GEM-DIPHOSPHONIC ACIDS, A PROCESS FOR THE PREPARATION THEREOF AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM

Abstract: Diphosphonic acids of formula (I), wherein R1 and R2 are hydrogen or C1-C4 alkyl; (A) is hydrogen, halogen, hydroxy or C1-C12 alkyl; (B) is a bond, a C1-C8 alkylene chain, a cycloalkylalkylene chain, an alkylene chain substituted by cyclohexyl or cyclopentyl groups, or an aralkyl chain, an alkyl chain containing an heteroatom (O, S or N-CH3) or an ureido residue -(CH2)n1-NHCONH -(CH2)n- with n ranging from 1 to 5, R3 is hydrogen, C1-C9 alkyl, C3-C6 cycloalkyl, benzyl, phenyl or p-methoxybenzyl; (C) is C1-C5 alkyl, phenyl or an aralkyl chain; R4 is hydrogen, C1-C4 alkyl or an amino group optionally substituted by alkyl, phenyl, benzyl, p-methoxybenzyl, acyl, amminoacidic or peptide groups; R5 and R6 are 2-haloethyl or together form a 1-aziridinyl residue, are useful as antitumor agent.

Description: GEM-DIPHOSPHONIC ACIDS, A PROCESS FOR THE PREPARATION THEREOF AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM. The present invention relates to diphosphonic acids having antitumor activity, a process for the preparation thereof and pharmaceutical compositions containing them. Gem-diphosphonic acids and the salts thereof are known and used in the treatment of osteoporosis and bone reεorption (EP 96.931, EP 252.504, BE 896.453, BE 903.519, DE 3.016.289, DE 3.540.150, DE 2.534.391). Moreover, diphosphonic acid esters having pesticide ac- tivity are disclosed in US-Pat. 3.906.062. However, no compounds described in the above mentioned patents have been reported to have intrinsic antitumor activity. DE 3.425.812 discloses 1,1-diphosphonic acid derivatives having a bis[ (haloalkyl)amino]phenyl resi- due as agents useful for the treatment of bone tumours. In fact, beside having the bone tropism characteristic of diphosphonic acids , they also have the typical cyto- toxic activity of molecules bearing dialkylating func¬ tions. It has now been found that diphosphonic acid deri¬ vatives characterized by the presence of a bond which can be physiologically hydrolyzed, connecting the dipho.? .onic derivative with a dialkylating residue, have, _ompared with the above cited compounds, advanta- geous antitumor and antimetastatic properties, which could not be predicted on the basis of their chemical structure and of the presumed bioconversion thereof into the separate components (diphosphonic derivative and alkylating derivative) . The present invention, therefore, provides com¬ pounds of formula (I) wherein : R, and R„, which can be the same or different, are hy- drogen or C.-C. alkyl; (A) is hydrogen, halogen (chlorine, bromine or iodine), hydroxy, straight or branched C--C,-, alkyl; (B) is a covalent bond, a straight or branched C,-Cg alkylene or, together with the adjacent nitrogen atom, a group of formula -N ( R-, is optionally gem 1,1; 1,2; 1,3 or 1,4 disubstituted; an ortho, meta or para -substituted aralkyl of formula -N(R 3 )— _ —(CH 2 ) -; an alkylene chain containing at least one hetero-ato of formula -[CH(CH 3 )] -(CH-,) nl -X- (CH 2 ) -; m is the integer 5 or 6; n and n, are an inte¬ ger from 1 to 5; p is zero or 1 and X is O, S, N-CH-, or I J the ureido group -NH-C0-NH-; R-. is hydrogen, straight or branched C,-C 8 alkyl, C 3 -C 8 cycloalkyl, benzyl, phe- nyl or p-methoxybenzyl; (C) is straight or branched C ^ -C-. alkyl, phenyl, an aralkyl chain of formula (CH 2 ) n - in which n is as above defined; R. is hydrogen, straight or branched C,-C. alkyl, or it is a group of formula in which R_ and R R , which are the same or different, are hydrogen, straight or branched C--C fi alkyl, phenyl, benzyl, p-methoxybenzyl, or one of R 7 and R fi is as above defined and the other one is a group of formula in which R_ is hydrogen, straight or branched C,-C 4 alkyl, phenyl, benzyl, p-methoxyphenyl, straight or branched C.-C. alkoxy, halo-C,-C.-alkoxy; R 5 and R g are haloethyl (2-chloroethyl, 2-bromoethyl, 2-iodoethyl) or Rr and R g , together with the nitrogen atom to which they are bound, are a 1-aziridinyl residue of formula The present invention also includes racemic and diastereoisomeric mixtures as well as the single enan- tiomers and diastereoisomers of the compounds of formula (I). The present invention further comprises the phar¬ maceutically acceptable salts of the compounds of formula (I), for example with inorganic bases, such as the salts with alkali metals (e.g. sodium or potassium) or with alkaline-earth metals (e.g. calcium or ma¬ gnesium), or the ammonium salts; the salts with organic bases, such as methylamine, ethylamine, propylamine. isopropylamine, butylamine, t-butylamine, dimethylami- ne, diethylamine, diethanolamine, trimethylamine, trie- thyla ine, piperidine, pyridine, picoline, dicyclo- hexylamine; the organic acid addition salts, such as: formate, acetate, trifluoroacetate, maleate, fu arate, tartrate, methanesulfonate, benzenesulfonate, toluene- sulfonate salts; the inorganic acid addition salts, such as hydrochloride, hydrobromide, sulphate, hydrogen sulphate, phosphate salts; or the salts with amino acids, such as aspartates, glutamates or the salts with lysine or arginine. C,-C. Alkyl includes methyl, ethyl, n-propyl, iso- propyl, n-butyl, t-butyl or isobutyl; particularly pre¬ ferred are methyl and ethyl. The C,-C, 2 alkyl group can be, beside the meanings above precised for C.-C. alkyl, n-pentyl, n-hexyl, n-decyl and the like; particularly preferred are methyl and ethyl. The alkylene chain (B) is preferably -(CH„) -, wherein q is an integer from 2 to 5, -CH-(CH 7 ) - wherein r is an integer from 2 CH 3 to 5, or one of the groups of formulae wherein s is an integer from 1 to 4. R 3 is preferably hydrogen or methyl, whilst (C) is preferably benzyl or When (C) is benzyl, R. is preferably a group of formula wherein R_ and R g are preferably hydrogen, or one of them is R g -C0- wherein R g is hydrogen, methyl, tert-bu- toxy , trichloromethox , (2,2,2) richloroethoxy, benzy- loxy, ethoxy. When (C) is a residue of formula — θ)-( CH 2'2"' R 4 is preferably hydrogen. R 5 and Rg are preferably 2-chloroethyl. The compounds according to formula (I) are prepared by reaction of a compound of formula (II) wherein R ? , R, and (C) are as above defined and R\ is the same as R. or a group which can be transformed into R. by removal of any protecting groups present, T is hydroxy or a group which activates the carboxylic function with a compound of formula (III) wherein R, , R 2 , R 3 , (A) and (B). are as above defined, to give a compound of formula (la) wherein R-, R_, R,, R. , R 5 , R g , (C) , (B) and (A) are as above defined, which compound can in its turn be tran- sformed into a compound of formula (I) by means of well-known reactions for the selective elimination of the protecting groups, or by alkylating or acylating the amino groups, and the like. When in the reaction of compounds (III) with com- pounds (II) the latter are used in form of carboxylic acids (T = OH) , the reaction is generally carried out in the presence of a condensing agent such as N,N'- dicyclohexylcarbodiimide, N-cyclohexyl-N*-morpholino- ethylcarbodiimide, N-ethyl- '-(3-dimethylaminopropyl)- carbodiimide; N, '-carbonyl-bis(imidazole) ; phosphorus oxychloride; phosphorus trichloride; thionyl chloride; oxalyl chloride; ethyl chloroformate; isobutyl chloro- for ate; morpholinoethylisonitrile and the like. Exam¬ ples of activated forms of carboxylic acids, according to formula (III), are acid halides, symmetrical or mixed anhydrides (e.g. with methanesulfonic, acetic, isobutyric, pivalic, trichloroacetic acids); activated amides (e.g. with imidazole or triazole) ; acylazide; activated esters (e.g. p-nitrophenyl ester, methoxy- methyl ester, 2,4-dinitrophenyl ester, penta- chlorophenyl ester, hydroxysuccinimido ester, 1-hy- droxy-2-(lH)-pyridone ester, 1-hydroxybenzotriazole ester) and the like. The reaction can be carried out in the presence of an inorganic base, such as an alkali carbonate or hydrogen carbonate, an alkaline or alka¬ line-earth hydroxide or an organic base, such as triethylamine, tributylamine, pyridine, dimethy1amino- pyridine, N-alkylmorpholine, N,N-dialkyl-aniline and the like. The reaction can be carried out at a temperature ranging from -40 β C to the reflux temperature of the solvent, preferably using a slight molar excess of com¬ pound (II) to compound (III), in a solvent such as wa- ter, pyridine or N,N-dimethylformamide or mixtures the¬ reof. The reaction temperature preferably ranges from -10 β C to room temperature and the reaction time ranges from 1 to 48 hours, but generally the reaction is complete within 2-12 hours. Alternatively, the mixed anhydrides or the acid chlorides of the compounds of formula (II) can be reacted with the compounds of formula (III) in heterogeneous phase, in a solvent such as an ester, e.g. methyl or ethyl formates; ethyl, methyl or iso- propyl acetates; or in a halogenated solvent such as dichloromethane or chloroform, or in a straight or cy¬ clic ether such as dioxane, tetrahydrofuran, ethyl ether, tert-butylmethyl ether and mixtures thereof. Temperature conditions and reaction times are the same as above reported. Compounds of general formula (II) are known com¬ pounds, which are commercially available and/or can be prepared by means of conventional methods, such as those described in : J. Med. Chem. 2_4, 1304, (1981); CA 5JL: 8066d, (1957); BE 905,974; CA 1O4_: 141897 (1986); J. Med. Chem. 1_, 468, (1964); J. Med. Chem. 6_, 85, (1963); Cancer Chem. Rep. 5fj, 685, (1966); J. Med. Chem. 20., 16, (1977); J. Org. Chem. 2_6, 1554, (1961); J. Org. Chem. 2_6, 1674, (1961); CA 6± 10267g, (1966); J. Chem. Soc. , 2994, (1960); Biochem. Pharmacol. 11, 847, (1962); Biochem. Pharmacol. 12^ 833, (1963); CA 73: 131293c, (1970); Biochem. Pharmacol. 5_, 192, (1960). Compounds of general formula (III) are also known and/or can be prepared according to known methods. See, for instance, EP 96,931, EP 252,504, BE 903,519, DE 3,016,289, EP 224,751, DE 2,534,391, EP 197,478. If desired, compounds of general formula (I) whe¬ rein R- and R 2 are different from hydrogen, can optio- nally be transformed into the corresponding gem-dipho- sphonic acids by treatment with a molar excess of trialkylsilyl-chloride, -iodide or -bromide in a halo- genated solvent such as dichloromethane, 1,2-dichloro- ethane, 1,1,2-trichloroethane and the like. Trimethyl- silyl iodide is preferably used. The reaction times ranges from a few minutes to 72 hours; the reaction temperatures range from 0 C C to the solvent's reflux temperature; preferred reaction condi¬ tions are those according to J. Org. Chem: 2j$, 2975-78, (1963). The removal of the secondary- or primary-amine protecting groups optionally present in the compounds of general formula (I) can be carried out according to well-known techniques, particularly those used in pep- tide synthesis. The compounds of the invention have high cytotoxic activity against tumour cells, as it can be evidenced by means of "in vitro" tests carried out, for instance, according to the procedure described by M.P. Hacker, Cancer Res. 4_5, 4748, (1985). The ID 50 (i.e. the compound dose which can inhibit by 50% the growth of "in vitro" cultured murine and human tumour cells of both solid and hematic tumors) of the compounds of the invention were found to be comprised from 0.1 to 5 μg/ml of culture medium. Under these test conditions, the compounds of the invention: N-{4-[bis(2-chloro- ethy1)a ino]phenyl-(L)-alany1}-5-amino-l-hydroxypenta- ne-l,l-diphosphonic acid monohydrochloride and N-{4- [bis(2-chloroethyl)amino]phenyl-butirroyl}-4-amino-l- hydroxy-butane-l,l-diphosphonic acid have an I D cn of 0.1 μg/ml and 0.5 μg/ml, respectively, against murine leukemia L1210. "In vitro" studies on the compound of the invention N-{4-[bis(2-chloroethyl)aminojphenyl-(L)-ala¬ ny1}-4-amino-l-hydroxy-butane-l,1-diphosphonic, using different human tumor cell lines (for example fibrosarcoma HT 1080, osteosarcoma H2-OS etc.) showed ID 50 ranging from 13.6 to 16.5 μg/ml; the substance appears to be less cytotoxic than the related starting material 4-amino-l-hydroxy-l,l-diphosphonic acid, whose ID 50 range from 2.7 to 8.2 μ/ml. The same compound, when tested "in vivo" by i.v. administration (on days 2 - 6 .- 9 - 13 after tumor implantation) in rats bearing Walker B mammary carcinoma (implanted on day 0 intramuscolarly 10 cells) causes complete regression of tumor growth combined with normalization of tumor induced hypercalcemia. Under the same experimental conditions, the related starting diphosphonate (i.e. 4-amino-l- hydroxy-butane-1,1-diphosphonic acid) even if endowed with higher "in vitro" cytotoxicicy, is surprisingly ineffective in retarding tumor growth, also when administered at its maximum tolerated dosage. The compounds of the invention are characterized by a low acute toxicity and are well tolerated by the animals. The compounds of the invention have a high thera¬ peutical index, in light of the low toxicity and the effective antitumor activity thereof. Moreover, the high water-solubility of the compounds of the present invention allows the easy preparation of parenteral and oral pharmaceutical forms. The compounds of formula (I), when administered to humans and animals affected with tumours which can be treated with alkylating agents, at doses ranging from 1 2 mg to 1.2 g/m body area, can induce the regression of the above mentioned tumoral forms. The effectj.ve dosage for the compounds of the in¬ vention can be determined by the expert clinicians ac¬ cording to conventional methods. The relationship between the dosages used for various animal species and 2 those for humans (on the basis of mg/m body area) is described by Freireich, E.J. , et al., Quantitative Comparison of Toxicity of Antic^ncer Agents in Mouse, Rat, Hamster, Dog, Monkey and Man, Cancer Chemother. Rep. , ^ , n. 4, 219-244, May 1966. Nevertheless, the patient will be administered ge¬ nerally with doses from 1 to 1,200 mg/kg body weight of the compounds of the invention, with a dosage regimen which will vary depending on various factors, which are well known to the expert clinicians. Some compounds of the invention may have such an high toxicity, or such an unfavourable therapeutical index, so as to be unsuited for an antitumor treatment in the patients. Nevertheless, those parameters can ea¬ sily be determined by means of conventional toxycologi- cal tests, such as acute and subacute DL 50 in mouse. Of course, those compounds which turn out to be toxic will be avoided. The compounds of the invention will be used in the treatment of those tumours which can be treated with alkylating agents. Particularly, multiple myeloma, osteosarcoma, bone-metastasis; breast, ovarian and testis carcinomas can also be advantageously treated. Moreover, the compounds of the invention can ad¬ vantageously be used ' in the therapy of other solid and hematic neoplasias, such as lymphomas and leukemias in humans and animals, according to treatment protocols which can be easily determined by those skilled in the art. The compounds of the invention are preferably ad- ministered by the intravenous or intraarterial routes, even though other administration forms can be envisaged in particular cases. The pharmaceutical forms which can be used for pa- renteral administration include sterile aqueous solu- tions or sterile powders for the extemporary prepara¬ tion of solutions, as well as oily preparations for in- tramuscular or intraperitoneal administrations. Other useful pharmaceutical forms are syrups or similar liquid forms, as well as solid forms such as tablets, capsules and the like. The following examples illustrate the invention in more detail. EXAMPLE 1 a) A solution of bis(tert-butoxy)carbonate (450 mg) in tetrahydrofuran (THF; 4 ml) is quickly added to a solution of 4-[bis(2-chloroethyl)amino]-(L)-phenyl- alanine (300 mg) in THF (10 ml) and IN NaOH (1 ml). The resulting mixture is stirred at room tempera¬ ture for 2 hours, adjusting pH to about 9 by repeated additions of IN NaOH. Then the solvent is removed under vacuum and the residue is partitioned between water (3 ml) and ethyl ether (5 ml) . The organic phase is discharged and the aqueous one is acidified with HC1 and repeatedly extracted with ethyl ether. The combined organic extracts are dried over Ma 2 SO. and evaporated to dryness under vacuum, to obtain a foamy residue of '-(tert-butoxycarbonyl)-4- [bis(2-chloroethyl)amino]-(L)phenylalanine (290 mg) . NMR (CDC1 3 , TMS) S = 1.4 (s, 9H) ; 3.1 (m, 2H) ; 3.65 (m 8H); 4.55 (m, 1H) ; 4.99 (d, 1H) ; 6.62 (d, 2H) ; 7.12 (d, 2H); 8.1 (s, 1H) . b) N-Hydroxysuccinimide (173 mg) and morpholino- ethylisonitrile (0.133 ml) are added to a solution of the compound from step a) (290 mg). The mixture is stirred at room temperature for one hour, then concen- trated to dryness under vacuum. The residue is taken up into 2N HC1 (5 ml) and repeatedly extracted with ethyl ether (3 x 5 ml). The organic phases are collected, washed with 5% aqueous NaHC0 3 , with water (5 ml) and then dried over Na_SO.. Upon removing the solvent under vacuum, an oily residue is obtained which is crystalli- zed from an ethyl ether - isopropyl ether mixture to give 260 mg of N'-(tert-butoxycarbonyl)-4-[bis(2-chlo- roethy1)amino]-(L)-phenylalanine hydroxysuccinimido ester, melting point 140-143°C; NMR (TMS, CDC1 3 ) S " = 1.45 (s, 9H) ; 2.85 (s, 4H) ; 3.15 ( , 2H); 3.69 (m, 8H) ; 4.9 (m, IH) ; 6.61 (d, 2H) ; 7,15 (d, 2H). c) A solution of the obtained activated ester (50 mg) in dimethylformamide (DMF, 2.5 ml) is slowly drop¬ ped into a solution of 5-amino-l-hydroxypentane-l,1- diphosphonic acid (21.8 mg) dissolved in a mixture of water (2.5 ml), IN NaOH (0.331 ml) and DMF (2 ml). DMF is added to the reaction mixture simultaneously to the ester solution, until complete dissolution of the rea¬ gents is obtained (2.5 ml). The obtained slightly opalescent solution is centrifuged; the supernatant liquid is collected and concentrated under reduced pressure. The residue is di¬ luted with DMF (5 ml) to separate a precipitate which is collected by centrifugation and washed with ethyl acetate, yielding 35 mg of N-{N'-(tert-butoxycarbonyl)- 4-[bis(2-chloroethy1)amino]-(L)-phenylalanyl}-5-amino- ..- ydroxypentane-l,l-diphosphonic acid trisodium salt, m.p. >250°C. NMR (TMS, D 2 0): <_T l.3 (s, 9H) ; 1.4-1.7 (m, 4H) ; 1.8- 2.05 (m, 2H); 2.62-3.1 (m, 2H) ; 3.2 (t, 2H) ; 3.75 (s, 8H); 4.2 (t, IH) ; 6.85 (d, 2H) ; 7.15 (d, 2H) . EXAMPLE 2 Following the same procedure as described in Example 1, by reacting N'-(tertbutoxycarbonyl)-4-[bis- (2-chloroethy1)amino]-(L)-phenylalanine hydroxysuc- cinimido ester with the appropriate aminoalkyl-1-hydro- xy-l,l-diphosphonic acids, the following compounds are obtained: N-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)ami¬ no]-(L)-phenylalany1}-4-amino-l-hydroxybutane-l,1-di- phosphonic acid (trisodium salt) , NMR (D 2 0, TMS): S " = 1.3 (s, 9H) ; 1.6 (m, 2H) , 1.92 (m, 2H) ; 2.62 ÷ 3.1 (m, 2H) ; 3.2 (t, 2H) ; 3.75 (s, 8H) ; 4.2 (t, IH) ; 6.8 (d, 2H) ; 7.15 (d, 2H) ; N-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)ami- no]-(L)-phenylalanyl}-3-amino-l-hydroxypropane-1,1-di- phosphonic acid (trisodium salt) , NMR (D 2 0, TMS): S * = 1.32 (s, 9H) ; 2.1 (m, 2H) ; 2.65 ■_- 3.1 (m, 2H) ; 3.21 (t, 2H) ; 3.75 (s, 8H) ; 4.2 (t, IH) ; 6.8 (d, 2H) ; 7.15 (d, 2H) ; N-{N' -(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)ami¬ no]-(L)-phenylalanyl}-6-amino-l-hydroxyhexane-1,1-di- phosphonic acid (trisodium salt) , NMR (D 2 0, TMS): S = 1.3 (s, 9H) ; 1.5 (m, 2H) ; 1.6 (m, 4H); 1.9 (m, 2H) ; 2.65 ÷ 3.1 (m, 2H) ; 3.20 (t, 2H) ; 3.75 (s, 8H); 4.2 (t, IH) ; 6.8 (d, 2H) ; 7.15 (d 7 2H) ; N-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)ami¬ no) ]-(L)-phenylalanyl}-4-amino-l-hydroxypentane-l,l-di- phosphonic acid (trisodium salt) , NMR (D 2 0, TMS): ,_ = 1.32 (s, 9H) ; 1.4 (d, 3H) ; 1.7 (m, 2H) ; 1.9 (m, 2H) ; 2.6 ÷ 3.05 ( , 2H) ; 3.15 (d, IH) ; 3.75 (s, 8H); 4.2 (t, IH) ; 6.85 (d, 2H) ; 7.15 (d, 2H) ; N-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)ami¬ no]-(L)-phenylalany1}-3-(2-aminocyclopent-l-y1)-1-hy- droxypropane-l,l-diphosphonic acid (trisodium salt), NMR (D 2 0, TMS): cT= 1.3 (s, 9H) ; 1.42*1-75 (m, 9H) ; 1.9 (m, 2H); 2.62 ÷ 3.1 (m, 2H) ; 3.3 (m, IH) ; 3.75 (s, 8H);4.2 (t, 1H);6.85 (d, 2H) ; 7.15 (d, 2H) ; N-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethy1)ami¬ no]-(L)-phenylalanyl}-4-amino-4,4-pentamethylene-1-hy- droxybutane-l,l-diphosphonic acid (trisodium salt), NMR (D 2 0, TMS): S ~ = 1.3 (s, 9H) : 1.55 (m, 10H) ; 1.6 (m, 2H) ; 1.9 ( , 2H) ; 2.62 ÷ 3.1 (m. 2H) ; 3.75 (s, 8H) ; 4.2 (t, IH); 6.85 (d, 2H);7.15 (d, 2H) ; N-methyl-N-{N » -(tert-butoxycarbonyl)-4-[bis(2-chloro- ethy1)amino]-(L)-phenylalanyl}-5-amino-l-hydroxypenta- ne-l,l-diphosphonic acid (trisodium salt), NMR (D 2 0, TMS): _> = 1.3 (s, 9H) ; 1.55 ( , 4H) ; 1.9 (m, 2H); 2. ' 1 (s, 3H); 2.62 ÷ 3.1 (m, 2H) ; 3.2 (t, 2H) ; 3.75 (s, 8H); 4.2 (t, IH) ; 6.85 (d, 2H) ; 7.15 (d, 2H) ; N-{ '-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)ami- no) ]-(L)-phenylalanyl}-4-amino-4,4-tetramethylene-l-hy- droxybutane-l,l-diphosphonic acid (trisodium salt), NMR (D 2 0, TMS): £ " = 1.3 (s, 9H) ; 1.5 (m, 8H) ; 1.6 (m, 2H); 1.9 (m, 2H) ; 2.62 ÷ 3.1 (m, 2H) ; 3.75 (s, 8H) ; 4.2 (t, IH); 6.85 (d, 2H) ; 7.15 (d, 2H) . EXAMPLE 3 Following the same procedure as described in Exam¬ ple lb), by reacting 4-[4-[bis(2-chloroethyl)amino]- phenyl]butyric acid (300 mg) with N-hy roxysuccinimide (245 mg) and N-morpholinoethylisonitrile (0.18 ml), 4- [4-[bis(2-chloroethyl)amino]phenyl]butyric acid N-hy- droxysuccinimido ester (360 mg) is prepared, m.p. 80- 82 °C . Following the same procedure as described in Exam¬ ple lc), the above hydroxysuccinimido ester (112 mg) is reacted with 5-amino-l-hydroxypentane-l,l-diphosphonic acid (61.2 mg) to obtain N-{4-[4-[bis(2-chloro- ethy1)amino]pheny1]butyroy1}-5-amino-l-hydroxypentane- 1,1-diphosphonic acid trisodium salt (80 mg) , m.p. >260°C. NMR (TMS, D 2 0) : S = 1.4-1.7 (m, 4H) ; 1.8 ÷ 2.05 (m, 4H); 2.15 (t, 2H) ; 2.55 (t, 2H) ; 3.18 (t, 2H) ; 3.75 (s, 8H) ; 6.8 (d, 2H) ; 7.15 (d, 2H) . HPLC : Partisfere R C.g, 150 x 4.6 mm; 0.025 M sodium heptanesulfonate in water/acetonitrile/dioxane 70:20:10, pH -.2.5 with H-.P0 4 ; flow : 1.3 ml/min; 7. = 255 nm. Retention time : 6.86'. EXAMPLE 4 Following the procedure described in Example 3, by reacting N-4-[4-[bis(2-chloroethy1)amino]phenyl]butyric acid hydroxysuccinimido ester with the appropriate ami- noalkyl-l-hydroxy-l,l-diphosphonic acids, the following compounds are obtained: N-{4-[4-[bis(2-chloroethyl)amino]phenyl]butyroyl}-4- amino-l-hydroxybutane-l,l-diphosphonic acid (trisodium salt) , NMR (TMS, D 2 0) : S = 1.7 ÷ 2.05 (m, 6H) ; 2.25 (t, 2H) ; 2.55 (t, 2H); 3.18 (t, 2H) ; 3.75 (s, 8H) ; 6.85 (d, 2H) ; 7.2 (d, 2H); HPLC : Partisfere C, g , 150 x 4.6 mm; 0.025 M sodium heptanesulfonate in water/acetonitrile/dioxane 70:20:10, pH _. 2.5 with H 3 P0 4 ; flow 1.3 ml/min.; ^ = 255 nm. Retention time : 5.96 1 . N-{4-[4-[bis(2-chloroethyl)amino]phenyl]butyroy1}-3- amino-l-hydroxypropane-l,l-diphosphonic acid (trisodium salt) , NMR (TMS, D 2 0) : ζ = 1.8 ÷ 2.05 (m, 4H) ; 2.25 (t, 2H) ; 2.55 (t, 2H);3.18 (t, 2H);3.75 (s, 8H) ; 6.8 (d, 2H) ; 7.15 (d, 2H); N-[4-[4-[bis(2-chloroethyl)amino]pheny1]butyroyl}-6- amino-l-hydroxyhexane-1,1-diphosphonic acid (trisodium salt) , NMR (TMS, D 2 0) : 6 - 1.4 (m, 2H) ; 1.6 (m, 4H) ; 1.9 (m, 4H) ; 2.25 (t, 2H) ; 2.55 (t, 2H);3.18 (t, 2H);3.75 (s, 8H); 6.8 (d, 2H) ; 7.15 (d, 2H) ; N-{4-[4-[bis(2-chloroethy1)amino]phenyl]butyroy1}-4- amino-l-hydroxypentane-1,1-diphosphonic acid (trisodium salt) , NMR (TMS, D 2 0) : cT _= 1.4 (d, 3H) ; 1.7 (m, 2H) ; 1.9 (m, 4H); 2.25 (t, 2H) ; 2.55 (t, 2H);3.18 (d, 1H);3.75 (s, 8H) ; 6. ' 85 (d, 2H) ; 7.2 (d, 2H) ; N-{4-[4-[bis(2-chloroethy1)amino]phenyl]butyroy1}-3-(2- aminocyclopent-1-y1)-1-hydroxypropane-1,1-diphosphonic acid (trisodium salt), NMR (TMS, D 2 0): S - 1.42 ÷ 1.75 (m, 9H) ; 1.9 (m, 4H) ; 2.25 (t, 2H) ; 2.55 (t, 2H);3.2 (m, 1H);3.75 (s, 8H) ; 6.8 (d, 2H); 7.15 (d, 2H) ; N-{4-[4-[bis(2-chloroethy1)amino]phenyl]buty oyl}-4- amino-4,4-pentamethylene-l-hydroxybutane-l,1-dipho- sphonic acid (trisodium salt), NMR (TMS, D 2 0): S = 1.55 (m, 10H) ; 1.6 (m, 2H) ; 1.9 (m, 4H); 2.15 (t, 2H) ; 2.55 (t, 2H);3.75 (s, 8H) ; 6.8 (d, 2H); 7.15 (d, 2H) ; N-methy1-N-{4-[4-[bis(2-chloroethy1) mino]phenyl]bu- tyroyl}-5-amino-l-hydroxypentane-l,1-diphosphonic acid (trisodium salt), NMR (TMS, D 2 0): ζ = 1.55 (m, 4H) ; 1.9 (m, 4H) ; 2.1 (s, 3H) ; 2.15 (t, 2H) ; 2.55 (t, 2H) ; 3.18 (t, 2H) ; 3.75 (s, 8H); 6.8 (d, 2H) ; 7.15 (d, 2H) ; N-{4-[4-[bis(2-chloroethyl)amino]phenyl]butyroyl}-4- amino-4,4-tetramethylene-l-hydroxybutane-1,1-dipho- sphonic acid (trisodium salt) , NMR (TMS, D 2 0): 6 " = 1.5 (m, 8H) ; 1.7 ÷ 2.05 (m, 6H) ; 2.15 (t, 2H); 2.55 (t, 2H) ; 3.75 (s, 8H) ; 6.8 (d, 2H) ; 7.15 (d, 2H). EXAMPLE 5 A solution of N-{N' -(tert-butoxycarbonyl)-4- [bis (2-chloroethy1)amino]-(L)-phenylalany1}-5-amino-l- hydroxypentane-l,l-diphosphonic acid trisodium salt (35 mg) in methanol saturated with hydrochloric acid (2 ml) is heated to 40-50 c C for 4 hours, then the solution is concentrated to reduced volume and the precipitated sodium chloride is filtered. The filtrate is evaporated to dryness and the residue is triturated with acetone and filtered. After recrystallization from ethanol/ethyl ether, N-{4-[bis(2-chloroethy1)amino]- (L)-phenylalanyl}-5-amino-l-hydroxypentane-l,1-dipho- sphonic acid monohydrochloride (25 mg) is obtained, m.p. > 260°C, [*]_. = + 7.9 (c = 2, methanol), NMR (TMS, D 2 0): 1.25 ÷ 1.40 ( , 2H) , 1.42 ÷ 1.72 (m, 2H) ; 1.8 i 2.06 (m, 2H); 2.9 ÷ 3.2 (m, 4H) ; 3.65 (t, 4H) ; 4.0 (t, 4H); 4.1 (t, IH) ; 7.35 (s, 4H) . HPLC : Partisfere R C, 0/ 150 x 4.6 mm; 0.025 M sodium heptanesulfonate in water/acetonitrile/dioxane 70:20:10, pH ._. 2.5 with H 3 P0. ; flow 1.3 ml/min.; ~\ = 255 nm. Retention time : 4.61'. EXAMPLE 6 Following the same procedure as described in Exam¬ ple 5, by reacting the trisodium salts of the acids de- scribed in Example 2, the following acid monohydrochlo- ride salts are obtained : N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-ami- no-l-hydroxybutane-l,l-diphosphonic acid: NMR ( D 2 0 ' TMS): ζ = 1.6 (m, 2H) ; 1.92 (m, 2H) ; 3.15 (m, 4H) ; 3.65 (t, 4H); 4.0 (t, 4H) ; 4.1 (m, IH) ; 7.35 (s, 4H) ; N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-3-ami- no-l-hydroxypropane-l,l-diphosphonic acid: NMR ( D 2 0 ' TMS): - 2,1 (m, 2H) ; 3,15 (m, 4H) ; 3,65 (t, 4H) ; 4,0 (t, 4H); 4,1 ( , IH) ; 7,35 (s, 4H) ; N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-6- amino-l-hydroxyhexane-l,l-diphosphonic acid: NMR ( D 2 0 ' TMS): £ = 1.5 (m, 2H); 1.6 (m, 4H) ; 1.9 (m, 2H) ; 3.15 (m, 4H) ; 3.65 (t, 4H) ; 4.0 (t, 4H) ; 4.1 (m, IH) ; 7.35 (s, 4H); N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-ami- no-l-hydroxypentane-l,l-diphosphonic acid: NMR (D O, TMS): ζ = 1.4 (d, 3H) ; 1.7 ( , 2H) ; 1.9 (m, 2H) ; 3.15 (m, 3H); 3.65 (t, 4H) ; 4.0 (t, 4H) ; 4.1 (m, IH) ; 7.35 (s, 4H); N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-3-(2- aminocyclopent-1-yl)-1-hydroxypropane-l,1-diphosphonic acid: NMR (D 2 0, TMS) : <_T = 1.5 (m, 8H) ; 1.6 (m, 2H) ; 1.9 (m, 2H); 3.15 (t, 2H) ; 3.65 (t, 4H) ; 4.0 (t, 4H) ; 4.1 ( , IH) ; 7.35 (s, 4H) ; N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-ami- no-4,4-ρentamethylene-l-hydroxybutane-l,1-diphosphonic acid: NMR (D 2 0 , TMS ) : cT = 1.45 (m, 10H) ; 1. 6 (m, 2H) ; 1. 9 (m, 2H) ; 3 .15 (t, 2H) ; 3. 65 (t , 4H) ; 4.0 (t , 4H) ; 4 . 1 (m , IH) ; 7 .35 ( s , 4H) ; N-methy1-N-{4-[bis(2-chloroethyl)amino]-(L)-phenylala- nyl}-5-amino-l-hydroxypentane-l,l-diphosphonic acid: NMR (D 2 0, TMS): cT = 1.55 (m, 4H) ; 1.9 (m, 2H) ; 2.1 (s, 3H) ; 3.05 (t, 2H) ; 3.15 (t, 2H) ; 3.65 (t, 4H) ; 4.0 (t, 4H) ; 4.1 (m, IH) ; 7.35 (s, 4H) ; N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-ami- no-4,4-tetramethylene-l-hydroxybutane-l,1-diphosphonic acid: NMR (D 2 0, TMS): S = 1.5 (m, 8H) ; 1.6 (m, 2H) ; 1.9 (m, 2H); 3.15 (t, 2H) ; 3.65 (t, 4H) ; 4.0 (t, 4H);4.1 ( , IH) ; 7.35 (s, 4H) . EXAMPLE 7 A solution of 4-[bis(2-chloroethyl)amino]-(L)- phenylalanine (150 mg) in formic acid (1.82 ml) and acetic ' anhydride (0.64 ml) is stirred at room tempera¬ ture for 3 hours. The reaction mixture is concentrated to reduced volume under reduced pressure and partitio- ned between water (3 ml) and ethyl acetate (2 x 50 ml). The organic phase is dried over sodium sulphate and the solvent is evaporated under vacuum, to give N-formyl-4- [bis(2-chloroethyl)amino]-(L)-phenylalanine (160 mg) as a yellow foam, [σ.] = +67° (C = 2, ethanol) . NMR (CDC1 3 , TMS): = 2.9 ÷ 3.1 (m, 2H) ; 3.6 (m, 8H) ; 4.68 ÷ 4.79 ( , IH) ; 6.5 (m, 2H+IH) ; 6.69 (d, 2H) ; 8.1 (s, IH). Following the same procedure as described in Exam¬ ple lb) and lc), this compound is transformed into the hydroxysuccinimido ester (50 mg) and then is reacted with 4-amino-l-hydroxybutane-l,l-diphosphonic acid (24.1 mg) , to obtain N-{N'-formyl-4-[bis(2-chloro- ethyl)amino]-(L)-phenylalanyl}-4-amino-l-hydroxybutane- 1,1-diphosphonic acid trisodium salt (32 mg) ; [A] D = +7.5° (c = 2, water) NMR (D 2 0, TMS): £ ~ = 1.7 ÷ 2.0 (m, 4H) ; 2.9 ÷ 3.1 (m, 2H); 3.2 ( , 2H) ; 3.75 (s, 8H) ; 4.55 (t, IH) ; 6.85 (d, 2H); 7.15 (d, 2H) ; 8.05 (s, IH) . HPLC : Partisfere R C- 8 , 150 x 4.6 mm; 0.025 M sodium heptanesulfonate in water/acetonitrile/dioxane 70:20:10, pH - 2.5 with H.,P0 4 ; flow 1.3 ml/min.; N. = 255 nm. Retention time : 2.9'. EXAMPLE 8 Following the same procedure as described in Exam¬ ple lc), by reacting N-formyl-4-[bis(2-chloroethyl)- amino]-(L)-phenylalanine hydroxysuccinimido ester with the appropriate aminoalkyl-l-hydroxy-l,l-diphosphonic acids, the following compounds are obtained : - N-(N'-formyl-4-[bis(2-chloroethyl)amino]-(L)-pheny1- alanyl}-5-amino-l-hydroxypentane-l,l-diphosphonic acid trisodium salt; NMR (D 2 0, TMS): ζ = 1.4 ÷ 1.6 (m, 4H) ; 1.92 (m, 2H) ; 2.9 ÷ 3.1 (m, 2H); 3.2 (m, 2H) ; 3.75 (s, 8H) ; 4.55 (t, IH) ; 6.85 (d, 2H);7.15 (d, 2H) ; 8.05 (s, IH) ; - N-{N'-formyl-[4-bis(2-chloroethyl)amino]-( )-pheny1- alanyl}-3-amino-l-hydroxypropane-l,l-diphosphonic acid trisodium salt; NMR (D 2 0, TMS): - 2,1 (m, 2H) ;.2,9 ÷ 3,1 (m, 2H) ; 3,2 (m, 2H); 3,75 (s, 8H) ; 4,55 (t, IH) ; 6,85 (d, 2H) ; 7,15 (d, 2H); 8,05 (s, IH) ; - N-{N'-formyl-4-[bis(2-chloroethyl)amino]-(L)-phenyl- alanyl}-6-amino-l-hydroxyhexane-l,1-diphosphonic acid trisodium salt; NMR (D 2 0, TMS): = 1.5 (m, 2H) ; 1.6 (m, 4H) ; 1.9 (m, 2H); 2.9 ÷ 3.1 (m, 2H) ; 3.2 (m, 2H) ; 3.75 (s, 8H) ; 4.2 (t, IH); 6.85 (d, 2H) ; 7.15 (d, 2H) ; 8.05 (ε, IH) ; - N-{N'-formyl-4-[bis(2-chloroethyl)amino]-(L)-pheny1- alanyl}-4-amino-l-hydroxypentane-l,l-diphosphonic acid trisodium salt; NMR (D 2 0, TMS): = 1.4 (d, 3H) ; 1.7 (m, 2H) ; 1.9 (m, 2H); 2.9 ÷ 3.1 ( , 2H) ; 3.2 (m, 2H) ; 3.75 (s, 8H) ; 4.55 (t, IH); 6.85 (d, 2H) ; 7.15 (d, 2H) ; 8.05 (s, IH) ; - N-{N'-formy1-4-[bis(2-chloroethy1)amino]-(L)-phenyl¬ alanyl}-3-(2-aminocyclopent-l-yl)-1-hydroxypropane-l,1- diphosphonic acid trisodium salt; NMR (D 2 0, TMS): £T = 1.42 ÷ 1.75 (m, 9H) : 1.9 (m, 2H) ; 2.9 ÷ 3.1 (m, 2H) ; 3.3 (m, IH) ; 3.75 (s, 8H) ; 4.55 (t, IH) ; 6.85 (d, 2H) ; 7.15 (d, 2H) ; 8.05 (s, IH) ; - N-{N'-formy1-4-[bis(2-chloroethyl)amino]-(L)-pheny1- alanyl}-4-amino-4 ,4-tetramethylene-l-hydroxybutane-l,l- diphosphonic acid trisodium salt; NMR (D 2 0, TMS): _T = 1.5 (m, 8H) ; 1.6 (m, 2H) ; 1.9 (m, 2H) ; 2.9 ÷ 3.1 (m, 2H) ; 3.75 (s, 8H) ; 4.55 (t, IH) ; 6.85 (d, 2H) ; 7.15 (d, 2H) ; 8.05 (s, IH) ; - N-{N'-formy1-4-[bis(2-chloroethyl)amino]-(L)-phenyl¬ alanyl}-4-amino-4 ,4-pentamethylene-l-hydroxybutane-1,1- diphosphonic acid trisodium salt; NMR (D 2 0, TMS): cT = 1.45 (m, 10H) ; 1.6 (m, 2H) ; 1.9 (m, 2H); 2.9 ÷ 3.1 (m, 2H) ; 3.75 (s, 8H) ; 4.55 (t, IH) ; 6.85 (d, 2H); 7.15 (d, 2H) ; 8.05 (s, IH) ; - N-methy1-N-[N'-formy1-4-[bis(2-chloroethyl)amino]- (L)-phenylalanyl}-5-amino-l-hydroxypentane-1,1-dipho- sphonic acid trisodium salt; NMR (D 2 0, TMS): cT = 1.55 ( m . 4H) ; 1.9 (m, 2H) ; 2.1 (s, 3H); 2.9 -r 3.1 (m, 2H) ; 3.2 ( , 2H) ; 3.75 (s, 8H) ; 4.55 (t, IH); 6.85 (d, 2H) ; 7.15 (d, 2H) ; 8.05 (s, IH) . EXAMPLE 9 A solution of N-{N'-formyl-4-[bis(2-chloro- eth 1)amino]-(L) henylalanyl}-4-amino-l-hydroxybutane- 1,1-diphosphonic acid trisodium salt (32 mg) in metha¬ nol saturated with hydrochloric acid (1 ml) is heated to 40-50°C for 4 hours, then the solution is concentra- ted to reduced volume and the precipitated sodium chlo¬ ride is filtered. The filtrate is evaporated to dryness and the residue is triturated with acetone and filte¬ red. After recrystallization from ethanol/ethyl ether, N-{ -[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4- amino-l-hydroxybutane-l,l-diphosphonic acid monohydro- chloride (25 mg) is obtained, m.p. > 260°C; [ Λ _ D = + 7.8 (c ' = 2, methanol). NMR (D 2 0, TMS): " = 1.6 (m, 2H) ; 1.92 (m, 2H) ; 3.15 (m, 4H); 3.65 (t, 4H) ; 4.0 (t, 4H) ; 4.1 (m, IH) ; 7.35 (s, 4H). HPLC : Partisfere R C.g, 150 x 4.6 mm; 0.025 M sodium heptanesulfonate in water/acetonitrile/dioxane 70:-20:10, pH - 2.5 with H 3 P0 4 ; flow 1.3 ml/min.; ? = 255 nm. Retention time : 4.24'. EXAMPLE 10 A solution of 4-[4-[bis(2-chloroethyl)amino]phe- nyl]bu.yric acid N-hydroxysuccinimido ester (73 mg) in DMF (1 ml) is dropped at room temperature into a stir¬ red solution of 4-aminobutane-l,l-diphosphonic acid te- traethyl ester (52 mg) in DMF/water 5/1 (0.6 ml). After 3 hours, the mixture is concentrated under reduced pressure and partitioned between a 5% sodium hydrogen carbonate solution (5 ml) and ethyl acetate (5 ml) . After separation of the organic phase, the aqueous phase is further extracted with ethyl acetate (2 x 5 ml) and discarded; the combined organic extracts are dried over sodium sulphate and solvent is evaporated off under reduced pressure, to yield N-[4-[4-[bis(2- chloroethy1)amino]phenyl]butyroyl]-4-aminobutane-1,1- diphosphonic acid tetraethyl ester (90 mg) as a clear oil; NMR (CDC1 3 , TMS): S ~ = 1.35 (t, 12H) ; 1.90 (t, 2H) ; 2.2 (t, 2H) ; 2.0 ÷ 2.5 (m, 5H) ; 2.55 (t, 2H) ; 3.45 (q, 2H) ; 3.65 ( , 8H); 4.20 (m, 8H) ; 6.51 (t, IH) ; 6.61 (d, 2H) ; 7.1 (d, 2H). EXAMPLE 11 Under a nitrogen atmosphere, a solution of iodo- trimethylsilane (92 μl) in anhydrous dichloromethane (1 ml) is dropped at 0 β C into a solution of N-{4-[4- [bis (2-chloroethy1)amino] henylbutyroyl}-4-aminobutane- 1,1-diphosphonic acid tetraethyl ester (72 mg) in anhy¬ drous dichloromethane (1 ml). The mixture is stirred for 2 hours at 0°C, then it is allowed to room tempera¬ ture and subsequently treated with methanol (1 ml). Af¬ ter 15 minutes, the solvent is evaporated off under reduced pressure and the residue is dissolved in water (2 ml), treated with IN NaOH (0.228 ml) and extracted with ethyl acetate (2 x 2 ml). The organic extracts are discarded. The aqueous phase is diluted with DMF to yield a white solid which is recovered by centri- fugation, to give N-{4-[4-[bis(2-chloro- ethy1)amino]phenyl]butyroyl}-4-aminobutane-l,1-dipho- sphonic acid disodiu salt (45 mg); NMR (D 2 0, TMS): % = 1.9 (m, 6H) ; 2.25 (t, 2H) ; 2.55 (t, 2H); 3.38 (m, 2H) ; 3.72 (s, 8H) ; 6.85 (d, 2H) ; 7.2 (d, 2H). EXAMPLE 12 Following the same procedure as described in Exam¬ ple 10, N-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)- amino]-(L)-phenylalanine-N-hydroxysuccinimido ester (87,5 mg) is reacted with 4-aminobutane-l,l-diphospho- nic acid tetraethyl ester (50 mg) to obtain N-{N'- (tert-butoxycarbonyl)-4-[bis(2-chloroethyl)amino]-(L)- phenylalanyl}-4-aminobutane-l,l-diphosρhonic acid te¬ traethyl ester (87 mg) as a clear oil. NMR (CDC1 3 , TMS): ό " = 1.38 (m, 21H) ; 2.2 ( , 5H) ; 3.45 (m, 4H) ; 3.65 (m, 8H) ; 4.18 (m, 8H) ; 5.05 (m, IH) ; 6.51 (t, IH); 6.61 (d, 2H); 7.1 (d, 2H) . EXAMPLE 13 A solution of N-{N'-(tert-butoxycarbonyl)-4-[bis- (2-chloroethyl)amino]-( )-phenylalanyl}-4-aminobutane- 1,1-diphosphonic acid tetraethyl ester (59 mg) in 10% HC1 (1 ml) is heated to 45°C for one hour, then evapo¬ rated to dryness under reduced pressure. The residue is suspended in ethyl ether (2 ml) and recovered by cen- trifugation, to give N-{ [4-[bis(2-chloroethyl)amino]- (L)-phenylalanyl}-4-aminobutane-l,l-diphosphonic acid tetraethyl ester dihydrochloride (45 mg) ; NMP (TMS, D 2 0): ζ = 1.22 (t, 12H) ; 1.95 (m, 4H) ; 3.2 ( , 2H); 3.4 ( , 2H) ; 3.7 (t, 4H) ; 3.95 (m, 12H) ; 4.1 (t, IH); 7.38 (s, 4H) . EXAMPLE 14 Under a nitrogen atmosphere, a solution of iodo- trimethylsilane (134 μl) in anhydrous dichloromethane (1 ml) is dropped into a stirred solution of N-{N'- (tert-butoxycarbonyl)-4-[bis(2-chloroethyl) mino]-(L)- phenylalanyl}-4-aminobutane-l,l-diphosphonic acid te- traethyl ester (93 mg) in anhydrous dichloromethane (1 ml), cooled to 0 C C. The mixture is stirred for 2 hours, allowing it to warm to room temperature, then it is treated with methanol (1 ml) . After 15 minutes, solvent is evapora- ted off under reduced pressure, the residue is dissol¬ ved in water, treated with IN NaOH (0.254 ml) and ex¬ tracted with ethyl acetate (2 x 3 ml). The organic ex¬ tracts are discarded. The aqueous phase is diluted with DMF to precipitate a solid, which is recovered by cen- trif gation and washed with ethyl acetate. 45 mg of N-{4-[bis(2-chloroethyl)amino]-(L)-phe¬ nylalanyl}-4-aminobutane-l,l-diphosphonic acid disodium salt are obtained, NMR (D 2 0, TMS): G = 1.95 ( , 4H) ; 2.9 (m, 2H) ; 3.4 (m, 2H); 3.65 (m, IH) ; 3.75 (s, 8H) ; 6.88 (d, 2H) ; 7.18 (d, 2H) . EXAMPLE 15 Following the same procedures as described in Examples 12 and 13, using N-(tert-butoxycarbonyl)-4- [bis(2-chloroethyl)amino]-(L)-phenylalanine hydroxysuc¬ cinimido ester and the appropriate tetraalkylesters of aminoalkyl-l,l-diphosphonic acids the following com¬ pounds are prepared: - N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-3-a- minopropane-l,l-diphosphonic acid tetraethylester dihydrochloride; - N-[4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-5- aminopentane-l,l-diphosphonic acid tetraethylester dihydrochloride; - N-{4-[bis(2-chloroethyl)amino]-(L)-phen lalanyl}-6-a- minohexane-1,1-diphosphonic acid tetraethylester dihydrochloride; - N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4- aminopentane-l,l-diphosphonic acid tetraethylester dihydrochloride; - N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-3- (2-aminocyclopent-l-yl)propane-l,1-diphosphonic acid tetraethylester dihydrochloride; - N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4-a- mino-4,4-tetramethylene-butane-l,1-diphosphonic acid tetraethylester dihydrochloride; - N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-4- amino-4 ; 4-pentamethylene-butane-l,1-diphosphonic acid tetraethylester dihydrochloride; - N-methyl-N-4-[bis(2-chloroethyl)amino]-(L)-phenylala- nyl}-5-aminopentane-l,1-diphosphonic acid tetraethyle¬ ster dihydrochloride. EXAMPLE 16 Following the same procedures as described in Example la) and lb), 3-[bis(2-chloroethyl)amino]-(L)- phenylalanine (J. Med. Chem., 6_, 85, (1963)) is tran¬ sformed into N'-(tert-butoxycarbonyl)-3-[bis(2-chloro- ethyl)ami. ,>]-(L)-phenylalanine .hydroxysuccinimido e- ster, which is reacted with 5-amino-l-hydroxypentane- 1,1-diphosphonic acid following the procedure described in Example lc), to give N-{N'-(tert-butoxycarbonyl)-3- [bis(2-chloroethyl)amino)-(L)-phenylalanyl}-5-amino-l- hydroxypentane-l,l-diphosphonic acid trisodium salt. This compound is subsequently transformed into N- (3-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-5-amino- l-hydroxypentane-l,l-diphosphonic acid dihydrochloride, following the procedure described in Example 5. EXAMPLE 17 Following the procedure described in Example la) and lb), N 6 ,N 6 -bis(2-chloroethyl)-(DL)-lysine (J. Med. 2 Chem. 1_, 468, (1964)) is transformed into N -(tert-bu- toxycarbonyl)-N ,N -bis(2-chloroethyl)-(DL)-lysine hy¬ droxysuccinimido ester which is reacted with 4-amino-l- hydroxybutane-l,l-diphosphonic acid, following the pro- 2 cedure described in Example lc), to give N-[N -(tert- butoxycarbonyl)-N 6,Nfi-bis(2-chloroethyl)-(DL)-lysinyl]- 4-amino-l-hydroxybutane-l,l-diphosphonic acid trisodium 6 6 salt, which is in its turn transformed into N-[N ,N - bis(2-chloroethyl)-(DL)-lysinyl]-4-amino-l-hydro- xybutane-l,l-diphosphonic acid dihydrochloride, fol¬ lowing the procedure described in Example 5. EXAMPLE 18 Following the procedure described in Example lb) , by reacting 4-[2-[bis(2-chloroethyl)amino]phenyl]buty- ric acid (J. Org. Chem. 2 _, 1554, (1961)) with N-hydro- xysuccinimide and N-morpholinoethylisonitrile, 4-[2- [bis(2-chloroethyl)amino]phenyl]butyric acid N-hydroxy¬ succinimido ester is prepared. Said N-hydroxysuccini¬ mido ester is reacted with 4-aminobutane-l,l-diphospho- nic acid tetraethyl ester, according to the procedure described in Example 10, to give N-{4- [2-[bis(2-chlo- roethyl)amino]phenyl]butyroyl}-4-aminobutane-1,1-dipho- sphonic acid tetraethyl ester, which is reacted with iodotrimethylsilane, following the procedure described in Example 11, to obtain N-{4-[2-[bis(2-chloro- ethy1)amino]phenyl]butyroyl}-4-aminobutane-l,1-dipho- sphonic acid disodium salt.

Claims: CLAIMS 1. Compounds of formula (I) wherein : R, and R 2 , which can be the same or different, are hy¬ drogen or C--C. alkyl; (A) is hydrogen, halogen (chlorine, bromine or iodine), hydroxy, straight or branched C--C, 2 alkyl; (B) is a covalent bond, a straight or branched C -Cr. alkylene or, together with the adjacent nitrogen atom, a group of formula -N(R 3 ) "' wll i ch ^ s optionally gem(l,l); 1,2; 1,3 or 1,4 disubstituted; an ortho, eta or para - substituted aralkyl of formula -N(R 3 )—rO -(CH 2 ) -; an alkylene chain containing at least one hetero-atom of formula ~[CH(CH 3 )] -( CH 2 ^n l ~ X ~ (CH») -, m is the integer 5 or 6; n and n, are an inte- ger 1 to 5; p is zero or 1 and X is 0, S, N-CH 3 or the ureido group -NH-CO-NH-; R, is hydrogen, straight or branched C.-C 8 alkyl, C 3 -C 8 cycloalkyl, benzyl, phenyl or p-methoxybenzyl; (C) is straight or branched C,-C-. alkyl, phenyl, an aralkyl chain of formula in which n is as above defined; R 4 is hydrogen, straight or branched C 1 -C 4 alkyl, or it is a group of formula .R. -N * R, in which R_ and R R , which are the same or different, are hydrogen, straight or branched C.-C 8 alkyl, phenyl, benzyl, p-methoxybenzyl, or one of R_ and R g is as above defined and the other one is a group of formula R 0 -C- 9 II 0 in which R g is hydrogen, straight or branched C,-C 4 alkyl, phenyl, benzyl, p-methoxyphenyl, straight or branched C,-C 4 alkoxy, halo-C.-C.-alkoxy; R g and R g are haloethyl (2-chloroethyl, 2-bromoethyl, 2-iodoethyl) or R-. and R g , together with the nitrogen atom to which they are bound, are a 1-aziridinyl residue of formula and isom -ers-, diastereisomers and pharmaceutically ac- ceptable salts thereof. 2. Compounds as claimed in claim 1, wherein A is hy¬ droxy. 3. Compounds as claimed in claim- 1, wherein A is hydro¬ gen. 4. Compounds as claimed in any one of claims 1 to 3, wherein R. and R 2 are hydrogen. 5. Compounds as claimed in any one of claims 1 to 3, wherein R. and R 2 are C,-C. alkyl. 6. Compounds as claimed in any one of claims 1 to 5, wherein (B) is a C 2 -C 8 alkylene chain and R 3 is hydro¬ gen. 7. Compounds as claimed in any one of claims 1 to 5, wherein (B) is a chain of formula wherein s is from 1 to 4. 8. Compounds as claimed in any one of claims 1 to 7, wherein (C) is phenyl or benzyl and 5 and R g are 2- chloroethyl. 9. Compounds as claimed in claim 8, wherein (C) is ben- zyl and R. is amino, t-butoxycarbonylamino, formyla- ino, acetylamino, benzyloxycarbonylamino or ethoxycar- bonylami o. 10. Compounds as claimed in claim 8, wherein (C) is phenetyl and R. is hydrogen. 11. A process for the preparation of compounds of for¬ mula (I), which process comprises reacting a compound of general formula (II) wherein R_, R g and (C) are as above defined and R'. is the same as R 4 or a group which can be transformed into R 4 by removal of protecting groups possibly present, T is hydroxy or a carboxy-activating group, with a compound of formula (III) wherein R n , , R _. 2 , , R -3 . , (A) and (B) are as above defined, to give a compound of formula ( la) wherein R, , R 2 , R 3 , R 4 , R 5 , R g , (C) , (B) and (A) are as above defined, which compound can in its turn be tran¬ sformed into a compound of formula (I) by selective elimination of the protecting groups, or by alkylating or acylating the amino groups. 12. Pharmaceutical compositions having anitumor acti¬ vity, containing as the active ingredient one compound of formula (I) in mixture with a non toxic carrier. 13. The use of a compound of formula (I) for the prepa- ration of an antitumor medicament.

Inventors: LIVI VALERIA, MENTA ERNESTO, SPINELLI SILVANO, TOGNELLA SERGIO, LIVI, VALERIA, MENTA, ERNESTO, SPINELLI, SILVANO, TOGNELLA, SERGIO
Applicants: BOEHRINGER BIOCHEMIA SRL, BOEHRINGER BIOCHEMIA ROBIN S.P.A.
Application year: 1990
Main classification: A61K31
IPC codes: A61K31, A61P35, C07F9