ITMI961290A1 - CARBOSS DERIVATIVES GEM-DIPHOSPHONATES WITH ANTI-TUMOR ACTIVITY, A PROCESS FOR THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS THAT - Google Patents

Description

Description of the industrial invention entitled: "DERIVATIVES OF CARBOSS GEM-DIPHOSPHONATES WITH ANTI-TUMOR ACTIVITY, A PROCESS FOR THEIR PREPARATION

AND PHARMACEUTICAL COMPOSITIONS THAT CONTAIN THEM ''

The present invention relates to conjugates of gem-diphosphonic carboxy acids with alkylating agents. These derivatives have a remarkable antitumor activity and a specific activity on bone resorption. The present invention also relates to a process for their preparation and pharmaceutical compositions containing them.

The skeletal system is the third most common site of metastasis, and more than 80% of all cancer patients have bone tumors at autopsy.

Bone metastases largely affect the pathology of cancer patients, causing imbalances in calcium metabolism and damage to the bone marrow, and are responsible for serious consequences in patients, such as pain, pathological fractures, spinal compression and hypercalcemia (Drew et al., Osseous complication of malignancy, Lokich J.J. ed. Clinical cancer medicine: treatment tactics Boston: G.K. Hall Medicai Publisher, 1980, 97-112).

Bone lesions are therefore one of the most important features associated with bone tumors. It is known that most local lesions caused by bone metastasis are due to direct effects on the mineralized matrix or an indirect effect of tumor-stimulated bone resorption. The bone resorption process is predominantly mediated by multinucleated osteoclasts and causes the release of bone mineral and the degradation of the bone matrix. Osteoclasts resorb bone through a specialized area of the cell membrane known as the "ruffled border". Bone resorption is associated with the release of lysosomal enzymes and collagenase from osteoclasts, as well as with the local production of acid which is responsible for the release of mineral from bone (Mundy, G.R., Bone resorption and turnover in health and disease, Bone , 1987, 8, S9-S16).

It is therefore evident that the discovery of a drug capable of inhibiting both tumor growth and bone destruction is a primary goal in anticancer research.

Gem-diphosphonic acids and their salts 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, DE 3.512.536). However, for none of the above compounds an antitumor activity is described.

DE 3,425,812 (Blum et al.) Describes derivatives of 1,1-diphosphonic acids, characterized by a bis [(haloalkyl) amino] phenyl residue, which are useful agents for the treatment of bone tumors. In fact, the typical cytotoxic activity of molecules having dialkylating functions is added to the bone tropism characteristic of diphosphonic acids. However, no activity on bone resorption is described.

In WO 88/06158, diphosphonic analogs of methotrexate are also described as useful agents for the treatment of bone problems.

It is therefore evident that none of the compounds mentioned above has been shown to be effective in acting simultaneously both as an antitumor agent and as an inhibitor of bone resorption. In particular, the compounds described in DE 3,425,812, despite being conjugated between a bisphosphonate, active on bone resorption, and an alkylating agent, active as an antitumor, only retained this latter activity. This shows that for them the activity of the final compound cannot be automatically determined by simply sowing the respective activities of the two starting intermediates.

WO 92/18512, on the other hand, claims conjugates between bis-phosphonic acids and alkylating agents characterized in that the two molecules are joined by amino acid groups. The amide bond, presumably hydrolysable by metabolism, is said to be responsible for the activity of these compounds, which retain their efficacy both as anticancer and as bone resorption inhibitors. However, these molecules are not able to completely inhibit bone resorption, especially long after treatment (see Table).

We have now found that the compounds of general formula (I):

NHCO (CH2) n

in which:

R and R1 represent halogen-ethyl (2-chloroethyl, 2-bromoethyl, 2-iodoethyl) or, taken together with the nitrogen atom to which they are bound, represent a 1-aziridinyl residue of formula

(A) is (C1-C5) linear or branched alkylene, phenylene or an aralkyl chain of formula

where m is an integer between 1 and 5;

n is an integer between 1 and 6;

R2 is hydrogen or hydroxyl;

R3 is hydrogen or (C1-C4) alkyl;

R4 represents hydrogen or (C1-C4) alkyl,

they are endowed with remarkable antitumor activity and at the same time they are able to totally inhibit bone resorption even long after treatment.

Scalo included in the scope of the present invention are the diastereomers, the racemates and the pure enantiomers of the compounds of formula (I).

Also included in the present invention are the pharmaceutically acceptable salts of the compounds of formula (I), for example with inorganic bases such as the salts of alkaline metals (for example sodium or potassium) or alkaline earth metals (for example calcium or magnesium) or the salts of ammonium; salts with organic bases such as methylamine, ethylamine, propylamine, isopropylamine, butylamine, t-butylamine, dimethylamine, diethylamine, diethanolamine, trimethylamine, triethylamine, piperidine, pyridine, picoline, dicyclohexylamine; salts with inorganic or organic acids such as for example hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, formic, acetic, trifluoroacetic, maleic, fumaric, tartaric, methanesulfonic or paratoluenesulfonic acids; salts with amino acids such as for example segregates, glutamates or salts of lysine or arginine.

R and R1 are preferably a 2-halogenethyl group;

(A) is preferably a group of formula

more preferably a formula group

n is preferably the integer 2 or 3;

R2 is preferably hydroxyl;

R3 and R4 are preferably hydrogen.

Particularly preferred compounds are those in which (A) is a group of formula

R3 and R4 are hydrogen.

Even more particularly preferred compounds are those in which R e are a 2-chloroethyl group, (A) is a group of formula

n is the integer 2 or 3 and R3 and R4 are hydrogen.

The compounds of general formula (I) can be prepared according to a process which involves the condensation reaction of a compound of formula (II):

in which R, R1, (A) and R4 have the meanings reported above, with a bisphosphonate of formula (III):

in which n has the above reported meaning, R2 'has the above reported meanings or is an O-G group, in which G is a suitable protective group of a tertiary alcohol; R3 'has the above meanings with the exception of hydrogen; T is hydroxyl or a group activating the carboxyl function, obtaining compounds of formula (I ');

which can then be converted into the compounds of formyl (I) by means of unblocking reactions of the optionally present protective groups and / or by possible hydrolysis of the phosphonic esters to give the corresponding phosphonic acids and possible salification of the compounds obtained with pharmaceutically acceptable acids or bases.

Any diastereomers of the compounds of formula (I) can be separated by selective crystallization or by purification by liquid chromatography.

Any enantiomers of the compounds of formula (I) can be separated from racemic mixtures according to optical resolution methods known to the skilled in the art, when not directly obtained by synthesis starting from optically active reagents.

The protective groups referred to in the present invention are all the protective groups of an alcoholic or carboxylic oxygen atom, such as for example ethers, esters and silyl derivatives.

Preferred species of protective groups G are silyl ethers and in particular tert-yldimethyl silyl ether.

These protective groups can be removed by means of reactions well known to those skilled in the art, such as release under basic conditions in the case of esters and release under acid conditions in the case of ethers and silyl derivatives. The phosphonic esters can be hydrolyzed with particular and selective agents, such as for example trimethylsilyliliodide.

When in the reaction of the compounds of formula (II) with the compounds of formula (III) the latter are used in the form of carboxylic acids (T = OH), the reaction is generally carried out in the presence of a condensing agent such as N, N'- dicyclohexylcarbodiinmide, N-cyclohexyl-N '-morpholinoethyl-carbodiimide, N-ethyl-N' - (3-dimethylamino) -propyl-carbodimide, N, N'-carbonyl-bis (imidazole), phosphorus oxychloride, phosphorus trichloride, thionyl chloride, oxalyl chloride, ethyl chloroformate, isobutyl chloroformate, morpholinoethyl isonitrile or the like.

When T is instead a group activating the carboxyl function, examples of -C (= 0) T groups are acyl halides, symmetrical or mixed anhydrides (for example with methanesulfonic, acetic, isobutyric, pivalic, trifluoroacetic acid); activated amides (e.g. with imidazole, 1,2,4-triazole); azide; activated esters (e.g., paranitrophenyl ester, methoxy methyl ester, 2,4-dinitrophenyl ester, pentachlorophenyl ester, hydroxysuccinimido ester, 1-hydroxy-2- (1H) -pyridone ester, 1-hydroxybenzotriazole ester) and the like.

A particularly preferred activating group T is the hydroxysuccinimidyl group of formula

The condensation reaction of the compounds of formula (II) with the compounds of formula (III) can be carried out in the presence of an inorganic base such as an alkaline carbonate or bicarbonate, an alkaline or alkaline-earth hydroxide or an organic base such as triethylamine, tributylamine, pyridine, 4-dimethylamino-pyridine, N-alkylmorpholine, Ν, Ν-dialkylaniline or the like. The pH is preferably maintained not above pH = 9.

The reaction temperature can vary between -40 ° C and the boiling temperature of the solvent, depending on the activating group selected, preferably between -10 ° C and 50 ° C.

The preferred solvents are inert organic solvents, such as pyridine, Ν,-dìmethylformamide or acetonitrile, or their mixtures with water in various proportions.

Reaction times vary according to the activating group and substrate selected and can range from 30 minutes to 48 hours.

Particularly preferred reaction conditions are those which provide for the use of triethylamine, in molar excess with respect to the reactants, in a water / acetonitrile mixture in a ratio of 1:10 and at a temperature between 0 ° C and room temperature.

The compounds of formula (II) are known compounds, commercially available and / or prepared with methods known to those skilled in the art, such as for example those described in: J. Med. Chem., 24, 1304 (1981); CA 51: 8066d (1957); BE 905.974; CA 104: 141897 (1986); J. Med. Chem., 7, 468 (1964); J. Med. Chem., 6, 85 (1963); Cancer Chem. Rep., 50, 685 (1966); J. Med. Chem., 21, 16 (1977); J. Org. Chem., 26, 1554 (1961); J. Org. Chem., 26, 1674 (1961); CA 64: 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); Int. J. Pept. Protein Res., 36, 308 (1990).

The compounds of formula (III) in which R2 'is hydrogen and T is an OH group are known compounds or can be prepared according to experimental methods known to the skilled in the art [Synthesis, 661 (1991); Phosphorus, Sulfur, Silicon Relat. Blem., 88 (1-4), 1-13 (1994); FR 2683527; Zh. Obshch. Khim., 61 (12), 2698 (1991); Bioorg. Khim., 12 (9), 1282 (1986); J. Organometal. Chem., 13 (1), 199 (1968)].

The compounds of formula (III) in which R2 'is O-G (in which G is hydrogen or a suitable protective group) and T is an OH group can be synthesized according to the following procedure which comprises the steps of:

(a) reacting equimolar quantities of a cyclic anhydride of a suitable dicarboxylic acid and of an alcohol of formula R5-OH, in which Rg is an alkyl with from 1 to 4 carbon atoms or is a benzyl group, possibly substituted, or allyl, obtaining the intermediate of formula (IV):

The reaction is carried out in an inert solvent, preferably dimethylformamide, in the presence of a base, preferably pyridine, and at temperatures between room temperature and the boiling point of the reaction mixture;

(b) suitably activating the non-esterified carboxylic group present in the intermediate (IV), transforming it, for example, into an acyl halide, an anhydride or an activated ester such as hydroxy-succinimido ester. A preferred example of activation of the carboxy group of intermediate (IV) is its transformation into acyl chloride by means of thionyl chloride;

(c) reacting the intermediate obtained in point (b) with a trialkyl phosphite P (OR3 ') 3 (preferably trimethyl phosphite) in an inert solvent such as chloroform and at a temperature between -10 ° C and room temperature, obtaining the intermediate of formula (V):

(d) reacting the intermediate of formula (V) with a dialkyl phosphite HP (O) (OR3 ') 2 (preferably dimethyl phosphite) in the presence of a base, preferably an organic base such as dialkyl or trialkyl amines, obtaining the intermediate of formula (VI):

(e) possibly protecting the OH group with an appropriate protective group selected from those suitable for a tertiary alcohol; (f) deprotect the -COOR5 ester group by hydrolysis reaction in a basic or acid environment and in conditions in which the other protective groups R3 'and the one possibly present on the alcoholic function are not touched or, in the case in which R5 is a benzyl group or allyl, by catalytic hydrogenation reaction or in the presence of hydrogen donors such as ammonium formed or sodium hypophosphite.

A particularly advantageous alternative process for obtaining the intermediates of formula (III) in which R2 'is a -0-silyl group is that which envisages in step (d) the use, instead of the dialkyl phosphite, of a dialkyl silyl phosphite of formula (VII):

obtaining directly the intermediate of formula (III '):

in which the groups R6 can be the same or different. A particularly preferred silyl group is the tert-butyl dimethyl silyl group.

This procedure allows to obtain with a single reaction the intermediate already protected on the alcoholic oxygen, avoiding the oxygen protection passage (s).

The intermediates of formula (III) in which both T and R2 'represent OH groups are known (DE 2117880; NL 6610762).

The intermediates of formula (III) in which T is an OH group and R2 'is a -O-Si (R6) 3 group are new.

A further object of the present invention are therefore the intermediates of formula (III) with T = OH and R2 '= -O-Si (R6) 3, or the intermediates of formula (VIII):

in which n and R3 'have the meanings reported above and the groups R6, which can be the same or different, are selected from the group comprising (C1-C4) alkyl, linear or branched, or phenyl.

A particularly preferred meaning of Si (R6) 3 group is terzbutyl dimethyl silyl.

The derivatives of formula (III) in which T is a suitable activating group are obtained from the derivatives with T = OH by means of reactions well known to those skilled in the art.

The compounds of the invention were tested "in vivo" on the Walker 256 / B breast carcinoma of the rat implanted intratibia (it). Inoculated in the bone marrow cavity of the tibia, this tumor grows inside the bone giving osteolytic lesions, paraneoplastic hypercalcemia and invades the surrounding tissues, producing a measurable tumor mass. It is therefore possible to measure both the anti-tumor activity on the extraosseous tumor and the anti-osteolytic effect (Cancer, 72 (1), 91 (1993)).

Walker 256 / B tumor was obtained from NCI Frederick Cancer Facility and maintained in CD1 male rats by subcutaneous transplantation of tumor fragments of approximately 1 era in diameter every 10 days.

To carry out the experiment, each rat was injected with 2.5x10® tumor cells; the animals were anesthetized with a mixture of 10 mg / kg of Ketalar (Park-Davis) and 5 mg / kg of Rompun (Bayer). Thus transplanted into the tibia, the tumor grows producing osteolytic lesions associated with increased osteoclast activity. To obtain an injectable cell suspension, the tumor fragments were disrupted by enzymatic digestion using collagenase type IV (Sigma) at a concentration of 400 U / ml for 20 minutes at 37 ° C.

The compounds of the invention were administered i.v. on days 1, 4 and 7 following and tumor transplantation. Extraosseous tumor mass was measured at day 14 after tumor transplantation. The antitumor activity was determined as TWI% (tumor weight inhibition%) calculated according to the following formula:

TWI% = (100 - TW average treated anim. / TW averagecontrolli) x 100

where the TW for each animal is given by the formula:

where a and b are respectively the maximum and minimum diameter of the tumor mass in mm.

Osteolytic lesions were radiologically assessed at days 8 and 16 following tumor transplantation. The animals were anesthetized, placed in the prone position on an X-ray film (X-OMAT MA, Kodak) and exposed to X-rays at 25 Kv for 30 seconds using a Radiolight instrument (Gilardoni S.p.A.). The degree of bone lesions was determined based on the following score:

absence of injuries

+/- low inhomogeneity

+ high inhomogeneity

++ isolated lesions

+++ extensive injuries

Based on this score, the Destruction Index (DI) was calculated:

DI = no. of injuries x degree of injury / no. total animals

DI values less than or equal to 1 indicate good bone protection. A value of DI = 0, on the other hand, indicates a complete absence of injuries and therefore total protection or shelter.

The compounds of the invention were tested on this experimental model in comparison with the compounds of the prior art and proved to be more active not only as regards the antitumor activity on the extraosseous tumor, but also as anti-osteolytic agents. The table shows the comparison data for a representative molecule of the compounds of formula (I).

Table

Antitumor activity of 4- [bis (2-chloroethyl) amino] -N- (5,5-diphospho-5-hydroxy-1-pentanoyl) - (L) -phenylalanine trisodium salt (example 1), 4- [4- (bis (2-chloroethyl) amino) phenyl] -1-hydroxybutane-1, 1-diphosphonic acid, trisodium salt (compound A, Blum et al.), N - [[4- (bis (2-chloroethylamino)) ) - (L) -phenylalanyl] - (L) -alanyl] -4-amino-1-hydroxybutane-1,1-diphosphonic acid trisodium salt (compound B, WO 92/18512) on rat breast cancer Walker 256 / B (it / iv; days 1,4,7).

Already at a dose of 20 mg / kg, the compound of the invention shows a

continuous tendency to shelter from bone lesions, with values of DI which

they pass from 0.75 after 8 days to 0.25 after 16 days. At the same dose,

on the other hand, compound B shows an opposite trend with the passage of

time, resulting in an increase in the destruction rate on day 16.

This goes to show that compound B is unable to cause a

real long-lasting repair of the bone tissue.

The compounds of the invention were also found to be active in a

experimental model of human multiple myeloma on the animal from

laboratory.

Human multiple myeloma is a cancer that affects the cells of the

plasma, sensitive to alkylating agents such as melphalan. Now I am

often associated with bone damage that causes pain and fractures.

It is therefore evident that a drug capable on the one hand of delivering

the alkylating agent to the bone and on the other hand to preserve or repair the bone from the damage produced by the tumor is an extremely interesting goal.

The experiment is performed by intravenous (i.v.) inoculating HS-Sultan human multiple myeloma cells into SCID (immunodeficient) mice on day 0, followed by i.v. with a compound of the invention on days 15, 18 and 21.

The activity of the compounds of the invention was evaluated as a function of the following parameters:

Mean Survival Time (MST) compared to untreated mice (which have a mean survival time of 30 days from tumor inoculation);

evaluation of histological parameters (spinal cord invasion and extent of osteolytic lesions) with the methods described above. The compounds of the invention are endowed with a not high acute toxicity and are well tolerated in animals.

The high solubility in water of the compounds of the invention allows the preparation of the parenteral and oral pharmaceutical forms.

The compounds of formula (I), when administered to humans and animals carrying tumors susceptible to treatment with alkylating agents, in doses ranging from 1 mg to 1200 mg per square meter of body surface, are capable of inducing the regression of the aforementioned tumor forms. and to favor the repair of the bone tissue, thus avoiding the pathological manifestations connected with bone lesions.

The effective dosage of the compounds of the invention can be determined by an expert clinician with conventional and known methods.

The correlation between the dosages used for animals of various species and humans (based on mg / m <2> of body surface area) is described by Freirich, E.J. et al., Cancer Chemother. Rep., 50, no.4, 219-244, May 1966.

Tumors treatable with the compounds of the present invention are those sensitive to therapy with alkylating agents.

In particular, multiple myeloma, osteosarcoma, bone metastases, carcinoma of the breast, ovaries and testis can be advantageously treated.

The pharmaceutical compositions containing the compounds of formula (I) are included in the invention. These pharmaceutical compositions can contain any quantity of compounds of formula (I) capable of exerting an antitumor activity in mammals against tumors sensitive to therapy with alkylating agents.

The pharmaceutical compositions may contain, in addition to at least one compound of formula (I), pharmaceutically compatible excipients, so as to allow administration by any route, such as oral, parenteral, intravenous, endodermal, subcutaneous or topical, in liquid or solid.

One method of administration of the compounds of formula (I) is by the oral route. Oral compositions will generally include an inert diluent or an edible carrier. They can be included in gelatin capsules or tablets in tablets. Other forms of oral administration are capsules, pills, elixirs, suspensions or syrups.

The tablets, pills, capsules and similar compositions may contain the following ingredients (in addition to the active ingredient): a binder such as microcrystalline cellulose, tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, primogel, corn starch and the like; a lubricant such as magnesium stearate; a fluidifier such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin or a flavoring agent such as mint flavor, methyl salicylate or orange flavor. When the selected composition is in capsule form, it may additionally contain a liquid carrier such as a fatty oil. Other compositions may contain various materials which alter their physical form, such as coating agents (for tablets and pills) like sugar or shellac. The materials used in the preparation of the compositions must be pharmaceutically pure and non-toxic at the dosages used.

For the preparation of pharmaceutical compositions for parenteral administration, the active ingredient can be incorporated in solutions or suspensions, which may additionally include the following components: a sterile diluent such as water for injections, saline, oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for adjusting the tonicity of the solution such as sodium chloride or dextrose. The parenteral preparation can be included in ampoules, disposable syringes, or glass or plastic vials.

The invention is further described by the following examples and preparations.

PREPARATION 1 - mono-benzyl glutaric acid

A solution of glutaric anhydride (22.8 g) in benzyl alcohol (20.7 ml), anhydrous pyridine (250 ml) and dimethylformamide (25 ml) is heated under reflux for about 4 hours and 30 minutes, then the solvent is evaporated under reduced pressure and the residue is taken up with 500 ml of ethyl acetate. The organic phase is then washed with 1 N hydrochloric acid (2 x 100 ml) and with water (3 x 100 ml), then it is basified with 200 ml of 1 N sodium hydroxide and with 200 ml of sodium bicarbonate in saturated solution. The organic phase is separated, while the aqueous phase is neutralized by the addition of 37% hydrochloric acid and extracted with methylene chloride (400 ml). The organic extracts are dried on sodium sulphate and the solvent is evaporated under reduced pressure, obtaining 32.8 g of product.

PREPARATION 2 - mono-benzyl glutaroyl chloride

A solution of mono-benzyl glutaric acid (32.8 g) in 250 ml of anhydrous toluene is added with 21.5 ml of thionyl chloride, then heated at 65 ° C for 1 hour. The solvent is evaporated under reduced pressure and the residue is taken up twice with toluene and brought to dryness. 35 g of product are obtained as rosé oil.

PREPARATION 3 - 5- (dimethoxyphosphinoyl) -5-ketopentanoic acid, benzyl ester

35 g of mono-benzyl glutaroyl chloride are dissolved in 150 ml of anhydrous chloroform and the solution is cooled to 0 ° C. 18 ml of phosphite trimeters are dripped slowly (over the course of 1 hour and 20 minutes), then the temperature is brought to room temperature. After another hour the solvent is evaporated under reduced pressure obtaining 45 g of product as oil.

PREPARATION 4 - (tert-butyldimethylsilyl) -dimethyl phosphite

To a suspension of 6.4 g of sodium hydride (60% in oil) in 255 ml of anhydrous tetrahydrofuran, cooled to 0 ° C, 13.4 ml of dimethyl phosphite are added dropwise, then the reaction mixture is heated under reflux for 2 hours and 30 minutes. After this time, the temperature is brought to room temperature and 20 g of tert-butyl dimethylsilyl chloride are added. The reaction mixture is again heated under reflux for a total of 16 hours, then the separated sodium chloride is filtered and the solvent is evaporated under reduced pressure. 23.62 g of product are obtained.

PREPARATION 5 - 5,5- (bis-dimethoxyiosphinoyl) -5- (terzbutyldimethylsilyloxy) pentanoic acid, benzyl ester

12.8 g of (tert-butyldimethylsilyl) -dimethyl phosphite are dropped into 5- (dimethoxyphosphinoyl) -5-ketopentanoic acid, benzyl ester (18 g), cooled to 10 ° C. Once the addition is complete, it is brought to room temperature. After 4 hours the reaction mixture is dissolved in 500 ml of ethyl acetate and the organic phase thus obtained is washed first with 5% sodium bicarbonate (3 x 70 ml), then with water (3 x 70 ml), then with potassium 5% carbonate (3 x 70 ml) and finally with saturated sodium chloride solution (1 x 70 ml). The organic phase is dried on sodium sulphate and the solvent is evaporated under reduced pressure, obtaining 25.9 g of crude which is purified by chromatography on silica gel (eluent AcOEt - AcOEt / acetone 1: 1) to give 17 g of product pure as oil.

PREPARATION 6

According to the methods described in preparations 1-5, starting from the appropriate reagents and using the appropriate trialkylsilyl halides, the following esters are prepared:

5,5- (bis-dimethoxyphosphinoyl) -5- (tert-butyldimethylsilyloxy) pentanoic acid, allyl ester;

4,4- (bis-dimethoxyphosphinoyl) -4- (tert-butyldimethylsilyloxy) butanoic acid, benzyl ester;

5,5- (bis-diethoxyphosphinoyl) -5- (tert-butyldimethylsilyloxy) pentanoic acid, benzyl ester;

5,5- (bis-dimethoxyphosphinoyl) -5- (trimethylsilyloxy) pentanoic acid, benzyl ester;

3,3- (bis-dimethoxyphosphinoyl) -3- (tert-butyldimethylsilyloxy) propanoic acid, allyl ester;

8.8- (bis-dimethoxyphosphinoyl) -8- (tert-butyldimethylsilyloxy) octanoic acid, benzyl ester;

7,7- (bis-dimethoxyphosphinoyl) -7- (trimethylsilyloxy) heptanoic acid, benzyl ester;

5,5- (bis-diethoxyphosphinoyl) -5- (phenyldimethylsilyloxy) pentanoic acid, benzyl ester;

6,6- (bis-dimethoxyphosphinoyl) -6- (phenyldimethylsilyloxy) hexanoic acid, benzyl ester.

PREPARATION 7 - 4,4-bis (diethoxy phosphinoyl) butanoic acid, N-hydroxysurcinimido ester

To a solution of 4,4-bis (diethoxy phosphinoyl) butanoic acid (1.28 g; prepared as described in Synthesis, 661-2 (1991)) and N-hydroxy succinimide (0.613 g) in 35 ml of tetrahydrofuran 0.636 ml of morpholinoethyl isonitrile are added dropwise, maintaining the temperature at about 0 ° C. The temperature is brought to room temperature and the reaction mixture is left under stirring for 20 hours. After this time, the solvent is evaporated under reduced pressure and the residue is redissolved in 70 ml of ethyl acetate and washed first with water (1 x 40 ml), then with 1 N hydrochloric acid (1 x 40 ml) and with a saturated solution of sodium chloride (2 x 40 ml). The organic phase is dried over sodium sulphate and the solvent is evaporated under reduced pressure to give 1.3 g of product.

EXAMPLE 1 - 5,5-bis (dimethoxyphosphinoyl) -5- (tert-butyldimethylsilyloxy) pentanoic acid

To a solution of 5,5- (bis-dimethoxyphosphinoyl) -5- (terzbuthyldimethylsilyloxy) pentanoic acid, benzyl ester (4.5 g; preparation 5) in 110 ml of dimethoxyethane are added in 0.9 g portions of palladium on carbon at 10%. The reaction mixture is hydrogenated at atmospheric pressure and at room temperature for 2 hours 45 minutes (consumption of 0.372 ml of hydrogen). The catalyst is then removed by filtration on celite and the solvent is evaporated under reduced pressure. 4 g of product are obtained as colorless oil.

N.M.R. (200 MHz) in CDCl3: 3.85 ppm (m, 6H); 1.85-2.4 ppm (m, 6H); 0.9 ppm (s, 9H); 0.2 ppm (s, 6H).

EXAMPLE 2

According to the method described in example 1, starting from the esters of preparation 6, the following acids are obtained:

4,4- (bis-dimethoxyphosphinoyl) -4- (tert-butyldimethylsilyloxy) butanoic acid;

5,5- (bis-diethoxyphosphate) -5- (tert-butyldimethylsilyloxy) pentanoic acid;

5,5- (bis-dimethoxyphosphinoyl) -5- (trimethylsilyloxy) pentanoic acid; 3,3- (bis-dimethoxyphosphinoyl) -3- (tert-butyldimethylsilyloxy) propanoic acid;

8.8- (bis-dimethoxyphosphinoyl) -8- (tert-butyldimethylsilyloxy) octanoic acid;

7,7- (bis-dimethoxyloxy) -7- (trimethylsilyloxy) heptanoic acid; 5,5- (bis-diethoxyphosphate) -5- (phenyldimethylsilyloxy) pentanoic acid; 6,6- {bis-dimethoxyphosphinoyl) -6- (phenyldimethylsilyloxy) hexanoic acid. ESBMPIUM 3 - 4- [bis (2-chloroethyl) amine] -N- [4 ', 4'-bis (diethoxyphosphinoyl) -1' -butanoyl] - (L) -phenylalanine

To a suspension of 4- [bis (2-chloroethyl) ansnino] - (L) -phenylalanine (melphalan; 0.78 g) in 50 ml of dimethoxyethane and 0.575 ml of triethylamine an acid solution 4.4 is added dropwise -bis (diethoxy phosph inoyl) butanoic, N-hydroxysuccinine starter ester (1.3 g; preparation 7) in 10 ml of dimethoxyethane. After 24 hours at room temperature under stirring, all the solid passed into solution. The solvent is evaporated under reduced pressure and the residue is redissolved in ethyl acetate (100 ml) and washed with 0.5 N hydrochloric acid (2 x 60 ml) and with saturated sodium chloride solution (2 x 60 ml). The organic phase is dried over sodium sulphate and the solvent is evaporated under reduced pressure, obtaining 1.6 g of residue, which is purified by chromatography on silica gel (eluents methylene chloride / methanol / acetic acid 95: 5: 1, then methylene chloride / methanol / acetic acid 90: 10: 1). 1.25 g of product are obtained.

N.M.R. (200 MHz) in CDCl3: 7.12 ppro (d, 1H); 7 ppm (d, 2H); 6.55 ppm (d, 2H); 4.72 ppm (dd, 1H); 4.2 ppm (m, 8H); 3.6 ppm (m, 8H); 3 ppm (dd, 1H); 2.8 ppm (dd, 1H); 2.1-2.7 ppm (m, 4H); 1.35 ppm (m, 12H).

EXAMPLE 4 - 4- [bis (2-chloroethyl) amino] -N- [4 <,> 4 '-diphospho-1'-butanoyl] - (L) -phenylalanine disodium salt

2.24 g of trimethylsilyl iodide are added dropwise to a solution of the product of Example 3 (1.1 g) in 35 ml of chloroform, maintaining the temperature at about 0 ° C. After 2 hours at 0 ° C and 1 hour at room temperature, the solvent is evaporated under reduced pressure and the residue is redissolved in water (80 ml). The pH of the solution is adjusted up to pH = 5 by the addition of 1N sodium hydroxide, then by addition of ethanol an oil is separated which is separated by decantation. The oil thus obtained is crystallized from methanol, obtaining 0.9 g of product.

[a] D = -2.6 ° (c = 1.05% in HCl 1 N)

N.M.R. (200 MHz) in D2O: 7.2 ppm (d, 2H); 6.85 ppm (d, 2H); 4.37 ppm (dd, 1H); 3.75 ppm (s, 8H); 3.07 ppm (dd, 1H); 2.85 ppm (dd, 1H); 2,452.6 ppm (m, 2H); 1.75-2.25 ppm (m, 2H).

EXAMPLE 5

By following the methods described in preparation 7 and in examples 3-4, starting from the appropriate reagents, the following gerrvdiphosphonic acids are obtained:

4- [bis (2-chloroethyl) amino] -N- [5 ', 5'-diphospho-1' -pentanoyl] - (L) -phenylalanine disodium salt;

4- [bis {2-chloroethyl) amino] -N- [8 ', 8'-diphospho-1-octanoyl] - (L) -phenylalanine disodium salt;

4- [bis {2-bromoethyl) amino] -N- [4 ', 4'-diphospho-1 '-butanoyl] - (L) -f enylalanine disodium salt;

4- [bis (2-chloroethyl) amino] -N- [3 ', 3'-diphospho-1 '-propanoyl] - (L) -phenylalanine disodium salt;

4- [N '-aziridinyl] -N- [4', 4 '-diphospho-1'-butanoyl] - (L) -phenylalanine disodium salt.

EXAMPLE 6 - 5,5-bis (dimethoxyphosph inols) -5- (tert-butyldimethylsilyloxy-pentanoic acid, K-hydroxysuccin starch ester

To a solution of 5, 5-bis (dimethoxyphosphineoyl) -5- (terzbuthyldimethylsilyloxy) pentanoic acid (3.7 g; example 1) in 82 ml of anhydrous tetrahydrof urane are added, in a nitrogen atmosphere and at 0 ° C, 1.42 g of N-hydroxysuccinimide. A solution of morpholinoethyl isonitri le (1.73 g) in 1 ml of tetrahydrofuran is added by dropping, then the temperature is brought to room temperature and the reaction mixture is kept for 20 hours under stirring. The solvent is then evaporated under reduced pressure and the residue is redissolved in ethyl acetate (100 ml), then the organic phase is washed first with hydrochloric acid I N (1 x 50 ml), then with saturated sodium bicarbonate solution (1 x 50 ml), finally with saturated sodium chloride solution (1 x 50 ml). The organic phase is dried over sodium sulphate and the solvent is evaporated under reduced pressure, obtaining 4 g of product.

ESSÌPIO 7 - 4- [bis (2-chloroethyl) amine] -N- [5 ', 5' -bis (dimethoxyphosphinoyl) -5 '- (tert-butyldimethylsilyloxy) -1' -pentanoyl] - (L) -phenylalanine

To a suspension of 4- [bis (2-chloroethyl) amino] - (L) -f enylalanine (1.85 g; melphalan) in 5.6 ml of water, 28 ml diacetonitrile and 1.24 g of sad amine is added by slow dripping (about 1 hour) a solution of the product of Example 6 (3 g) in 28 ml of acetonitrile. At the end of the dripping, the reaction mixture is kept under stirring at room temperature for 40 minutes, then the solvent is evaporated under reduced pressure and the residue is redissolved in 100 ml diethyl acetate. The organic phase is washed with 1 N hydrochloric acid (2 x 40 ml) and with water (2 x 40 ml), then it is anhydrified on sodium sulphate and the solvent is evaporated under reduced pressure. 3.7 g of product are obtained.

N.M.R. (200 MHz) in d5 ~ DMS0: 12 ppm (b, 1H); 8.1 ppm (d, 1H); 7.05 ppm (d, 2H); 6.65 ppm (d, 2H); 4.32 ppm (m, 1H); 3.6-3.85 ppm (m, 20H); 2.65-3 ppm (m, 2H); 1.6-2.2 ppm (m, 6H); 0.85 ppm (s, 9H); 0.15 ppm (s, 6H).

EXAMPLE 8 - 4- [bis (2-chloroethyl) amino] -ii- [5 ', 5'-diphospho-5' - (terzbuthyldimethylsilyloxy) -1 '-pentanoyl] - (L) -phenylalanine

To a solution of the product of Example 7 (2.7 g) in 20 ml of anhydrous acetone tryl, kept under stirring in a nitrogen atmosphere and at 0 ° C, 5 g of trimethylsilyl bromide are added. The temperature is then brought to room temperature and the stirring is maintained for a total of 4 hours 30 minutes. The solvent is evaporated under reduced pressure and the residue is partitioned into water (100 ml) and ethyl acetate (100 ml). The organic phase is separated and anhydrified on sodium sulphate, then the solvent is evaporated under reduced pressure, obtaining 1.88 g of product.

N.M.R. (200 MHz) in d6-DMS0: 8.06 ppm (d, 1H); 7.05 ppm (d, 2H); 6.65 ppm (d, 2H); 4.3 ppm (m, 1H); 3.7 ppm (s, 8H); 2.65-3 ppm (m, 2H); 1.65-2.2 ppm (m, 6H); 0.9 ppm (s, 9H); 0.2 ppm (s, 6H).

EXAMPLE 9 - 4- [bis {2-chloroethyl) amino] -N- [5 ', 5'-diphospho-5' -hydroxy-1 '-pentanoyl] - (L) -fem Ίalanine trisodium salt

1.65 ml of 37% hydrochloric acid are added to a solution of 2.7 g of the product of Example 8 in 105 ml of acetonitrile and the reaction mixture is left under stirring at room temperature for 28 hours. The reaction mixture is brought to dryness and the residue is divided between 90 ml of water and 100 ml of ethyl acetate. The separated aqueous phase is treated with a 20% sodium hydroxide solution up to pH = 4.8, then the opalescent solution is cooled to 0 ° C and added with 250 ml of ethanol.

A white solid is separated and left at 0 ° C under stirring for about 50 minutes. After filtration and drying under vacuum at 50 ° C, 1.55 g of product are obtained.

N.M.R. (200 MHz) in D2O DC1 (pH = 1): 7.55 ppm (s, 4H); 4.67 ppm (dd, 1H); 4.1 ppm (t, 4H); 3.65 ppm (t, 4H); 3.35 ppm (dd, 1H); 3.05 ppm (dd, 1H); 1.7-2.35 ppm (m, 6H).

EXAMPLE 10

According to the methods described in Examples 6-9, starting from the intermediates of Example 2 and the suitable substituted amino acids, the following gem-diphosphonic acids are prepared:

4- [bis (2-chloroethyl) amino] -N- [4 ', 4'-diphospho-4'-hydroxy-1'-butanoyl] - (L) -phenylalanine, trisodium salt;

4- [bis (2-chloroethyl) amino] -N- [3 ', 3'-diphospho-3'-hydroxy-1'-propanoyl] - (L) -phenylalanine, trisodium salt;

4- [bis (2-chloroethyl) amino] -N- [8 ', 8'-diphospho-8'-hydroxy-1'-octanoyl] - (L) -phenylalanine, trisodium salt;

4- [bis (2-chloroethyl) amino] -N- [7 ', 7'-diphospho-7'-hydroxy-1'-heptanoyl] - (L) -phenylalanine, trisodium salt;

4- [bis (2-chloroethyl) amino] -N- [6 ', 6'-diphospho-6'-hydroxy-1'-hexanoyl] - (L) -phenylalanine, trisodium salt;

4- [N'-aziridinyl] -N- [5 ', 5'-diphospho-5'-hydroxy-1'-pentanoyl] - (L) -phenylalanine, trisodium salt;

4- [bis {2-chloroethyl) amino] -N- [4 ', 4'-diphospho-4'-hydroxy-1'-butanoyl] - (L) -phenylalanine, methyl ester, disodium salt;

4- [bis (2-chloroethyl) amino] -N- [3 ', 3'-diphospho-3'-hydroxy-1'-propanoyl] - (L) -phenylalanine, ethyl ester, disodium salt.

EXAMPLE 11

In accordance with the methods described in Examples 6, 7 and 9, starting from the intermediates of Example 2 and the appropriate substituted amino acids, the following esters of bis-phosphonic acids are prepared:

4- [bis (2-chloroethyl) amino] -N- [3 ', 3' - (bis-dimethoxyphosph inoyl) -3'-hydroxy-1 '-propanoyl] - (L) -phenylalanine;

4- [bis (2-chloroethyl) amino] -N- [8 ', 8' - (bis-dimethoxyphosphoyl) -8 '-hydroxy-1' -octanoyl] - (L) -phenylalanine;

4- [bis (2-chloroethyl) amino] -N- [7 ', 7' - (bis-dimethoxyphosphinoyl) -7 '-hydroxy-1' -heptanoyl] - (L) -phenylalanine;

4- [bis (2-chloroethyl) amino] -N- [6 ', 6' - (bis-dimethoxyphosphinoyl) -6'-hydroxy-1 '-hexanoyl] - (L) -phenylalanine;

4- [N '-aziridinyl] -N- [5', 5 '- (bis-dimethoxyphosphinoyl) -5' -hydroxy-1 '-pentanoyl] - (L) -phenylalanine;

4- [bis (2-chloroethyl) amino] -N- [4 ', 4' - (bis-dime toxifosphinyl) -4'-hydroxyl'-butanoyl] - (L) -phenylalanine, methyl ester;

4- [bis (2-chloroethyl) amino] -N- [3 ', 3' - {bis-dimethoxyphosphinoyl) -3'-hydroxyl'-propanoyl] - (L) -phenylalanine, ethyl ester;

4- [bis (2-chloroethyl} amino] -N- [5 ', 5' - (bis-diethoxyphosphoyl) -5'-hydroxy-1 '-pentanoyl] - (L) -phenylalanine.

Claims (16)

CLAIMS Compounds of general formula (I): in which: R, R1 represent halogen-ethyl (2-chloroethyl, 2-bromoethyl, 2-iodoethyl) or, taken together with the nitrogen atom to which they are bound, they represent a 1-aziridinyl residue; (A) is (C1-C5) linear or branched alkylene, phenylene or an aralkyl group of formula where m is an integer from 1 to 5; n is an integer from 1 to 6; R2 is hydrogen or hydroxyl; R3 is hydrogen or (C1-C4) alkyl; R4 is hydrogen or (C1-C4) alkyl, their diastereomers, racemic mixtures and pure enantiomers and their salts with pharmaceutically acceptable acids or bases. Compounds according to claim 1, wherein (A) is a group of formula Compounds according to claim 2, wherein R3 and R4 are hydrogen. 4. Compounds according to claim 3, wherein n is the integer 2 or 3 and R and R1 are a 2-chloroethyl group. 5. Compounds according to any one of claims 1-4, wherein R2 is a hydroxyl group. 6. Compound according to claim 1, selected from the group comprising: 4- [bis (2-chloroethyl) amino] -N- [4 ', 4'-diphospho-1' -butanoyl] - (L) -phenylalanine disodium salt; 4- [bis {2-chloroethyl) amino] -N- [5 ', 5'-diphospho-1' -pentanoyl] - (L) -phenfiaianine disodium salt; 4- [bis (2-chloroethyl) amino] -N- [8 ', 8'-diphospho-1' -octanoyl] - (L) -phenylalanine disodium salt; 4- [bis (2-bromoethyl) amino] -N- [4 ', 4'-diphospho-1' -butanoyl] - (L) -phenylalanine disodium salt; 4- [bis (2-chloroethyl) amino] -N- [3 ', 3'-diphospho-1' -propanoyl] - (L) -phenylalanine disodium salt; 4- [N'-aziridinyl] -N- [4 ', 4'-diphospho-1' -butanoyl] - (L) -phenylalanine disodium salt; 4- [bis (2-chloroethyl) amino] -N- [5 ', 5'-diphospho-5' -hydroxy-1 '-pentanoyl] (L) -phenylalanine trisodium salt; 4- [bis (2-chloroethyl) amino] -N- [4 ', 4'-diphospho-4 '-hydroxy-1' -butanoyl] - (L) -phenylalanine trisodium salt; 4- [bis (2-chloroethyl) amino] -N- [3 ', 3'-diphospho-3 '-hydroxy-1' -propanoyl] - (L) -phenylalanine trisodium salt; 4- [bis (2-chloroethyl) amino] -N- [8 ', 8'-diphospho-8 '-hydroxy-1' -octanoyl] - (L) -phenylalanine trisodium salt; 4- [bis (2-chloroethyl) amino] -N- [7 ', 7'-diphospho-7' -hydroxy-1 '-heptanoyl] - (L) -phenylalanine trisodium salt; 4- [bis (2-chloroethyl) amino] -N- [6 ', 6'-diphospho-6 '-hydroxy-1' -hexanoyl] - {L) -phenylalanine trisodium salt; 4- [N '-aziridinyl] -N- [5', 5'-diphospho-5 '-hydroxy-1' -pentanoyl] - (L) -phenylalanine trisodium salt; 4- [bis (2-chloroethyl) amino] -N- [4 ', 4'-diphospho-4'-hydroxy-1 '-butanoyl] - (L) -phenylalanine, methyl ester disodium salt; 4- [bis (2-chloroethyl) amino] -N- [3 ', 3'-diphospho-3' -hydroxy-1 '-propanoyl] - (L) -phenylalanine, ethyl ester disodium salt; 4- [bis (2-chloroethyl) amino] -N- [3 ', 3' - (bis-dimethoxyiosphinoyl) -3 '-hydroxy-1' -propanoyl] - (L) -phenylalanine; 4- [bis (2-chloroethyl) amino] -N- [8 ', 8' - {bis-dimethoxyphosphinoyl) -8'-hydroxy-1 '-octanoyl] - {L) -phenylalanine; 4- [bis (2-chloroethyl) amino] -N- [7 ', 7' - (bis-dimethoxyphosphinoyl) -7 '-hydroxy-1' -heptanoyl] - (L) -phenylalanine; 4- [bis (2-chloroethyl) amino] -N- [6 ', 6' - (bis-dimethoxyphosphinoyl) -6 '-hydroxy-1' -hexanoyl] - (L) -phenylalanine; 4- [N'-aziridinyl] -N- [5 ', 5' - (bis-dimethoxyphosphinoyl) -5'-hydroxy-1'-pentanoyl] - (L) -phenylalanine; 4- [bis (2-chloroethyl) amino] -N- [4 ', 4' - (bis-dimethoxyphosphinoyl) -4'-hydroxy-1 '-butanoyl] - (L) -phenylalanine, methyl ester; 4- [bis (2-chloroethyl) amino] -N- [3 ', 3 (bis-dimethoxyiosphinoyl) -3'-hydroxyl'-propanoyl] - (L) -phenylalanine, ethyl ester; 4- [bis (2-chloroethyl) amino] -N- [5 ', 5' - (bis-diethoxyphosphinoyl) -5'-hydroxy-1 '-pentanoyl] - (L) -phenylalanine. 7. Compound according to claim 6, wherein said compound is: 4- [bis (2-chloroethyl) aneline] -N- [5 ', 5'-diphospho-5'-hydroxy-1'-pentanoyl] - (L) -phenylalanine trisodium salt. 8. Process for the preparation of the compounds of formula (I): in which: R, R1 represent halogen-ethyl (2-chloroethyl, 2-bromoethyl, 2-iodoethyl) or, taken together with the nitrogen atom to which they are bound, they represent a 1-aziridinyl residue; (A) is (C1-C5) linear or branched alkylene, phenylene or an aralkyl group of formula V v (CH2) m where m is an integer from 1 to 5; n is an integer from 1 to 6; R2 is hydrogen or hydroxyl; R3 is hydrogen or (C1-C4) alkyl; R4 is hydrogen or (C1-C4) alkyl, their diastereomers, racemic mixtures and pure enantiomers and their salts with pharmaceutically acceptable acids or bases, comprising the following steps: (a) condensation reaction of the compounds of formula (II): n which R, R1, (A) and R4 have the meanings reported above, with a bis-phosphonate of formula (III): where n has the above meaning, has the above meanings or is an O-G group, where G is a suitable protective group of a tertiary alcohol, R3 'has the above meanings except for hydrogen, T is hydroxyl or a group activating the carboxyl function; (b) selective unblocking of the optionally present protective groups and / or possible hydrolysis of the phosphonic esters to give the corresponding acids; (c) optional salification with pharmaceutically acceptable acids or bases; (d) possible separation of diastereomers or enantiomers. 9. Compounds of formula (VIII): where n is an integer from 1 to 6, R3 'is (C1-C4) alkyl and the R5 groups, which may be the same or different, are selected from the group comprising (C1-C4) alkyl, linear or branched, and phenyl, as intermediates. 10. As a compound according to claim 9, the 5,5-bis (dimethoxyphosphinoyl) -5- (tert-butyldimethylsilyloxy) pentanoic acid. 11. Process for the preparation of the compounds of formula (VIII): where n is an integer from 1 to 6, R3 'is (C1-C4) alkyl and the R6 groups, which may be the same or different # are selected from the group comprising (C1-C4) alkyl, linear or branched, and phenyl, comprising the following steps: (a) reaction of a cyclic anhydride of a suitable dicarboxylic acid with an alcohol of formula R5-OH, wherein R5 is (C1-C4) alkyl, optionally substituted benzyl or allyl; (b) activation of the free carboxyl group of the compound obtained in step (a); (c) reaction of the intermediate obtained in step (b) with a trialkylphosphite P (OR3 ') 3, in which R3' has the meanings reported above, to give a keto-phosphonic ester; (d) reaction of the compound obtained in step (c) with a dialkyl silyl phosphite of formula (VII): in which R3 'and R6 have the meanings indicated above, followed by the hydrolysis of the ester of the carboxylic group, to give the compounds of formula (VIII). 12. Process according to claim 11, wherein the Rj group is a benzyl and wherein the hydrolysis of the carboxylic group ester in step (d) is achieved by catalytic hydrogenation. 13. Process according to claim 12, wherein the -Si (R6) 3 group is a tert-butyl dimethyl silyl group. 14. Pharmaceutical compositions containing at least one compound of claims 1-7 as active ingredient, together with pharmaceutically acceptable excipients. 15. Compounds according to claims 1-7 as anticancer and anti-osteolytic agents. 16. Use of the compounds of claims 1-7 for the preparation of medicaments for the treatment of multiple myeloma, osteosarcoma, bone metastases, breast, ovarian and testicular carcinoma.