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

Abstract: Diphosphonic acids of formula (I), wherein groups R1-R11, A, B, C, x, y and z have the meanings defined in the specification, are useful as antitumour agents.

Description: AMINOACYL DERIVATIVES OF GEM-DIPHOSPHONIC ACIDS- A PROCESS FOR THE PREPARATION THEREOF AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM The present invention relates to diphosphonic acid having a marked antitumour activity, to a process for the preparation thereof and to pharmaceutical composi¬ tions containing them. Gem-diphosphonic acids and the salts thereof are known and used in the therapy of osteoporosis and in the treatment of bone resorption (see EP 96.931, EP 252.504, BE 896.453, BE 903.519, DE 3.016.289, DE 3.540.150, DE 2.534.391) . i0 Moreover, diphosphonic acid esters having pesticide activity are disclosed in US-Pat. 3.906.062. However, no compounds described in the above mentioned patents have been reported to have intrinsic antitumour activity. 5 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- 0 toxic activity of molecules bearing dialkylating functions. Metotrexate diphosphonic analogs are described in WO 88/06158 to be useful agents in the treatment of bone tumours. 5 It has now been found that aminoacyl diphosphonic acid derivatives characterized by the presence of a bond which can be physiologically hydrolyzed, connecting the diphosphonic derivative with a dialkylating residue, have, compared with the above cited compounds, advantageous antitumour and antimeta- static 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 relates to compounds of for¬ mula (I) wherein: R^ and R2, which can be the same or different, are hy¬ drogen or C 1 _c 4 alkyl; (A) is hydrogen, halogen (chlorine, bromine or iodine), hydroxy, straight or branched C. ^ c.- alkyl; (B) is a covalent bond, straight or branched C,-c ft alkylene, an alkylene chain containing at least one he- tero-atom of formula -[CH(CH 3 )] p -(CH 2 ) n -χ_(CH 2 ) n - or, together with the adjacent nitrogen atom, a group of formula heterocyclic rings of formulae an ortho, meta or para substituted aralkyl of formula in which chain or groups X is 0, S, N-CH,; m is zero or the integer 1 or 2; m, is the integer 1, 2, 3 or 4; n and n, are an integer from 1 to 5; p is zero or the integer 1; R 3 is hydrogen, straight or branched C.-C, Q alkyl, C,-C, cycloalkyl, benzyl, phenyl, or p- methoxybenzyl; (C) is straight or branched C--C-. alkylene, phenylene, an aralkyl chain of formula wherein n is as defined above; R 4 is selected from hydrogen, straight or branched C j^ - C 4 alkyl, or R represents a group wherein 12 and R^, which are the same or different, are hydrogen, straight or branched C,-Cg alkyl, phenyl, benzyl, para-methoxybenzyl, or one of R- 2 and 13 is as defined above and the other one is a group of formula: R 14 -C- 0 wherein R^ 4 is hydrogen, straight or branched C^-C 4 alkyl, phenyl, benzyl, para-methoxybenzyl, straight or branched C 1 -C 4 alkoxyl, halo-C 1 -C 4 alkoxyl; 5 and R 6 are haloethyl (2-chloroethyl, 2-bromoethyl, 2-iodoethyl) or R 5 and R g , taken together with the ni¬ trogen atom to which they are linked, are a 1-azyri- dinyl group of formula 7 , Rg, Rg e ^ Q - which are the same or different, 1 1 taken together with the -N-CH-CO- group to which they are linked, form the residue from a D or L amino acid; alkyl chain; x is zero or the integers 1 and 2; y and z are zero or the integer 1, with the proviso that : when x, y and z are at the same time zero, (B) is one of the residues of formula o relates to racemates, diastereoisomers and optically pure forms of compounds of general formula I. The present invention also relates to the pharma¬ ceutically acceptable salts of compounds of general formula (I), for example with inorganic bases, such as salts with alkali metals (such as sodium or potassium) or alkaline-earth metals (such as calcium or magnesium) or ammonium salts; salts with organic bases such as methylamine, ethylamine, propylamine, isopropylamine, butylamine, t-butylamine, dimethylamine, diethylamine, diethanolamine, trimethylamine, triethylamine, piperi¬ dine, pyridine, picoline, dicyclohexylamine; organic acid addition salts, such as : formate, acetate, tri- fluoroacetate, maleate, fumarate, tartrate, methanesul- fonate, benzenesulfonate, toluenesulfonate salts; inor- ganic acid addition salts, such as hydrochloride, hy- drobromide, sulfate, hydrogen sulfate, phosphate salts, or with amino acids, such as aspartate, glutamate or lysine or arginine salts. C 1~ C 4 Alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl; methyl and ethyl being particularly preferred. C,-C, 2 Alkyl, besides the meanings precised above for C- . -c 4 alkyl, can be n-pentyl, n-hexyl, n-decyl and the like; methyl and ethyl being particularly preferred. A is preferably hydroxy. The alkylene chain B is preferably - - CH ) n - ; with n comprised from 2 to 5 , -CH- (CH 5 ) _ with r comprised I Δ r CH 3 from 2 to 5, or one of the chains of formula with n compris δ ed fr * om 1 * to 4, or, considered together with the adjacent nitrogen atom, one of the chains of formula with n comprised from 1 to 3. ^ and R 2 are preferably hydrogen. 3 is preferably hydrogen or methyl, whereas (C) is 2 -. preferably a group of formula -N " wherein R^ 2 and R,, are preferably hydrogen or one of them is R 14 _co-, wherein R^ 4 is hydrogen, methyl, tert- butyloxy, trichloromethoxy, trichloroethoxy, benzyloxy, ethoxy. When (C) is a residue of formula - θ>-(CH 2 )—., R is preferably hydrogen. 5 and Rg are preferably 2-chloroethyl. R 7D, R g , R 9 and R 1Q , taken together with the -'N-CIH-CO- group to which they are linked are preferably the resi- due from the D or L amino acids glycine, sarcosine, alanine, phenylalanine, valine, leucine, isoleucine, methionine, proline. is preferably hydrogen, methyl, ethyl. Preferred compounds are those in which : z is 1 and x and y are zero; x and y are different from zero and z is zero; particularly preferred compounds are those in which x is 1 and y and z are zero. Examples of com¬ pounds of the invention are: N-[ [4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl]-(L)- alanyl]-4-amino-l-hydroxybutane-l,l-diphosphonic acid; N-[ [4-[bis(2-chloroethyl)amino3-(L)-phenylalanyl]-(D)- alanyl]-4-amino-l-hydroxybutane-l,1-diphosphonic acid; N-[ [4-[bis(2-chloroethyl)amino3-(L)-phenylalanyl]-(L)- alanyl3-5-amino-l-hydroxypentane-l,1-diphosphonic acid; N-[ [4-[bis(2-chloroethyl)amino3-(L)-phenylalanyl]-(D)- alanyl3-5-amino-l-hydroxypentane-l,1-diphosphonic acid; N-[ [4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl]gly- cyl]-4-amino-l-hydroxybutane-l,l-diphosphonic acid; N-[ [4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl]gly- cyl3-5-amino-l-hydroxypentane-l,l-diphosphonic acid; N-methy1-N-[ [4-[bis(2-chloroethyl)amino]-(L)-phenylala¬ nyl3glycyl3-4-amino-l-hydroxybutane-l,l-diphosphonic acid; N-[ [4-[bis(2-chloroethyl)amino3-( )-phenylalanyl]-(L)- prolyl3-3-amino-l-hydroxypropane-l,l-diphosphonic acid; N-[ [4-[bis(2-chloroethyl)amino3-( )-phenylalanyl3-(L)- prolyl3-5-amino-l-hydroxypentane-l,1-diphosphonic acid; N-[[4-[bis(2-chloroethyl)amino3-( )-phenylalanyl]-(L)- V-glutamyl3-5-amino-l-hydroxypentane-l,1-diphosphonic acid; N-[[4-[bis(2-chloroethyl)amino3-( )-phenylalanyl]-(L)- ^-glutamy13-4-amino-l-hydroxybutane-l,1-diphosphonic acid; 3_[4_[[4-[bis(2-chloroethyl)amino3-( )-phenylalanyl]- (L)-alanyl3piperazin-l-yl]-1-hydroxypropane-l,1-dipho- sphonic acid; 4-[4-[[4-[bis(2-chloroethyl)amino3-( )-phenylalanyl3- (L)-alanyl3piperazin-l~yl -l-hydroxybutane-l,l-dipho- sphonic acid; N-[[4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl3-( )- leucyl3-3-amino-l-hydroxypropane-l,l-diphosphonic acid; N-[[4-[bis(2-chloroethyl)amino3-( )-phenylalanyl3sarco- syl3-4-amino-l-hydroxybutane-l,1-diphosphonic acid; N-[[4-[bis(2-chloroethyl)amino3-( )-phenylalanyl]-(L)- valyl]-4-amino-l-hydroxybutane-l,1-diphosphonic acid; N-[[4-[bis(2-chloroethyl)amino3-( )-phenylalanyl3gly- cylglycyl3-5-amino-l-hydroxypentane-l,1-diphosphonic acid; N-[[4-[bis(2-chloroethyl)amino3-( )-phenylalanyl3-( )- alanylglycyl3-4-amino-l-hydroxybutane-l,1-diphosphonic acid; N-[[4-[bis(2-chloroethyl)amino3-(L)-phenylalanyl]gly- cylglycylglycyl3-4-amino-l-hydroxybutane-l,1-dipho- sphonic acid; N-[[4-[bis(2-chloroethyl)amino3-( )-phenylalanyl]-(L)- alanyl-(L)-alanyl3-5-amino-l-hydroxypentane-l,1-dipho- sphonic acid; N-[[4-[4-[bis(2-chloroethyl)aminoJphenyl] utyroyl]-(L)- alanyl3-4-amino-l-hydroxybutane-l,l-diphosphonic acid; N-[ [4-[4-[bis(2-chloroethyl)aminoJphenyl]butyroyl3-(D)- alanyl3-4-amino-l-hydroxybutane-l,l-diphosphonic acid; N-[ [4-[4-[bis(2-chloroethyl)amino]phenyl]butyroyl3-(L)- alanyl3-5-amino-l-hydroxypentane-l,l-diphosphonic acid; N-[[4-[4-[bis(2-chloroethyl)aminoJphenyl3butyroyl3-(D)- alanyl]-5-amino-l-hydroxypentane-1,1-diphosphonic acid; N-[[4-[4-[bis(2-chloroethyl)amino3phenyl3butyroyl]gly- cyl3-4-amino-l-hydroxybutane-l,l-diphosphonic acid; N-[ [4-[4-[bis(2-chloroethyl)amino]phenyl}butyroyl]gly- cyl]-5-amino-l-hydroxypentane-l,l-diphosphonic acid; N-methyl-N-[[4-[4-[bis(2-chloroethyl)amino3phenyl3 uty¬ royl3glycyl3-4-amino-l-hydroxybutane-l,1-diphosphonic acid; N-[ [4-[4-[bis(2-chloroethyl)amino]phenyl]butyroyl]-(L)- prolyl3-3-amino-l-hydroxypropane-l ,1-diphosphonic acid; N-[[4-[4-[bis (2-chloroethyl)amino3phenyl3butyroyl3-( )- ζ-glutamyl]-5-amino-l-hydroxypentane-l,1-diphosphonic acid; N-[ [4-[4-[bis(2-chloroethyl)amino3phenyl]butyroyl]-(L)- ^--glutamyl]-4-amino-l-hydroxybutane-l,1-diphosphonic acid; N-[[4-[4-[bis(2-chloroethyl)amino3phenylJbutyroyl3-(L)- prolyl3-5-amino-l-hydroxypentane-l,1-diphosphonic acid; 3-[4-[[4-[4-[bis(2-chloroethyl)amino3phenyl]butyroyl3- (L)-alanyl]-piperazin-l-yl3-1-hydroxypropane-l,1-dipho- sphonic acid; 4-[4-[[4-[4-[bis(2-chloroethyl)amino3phenyl3butyroyl3- (L)-alanyl3-piperazin-l-yl]-l-hydroxybutane-l,1-dipho- sphonic acid; N-[[4-[4-[bis(2-chloroeth l)amino]phenyl3butyroyl]-(L)- valylj-5-amino-l-hydroxypentane-l,1-diphosphonic acid; N-[[4-[4-[bis(2-chloroethyl)amino3phenyl]butyroyl3gly- cylglycyl3-5-amino-l-hydroxypentane-l,1-diphosphonic acid; N-[[4-[4-[bis(2-chloroethyl)amino3phenyl3butyroyl]-(D- alanyl-glycyl3-4-amino-l-hydroxybutane-1,1-diphosphonic acid; N-[[4-[4-[bis(2-chloroethyl)amino3phenyl3butyroyl3-(L)- alanyl-(L)-alanyl-(L)-alanyl3-5-amino-l-hydroxypentane- 1,1-diphosphonic acid; 3_[4_[ -[bis(2-chloroethyl)amino]-(L)-phenylalanyl]-pi- perazin-l-yl3-l-hydroxypropane-l,l-diphosphonic acid; 4_[4_[4_[bis(2-chloroethyl)amino3-(L)-phenylalanyl3-pi- perazin-l-yl3-l-hydroxybutane-l,l-diphosphonic acid; N-[4-[bis(2-chloroethyl)amino3-(L)-phenylalanyl3-3-(pi- rrolidin-2-yl)-l-hydroxypropane-l,l-diphosphonic acid; N-[4-[bis(2-chloroethyl)amino3-( )-phenylalanyl]-3-(pi- peridin-4-yl)-1-hydroxypropane-l,1-diphosphonic acid; N-[4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl3-3-(pi- peridin-2-yl)-l-hydroxypropane-l,l-diphosphonic acid; N-[4-[bis(2-chloroethyl)amino]-( )-phenylalanyl]-2-(pi- peridin-2-yl)-1-hydroxyethane-1,1-diphosphonic acid; N-[[4-[4-[bis(2-chloroethyl)amino3phenyl3butyroyloxy3- acetyl3-4-amino-l-hydroxybutane-l,l-diphosphonic acid; N-[[4-[4-[bis(2-chloroethyl)amino3phenyl3butyroyloxyJ- acetyl3-3-(piperidin-2-yl)-1-hydroxypropane-l,1-dipho- sphonic acid; N-[[ -[4-[bis(2-chloroethyl)amino3phenyl3butyroyloxy]- acetyl]-5-amino-l-hydroxypentane-l,l-diphosphonic acid; N-[[4-[bis(2-chloroethy1)amino3-( )-phenylalanyloxy]- acetyl]-4-amino-l-hydroxybutane-l,l-diphosphonic acid; N-[ [4-[bis(2-chloroethyl)amino3-(L)-phenylalanyloxy3- acetyl]-3-(pirrolidin-2-yl)-1-hydroxypropane-l,1-dipho- sphonic acid. The compounds of the present invention are prepa¬ red by reacting a compound of formula (II) wherein R^ ; Rg and (C) have the above mentioned mea¬ nings and R' is the same as R or it is a group which can be converted into R 4 by removing any protecting groups, T is hydroxy or a carboxy-activating group, with a compound of formula (III) Q wherein R 1 , R 2 , R 3 , R g , 1Q , 1;L , (A), (B) , x, y and z have the above mentioned meanings and R'_ and R' fi have the same meanings as R-_ and Rg or they can be converted into R_ and Rg by removing the amino-, hydroxy-, thio- and carboxy-protecting groups which can optionally be present, to give a compound of formula (la): Ia wherein R χ , R 2 , 3 , R- 4 , R 5 , R g , R ? «, R g « , R 9 , R 1Q , R ll- f ), (B) , (C), x, y and z have the above mentioned meanings, which compound in its turn can be transformed into a compound of formula (I) by means of known reac¬ tions for the selective removal of protecting groups, alkylation or acylation of amino groups and the like. When, in the reaction of compounds of formula (II) with compounds of formula (III), compounds of formula (II) are used in form of carboxylic acids (T - OH), the reaction is usually carried out in the presence of a condensing agent, such as N,N'-dicyclohexylcarbodiimi- de, N-cyclohexyl-N'-morpholinoethyl-carbodiimide, N- ethy1-N'-(3-dimethylamino)-propyl-carbodiimide, N,N'- carbonyl-bis-(imidazole) , phosphorous oxychloride, pho¬ sphorous trichloride, thionyl chloride, oxalyl chlo¬ ride, ethyl chloroformate, isobutyl chloroformate, morpholinoethyl isocyanide and the like. Examples of carboxy-activating groups are acyl halides, symmetric or mixed anhydrides (such as with methanesulfonic, acetic, isobutyric, pivalic, trichloroacetic acids); reactive amides (for example with imidazole, 1,2,4- triazole); azide; reactive esters (such as para- nitrophenyl ester, methoxymethyl ester, 2,4- dinitrophenyl ester, pentachlorophenyl ester, hydroxysuccinimide ester, l-hydroxy-2-(lH)-pyridone ester, 1-hydroxybenzotriazole ester) and the like. The reaction can be performed in the presence of an inorganic base, such as an alkali carbonate or bicarbonate, an alkali or alkaline-earth hydroxide or an organic base such as triethylamine, tributylamine, pyridine, 4-dimethylamino-pyridine, N-alkylmorpholine, N,N-dialkyl-aniline and the like. The reaction can be effected at a temperature ran¬ ging from -40 β C to the reflux temperature of the solvent preferably using a slight molar excess of compound (II) compared to (III) in a solvent such as water, N,N-dimethylformamide or mixtures thereof. The reaction temperature preferably ranges from -10°C to room temperature and, in this case, the reac- tion time ranges from 1 to 48 hours, but generally the reaction is complete within a time from 2 to 12 hours. Compounds of general formula (II) are known com¬ pounds, which are commercially available and/or can be prepared by known methods to those skilled in the art, such as those described in: J. Med. Chem. 2! , 1304, (1981); CA 1: 8066(d), (1957); BE 905,974; CA 104: 141897 (1986); J. Med. Chem. 7_, 468, (1964); J. Med. Chem. j, 85, (1963); Cancer Chem. Rep. J50, 685, (1966); J. Med. Chem. 21 , 16, (1977); J. Org. Chem. 2 _, 1554, (1961); J. Org. Chem. 2 , 1674, (1961); CA j64_; 10267 g, (1966); J. Chem. Soc, 2994, (1960); Biochem. Pharma¬ col. 11, 847, (1962); Biochem. Pharmacol. .12, 833, (1963); CA 73_: 131293c, (1970); Biochem. Pharmacol. 5_, 192, (1960); Int. J. Pept. Protein Res., 36, 308, (1990). Compounds of formula (III) wherein z is zero and x and y are as defined above are also known or they can be prepared according to known methods; see, for instance, EP 96931, EP 252504, BE 903519, DE 3016289, EP 224751, DE 2534391, EP 197478, DE 3512536 e DE 3623397. Compounds of formula (III) wherein z is 1 are not known, but they can be prepared by reacting a compound of f IV wherein R χ , R 2 , R 3 , iV 7 ? ', R Λ ' g , « - 99 ,, ΛlQ » (A) , (B) , x and y have the above mentioned meanings, with a compound of formula (V) : wherein R^ and T have the above mentioned meanings and j _5 is a hydroxy -protecting group, to give a compound of formula (VI) : O which, by removing the protective group R.,.. gives a compound of formula (III) wherein z is 1. 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 dichlorome hane, 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 β C to the solvent's reflux temperature; preferred reaction condi¬ tions are those according to J. Org. Chem: 8, 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 5Q (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- ethyl)amino]-(L)-phenylalanyl3-( )-alanyl3-4-amino-l- hydroxybutane-l,l-diphosphonic acid monohydrochloride and N-[[4-[4-[bis(2-chloroethyl)amino3phenyl]butyroyl]- (L)-alanyl3-4-amino-l-hydroxybutane-l,1-diphosphonic acid disodium salt have an ID 5Q of 0,1 /ml and 0,5 ^ ml, respectively, against murine leukemia L1210. 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 mg/m to 1.2 g/m body area, can induce the regression of the above mentioned tumoral forms. The effective 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 those for humans (on the basis of mg/ιrr ; body area) is described by Freireich, E.J. , et al., Quantitative Comparison of Toxicity of Anticancer Agents in Mouse, Rat, Hamster, Dog, Monkey and Man, Cancer Chemother. Rep., 5 , n. 4, 219-244, May 1966. The tumours which can be treated with the compounds of the present invention are those susceptible to therapy with alkylating agents. Particularly, multiple myeloma, osteosarcoma, bone metastatis and breast, ovarian and testis carcinomas can be advantageously treated. Moreover, the compounds of the invention can ad¬ vantageously be used in the therapy of other solid and liquid tumors, such as lymphomas and leukemias. The compounds of the invention can be administered by the parenteral route (intravenously, intramuscu¬ larly, intraarterially, intraperitoneally) in form of sterile aqueous solutions or sterile powders for the extemporary preparation of solutions, oily preparations for the intramuscular or intraperitoneal administra¬ tions. The compounds of the invention can also be admini¬ stered by the oral route : in this instance, useful pharmaceutical formulations can be syrups or similar liquid formulations, as well as solid formulations, such as tablets, capsules and the like. The following Examples further illustrate the in¬ vention. EXAMPLE 1 a) A solution of di(tert-butyl)dicarbonate (900 mg) in tetrahydrofuran (THF; 8 ml) is quickly added to a solu¬ tion of 4-[bis(2-chloroethyl)amino) 3-(L)-phenylalanine (600 mg) in THF (20 ml) and IN NaOH (2 ml). The resul- ting mixture is stirred at room temperature for 2 hours, adjusting pH to about 9 by repeated additions of IN NaOH. After that, solvent is distilled off under va¬ cuum and the residue is partitioned between water (6 ml) and ethyl ether (10 ml). The organic phase is discarded and the aqueous one is acidified with HCl and repeatedly extracted with SUBSTITUTES ethyl ether. The combined organic extracts are dried over sodium sulfate and concentrated to dryness under vacuum, to obtain a white foamy residue of N*-(tert- butoxycarbonyl)-4-[bis(2-chloroethyl)amino]-(L)-phenyl- alanine (mg 580). NMR (CDC1 3 , TMS) - 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) A stirred solution of the compound obtained in a) (580 mg) in anhydrous THF (10 ml) is added with N- hydroxysuccinimide (346 mg) and morpholinoethyl isocyanide (0.266 ml). The mixture is stirred for one hour at room temperature, then concentrated to dryness under vacuum. The resulting residue is taken up into 2N HC1 (10 ml) and repeatedly extracted with ethyl acetate (3x10 ml). The organic phases are combined, washed with 5% aqueous NaHC0 3 , with water (10 ml) and dried over sodium sulfate. Solvent is evaporated off under vacuum, to obtain an oily residue which is crystallized from an ethyl ether/isopropyl ether mixture, to give 520 mg of N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)amino3- (L)-phenylalanine hydroxysuccinimide ester (melting point 140-143°C); [&3 D - + 3,5 (c - 2 chloroform) NMR (TMS, CDC1 3 ) δ ' - 1,45 (s, 9H) ; 2,85 (s, 4H) ; 3,15 (m, 2H) ; 3,69 ( , 8H) ; 4,9 (m, 1H) ; 6,61 (d, 2H) ; 7,15 (d, 2H) . c) A solution of the ester obtained in b) (203 mg) in dimethylformammide (DMF; 1 ml) is slowly dropped into a solution of N-[(L)-alanyl3-4-amino-l-hydroxybutane-l,l- diphosphonic acid disodium salt (100 mg) in a mixture of water (4 ml) and DMF (3 ml) adjusted to pH 10.5 by addition of IN NaOH. During the dropping of the hydroxysuccinimide ester the pH is maintained between 9-10 by repeated additions of IN NaOH. At the end of the addition, the pH is brought to 5-5.4 by 2N HCl and the resulting mixture is diluted with methanol (50 ml) and refrigerated (5 minutes); a precipitate separates which is recovered by filtration to yield 120 mg of N-{[N'-(tert-butoxycarbonyl)-4- [bis(2-chloroethyl)amino]-(L)-phenylalanyl3-( )-ala- nyl}-4-amino-l-hydroxybutane-l,l-diphosphonic acid di¬ sodium salt m.p. > 260°C [&] Ώ - 4 (c - 2 in water). ■ 'Ή-NMR (TMS, D 2 0): 1,35 (m, 12H) ; 1,85 (m, 4H) ; 2,9 (m, 2H); 3,2 (m, 2H) ; 3,75 (s, 8H) ; 4,3 (m, 2H) ; 6,85 (d, 2H) ; 7,15 (d, 2H) HPLC: Partisphere C,g 150 x 4,6 mm; sodium heptanesulfo- nate 0,025 M in water/acetonitrile/dioxane 70:20:10, pH 2,5 with HJ?0.; flow 0,8 ml/min; X. » 255 nm Retention time 12,86*. EXAMPLE 2 Following the procedure described in Example 1, by reacting N*-(tert-butoxycarbonyl)-4-[bis(2-chloroe- thyl)amino3-(L)-phenylalanine hydroxysuccinimide ester with the appropriate N-(aminoacyl)-aminoalkyl-l-hydro- xy-l,l-diphosphonic acids, the disodium salts of the following compounds are obtained: N-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)ami¬ no]-(L)-phenylalanyl-(D)-alanyl}-4-amino-l-hydroxybu- tane-l,l-diphosphonic acid; N-{N•-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)ami- no]-(L)-phenylalanyl-(L)-alanyl}-5-amino-l-hydroxypen- tane-l,l-diphosphonic acid; N-{N 1 -(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)ami¬ no3-(L)-phenylalanyl-(D)-alanyl}-5-amino-l-hydroxypen- tane-l,l-diphosphonic acid; N-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)ami- no3-(L)-phenylalanyl-glycyl}-4-amino-l-hydroxybutane- 1,1-diphosphonic acid; N-{N--(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)ami¬ no3-(L)-phenylalanyl-glycyl}-5-amino-l-hydroxypentane- 1,1-diphosphonic acid; N-methy1-N-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloro- ethyl)amino3-(L)-phenylalanyl-glycyl}-4-amino-l-hy- droxybutane-1,1-diphosphonic acid; N-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)ami¬ no]-(L)-phenylalanyl-(L)-prolyl}-3-amino-1-hydroxypro- pane-l,l-diphosphonic acid; N-{N*-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)ami¬ no3-( )-phenylalanyl-(L)-prolyl}-5-amino-l-hydroxypen- tane-l,l-diphosphonic acid; 3-{4-{N 1 -tert-butoxycarbonyl)-4-[bis(2-chloroethyl)ami- no3-( )-phenylalanyl-(L)-alanyl}-piperazin-1-yl}-1-hy- droxypropane-1,1-diphosphonic acid; 4-{4-{N*-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)- amino3-( )-phenylalanyl-(L)-alanyl}-piperazin-1-yl}-1- hydroxybutane-1,1-diphosphonic acid; N-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)ami- no3-( )-phenylalanylglycylglycyl}-5-amino-l-hydroxypen- tane-l,l-diphosphonic acid; N-{ •-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)ami¬ no3-(L)-phenylalanylglycylglycyl}-4-amino-l-hydroxybu- tane-1,1-diphosphonic acid; N-{N'-(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)ami- no]-(L)-phenylalanyl-(L)-alanylglycyl}-4-amino-l-hy- droxybutane-1,1-diphosphonic acid; N-{N 1 -(tert-butoxycarbonyl)-4-[bis(2-chloroethyl)ami¬ no]-(L)-phenylalanylglycylglycylglycyl}-4-amino-l-hy- droxybutane-l,l-diphosphonic acid. EXAMPLE 3 A solution of N-{N'-(tert-butoxycarbonyl)-4-[bis- (2-chloroethyl)amino]-(L)-phenylalanyl-(L)-alanyl}-4-a- mino-l-hydroxybutane-l,l-diphosphonic acid disodium salt (50 mg) in methanol (1 ml) and 6.9M HC1 in ethanol (0.1 ml) is stirred at room temperature for 20 hours, then the reaction mixture is concentrated to small volume and the separated sodium chloride is filtered off. By diluting the filtrate with ethyl ether (10 ml), N-{[4-[bis(2-chloroethylamino) ]-(L)-phenylalanyl]-( )- alanyl}-4-amino-l-hydroxybutane-l,1-diphosphonic acid monohydrochloride precipitates (35 mg). m.p. = 190 β C (decomp.) [ot] = + 4,8 (c = 2 in methanol) X H-NMR (TMS, HC1 0,1 N in D 2 0) : δ " = 1,3 (d, 3H) ; 1,75- 2,1 ( , 4H) ; 3,25 (m, 4H) ; 3,62 (t, 4H) ; 4,15 (t, 4H) ; 4,25 (t, 2H); 7,55 (q, 4H) HPCL: Partisphere C lg , 150 x 4,6 mm; sodium heptanesul- fonate 0,025 M in water/acetonitrile/dioxane 70:20:10, pH ^2,5 with H 3 P0 4 ; flow 0,8 ml/min; A,- 255 nm Retention time: 4,66' EXAMPLE 4 Following the procedure described in Example 3, by reacting the disodium salts of the acids described in Example 2, the monohydrochloride salts of the following acids are obtained: N-{ -[bis(2-chloroethyl)amino -( )-phenylalanyl-(D)- alanyl}-4-amino-l-hydroxybutane-l,1-diphosphonic acid; N-{4-[bis(2-chloroethyl)amino3-( )-phenylalanyl-(L)- alanyl}-5-amino-l-hydroxypentane-l,l-diphosphonic acid; N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl-(D)- alanyl}-5-amino-l-hydroxypentane-l,1-diphosphonic acid (trisodium salt); N-{4-[bis(2-chloroethyl)amino3-( )-phenylalanyl-gly- cyl}-4-amino-l-hydroxybutane-l,l-diphosphonic acid; N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl-gly¬ cyl}-5-amino-l-hydroxypentane-1 ,1-diphosphonic acid; N-methyl-N-{4-[bis(2-chloroethyl)amino3-(L)-phenylala¬ nyl-glycyl}-4-amino-l-hydroxybutane-l,1-diphosphonic acid; N-{4-[bis(2-chloroethyl)amino3-(L)-phenylalanyl-( )- prolyl}-3-amino-l-hydroxypropane-l,1-diphosphonic acid; N-{4-[bis(2-chloroethyl)amino -( )-phenylalanyl-(L)- prolyl}-5-amino-l-hydroxypentane-l,1-diphosphonic acid; 3-{4-[bis(2-chloroethyl)amino -( )-phenylalanyl-(L)- alanyl}-piperazin-l-yl}-l-hydroxypropane-l,l-diphospho- nic acid; 4-{4-{4-[bis(2-chloroethyl)amino3-( )-phenylalanyl-(L)- alanyl}-piperazin-1-yl}-1-hydroxybutane-l,1-diphospho- nic acid; N-{4-[bis(2-chloroethyl(amino) 3-( )-phenylalanylglycyl¬ glycyl}-5-amino-l-hydroxypentane-l,1-diphosphonic acid; N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanylglycyl¬ glycyl}-4-amino-l-hydroxybutane-l,1-diphosphonic acid; N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl-( )- alanylglycyl}-4-amino-l-hydroxybutane-l,1-diphosphonic acid; N-{4-[bis(2-chloroethyl)amino3-( )-phenylalanylglycyl- glycylglycyl}-4-amino-l-hydroxybutane-l,1-diphosphonic acid. EXAMPLE 5 Following the procedure described in Example 1, step b) , by reacting 4-[4-[bis(2-chloroethyl)amino3- phenyljbutyric acid (1.5 g) with N-hydroxysuccinimide (1.2 g) and N-morpholinoethylisocyanide (0.9 ml), 4-[4- [bis(2-chloroethyl)amino3phenyl]butyric acid N-hy¬ droxysuccinimide ester is obtained (1.88 g) , m.p. 80- 82 β C. According to the procedure described in Example 1, step c), the above hydroxysuccinimide ester (830 mg) is reacted with N-[ (L)-alanyl3-4-amino-l-hydroxybutane- 1,1-diphosphonic acid disodium salt (500 mg), to give N-{4-[4-[bis(2-chloroethyl)aminoJphenyl3butyroyl-(L)- alanyl}-4-amino-l-hydroxybutane-l,1-diphosphonic acid disodium salt (820 mg), m.p. > 260°C. 1 H-NMR (D 2 0, TMS): <5 = 1.35 (d, 3H) ; 1.9 (m, 6H) ; 2.27 (t, 3H); 2.56 (t, 2H) ; 3.22 (t, 2H) ; 3.75 (s, 8H) ; 4.15 (q, 1H); 6.89 (d, 2H) ; 7.15 (d, 2H) . EXAMPLE 6 Following the procedure described in Example 5, by reacting 4-[4-[bis(2-chloroethyl)amino3phenyl3butyric acid hydroxysuccinimide ester with the appropriate N- (aminoacyl)-aminoalkyl-l-hydroxy-1,1-diphosphonic acids or disodium salts thereof, the disodium salts of the following compounds are obtained: N-{ [4-[4-[bis(2-chloroethyl)amino3phenylJbutyroyl]-(D)- alanyl}-4-amino-l-hydroxybutane-l,1-diphosphonic acid; N-{[4-[4-[bis(2-chloroethyl)aminoJphenyl]butyroyl3-(L)- alanyl}-5-amino-l-hydroxypentane-l,l-diphosphonic acid; N-{[4-[4-[bis(2-chloroethyl)amino]phenyl]butyroyl3-(D)- alanyl}-5-amino-l-hydroxypentane-l,1-diphosphonic acid; N-{[4-[4-[bis(2-chloroethyl)amino]phenyl]butyroyl]gly- cyl}-4-amino-l-hydroxybutane-l,l-diphosphonic acid; N-{[4-[4-[bis(2-chloroethyl)amino]phenyl3butyroyl]gly- cyl}-5-amino-l-hydroxypentane-l,l-diphosphonic acid; N-methyl-N-{[4-[4-[bis(2-chloroethyl)amino3phenyl]buty- roylJglycyl}-4-amino-l-hydroxybutane-l,1-diphosphonic acid; N-{[4-[4-[bis(2-chloroethyl)amino3phenyl]butyroyl3-( )- prolyl}-3-amino-l-hydroxypropane-l,1-diphosphonic acid; N-{[4-[4-[bis(2-chloroethyl)amino3phenyl3butyroyl]-(L)- prolyl}-5-amino-l-hydroxypentane-l,l-diphosphonic acid; 3-{{4-[4-[4-[bis(2-chloroethyl)amino3phenyl]butyroyl]- (L)-alanyl}piperazin-l-yl}-1-hydroxypropane-l,1-dipho- sphonic acid; 4-{{4-[4-[4-[bis(2-chloroethyl)amino3phenyl]butyroyl3- (L)-alanyl}piperazin-l-yl}-1-hydroxybutane-l,1-dipho- sphonic acid; N-{[4-[4-[bis(2-chloroethyl)amino]phenyl3butyroyl]gly- cylglycyl}-5-amino-l-hydroxypentane-1,1-diphosphonic acid; N-{[4-[4-[bis(2-chloroethyl)aminoJphenyl]butyroyl3gly- cylglycyl}-4-amino-l-hydroxybutane-l,l-diphosphonic acid; N-{[4-[4-[bis(2-chloroethyl)amino]phenyl]butyroyl3-( )- alanylglycyl}-4-amino-l-hydroxybutane-l,l-diphosphonic acid; N-{[4-[4-[bis(2-chloroethyl)amino]phenyl]butyroyl]gly- cylglycylglycyl}-4-amino-l-hydroxybutane-l,1-diphospho- nic acid. EXAMPLE 7 A solution of 4-[bis(2-chloroethyl)amino]-(L)-phe- nylalanine (150 mg) in formic acid (1.82 ml) and acetic anhydride (0.64 ml) is stirred at room temperature for 3 hours. The reaction mixture is concentrated under re¬ duced pressure and partitioned between water (3 ml) and ethyl acetate (2x50 ml). The organic phase is dried over sodium sulfate and solvent is evaporated off under vacuum to give N-formyl-4-[bis(2-chloroethyl)amino3- (L)-phenylalanine (160 mg) as a yellow foam, [θU D - + 67 β C (c - 2 ethanol) -^Ϊ-NMR (CDC1 3 , TMS): 67 = 2,9 * 3,1 (m, 2H) ; 3,6 (m, 8H) ; 4,68 - 4,79 (m, 1H) ; 6,5 (d, 2H+1H) ; 6,69 (d, 2H) ; 8,1 (s, 1H). Following the procedure described in Example 1, steps b) and c), this compound is converted into the hydroxysuccinimide ester thereof (50 mg) , which is re¬ acted with N-[ (L)-alanyl3-4-amino-l-hydroxybutane-l,l- diphosphonic acid disodium salt (30 mg) to give N-{[N'- formyl-4-[bis(2-chloroethyl)amino3-( )-phenylalanyl3- (L)-alanyl}-4-amino-l-hydroxybutane-l,1-diphosphonic acid disodium salt (55 mg). EXAMPLE 8 Following the procedure described in Example 1, step c), by reaction of N-formyl-4-[bis(2- chloroethyl)amino]-(L)-phenylalanine hydroxysuccinimide ester with the appropriate N-(aminoacyl)-aminoalkyl-l- hydroxy-l,l-diphosphonic acids or disodium salts thereof, the disodium salts of the following compounds are obtained: N-{N*-formyl-4-[bis(2-chloroethyl)amino3-( )-phenylala¬ nyl-(D)-alanyl}-4-amino-l-hydroxybutane-l,1-diphospho- nic acid; N-{N'-formyl-4-[bis(2-chloroethyl)amino3-( )-phenylala¬ nyl-(L)-alanyl}-5-amino-l-hydroxypentane-l,1-diphospho- nic acid; N-{N*-formyl-4-[bis(2-chloroethyl)amino3-(L)-phenylala- nyl-(D)-alanyl}-5-amino-l-hydroxypentane-l,1-diphospho¬ nic acid; N-{N'-formyl-4-[bis(2-chloroethyl)amino3-(L)-phenylala¬ nyl-glycyl}-4-amino-l-hydroxybutane-l,1-diphosphonic acid; N-{N*-formyl-4-[bis(2-chloroethyl)amino]-(L)-phenylala- nyl-glycyl}-5-amino-l-hydroxypentane-l,1-diphosphonic acid; N-methyl-N-{N 1 -formyl-4-[bis(2-chloroethyl)amino3-(L)- phenylalanyl-glycyl}-4-amino-l-hydroxybutane-l,1-di- phosphonic acid; N-{N 1 -formyl-4-[bis(2-chloroethyl)amino3-(L)-phenylala¬ nyl-(L)-prolyl}-3-amino-l-hydroxypropane-l,1-diphospho- nic acid; N-{N'-formyl-4-[bis(2-chloroethyl)amino3-( )-phenylala- nyl-(L)-prolyl}-5-amino-l-hydroxypentane-l,1-diphospho- nic acid; 3-{4-{N'-formyl-4-[bis(2-chloroethyl)amino3-(L)-phenyl¬ alanyl-( )-alanyl}-piperazin-l-yl}-1-hydroxypropane- 1,1-diphosphonic acid; 4-{4-{N'-formyl-4-[bis(2-chloroethyl)amino]-(L)-phenyl¬ alanyl-(L)-alanyl}-piperazin-l-yl}-1-hydroxybutane-l,1- diphosphonic acid; N-{N'-formyl-4-[bis(2-chloroethyl)amino]-(L)-phenylala¬ nylglycylglycyl}-5-amino-l-hydroxypentane-l,1-dipho- sphonic acid; N-{N'-formyl-4-[bis(2-chloroethyl)amino3-(L)-phenylala- nylglycylglycyl}-4-amino-l-hydroxybutane-l,1-diphospho- nic acid; N-{N'-formyl-4-[bis(2-chloroethyl)amino3-(L)-phenylala¬ nyl-(L)-alanylglycyl}-4-amino-l-hydroxybutane-l,1-di- phosphonic acid; N-{N 1 -formyl-4-[bis(2-chloroethyl)amino]-(L)-phenylala- nylglycylglycylglycyl}-4-amino-l-hydroxybutane-l,1-di- phosphonic acid. EXAMPLE 9 Following the procedure described in Example 1, steps a) and b) , N 6 ,N 6 -bis(2-chloroethyl)-(DL)-lysine (J. Med. Chem. 7_, 468, (1964)) is transformed into N 2 - (tert-butoxycarbonyl)-N 6 ,N 6 -bis(2-chloroethyl)-(DL)- lysine hydroxysuccinimide ester which, according to the procedure described in Example 1, step c), is reacted with N-[ (L)-alanyl3-4-amino-l-hydroxybutane-l,l-dipho- sphonic acid disodium salt to give N-{[N -( ert- butoxycarbonyl)-N ,N -bis(2-chloroethyl)-(DL)-lysinyl- (L)-alanyl]}-4-amino-l-hydroxybutane-l,l-diphosphonic acid disodium salt which, following the procedure described in Example 3, is transformed into N-{[N 6 ,N - bis(2-chloroethyl)-(DL)-lysinyl-(L)-alanyl]}-4-amino-l- hydroxybutane-l,l-diphosphonic acid dihydrochloride. EXAMPLE 10 Following the procedure described in Example 1, step c) , N'-(tert-butoxycarbonyl)-4-[bis(2-chloroetyl)ami¬ no3-(L)-phenylalanine hydroxysuccinimide ester is reac¬ ted with 3-(piperazin-l-yl)-l-hydroypropane-l,l-dipho- sphonic acid to give 3-{4-{N'-(tert-butoxycarbonyl)-4- [bis(2-chloroethyl)amino3-(L)phenylalanyl}-piperazin-l- yl}-l-hydroxypropane-l,l-diphosphonic acid disodium salt. Following the procedure described in Example 3, this compound is subsequently transformed into 3-{4-{4- [bis(2-chloroethyl)amino}-(-->)-phenylalanyl}-piperazin- l-yl}-l-hydroxypropane-l,l-diphosphonic acid monohydro¬ chloride. ^-NMR (D 2 o, TMS): 6 * = 2.1 ( , 4H) ; 3.2 (m, 4H) ; 3.7 (m, 4H); 3.75 (s, 8H) ; 3.92 (m, 4H); 4.6 (m, 1H); 7.15 (d, 2H); 7.32 (d, 2H) . EXAMPLE 11 Following the procedure described in Example 10, by reacting the appropriate l-hydroxy-l,l-diphosphonic acids with N'-(tert-butoxycarbonyl)-4-[bis(2-chloroe- thyl)amino3-(L)-phenylalanine hydroxysuccinimide ester, the following compounds are obtained: 4-{4-{4-[bis(2-chloroethyl)amino3-( )-phenylalanyl}-pi- perazin-l-yl}-l-hydroxybutane-l,1-diphosphonic acid mo¬ nohydrochloride; ^-NMR D 2 0, TMS):£ " = 2.1 (m, 4H) ; 3.2 (m, 4H); 3.75 (s, 8H) ; 3.92 (m, 4H) ; 4.6 ( , 1H) ; 7.15 (d, 2H) ; 7.32 (d, 2H) . N-{4-[bis(2-chloroethyl)amino3-( )-phenylalanyl}-3- (pirrolidin-2-yl)-1-hydroxypropane-l,1-diphosphonic acid monohydrochloride; N-{4-[bis(2-chloroethyl)amino3-( )-phenylalanyl}-3-(pi- peridin-4-yl)-l-hydroxypropane-l,l-diphosphonic acid monohydrochloride; N-{4-[bis(2-chloroethyl)amino]-(L)-phenylalanyl}-3-(pi- peridin-2-yl)-1-hydroxypropane-l,1-diphosphonic acid monohydrochloride; N-{4-[bis(2-chloroethyl)amino]-( )-phenylalanyl}-2-(pi¬ peridin-2-yl)-l-hydroxyethane-l,l-diphosphonic acid mo¬ nohydrochloride. EXAMPLE 12 Following the procedure described in example 1, step C, N-formyl-4-[bis(2-chloroethyl)amino]-(L)-phe- nylalanine hydroxysuccinimide ester is reacted with 4- (piperazin-1-yl)-1-hydroxybutane-l,1-diphosphonic acid to give 4-{4-{N'-formyl-4-[bis(2-chloroethyl)amino3- (L)-phenylalanyl}piperazin-1-yl}-1-hydroxybutane-l,1- diphosphonic acid disodium salt. ^ ■ H-NM (D 2 0, TMS): <$ = 1.85 (m, 4H) ; 2.30 (m, 3H) 2.65 (m, 3H); 2.85 (m, 2H) ; 3.05 (m, 2H) ; 3.50 (m, 2H) 3.75 (s, 8H); 4.55 (q, 1H) ; 6.85 (d, 2H) ; 7.15 (d, 2H) 8.05 (s, 1H) . EXAMPLE 13 Following the procedure described in example 1, step C, 4-[4-[bis(2-chloroethyl)amino]phenyl3butyric acid hydroxysuccinimide ester is reacted with 4- (piperazin-l-yl)-l-hydroxybutane-l,l-diphosphonic acid to give 4-{4-{[4-[bis(2-chloroethyl)aminoJphenyljbu- tyroyl}piperazin-1-yl}-1-hydroxybutane-l,1-diphosphonic acid disodium salt. 1 H-NMR (D 2 0, TMS): <^= 1.85 (m, 6H) ; 2.10 (t, 2H) ; 2.30 (m, 2H); 2.55 (t, 2H) ; 2.70 (m, 2H) ; 2.85 (m, 2H) ; 3.05 (m, 2H); 3.48 (m, 2H) ; 3.80 (s, 8H) ; 6.85 (d, 2H) ; 7.20 (d, 2H). EXAMPLE 14 Acetoxyacetyl chloride (0.324 ml) is dropped into a solution of 4-amino-l-hydroxybutane-l,l-diphosphonic acid (500 mg) in water (5 ml) and IN NaOH (8 ml), si- multaneously adding IN NaOH (3 ml) to maintain pH between 11 and 12. When the additions are completed, the reaction mixture is stirred at room temperature for 2 hours, then it is diluted with DMF : a precipitate separates which is recovered by centrifugation and purified by redissolution in water (3 ml) and reprecipitated with acetone (30 ml), to give 720 mg of N-(hydroxyacetyl)-4-amino-l-hydroxybutane-l,1-dipho- sphonic acid trisodium salt; m.p. > 260° ■ ^-NMR: (D 2 0, TMS): θ~ = 2,0 ( - 4H) ; 3,05 (t, 2H) ; 4,9 (s, 2H) EXAMPLE 15 Following the procedure described in Example 14, by reacting the appropriate l-hydroxy-l,l-diphosphonic acids with acetoxyacetyl chloride, the trisodium salts of the following compounds are obtained: N-hydroxyacetyl-5-amino-l-hydroxypentane-l,1-diphospho- nic acid; 3-(4-hydroxyacetyl-piperazin-l-yl)-1-hydroxypropane- 1,1-diphosphonic acid; 4-(4-hydroxyacetyl-piperazin-l-yl)-1-hydroxybutane-l,1- diphosphonic acid; 3-(N-hydroxyacetyl-pirrolidin-2-yl)-l-hydroxypropane- 1,1-diphosphonic acid; 3-(N-hydroxyacetyl-piperidin-4-yl)-l-hydroxypropane- 1,1-diphosphonic acid; 3-(N-hydroxyacetyl-piperidin-2-yl)-l-hydroxypropane- 1,1-diphosphonic acid; 2-(N-hydroxyacetyl-piperidin-2-yl)-1-hydroxyethane-l,1- diphosphonic acid. EXAMPLE 16 A solution of 4-[4-[bis(2-chloroethyl)amino3phe¬ nyl3butyric acid hydroxysuccinimide ester (240 mg) in DMF (5 ml), prepared according to the procedure de¬ scribed in Example 5, is slowly dropped into a solution of N-hydroxyacetyl-4-amino-l-hydroxybutane-l,1-dipho- sphonic acid trisodium salt (200 mg) dissolved in water (5 ml) and IN NaOH (0.5 ml), heated to 40°C. At the end of the addition, heating is continued for 30 minutes, then the reaction mixture is cooled and diluted with DMF (10 ml) to obtain a precipitate which is recovered by centrifugation and purified by redissolution in wa¬ ter and reprecipitation with acetone, to give 350 mg of N-{[4-[4-[bis (2-chloroethyl)aminoJphenylJbutyroyloxy3- acetyl}-4-amino-l-hydroxybutane-l,1-diphosphonic acid (trisodium salt); m.p. > 260° ^-NMR (D 2 0, TMS): S = 1,85 (m, 4H) ; 2,25 (t, 2H) ; 2,55 (t, 2H); 3,2 (t, 2H); 3,75 (s, 8H) ; 5,05 (s, 2H) ; 6,85 (d, 2H); 7,2 (d, 2H) . EXAMPLE 17 Following the procedure described in Example 16 , by reacting the 1-hydroxy-l , 1-diphosphonic acids de¬ scribed in Example 13 with the hydroxysuccinimide esters of 4- [ 4- [bis ( 2-chloroethyl ) amino 3phenyl 3 butyric acid and of N 1 - ( tert-butoxycarbonyl ) -4- [bis ( 2-chloro- ethyl ) amino 3 - (L ) -phenylalanine , the following optional- ly deprotected compounds are obtained : N- { [ 4- [ 4- [bis ( 2-chloroethyl ) amino 3phenyl Jbutyroyloxy 3 - acetyl}-5-amino-l-hydroxypentane-l,1-diphosphonic acid (trisodium salt) ; 3_{[4_[4_[4_[bis(2-chloroethyl)aminoJphenylJbutyroyl- oxy3acetyl3-piperazin-l-yl}-1-hydroxypropane-l,1-dipho- sphonic acid (trisodium salt); 4_{[4_[4_[4-[bis(2-chloroethyl)amino3phenyl3butyroyl- oxy3acetyl]-piperazin-1-yl}-1-hydroxybutane-l,1-dipho- sphonic acid (trisodium salt); N-{[4-[4-[bis(2-chloroethyl)amino3phenyl3butyroyloxy3- acetyl}-3-(pyrrolidin-2-yl)-1-hydroxypropane-l,1-dipho- sphonic acid (trisodium salt); N-{[4-[4-[bis(2-chloroethyl)amino3phenyl3butyroyloxy3- acetyl}-3-(piperidin-4-yl}-1-hydroxypropane-l,1-dipho- sphonic acid (trisodium salt); N-{[4-[4-[bis(2-σhloroethyl)amino3phenyl3butyroyloxy3- acetyl}-3-(piperidin-2-yl)-1-hydroxypropane-l ,1-dipho- sphonic acid (trisodium salt); N-{[4-[4-[bis(2-chloroethyl)amino3phenyl3butyroyloxy3- acetyl}-2-(piperidin-2-yl)-1-hydroxyethane-l,1-dipho- sphonic acid (trisodium salt); N-{[4-[bis(2-chloroethyl)amino3-(L)-phenylalanyloxy3- acetyl}-5-amino-l-hydroxypentane-l,1-diphosphonic acid (monohydrochloride) ; 3-{{4-[4-[bis(2-chloroethyl)amino3-( )-phenylalanylo- xy3acetyl}-piperazin-1-yl}-1-hydroxypropane-l,1-dipho- sphonic acid (monohydrochloride) ; 4-{{4-[4-[bis(2-chloroethyl)amino3-(L)-phenylalanyl¬ oxy3acetyl}-piperazin-1-yl}-1-hydroxybutane-l,1-dipho- sphonic acid (monohydrochloride) ; N-{[[4-bis(2-chloroethyl)amino}-(L)-phenylalanyloxy]- acetyl}-3-(pirrolidin-2-yl)-l-hydroxypropane-l,l-di- phosphonic acid (monohydrochloride); N-{[ [4-bis(2-chloroethyl)amino]-(L)-phenylalanyloxy]- acetyl}-3-(piperidin-4-yl)-1-hydroxypropane-l,1-di- phosphonic acid (monohydrochloride); N-{ [ [4-bis(2-chloroethyl)amino}-(L)-phenylalanyloxy3- acetyl}-3-(piperidin-2-yl)-1-hydroxypropane-l,1-dipho- sphonic acid (monohydrochloride); N-{[ [4-bis(2-chloroethyl)aminoJ-(L)-phenylalanyloxy3- acetyl}-2-amino-l-hydroxyethane-l,1-diphosphonic acid (monohydrochloride).

Claims: CLAIMS 1. Compounds of formula (I) wherein: R, and R 2 , which can be the same or different, are hy¬ drogen or C 1 _c 4 alkyl; (A) is hydrogen, halogen (chlorine, bromine or iodine), .hydroxy, straight or branched C 1 -c 12 alkyl; (B) is a covalent bond, straight or branched C,_c 8 alkylene, an alkylene chain containing at least one he- tero-ato of formula -[CH(CH 3 )] _(CH 2 ) n -X-(CH 2 ) n - or, together with the adjacent nitrogen atom, a group of heterocyclic rings of formulae an ortho, meta or para substituted aralkyl of formula in which chain or groups X is 0, S, N-CH 3 ; m is zero or the integer 1 or 2; m. is the integer 1, 2, 3 or 4; n and n, are an integer from 1 to 5; p is zero or the integer 1; R 3 is hydrogen, straight or branched C,-C,_ alkyl, C 3 ~ c _g cycloalkyl, benzyl, phenyl, or p- methoxybenzyl; (C) is straight or branched C,-C 5 alkylene, phenylene, an aralkyl chain of formula wherein n is as defined above; R 4 is selected from hydrogen, straight or branched C.- C . alkyl, or R. represents a group '12 -N ~ R 13 wherein R._ and which are the same or different, are hydrogen, straight or branched C,-C 8 alkyl, phenyl, benzyl, para-methoxybenzyl, or one of R. 2 and R._ is as defined above and the other one is a group of formula: wherein R, 4 is hydrogen, straight or branched C j -C 4 alkyl, phenyl, benzyl, para-methoxybenzyl, straight or branched C χ _c 4 alkoxyl, halo-C 1 -C 4 alkoxyl; R 5 and Rg are haloethyl (2-chloroethyl, 2-bromoethyl, 2-iodoethyl) or R 5 and Rg, taken together with the ni¬ trogen atom to which they are linked, are a 1-azyri- dinyl group of formula , Rg, Rg e Ri Q ' which are the same or different, taken together with the -N-CH-CO- group to which they are linked, form the residue from a D or L amino acid; - j ^ is hydrogen or straight or branched C 1 -C^ 2 alkyl chain; x is zero or the integers 1 and 2; y and z are zero or the integer 1, with the proviso that: when x, y and z are at the same time zero, (B) is one of the residues of formula and the pharmaceutically acceptable acids, enantiomers, diastereoisomers and racemates thereof. 2. Compounds according to claim 1, wherein R_, R g , R Q and R 10 , together with the -N-CH-CO- group to which they are linked, form the residue from a D or L amino acid selected from: glycine, sarcosine, alanine, phenylalanine, valine, leucine, isoleucine, methionine, proline. 3. Compounds according to claim 1 or 2, wherein (A) is hydroxy. 4. Compounds according to any one of the preceding claims, wherein (B) is a straight C 2 _c 5 alkylene, a branched alkylene of formula -CH-(CH 2 ) r _ wherein r is from 2 to 5, or one of the CH, chains of formula wherein n is from 1 to 4, or, taken together with the adjacent nitrogen atom, one of the chains of formula wherein n is from 1 to 3. 5. Compounds according to any one of the preceding claims, wherein R^ and R 2 are hydrogen. 6. Compounds according to any one of the preceding claims, wherein z is 1 and x and y are zero. 7. Compounds according to any one of claims 1-5, whe¬ rein x and y are different from zero and z is zero. 8. Compounds according to any one of claims 1-5, wherein x is 1 and y and z are zero. 9. A process for the preparation of the compounds ac¬ cording to claims 1-8, which process comprises reacting 38 a compound of formula (II) : R 5 R 4 \ I N-(C)-CH-C—T ^ / II π ° wherein 5 , R g and (C) have the above mentioned mea¬ nings and R' 4 is the same as R 4 or it is a group which can be converted into 4 by removing any protecting groups, T is hydroxy or a carboxy-activating group, with a compound of formula (III) ra wherein R χ , R 2 , R 3 , R g , R 1Q , R^, (A), (B) , x, y and z have the above mentioned meanings and R'_ and R' fl have the same meanings as R ? and g or they can be converted into R 7 and R g by removing any amino-, hydroxy-, thio- and carboxy-protecting groups optionally present, to give a compound of formula (Ia): Ia wherein R 1' c 2 , κ 3 , R« R 7 ', Rg' , Rg, Rχ 0 , R ll' (A), (B), (C), x, y and z have the above mentioned meanings, which compound in its turn can be transformed into a compound of formula (I) by means of known reac¬ tions for the selective removal of protecting groups, alkylation or acylation of amino groups. 10. Pharmaceutical compositions containing as the ac¬ tive ingredient a compound according to claims 1-8, in admixture with a suitable carrier. 11. The use of the compounds according to claims 1-8 for the preparation of an antitumour medicament.

Inventors: LIVI VALERIA, MENTA ERNESTO, SPINELLI SILVANO, TOGNELLA SERGIO, LIVI, VALERIA, MENTA, ERNESTO, SPINELLI, SILVANO, TOGNELLA, SERGIO
Applicants: BOEHRINGER MANNHEIM ITALIA, BOEHRINGER MANNHEIM ITALIA S.P.A.
Application year: 1992
Main classification: C07K5
IPC codes: A61K31, A61K38, A61P35, C07F9, C07K1, C07K5