.

Found: C, 52.49; H, 5.56; N, 10.61.

Example 18

6, 9- bis - T r2-( 2- methoxyethylamino) ethylaminoT- benzoral isoquinoline- 5. 10-dione ( lOm)

A solution of 11b (0.15 g, 0.21 mmol) and 2-methoxyethylamine (0.47 g, 6.20 mmol) in pyridine (2.0 mL) was stirred at room temperature under a nitrogen blanket for 48 h . The pyridine and excess amine were removed under vacuum. The residue was dissolved in methylene chloride, washed with a saturated sodium bicarbonate solution and dried over sodium sulfate. After removal of the solvent, the residue was purified by column chromatography on silica gel eluting with 1:1 CH 3 OH:CHCl 2 . Crystallization from an ethanol and pentane mixture gave 10m (0.091 g, 66%); m.p. 174-175°C; 1 U NMR (CDCl 3 ) 11.08 (br, 1H) , 10.97 (br, 1H) , 9.56 (s, 1H) , 8.90 (d, 1H) , 8.05 (d, 1H) , 7.31 (S, 2H) , 3.62 (m, 8H), 3.43 (s, 6H), 3.08 (t, 4H) , 2.94 (t, 4H), 2.66 (br s, 2H); mass spectrum m/z (relative intensity) 441 (100, M<+>), 354 (57.4), 266 (50.5).

Calculated for C^HjiNjCV C, 62.57; H, 7.09; N, 15.86.

Found: C, 62.51; H, 6.92; N, 15.47.

Example 19

6. 9- bis- f f 2- f 2- hydroxyethoxy) ethyl 1 amino>benzo (" q] isoquinoline-5. 10- dione flic)

A solution of 2-(2-aminoethoxy)ethanol (446 mg, 4.25 mmol) in pyridine (1 mL) was added to compound 9 (100 mg, 0.41 mmol) in pyridine (1 mL). The mixture was stirred at room temperature for 45 hours. The excess amine and pyridine were removed via a slow stream of nitrogen gas, and the remaining solid was placed under vacuum overnight. A cold saturated sodium chloride solution was added to the solid and the product was extracted with chloroform (6 x 25 mL). The extracts were dried over magnesium sulfate and concentrated on the rotary evaporator to give 140 mg (82%) of product lic; mp 97-99°C.<t>ø NMR (CDCl 3 ) : 11.15-11.35 (2m, 2H), 9.60 (s, 1H), 8.85 (d, 1H), 8.08 (d, 1H), 7.30 (m, 2H), 3.85 (m, 8H), 3.57-3.75 (2m, 9H), 2.75-3.25 (br S, 1H) .

Example 20

6. 9- bis- f r 3- f amino) propy11aminoIbenzofq] isoquinoline- 5. 10-dione

(12a)

1) Free amine: A solution of 1,3-diaminepropane (870 mg, 11.76 mmol) in chloroform (3 mL) was added to 9 (300 mg, 1.22 mmol) in chloroform (6 mL). The purple colored mixture was allowed to stand under stirring for 166 hours at room temperature. The mixture was filtered and the salts were washed with chloroform. The solvent was removed by rotary evaporation and the residue was placed under vacuum overnight, which

gave 290 mg (93%) of 12a; TLC (silica gel, 25% MeOH/75% chloroform/a few drops of aqueous ammonium hydroxide) indicated a blue spot; m.p. 105°C (becomes soft) 112-115°C.<t>ø NMR (DMSO-d6) 11.15 (m, 1H) , 11.05 (m, 1H) , 9.45 (S, 1H) , 8.90 (d, 1H) , 8.0 (d, 1H), 7.55 (br S, 2H), 3.55 (br m, 4H) , 4.65

(t, 4H) , 3.75 (t, 4H), 1.75 (t, 4H).

Calculated for C 2 H 2 N 2 O 2 : C, 64.57; H, 6.56; N, 19.81. Found: C, 65.00; H, 6.50; N, 19.78. 2) Maleate salt: A solution of maleic acid (11 mg, 0.86 mmol) in methanol was added to 12a (100 mg, 0.28 mmol) undissolved in methanol (2 mL) and ethyl acetate (2 mL). Addition of more ethyl acetate gave a blue precipitate which was filtered and dried under vacuum (120 mg, 79%), <t>ø NRM (DMSO-d*) 11.10 (t, 1H), 11.00 (t , 1H), 9.45 (S, 1H) , 8.95 (d, 1H), 8.05 (d, 1H) , 7.75 (br, 4H), 7.60 (s, 2H), 6 .0 (s, 4H), 3.60 (m, 4H), 2.95 (t, 4H), 1.95 (m, 4H).

Calcd for Ca 2 H 5 H 5 O 6 : C, 58.84; H, 5.80; N, 14.92. Found: C, 58.75; H, 5.70; N, 14.85.

Example 21

6. 9- bis- f f 4- f amino) butyl] amino > benzo Tg] isoquinoline- 5. 10-dione

(12b)

1) Free amine: A solution of 1,4-diaminebutane (960 mg, 10.9 mmol) in chloroform (3 mL) was added to 9 (319 mg, 1.3 mmol) in chloroform (6 mL). The purple reaction mixture was stirred at room temperature for 217 hours (9 days). The precipitated salts were removed by filtration and the filtrate was concentrated under a slow stream of nitrogen to give the free amine 12b (400 mg, 80%); TLC (silica gel, 25% MeOH/75% CHCl 2 /a few drops of aqueous ammonium hydroxide); m.p. 80°C, (becomes soft) 90-92°C.<t>ø NRM (DMSO-dg) 11.15 (m, 1H) , 11.05 (m, 1H), 9.42 (s, 1H) , 8.90 (d, 1H), 8.0 (d, 1H), 7.5 (br s, 2H), 3.45 (m, 4H), 2.60 (m, 4H), 1.70 (m, 4H), 1.45 (m, 4H) .

Calcd for C2iH27N5O2: C, 66.12; H, 7.13; N, 18.36. Found: C, 64.26; H, 6.95; N, 17.42.

2) Maleate salt: A solution of maleic acid (116 mg, 1 mmol) in ethyl acetate (4 mL) was added to 12b (124 mg, 0.40 mmol) in a methanol (6 mL) and ethyl acetate (8 mL) solution. A blue oil separated and the mixture was refrigerated overnight. The solvents were removed by decantation and the remaining solid was washed thoroughly with ethyl acetate to give a blue, highly hygroscopic solid, 125 mg (63%).<t>ø NMR (DMSO-d6) 11.20 (m, 1H) , 11.10 (m, 1H) , 9.45 (S, 1H) , 8.95 (d, 1H) , 8.05 (d, 1H), 7.70 (br S, 4H), 6 .00 (s, 4H), 3.55 (m, 4H), 2.85 (m, 4H), 1.70 (m, 8H). Calculated for CjsHjjNjO^: C, 56.76; H, 5.75; N, 11.41. Found: C, 53.65; H, 6.10; N, 10.05.

Example 22

6. 9- bis-f r3 - (dimethylamino) propyl] aminolbenzof] isoquinoline-5. 10-dione f12c)

A solution of 3-dimethylaminopropylamine (700 mg, 6.9 mmol) in pyridine (2 mL) was added to compound 9 (100 mg, 0.41 mmol) in pyridine (2 mL). The mixture was stirred at room temperature for 120 hours. The excess diamine and pyridine were removed under a slow stream of nitrogen gas, and the residue was placed under vacuum overnight. The residue was added to cold water and extracted with chloroform (4 x 20 ml). The extract was dried over Na 2 SO 4 and concentrated by rotary evaporation. The blue solid was purified by column chromatography over silica gel. The first eluent was 10% methanol/90% chloroform followed by 25% methanol/75% chloroform. The product was eluted by adding small amounts of ammonium hydroxide to 25% methanol/75% chloroform. Concentration of these eluents led to 92 mg (55%) of the desired product 12c, m.p. 107-109°C.<t>ø NMR (CDCl1) : 11.00-11.20 (2m, 2H) , 9.63 (s, 1H) , 8.93 (d, 1H) , 8.12 ( d, 1H), 7.37 (s, 2H), 3.55 (q, 4H), 2.55 (m, 4H), 2.35 (S, 12H), 2.00 (m, 4H).

Example 23

6, 9- bis- f r 2- ( ethylamino) ethyl] aminoT- benzo rg] isoquinoline- 5. 10-dione ( 10n)

A solution of N-ethylethylenediamine (400 mg, 4.5 mmol) in pyridine (1 mL) was added to compound 9 (98 mg, 0.40 mmol) in pyridine (1 mL). The mixture was stirred at room temperature. temperature for 66 hours. The pyridine and excess diamine were removed under a slow stream of nitrogen, and the remaining material was placed under vacuum overnight. Chloroform was then added, and the chloroform was washed twice with cold water. The chloroform extract was dried over MgSO 4 , and the solvent was removed by rotary evaporation. The resulting blue solid was purified by column chromatography over silica gel using 5% methanol/95% chloroform as the first eluent. The eluent was gradually changed to 5%, 10%<p>g then 50% methanol in chloroform. The desired product was eluted using 50% methanol/48% chloroform/2% ammonium hydroxide. Removal of the solvent gave 32 mg (21%) of the product (10n). mp 101-102°C.

<t>ø NMR (CDCl1) 11.10 (m, 1H) , 11.00 (m, 1H) , 9.6 (s, 1H) , 8.9 (d, 1H), 8.05 (d, 1H), 7.3 (m, 2H), 3.55 (m, 4H), 3.0 (t, 4H), 2.8 (q, 4H), 1.15 (t, 6H).

Example 24

6. 9- bis- f r2- (propylamino) ethyl] aminoVbenzofa] isoquinoline- 5. 10-dione flOo)

A solution of N-propylethylenediamine (403 mg, 4.0 mmol) in pyridine (1 mL) was added to compound 9 (98 mg, 0.40 mmol) in pyridine (1 mL). The reaction mixture was stirred at room temperature for 24 hours and concentrated under a slow stream of nitrogen. The remaining material was placed under vacuum overnight. Ice water was added to the residue and the aqueous layer was extracted with chloroform (5 x 40 ml). The extracts were dried over MgSO 4 , and the chloroform was removed by rotary evaporation. The blue solid was purified by column chromatography over silica gel. The first eluent was 5% methanol/95% chloroform followed by increasing the methanol content gradually to 10%, 20%, 30%, 40% and 50%. The desired compound could be eluted with 60% ethanol/40% chloroform containing some ammonium hydroxide. Removal of the eluent gave 50 mg (30% of the product (10o). mp 105-106°C.

1 H NMR (CDCl 3 ) 11.15 (m, 1H), 11.05 (m, 1H), 9.6 (s, 1H), 8.9 (d, 1H), 8.10 (d, 1H) , 7.3 (m, 2H), 3.6 (m, m, 4H), 3.0 (t, 4H) , 2.75 (t, 4H) , 1.6 (m, 4H, H20 peak added), 0.95 (t, 6H).

Example 25

6. 9- bis- f [ 2- f isopropylamino) ethyl laminoT- benzo [ al isoquinolone-5. 10-dione (IOp)

A solution of N-isopropylethylenediamine (450 mg, 4.5 mmol) in pyridine (2 mL) was added to compound 9 (110 mg, 0.45 mmol) in pyridine (1 mL). This resulted in an instant permanganate-like color. The mixture was stirred at room temperature for 40 hours. The pyridine and excess diamine were removed under a slow stream of nitrogen and the residue placed under vacuum overnight. Chloroform (20 mL) was added followed by ice water. Since the blue color appeared in the aqueous layer, this was extracted with chloroform (4 x 25 ml). The combined extracts were dried over MgSO 4 , and the chloroform was removed by rotary evaporation. The resulting blue solid was purified by column chromatography over silica gel using chloroform as the first eluent followed by stepwise changes to 2%, 5%, 10%, 20%, 40% and 50% methanol in chloroform. The desired product was eluted from the column with 50% ethanol/49% chloroform/1% ammonium hydroxide. Removal of the eluents afforded 56 mg (30%) of the desired product (10p). Temp. 136-137°C.

<t>ø NMR (CDCl1) 11.1 (t, 1H) , 11.0 (t, 1H) , 9.6 (s, 1H) , 8.9

(d, 1H),8.1 (d, 1H), 7.30 (m, 2H), 3.6 (m, 4HY), 3.0 (t, 4H), 2.9 (m, 2H) , 1.05 (d, 6H).

Example 26

In vitro biological evaluation: MTT assay on human colon adenocarcinoma Lovo cells.

The MTT assay was performed according to T. Mosmann, J. Immunol. Methods, (1983), 65, 55-63 and L.M. Green, J. Immunol. Methods, (1984), 70, 257-268.

Immediately before use, the compounds and reference standards are dissolved in a suitable solvent, and then further diluted in a complete culture medium. Human colon adenocarcinoma Lovo cells and the subline resistant to doxorubicin (Lovo/DX) (2.5 10 cells/ml) are plated on 96-well plates and pre-incubated for 24 hours in the culture medium. After this time, the cells are exposed to medication for 144 hours, then the blue thiazolyl-tetrazolium bromide solution (MTT solution) is added, and the cells are incubated again for 4 hours. The supernatant is removed and 150/L of dimethylsulfoxide is added to dissolve formazan crystals. The plate is read at 570 nm with a microplate reader, and the inhibitory concentration for 50% cell growth (IC^;/ug/ml) is calculated.

The results are shown in Table VI.

IN VITRO CYTOTOXIC ACTIVITY OF REPRESENTATIVE COMPOUNDS OF THE INVENTION ON LOVO AND LOVO/DX CELLS BY

MTT ASSAY

Example 27

In vitro biological assessment: L1210 murine leukemia

L1210 murine leukemia cells were routinely maintained in suspension cultures in McCoy</>'s 5A medium supplemented with 10% horse serum, glutamine, penicillin and streptomycin, and were grown in a humid environment of 10% carbon dioxide and 90% air at 37°C.

To determine the in vitro toxicity, each compound was dissolved in dimethylsulfoxide and added to 1 ml of L1210 cells (10<5>cells/tube) to obtain the final concentration of 0.01, 0.1 and 1 /xg drug/ml culture. After 72 hours of continuous exposure to the drug, the cell concentration was determined with a Coulter counter. Growth inhibition was calculated for each medincin using the following formula:

The growth inhibition data were then used to calculate the ICjq value (the calculated drug concentration required to inhibit cell growth by 50% compared to the control).

The results are given in Table VII

TABLE VII - IN VITRO CYTOTOXIC ACTIVITIES OF REPRESENTATIVE COMPOUNDS OF THE INVENTION AGAINST L1210 LEUKEMIA COMPARED TO THE PRIORLY KNOWN COMPOUND 5a

Example 28

In vivo biological investigations: P388- murine leukemia

(iv/iv, d lf 4, 7)

P388 murine leukemia cells were maintained in vivo by serial intraperitoneal (i.p.) injections of 10<6> cells into DBA2 mice. In the experiment, CDF1 mice were inoculated intravenously (i.v.) with IO<6>P388 cells, and treatment was started 24 hours later. The intravenous dose of the medication was administered on days 1, 4 and 7. The mice were observed daily for signs of toxicity and survival. The date of death was recorded for each animal that died or was euthanized during the 60-day study. The Median Survival Time (MST) for each treatment group was calculated and the %T/C was determined using the following formula:

The results of the antileukemic activity of 6,9-bis{[2-(dimethylamino)ethyl]amino}benzo[g]isoquinoline-5,10-dione (10a) from Example 4 and 6,9-bis{[2-( amino)-ethyl]amino}benzo[g]-isoquinoline-5,10-dione-dimaleate (1Oimaleate) of Example 12 compared to mitoxantrone is set forth in Table VIII.

Example 29

In vivo biological studies: P388- murine leukemia

(ip/i<p>, d l. 5, 9)

P388 murine leukemia cells were maintained in vivo by serial intraperitoneal (ip) injections of 10<6> cells into DBA2 mice. In the experiment, CDFl mice were inoculated i.p. with IO<6>P388 cells, and treatment was initiated 24 hours later. The i.p. dose of the drug was administered on days 1, 5 and 9. The mice were observed daily for signs of toxicity and survival. The date of death was recorded for each animal that died or was euthanized during the 60-day study. The median survival time (MST) for each treatment group was calculated, and the % T/C was determined using the following formula:

The results of the anti-leukemic activity of 6,9-bis{[2-(amino)ethyl]amino}benzo[g]isoquinoline-5,10-dione (10k) as dihydrochloride (lOiHCl) and dimaleate (lOimaleate) salts (Example 12) compared to mitoxantrone is set forth in Table IX. 3) Median survival time for treated mice/median survival time for controls x 100

4) Number of toxic deaths/total number of mice

5) Long-term survivors at the end of the experiment (60 days)

Example 30

In vivo biological studies: L1210- murine leukemia

(ip/ipf day 1, 5, 9)

a) L1210 murine leukemia cells were maintained in vivo by weekly intraperitoneal (ip) injections of 10<6> cells in BDF, mice. In the experiment, the mice were inoculated i.p. with 10<6>L1210 cells, and treatment was initiated 24 hours later. The desired dose of the drug was administered on days 1, 5 and 9. The mice were observed daily for signs of toxicity and survival. The date of death was recorded for each animal that died or was euthanized during the 60-day study. The mean survival time (MST) for each treatment group was calculated, and the % T/C was determined by the following formula:

The results are given in Table X

b) L1210 murine leukemia cells were maintained in vivo by weekly intraperitoneal (ip) injections of 10<5> cells

in DBA2 mice. In the experiment, CDFl mice were inoculated i.p. with 10<5>L1210 cells, and treatment was initiated 24 hours later. The desired dose of the drug was administered on days 1, 5 and 9. The mice were observed daily for signs of toxicity and survival. The date of death was recorded for each animal that died or was euthanized during the 60-day study. The Median Survival Time (MST) for each treatment group was calculated and the %T/C was determined using the following formula:

The results are given in Table XI.

Example 31

In vivo biological studies: antitumor activity against murine Lewis lung carcinoma and human MX-1 breast carcinoma

a) Murine Lewis lung carcinoma

Female C57bl/6 mice were transplanted im (between the legs)

with 10<5> cells. The treatment was given iv (intravenously) on days 1, 7, 15, after the tumor transplantation (day 0). The mean tumor weight for each treatment group was calculated according to the R.I. Geran et al, Cancer Chemother. Rep., (1972), 3, 51-61, and the TWI value in % was calculated 7 days after the last drug treatment using the formula:

The results are given in Table XII for the representative compound according to the invention 6,9-bis{[2-(amino)-ethyl]amino}benzo[g]isoquinoline-5,10-dione (10i) as the dimaleate salt (1Oimaleate) from example 12.

b) Human MX-1 breast carcinoma

Female CD1 nu/nu mice were transplanted sc (subcutaneously) with

tumor fragments (calmmxlmmxl mm). The treatment was given IV once a week for 3 weeks, and then the tumor weight reached an average of 150 mg. For each individual tumor, the weight change (the relative tumor weight) for the beginning of the treatment (VG) was expressed as Vt/Vc at each measurement day (Vt). TWI% was calculated 7 days after the last drug treatment using the formula:

The results are given in Table XII for the representative compound according to the invention 6,9-bis{[2-(amino)ethyl]-amino}benzo[g]isoquinoline-5,10-dione (10i) as the dimaleate salt (10i«maleate) from example 12.

Claims (12)

1. Compound characterized by the formula (I): where R is C2-C10-alkyl having a substituent selected from the group consisting of OR, and -NR2R3, where Rt is selected from the group consisting of hydrogen, C1-C1-alkyl, -S(O2)R5 and C-C6- alkyl, optionally substituted with OH; Rj and Rj may be the same or different and are selected from the group consisting of hydrogen, C 1 -C 10 alkyl, C 1 -C 2 alkyl substituted with an OH group, C 1 -C 1 alkoxy, -CORs, -COORj, or R 2 and R 3 together with the nitrogen atom to which they are attached form an aziridine, morpholine or pyrrolidine ring; R 5 is C 1 -C 10 alkyl, as free bases and their salts with pharmaceutically acceptable acids, preferably maleate salts or hydrochloride salts. 2. Compound according to claim 1, characterized in that R is -(CH2)p-NH2 and p is 2, 3 or 4. 3. Compound according to claim 1, characterized in that R is - (CH 2 ) p-NR^, and p is 2, 3 or 4, and where R 2 and R 3 are a C 1 -C 8 alkyl group or together with the nitrogen atom they form a hetero -cyclic ring selected from the group consisting of 1-aziridine, 1-pyrrolidine and 1-morpholine. 4. Compound according to claim 1, characterized in that R is -(CH2)p-NR2R3, where p is 2, 3 or 4, R 2 is hydrogen, and R 3 is C-a-alkyl. 5. Compound according to claim 1, characterized in that R is - (CH2)p-NH-(CH2)qOH, and p and q are independently 2, 3 or 4. 6. Compound according to claim 1, characterized in that R is -(CH2)p-OH, and p is 2, 3 or 4. 7. Compound according to claim 1, characterized in that R is -(CH2)p-0-(CH2)q-OH, and p and q are independently 2, 3 or 4. 8. Compound according to claim 1, characterized in that it is selected from the group consisting of: 6,9-bis{[2-(amino)ethyl]amino}benzo[g]isoquinoline-5,10-dione; 6,9-bis{[2-(4<7->morpholine)ethyl]amino}benzo[g]isoquinoline-5,10-dione; 6.9- bis{[2-(dimethylamino)ethyl]amino}benzo[g]isoquinoline- 5.10- dione; 6,9-bis{[2-(diethylamino)ethyl]amino}benzo[g]isoquinoline-5,10-dione; 6,9-bis{[2-[di(sec-propyl)amino]ethyl]amino}benzo[g]isoquinoline-5,10-dione; 6.9- bis{[2-(1'-pyrrolidine)ethyl]amino}benzo[g]isoquinoline- 5.10- dione; 6,9-Bis{[2-(1</->aziridine)ethyl]amino}benzo[g]isoquinoline-5,10-dione; 6,9-bis{[2-(methanesulfonyloxy)ethyl]amino}benzo[g]isoquinoline-5,10-dione; 6,9-bis{[4-(amino)butyl]amino}benzo[g]isoquinoline-5,10-dione; 6,9-bis{[3-(amino)propyl]amino}benzo[g]isoquinoline-5,10-dione; 6,9-bis{[2-[(2-hydroxyethyl)amino]ethyl]amino}benzo[g]isoquinoline-5,10-dione; 6,9-bis{[3-(dimethylamino)propyl]amino}benzo[g]isoquinoline-5,10-dione; 6, 9-bis{[(2-hydroxy)ethyl]amino}benzo[g]isoquinoline-5,10-dione; 6, 9-bis{[(2-(2-hydroxyethoxy)ethyl]amino}benzo[g]isoquinoline-5,10-dione; 6,9-bis{[2-(methylamino)ethyl]amino}benzo[g ]isoquinoline-5,10-dione; 6,9-bis{[2-(ethylamino)ethyl]amino}benzo[g]isoquinoline-5,10-dione; 6,9-bis{[2-(n-propylamino)ethyl ]amino}benzo[g]isoquinoline-5,10-dione;6,9-bis{[2-(sec-propylamino)ethyl]amino}benzo[g]isoquinoline-5,10-dione;6,9-bis{[2-( guanidine)ethyl]amino}benzo[g]isoquinoline-5,10-dione;6,9-bis{[(2-amino-2,2-dimethyl)ethyl]amino}benzo[g]isoquinoline-5,10- dion. 9. Compound according to claim 1, characterized in that it is selected from the group consisting of: 6,9-bis{[2-(amino)ethyl]amino}benzo[g]isoquinoline-5,10-dione, 6,9-bis {[2-(1'-pyrrolidine)ethyl]amino}benzo[g]isoquinoline-5,10-dione, 6,9-bis{[4-(amino)butyl]amino}benzo[g]isoquinoline-5,10- dione, 6,9-bis{[3-(amino)propyl]amino}benzo[g]isoquinoline-5,10-dione, 6,9-bis{[2-[(2-hydroxyethyl)amino]ethyl]amino }benzo[g]isoquinoline-5,10-dione, and a salt of any of these compounds. 10. Compound according to claim 1, characterized in that it is 6,9-bis{[2-(amino)ethyl]amino}benzo[g]isoquinoline-5,10-dione or a salt thereof. 11. Pharmaceutical composition, characterized in that it comprises a compound according to claims 1-10 and a pharmaceutically acceptable diluent or excipient. 12. Use of a compound according to claims 1-10 for the production of pharmaceutical compositions with antitumor activity.