LU86212A1 - NOVEL CONJUGATES OF VINBLASTINE AND DERIVATIVES THEREOF, PROCESS FOR THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM - Google Patents

Description

"" NEW CONJUGATES OF VINBLASTINE AND ITS DERIVATIVES, PROCESS FOR THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM.

The present invention relates to new conjugates of vinca alkaloids of the indole-dihydroindole type with proteins or protein fragments endowed with pharmaceutical properties and, in particular, | of cytostatic activity. The vinca alkaloids of the indole-dihydroindole type,. in particular Vinblastine, vincristine and vindésine, are used i; . in the treatment of cancer. The chemotherapeutic use of these derivatives; is however limited in its effectiveness by their side effects, i. This is why many derivatives have been synthesized in order to reduce these side effects.

U

Vinblastine and some of its derivatives, especially vincristine or | - vindesin, have already been coupled to proteins, for example albumin! or various immunoglobulins. This results in coupling products or the so-called "conjugate" compounds.

\ Γ "2 I" <* ·

We note in particular the following references from the literature: - J.D.Teale, Jacqueline M.CIough and V.Marks, Br.J.Clin.Pharmac. 4, 169-172, 1977 - CHJFord, CENewman, JRJohnson, CSWoodhouse, TAReeder, GFRowland, RGSimmonds, Br.J. Cancer 47, 35-42, 1983 - MJEmbleton, GFRowland, RGSimmonds , E.Jacobs, CHMarsden, RWBaldwin, Br.J. Cancer 47,43-49, 1983 - JRJohnson, CHJFord, CENewman, CSWoodhouse, GF

Rowland, R.G. Simmonds, Br.J. Cancer, 44, 472-475, 1981 - Eli Li ,, y> European patent application n ° 56.322 dated 21.07.82 - R.A. ' Conrad, G.J. Cullinan, K. Gerzon, G.A. Poore, J. Med. Chem. 22, 391, 1979 - Eli Lilly ,, British patent application n ° 2.137210 dated 03.11.84 - OmniChem, · European patent application n ° 124.502 dated 07.11.84 'The coupling of these indole-dihvdroindole $ dimers has been undertaken not i only for the purpose of developing new immunological reagents but above all for the preparation of more active, more selective and less toxic anti-tumor substances.

f

Two types of coupling have so far been used.

al C ^ coupling via an azide derivative (European patent application No. 56.322).

b) C 4 coupling via an ester group derived from the hydroxyl group of carbon 4 of the skeleton of the vinca alkaloid (British patent application No. 2,137,210; European patent application No. 124,502).

The present invention relates to novel products which are conjugates of vinca alkaloids of the indole-dihydroindole type with proteins, protein fragments characterized in that the coupling is also carried out via an ester group derived from the hydroxyl group. carbon 4 from the skeleton of the vinca alkaloid.

? h / ν '· «« * 3. The subject of the invention consists more particularly of conjugates of vinca alkaloid of the in-dole-dihydroindole type with a protein or a protein fragment, corresponding to the general formula 05 / ["f *> Q 1 y ·

10 S..A J

, X

CÜOQI ^ s • '/, \ 15 [JT ··. / · ^

f: T

it o> '\ r V,'

I ·· J

1! 2 wherein represents a protein or protein fragment; R2 is C00Ci_3 alkyl, or CO-R7 where R7 is NH2 / an amino acid or peptide ester; R3 is H, CH3, CHO; when R5 and Rg are taken separately, Rg is H and one of R4 and R5 is ethyl and the other is H or OH; when R5 and Rg are taken together with the carbons to which they are attached, they form an oxirane ring and R4 is ethyl and '35 1 A is a residue of bifunctional organic derivative of the maleoylaminoid or maleic type - Koylpeptide or maleoylphenoxy .

β »0 'it

These conjugates, according to the invention, are characterized in that the protein or the protein fragment is coupled to the vinca compound via an arm, by prior condensation of the vinca 4-deacetyl indole dihydroindole alkaloid with a derivative bifunctional organic maleoylaminoacid type or maleoylpeptide type of general formula 0 ti

HOOC - X - N

yJ

0 - In the case of maleoylamino acids X represents a linear alkylene chain of 1 to 6 carbon atoms.

a branched alkvlene chain of 2 to 5 carbon atoms.

. a cycloalkylene chain of 3 to 6 carbon atoms, a phenylene chain of 3 to 6 carbon atoms.

<

In addition, when the bifunctional derivative is of the maleoylamino-acid type, X represents, in addition to the values mentioned above, the R-CH group of natural amino acids R-CH-COOH. In the latter NH2 case, it goes without saying that, when the R-CH group is functionalized, the functions can be protected by the protective groups usually used in peptide synthesis.

v - In the case of maleoylpeptides X represents a fragment of peptide chain - (X. | -NH-CO) -X -where n = 1,2,3,4 and X has the same meaning as the X described above .

- X can also represent a pheny radical! :

When the bifunctional derivative defined above represents centers of asymmetry, it can be used in its racemic form or in one of its optically active forms.

λ * "· ·" j

More particularly, the conjugates of the invention can be represented by the following formula: "K5 H ^ ^ _ UOULt * ..

* j y s 1 / Λ

HrV ‘ji ·

3 | Y O-C-X-N

R. '' \ j, s;

K; ·} -V

0 Ri t in which represents a protein or a protein fragment.

X is as defined above R2 is COOC ^ _3 alkyl3 or CO-R7 where R ^ is NH ^^ an amino acid or peptide ester.

'R3 is H, CH3, CHO.

when R ^ and R ^ are taken separately, R ^ is H and one of 'Ri, and r5 and the other esc H had CH. when Rr and Rr are * taken together with the carbons j υ to which they are attached, they form an oxirane ring and R ^ is ethyl.

—Y e * 6 • The compounds having the preceding formula can be described generically either as derivatives of vinblastihe where R ^ is COOCH ^, R3 is methyl, R ^ is hydroxyl ^ is ethyfe, Rg is hydrogen.

derivatives of vindesine where R ^ is CO-NH ^, R ^, R ^, R ^ and Rg have the meaning described for the derivatives of Vinblastine.

dice derived from vinblastinoyl-23 amino acid where R2 is CO-Ry, Ry being an amino acid or peptide ester, R ^, R ^, R ,. and Rg have the meaning described for the Vinblastine derivatives.

derivatives of vincristine where Ry is COOCH ^, is formyiejR ^ is hydroxyl e R ,. is ethyl, Rg is hydrogen.

derivatives of leurosidine where R ^ is COOCH ^, R3 is methylJR ^ is ethyl R5 is hydroxyfe, Rg is hydrogen.

derivatives of VLB "A" (il'-deoxyVinblastine) where R2 is COOCH3. R3 is methyl, R ^ and Rfi are hydrogens, 'R ,, is ethyl · derivatives of VLB "B" (H'-deoxyleurosidine) where R2 is COOCH3, R3 is methvlejR ^ is ethyl, Rg and Rg are hydrogens. 'derivatives of leurosine where R2 is COOCHg, R3 is methyl, R4 .is ethyl Rg and Rg together form an a -epoxide bond.

The bifunctional derivative of the maleoylamino acid type can result from the condensation of an N-alkoxymaleimide with a natural amino acid OR not, in the case of condensation with the glycine X = Cl · ^; with the alkaline X = CH-CH3; with ß -alanine X = (CH ^; with phenylalanine X = CH-CH - C.Hc; with α-butyric acid X = CH-CH - CH * with valine X = CH-CH -fCH ^^; with norvaline X = CH-CHy-CH ^ -Cf · ^; with leucine X = -CH-Cl - ^ - CH-fCH ^; with XC9H5.

isoleucine X = -CH-CHN “-with norleucine X = CH3 '” CH-fCH ^ -Cl · ^; with 6-amino caprolic acid X = fCHylg ...

r j -. F 7 m

The bifunctional derivative of maleoylpeptide type can also result from the condensation of an N-alkoxy maleimide with a dipeptide, a tripeptide to give a derivative - (X-NH-CO-) X, where X, can have the same meaning as X described above.

i

Proteins which can be advantageously used are in particular bovine or human serum albumin, fetuin or immunoglobulins.

The proteins used can also be processed in order to be selectively modified. These modifications make it possible to obtain protein conjugates which will, during their therapeutic use, preferably be concentrated in certain tissues, for example, in the liver. It is thus possible, prior to the condensation of the derivative of the alkaloid .vinca with the protein, to galactosylate the latter.

Immunoglobulins specific to surface antigens of malignant cells and techniques for their production from serum from immunized animals or by culture of hybridomas secreting monoclonal antibodies are well known. The preferred type of antibody for use in the invention is an IgG class immunoqlobulin of human origin.

However, immunoglobulins of other species are also included in this invention. Some representative immunoglobulins are as follows: - Goat or sheep Ig immunized with carcinoembryonic antigen.

- Rabbit Ig anti-LLA

[- Different monkey Ig anti-LLA, anti-LMA, anti-LLC, anti-CML.

; r 8 * »- lg of goat or sheep immunized with lung carcinoma membranes.

- Monoclonal Ig from mouse hybridomas secreting antibodies against human colorectal carcinoma.

- Ig monoclonal from hybridomas of mice secreting antibodies against human melanoma.

- Ig monoclonal from hybridomas of mice secreting antibodies reacting with human leukemia cells.

- Ig monoclonals from hybridomas of mice secreting antibodies reacting with human neuroblastoma cells.

- Ig monoclonals from hybridomas of mice secreting antibodies reacting with antigens of human breast cancer.

- Ig monoclonal from hybridomas of mice secreting antibodies reacting with human ovarian carcinoma cells.

- Ig monoclonal from hybridomas of mice secreting antibodies reacting with human osteosarcoma cells.

- the monoclonals from hybridomas of mice secreting antibodies to lung cancer.

The conjugates can also be prepared with immunoglobulin fragments, namely the Fab, Fab 'or F (ab') 2 or IgM monomer fragments obtained from an antibody by digestion with a proteolytic enzyme.

The conjugates of the present invention are obtained, in a first step, by condensation of a maleoylamino acid or a maleoylpeptide on the hydroxyl into an alkaloid vinca 4-deacetyl indole-dihydroindole ·. formula ll 'to give a derivative of formula III,

L

s Ά-Α ^ uuu fl ^ C00C113 -> · H ^ ^> <vcooch3 ^ 0, _> 1

Mgl -, R3: tt R3: "y * J II 3. 00

flj R

1 III R2 in which X, R ^, R ^, R ^, Rg, Rg have the meaning mentioned above.

In a second step, the vinca-4-carboxy maleoyl III derivatives are then condensed with a protein or a protein fragment by adding either free thiol groups or free amino groups of the protein, for example, the amino groups derived lysine residues of the protein on the olefinic double bond of maleimide according to a Micha'el-type addition mechanism (Means, GE and Feeney, RE; Chemical modification of proteins, 1971, p.110-138, Holden Day Inc ., San Francisco). J

From a chemical point of view, - the maleoylamino acids or maleoylpeptides are obtained according to the methods described by 0. Keller and J. Rudinger, Helv. 58, 531 f 1975), by D.H. Rieh, P.D. Cesselchen, "A. Tong, A. Cheung and C.K. Buckner; J. Med. Chem., 18, 1004 (1975) and by A ". Sato and M. Nakao; J. Biochem., 90, 1117 (1981).

the condensation of the maleoy derivatives with the vinca alkaloid can be carried out in a conventional manner with an alkyl chioro-formate, preferably ethyl or isobutyl chloroformate, in the presence of an amino base such as triethylamine, N-methyl piperidine, N-methyl morpholine, in an organic solvent such as ethyl acetate. I tetrahydrofuran, methylene chloride.

i * * 10! .

The derivative obtained is isolated from the reaction medium and purified by means of conventional methods used in chemistry.

Any other method of activating the carboxyl group of maleoylamino acid or maleoylpeptide to achieve condensation with the alkaloid yinca, in particular the methods used in peptide chemistry, can be applied to this type of condensation.

In particular, in the case where X is phenyl, the condensation is carried out with N-succinimidyI-3 maleimidobenzoate, obtained according to the methods i described by T. Aikawa; J. Biochem., 79, 233 (1976) and M.J. O'Sullivan et al., Anal. Biochem., 100, 100 (1979).

The conjugates can be obtained by reacting the protein, the polyclonal or monoclonal antibody with the vinca compound of formula III under conventional conditions, for example in an aqueous medium, at a temperature between U ° C and 25 ° C and a pH of 7.5 to 9.5.

((

\ I

The number of residues attached may depend on the concentration of the reactants and the duration of the reaction, but the average number is! Generally between 5 and 20.

For example, a solution of compound III in an organic solvent such as dioxane is added dropwise to a buffered solution of ί protein, for example a 0.1 M phosphate buffer at pH 8.2. After overnight at room temperature, the conjugate is isolated by filtration through qel, concentrated by ultrafiltration and sterilized. The protein content is * * 'measured by the Lowry method and the alkaloid content estimated by y' measurement of radioactivity.

"R I The" in vitro "and" in vivo "tests carried out with the compounds of | the invention to demonstrate their anti-tumor activity indicate that the

jv formation of the conjugate via a maleoyl link O-CO-X-N

|| | may be more advantageous than by an O-CO-X-CO- 0H link

I 1 11

Indeed, the maleoyl link allows both the conjugation of thiol groups and free amino groups of the protein or protein fragment. In addition, digestion with lysosomal enzymes indicates a much greater release of the vinc alkaloid. in the case of the maleoyl link. The conjugates are also stable in serum and at acidic pH.

The compounds of the invention were tested on BDF ^ mice to which P388 leukemia was implanted intraperitoneally. The first results indicate that the compounds exhibit significant activity on this experimental model since they induce an increase in survival time.

The new conjugates of the invention have particularly interesting antitumor properties which can be used in human therapy.

For their therapeutic application, the compounds of the invention are preferably administered parenterally, dissolved in a pharmaceutically acceptable solvent. Physiological water or other buffered solutions, for example with phosphate are suitable solvents. The active substance is generally administered at a dosage which can vary from 50 mq to several grams.

; | The compounds of the invention can also be used in combination with other anti-tumor agents.

Ί I "! ..

[- 12

The following examples illustrate, without limitation, the process leading to the compounds of the invention.

Example T. N-carbomethoxy maleimide.

A solution of 3.8 g. (0.04 M) of maleimide and 4 g (0.04 M) of triethylamine in 150 ml of ethyl acetate is treated at 0 ° C with 3 ml (0.04 M) of methyl chloroformate dissolved in 20 ml d 'ethyl acetate. After one hour of stirring, the precipitate is filtered, washed with ethyl acetate. The organic phases are combined, washed with water, dried over anhydrous sodium sulfate and evaporated to dryness under vacuum.

The residue, crystallized from an ethyl acetate-isopropyl ether mixture, gives 4.19 g of N-carbomethoxy maleimide.

NMR spectrum: (CDCI3.6): 6.75 (2H, s), 3.9 (3H, s) i

Example 2. N-Maleoyl L-alanine. !

A solution of 2.3 g (0.025 M) of L-alanine in 100 ml of a saturated sodium bicarbonate solution is treated at 0 ° C, with vigorous stirring, with 3.87 g (0.025 M) of N- carbomethoxymaleimide.

After one hour, the solution is diluted with 200 ml of water and stirred at 40 ° C for one hour. The pH of the solution (8.2) is then brought to 6.4 by addition of concentrated sulfuric acid. The solution is then concentrated to 100 ml and acidified to pH 2 by addition of 1 M sulfuric acid and extracted three times with ethyl acetate. The organic phases are combined, dried over anhydrous sodium sulfate and evaporated to dryness under vacuum.

The residue obtained is purified by chromatography on a silica column (chloroform / acetic acid 95/5). 1.2 g of N-maleoyl L-alanine are obtained in this way.

Yield 48%.

J

-, 13

Spectrum I.R. (KBr) cm: 3400, 3100, 2930, 1780, 1740, 1700, 1460, 1420, 1390, 1370, 1345, 1220, 1175, 1118, 1080, 1068, 1015, 970, 835, 700.

R.M.N spectrum (CDCI) 6: 6.7 (2H, s); 4.8 (1H, g); 1.65 '(3H, d)

Example 3. 4- (N-maleoyl) -L-alanyl-vinblastine.

To a solution of 1.52 g (0.009 M) of N-maleoylalanine and 1.25 ml (0.009 M) of triethylamine in 10 ml of ethyl acetate cooled to 0 ° C., a solution of 1.17 ml (0.009 M) of isobutylchloroformate in 5 ml of ethyl acetate. The mixture is stirred for 1 hour 30 minutes at 0 ° C., then 2.3 g (0.003 M) of 0-4 deacetyl Vinblastine in solution in | 20 ml of ethyl acetate, keeping the temperature at 0 ° C. It is left to return to ambient temperature and the mixture is stirred for 10 hours. 50 ml of ethyl acetate and 50 (ml of a 10% aqueous solution of sodium carbonate are added. decant and separate the organic phase.

ïi | The aqueous phase is extracted three times with ethyl acetate. The combined organic phases are washed with an aqueous solution, dried! over magnesium sulfate and evaporated to dryness under vacuum. The residue obtained! is purified by chromatography on a silica column "(elution dichloro [methane / methanol 92/8). 1.86 g of pure product are thus obtained.

: Yield: 67.6%

Mass spectrum (DCI, isobutane); 935 (M + 14), 922 (M + 1), 921 (M), 751, | '693, 519, 445, 371, 133.

| (NMR spectrum (CDC13.360 MHz, ppm): 8 (NH, 1 H); 7.5-7 (4H, H-9 ', H-10; H-11', AH-12 '); 6 , 7 (2H, anhydride); 6.55 (1H, H-14); 6.05 (1H, H-17); 5.85 (1H, H-7); 5.45 (1H, H- 4); 5.15 (1H, H-6); m 4.8 ΠΗ, CH *); 3.95 (ΐΗ, Η-17 '); 3.85 (3Η, -OMe); 3.78 (3H, -OMe); 3.7 (1H.H-2); 3.6 '(3H, -Ome); 2.7 (3H, NMe); 1.72 (3H, CH3 alanine); 0.9-0.8 (6H, CH3-21, CH3-2T).

Example 4. Coupling of 4- (N-maleoyl) -L-alanyl Vinblastine with galactosylated human albumin (AHgal). (V ^ -Ala-Mal-AHg) 88.4 mg of 4- (N-maIéoyl) -L-alanyl Vinblastine are dissolved in 1 ml of dioxane. On the other hand, a solution of 50 mg of AHgal in 5 ml of 0.1 M phosphate buffer pH 8.2 is prepared. The pH is adjusted to 8.5 with IN sodium hydroxide solution. After one hour, the solution of 4- (N-maleoyl) -L-alanyl Vinblastine is added to the AHgal solution. The mixture is stirred at room temperature for 5 hours and then clarified by centrifugation. The solution is purified by filtration on Sephadex G-25 gel (3 x 90 cm) equilibrated in a solution of NH ^ HCC ^ 0.1 M pH 7.8.

The excluded peak is collected (96 m!) And concentrated by ultrafiltration and sterilized.

The protein content is measured by the Lowry method and the alkaloid content estimated by measuring the radioactivity.

The conjugate obtained contains 13.5 moles of Vinblastine per mole of galactosylated human albumin.

Example ~ 5. Sensitivity to lysosomal enzymes.

3 hrs

The sensitivity of the -Ala-Mai-AHgal conjugate to lysosomal enzymes was studied by incubation, for 48 hours at 37 ° C, of this conjugate in the presence of 5 mM cysteine, 40 mM acetate buffer and lysosomal enzymes. Over time, aliquots are taken and the non-degraded proteins are precipitated by adding a volume of trichloroacetic acid (TCA 401).

S- is] h; . After incubating the samples <4 ° C for one hour, they are centrifuged and the radioactivity of the supernatant is estimated by counting an aliquot part in liquid scintillation.

Soluble radioactivity is a measure of the digestion of the conjugate. Experience shows that 851 of the conjugate are digested after 24 hours. No further development is observed until 48 hours.

Example 6. Stability in serum.

! The stability of the V ^ '- Ala-Mal-AHgal conjugate in the presence of serum was studied by incubation of the conjugate in the presence of 62% fetal calf serum, at 37! C for 48 hours. The aliquots are taken at •; over time and the percentage of digestion is measured by the T CA precipitation technique described above. The results ; show that the conjugate is stable over the 48 hours.

f f

Example 7. Stability at acid pH.

! (- -

'· 3H

The stability of the -Ala-Mnl-AHqal conjugate at acidic pH has been studied by | incubation of the conjugate in the presence of acetate / 40 mM buffer, pH 4.5, at 37 ° C., for 48 hours. Digestion is estimated by the technique of f,, I precipitation with TCA. The results show that the conjugate is stable! in these conditions.

! Example 8. Antitumor activity.

j I! I The activity *; chemotherapy- of the conjunct Y ^ ‘-Aia-Mai-AHaa! was assessed on P388 leukemia administered i.p. in female BDF ^ mice: 10, tumor cells are inoculated i.p. on day 0. The conjugate is administered i.p. on day 1,

The first results demonstrate that the conjugate has significant activity on this experimental model since it induces an increase! Ii. 210% survival rate when administered at a dose of i00mg / kg.

I. J c r. vrv k \ i · /

H

Claims (25)

1. Conjugates of vinca alkaloid of the indole-dihydroindole type with a protein or a protein fragment, corresponding to the general formula 05 .x,; f io II \ A :; / k J .. - *>, CUUCI! ^ 15, |! 4. I \ S.s-. r JL. . , -C "" ·: I N /.j '1 I. 20; i ", i in which R_l represents a protein or a 1 · * J fragment of protein; i 2. R2 is C00Ci_3 alkyl, or CO-R7 where R7 is NH2 / an amino acid or peptide ester; 'R3 is H, CH3, CHO; when R5 and Rg are taken separately, Rg, · is H and one of R4 and R5 is ethyl and the other is H or OH; when R5 and Rg are taken together with the. carbons to which they are attached,) they form an oxirane nucleus and R4 is ethyl and 35. is a bifunctional organic derivative of. maleoylamino acid or maleoylpeptide i tfk or maleoylphenoxy type. "4 17 2. Conjugate according to claim 1 characterized in that A corresponds to the general formula υ \ '> --—- 1 05 HOCH. - X - - \ * \ AU 10 in which X represents a linear alkylene chain of 1 to 6 carbon atoms, a branched alkylene chain of 2 to 5 carbon atoms, a cycloalkylene chain of 3 to 6 carbon atoms, a chain phenylene of 3 to 6 carbon atoms, or the R-CH group of natural friend-15 acids R-CH-COOH; nh2 a fragment of peptide chain of the type - (Χχ-ΝΗ-CO) η-Χχ- in which n = 1, 2, 3, 4 and Χχ is a linear alkylene chain! of 1 to 6 carbon atoms, 20 a chain branched alkylene (from 2 to 5 carbon atoms, a cycloalkylene chain from 3 to 6 carbon atoms, a phenylene chain from 3 to 6 carbon atoms, or the R-CH group of natural amino acids; in its racemic form or in one of its optically active forms or a phenyl radical. 3. Conjugate according to claim 1 characterized in that the functionalized group or groups RCH des | acids, natural amino, represented by X or Χχ, are | protected by protective groups known per se. 4. Conjugate according to any one of claims 1 to 3 characterized in that R2 is COOCH3, R3 is methyl, R4 is hydroxyl, R5 is ethyl, Rg is hydro- .gene. 5. Conjugate according to any one of the claims: 35 1 to 3 characterized in that R2 is C0-NH2, R3, R4, J R5, R5 have the meaning described for derivatives of! 1 Vinblastine. i „i ιδ 6. Conjugate according to any one of claims 1 to 3 characterized in that R2 is CO-R7 with R7 being an amino acid or peptide ester, R3, R4, R5 and Rg have the meaning described for the derivatives of 05 Vinblastine. 7. Conjugate according to any one of claims 1 to 3 characterized in that R2 is COOCH3, R3 is formyl, R4 is hydroxyl, R5 is ethyl, Rg is hydrogen. 8. Conjugate according to any one of claims 1 to 3, characterized in that R2 is COOCH3, R3 is methyl, R4 is ethyl, R5 is hydroxyl, Rg is hydrogen. 9. Conjugate according to any one of the claims 15 to 1 to 3 characterized in that R2 is COOCH3, R3 is methyl, R4 and Rg are hydrogen, R5 is ethyl. , 10. Conjugate according to any one of claims 1 to 3 characterized in that R2 is COOCH3, R3 ^ st methyl, R4 is ethyl, R5 and Rg are hydrogenated. { 11. Conjugate according to any one of claims 1 to 3 characterized in that R2 is COOCH3, R3 is methyl, R4 is ethyl, R5 and Rg form a bond & -epoxide. 12. Conjugate according to any one of the preceding claims, characterized in that the protein Rg is bovine or human serum albumin, fetuin or £ immunoglobulins. 13. Conjugate according to any one of the preceding claims, characterized in that the protein Rj ^ is treated selectively. 14. Conjugate according to any one of the preceding claims, characterized in that R ^ is an immunoglobulin chosen from the group consisting of 35. Ig of goat or sheep immunized with carcinoembryonic antigen; f · anti-LLA rabbit Ig; „<“ 15 - different anti-LLA, anti-LMA, anti-LLC, anti-CML monkey Ig; - Goat or sheep Ig immunized with lung carcinoma membranes; 05. Monoclonal Ig from mouse hybridomas secreted both antibodies to human colorectal carcinoma; · - Monoclonal Ig from mouse hybridomas secreting antibodies against human melanoma; 10. Monoclonal Ig from secreted mouse hybridomas as well as antibodies reacting with human leukemia cells; ; - Monoclonal Ig from secreted mouse hybridomas! both antibodies reacting with human neuroblastoma cells; j - Monoclonal Ig from secreted mouse hybridomas | both antibodies reacting with human breast cancer antigens; | j - Monoclonal Ig from mouse hybridomas secreting antibodies reactive with human ovarian carcinoma cells; | - monoclonal Ig from hybridomas of mice secreting antibodies reacting with human osteosarcoma cells; 25. Monoclonal Ig from secret mouse hybridomas! | so many lung cancer antibodies; | or an immunoglobulin fragment, namely Fab, Fab * or f F (ab ') 2 or IgM monomer obtained from an antibody j; by digestion with a proteolytic enzyme, j 30 15. Process for the preparation of a vinca alkaloid conjugate with a protein or a protein fragment corresponding to the formula (I) 1 -Λ A 20 05 - \ K <. MjJ> - H COOC11 -J s 1: 10 / A '"A'! A, _γΝΟ- c! I", I? - ί ";" Ά.Α ,. Αρ j I 15 *>,: “VA I 0 ". Ί! I in which Rj, R2, R3, R4, R5, Rg and X have the meanings mentioned in claims 1 and 2, characterized in that a maleoylamino acid or ma- leoylpeptide of formula I t 1 25. - i - p 1 I ί: 1-1 (- -> lii 30 î 35 i 1 · î: ir. "* * 9 '21 on the C4 hydroxyl of a compound of general formula (II) 05 ûdi? ·' H ^ COOCH, Z '/ / jO 15 1 „„ »3C0 i 3 11; * 2 - 1 i 20 f in which R2, R3, R4, R5, Hg and X have the meanings mentioned, and in that condensing the intermediate product obtained with a protein or a protein fragment. 16. The method of claim 15 characterized in that the first condensation step is carried out using 'an alkyl chloroformate, preferably ethyl chloroformate or isobutyl, in the presence of an amino base, such as triethylamine, N-methyl-piperidine or N-methylmorpholine, in an organic solvent such as ethyl acetate, tetrahydrofuran or methylene chloride, and in that it is isolated the intermediate compound obtained from the reaction medium, in that it is purified and that the second condensation step is carried out in a conventional manner, in aqueous medium, at a temperature between 4 ° C and D25 ° C and a pH from 7.5 to 9.5. . - - 22 17. As intermediate compound, the compound of general formula (III) 05 _ί Ιχγ ^ ·· »1 10 N 'H ^ "^ COOCH V *» - JL / x) LI ° H 1 ° v_ τΛΑιΛιΑ ,, η' ir,: Il> - 3 ·; -oo kj • ί <20 in which R2 / R3, R4 , R5, R5 and X have the meanings mentioned in claims 1 and 2. 18. Pharmaceutical composition comprising, as active substance, the conjugate according to any one of claims 1 to 14 in combination with pharmaceutically acceptable carriers and excipients in a unit dose ranging from 50 mg to several grams, optionally in combination with other "active substances. 19. A pharmaceutical composition according to claim 18 comprising the conjugate according to any one of claims 1 to 14 in the dissolved state in a pharmaceutically acceptable solvent, such as physiological water or solutions buffered by means of a phosphate buffer. 20. Use of the conjugate according to any one of claims 1 to 14 for the preparation of medicaments with anti-tumor and cytostatic activity. _