DD262998A5 - METHOD FOR PRODUCING A CONJUGATE OF A VINCA ALKALOID WITH A PROTEIN - Google Patents

Abstract

The invention relates to a process for the preparation of a conjugate of a vinca alkaloid with a protein. According to the invention, conjugates of a vinca alkaloid of the indole-dehydroindole type are prepared with a protein or a protein fragment, of the general formula I in which, for example: R 1 is a protein or a protein fragment, R 2 COOC 1-3 -alkyl or COR 7 wherein R7 is NH2, an ester of an amino acid or a peptide, R3, H, CH3, CHO; when R5 and R6 are considered individually, R6 is H and one of R4 and R5 is ethyl and the other is H or OH; when R5 and R6 are considered together with the carbon atoms to which they are attached, they together form an oxirane nucleus and R4 is ethyl, and A represents a bifunctional organic derivative of the maleoyl amino acid, maleoyl peptide or maleoylphenoxy type. Formula I

Description

Field of application of the invention

The present invention relates to a process for the preparation of novel conjugates of vinca alkaloids of the indole-dihydromdol type with proteins or protein fragments which have pharmaceutical properties, in particular a cytostatic activity. The vinca alkaloids IndoJ dihydroindole, especially vinblastine, vincristine and vindesine, are used in the treatment of cancer. The chemotherapeutic use of these derivatives, however, is limited in effectiveness by their secondary effects. Therefore, many derivatives have been synthesized to reduce these secondary effects.

Characteristic of the known state of the art

The vinblastine and some of its derivatives, in particular vincrisin or vindesine, have already been coupled with proteins, for example with albumin or with various immunoglobulins. This resulted in coupling products or compounds, so-called "conjugates"

 We refer in particular to the following references in the literature:

J.D. Teale, Jacqueline M. Cleugh and V. Marks, Br. J. CHn. Pharm.4,169-172,1977

, Ford, C.E. Newmann, J.R. Johnsen, C.S. Woodhouse, T.A. Reeder, G.F. Rowland, R.G. Simmends, Br. J. Cancer, 47, 35-42, 1983. "

M.J. Embleton, G.F. Rowland, R.G. Simimmonds, E. Jacobs, C.H.Marsden, R.W. Balwin, Br. J. Cancer, 47, 43-49, 1983

J.R.Johnson, C.H.J. Ford, C.E. Newman, C.S. Woodhouse, G.F. Rowland, R.G.Simmonds, Br. J. Cancer, 44, 472-475, 1981.

- Eli Lilly Eur. Pat. Applic, Publ. N ° 56.322, 21.7.82

R.A. Conrad, G.J. Cullinan, K.Gerzan, G.A.Poore, J. Med. Chem. 22, 391, 1979

- Eii Lilly, U.K. Pat. Applic., Publ. N ° 2.137210,3.11.84

- OnmiChem., Eur. Pat. Applic., Publ. N ° 124.502. 11.07.84.

The coupling of these indole-dihydroindole derivatives has been carried out not only with the aim of providing new immunological reagents, but above all with regard to the production of more effective, more selective and less toxic anti-tumor substances.

To date, two types of coupling have been considered:

a) the coupling to C ₃ via the intermediate of an azide derivative (EP-PS 56322).

b) The coupling at C ₄ via the intermediate of an ester group derived from the hydroxyl group of the carbon in the 4-position of the Vinca alkaloid skeleton (GB-PS 2137210, EP-PS 124502).

Object of the invention

The aim of the invention is the provision of new vinblastine derivatives with strong antitumor effect, which do not cause any unwanted side effects.

Explanation of the essence of the invention. '

The invention has for its object to find new conjugates of a vinca alkaloid with a protein having the desired properties and processes for their preparation.

According to the invention, new products are prepared which are conjugates of vinca alkaloids of the indole-dihydroindole type with proteins or protein fragments. According to the invention, the coupling also takes place via the intermediate of an ester group which is derived from the hydroxyl group of the carbon in the 4-position of the vinca alkaloid skeleton. The object of the present invention consists in particular in conjugates of vinca alkaloids of the indole-dihydroindole type with a protein or a protein fragment, corresponding to the general formula I.

AR ₁

in the

R _{1 represents} a protein or a protein fragment; R ₂ is COOC ^ alkyl or Co-R ₇ , wherein R _{7 is} NH ₂ , an ester of an amino acid or a peptide; , R ₃ is H, CH ₃ , CHO;

when R ₅ and R ₆ are considered individually, R _{6 is} H and one of R ₄ and R ₅ is ethyl and the other is H or OH; when R ₅ and R _{6 are considered} together with the carbon atoms to which they are attached, they together form an oxivane core and A represents a bifunctional organic derivative of the maleoyl amino acid, maleoyl peptide or maleoylphenoxy type.

These conjugates of the invention are characterized in that the protein or protein fragment is coupled with the vinca compound via the intermediate of an arm compound by prior condensation of the 4-deacetylindole dihydroindole-vinca alkaloid with a bifunctional organic derivative of the type Maleoylamino acid or maleoyl peptide of the general formula

HOOC- Σ - Έ

Wherein, in the case of maleoylamino acids, X means: a linear alkylene chain having 1 to 12 carbon atoms; a branched alkylene chain of 2 to 5 carbon atoms; a cycloalkylene chain having 3 to 6 carbon atoms;

a phenyl chain of 3 to 6 carbon atoms.

When the bifunctional derivative is of the maleoylamino acid type, X means, in addition to the above values, the group R-CH of the natural amino acids R-CH-COOH. In this last case, it goes without saying that the functions

NH ₂ can be protected by the usual in the peptide synthesis protecting groups, when the R-CH group is reactive.

Wherein in the case of the maleoyl peptides X means:

a fragment of the PePtId-KeWe- (X ₁ -NH-CO) _n -Xi-wherein η = 1,2,3 or 4 and X _{1 has} the same meaning as X in the above-described form.

Wherein X may also be a phenyl radical: when the above-defined bifunctional derivative has asymmetric centers, it may be used in the racemic form or in one of its optically active forms.

In particular, the conjugates of the invention may be represented by the following formula:

/ Y

'Kr

Il, -

COUCH..

O-C-X-N

in the

Ri represents a protein or a protein fragment;

X is as defined above;

R ₂ is COOCi_ ₃ alkyl or CO-R7, wherein _R7 is _NH2 or the ester of an amino acid or a peptide; R ₃ is H, CH _3, CHO;

when R ₅ and R ₆ are considered individually, R _{6 is} H and one of R ₄ and R ₅ is ethyl and the other is H or OH; when R ₅ and R _{6 are considered} together with the carbon atoms to which they are attached, they together form an oxivane core and R ₄ is ethyl.

The compounds having the above formula can be generically described as

- Vinblastine derivatives in which R _{2 is} COOCH ₃ , R _{3 is} methyl, R _{4 is} hydroxyl, R _{5 is} ethyl and R _{6 is} hydrogen;

- Vindesine derivatives in which R ₂ CO-NH ₂ JSt and R ₃ , R ₄ , R ₅ and R _{6 have} the abovementioned meanings for the vinblasin derivatives;

- 23-Vinblastinoyl-amino acid derivatives in which R _{2 is} CO-R ₇ , wherein R _{7 represents} the ester of an amino acid or a peptide and R ₃ , R ₄ , R ₅ and R _{6 have} the abovementioned meanings for the vinblastine derivatives have;

Vincristine derivatives in which R _{2 is} COOCH ₂ ;

R _{3 is} formyl, R _{4 is} hydroxyl, R _{5 is} ethyl and R _{6 is} hydrogen;

Leurosidine derivatives wherein R _{2 is} COOCH ₃ ,

R _{3 is} methyl, R _{4 is} ethyl, R _{5 is} hydroxyl and R _{6 is} hydrogen;

4-deoxy-VLB "A" derivatives in which R _{2 is} COOCH ₃ , R _{3 is} methyl, R ₄ and R _{6 are} hydrogen and R _{5 is} ethyl,

4-deoxy-VLB "B" derivatives in which R _{2 is} COOCH ₃ , R _{3 is} methyl, R _{4 is} ethyl and R ₅ and R _{6 are} hydrogen;

Leurosine derivatives in which R _{2 is} COOCH ₃ ,

R _{3 is} methyl, R _{4 is} ethyl and R ₅ and R ₆ together form an epoxide bond.

The bifunctional derivative of the maleoyl amino acid type can be derived from the condensation of an N-alkoxymaleimide with a natural or non-natural amino acid. In the case of condensation with glycine, X = CH ₃ ; with alanine X = CH-CH ₃ ; mitß-alanineX = (CH ₂ ) ₂ ; with phenylalanine X = CH-CH ₂ -C ₆ H ₅ ; with α-amino-butyric acid X = CH-CH ₂ -CH ₃ ; with valine X = CH-CH- (CH ₃ ) ₂ ; with norvaline X = CH-CH ₂ -CH ₂ -CH ₃ ; with leucine X = -CH-CH ₂ -CH- (CHs) ₂ ; with isoleucine X =

-CH - ClT ^G 2 ^H 5

with norleucine X = CH- (CH ₂ I ₃ -CH ₃ ; with 6-aminocaproic acid X = (CH ₂ ) ₅ ; with 11-amino undecanoic acid X = (CH ₂ ) ₁₀ ; with 12-amidodecanoic acid X = (CH ₂ J ₁₁ .

The bifunctional maleoylpeptide derivative may also be derived from the condensation of an N-alkoxymaleimide with a dipeptide or a tripeptide to give a derivative (X 1 NH-CO-in X-i wherein X _{1 may have} the same meaning as above for X described.

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

The proteins used can also be treated to selectively modify them. These modifications make it possible to obtain protein conjugates which, in their therapeutic application, are preferably concentrated in the area of certain tissues, for example in the liver. It is therefore possible to galactosylate the latter prior to the condensation of the vinca alkaloid derivative with the protein.

The immunoglobulins specific to the antigens of the surface of malignant cells and the techniques for their production, from immunized animal serum or by culture of hybrids secreting monoclonal antibodies are known.

The preferred type of antibody for use according to the invention is an immunoglobulin of class IgG of human origin.

However, the immunoglobulins of other types are also included in the present invention. Some representative immunoglobulins are the following:

Ig of goat or sheep immunized with carcinomembryonic antigen,

- Ig from rabbit anti-LLA,

- different Ig from the monkey, anti-LLa, anti-LMA, anti-LLC, anti-LMC,

Ig of goat or sheep immunized with membranes of lung carcinoma,

Monoclonal IG, starting from mouse hybrids secreting antibodies against the human colo-rectal carcium,

Monoclonal IG, starting from mouse hybrids secreting antibodies to human melanoma,

Monoclonal Ig, starting from mouse hybrids secreting antibodies reactive with leukemic human cells,

- Monoclonal Ig derived from mouse hybrids secreting antibodies that react with human neuroblastoma cells. *.

Monoclonal Ig starting from mouse hybrids secreting antibodies that react with human mammary carcinoma antigens

Monoclonal Ig, starting from mouse hybrids secreting antibodies that react with human ovarian carcinoma cells,

Monoclonal Ig, starting from mouse hybrids secreting antibodies that react with human osteosarcoma cells,

- monoclonal Ig, starting from mouse hybrids secreting lung carcinoma antibodies.

The conjugates can also be prepared with immunoglobulin fragments, namely the fragments Fab, Fab 'or F (ab') ₂ or monomeric IgM, obtained from an antibody by digestion with the aid of a proteolytic enzyme. The conjugates of the present invention are obtained in a first step by condensation of a maleoyl amino acid or maleoyl peptide with the 4-position hydroxyl of a 4-deacetyl-indole dihydroindole-vinca alkaloid of formula II to give a derivative of formula III.

in which X, R ₂ , R ₃ , R ₄ , R ₅ and R _{6 have} the abovementioned meaning.

In a second step, the vinca-4-carboxy-maleoyl derivatives of formula III are then condensed with a protein or a protein fragment by addition of either free thiol groups or free amino groups of the protein, e.g. Amino groups derived from the lysine residue of the protein, having the olefinic double bond of the maleimide, according to a Michael addition mechanism (Means, GE et Feeney, RE; Chemical modification of proteins, 1971, pp. 110-138, Holden, Day Inc., San Francisco) to give the formula I conjugates. From the chemical point of view, the preparation of the maleoylamino acids or the maleoyl peptides according to the methods described by O. Keller and J. Rudinger, HeIv. 58,531 (1975), D.H. Rich, P.D. Gesselchen, A.Tong, A. Cheung, and C.K. Buckner; J. med. Chem., 18, 1004 (1975) and A. Sato and M. Nakao; J. Biochem., 90,1117 (1981). The condensation of the maleoyl derivatives with the vinca alkaloid can be carried out in the classical manner with the aid of a chloroformic acid alkyl ester, preferably with ethyl chloroformate or isobutyl ester, in the presence of an amine base such as triethylamine, N-methylpiperidine, N-butyl ester. Methyl-morpholine, and in an organic solvent such as ethyl acetate, tetrahydrofuran or methylene chloride.

The resulting derivative is isolated from the reaction medium and purified by classical methods used in chemistry.

Any other method for activating the carboxylic acid group of the maleoylamino acid or the maleoyl peptide, in particular the methods customary in peptide chemistry, can also be used in this type of condensation in order to carry out the condensation with the vinca alkaloid.

In particular, in the case where X is phenyl, the condensation is carried out with 3-N-succinimidyl maleimidobenzoate, obtained according to the method described by T. Alkawa, J. Biochem., 79, 233 (1976) and MJO'Sullivan et al Anal Biochem., 100, 100 (1979). ,

The preparation of the conjugates can be carried out by reaction of the protein, monoclonal or polyclonal antibody with the vinca compound of formula III under classical conditions, for example in an aqueous medium at a temperature between 4 ° C and 40 ⁰ C and a pH of 7.5 to 9.5.

The number of linked radicals may depend on the concentration of the reactants and the duration of the reaction, but in general the number is between 5 and 20.

For example, a solution of the compound of formula III, in an organic solvent such as dioxane, is added dropwise to a buffered solution of the protein, e.g. Using a 0.1 M phosphate buffer of pH 8.2. After one night at ordinary temperature, the conjugate is isolated by gel filtration, concentrated by ultrafiltration and sterilized. The content of proteins is measured by the method of Lowry and estimated the content of alkaloids by measuring the radioactivity.

The "in vitro" and "in vivo" experiments to demonstrate their anti-tumor activity with the compounds of the invention show that the formation of the conjugate via the maleoyl-binding intermediate

0 Q-CO-Z-H

' ⁴

may be more advantageous than via a bond OCO-X-Co. Namely, the maleoyl bond simultaneously allows the conjugation of the free thiol groups and the amino groups of the protein or protein fragment. In addition, the reaction by lysosomal enzymes shows a much larger release of the vinca alkaloid in the case of maleoyl binding. The conjugates are also more stable in the serum and in the acidic pH range.

The compounds of the invention were tested on BDF- mice in which a leukemia P388 was implanted by intraperitoneal route. The first results show that the compounds on this experimental model a significant

Have activity as they induce an increase in survival time.

The novel conjugates according to the invention have in particular interesting anti-tumor properties and are therefore suitable for use in human therapeutics.

In their therapeutic use, the compounds of the invention are preferably administered by parenteral route, dissolved in a pharmaceutically acceptable solvent. Physiological water or other buffer solutions, for example with a phosphate, are suitable solvents. The active ingredient is generally administered at a dosage that can vary from 50 mg to several grams.

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

embodiment

The following examples illustrate, by way of non-limiting example, the process that results in the compounds of this invention.

Example 1: N-carbomethoxy-maleimide

A solution of 3.8 g (0.04 mol) maleimide and 4 g (0.04 M) triethylamine in 150 mL ethyl acetate is treated at ⁰ ° C with 3 mL (0.04 mol) of methyl chloroformate dissolved in 20 mL ethyl acetate. After stirring for 1 hour, the precipitate is filtered off and 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 in an ethyl acetate-isopropyl ether mixture, gives 4.19 g of N-carbomethoxy-maleimide. Nuclear Magnetic Resonance Spectrum: (CDCl ₃ , 5): 6.75 (2H, S) f, 3.9 (3H, S).

Example 2: IM-maleoyl-L-analine

A solution of 2.3g (0.025M) L-alanine in 100ml saturated sodium bicarbonate solution is treated with 3.87g (0.026M) N-carbomethoxymaleimide at a temperature of ⁰ ° C with vigorous stirring. After one hour, the solution is diluted with 200 ml of water and stirred at a temperature of 40 ^° C. for one hour. The pH of the solution (8.2) is then brought to 6.4 by the addition of concentrated sulfuric acid. The solution is then concentrated to 100 ml, 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 (elution chloroform / acetic acid 95/5).

cleaned. This gives 1.2 g of N-meleoyl-L-alanine. ·

Yield 48%

IR spectrum (kBr) cnTV 3400,3100,2930,1780,1740,

1700,1460,1420,1390,1370,

1345,1220,1175,1118,1080,

1068, 1015, 970, 835, 700

NMR spectrum (CDCl ₃ ) 5: 6.7 (2H, S); 4.8; (1H, g); 1.65 (3H, d).

Example 3: IM-Maleoyl-e-aminocabronic acid

A solution of 4.2 g (32,2mM) of 6-amino-capronsäurein 163ml of a saturated solution of sodium bicarbonate is treated at a temperature of 0 ⁰ C under vigorous stirring with 5 g (32.3 mM) of N-Carbomethoxymaleimid. After one hour the solution is diluted with 256 ml of water and stirred at ambient temperature for one hour. The pH of the solution (8.2) is then brought to 6.4 by the addition of concentrated sulfuric acid. The solution is then concentrated to 100 ml, acidified to pH 2 by adding 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 (elution chloroform / acetic acid 95/5). This gives 4.7 g of N-maleoyl-6-amino-caproic acid.

Yield: 51%

IR spectrum (kBr, cm ^-1 ): 3090, 2940, 2880, 1770, 1710, 1440, 1450, 13, 31, 12, 12, 12, 110, 110, 110, 110, 110, 110, 150, 110, 150, 311 , 1015,1000,815,840,735,700.

Example 4: methyl N-Maleoyo-L-glutamate

A solution of 1 g (6.2 mm) y ^ L-glutamic acid-methylester in 31.2 ml of a saturated sodium bicarbonate solution is at a temperature of 0 ⁰ C under vigorous stirring with 961 mg (6.2 mm) of N-carbomethoxy-maleimide treated. After one hour the solution is diluted with 126 ml of water and stirred at a temperature of 4O ⁰ C for one hour. The pH of the solution (8.2) is then brought to 6.4 by the addition of concentrated sulfuric acid. The solution is then concentrated to 50 ml, acidified to pH 2 by the 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 (elution chloroform / acetic acid 95/5). This gives 956 mg of pure product. Yield: 49% NMR spectrum: 60: 7.9 (S, H acid); 6.5 (S, maleimide);

4.6 (m, CH *); 3.5 (S, OCH ₃ );

2,3 (m, CH ₂ Glu) IR spectrum (kBr) cm ^-1 : 3470,3110,2960,1775,1 750-1 690,1440,1410,1 265-1150,1 090,1 015,830,700.

Example 5: N-Maleoyl-L-isoleucine

Following the procedure of Example 2, N-maleoyl-L-isoleucine is obtained by treating 424 mg (3.22 mM) of L-isoleucine with 500 ml (3.2 mM) of N-carbomethoxy-maleimide.

Yield: 20%

Mass spectrum (CDI, isobutane): 432 (2M + 1), 352,270,212 (M ⁺ + 1), 188,166,132,123.

Nuclear Magnetic Resonance Spectrum (CDCl ₃ ) δ: 8.55 (Ep., OH); 6.85 (2, S, maleimide double bond); 4.65 (1, d, CH); 2.6 (m, 1, CH); 1.28 (d, _CH3); 1.1

Example 6: a- (N-Maleoyl) -L-aspartic acid benzyl ester

A solution of 1 g (4.48 mM) of α-L-aspartic acid benzyl ester in 22.5 ml of a saturated sodium bicarbonate solution is treated with 694 mg (4.48 mM) of N-carbomethoxymaleimide at 0 ° C. with vigorous stirring.

After one hour, the solution is diluted with 91 ml of water and passed at a temperature of 40 ° C for 1 hour. The pH of the solution (8.2) is then brought to 6.4 by the addition of concentrated sulfuric acid. The solution is then concentrated to 50 ml, 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 (elution chloroform / acetic acid 95/5). This gives 267 mg of pure product.

Yield: 20%

NMR spectrum (CD ₃ OD, 6OmH ₂ , ppm):

7.1 (5H, m, H benzyl); 6,6 (2H, S, D ₁ , maleimide); 5 (2H, S, CH ₂ benzyl); 3.1 (2H, m CH ₂ ).

IR spectrum (kBr, cm ^-1 );

3,060,1765,1745,1 720,1410,1270, 830,740, 700.

Example 7: N-Maleoyl-H-amino undecanoic acid

A solution of maleic anhydride (2.4 g, 24.8 mmol) in 10.1 ml of acetic acid is added to a solution of 11-aminoundecanoic acid (5 g, 24.8 mmol) in 30 ml of acetic acid and the mixture stirred vigorously for 3 hours at ambient temperature.

The white precipitate is filtered off, washed with cold acetic acid and dried (6.3 g, 21.1 mM, 85%). 3 g of the precipitate (10.0 mM) are dissolved in dry toluene (300 ml) and treated with triethylamine (2 g, 22.2 mM). The solution is refluxed in a Dean-Stark apparatus by vigorous stirring until the toluene evaporates.

The product is purified on a silica column (elution chloroform / acetic acid 95/5). This gives 1.06 g of N-maleoyl-11-amino undecanoic acid.

Yield: 37%

NMR spectrum (6OmH ₂ , CDCl ₃ , ppm);

8.8 (bp, COOH); 6.5 (2H, S, d _{1 #} maleimide); 3.4 (2H, m, CH ₂ ); 2,3 (2H, m, CH ₂ ); 1,2 (16H, bp, 8 CH ₂ ).

IR spectrum (kBr, cm- ¹ ): 3450, 31100, 2910, 2850, 1770, 1700, 1610, 1515, 1470, 1450, 1241, 375, 1340, 1310, 180, 240, 240, 180 , 1125,840,700.

Example 8: IM-maleoyl-12-amino-dodecanoic acid

A solution of maleic anhydride (2.28g, 23.22mM) in 9.6mL acetic acid is added to a solution of 12-amino-dodecanoic acid (5g, 23.22mM) in 28mL acetic acid and the mixture stirred vigorously for 3 hours at ambient temperature ! The white precipitate is filtered off, washed with cold acetic acid and dried (6.2 g, 20.9 mM, 86%). 4.9g (15.9mM) of the precipitate is dissolved in dry toluene (500ml) and treated with triethylamine (4.9ml, 35mM). The solution is refluxed in a Dean-Stark apparatus by vigorous stirring until the toluene evaporates. The product is purified on a silica column (elution chloroform / acetic acid 95/5). This gives 1.38 g of N-maleoyl-12-amino-dodecanoic acid.

Yield: 29%

NMR spectrum (6OmHz, CDCl ₃ , ppm); 6.52 (2H, s, d1, maleimide); 3.42 (2H, m, CH ₂ ); 2,3 (2H, m, CH ₂ ); 1.25 (18H, broad peak,

IR spectrum (kBr, cm ^-1 ):

3450, 3080, 2920, 2825, 1770, 14170, 450.1, 440, 141, 380, 340, 300, 260, 121, 250, 205, 120, 920, 840, 700.

Example 9: 4- (N-Maleoyl) -L-alanyl-vinblastine

To a solution of 1.52 g (0,009M) N-Maieoylalanin and 1,25ml (0,009M) of triethylamine in 10 ml of ethyl acetate, cooled to a temperature of 0 ⁰ C, was added dropwise a solution of 1,17ml (0,009M) Isobutyl chloroformate in 5 ml of ethyl acetate.

The mixture is stirred for a further 2 hours at a temperature of ⁰ ° C. and then 2.3 g (0.003M) of 0-4-deacetyl-vinblastine are added in solution of 20 ml of ethyl acetate, keeping the temperature at 0 ^° C. Then allowed to rise to ambient temperature and the mixture is stirred for 10 hours. Then, 50 ml of ethyl acetate and 50 ml of an aqueous 10% sodium carbonate solution are added. The mixture is stirred, decanted and the organic phase is separated off. 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 dichloromethane / methanol 92/8). This gives 1.86 g of the pure product.

Yield: 67.6%

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

NMR spectrum (CDCl ₃ , 360MHz, ppm):

8 (NH, 1H); 7.5-7 (4H, H-p ', Η-10Ή-11', H-12 '); 6,7 (2H, anhydride); 6.55 (1H, H-14); 605 (1H, H-17); 5.85 (1H, H-7); 5.45 (1H, H-4); 5.15 (1H, H-6); 4.8 (1H, CH); 3.95 (1H, H-17 '); 3.85 (3H, -OMe); 3.78 (3H ₁ -OMe); 3.7 (1H, H-2); 3.6 (3H ₁ -OMe); 2.7 (3H, NMe); 1.72 (3H, _CH3 alanine); 0.9-0.8 (6H, CH 3-21, CH ₃ -2T).

Example 10: 4- {1-Maleoyl) -6-amino-caproyl-Vinblastiri

To a solution of 604mg (2.861 mM) of N-maleoyl-6-amino-caproic acid and 514μΙ (4,65mM) N-methylmorpholine in 4 ml of ethyl acetate, cooled to a temperature of 0 ⁰ C, was added dropwise a solution of 371 μΙ ( 2.861 mmol of isobutyl chloroformate in 1 ml of ethyl acetate are stirred for 3 minutes at a temperature of ⁰ ° C. and then 550 mg (0.715 mmol) of O-4-deacetylvinblastine in solution of 1 ml of ethyl acetate are added, the temperature being ⁰ C. The mixture is then allowed to rise to ambient temperature and the mixture is stirred for 10 hours The solution is filtered and the ethyl acetate phase is evaporated to dryness in vacuo The residue obtained is purified by chromatography on a silica column (elution dichloromethane / methanol 94/4), giving 263 mg of the pure product in this way Yield: 23%

Mass spectrum (DCI, isobutane): 993.979; 965 (M ⁺ + 4); 963 (M ⁺ + 2); 961 (M ⁺ ); 946; 933; 920; 906; 812; 754; 693. IR spectrum (kBr, cm ^-1 ): 3400, 350, 2940, 1740, 1700, 1615, 1500, 1460, 1440, 1410, 1370, 1220, 1170, 1040. NMR spectrum (CDCI ₃ , 360 MHz , ppm):

7,47-712 (4H, m, H ¹¹ ', H ¹² ', H ¹³ ', 1 H ¹⁴ '); 6.65 (2H, s, d 1 maleimide); 6.55 (14, s, H ¹⁴ ); 6.05 (1 H, s, H ^17); 5.82 (1 H, m, H ^7); 5.42 (1H, s, H ⁴ ); 5.25 (1H, m, H ⁶ ); 3.92 (1 H, m, H ^17); 3.77 (6H, s, OCH ₃ C ²³ OOCH ₃ ); 3.70 (1 H, s, Hz); 3.6 (3 H, s, C ¹⁸ OOCH _3); 2.7 (3H, s, NCH ₃ ); 2.32 (m, CH ₂ aminocapron); 1.62 (m, CH ₂ aminocapron); 0.9-0.8 (6H, t, CH ₃ CH ₃ 21 + 21 ').

Example 11: 4- (N-Maleoyl) -L-glutamyl- ^: metgyl ester vinblastine

To a solution of 282mg (1,17OmM) N-maleoyl-L-glutamic acid methyl ester and 163μΙ ^ (1,17OmM) of triethylamine in 1.4 ml of ethyl acetate, cooled to a temperature of 0 ⁰ C, was added dropwise a solution of 152 μΙ (1.17 Omm) isobutyl chloroformate in 1 ml of ethyl acetate. The mixture is stirred for 4 minutes at a temperature of 0 ⁰ C and then 300 mg (0.390mM) 0-4-deacetyl-Vinb | Astin added in solution of 1 ml of ethyl acetate, keeping the temperature at ^{0 0} C. Then allowed to rise to ambient temperature and the mixture is stirred for 10 hours. The solution is filtered and the organic phase is concentrated under vacuum. The residue obtained is purified by chromatography on a silica column (elution ethanol / ethyl acetate 30/90). This gives 22 mg of the pure product. Yield: 38%

Mass spectrum (DCI, isobutane): 1,036.1 022.1 009, 995 (M ⁺ + 4), 992 (M ⁺ + 1), 937, 885, 811, 751, 694, 635, 541. NMR spectrum ( CDCl ₃ , 360MHz, ppm): 9.4 (1H, m, OH); 8 (1H, s, NH, Ind.); 7.5-7.10 (4H, m, H ¹¹ , H ¹² , H ¹³ , H ¹⁴ ); 6.7 (2H, s, d1 maleimide); 6.58 (1 H, s H ^14); 6.05 (1 H, s, H ^17); 5.80 (1H, m, H ⁷ ); 5.48 (1H, s, H ⁴ ); 5.25 (1H, m, H ⁶ ); 4.73 (1H, m, CH ^x Glu); 3.95 (1 H, m, H ^17); 3.85 (3H, s, OCH ₃ Ar); 3.75 (3H, s, C ²³ OOCH _3); 3.63 (3H, s, C ¹⁸ OOCH ₃ ); 3.58 (3H, s, OCH ₃ Glu); 2.80 (3H, s, NCH ₃ ); 2.238 (m, CH ₂ , Glu); 0.9-0.8 (6H, t, CH ₃ 21 CH ₃ + 2T)

IR spectrum: (kBr, cm ^-1 ): 3430.1 740.1715.1 615.1 500.1460.1 430.1405.1 385.1 250.1 225.1 030.1 005.825.

Example 12: 4- (N-Maleoyl) -L-isoleucyl-Vinblastine

To a solution of 150mg (0,7109mM) N-maleoyl-L-isoleucine and 99μΙ (0,7109mM) of triethylamine in 1 ml of ethyl acetate, cooled to a temperature of 0 ⁰ C, was added dropwise a solution of 92 μΙ (0, 7109 mM) isobutyl chloroformate in 1 ml of ethyl acetate. The mixture is stirred for 3 minutes at a temperature of 0 ° C and then 182 mg (0.237 mM) 0-4-deacetyl-vinblastine in solution of 1 ml of ethyl acetate, keeping the temperature at ^{0 0} C. Then allowed to rise to ambient temperature and the mixture is stirred for 10 hours. The solution is filtered and the ethyl acetate phase is evaporated to dryness under vacuum. The residue obtained is purified by chromatography on a silica column (elution ethanol / ethyl acetate 30/90). This gives 133 mg of the pure product.

Yield: 40%

Mass spectrum (DCI, acetone): 1039.963 (M ⁺ + 2); 904; 812; 769; 728; 637; 593; 549th

IR spectrum (kBr, cm "!: 3480-3400; 2960; 2920; 2880; 1 775; 1740; 1710; 1 610; 1 500; 1460; 1 430; 1 380; 1 250; 1 220; 1 040; 1000, 830, 740.

Nuclear Magnetic Resonance Spectrum (CDCl ₃ , 360MHz, ppm): 9.4 (m, OH); 7.5-7.1 (4H, m, CH arom. 11'-12'-13'-14 '); 6.65 (2H, s, d, maleimide linkage); 6.6 (1 H, S, C ¹⁴ H); 6.05 (1H, s, C ¹⁷ -H); 5.8 (1 H, s, C ⁷ -H); 5.45 (1 H, S, C ⁴ -H); 5.25 (1 H, m, C ⁶ H); 4.5 (1H, d, CH); 3.95 (1 H, m, H ^17); 3.8 (3H, s, OCH ₃ Ar); 3.75 (3H, s, C ²³ OOCH _3); 3.7 (1H, s _; H ₂ ); 3.6 (3H, s, ^C-18 O _OCH3); 2.65 (3H, s, NCH ₃ ); 1.1 (d, 3 H, _CH3 isoleucine); 0.9-0.8 (9H, m, CH ₃ + CH ₃ Isoleucine 2V + CH, 21).

Example 13: 4- (N-Maleoyl) -L-aspartyl-a-benzyl-ester-Vinblastine

To a solution of 267 mg (0,88mM) N-maleoyl-L-aspartic acid-a-benzylesterund 118μΙ (0,88mM) of triethylamine in 1 ml of ethyl acetate, cooled to a temperature of 0 ⁰ C, was added dropwise a solution of 114μΙ ( 0.88 mM) isobutyl chloroformate in 1 ml of ethyl acetate.

The mixture is stirred for 4 minutes at a temperature of ⁰ ° C and then 227 mg (0.29 mM) O-4-deacetyl-Vinblatin in solution of 1 mL of ethyl acetate added, keeping the temperature at 0 ⁰ C. Then allowed to rise to ambient temperature and the mixture is stirred overnight.

The solution is filtered and the organic phase is concentrated under vacuum. The residue obtained is purified by chromatography on a silica column (elution ethanol / ethyl acetate 30/90). This gives 105 mg of the pure product.

Yield: 34%

NMR spectrum (CDCl ₃ , 360 MHz, ppm);

805 (1H, s, NH Ind ¹⁶ ); 7.5-7.17 (4H, m, H ¹¹ ', H ¹² ', H ¹³ ', H ¹⁴ '); 7.37-7.3 (5H, m, H benzyl); 6.75 (2H, s, d 1 maleimide); 6.60 (1 H, s, H ^14); 6.10 (1H, s, H ¹⁷ ); 5.87 (1H, m, H ⁷ ); 5.45 (1H, s, H ⁴ ); 5.30 (1H, m, H ⁶ ); 5.20 (2H, m, CH ₂ benzyl); 3.95 (1 H, m, H ^17); 3.82-3.75 (6H, s, OCH ₃ + COOCH ₃ ²³ ); 3.70 (1H, s, H ₂ ); 3.62 (3H, s, C ¹⁸ OOCH ₃ ); 2.60 (3H, s, NCH ₃ ); 0.9-0.8 (6H, CH ₃ 21 ') · IR spectrum (kBr, cm ^-1 )

3 460.3 030.2 960.2 880.1 770.1740.1715.1 610.1 600.1 460.1 430.1 410.1380.1 260.1 220.1175.1110.1 025.910.800.730.700.

Mass spectrum (DCI, isobutane);

1 085.1 071.1 057.1 054.1 039.1 023, 1013, 768, 708, 542, 158.

Example 14: 4- (N-Maleoyi) -11-amino-undecanoyl-vinblastine

To a solution of 476 mg (1.69 mM) of N-maleoyl-11-aminoundecanoic acid and 326 μM (2.8 mM) of N-methylmorpholine in 3 ml of ethyl acetate, cooled to a temperature of 0 ° C., is added dropwise a solution of 218 μM (1.69 mM Isobutyl chloroformate in 1 ml of ethyl acetate. The mixture is stirred for 3 minutes at a temperature of 0 ° C. and then 433 mg (0.56 mmol) of 0-4-deacetyl-vinblastine in 1 ml of ethyl acetate are added, keeping the temperature at 0 ° C. , Then allowed to rise to ambient temperature and the mixture is stirred overnight.

The solution is filtered and the ethyl acetate phase is evaporated to dryness under vacuum. The residue obtained is purified by chromatography on a silica column (elution ethanol / ethyl acetate 30/90). This gives 240 mg of the pure product.

Yield: 41%

NMR spectrum (CDCl ₃ , 360MHz, ppm):

8 (1H, s, NH Ind ¹⁶ '); 7.5-7.13 (4H, m, H ¹¹ ', H ¹² ', H ¹³ ', H ¹⁴ '); 6.68 (2H, s, d 1 maleimide); 6.63 (1 H, s, H ^14); 6.10 (1H, s, H ¹⁷ ); 5.83 (1 H, m, H ^7); 5.48 (1H, s, H ⁴ ); 5.25 (1H, m, H ⁶ ); 3.95 (1 H, m, H ¹⁷ '); 3.80 (6H, s, OCH ₃ + C ²³ OOCH ₃ ); 3.73 (1H, S, H ² ); 3.6 (3H, s, C ¹⁸ OOCH ₃ ); 2.7 (3H, s, NCH ₃ ); 1.28 (16H, broad peak, CH ₂ -); 0.88-0.8 (6H, tCH ₃ 21 + tCH ₃ 21 ').

IR spectrum (kBr, cm ^-1 ):

3430,3040,2930,2860,1770,1735,1710,1615,1 505,1460,1410,1 370,1230-1 250,1000-1100.

Mass spectrum (DCI, isobutane):

1089,1064.1 048.1 034.1 000.990, 976, 960.

Example 15: 4- (N-Maleoyl) -12-amino-dodecanoyl-Vinblastine

To a solution of 699mg (2.37 mM) Ν-Μ3ΐβονΙ-12-3Γηίηοαοαβ03η83υΓβυηα433μΙ (3.95 mM) of N-Methylenmorpholin in 4.2 ml of ethyl acetate, cooled to a temperature of 0 ⁰ C, was added dropwise a solution of 307 μΙ (2.37 mM) isobutyl chloroformate in 1 ml of ethyl acetate.

The mixture is stirred for 3 minutes at einerTemperaturvonO ° C and then is added 607 mg (O, 79mm) O-4-deacetyl vinblastine in solution of 1 ml of ethyl acetate, keeping the temperature at 0 ⁰ C. Then allowed to rise to ambient temperature and the mixture is stirred overnight. The solution is filtered and the ethyl acetate phase is evaporated to dryness under vacuum. The residue obtained is purified by chromatography on a silica column (elution ethanol / ethyl acetate 30/90). This gives 271 mg of the pure product.

Yield: 33%

NMR spectrum (CDCl ₃ , 36 ohms, ppm):

8 (1H, s, NH Ind ¹⁶ '); 7.5-7.10 (4H, m, H ¹¹ ', H ¹² ', H ¹³ ', H ¹⁴ '); 6.65 (2H, s, d1 maleiide); 6.60 (1 H, s, H ^14); 6.08 (1 H, s, H ^17); 5.83 (1 H, m, H ^7); 5.48 (1H, s, H ⁴ ); 5.25 (1 H, d, H ^6); 3.95 (1 H, m, H ¹⁷ '); 3.78 (6H, S ₁ -OCH ₃ + C ²³ OOCH ₃ ); 3.73 (1H, S, H ₂ ); 3.6 (3H, s, C ¹⁸ OOCH ₃ ); 2.7 (3H, s, NH ₃ ); 1.25 (2OH, Vn ₁ -CH ₂ -); 0.9-0.8 (6H, 6, CH ₃ 21 CH ₃ + 21 ').

IR spectrum (kBr, cm ^-1 ):

3460.3040, 2930.2860.1 735.1700.1 610.1 500.1 460.1 430, 1410.1 365.1 245.1 220.

Mass spectrum (CDI, isobutane):

1 063.1 046 (M ⁺ + 1), 1 037.1 028.1 016, 312.117.

Example 16: 4- (N-Maleoyi) -L-alanyl-lysyl-ethyl ester-vinblastine

To a solution of 40.26 mg (0.163 mM) lysine ethyl ester hydrochloride and 150 μΙ (0.978 mM) triethylamine in 7.5 ml absolute ethanol, is added dropwise a solution of 150 mg (0.163 mM) of 4- (N- Maleoyl) -L-alanyl-vinblastine in 1 ml of ethanol. The mixture is stirred overnight at ambient temperature and then the solution is evaporated. The residue obtained is purified by chromatography on a silica column (elution 14% MeOH / NH ₃ in ether). This gives 101.2 mg of the pure product.

Yield: 67%

Mass spectrum (CDI, isobutane):

1095 (M ⁺ + 2); 1 094 (M ⁺ + 1); 1 037; 921; 839; 769; 693; 615; 574; 532nd

IR spectrum (kBr, cm ^-1 ):

3460; 2940; 1740; 1 710; 1 615; 1,500; 1460.1430; 1 590; 1 250; 1 225; 1120; 1 010; 735th

NMR spectrum (CDCl ₃ , 36 ohms, ppm):

8 (1H, NH, 16 '); 7.5-7.1 (4H, H ¹¹ ', H ¹² ', H ¹³ ', H ¹⁴ '); 6.6 (1 H, s, H ^14); 6.08 (1 H, s, H ^17); 5.8 (1H, m, H ⁷ ); 5.45 (1H, s, H ⁴ ); 5.3 (1H, m, H ⁶ ); 4.8 (1H, m, CH ^x ); 4.18 (2H, q, -OCH ₂ ); 3.83 (3H, s, OCH ₃ ); 3.78 (3H, s, OCH ₃ ); 3.6 (3H, s, OMe); 2.68 (3H, s, NCH ₃ ); 1.65 (3H, d, CH ₃ Ala); 1.43 (2H, s); 1.28 (CH ₃ , OCH ₂ CH ₃ ); 0.9-0.8 (6H, CH ₃ 'CH ²¹ + ₃ ^"21').

Example 17: Coupling of 4- (N-maleoyl) -L-alanyl-vinblastine with galactosylated human albumin (AHGaI) (V ₄ -Ala-MaI-AHg)

44 mg of 4- (N-maleoyl) -L-alanoyl-vinblastine are dissolved in 1 ml of dioxane. On the other hand, a solution of 50 mg AHGaI in 5 ml of phosphate buffer (0.2 M) of pH 8.5 is prepared. Then, the 4- (N-maleoyl) -L-alanyl-vinblastine solution is added to the AHGal solution. The mixture is stirred at ordinary temperature overnight and purified by filtration over Sephadex gel G-25 (2.6 x 96 cm), in equilibrium with a NaCl solution (9%, pH 7.5). The discarded pie is collected (96 ml), concentrated by ultrafiltration and sterilized.

The content of proteins is measured by the method of Lowry and estimated the content of alkaloids by measuring the radioactivity. The resulting conjugate contains 13.5 moles of alkaloid per mole of galactosylated human albumin.

Example 18: Coupling of 4- (N-Maleoyl) -6-amino-caproyl-Vinblastine with Galactosylated Human Albumin (AHg) · (V ₄ -C ₆ -laI-AHg)

234 mg of 4- (N-maleoyl) -6-aminocaproyl-vinblastine are dissolved in 1 ml of dioxane. On the other hand, a solution of 50 mg AGGal in 5 ml of phosphate buffer (0.2M) of pH 8.5 is prepared. Then the 4- (N-maleylol) -6-amino-caproyl-vinblastine solution is added to the AH-Gal solution. The mixture is stirred at ordinary temperature overnight and purified by filtration over Sephadex gel G-25 (2.6 X 96 cm) in equilibrium with a NaCl solution (9%, pH 7.5). The discarded pie is collected (100ml), concentrated by ultrafiltration and sterilized.

The content of proteins is measured by the method of Lowry and estimated the content of alkaloid by measuring the radioactivity. The resulting conjugate contains 12.7 moles of alkaloid per mole of galactosylated human albumin.

Example 19: Coupling of 4- (N-maleoyl) -L-glutamyl-y'-methyl ester -vinblastine with galactosylated human albumin (AHg) (V ₄ -Glu-Y-methylester-Mal-AHg)

24 mg of 4- (N-maleoyl) -L-glutamyl-y = methyl ester -vinblastine are dissolved in 1 ml of dioxane. On the other hand, a solution of 50 mg AHGaI in 5 ml phosphate buffer (0.2 M) of pH 8.5 is prepared. Then the 4- given (N-maleoyl) ^{-L-glutamyl-y-r} methylester vinblastine solution to the AHGal solution. The mixture is stirred at ordinary temperature overnight and purified by filtration over Sephadex gel G-25 (2.6 X 96 cm) in equilibrium with a NaCl solution (9%, pH 7.5). The discarded pie is collected (100 ml), concentrated by ultrafiltration and sterilized. The content of proteins is measured by the method of Lowry and estimated the content of alkaloid by measuring the radioactivity. The resulting conjugate contains 11.8 moles of alkaloid per mole of galactosylated human albumin.

Example 20 Coupling of 4- (N-Maleoyl) -L-Isoleucyl-Vinblastine with Galactosylated Human Albumin (AHGaI) (V ₄ -IIe-MaI-AHG)

250 mg of 4- (N-maleoyl) -L-isoleucyl-vinblastine are dissolved in 20 ml of dioxane. On the other hand, a solution of 591 mg AHGaI in 16.5 ml of phosphate buffer (0.4M) of pH 8.5 is prepared. Then the 4- (N-maleoyl) -L-isoleucyl-vinblastine solution is added to the AHGal solution. The mixture is stirred overnight at ordinary temperature and purified by filtration over Sephadex Gel G-25 (2.6 x 96 cm), in equilibrium with a NaCl solution (9%, pH 7.5). The discarded pie is collected (180ml), concentrated by ultrafiltration and sterilized.

The content of proteins is measured by the method of Lowry and estimated the content of alkaloid by measuring the radioactivity. The resulting conjugate contains 8.8 moles of alkaloid per mole of galactosylated human albumin.

Example 21: Coupling of 4- (N-maleoyl) -L-aspartyl-a-benzyl ester -vinblastine with galactosylated human albumin (AHGaI) (V ₄ -ASp-OB, E.-Mal-AHg)

105 mg of 4- (N-maleoyl) -L-aspartyl-a-benzyl ester-vinblastine are dissolved in 7.9 ml of dioxane. On the other hand, a solution of 226 mg AHGaI in 6.3 ml of phosphate buffer (0.4M) of pH 8.5 is prepared. Then, the 4- (N-maleoyl) -L-aspartyl-a-benzyl ester vinblastine solution is added to the AHGal solution. The mixture is stirred at ordinary temperature overnight and purified by filtration over Sephadex gel G-25 (2.6 X 96 cm) in equilibrium with a NaCl solution (9%, pH 7.5). The discarded pie is collected (60 ml), concentrated by ultrafiltration and sterilized.

The content of proteins is measured by the method of Lowry and estimated the content of alkaloid by measuring the radioactivity. The resulting conjugate contains 5.7 moles of alkaloid per mole of galactosylated human albumin.

Example 22: Coupling of 4- (4-maleoyi) -11-amino-undecanoyl-vinblastine with galactosylated human albumin (AHGaI) · (V ₄ -C ₁₁ -laI-AHg).

440 mg of 4- (N-maleoyl) -11-amino-undecanoyl-vinblastine are dissolved in 46 ml of dioxane. On the other hand, a solution of 969 mg of AHGaI in 27 ml of phosphate buffer (0.4 M) of pH 8.5 is prepared. Then, the 4- (N-maleoyl) -11-amino-undecanoyl-vinblastine solution is added to the AHGal solution. The mixture is stirred at a temperature of 40 ° C for 5 hours and purified by filtration over Trisacryl gel (5.6 x 50 cm), in equilibrium with a NaCl solution (9%, pH 7.5). The discarded pie is collected (250 ml), concentrated by ultrafiltration and sterilized.

The content of proteins is measured by the method of Lowry and estimated the content of alkaloid by measuring the radioactivity. The resulting conjugate contains 10.2 moles of alkaloid per mole of galactosylated human albumin.

Example 23: Coupling of 4- (N-maleoyl) -12-amino-dodecanoyl-vinblastine with galactosylated human albumin (AHGaI) - (V ₄ -C ₁₂ -laI-AHg).

120 mg of 4- (N-maleoyl) -12-amino-dodecanoyl-vinblastine are dissolved in 16 ml of dioxane. On the other hand, a solution of 339 mg of AHGaI in 9.4 ml of phosphate buffer (0.4 M) of pH 8.5 is prepared. Then, the 4- (N-maleoyl) -12-amino-dodecanoyl-vinblastine solution is added to the AGGal solution. The mixture is stirred at a temperature of 40 ° C for 6 hours and purified by filtration over triacrylic gel (5.6 x 50 cm), in equilibrium with a NaCl solution (9%, pH 7.5). The discarded pie is collected (240 ml), concentrated by ultrafiltration and sterilized.

The content of proteins is measured by the method of Lowry and estimated the content of alkaloid by measuring the radioactivity. The resulting conjugate contains 9 moles of alkaloid per mole of galactosylated human albumin.

Example 24: Coupling of 4- (N-maleoyl) -L-alanyl-vinblastine with non-specific immunoglobulin (IgG) (V ₄ -Ala-MaI-IgG).

4mg of 4- (N-maleoyl) -L-alanyl-vinblastine are dissolved in 193μΙ ethanol. On the other hand, a solution of 10mg IgG (626μΙ) in 357μΙ phosphate buffer (0.4M) of pH 8.5 and 447μΙ water (7mg Prot / ml) ago. Then, the 4- (N-maleoyl) -L-alanyl-vinblastine solution is added to the IgG solution. The mixture is stirred overnight at a temperature of 35 ° C and purified by HPLC through a column (gel filtration) of Dupont de Nemours GF450x250, adjusted in phosphate buffer 0.2M of pH 7.5.

The discarded pie is collected and dosed. The content of proteins is measured by the method of Lowry and estimated the content of alkaloid by measuring the radioactivity. The conjugate contains 6.6 moles of alkaloid per mole of immunoglobulin.

The compounds according to the invention were subjected to a pharmacological examination.

1. Sensitivity to lysosomal enzymes

The sensitivity of the conjugates to lysosomal enzymes was investigated by incubating these conjugates in the presence of 5 mM Cyctein, 4 mM acetate buffer and lysosomal enzymes over a period of 48 hours and at a temperature of 37 ° C. After 48 hours of incubation, the undamaged proteins are precipitated by addition of 5 mg albumin serum through 2 volumes of acetonitrile.

After incubation of the samples at a temperature of 4 ° C for 40 'and centrifuging at 3000 revolutions per minute for 40', the radioactivity of the floating portion is determined by counting an aliquot in liquid scintillation. The soluble radioactivity is a measure of the conversion of the conjugate.

The percentages for the reactions obtained are as follows:

V ₄ -Ala MaIAHg: 92%

V ₄ -C ₆ -MaIAHg: 100%

V ₄ -IIe-MaIAHg: 75%

V ₄ -C ₁₁ -MaIAHg: 79%

V ₄ -C ₁₂ -MaIAHg: 92%

2. Stability in serum

The stability of the conjugates in the presence of serum was examined by incubating them in the presence of 62% fetal calf serum for a period of 48 hours and at a temperature of 37 ^° C.

After 48 hours of incubation, the undamaged proteins are precipitated by adding one volume of trichloroacetic acid (TCA 40%).

After incubation of the samples at a temperature of 4 ° C for one hour, they are centrifuged and the radioactivity of the floating portion is determined by counting an aliquot in liquid scintillation. The soluble radioactivity is a measure of the conversion of the conjugates

 The results show that the conjugates are stable over 49 hours.

3. Stability at acidic pH

The stability of the conjugates in the acidic pH range was investigated by incubation of the conjugates in the presence of 40 mM acetate buffer of pH 4.5 for 48 hours and at a temperature of 37 ° C. The reaction is determined by the technique of precipitation by means of TCA

 The results show that the conjugates are stable under these conditions.

4. Chemotherapeutic activity in leukemia P388

The chemotherapeutic activity of the intermediate derivatives and the conjugates was determined on leukemia P388, verabfolgtauf intraperitoneal route in the mouse female BDF _1: 10 ^e tumor cells are inoculated by intraperitoneal route on day zero. The conjugates are then administered by day 1 intraperitoneal route. The ILS value represents the percentage of survival time of the treated mice compared to untreated mice.

The molar ratio indicates the number of alkaloid moles per mole of galactosylated human albumin.

The number of mice surviving on day 60 is given as well as the dose in mg / kg of the alkaloid and the protein.

3.1. Intermediate derivatives

product dose ILS survivor mg / kg / day % on day 60 VBL (vinblastine) 3 77 0/10 VCR (Vincristine) 2.7 64 0/11 V ₄ -Ala-MaI 5 (Example 9) 25 100 0/7 50 127 0/7 75 -70 0/6 V ₄ -Glu-y ^ methyl ester times 50 88 0/10 100 111 0/10 V ₄ -IIe-MaI (Example 12) 50 148 0/7 100 -79 0/7 V ₄ -C ₆ -MaI (Example 10) 25 102 0/8 50 218 1.7 100 -83 0/7

product dose ILS survivor ILS survivor mg / kg / day % on day 60 % on day 60 V ₄ -C ₁₁ -MaI (BeISpIeIM) 50 37 1.7 100 -19 1.5 77 0/10 V ₄ -C ₁₂ -MaI (Example 5) 25 185 1.5 64 0/11 50 > 633 6.8 69 0/5 100 7 1.7 133 1.5 3.2. ! Conjugate 201 0/5 dose molar 175 3.1 product mg / kg / day relationship 138 0/7 Vinca protein > 552 3.5 VBL 3 > 574 3.5 VCR 2.7 47 0/5 V ₄ -Ala-MAIAHg 60 374-466 13.5 to 10.7 67 0/5 80 450 15 75 0/5 100 562 15 88 0/5 130 2766 6.6 51 0/5 150 3035 4.1 48 0/5 150 1276 9.8 79 0/5 150 913 13.7 95 0/5 V ₄ -Asp-a-benzyl ester MaI-AHg 60 768 5.7 137 1.5 V ₄ -Glu-y ⁱ methyl ester-May AHg 60 987 4.7 147 1.5 100 1645 4.7 153 1.5 150 2468 4.7 V ₄ -IIe-MaIAHg 60 545 8.8 227 , 100 908 8.8 > 606 3.5 150 1362 8.8 186 3.10 V ₄ -C ₆ -MaIAHg 60 400 12 -45 0/5 150 995 12 > 769 5.5 V ₄ -C ₁₁ -MaIAHg 60 378 11.8 80 504 11.8 753 7.9 90 658 10.2 100 630 11.8 100 646 7.9 150 945 11.8 V ₄ -C ₁₅ -MaIAHg 90 741 9

Claims (6)

claims: 1. A process for the preparation of a conjugate of a vinca alkaloid with a protein or a protein fragment, the general formula I. CCCCH. AR, and optionally a pharmaceutical composition ₍ in the R _{1 represents} a protein or a protein fragment; R ₂ is COOC ^ alkyl or CO-R7 where R _{7 is} NH ₂ , an ester of an amino acid or a peptide, R ₃ is H, CH ₃ , CHO; when R ₅ and R ₆ are considered individually, R _{6 is} H and one of R ₄ and R ₅ is ethyl and the other is H or OH; when R ₅ and R _{6 are considered} together with the carbon atoms to which they are attached, they together form an oxirane nucleus and R ₄ is ethyl, and A is a bifunctional organic derivative of the maieoyl amino acid, maleoyl peptide or maleoylphenoxy type characterized in that one comprises a maieoyl amino acid, a maleoyl peptide or maleoylphenoxy of the formula. HOOC - X - M wherein X represents a linear alkylene chain of 1 to 12 carbon atoms, or a branched alkylene chain of 2 to 5 carbon atoms, a cycloalkylene chain of 3 to 6 carbon atoms, a phenyl chain of 3 to 6 carbon atoms, or the group R-CH of the natural amino acids R-CH-COOH; or the fragment of a peptide chain of the type - (Xi-NH-CO) _n -Xi-, in which η = 1, 2, 3 or 4 and X _{1 is} a linear alkylene chain of 1 to 12 carbon atoms, a branched alkylene chain of 2 to 5 carbon atoms, a cycloalkyl chain of 3 to 6 carbon atoms, a phenyl chain of 3 to 6 carbon atoms, or the group R-CH of the natural amino acids , or is also a phenyl radical; in its racemic form or in one of its optically active forms, with the hydroxyl in the 4-position of a compound of the general formula condensed; in the R2 f R3 / R4f Rs * R6 ^ur > d X have the abovementioned meanings, and in that the resulting intermediate is condensed with a protein or a protein fragment, and optionally the compound of general formula I with pharmaceutically acceptable carriers and fillers, according to Unit dose of 50 mg up to several grams, optionally in combination with other active substances. 2. A process as claimed in claim 1, wherein the first stage of the condensation is carried out with the aid of a chloroformic acid alkyl ester, preferably with ethyl chloroformate or isobutyl chloroformate, in the presence of an amine base, such as triethylamine, N-methylpiperidine or N-methylmorpholine, in an organic solvent such as ethyl acetate, tetrahydrofuran or methylene chloride, by isolating the resulting intermediate from the reaction medium, purifying it, and carrying out the second stage of condensation in a conventional manner, and in the aqueous medium at a temperature between 4 ⁰ C and 25 ° C and a pH of 7.5 to 9.5. 3. The method according to claim 1 or 2, characterized in that the one or more RCH functional groups of the natural amino acids, represented by X or X ₁ , are protected by means of known protecting groups. A method according to any one of the preceding claims, characterized in that the protein R _{1 is} albumin from bovine serum or human serum, fetuin or immunoglobulins. Process according to any one of the preceding claims, characterized in that the protein Ri is selectively treated. A method according to any one of the preceding claims, characterized in that R _{1 is} an immunoglobulin selected from the following group formed by: Ig of goat or sheep immunized with carcinoembryonic antigen; - Ig from rabbit anti-LLA; - Ig of goat or sheep, immunized with carcinoembryonic antigen; - different Ig from the monkey, anti-LLA, anti-LMA, anti-LLC, anti-LMC; - Ig of goat or sheep, immunized with membranes from lung carcinoma; ^; - monoclonal Ig, starting from mouse hybrids, which / -r- secrete antibodies against human Colo-rectal carcinoma; Monoclonal Ig from mouse hybrids secreting antibodies to human melanoma; - monoclonal Ig from murine hybrids secreting antibodies reactive with human meukemic cells; - monoclonal Ig from murine hybrids secreting antibodies that react with human neuroblastoma cells; - monoclonal Ig, starting from mouse hybrids secreting antibodies that react with human mammary carcinoma antigens; - monoclonal Ig, starting from mouse hybrids secreting antibodies that react with the cells of human ovarian carcinoma; Monoclonal Ig from murine hybrids secreting antibodies that react with human osteosarcoma cells; Monoclonal Ig, starting from mouse hybrids secreting lung carcinoma antibodies; or an immunoglobulin fragment, namely Fab, Fab 'or F (ab'> 2 or monomeric IgM, obtained from an antibody by digestion with the aid of a proteolytic enzyme.