6,9-BIS(AMINO SUBSTITUTED)BENZO(G)PHTHALAZINE-5,10-DIONES AS ANTITUMOR AGENTS

Abstract: Compounds of formula (I) wherein X and R1 have the meanings described in the specification, useful as antitumor agents.

Description: 6,9-BIS(AMINO SUBSTITUTED)BB ZO[g1PHTHAIAZINE-5.10-DIO- NES AS ANTITUMOR AGENTS Certain 1,4-bis[ (aminoalkyl)amino]anthracene-9,10- dioneε have been reported which show antitumor activity in clinical trials. Of particular interest has been ametantrone, 1, -bis{[2-(2-hydroxyethylamino)ethyl]ami- no]anthracene-9,10-dione and mitoxantrone, 5,8-dihy- droxy-1,4-bis{[2-(2-hydroxyethylamino)ethyl]amino]an- thracene-9,10-dione. (Zee-Cheng et al. , J. Med. Chem. , (1978), 21, -291-4; Cheng et al., "Progress in Medicinal Chemistry", Ellis, G.P. and West, G.B., eds.; Elsevier: Amsterdam, 1983; pp. 20, 83 and references cited the¬ rein). Mitoxantrone is a broad spectrum oncolytic agent, whose activity is similar to that of the anthracycline antibiotic doxorubicin. Clinical trials have demonstrated that mitoxantrone has particularly promising activity in the treatment of advanced breast cancer, acute leukemia and lymphoma (Legha, Drugs of Today, (1984), 2_\, 629). Although animal studies have demonstrated a diminished cardiotoxicity in comparison to doxorubicin, some clinical cardiotoxicity has been observed also with mitoxantrone, mostly in patients previously treated with doxorubicin (R. Stuart Harris et al. , Lancet, (1984), 219, and references cited the¬ rein). Ametantrone has been reported to be, in animals, about 10-fold less potent and cardiotoxic than mitoxan¬ trone. Because a delayed toxicity is observed only with mitoxantrone after administration of the two drugs by the i.p. route to non-tumor bearing rats at equieffec- tive antitumor dosages, it is suggested that the pre¬ sence of the 5,8-dihydroxy substitution in mitoxantrone might be implicated in the delayed deaths (Corbet et al. , Cancer Chemother. Pharmacol., (1981), ]5, 161). In addition, both mitoxantrone and ametantrone have a remarkable myelodepressive toxicity and both compounds show cross-resistance to cell hystotypes de¬ veloping resistance against doxorubicin mediated by overexpression of glycoprotein P. Such a resistance, which is named multidrug resistance, involves a number of antitumor antibiotics, among which amsacrine and po- • dophyllotoxinic derivatives, and it is one of the main reasons for therapeutical failures in the treatment of solid tumors with said antibiotics. Therefore, search is required for novel anthrace- nedione antitumor agents, having a higher therapeutical index than mitoxantrone and being effective both in inhibiting or delaying the growth of those solid tumors which are more refractory to chemotherapeutic treatment (such as lung, breast and colon tumors) and against tu¬ mor histotypes developing multidrug resistance. In the search for safer, active analogues of anthracenedioneε, compounds bearing hydroxysubstituents and/or (aminoalkyl)amino side chains at various posi- tions on the anthracene-9,10-dione nucleus have been studied (C.C. Cheng et al. , Drugs of the Future, (1983), J3, 229) without notable 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 ( ), were disclosed by Potts et al. (Synthesis, (1983, 31). These compounds were repor¬ ted to be related to the antitumor 1,4- bis[ (aminoalkyl)amino]anthracene-9,10-diones; however no antitumor activity data was reported for any of the above compounds. l-[ (aminoalkyl)amino] derivatives (4_) of 6,9-dihydroxybenzo[g]isoquinoline-5,10-dione related to mitoxantrone have been disclosed as DNA intercala- tors but they were completely devoid of any "in vitro" or "in vivo" antitumor activity (Croisy-Delcey et al. , Eur. J. Med. Chem. , (1988), Z3, 101-106). The 6,9-bis[ (aminoalkyl)amino]benzo[g]quinoline- 5,10-diones of formulas ^ - (see Table I) were disclo¬ sed in J. Med. Chem. (1985), 2]_, 1124-26, where they were referred to as 5,8-bis[ (aminoakyl)amino]-l-azaan- thracenediones. As reported in table II hereinbelow reported the prior art compounds j> are clearly less active than the corresponding carbocyclic analogues (j6). In fact, as reported in table II, the compound j>a and S are less cytotoxic than the analogues 6a and jϊb. Moreover the compound J5a, which is poorly cytotoxic in vitro, is inactive in vivo, and it is both less active and less potent than the carbocyclic analogue a. Even though the introduction of an heteroatom is a common process in the medicinal chemistry, its effect must be evaluated case by case. In this particular case of aza-analogues of anthracenediones the prior art teachings clearly show that the introduction of a nitrogen atom into the anthracenedione skeleton is detrimental for antitumor activity. In fact the aza-substituted anthracenediones are less cytotoxic than the corresponding anthracene¬ diones and inactive "in vivo". Thus a person skilled in the art, would have not considered the introduction of heteroatoms into the anthracenedione skeleton as a possible way to obtain more effective antitumor agents. It is well known that the screening for the disco¬ very of the antitumor agent mitoxantrone (J. Med. Chem., (1978), - » 291-4) among a number of analogues has been carried out by using the experimental model of murine 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 murine leukemias P388 and L1210 such as m-amsacrine and doxorubicin. Moreover mitoxantrone has shown good activity in other significative experimental tumors such as murine Lewis lung carcinoma and MXl human mammary carcinoma. We have now discovered that the compounds of the invention 6,9-bis[ (aminoalkyl)amino]benzo[g]isoquinoli- ne-5,10-diones are active as antitumor agents: they are highly effective against murine leukemias L1210 and P388 and against Lewis lung carcinoma and MXl human mammary carcinoma. Table I Prior art compounds 5ι =CH2CH N(CH3)2 6JR=CH 2 CH 2 N(CH3) 5b R= CH 2 CH2N(Et)2 6bR-CH 2 CH 2 N(Eι>2 5c R» CH 2 CH2CH 2 N(CH3) 2 ξΛ K CH CH 2 NHCXX:H3 Table II Antitumor activity of prior art compounds J5 and 6_ against murine L 1210 leukemia ( from J. Med. Chem . ( 1985 ) , 28 , 1124-1126 ) _5a 5b .5c 1.7 a 0.08 30 150 6b 0.30 Contrarily to the teachings of the prior art, now it has surprisingly been found that benzo[g]phthala- zine-5,10-dione analogues substituted at the 6- and 9- positions by variously substituted amino groups, have a remarkable in vivo antitumor activity. The compounds of the invention have the following formula (I) wherein R, and R-, which can be the same or different, are hydrogen; (C^C^J-alkyl; phenyl; (C-,-C. Q )-aralkyl; (C 2 -C, 0 )-alkyl containing at least one of the following substituents: -O-R. said (C 2 -C. 0 )-alkyl chain being optionally interrupted by one or more oxygen atoms, by one or more groups of formula -N- and optionally containing one or more dou- ble or tr ple bonds or hydroxy groups; R a is hydrogen; (C 1 -C 1Q )-alkyl; phenyl; ( C 7- ι 0 ^~ aralkyl; (C 2 -C, 0 )-alkyl substituted with one or more hydroxy groups and/or ; - -alkyl, (C 6 -C 10 )-aralkyl; phenyl; R D. and RC. which are the same or different, are hydrogen; (C.-C. Q )-alkyl; (C--C...)-aralkyl; phenyl; (C 2 -C, 0 )-alkyl substituted with one or more hydroxy groups; one of R, and R is a -CH, -C-R,, -C-O-R,, r c ' b c II l l II 0 0 o -C-NH-R, group, R, being as defined above; or R fe and 0 R , taken together with the nitrogen atom which they are linked to, form an aziridine ring or a 5-6 membered heterocyclic aromatic or non aromatic ring, optionally containing one or more heteroatoms such as nitrogen, oxygen and sulfur; n and m are independently zero or the integers 1 and 2, with the proviso that and n cannot be zero at the same time; in form of free bases or pharmaceutically acceptable salts thereof. The present invention also relates to the tautome- ric forms, the racemic and diastereoisomeric mixtures of the compounds of formula (I) as well as to the sin- gle enantiomers and diastereoisomers thereof. Examples of pharmaceutically acceptable acids which can salify compounds (I) are inorganic acids, such as hydrochloric, hydrobromic, sulfuric, phospho- ric, pyrophosphoric acids or organic acids, such as acetic, propionic, citric, benzoic, lactic, maleic, ma¬ lic, fumaric, succinic, tartaric, glutamic, aspartic, gluconic, ascorbic acids or other conventionally used acids. In compounds (I), the words "phenyl" and "5-6 mem¬ bered heterocyclic ring" mean phenyl rings or heterocy¬ clic rings which can optionally contain substituents such as (C,-C 4 )-alkyl groups; CF-; halogen atoms; ni- tro, amino, acetylamino, formylamino, dimethylamino, dieth lamino, hydroxy, methoxy and ethoxy groups. Preferred examples of 5-6 membered aromatic or non aromatic heterocyclic rings containing at least an oxy¬ gen or nitrogen atom are, for example, 1-imidazolyl, 1- pyrrolyl, 1-tetrahydropyrrolyl, 1-pyrazolyl, 1-aziri- dinyl, 4-morpholinyl, 1-piperidinyl, 1-piperazinyl, 1- (4-methyl)piperazinyl, l-(4-benzyl)piperazinyl, 4-(3- methoxy)morpholiny1, 4-(3-cyano)morpholiny1. Preferred examples of (C--C-.-)-aralkyl groups are benzyl and 4-methoxybenzyl. When in compounds (I) one of R. and R 2 is a strai¬ ght or branched (C 2 -C, Q )-alkyl group, optionally inter¬ rupted by one or more hetero-atoms, at least 2 carbon atoms are preferably present between said hetero-atoms. Preferred examples of straight or branched (C.-C. n )-alkyl groups are methyl, ethyl, n-propyl, sec- propyl, n-butyl, εec-butyl, tert-butyl, n-hexyl, n- pentyl. Particularly preferred are compounds (I) in which R, is a substituted (C 2 -C, Q )-alkyl group, selected from the group consisting of : - residues of formula -(CH 2 ) -OH, wherein p is an inte¬ ger from 2 to 4; - residues of formula -(CH 2 ) -NH 2 wherein p is as above defined; - residues of formula -(CH 2 ) -NH-(CH-) -OH, wherein p and q are independently an integer from 2 to 4; 0 - residues of formula -(CH_) -NH- ICI-NH-R-, wherein p is as defined above and Rd is (C.-Cg)-alkyl or phenyl; - residues of formula -(CH 2)pNR.bRc, wherein p is as de- fined above and R, and R are (C,-C 8 )-alkyl groups or, taken together with the nitrogen atom, they form an aziridino, pyrrolidino, morpholino, piperazino, N- methylpiperazino, N-benzylpiperazino, piperidino ring; - residues of formula and n being preferably 1; - X is OH or NHR 2 , wherein R has the same meanings as R, , defined above. Specific examples of preferred compounds of the invention are the following ones: 6,9-bis[N-(2-dimethylaminoethyl) mino]benzo[g]phthala- zine-5,10-dione; 6,9-bis[N-(2-aminoethyl)amino]benzo[g]phthalazine-5,10- dione; 6,9-bis[N-(3-aminopropyl)amino]benzo[g]phthalazine- 5,10-dione; 6,9-bis[N-(4-aminobutyl)amino]benzo[g]phthalazine-5,10- dione; 6,9-bis[N-(2-diethylaminoethyl)amino]benzo[g]phthalazi- ne-5,10-dione; 6,9-bis[N-{2-(2-hydroxyethylamino)ethyl)amino]benzo[g]- phthalazine-5 , 10-dione ; 6-[N-(2-(2-hydroxyethylamino)ethyl)amino]-9-[N-(2-dime- thylaminoethyl)amino]benzo[g]phthalazine-5,10-dione; 6-[N-(2-(2-hydroxyethylamino)ethyl)amino]-9-[N-(2-ami- noethyl)amino]benzo[g]phthalazine-5,10-dione; 6-[N-(1,1-bis(hydroxymethyl)methyl)amino]-9-[N-(2-dime- thylaminoethyl)amino]benzo[g]phthalazine-5,10-dione; 6-[N-(2-dimethylaminoethyl)amino]-9-[N-(2-aminoethyl)- amino]benzo[g]phthalazine-5,10-dione; 6,9-bis[N-(2-(4-morpholinyl)ethyl)amino]benzo[g]phtha- lazine-5,10-dione; 6,9-bis[N-(2-(1-pyrrolidinyl)ethyl)amino]benzotg]phtha- lazine-5,10-dione; 6,9-bis[N-(2-(1-aziridinyl)ethyl)amino]benzo[g.phthala- zine-5 , 10-dione ; 6,9-bis[N-(2-hydroxypropyl)amino]benzo[g]phthalazine- 5,10-dione; 6-[N-( (2-oxotetrahydropyran-4-yl)methyl)amino]-9-[N-(2- di eth laminoethyl)amino]benzo[g]phthalazine-5,10-dio- ne; €,9-bis[N-[2-(methylcarbamoylamino)ethyl] mino]benzo- [g]phthalazine-5,10-dione; 6,9-biε[N-[2-(phenylcarbamoylamino)ethyl] minoJbenzo- [g]phthalazine-5,10-dione; 6-[N-(2-dimethylaminoethyl)amino]-9-hydroxybenzo[g]- phthalazine-5,10-dione; 6-[N-(2-(2-hydroxyethylamino)ethyl)amino]-9-hydroxyben¬ zo[g]phthalazine-5,10-dione; The compounds of the invention wherein X is the -NHR_ group can be prepared by reacting a compound of formula (II) wherein R, is as defined above, with a compound of for¬ mula (III) H 2 N_R x (III) wherein R is indifferently one of R. and R_, or a group which can be converted into R. and R_ by removing the amino- or hydroxy-protecting groups which can op¬ tionally be present, to give a compound of formula (la) which can subsequently be subjected to one or more of the following reaction steps: a) when R χ is different from R. and R 2 , R is tran¬ sformed into R. and R 2 , to give a compound of formu¬ la (I); b) when R is one of the groups defined above for R. and R 2 , R can be converted into another R. and R_ group to give another compound of formula (I); c) salification and/or solvation of the obtained com¬ pound of formula (I) or separation of the isomers thereof. Alternatively, compounds (I) wherein X= NHR 2 with R_ different from R 1 , can be prepared by reacting a compound of formula (II) with an equivalent of a com¬ pound of formula (IV) H 2 N-R- 1 (IV) wherein R', has the same meanings as R Λ ,, or it is a group which can be converted into R, by removing the amino- or hydroxy-protecting groups which are optional¬ ly present, to give an intermediate of formula (V) wherein R 1 . and R, are as above defined, which in its turn can be reacted with a compound of formula (VI) H 2 N-R « 2 (VI) wherein R' 2 has the same meanings as R 2 or it is a group which can be converted into R~ by removing the amino- or hydroxy-protecting groups which are optional¬ ly present, to give an intermediate of formula (lb) wherein R*. and R' 2 are as above defined, which can be subjected to one or more of the following reaction steps: a) when either or both of R' and R' is different from R, and R 2 , R', and R' 2 are transformed into R, and R 2 ; b) when R', and R' 2 are the same as R, and R 2 , R', and/or R' 2 can be converted into another R. and R_ group, to give another compound of formula (I); c) salification and/or solvation of the obtained com¬ pound of formula (I) or separation of the isomerε thereof. Alternatively, if desired, compounds (I) wherein X is the -NHR 2 group, can be prepared by reacting a leuco compound of formula (VII) with a compound of formula (III), followed by spontane¬ ous oxidation during the working-up of the reaction mixture, to give a compound of formula (la) which then can be subjected to one or more of the above described steps, to give a compound of formula (I) wherein X is the -NHR 2 group. Compounds (I) wherein X is the -OH group can be prepared by hydrolysis of intermediate (V) with an al¬ kali or alkaline-earth metal hydroxide, carbonate or hydrogen carbonate, to give a compound of formula (Ic) wherein R'. iε as above defined, which compound can be subjected to one or more of the following reaction steps : a) when R'. is different from R, , R', is tranεformed into R. , to give a compound (I) wherein X iε -OH; b) when R'. iε the same as R,, R « , can be converted in¬ to another R, group to give another compound of formula (I) wherein X is -OH; c) salification and/or solvation of the compound of formula (I) or separation of the isomers thereof. Compounds of formula (II) can be obtained by the multi-step process shown in scheme I below Scheme I The reaction of compounds (II) with amines (III) can be carried out in the presence of a stoichiometric amount or an excess of compoundε (III) in the presence of a solvent such as the compound (III) itself, pyri- dine, picoline, quinoline, N,N,N' ,N'-tetramethylethy- lenediamine and the like, chloroform, methylene chlo¬ ride, 1,1,1-trichloroethane and the like, optionally in the presence of an inorganic base such as an alkali or alkaline-earth carbonate or hydrogen carbonate or an organic base εuch aε a trialkylamine, e.g. triethyla- mine, at a temperature from 0°C to the reflux tempera¬ ture of the solvent and for a time ranging from a few hours to some days, depending on the used experimental conditions. Preferably, the reaction iε carried out by reacting compounds (II) in the presence of a molar ex¬ cess of a ine (III) in pyridine at room temperature. If compounds of formula (I) are desired in which X iε the -NHR 2 group and R. is different from R 2 , it is sufficient reacting compounds (II) with at least an equivalent of an amine (IV), to obtain intermediate (V) which can in its turn be reacted with at least one more equivalent of an amine (VI). Also in this caεe the re¬ action iε generally carried out in pyridine at room temperature. Reaction of leuco compounds (VII), which can be obtained iri situ from compoundε (IX) by reduction with a reducing agent such as sodium dithionite in the pre¬ sence of an inorganic base such as an alkali or alka¬ line-earth metal hydroxide or carbonate, with amines (III), can be carried out by reacting a stoichiometric amount or an excesε of amine (III) in a εolvent εuch aε a C.-C. alcohol, water or mixtures thereof. The reac¬ tion can be performed at a temperature from 0 β C to the reflux temperature of the solvent. Reaction of intermediate (V) with of an alkali or alkaline-earth metal hydroxide or carbonate, to give a compound of formula (Ic), is generally carried out using a stoichiometric amount or an excesε of hydroxide or carbonate, in a solvent such as water, a C.-C. al¬ cohol or mixtures thereof, at a temperature from 0 β C to the solvent's reflux temperature. The reaction is preferably performed in ethanol, using sodium hydroxide, at reflux temperature. The removal of primary- or secondary-amine protec¬ ting groups or hydroxy-protecting groups optionally present in compounds of formulae (Ia)^ (lb) or (Ic) can be carried out according to well-known methods, such as by hydrolysis of the esters and amides of compoundε of formulae (la), (lb) or (Ic) with organic or inorganic acids or baεeε, or by selective cleavage of the protec- ting groups, using suitable reducing and oxidizing agents; the preferred reagent of course depending on the nature of the protecting group. The multi-step process by which compounds of for¬ mula (II) are prepared includes reacting 5,8-dihydroxy- naphtho[2,3-c]furane-4,9-dione (VIII) with 80% hydra- zine at room temperature, to give 6,9-dihydroxy- benzo[g]phthalazine-5,10-dione (IX), which in its turn aralkyl or arylsulfonyl chlo- (X), to yield compounds of formula (II). Reaction of 6,9-dihydroxybenzo[g]ρhthalazine-5,10- dione (IX) with an alkyl, aralkyl or arylsulfonyl chlo¬ ride is generally carried out in the presence of a stoichiometric amount or a slight excesε of the sulfo- nyl chloride in a solvent such as chloroform, methylene chloride, 1,1,1-trichloroethane, pyridine, collidine, lutidine and the like, or mixtures thereof, optionally in the presence of an organic or inorganic base, or ac¬ cording to Schotten-Baumann method, at a temperature from room temperature to the reflux temperature of the solvent. The reaction iε preferably performed using p- toluenesulfonyl chloride in pyridine at room tempera¬ ture. Conpound (VIII) is known and can be prepared aε described in J. Cher.. Res. (!!), (1979), 3510-3518. Compounds (III) are commercially available or they can be prepared according to known methods. Useful tea¬ chings for preparing some compounds of formula (III) can be found in J. Org. Chem. (1959), 2_4_, 818-820; J. Med. Chem. (1980), 33_, 112-117; J. Med. Chem., (1981), 3_4, 184-189; Synthetic Comm. , (1990), 2_0(16), 2559- 2564. The compounds of the invention, when administered to animals bearing experimental tumors such as P388 mu- rine leukemia, P388/DOXO doxorubicin-resistant P388 mu¬ rine leukemia, Lewis lung carcinoma, MXl human mammary carcinoma, LOVO human colorectal tumor, L0V0/D0X0 doxo- rubicin-reεiεtant human colorectal tumor, induce a sta¬ tistically significant regression in the experimental tumors compared to the control animals treated with the only vehicle (water or physiological saline solution) when the compounds of the invention are administered in the form of pharmaceutically acceptable acid salts, or acid solutions tolerated for the intravenous admini¬ stration, such aε 1% citric acid, 5% lactic acid, when the compounds are used as the free bases). The activity of the induced regression is directly proportional to the used dosage (dose-related pharmacological effect) and to the dosage scheme (e.g. single administration in the day after tumor transplant or repeated administra- tionε at days 3, 5, 9 or 3, 5, 9, 14 after the tumor tranεplant). The compounds of the invention, when admi¬ nistered at the maximum tolerated doses, induce a hi¬ gher regression of experimental tumors than that caused by doxorubicin and mitoxantrone, at the maximum tolera- ted doses, under the same experimental conditions. Par¬ ticularly, the compounds of the invention are markedly more effective than the control drugs in inhibiting the growth of solid tumors which are doxorubicin- and mi¬ toxantrone-resistant. Therefore, the available experimental data prove that the compounds of the invention have the following features: - they have a higher therapeutic index than mitoxantro¬ ne, since the range of therapeutically effective to- lerated doses in the animal is markedly wider than that of mitoxantrone; - they are effective in inhibiting growth of hematolo- gical and solid experimental tumors, εuch .as murine leukemias, murine lung solid tumors, human mammary and colon solid tumors; - they are effective against experimental solid tumors which are doxorubicin- and itoxantrone-resiεtant ( ultidrug-reεistant). Therefore, due to these characteristics, the com¬ pounds of the invention can be used as the active in- gredients in therapeutical compositions for the re¬ gression and/or the treatment of tumors in mammalε, when adminiεtered in amountε ranging from 1 mg to about 0.4 g per kg body weight per day. A preferred doεage regimen for optimum reεults would be from about 1.0 to about 50 mg per kg body weight per day, using unitary dosages so as to administer from about 70 mg to about 3.5 g of active compound to a subject of about 70 kg body weight, in a 24 hour period. This dosage regimen can be adjusted to provide optimum therapeutical re- sponεe. For example, several divided doses can be admi¬ nistered according to the requirements of the therapeu¬ tical situation. A remarkable practical advantage is that the active compound can be administered by the oral, intravenous, intramuscular or subcutaneous rou- tes. The compoεitionε of the preεent invention contai¬ ning compoundε of formula (I) can be used for the tre¬ atment of blood cancer diseaεeε εuch as leukemia or other solid and non-εolid tumorε such as lung carci- noma, colon carcinoma, melanocarcinoma and mammary tu¬ mors. As uεed herein, by regression or treatment is me¬ ant arresting or delaying the growth of the tumor. Sui¬ table adminiεtration forms for intravenous injection are physiological sterile solutions. For the intramu- scular or intraperitoneal administrations, oily or aqueous injectable preparations are alεo suitable, whe- reas suitable forms for the oral administrations are liquid or solid preparations such as syrups, capsuleε, tablets, and the like. The invention is further illustrated by the fol- lowing examples. EXAMPLE 1 At 0°C and under magnetic stirring 5,8-dihydroxy- naphtho[2,3-c]furane-4,9-dione (2.12 g) is added por- tionwise during 20 min to 80% hydrazine hydrate (18 ml). The reaction mixture is left under stirring at room temperature for 17 hrs, then it is carefully pou¬ red into 60 ml of 6N HCl, while cooling to 0 β C. The ob¬ tained precipitate is filtered, washed with water, then it is suspended in 40 ml of IN NaOH. The mixture is he- ated to 80 β C for 15 min, cooled to room temperature and left under stirring under a εlight air flow for 18 hrε. Upon addition of concentrated HCl (3.1 ml) and dilution with water (15 ml), a red precipitate is obtained which is filtered, washed firεt with water, then with some methanol, and finally dried to give 1.98 g of 6,9-dihy- droxybenzo[g]phthalazine-5,10-dione; m.p. >220 β C, H- NMR (CDC1 3 ) 8 : 7,47, s, 2H; 10,07, s, 2H; 12,65, s, 2H, (exchangeable with D 2 0) . EXAMPLE 2 Under a nitrogen atmoεphere, 4-toluenesulfonyl chloride (2.60 g) iε added portionwise during 2 min, to a stirred suspenεion of 6,9-dihydroxybenzo[g]phtha- lazine-5,10-dione (1.50 g) in anhydrouε pyridine (15 ml) and the obtained mixture is left under stirring at room temperature for 23 hrε. Subsequently the solvent iε distilled off under reduced pressure and the obtained residue is purified by column chromatography (Si0 2 ; eluent: dichloromethane/ethyl acetate 20:1) to give yellow-orange crystals of 6,9-bis(4-tolueneεul- fonyloxy)benzo[g]phthalazine-5,10-dione (1.57 g); .p. > 230 β C (from CHC1 3 ); X H-NMR (CDC1 3 ) S : 2,47, s, 6H; 7,40, d, 4H; 7,65, s, 2H; 7,90, d, 4H; 9,80, s, 2H. EXAMPLE 3 N,N-dimethylethylenediamine (1.0 ml) is added to a solution of 6,9-bis(4-tolueneεulfonyloxy)benzo[g]- phthalazine-5,10-dione (0.16 g) in anhydrous pyridine (10 ml) and the resulting mixture is left under stirring at room temperature under a nitrogen atmosphe¬ re for 24 hrs. After distilling off the solvent under reduced pressure, the obtained residue is redissolved in CHC1 3 (50 ml) and washed with a 3% NaHC0 3 solution (2 x 15 ml). The aqueous phaseε are discarded. The organic phase is dried over Na.SO, and, after diεtilling off the solvent under reduced presεure, the obtained reεidue iε purified by column chromatography (Si0 2 ; eluent: chloroform/methanol/triethylamine 95:4:1) and subsequent recrystallization from dichloro- methane/pentane to give blue crystals of 6,9-biε[N-(2- dimethylaminoethyl)amino]benzo[g]phthalazine-5,10-dio- ne, (36 mg); m.p. 188-189°C; X H-NMR (CDC1.,) : 2,35, s, 12H; 2,68,t, 4H; 3,57, q, 4H; 7,35, ε, 2H; 9,95, ε, 2H; 11,42, br.t., 2H (exchangeable with D 2 0); UV (HCl 0,1N): λ w (nm), E 1β/ (cm): 245 (475); 279 (337); 351 (106); 684 (337) H.P.L.C.: Lichrospher 100 RP18 (5 μm) ; eluent: 10 mM sodium heptanesulfonate + 10 mM KH-PO. in water/ace- tonitrile/dioxane (70:20:10), pH 3 (H 3 P0.); eluent flow rate: 1 ml/min; retention time: 4.50 min. EXAMPLE 4 Under a nitrogen atmosphere and with stirring a mixture of 6,9-dihydroxybenzo[g]phthalazine-5,10-dione (63 mg) , sodium dithionite (97 mg) and anhydrous sodium hydrogen carbonate (17 mg) in anhydrous degased ethanol (7 ml) is heated to reflux for 1.5 hrs. l-Amino-3-di- methylaminopropane (0.43 ml) is subsequently added and heating is continued for further 12 hours. After diεtilling off the εolvent under reduced preεεure, the obtained reεidue iε taken up into chloroform (15 ml) and left under stirring at room temperature, in an open flask, for 30 min. The solvent is distilled off again under reduced presεure and the reεidue iε purified by column chromatography (Si0 2 ; eluent: chloroform/metha- nol/triethylamine 95:5:1). Blue cryεtals of 6,9-bis[N- (3-dimethylaminopropyl)amino]benzo[g]phthalazine-5,10- dione (15 mg) are obtained; X H-NMR (CDC1 3 )$: 2,01, m, 4H; 2,35, s, 12H; 2,55, t, 4H; 3,55, q, 4H; 7,36, s, 2H; 9,96, s, 2H; 11,15, br.t., 2H (exchangeable with D 2 0). EXAMPLE 5 Under a nitrogen atmosphere N,N-dimethylethylene- diamine (0.11 ml) is added to a stirred suspension of 6,9-bis(4-tolueneεulfonyloxy)benzo[g]phthalazine-5,10- dione (300 mg) in anhydrous pyridine (5 ml), cooled to 0*C. After 6 hrs at 0 β C, the reaction mixture is allowed to warm to room temperature and after 17 hrε the solvent iε distilled off under reduced pressure and the residue is purified by column chromatography (SiO_; eluent: chloroform/methanol 100:5). Purple crystals of 9-[N-(2-dimethylaminoethyl)amino]-6-(4-tolueneεulfonyl- oxy)benzo[g]phthalazine-5,10-dione (118 mg) are obtai¬ ned; ^Η-NMR (CDC1 3 )£: 2,45, 8, 9H; 2,72, t, 2H; 3,47, q, 2H; 7,17, d, 1H; 7,32, d, 2H; 7,48, d, 1H; 7,85, d, 2H; 9,67, ε, 1H; 9,95, ε, 1H; 10,25, br.t., 1H. EXAMPLE 6 Following the procedure described in Example 5, the following compoundε are prepared: 9-[N-(2-(2-hydroxyethylamino)ethyl)amino]-6-(4-toluene- εuIfonyloxy)benzo[g]phthalazine-5,10-dione; 9-[N-(2-tert-butoxycarbonylaminoethyl)amino]-6-(4-to- lueneεulfonyloxy)benzo[g]phthalazine-5,10-dione. EXAMPLE 7 N-(Tert-butoxycarbonyl)ethylenediamine (Synthetic Comm., 2_ , 2559, (1990)) (340 mg) iε added to a suspen¬ sion of 9-[N-(2-dimethylaminoethyl)amino]-6-(4-toluene- εulfonyloxy)benzo[g]phthalazine-5,10-dione (195 mg) in anhydrouε pyridine (5 ml) and the reεulting mixture iε heated to 50°C for 8 hrε, under a nitrogen atmoεphere, with εtirring. The solvent is diεtilled off under reduced pressure and the residue is purified by column chromatography (Si0 2 ; eluent: chloroform/methanol/tri- ethylamine 95:4:1), to give blue crystalε of 6-[N-(2- tert-butoxycarbonylaminoethyl)amino]-9-[N-(2-dimethyl- aminoethyl)amino]benzo[g]phthalazine-5,10-dione (120 mg); X H-NMR (CDC1 3 )£: 1,53, ε, 9H; 2,34, s, 6H; 2,72, t, 2H; 3,51, q, 2H; 3,60÷3,78, m, 4H; 5,60, m, 1H; 7,30, d, 1H; 7,51, d, 1H; 9,85, s, 1H; 9,96, s, 1H; 10,40, br.t., 1H; 10,63, br.t., 1H. EXAMPLE 8 Following the procedure described in Example 3, using N-(tert-butoxycarbonyl)ethylenediamine, blue cry¬ stals of 6,9-bis[N-(2-tert-butoxycarbonylaminoethyl)- amino]benzo[g] hthalazine-5,10-dione are obtained, m.p. 209-210 β C; 1 H-NM (CDCl..)g: 1,55, s, 18H; 3,50, q, 4H; 3,70, q, 4H; 5,50, m, 2H (exchangeable with D 2 0) ; 7,49, s, 2H; 9,85, ε, 2H; 11,65, m, 2H (exchangeable with D 2 0). Gaseous HCl iε bubbled through a εolution of the above compound (245 mg) in anhydrous chloroform (200 ml) until the end of formation of a precipitate, which iε filtered under a nitrogen atmosphere, waεhed with dichloromethane and dried to give 175 mg of 6,9-bis[N- (2-aminoethyl)amino]benzo[g]phthalazine-5,10-dione trihydrochloridε. m.p. > 225 β C 1 H-:iriR (D 2 0) £ : 3,35, t, 411; 3,35, br.m, 4Tϊ; 7,40, br.s, 2H; 9,43, s, 2H. ϋ.V. (H 2 0): maχ (nn); E χ/0 (cm): 245(457); 279(300); 342(95); 669(334). H.P.L.C: lichrospher 100 RP 18, eluent: 2 g/1 sodium heptanesulfonate in water/acetonitrile/dioxane 75/25/5, pH 3 with K 3 PO ; eluent flow rate: 1 ml/min.; = 230 nm; retention time: 4.13 min. EXAMPLE 9 A solution of 6.9 N HCl in methanol (0.58 ml) iε added to a εolution of 6,9-bis[N-(2-dimethylaminoe- thyl)amino]benzo[g]phthalazine-5,10-dione (0,33 g) in chloroform (30 ml), while cooling with ice-water. After 30 min the mixture iε diluted with diethyl ether (30 ml) and the obtained precipitate iε filtered and washed with diethyl ether, to give 6,9-bis[N-(2-dimethylamino- ethyl)amino]benzo[g]phthalazine-5,10-dione trihydro- chloride (0.35 g). EXAMPLE 10 Following the procedures described in Examples 3, 4, 7 and 8, the following compounds are prepared, which are isolated as the free baseε: 6,9-biε[N-(4-aminobutyl)amino]benzo[g]phthalazine-5,10- dione 6,9-biε[N-(2-diethylaminoethyl)amino]benzo[ ]phthalazi- ne-5,10-dione ^ ■ H-NMR (CDC1 3 )^ : 1,12, t, 12H; 2,60, q, 8H; 2,74, t, 4H; 3,53, q, 4H; 7,31, ε, 2H; 9,55, s, 2H; 11,23, t, 2H; 6,9-bis[N-(2-(2-hydroxyethylamino)ethyl)amino]benzo[g]- phthalazine-5,10-dione 1 H-M R (CDC1 3 ) £: 2,96, t, 4H; 3,07, t, 4H; 3,52, q, 4E; 3,82, q, 4H; 7,05, ε, 2H; 9,53, s, 2H; 11,30, br.t., 2H; 6-[N-(2-(2-hydroxyethylamino)ethyl)amino]-9-[N-(2-a i- noeth 1)amino]benzo[g]phthalazine-5,10-dione 1 K- » IMS (CDC1 3 ) S: 2,97, t, 2H; 3,08, t, 2H; 3,17, t, 2H; 3,55, n, 4H; 3,83, t, 2H; 7,02, d, 1H; 7,25, d, 1H; 9,62, s, IK; 9,80, s, 1H; 11,21, t. Hi; 11,30, t, 1H; 6-[N-(1,1-bis(hydroxymethyl) ethyl)amino]-9-[N-(2-dime- thylaminoethyl)amino]benzo[g]ρhthalazine-5,10-dione 1 H-N!1R (CDC1 3 ) £ s 2,37, s, 6H; 2,70, t, 2H; 3,57, q, 2H; 3,80*3,92, m, 5H; 7,20, d, 1H; 7,35, d, 1H; 9,65, s, 1H; 9,88, s, 1H; 6,9-bis[IJ-(2-(4- orpholiny1)ethyl)amino]benzo[g]phtha- lazine-5,10-dione ^-H-N R (CDCl 3 )δ : 2,53, m, 8H; 2,78, t, 4H; 3,58, q, 4H; 3,75, m, 8H; 7,29, s, 2H; 9,58, ε, 2H; 11,30, br.t., 2H; 6 , 9-bis [N- ( 2 - ( 1-pyrr ol idinyl ) ethyl ) amino ] benzo [ g ]phtha- lazine-5 , 10-dione 1 H-NΪ4R (CDC1 3 ) & : 1,85, , 8H; 2,68, m, 8H; 2,88, t, 4E; 3,63, g, 4H; 7,26, ε, 2H; 9,55, s, 2H; 11,25, t, 2H; 6,9-bis[N-(2-(1-aziridinyl)ethyl)amino]benzo[g]phthala- zine-5,10-dione 1 H-NIIR (CDC1 3 ) 5: 1,62, s, 8H; 2,60, t, 4H; 3,69, q, 4H; 7,23, ε, 2H; 9,57, ε, 2H; 11,31, t, 2H; 6, 9-biε [N-(3-hydroxypropyl)amino]benzo[g]phthalazine- 5,10-dione 1 H-N:.-H (CDC1 3 ) S " : 2,06, ra, 4H; 3,58, q, 4H; 3,63, t, 4H; 4,37, br.s., 2H; 7,29, s, 2H; 9,45, ε, 2H; 11,60, t, 2H; 6-[ -(2-(2-hydroxyethylamino)ethyl)amino]-9-[N-(2-dime- thylaminoethyl)amino]benzo[g]phthalazine-5,10-dione 1 H-:riH (CDC1 3 ) B : 2,36, ε, 6H; 2,69, t, 2H; 2,99, ra, 2I-I; 3,10, t, 2H; 3,55, m, 411; 3,73, t, 2H; 7,28, d, 1H; 7,51, d, 1H; 9,70, ε, 1H; 9,93, ε, 1H; 10,25, br.t., 1H; 10,51, br.t., 1H; 6,9-bis[N-[3-(tert-butoxycarbonylamino)propyl]amino]- benzo[g]phthalazine-5,10-dione 6-[Tl-(2-dimethylaminoethyl)amino]-9-[ -[2-(tert-butoxy- carbonylamino)ethyl]amino]benzo[g]phthalazine-5,10-dio- ne 6,9-bis[N-[2-(methylcarbamoylamino)ethyl]amino]benzo- [g]phthalazine-5,10-dione 6,9-bis[N-[2-(phεnylcarbamoylamino)ethyl]amino]benzo- [g]phthalazine-5,10-dione EXAMPLE 11 Following the procedures described in Examples 8 and 9, using the appropriate free bases as the starting products, the following compounds are prepared: 6,9-biε[N-(3-aminopropyl)amino]benzo[g]phthalazine- 5,10-dione trihydrochloride 1 H-NMR (D 2 0) : 2,25, m, 4H; 3,25, t, 4H; 3,78, t, 4H; 7,30, s, 2H; 9,57, s, 2H; 6,9-bis[N-(4-aminobutyl)aminoJbenzo[g]phthalazine-5,10- dione trihydrochloride 1 H- Mn (D 2 0)£ : 2,15, m, 8H; 3,27, t, 4H; 3,76, t, 4H; 7,28, s, 2H; 9,58, s, 2H; 6-[!J-(2-dimethylaminoethyl)amino]-9-[N-(2-aminoethyl)- amino]benzo[g]phthalazine-5,10-dione trihydrochloride 1 H-NMR (D.,0) S : 2,60, ε, 6H; 2,90, t, 2H; 3,31, t, 2H; 3,85, n, 411; 7,32, d, 1H; 7,43, d, 1H; 9,50, s, 1H; 9,71, ε, IK. EXAMPLE 12 20% NaOH (0.1 ml) is added to a solution of 9-[N- (2-dimethylaminoethyl)amino]-6-(4-toluenesulfonyloxy)- benzo[g]phthalazine-5,10-dione (46 mg) in 95° ethanol (2 ml) and the obtained mixture is heated to 50°C for 4 hrs. After cooling to room temperature, the reaction mixture is concentrated to small volume, neutralized with IN HCl, saturated with sodium chloride and extrac¬ ted with chloroform (5 x 4 ml). The combined extracts are dried, the solvent is distilled off under reduced presεure and the obtained residue is purified by column chromatography (silica gel; eluent: chloroform/methanol 95/5) to give 6-[N-(2-dimethylaminoethyl)amino]-9-hy- droxybenzo[g]phthalazine-5,10-dione. EXAMPLE 13 Following the procedure described in Example 12, 6-[N-(2-(2-hydroxyethylamino)ethyl)amino]-9-hydroxyben- zo[g]phthalazine-5,10-dione iε prepared.

Claims: CLAIMS 1. ComDOunds of formula (I) wherein X iε -OH or -NHR ; R, and R_, which can be the same or different, are hydrogen; (C.-C 10 )-alkyl; phenyl; (C_-C-, 0 )-aralkyl; (C..-C, 0 )-alkyl containing at least one of the following R substituents: -O-R,; -N said (C 2 -C. 0 )-alkyl chain being optionally interrupted by one or more oxygen atoms, by one or more groups of formula -??- and optionally containing one or more dou- ' a ble or triple bonds or hydroxy groups; Pv is hydrogen; (C-^-C-^J-alkyl; phenyl; (C-,-C 10 )- aralkyl; (C 2 -C, Q )-alkyl subεtituted with one or more hydroxy groups and/or ; -CH; -C-Rjj -C-OR,; - -alkyl, ( 8C 6 -C 1 8 0 )-aral 8kyl; phenyl; R. and Re, which are the same or different, are hydrogen; (C 1 -C 1Q )-alkyl; (C 7 -C lfJ )-aralkyl; phenyl; (C 2 -C. 0 )-alkyl substituted with one or more hydroxy groups ; either -C-NH-R, group, R, being as defined above; or R. and O R , taken together with the nitrogen atom which they are linked to, form an aziridine ring or a 5-6 membered heterocyclic aromatic or non aromatic ring, optionally containing one or more heteroatoms εuch aε nitrogen, oxygen and εulfur; n and m are independently zero or the integerε 1 and 2, with the proviso that m and n cannot be zero at the • sane tine; in form of free bases or pharmaceutically acceptable salts thereof. 2. Compounds as claimed in claim 1, wherein R 1 is a substituted <C 2 -C, Q )-alk l group, selected from the group consiεting of s - residues of formula -(CH 2_)p-OH, wherein p r is an inte- ger from 2 to 4; - residues of formula -(CH 2.)p-IJH2- wherein p c is as above defined; - residueε of formula -(CH 2 ) -NH-(CH 2 ) -OH, wherein p and q are independently an integer from 2 to 4; - reεidueε of formula ,, wherein p is as defined above and R d is (C,-C 8 )-alkyl or phenyl; - residues of formula -(CH. 2,)pNRb.Rc,- wherein p is aε defined above and R. and R are (C,-C 8 )-alk l groups or, taken together with the nitrogen atom, they form an aziridino, pyrrolidino, morpholino, piperazino, N- methylpiperazino, N-benzylpiperazino, piperidino ring; - reεidues of formula -CH and n being preferably 1. 3. Compounds aε claimed in claims 1 and 2, wherein X is NHR 2 . 4. Compounds as claimed in claims 1 and 2, wherein X iε hydroxy. 5. A proceεs for the preparation of compounds (I), wherein X iε NHR 2 , which procesε compriεeε reacting a compound of wherein Rd is as defined above, with a compound of formula (III) H 2 N-R χ (III) wherein R is indifferently one of R, and R 2 ' or a group which can be converted into R. and R- by removing the amino- or hydroxy-protecting groups which can optionally be present, to give a compound of formula which can subsequently be subjected to one or more of the following reaction steps: a) when R is different from R, and R 2 , R χ is tran¬ sformed into R. and R_, to give a compound of formu¬ la (I); b) when R is one of the groups defined above for R. and R 2 , R can be converted into another R. and R 2 group to give another compound of formula (I); c) salification and/or solvation of the obtained com¬ pound of formula (I) or separation of the isomers thereof. 6. A proceεε for the preparation of compoundε (I), wherein X is NHR 2 and R 2 is different from R,, which process comprises reacting a compound of formula (II) with an equivalent of a compound of formula (IV) H^-R^ (IV) wherein R 1 , has the same meanings as R. or it is a group which can be converted into R. by removing the amino- or hydroxy-protecting groups which are optional- ly present, to give an intermediate of formula (V) wherein R', and R. are as above defined, which in its turn can be reacted with a compound of formula (VI) H 2 N-R' 2 (VI) wherein R' 2 has the same meanings as R 2 or it iε a group which can be converted into R 2 by removing the amino- or hydroxy-protecting groupε which are optional- ly preεent, to give an intermediate of formula (lb) wherein R 1 . and R' 2 are aε above defined, which can be εubjected to one or more of the following reaction εteps: a) when either or both of R', and R' 2 iε different from R, and R 2 , R', and R' 2 are tranεformed into R, and R 2 ; b) when R 1 . and R' 2 are the εame as R. and R 2 , R 1 . and/or R' 2 can be converted into another R. and R- group, to give another compound of formula (I); c) salification and/or solvation of the obtained com¬ pound of formula (I) or separation of the iεomers thereof. 7. A process for the preparation of compounds (I) wherein X is -TJHR 2 , which process comprises reacting a leuco compound of formula (VII) with a compound of formula (III), followed by spontaneous oxidation during the working-up of the reaction mixture, to give a compound of formula (la) which then can be εubjected to one or more of the following εteps: a) when R is different from R, and R 2 , R is tran¬ sformed into R, and R 2 , to give a compound of formu¬ la (I); b) when R is one of the groups defined above for R, and R 2 , R can be converted into another R, and R 2 group to give another compound of formula (I); c) salification and/or solvation of the obtained com¬ pound of formula (I) or separation of the isomers thereof. 8. A proceεε for the preparation of compoundε (I), wherein X is OH, in which process a compound of formula iε hydrolyzed to compound (Ic) wherein R', is as above defined, which compound can be subjected to one or more of the following reaction steps : a) when R 1 , is different from R., R 1 . is transformed into R., to give a compound (I) wherein X is -OH; b) when R'-. is the same as R,, R*, can be converted in¬ to another R, group to give another compound of formula (I) wherein X iε -OH; c) salification and/or solvation of the compound of formula (I) or separation of the isomers thereof. 9. Pharmaceutical compositions containing as the active ingredient one compound of claims from 1 to 4, in admixture with suitable excipients. 10. The use of the compounds as claimed in claims 1-4 for the preparation of an antitumor medicament.

Inventors: MENTA ERNESTO, OLIVA AMBROGIO, SPINELLI SILVANO, TOGNELLA SERGIO, MENTA, ERNESTO, OLIVA, AMBROGIO, SPINELLI, SILVANO, TOGNELLA, SERGIO
Applicants: BOEHRINGER MANNHEIM ITALIA, BOEHRINGER MANNHEIM ITALIA S.P.A.
Application year: 1992
Main classification: A61K31
IPC codes: A61K31, A61P35, C07D237, C07D403