CZ598A3 - PROCESS FOR PREPARING 6,9-BIS[(2-AMINOETHYL)AMINO]BENZO[g]ISOQUINOLINE-5-10-DIONEDIMALEATE - Google Patents

Abstract

In the search for novel heteroanalogs of anthracendiones, 6,9-bis[(2-aminoethyl)amino]benzo[g]isoquinoline-5,10-dione dimaleate salt (BBR 2778) was selected as the most promising compound. New methods of synthesis produce the compound in purity greater than 99 %.

Description

Process for the preparation of 6,9-bis [(2-aminoethyl) amino] benzo [g] isoquinoline-5,10-dione dimaleate

Technical field

The invention relates to a process for the preparation of 6,9-bis [(2-aminoethyl) amino] benzo [g] isoquinoline-5,10-dione dimaleate.

BACKGROUND OF THE INVENTION

Mitoxantrone (Mitox), an antitumor 1,4-bis (aminoalkylamino) anthracene-9 ₁ 10-dione, is currently gaining an important place in the clinical management of leukemias and lymphomas as well as in combination therapy of advanced breast and ovarian cancers. Although it has a better tolerance profile compared to doxorubicin (DX) and other Mitox anthracyclines, this drug does not lack significant toxic side effects, especially those associated with myelosuppression and cardiotoxicity. the therapeutic and toxicological profile of mitoxantrone is reported in Faulds, D. Langtry,

H.D.Mitoxantrone, and Review of its Pharmacodynaroic and Pharmacokinetic Properties, and Therapeutic »Potential in the Chemotherapy of Cancer, Drugs 1991, 41, 400449).

The mechanism of cellular degradation of Mitox is probably multimodal in nature: many studies assume that DNA is the main cellular event.

Nucleic acid compression and interference with DNA-Topoisomerase II activity, which results in disruption of the protein-associated DNA strand, is also reported to be the main cause of Mitox-induced cell death. Cellular destruction by antitumor anthracene-9,10-diones, including Mitox, is also attributed to oxidative metabolism, which leads to the formation of free radicals capable of alkylating and / or disrupting DNA, which induces disruption of the non-protein associated DNA strand. However, it is generally believed that the redox instability of the quinone moiety is likely more related to the cardiotoxic side effects of Mitox than to the mechanism of its antitumor activity. The cardiotoxicity of Mitox and DX has been associated with the ability of adjacent hydroxyl and quinone groups to form chelates with metals. Drug-metal complex formation can increase oxidative-reducing instability by metal-catalyzed type reactions (Shipp, NG; Dorr RTj Alberts, DS; Dawson, BV; Hendrix, M. rat heart cells, Canner Res, 1993, 53,550-556).

The significant clinical activity of Mitox makes the development of the second generation of Anthracenodion-like substances an attractive area of research. Meanwhile, much research has been devoted to exploring possibilities in the side chain and repository of hydroxide substituents and / or lateral side chains.

The introduction of heteroatoms into the anthraquinone chromophore is a relatively unexplored approach, but such a change may substantially affect the interaction of such a molecule with biological materials. In particular, the heterocyclic anthraquinone analogs a) may retain essentially the same spatial and planar characteristics as the parent drugs for the host molecular exploration as incorporation into DNA and b) introduce additional hydrogen bonds or base sites that may increase the affinity of the drug for DNA ^ and / or affect interaction with Topoisomerase II. In addition, heteroanalogs may have altered redox properties.

Several aza Analogs (Krapcho, AP, 6,9-bis (substituted-amino) benzo [g] isoguinoline-5,10-dione, PCT application WO 92/45300, September 17, 1992 (herein incorporated by reference) were prepared and observed. AP Krapcho et al.: 6.9-bis (2-aminoalkyl) aminobenzorq] isoquinoline4

5,10-dione. A novel class of chromophore-modified antitumor anthracene-9,10-dione; svnthesis and antitumor evaluation, J. Med. (1994), in the press, herein incorporated by reference.

Among these compounds, the dimalate salts of 6,9-bis [(2-aminoethyl) amino] benzo [g] isoquinoline-5,10 dione appeared to be the most active in antitumoral experimental models.

In addition, the 6,9-bis [(2-Aminoethyl) amino] benzo [g] isoquinoline-5,10 dione di maleate salt lacks some toxic effects on cardiac tissue after both single and multiple treatments in rats and mice. After simple treatment of rats at doses approximately equal to LD10 and LD50, the compound 6,9-bis [(2-aminoethyl) amino] benzo [g] isoquinoline-5,10-dione dimaleate induces erythropenia and throrabocytopenia to a lesser extent than Mitox. This favorable profile was confirmed in retreated mice as compared to Mitoxantrone at equiactive doses in leukemic mouse models.

Unfortunately, the chemical evolution of the compounds faces unforeseen problems due to lower purity levels of these compounds (purity less than 96%). In addition, analytical examination of these compounds revealed the presence of unknown impurities that are formed during the final preparation step and which cannot be removed from the compound by any of the available purification methods. Hitherto unknown impurities account for more than 2% of the content, and one of these unknown impurities alone 'v' represents 1.3%, so the development of compounds is seriously compromised, since supervisory authorities recommend a broad investigation of these impurities, when present in such substantial amounts.

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION The present invention provides a process for the preparation of 6,9-bis [(2-aminoethyl) amino] benzo (g) isoquinoline-5,10-dione dimaleate having a purity of more than 99% by weight, comprising: N-tert-butoxycarbonylaminoethyl) amino] benzo [g] isoquinoline-5,10-dione is deprotected with trifluoroacetic acid to give 6,9-bis [(2-aminoethyl) amino] benzo [g] isoquinoline-5,10-dione trifluoroacetate, which is subsequently converted to the desired dimalate by treatment with an aqueous solution of maleic acid.

In a preferred embodiment, the above process is carried out by converting 6,9-bis [(2-aminoethyl) amino] benzo [g] isoquinoline-5,10-diontrifluoroacetate to the desired 6,9-bis [(2-dimethylate) dimalate salt prior to conversion. (aminoethyl) amino] benzo [g] isoquinoline-5,10-dione trifluoroacetate is dissolved in water, the pH of the resulting aqueous solution is adjusted to an acidic value higher than 2.5, and then the solution obtained is filtered.

In an even more preferred embodiment, in the above preferred embodiment, the pH is adjusted to 4.2, while various reagents may be used to adjust the pH, but most preferably sodium hydroxide is used.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: HPLC analysis describing a mixture of a compound of the invention, peak 6 and identified impurities.

Figure 2: HPLC analysis of a particular sample of a compound of the invention produced by the method of Figure 1

Ί

state of the art - according to J. Med. or WO 92/15300 for bismaleate. Long retention peaks, peaks 7, 8 and 9 are unknown impurities.

Figure 3: HPLC analyzes of a compound of the invention prepared according to the invention by Method A.

Figure 4: HPLC analyzes of a compound of the invention prepared according to the invention by Method B.

6,9-bis ((2-Aminoethyl) amino] benzo [g] isoquinoline-5,10 dione as the free base is not stable as it cyclizes very rapidly in solution, resulting in the formation of compound 1a and 1b, which produces two different peaks due to different compounds (Figure 1)

O

2a: X-N, Y-C2b: X = C, Y = N

All the above decomposition products are present as impurities in

6,9-bis [(2-aminoethyl) amino] benzo [phi] isoquinoline-B, 10 dione (cfr FIG. 2, peaks 3,4,5). Dimaleate salt, on the other hand, is endowed with excellent internal stability.

Methods of synthesizing 6,9-bis [(2-aminoethyl) amino] benzo [g] isoquinoline-5,10-dione and its prior art dimaleate salts are disclosed in:

1) WO92 / 15300 and 2) A.P.Krapcho et al .: 6,9-bis [(2-aminoalkyl) amino] benzo [g] isoquinoline-5,10-diones. A novel class of chromophore-modified antitumo anthracene-9,10-diones: synthesis and antitumor evulations :, J. Med, Chem, (1994), in print.

These synthetic methods use as a key intermediate

6,9-Dufluorobenzo [g] isoquinoline-5,10-dione.

This compound is reacted with ethylenediamine to give the desired 6,9-bis (2-aminoethyl) amino] benzo [g] isoquinoline-5,10-dione.

Another method involves the reaction 6,9difluorobenzo [g] isoquinoline-5,10-dione with mono-tbutoxykarbonylethylendiaminem (BOC-ethylene diamine) and subsequent removal of the protecting BOC group with dry Sy ^ ^ <7 _of 8

HCl, resulting in the 6,9-bis [(2-aminoethyl) amino] benzo [g] isoquinoline-5,10-dione dihydrochloride salt. Conversion of the hydrochloric acid salt to a stable dimalate salt is not feasible because HCl cannot be displaced by weaker maleic acid. Moreover, the HCl salt decomposes by dissolving in water.

Moreover, the prior art method 1) does not lead to preparation

6,9-bis [(2-aminoethyl) amino] benzo [g] isoquinoline-5,10-dione and its dimalate salts of a purity greater than 96,1%

And, by the method 2), the presence of unidentified impurities formed during the last preparation step cannot be avoided. Giant. 2 shows the results of an HPLC analysis of a typical 6,9-bis [(2-aminoethyl) amino] benzo [g] isoquinoline-5,10-dione dimalate salt prepared according to the prior art method: three unknown impurities are present (peaks 7.8 and 9); which represent y 'more than 2% and the amount of some of them (e.g.

A y

8), is about 1.3¾. The purity of the compound cannot be increased A and the presence of unknown impurities cannot be avoided:

- repeated suspension or crystallization in various solvents or solvent mixtures, since the amount of unknown impurities remains constant

column chromatography or performing the last step of the synthesis in other solvents and at different temperatures.

In addition, according to prior art methods, during the final precipitation step of the compound, the dimalate salt is obtained in the presence of a crude dihydrochloride suspension. A complete solution is never obtained. Ve

For example, this suspension may further result in an inhomogeneous product which may absorb other salts and / or contain undesirable substances (e.g. insoluble substances, impurities). Complete dissolution of the final product, at least once during the last step, is an important prerequisite for obtaining a chemical compound which is intended for use in the treatment of mammals.

These drawbacks typical of the prior art synthesis processes present a serious obstacle to the production and expansion of the compound of the invention. Supervisory authorities usually do not allow the use in human medicine of a substance with the main active ingredient with a purity as low as in.

is 96%, especially if unknown impurities are present in A y more than 2½. It is highly preferred

Not to allow the presence of impurities in quantities greater than

The process according to the invention does not have these disadvantages. As mentioned above, 6,9-bis [2-N-tert-butoxycarbonylaminoethyl) amino] benzo [g] isoquinoline-5,10-dione is reacted with trifluoroacetic acid to form the present invention. Crude trifluoroacetate

1 '- [44'] X salts of 6,9-bis [(2-aminoethyl) amino] benzo [g] isoquinoline-5,10-dione. This compound is then completely dissolved in water, the pH of the resulting solution is preferably adjusted to 4.2 with sodium hydroxide, and the resulting solution is filtered. Thereafter, the crude trifluoroacetate salt solution thus obtained is treated with an aqueous solution of maleic acid, whereby the pure dimalate salt of 6,9-bis [(2-aminoethyl) amino] benzo [g] isoquinoline-5,10-dione crystallizes. Further details relating to the process of the invention are particularly described in Example 4.

In the context of the invention, it has surprisingly been found that the desired 6,9-bis [(2-aminoethyl) amino] benzo [g] isoquinoline-5,10-dione dimalate salt can be obtained by displacement from crude trifluoroacetate. Intermediate trifluoroacetate can be obtained by removal of BOC protecting groups by treatment with trifluoroacetic acid.

The key intermediate, 6,9-difluorobenzo [g] isoquinoline-5,10-dione, can be prepared by an improved multistage process. The improvements introduced make it possible to increase the yields so that the large-scale process can be carried out, which is an important prerequisite for the pharmaceutical application of 6,9-bis [(2-aminoethyl) amino] benzo [g] isoquinoline-5,10-dione.

Reaction of 1,4-difluorobenzene with pyridine-3,4-dicarboxylic anhydride in the presence of aluminum chloride resulting in an acid mixture

4- (2 ' _of 5'-difluorobenzoyl) nicotinic acid and 4- (2',

The 5'-difluorobenzoyl) isonicotin is performed as mentioned, with the modification in which the reaction mixture is diluted with nitrobenzene after removing the excess 1,4-difluorobenzene and then slowly poured into water. The procedure described in the prior art requires the addition of water to the entire semi-solid reaction mixture after removal of the excess 1,4-difluorobenzene: this procedure is extremely risky and not suitable for a larger scale because the reaction of hilinium chloride with water is highly exothermic. The new procedure allows to slowly add a solution of the remaining aluminum complexes and excess chloride to a large excess of water.

The resulting mixture of 4- (2 ^Z , 5 ^Z difluorobenzoyl) nicotinic acid and 4- (2 ', 5'-difluorobenzoyl) isonicotinic acid is then subjected to a cyclization reaction in 20% fuming sulfuric acid with the improvement that additional portions of added 20% fuming acid

The sulfuric acid increases the yield to 81%.

Λ

DETAILED DESCRIPTION OF THE INVENTION

Preparation Example 1;

Preparation of 3,4-pyridinedicarboxylic anhydride

A suspension of 97% pure 3,4-pyridinedicarboxylic acid (152g, 0 _{out of} 88mol) in acetic anhydride (450ml) was heated to reflux under nitrogen to give a complete solution. When the reflux temperature was reached, the solvent was removed by distillation at atmospheric pressure for about 1 hour (about 400 ml was obtained). An increase in vapor temperature from about 132 ° C to 140 ° C was observed, and the distillation was 1 ° C.

A. μΥ’Υ / Y terminated when the internal temperature reached 150-155 ° C.

The reaction mixture was cooled to 70 ° C and tert-BuOMe (450 mL) was added dropwise with stirring, while dark gray precipitated at a temperature of about 40 ° C while allowing the temperature to self-lower to 20-25 ° C. precipitate. The suspension was further cooled to 0-5 ° C and stirred for two hours. The dark gray precipitate was collected by filtration under a nitrogen atmosphere, washed with tert-BuOMe (100 mL), and dried under vacuum (2.67 kPa, 30 ° C, 2h d d-j). 3,4-pyridinedicarboxylic anhydride (100 g, yield 76%) was obtained. which was directly used in the following

And step.

3,4-Pyridinedicarboxylic anhydride is highly sensitive to atmospheric moisture, so it must be handled under a nitrogen atmosphere and stored with P2O5.

mp 72-74 ° C

Preparation example 2t

4- (2 ', 5'-Difluorobenzoyl) nicotinic acid and 4- (2', 5'-difluorobenzoyl) isonicotinic acid

3,4-Pyridinedicarboxylic anhydride (95.7g, 0.67 mol) and AlCl 3 (367.3 g, 2.67 mol) were added simultaneously, but separately, in five portions (once every 15 minutes) to the boiling 1, 4-difluorobenzene (650 mL, 90 ° C). About one hour after the last addition was the majority

1,4-difluorobenzene was removed by distillation at normal pressure until a thick mass was obtained. The temperature was lowered to 80 ° C and nitrobenzene (150 mL) was added to dissolve the remaining mass. While still hot, the resulting solution was carefully cooled in ice-cooled stirred water (1000 g ice and 530 g demineralized water) (i.e., a slow dropwise addition of the solution to ice water). Concentrated HCl (37%, 160 mL) was then added to the mixture at M 2 O and stirring was continued for about 3 hours. An oily beige precipitate (about 150 g wet) was obtained by filtration while the aqueous layer was separated from nitrobenzene and extracted with AcOEt (6 x 500 mL). The nititorbenzene layer was dissolved in petroleum ether (400 mL) and the resulting small amount of precipitate was concentrated in vacuo and the resulting crude solid (about 45 g), together with the previously obtained precipitate, was suspended in AcOEt / petroleum ether (1/1). (600 mL). After about 2 hours at room temperature, the suspension was filtered and dried under vacuum. A mixture of 4- (2 ', 5'-difluorobenzoyl) nicotinic acid and 4- (2', 5'-difluorobenzoyl) isonicotinic acid (147.3 g, 84% yield as a pale beige solid) was obtained.

mp 214-216 ° C

Preparation example 3:

6,9-difluorobenzo [g] isoquinoline-5,10-dione ‘<1

A solution of a mixture of 4- (2 ', 5' difluorobenzoyl) nicotinic acid and 4- (2 ', 5' difluorobenzoyl) isonicotinic acid (120 g, 0.456 mol) in 20% fuming H ₂ SO ₄ (180 mL) was heated to 140 Deň: 32 ° C. After about 30 minutes, an additional 20% fuming sulfuric acid was added to the hot reaction mixture in five portions of 30 ml each after 20 minutes. Twenty minutes after the last addition, the reaction mixture was cooled to about 80 ° C and then poured into ice demineralized water (3000 g ice and 3000 ml demineralized water). The pH of the cooled reaction mixture was adjusted to a value of tons. 40% NaOH (850 mL) ”and a yellow-brown precipitate began to form. After about one hour at 0-5 ^° C, the crude precipitate was filtered and dried under vacuum to give crude 6,9-difluoro-benzophenyl] isoquinoline-5,10-dione (98.5 g). The crude reaction product was dissolved in boiling THF (1000 mL) and the hot solution was decolorized with activated carbon (9g), filtered and concentrated to a volume of about 300 mL. After cooling to 0-5 ° C after two hours, the yellow solid was collected by filtration to give analytically pure 6,9-difluorobenzo [g] isoquinoline-5,10-dione (90.6 g, 81% yield).

Melting point: 197-199 ° C

Example 1:

6-Bis [(2-aminoethyl) amino] benzo [q] isoquinoline-5,10-dione dimalate

To a hot solution (55 ° C) of ethylenediamine (154.4 mL, 2.29 mol) in THF (1400 mL) was gradually added over 2.5 hours 6,9-difluorobenzo [g] isoquinoline-5,10 dione (70.1 g, 0.29 mol) (portion 4.6g / 10 min). The mixture was stirred at the same temperature for 3 hours with the gradual formation of a blue precipitate. The suspension was stirred overnight and then filtered under a nitrogen atmosphere, washed with THF (200 mL) and dried under vacuum (2 kVh, 60 ° C, 3 h | x) to give crude 6,9-bis [(218 aminoethyl). amino] benzo [g] isoquinoline-5,10-dione as dihydrofluoride salt (117g).

The crude product was directly dissolved in a mixture of water (2340 mL) and AcOH (40 mL) to give a dark urine solution having a pH of about 5, which was filtered on a glass fiber filter. This pH is very important to prevent degradation of the reaction product. The filtered solution was gradually treated at room temperature with a 3M aqueous filtered maleic acid solution (585 mL) to pH 3.5. After about min. at 40 ° C the suspension was stirred overnight at room temperature. The blue precipitate was filtered and prorayed by addition of water (3 x 60 mL) and EtOH (3 x 100 mL) and dried under vacuum (20 mbar, 60 ° C, 4 h |> d |) to give crude dimaleate 6.9 - bis [(2-aminoethyl) amino] benzo [g] isoquinoline-5,10-dione (168 g).

The crude dimethyl salt was suspended in water (3180 mL) and heated for 30 min. at 50 ° C. After stirring overnight at 25 ° C, the suspension was filtered again, the blue precipitate was washed with water (3 x 100 ml) and this wet material was resuspended in water (2400 ml) and kept at room temperature for 40 hours while stirring. .

The dimalphate salt was filtered, washed with water (3 x 100 mL) and EtOH (3 x 100 mL) and then vacuum dried (2-4: 1¾ _t 60 ° C, 4 h] then 40 ° C overnight) and 6,9-bis [(2-amino) benzo [g] isoquinoline-5,10-dione dimalate was obtained (146.7 g, 92% yield).

HPLC analysis (Waters, UV / Vis detector 486, Pump

510):

column t .a.

eluent flow detector: Lichrospher C16 (5 micrometer): H ₂ O / CH ₃ CN / dioxane 75/20/5 sodium heptanesulfonate (20 mMol) pH 3.0 with H 3 PO 4; 1 ml / min.

UV (245 nm)

C.

: 12 min.

HPLC purity (area%) 99.585%, Figure 3, peak

No. 4

1 H-NMR analysis (Brucker 200 spectrophotometer)

MHz, chemical shifts (δ) are given in ppm relative to the internal standard Me4Si).

(δ; D ₂ O): 3.30 (m, 4H); 3.70 (m, 4H); 6.05 (s,

4H); 7.05 (s, 2 H); 7.70 (d, IH); 8.70 (d, IH); 8.95 (s, 1 H).

Example 2

A solution of di-t-butyl dicarbonate (290.8 g, 1.32 mol) in anhydrous THP (1200 mL) was slowly added over about 3 hours to a cooled (0 ° C) and stirred 1,2-ethylenediamine solution under a nitrogen atmosphere. (268 mL, 4 mol) in THF (3600 mL). After 3 hours at 10 ° C and about 16 hours at room temperature, the solvent was removed in vacuo. The residual yellow oil (about 230g) was dissolved in isopropyl ether (460ml) and washed with brine (50ml). After drying with sodium sulfate (50 g), the organic solution was distilled under reduced pressure (119-111 ° C) to give N-t-butoxycarbonyl-1,2-ethylenediamine (161 g, 76% yield based on di-butyldicarbonate).

NMR (CDC1 _3, δ)

1.3 (s, 2H, D 2 O swap), 1.4 (s, 9H), 2.7 (bt, 2H),

3.1 (dd, 2H); 5.4 (bs, 1H).

Example 3

A solution of 6,9-difluorobenzo [g] isoquinoline-5,10-dione (15 g, 0.061 mol) and Nt-butoxycarbonyl-1,2-ethylenediamine (49 g, 0.305 mol) in N-methylpyrrolidone anhydride was heated under nitrogen atmosphere to 20 ° C. After about 4.5 hours, the reaction mixture was slightly cooled (50 ° C) and added to stirred demineralized water (1500 mL). Stirring was continued for 2 hours at room temperature and the dark blue solid precipitate was collected by filtration and washed with water. The crude filtered precipitate was resuspended in water (1500 mL), filtered and dried under vacuum. The product was dissolved in a hot mixture of methylene chloride and methanol (1/1, 500 mL), the hot solution was filtered on a glass wool filter and cooled to 10 ° C. 15 ° C for 2 hours. After a further 16 hours at room temperature, the crystallized dark blue product was collected by filtration and dried. 27 g of 6,9-bis [(2-N-t-butoxycarbonylaminoethyl) amino] benzo [g] isoquinoline-

5,10-dione (84% yield).

NMR (CDCl ₃ , δ): 1.4 (s, 18H), 3.4-3.7 (m, 8H), 5.4 (m, 2H), 7.3 (s, 2H), δ ( d, J = 6 Hz, 1H); 8.85 (d, J = 6 Hz, 1H); 9.5 (s, 1H); 11 (m, 2H).

Example 4

Trifluoroacetic acid (32 mL, 0.42 mol) was added to a suspension of 6,9-bis [(2-Nt-butoxycarbonylaroinoethyl) amino] benzo [g] isoquinoline-5,10 dione (22 g, 0.042 mol) in methylene chloride (330 mL). After stirring at room temperature for about 16 hours, the reaction mixture was diluted with EtOH (100 mL) and concentrated in vacuo. The resulting oil was re-diluted with ethanol (300 mL) and concentrated again to a small volume. To the oily residue was added demineralized water (220 mL), and the pH was adjusted to

4.2 using 20% aqueous KOH (45 mL). The obtained dark blue solution was filtered on a glass wool filter and treated with aqueous 3M maleic acid solution (45 mL, 0.135 mol). The pH of the solution was readjusted

3.4 with 20% KOH (24 mL) and the mixture was stirred at room temperature for 40 hours. The crude maleate was cooled by filtration and resuspended in demineralized water (200 mL), heated to 50 ° C for 1 hour and kept under stirring at room / telopt for 16 hours. The dark blue solid was filtered off, washed with water (2 x 50 ml) and ethanol (2 x 50 ml) and dried in vacuo. 18.6 g of 6,9-bis [(2-aminoethyl) amino] benzo [g] isoquinoline-5,10-dione dimalate were obtained

V '(80% yield).

HPLC analysis (Waters, UV / Vis detector 486, Pump

510):

column: Lichrospher C16 (5 micron) t .a.

eluent: H ₂ O / CH ₃ CN / dioxane 75/20/5 sodium heptanesulfonate

(20 mMol) pH 3.0 with H ₃ PO ₄ flow 1 ml / min. detector UV (245 nm)

row: 12 min.

HPLC purity (area%) 99.285%, Fig. 4, peak # 30 ^{0 1} H-NMR analysis (Brucker 200 spectrophotometer)

MHz, chemical shifts (δ) are given in ppm relative to the internal standard Me ^ Si).

(δ; D ₂ O): 3.30 (m, 4H); 3.70 (m, 4H); 6.05 (s,

4H); 7.05 (s, 2 H); 7.70 (d, IH); 8.70 (d, IH); 8.95 (s, 1 H).

The application made?

The compounds of the present invention can be used as active ingredients of therapeutic compositions to induce regression and / or alleviation of tumors in mammals at daily dosages ranging from about 1 mg to about 0.4 g / kg body weight. A preferred dosage range may be from about 1 mg to about 50 mg per kilogram of body weight per day. The unit dose may be used so that a subject having a body weight of about 70 kg is administered at 24 hour intervals from about 70 mg to about 3.5 g of the active compound. The dosage may be adjusted to be compatible with other therapies, such as radiotherapy.

The pharmaceutical preparations may be in the form of tablets, capsules, gel capsules, suppositories, lyophilisates and solutions for intravenous administration.

Claims (4)

A process for the preparation of 6,9-bis [(2-aminoethyl) amino] benzo [g] isoquinoline-5,10-dione dimaleate having a purity of more than 99% by weight, characterized in that 6,9-bis [2-N- target. butoxycarbonylaminoethyl) amino] benzo [g] isoquinoline-5,10-dione is deprotected with trifluoroacetic acid to give 6,9-bis [(2-aminoethyl) amino] benzo [g] isoquinoline-5,10-d trifluoroacetate it is subsequently converted to the desired dimalate by treatment with an aqueous solution of maleic acid. ion2. Process according to claim 1, characterized in that before the conversion of 6,9-bis [(2-aminoethyl) amino] benzoisoquinoline-5,10-diontrifluoroacetate to the trifluoroacetate salt, the desired dimalate salt is dissolved in the solution obtained with e [g]. 6,9-bis [(2-aminoethyl) amino] benzo [g] isoquinoline-5,10-dione, the pH of the resulting aqueous is adjusted to an acidic value higher than the solution is filtered. 2.5 then The method according to claim 2, characterized in that the pH is adjusted to 4.2. 3. The process according to claim 2 or 3, wherein the pH adjustment is carried out with an alkali metal hydroxide. -τις _ζ Giant. 1 Peak no. Area (%) RT Area BC 1 0.551 7.06 4741 01 2 · « _r . 10,759 8.22 92519 02 3 9,967 s, 1 85708 02 4 6,657 10.34 57244 02 5 28,166 11.02 '242210 03 6 43.9 13.38 377513 01 Total 100.0 859935 · Peak 6 and 6,9-bis (2-aminoethyl) axino-benzo [g] isoquinoline-5,10-dione. Giant. 2 maleic acid