DD219771A5 - METHOD FOR PRODUCING NEW CONJUGATED COMPOUNDS OF VINBLASTINE AND ITS DERIVATIVES - Google Patents

Abstract

Process for the preparation of novel conjugated compounds of vinblastine and its derivatives. The invention relates to a process for the preparation of novel conjugated compounds of vinblastine and certain known derivatives thereof with proteins, protein fragments, amino acids or amines which are useful as antitumor agents. These compounds correspond to the following general formula in which A is a "bridge" such as -COCH 2 -, -COCH 2 -CH 2 CO- or COCH 2 -CH 2 -CH 2 CO, R 1 is an optionally modified protein radical, an amino group NR 3 R 4, chlorine or an amino acid ester group bound via the amino function and R 2 represents a methoxy group, an amino group or an amino acid residue bound by an amide bond and having one to six carbon atoms of its ester group, wherein R 3 and R 4 are alkyl groups of one to three carbon atoms or together an alkanediyl group of three to five carbon atoms represent. formula

Description

W "i mm.

Process for the preparation of novel conjugated compounds of vinblastine and its derivatives

Field of application of the invention '_; /

The present invention relates to a process for preparing compounds of .ηθμθΓ.konjugierter vinblastine and known from certain :, derivatives thereof with proteins, protein fragments, amino acids or amines _a to be used as antitumor agents.

Characteristic of the known technical solutions ^; '

Vinblastine and certain of its derivatives, in particular vincristine or vindesine, have already been incorporated with proteins, for example. Coupled with albumin or various immunoglobulins. This results in coupling products or compounds called "conjugated compounds." We refer in particular to the following references:.

J.D. Teale, Jacqueline M. Cough et V.Marks, Br. J. Clin. Pharmac. 4, 169-172, 1977

-'C, HJFord, C.E. Newman, J.R. Johnson, C.S. Woodhouse, T.A. Reeder, G.F. Rowland, R.G., Simmonds, Br. J. Cancer 17, 35-42, 1983

M.J. Embleton, G.F. Rowland, R.G. Simmonds, E. Jacob, C.H.Marsden, R.W.Baldwin Br. J, Cancer 47, 43-49, 1983

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

- EU Lilly Eur.Pat.Applic. , Publ. N ° 56.322, 21.07.82

- R.A. Conrad, G. J. Cullinan, K. Gerzon, G. A. Poofe J. Med. 22, 391. 1979;

The coupling of these bis-indole derivatives has not been made solely for the purpose of developing new immunologically reactive substances,

took place '-., v,.

but especially with regard to the production of antitumor agents that are more active, more selective and less toxic,

'0 0 f ^{! ;} 4 ' '. ' ν · '"' i ν '

63 939 18

 '.. - 2 ~

For the latter purpose, numerous conjugated protein compounds have been studied with other antitumor agents heretofore. This is particularly true for conjugated compounds of an antibody and a ricin fragment or for conjugated compounds of albumin and methotrexate (French Patent Application Hr. 2 437 213, CM Industriesf BCF Ohu, SB Howell J. of Pharmacology and Ixp. Therapeutics, 219 (2) , 389-393, 1981). V

The monoclonal antibodies, especially those of human origin, which are coupled with known antitumour drugs / are in particular the subject of various studies.

Finally, it is emphasized that the use and evaluation of 1: 1 complexes of bis-indo alkaloids with tubulin has been described in Belgian Patent Specification No. 854,053. In certain cases, this may result in less toxicity and chemotherapeutic efficacy than the corresponding ones free alkaloids.

Object of the invention

The novel compounds produced by the method of the present invention are antitumor agents that are active, selective and low in toxicity.

Explanation of the essence of the invention

The object of the invention is to provide a process for the preparation of novel conjugated compounds of vinblastine. '

The present invention relates to a process for the preparation of novel products which are conjugated compounds of vinblastine or derivatives of vinblastine with proteins, jamming agents, amino acids or simple amines, characterized in that the coupling is effected via an ester group. " According to a preferred embodiment, the process according to the invention is characterized in that the protein, the protein fragment or the amine is coupled to the vinblastine compound via a bridge formed by the above-mentioned Condensation of Vinblastinderivats with a bifunktioneilen organicp compound, which is optionally activated, was prepared.

For example, the UO-desacetylvinblastine derivative may be the vindesine, 4-O-desacetylvinblastine coupled to an amino acid at 3-C, or the 4-O-desacetyl-1'-deoxy -vinblastine or a 23-vinylblastinoyl ester derivative Amino acid. , ^K ".

In particular, the process allows the preparation of compounds which correspond to the following general formula:

CH ₃ OOC

CH ₃ O

"*""A ^mmm κ ι

CO

in which A represents a "bridge" such as the -COCH ₂ - or -CO (CH ₂ ) _n -CO- •., wherein η varies from 1 to 5, PL represents an optionally modified protein residue, Ro represents a methoxy group, one Amino group or an alpha-amino acid residue which is bonded by an amide bond and whose ester group contains one to six carbon atoms, and R, a hydrogen atom or a hydroxyl group, which may be present in each of the two possible configurations, as well as of addition salts thereof with a mineral or organic acid. ·, ...

It will be appreciated that the acetate, hemi-succinate, hemiglutarate bridge may be substituted by a higher homologue, for example, by an alkyl or aminoin group, without losing the effect that characterizes the compounds prepared by the process of the present invention.

In the work of the prior art, binding was effected via an amide link in the region of the 23-vinblastoyl function of the bis-indole derivative. Surprisingly, we have found that the antitumour effect is better preserved. remains when the bond is made at the ^ carbon atom and not at the 23 carbon atom. ,.

According to the invention, in a first step, the derivatives are obtained by condensation of chloroacetic acid chloride or an anhydride, for example, the chloroacetic, succinic, glutaric anhydride or a higher homologue on the ^ -g-hydroxyl of 4-0-deacetylvinblastine or a derivative of Type of iJ-O-desacetylvinblastine J-carboxamide prepared.

The 4-chloroacetate, hemisuccinate or hemiglutarate derivatives prepared in this way are now condensed in a solvent in which these compounds are soluble with the protein, the amino acid or the simple amine. For this purpose, one can use a mixture of water and dioxane, which is kept at a corresponding pH with the aid of a buffer, for example a borate buffer. A confirmation of the condensation is carried out by chromatography or electrophoresis. In this latter case radioactive vinblastine v _; (eg tritiated) to facilitate the labeling. , , ''

Recovery of the chloroacetate on the 4-carbon of vinblastine is reported by W.W. Hargrove Lloydia, 27 (4), 3, ^ 2, 1964. It can also be obtained by the action of chloroacetic anhydride on the desacetylvinblastine in dichloromethane.

From a chemical point of view, condensation with proteins can be explained by obtaining covalent bonds formed by the reaction of the amino groups of the protein lysine with the activated chlorine of the chloroacetate function or with the. activated ester group of hemisuccinate or hemi. glutarate derivative arise. ',

The albumin from bovine serum has, for example, 56aminierte. Lysine residues on .The. The number of vinblastine molecules per protein will vary depending on the working conditions, but will generally be between 1 and 3 ^.

ι. '.

The activation can be carried out in a conventional manner by treatment with an alkyl chloroformate, preferably with ethyl or isobutyl chloroformate ₃ in the presence of an amino base, such as N-methyl piperidine or N-methyl morpholine. ._, ^l , """'""

ι ' ^λ '. ' , j

The condensation can be carried out in situ with the reaction mixture containing the activated anhydride. However, in most cases the activated anhydride can also be isolated. / ^! _;

The resulting conjugated compound becomes classical Weir-e. , as in chemistry or in the case of conjugated protein. isolated in biochemistry. The conjugated protein compound is thereby. Addition of acetone from the. Reaction medium precipitated dann'zentrifugiert, ^washed: lyophilically-ized and purified by gel filtration. Optionally, subjecting the derivative thus obtained to a classical succinylation reaction whereby ^; Aggregation problems that are characteristic of certain conjugated or non-conjugated proteins are avoided.

The proteins used to advantage are, in particular, albumin from bovine serum or human serum, the fetuin or immunoglobulins, the latter optionally being

In the latter case, the use of monoclonal antibody of human origin, which show a particular activity against human tumors, has proven to be of particular interest. '/ .. .. ..,' '.

The proteins used can also be selective. Modification to be treated. These modifications allow the recovery of conjugated protein compounds which, in their therapeutic use, are preferentially enriched in the area of certain tissues, for example in the region of the liver. Thus, it is possible, prior to the condensation of the 'vinblastine. derivat - ssylate the latter with the protein. The galactosylation is carried out, for example, by using the method described by G.Wilson in The Journal of Biochemistry, 253 (7) 2070-2072, 1978.

The in vitro experiments with these compounds of the invention to demonstrate their antitumor activity indicate that the formation of the conjugated compound via C-4 birefringence may be more advantageous when binding through C-3.

The coupling via the 3-carbon atom. of vinblastine or "one derivatives is carried out in conventional manner in that the hydrazide ¹ in., ¹ .One first stage at C-23 (carbohydrazide) and, then, the corresponding Nitrosiierung Azotierungsprodukt of the acid is formed. The azotization product is then coupled directly with the protein; , ^Λ .

K. ..:

However, this azotination product of the acid can also be condensed in a likewise known manner with an amino acid ester, for example a methyl ester. The ester function of the amino acid, in turn, may be subjected to hydrazinolysis and then nitrosation. The new acid acid quotation product. The coupling compound Amihosäure-Vinblastin is then condensed with the protein. In this case, the amino acid is the link. -

- 7 -

 that combines the protein with the vinblastine molecule.

In this way, condensation compounds were obtained, which are gekouuelt at C-3 or C -'4 and deacetylated at 0-4 and, for example, are constructed as follows:

a) Coupling via the C-3 Carbon " ⁵ Vinblastine / fetuin, coupled via C-3 (fetid-C-3) vinblastine / fetuin, succinylated and coupled via C-3 (FOt (S) -VDS-C- _?!) vinblastine-isoleucyl Fötuin (fot-Ile-VDS-C-3) vinblastine Tryptqphyl albumin from bovine serum (RSA TRPV-C-3) (example 1):

Bovine serum vinblastine-tryptophyl-succinylated albumin (RSA (S) -TrpV-C-3). , '.' '..

b) Coupling over the C-il-carbon "vindesine-4-O-chloroacetate (Example 2).

, yindesine - ^ - O-chloroacetate + albumin from bovine serum (RSA-VDS-C-'I) (Example 3a). ,

Vindesine 4-O-chloroacetate + succinylated albumin from bovine. serum · (RSA (S) -VDS-C-4h (Example 3b)

Vindesln - ^ - O-chloroacetate + albumin, from human serum (AH-VDS-C- * ¹ !) (Example Ha) '"^

Vindesine 4-O-chloroacetate. + Galactosylated albumin from human serum (AHgäl-VDS-C-4). (Example 4b). '

Vindesin-4-O-Chloracet.at + Pynrolidine. ".-.

, Vinblastine C-3-ethyl-lsp.leucinate '4-O-chloroacetate' (Example 5) Vinblastinoyl-23-ethyl-isoleucinate-4-Ό-chloroacetate + succinylated albumin from bovine serum (RSA (S) -VIle -C ~ 4) (Example 5) 'Vindesine 4-O-Hemisuccinate + Py'nsslidine (Example 6) Vindesine 4-O-Hemisuccinate + Bovine Serum Albumin (RSA-Suc VDS) (Example 7)

Vindesine 4-O-Hemisuccinate + Albumin from Human Serum (AH-Succ-VDS) (Example .8). > ...;

Vindesine 4-O-hemisuccinate + galactosylated albumin from human serum (AHgal-Succ-VDS) (Example 9)

; ^v '': / - ⁸ - 'Λ.' -Λ "'

yindesin - ^ - O-hemisuccinate + .unspecific immunoglobulins (IgG) (IgG-SuCC-VDS) (Example 10)

Vindesine 4-O-hemisuccinate + IgG anti-milk fat globules / (IgGanti-MFG) (IgG anti-MFG ^ Suc-VDS) (Example 11),, vinblastine - ^ - O-hemisuccinate + pyoollidine (Example 12) Vinblastine - ^ - O-hemisuccinate + ethyltryptophanate. (Example 13) Deoxyvinblastinoyl '- ^ - ethyltryptophanate ^ -O-hemisuccinate. + ·,. Pyrrolidine (Example 14). '·;'DesoxyvinbIastinoyl-23 ~ Äthyltryptöphanat-4-0-hemisuccinate albumin + Rihderserum (RSA-Succ-DesoxyVTrpE) (Example 15) Deso ^ xyvindesin-4-O-hemisuccinate Äthyltry.ptophanat + (Example 16) Vinblastinoyl-23 ^ Äthy tryptophanate 4-O hemisuccinate + pyrrolidine (Example 17)

Vinblastine 4-O-hemiglutarate + pyrrolidine (Example 18).

embodiments

The following examples illustrate, in a nonlimiting manner, the process according to the invention. .- '

A. coupling via the C-3 carbon _;

Example 1 . , ,; ·. ^;

Bovine serum vinblastine-tryptophyl-albumin (RSA-Trp V-C-3)

'.,'. ', '''

into a flask equipped with a reflux condenser is added the radioactive ^ -ethyl acetyl-VBL ^ -Trp methyl ester (250 mg, 0.26 mmol) in 5 ml of a 1: 1 (v / v) solution of anhydrous Hydrasin ir. Ethanol introduced. The solution is stirred at 60 ° C for five hours and at room temperature overnight under nitrogen. The mixture is cooled, diluted with 50 ml of deionized water and 50 ml of saturated sodium chloride solution and extracted three times with 50 ml of CHpClp. The organic phases are combined, washed successively with 50 ml of water and 100 ml of saturated NaCl solution, and Na ₂ SO ₄ . dried. After evaporation of the solvent, 217 mg (0.23 mmol)

" - , '., - 9 -:,: ·" ·. ^:

4-deacetyl-VBL ~~ Trp monohydrazide isolated, which is used without purification in the following reactions. , ,

The 4-deacetyl-VBL-Trp-M6nohydrazide (0.23 mmol) is dissolved in a solution of 5 ml of methanol and 16 ml of 1N HCl. The solution is cooled to -10 ° C and with vigorous stirring 45 mg NaNOp are added. After 10 minutes, the pH is Werr. The solution is extracted to 8.5 with saturated NaHCO-. solution. The azide is rapidly extracted with several portions of cold CHpCIp. The organic phases become crazy. agree, about Na ₂ SOj. -dried'and concentrated in a rotary evaporator. The CHpCIp is constantly being replaced by dioxane.

The dissolved in dioxane azide is added dropwise in an equimolar amount to a solution of BSA (269 mg) in ..0,1N Na ₂ Jipo ₁ .-, which is adjusted with dilute sodium hydroxide solution to pH 9 trains sets. When the addition is complete, the pH is adjusted to... With 1N NaOH. 9 set. The mixture is left at room temperature for 72 hours; touched. The excess of azide is destroyed by addition of 100 ul lO ^ ₁ ethyl NH, OH and stirring for one would. The Frotei: is precipitated by the addition of 6 V parts of cold acetone and centrifuged (Centrifugeuse International 30 min, 1500 rpm). The residue is rinsed twice with cold acetone and lyophilized. The conjugated compound is purified by chromatography over Sephadex G 2F> . The fractions containing the conjugated compound are combined and either lyophilized or by ultrafiltration with a Diaflo. .

Concentrated membrane. The yield is 270 mg RSA-Trp-V-C-3 ·

B. coupling via the C-4 carbon

Example 2 ·, "'-

Vindesine - ^ - O-chloroacetate

In a flask Ö _S introduced 92 mmol vindesine, 25 ml CHpCIp and 4.1 mmol Chloressigsäzreanhydrid. The solution is stirred in the sintering for 14 hours. Add 25 ml of methanol, and stir for two hours at normal temperature. The solvent is evaporated under vacuum, the residue dissolved in 500 ml of CHpClP and cold-washed with a dilute, aqueous

-ΙΟ-

Washed ammonia solution. After evaporation of the organic phase, the residue is dissolved in 47 ml of methanol containing 11.2 ml of water and 2.33 g of silica. The mixture is stirred for 6 hours at room temperature. The silica is separated by filtration and washed several times with hot methanol. The filtrate is evaporated under vacuum ^1, made alkaline with a dilute ammonia solution and extracted with CH Cl _{0 0} extracted: The extracts are combined, dried over Na ₀ SOU and evaporated to dryness. The purification is carried out on a silica column by elution with a mixture of CH ₂ Cl ₂ : 5% MeOH. Yield 88%,

IR spectrum: 3-470, 3040, 2970, 2880. 1740, 1690, 1615, 1510,

1500, 1430, 1225, 1190, 1010, 740 cm " ¹

Mass spectrum 830 (M + 1), 813, 796, 773, 754, 297, 277, 293, 108 NMR spectrum: _t · ppm 8 (lH, s, NHind),

7.45 (1H, d), 7.2-7.0 (4H, m), 6.9 (1H, d), 6.5 (1H, s), 6.05 (lH, s, 5, $ (lH, m), 5.5 (IH, m), 5.28 (1H, d), 4, O (2H, CH2C!).

3.7 (3H, s, -CO2CH3), 3j, 55 (3H, s, OCH3). 2.8 (3H, s, NCH3), 0.9-0.65 (8H, m).

Example 3 '

a) Coupling via C-4 of 4-0-deacetyl-vindesine (RSA-VDS-C-4)

The radioactive C-4-chloroacetate of O-4-desacetylvindesin GL50 mg), dissolved in 5 ml dioxane, is added to a solution of 2l6 ng RSA (albumin from bovine serum, CaI Biochem) in 5 ml borate buffer 0.4 « M pH 9.0 was added dropwise. The mixture is stirred at room temperature for 4 to 8 hours. The conjugated compound is precipitated by addition of 6V portions of acetone and centrifuged at 1300 rpm for 30 minutes. The residue is washed twice with acetone and centrifuged under the same conditions. After these two rinses, the precipitate is lyophilized and purified by filtration over G-25 gel (3x90 cm) equilibrated in a solution of NHhHCO-, -0.1 M pH 7.8. The excluded peak is recovered and lyophilized. The protein content is measured by the Lowry method and the alkaloid content is determined by measuring the radioactivity.

The resulting conjugated compound contains 2.5 moles of VindVsin per mole of RSA. Chromatography on agarose gel shows that the radioactivity is well bound to the proteins.

b) succinylation (RSA (S) -VDS-C-M)

The conjugated compound is dissolved in water at a concentration of 100 mg per 5 ml of water and the pH of the solution is adjusted to 7 with 0.1 N sodium hydroxide solution. Then 55.mg of succinylanhidride are added in small portions. By adding 0.1 N sodium hydroxide solution, the pH is kept at 7. When the pH becomes stable, 55 mg of succihyfenhidride are added. The solution is stirred for one hour at room temperature, dialyzed against water at 0 ° C. for one night and lyophilized. The protein and alkaloid contents are determined by the methods described under a). ^,,

Example 4 ^; ,

a) Coupling via C-1 'of, 4-O-deacetyl-vindesine (AH-VDS-C-4)

The C-4-chloroacetate of radioactive 4-O-deacetyl-vindesine (140 mg) dissolved in 3 ml dioxane is added to a solution of 200 mg human albumin (Merieux) in 2 ml borate buffer 0.34 M pH 9 '0 drip-drips'. The mixture is stirred for 24 hours at room temperature. The conjugated compound is precipitated by the addition of β V portions of acetone and centrifuged at 1300 rpm for 30 minutes. The precipitate is washed twice with acetone, and centrifuged to ¹ same conditions. After these two rinses, the precipitate is lyophilized and purified by filtration over G-25 gel (3x90 cm) which would be equilibrated in a solution of NH ₁ JHCO, 0.1 M pH 7, S. The excluded peak is recovered and lyophilized. The protein content is measured by the method of Lowry and the alkaloid content is determined by measuring the radioactivity. The resulting conjugated compound contains 2.6 moles of vindesine per mole of AH. , '.'

. :: - _; , · ·. - ⁱ² - ".;':.

b) Coupling over .C-4 "of 4-O-deacetyl-vindesine (AHgal-VDS-C-4)

The C 1-4 -chloroacetate of radioactive 4-O-deacetyl-vindesine (51 mg), dissolved in 2 ml dioxane, is added to a solution of 200 mg AHgael in 17 ml PBS and 3.1 ml borate 1.7 N pH 9 (Phosphate buffer) 'added dropwise. The mixture is stirred for 2'4 hours at room temperature. The conjugated compound is precipitated by the addition of 6V portions of acetone and centrifuged at 1300 rpm for 30 minutes. The residue is washed twice with acetone and centrifuged under the same conditions After these two rinses, the precipitate is lyophilized and purified by filtration over G-25 gel ( ₃ × 90 cm) suspended in a solution of NH ₁₁ HCO ₅ . The excluded peak is recovered and lyophilized - The protein content is measured by the Ldwry method and the alkaloid content is determined by measurement of the radioactivity The conjugated compound obtained contains 0, 0.1 M pH 7.8. 4 moles of vindesine per mole of oil. '

Example 5 - ... '. ,

il-O-chloroacetate of N (4-OrDesacetyl-3-decarbomethoxy-vinblastine-23-oyl) -L-ethylisoleucinate (RSA (S) -VIIe-C-)

Following the procedure of Example 2, starting from the corresponding vinblastine derivative (cf. Belgian Patent 889,136), the compound vinblastine C-3-ethyl-isoleucinate ¹ ! -0-chloroacetate is obtained in 85% yield.

IR spectrum: V 3470, 3H10, 3040, 2970/2880, 1740, 1680, 1615,

1500, 1460, 1430, 1300, 1225, 1190, 1150, 1010, 740 cm ^-1

Mass Spectrum: 988 (M + 17), 973 (M + 2), 939, 917, 391 _# 236, 94

NMR spectrum: ppm 9.85 (1H, s, OH),

8.6 (1H, s, NH), 7.55 (1H, d), 7.4 (1H, d). 7.2-7.0 (m, 3H), 6.65 (1H.s), 6.1 (1H.s), 5.85 (1hl, m), 5.58 (1H, s), 5.32 (1H, m) /4.6 (1H, q), 4.2 (2H, m), 4.0 (2H), 3.78 (3H, s), 3.1 (3H, s), 2 , 7 (3H, s), 1.28 (3H, t), 1.0-0.8 (14H, m).

 The procedure described in Example 3 gives the RSA (S) -VIle-C-4.

Example 6

Vindesine ~ 4-0 hemisuccinate -pyrrolidinamide

Using an analogous procedure to that of Example 12, starting from vindesine, the corresponding conjugated compound is obtained in 62% yield. ,

V. . · ';' ..

Mass spectrum (DCI, isobutane): 921 (M + 14), 907 (M) IR (CHCl ₃₅ 4%): 3400, 2975, 2882, 1732, 1693, 1632, 1503, 1462, 1380, 12147 cm ^-1 .

RMN (360 MHz, CDCl ₃ ) 8.05 (1H, H17), 5.46 (1H, H15), 3.96 (1H, H17'a), 3.80 (3H, OMe), 3.64 ( 3H, OMe), 3.45 (2 CH, -. pyrrolidine), 2.88 (3H.N-CH _3), 0.95 (3H, CH _3), 0.83 (3H ^ CH _3).

Example 7

Preparation of Vindesine 4-O-Hemisuccinate and Bovine Serum Albumin Coupling (RSA-Succ-VDS). /

In a. Flasks are 0.125 mmol Vindesin, 0.187 mmol. Succinylanhydrid and 4 ml of anhydrous methylene chloride introduced. The solution is stirred under exclusion of light for 14 hours at room temperature. Then the solution in an ice. bath dipped. Are added O, 25o mmol of triethylamine, O, 25o mmol of Xthylchloroformiat and 5 ml of dioxane. It is stirred for half an hour. Independently, a solution of 90 mg RSA in 17.3 ml is prepared; Water and dropwise-17.3 ml of dioxane. The pH is adjusted to 8.5 with 1 N / soda. The solution is cooled to 5 ° C. After, half an hour will be the activated. Ester added to the RSA solution. The solution will be four hours

, -. ".., .. - 14 -, ..:

stirred at 5 ° C, yobei the pH by adding IN. NaOH to 8.5. gehaiVen /. The precipitation of the conjugated compound and. their characterization is done as described above. The molar ratio is about 18 and remains constant after chromatography on Sepharose 6B in the presence of 6M guanidine. .

Example 8 _; >.

'· -.- ..' '"". , '.... ί Vindesine 4-O hemisuccinate and human albumin coupling.

0.28 mmol (210 mg) of vindesine, 42 mg (0.4 mmol) of succinic anhydride and 6 ml of anhydrous methylene chloride are placed in a flask. The solution is stirred under exclusion of light for 14 hours at room temperature. The solvent is evaporated. The residue is taken up in 4.7 ml of dioxane: ·. Then the solution is immersed in an ice bath. 0.56 ml of triethylamine in 3.5 ml of dioxane and 0.56 ml of isobutyl chloroformate in 3.5 ml of dioxane are added.

One stirs for one hour. Independently, a solution of 208 mg of AH in 38 ml of water is prepared and 26 ml of dioxane are added dropwise. The pH is with. 1N soda. to 8.5 is ~ _(provides Die'Lösung is cooled to 5 ° C After 1.0 hours, the activated ester is inlet to the solution of human albumin:... '' set The solution is stirred for 14 hours at 5 C, the pH is kept at 8.5 by the addition of 1N'NaOH, the precipitation of the conjugated compound and its purification are carried out in the manner described above, the McI ratio being 13 ,8th. ; ·

'. '' '. '/'. ' _% ''-''''.'

\. . ' _l '. , '.' ·,. ,

Example; 9 '' · ''. , '

Vindesine 4-O-hemisuccinate and coupling with galactosylated human albumin (AHgal) (AHgal-Succ-VDS)

 , - '\ '-    '- 15 - ->':.

i

0.16 mmol (119 mg) of vindesine, 0.24 mmol (24 mg) of succinic anhydride and 3 ml of anhydrous methylene chloride are placed in a flask. The solution is stirred for 16 hours at room temperature with exclusion of light. The solvent is evaporated. The residue is taken up in 2.7 ml of dioxane. Then the solution is immersed in an ice bath. You put 0.32. mmol of triethylamine in 2 ml of dioxane and 0.32 mmol of isobutyl _: elchloroformate in 2 ml of dioxane. Stir for an hour. Independently, a solution of 164 mg AHgal in 17.5 ml of water is prepared and 25 ml of dioxane are added dropwise. The pH is adjusted to 8.5 with 1 N sodium carbonate. The solution will open. 5 ° C cooled. After one hour, the activated ester is added to the AHgal solution. The solution is stirred for 14 hours at 5 ° C, with the pH being kept at 8.5 with 1N soda The precipitation of the conjugated compound and its purification are carried out in the manner described above , 6 ... ^; ..

Example 10 ^:

Vindesine 4-O-hemisuccinate and coupling with non-specific goat serum (IgG) immunoglobulins (IgG-Succ-VDS)

0.07 mmol (50 mg) of vindesine, 0.1 mmol of succinic anhydride and 4 ml of anhydrous methylene chloride are placed in a flask. The 'solution is stirred under exclusion of light for 14 hours at room temperature. The solvent is evaporated. The residue is taken up in 1 ml of dioxane. The solution is then immersed in an ice bath and 0.14 mmol of triethylamine and 0.14 mmol of isobutyl chloroformate are added. Stir for an hour. Independently, a solution of 66 mg of IgG in 12.7 ml of water is prepared and 12.7 ml of dioxane are added dropwise. The pH is adjusted to 8.5 with 1 N sodium carbonate. The solution is cooled to 5 ° C. After one hour, the activated ester is added to the IgG solution. The solution is stirred for 14 hours at 5 ° C, the pH being kept at 8.5 by the addition of IN NaOH.

The precipitation of the conjugated compound and its purification are carried out in the manner described above. The molar ratio is 9. "'

Example 11

Vindesine 4-O-hemisuccinate and polyclonal anti-fat-globule immunoglobulins (IgG-anti-MFG) (IgG-anti-MFG-Succ-VDS) _:

0.035 mmol of VDS (25 mg), 0.05 mmol of Süccinylahhydrid and 1 ml of anhydrous methylene chloride are placed in a flask. The solution is stirred with exclusion of light for 14 hours at room temperature. The solvent is evaporated and the residue is taken up in 0.25 ml of dioxane. The solution is then immersed in an ice bath. 0.07 mmol of triethylamine,, and 0.07 mmol of isobutyl chloroformate are added. Stir for an hour. Independently, a solution of 25 mg IgG anti-MFG in 21.3 ml water is prepared. The pH is adjusted to 10.5 with 1 N sodium carbonate. The solution is cooled to 5 ° C. After one hour, the activated ester is added to the IgG anti-MFG solution. The solution is stirred for 14 hours at 5 C, whereby the pH is kept at 8.5 by the addition of 1 N NaOH. The conjugated compound is dialysed against 9% NaCl and purified on Sepha-dex G 25 as described above. The molar ratio is 15 ·

Example 12 '; '-

Vinblastine il-O-hemisuccinate pyrrolidinamide (DAVI'iB-pyrrolidine)

97.6 mg (0.976 mmol) of succinyl anhydride are added to a solution of 500 mg (0.651 mmol) of 4-O-deacetylvinblastine in 15 ml of anhydrous dichloromethane. The solution is stirred for 20 hours at room temperature. Then it is cooled in an ice bath to 0 ° C and successively added dropwise, a solution of 131.5 mg (1, 303 mmol) 'triethylamine

in 8 ml of dichloromethane and a solution of 141.2 mg (1.302 mmol) of ethyl chloroformate in 8 ml of dichloromethane. The mixture is stirred for 30 h at 0 ° C. and a solution of 92.5 mg (1.322 mmol) of pyrrolidine in 8 ml of dichloromethane is added. The mixture is allowed to warm to room temperature and the mixture is stirred for two hours. Then, 50 ml of dichloromethane and 50 ml of a 10% aqueous sodium carbonate solution are added. The mixture is stirred, decanted and the organic phase is separated off. The organic phase is extracted three times with dichloromethane. The combined organic phases are washed with eerier saturated NaCl solution and dried over magnesium sulfate. The residue obtained after evaporation is purified by chromatography over a column of SiO 2 (elution with dichloromethane / methanol 92: 8). After trituration in a mixture of ethyl acetate / cyclohexane, 386 mg of the product are obtained in the form of an amorphous powder. Yield 6-456 ". ' · ''. · '

Mass spectrum (DCI, isobutane): 936 (M + 14), 922 (M) IR (CHCL, 5%): 3477, 3015, 2980, 2885, 1741, 1635, 1505, U50, 1250

NMR Spectrum:,. ':: (CDCI ₃ , 360 MHz, ppm) r

8.05 (NH, IH), 7.53 (IH), 7.16 (3H), 6.66 (1H), 6.10 (1H), 5.85 (1H) et S, US (IH) (HIU, H15). 5.51 (1H, H17) _S 3.95 (1H, H17'a), 3.78 (3H, OMe), 3.75 (3H, OMe), 3.60 (3H, OMe), 3.70 (1H _t H2), 3.43 (7H), 3.11 (2H, H5'b + H6'b), 2.78 (2H, H21A '+ H21 ^l _b)> 2.70 (3H, NMe) , 2.61 (IH, H21), 2.25 (ΙΗ, ΗΠ ¹ ), V, 90 (2H, CH 2 -CO), 1.80 (2H, CH 2 CO), 1.73 (2H, beta pyrrolidine), 1.28 (2H, ldem), 1.U5 (IH.H15'a), 1.37 (IH, H14'b), 0.86 (3H, CH3), 0.80 (3H, CH3), 0.76 (1H, HT * '). ;,. ", '.': ''. " J. ·

Example 13

Coupling of vinblastine-ii-O-hemisuccinate with ethyltryptophanate

To a solution of 0.310 g (0.403 mmol) of ^ -O-desacetylvinblastine in 7 ml of anhydrous dichloromethane is added '52 mg (0.524 mmol)

  , , , , - 18 -

Succinylanhydrid added. The solution is stirred for 15 hours at room temperature, then cooled to 0 ° C. After successive addition of a solution of 81 mg (0.806 mmol ×) of triethylamine in 5 ml of dichloromethane and a solution of 87 mg of ethyl chloroformate in 5 ml of dichloromethane, the reaction mixture is stirred at 0 ° C. for one and a half hours. Then 187 mg '(0.806 mmol) of ethyl L-tryptophanate, dissolved in 7 ml of dichloromethane, are added. The mixture is allowed to warm to room temperature and the mixture is stirred for 15 hours. The solution is finally treated as in Example 12. The residue obtained is purified by chromatography on a. SiO 2 - column (elution ether-NH, -saturated methanol 92: 8). In this way, 305 mg of the pure product are obtained in the form of a white powder after trituration with isopropanol. Yield 70%. ''. ' - .. '.-

Mass spectrum (DCI, isobutane): 1097 (M + 1U), 1083 (M) IR (CHCl ₃ ): 3460, 2995, 2960, 2870, 1735/1669, 1613, 1502, 1457, 1331, 1251, 1167, 1010 cm " ¹ UV (Mothanol max, log): 216 (A, 87), 265 (4.27), 288 (3.01)

RMN (360 MHz, CDCl ₃ ): 8.44 (1H, NH), 8, O8 (1H, NH), 7.52 (2H), 7.33 (1H), 7.10 (6H). 6.66 (1H, H9), 6.06 (1H, H12), 5.86 (1H, H14), 5.50 (1H, H17). 5.38 (1H, H15), 4.91 (1H), 4.10 (2H, CH2-O), 3.76 (3H, OMe), 3.72 (3H, OMe), 3.63 (3H, OMe ), 3.13 (2H, H5'b + H6'b), 2.80 (H21 ^l), 2.68 (NCH _3), 0.90 (CH ₃ .3H), 0.78 (CH _3, 3H), 0.74 (H14 ¹ ).

Example 1 * i. ,,

Coupling of N- (4-O-deacetyl- ⁱ ) -0-hemisuccinate-pesoxy-vinblastinoyl · 23) -ethyl-tryptophanate with pyrrolidine . ^] '\

A solution of 400 mg (0.419 mmol) of N- (4-O-deacetyl-deoxy-vinblestinoyl-23) -ethyl thyltryptophanate (cf. Belgian Patent 889,136), 105 mg (1.05 mmol) of succinic anhydride in 20 ml of a mixture of dioxane and toluene (50:50) is heated for three hours>'under reflux. After distilling off the solvents under vacuum, the residue is dissolved in 20 ml of anhydrous dichloromethane.

- 19 -,. ,

methane solved. After cooling to 0 ° C and successive addition of a solution of triethylamine (126 mg, 1.25 mmol) in 5 ml of dichloromethane and a solution of ethylchloroforrhine (136 mg, 1.25 mmol) in 5 ml of dichloromethane, the mixture is stirred for one hour touched. Then a solution of pyrrolidine (178 mg, 2.5 mmol) in 5 ml of dichloromethane at 0 ° C / is added. After warming to room temperature, the solution is stirred for four hours and then between 50 ml of dichloromethane and 50 ml of a 5 $ aqueous potassium carbonate solution advantageous. The phases are separated and the aqueous phase extracted with two 20 ml portions of dichloromethane. The organic extracts are combined, washed successively with 40 ml of water and 40 ml of a saturated aqueous NACl solution, dried over magnesium sulfate and evaporated under reduced pressure. The residue is purified by chromatography on SiO ₂ (elution with methylene chloride: methanol 95: 5) and trituration with ether. Yield: 346 mg (75%) of the desired compound. '"\ ^:

NMR spectrum ^ (CDCl ₃ , 360 MHz) bs = singlet

wide '

9.23 (1H, bs, NH), 7.95 (H, bs), 7.45-7.65 (3H, m), 7.33 (1H, m), 7.03-7.21 (6H, m) , 6, U6 (IH, s), 6.05 (1H, s), 5.83 (1H, m), 5.53 (1H, s), 5.33 (1H, m), 5.01 ( lH, m), 4.11 (2H, m), 3.76 (3H, s), 3.58 (3H, s), 3> 8 (UH, m), 2.75 (3H, s), 2.65 (1H, s), 2.30 (IH, m), 1.93 (2H _# rri),. 1, 85 (2H, m), 1.21 (3H, t), 0.88 (3H, t), 0.76 (3H, t) ₇ ;

IR spectrum (CHCl ₃ , 5% v: v): 3U70, 3003, 2973, 2881, 1732, 1679, 1617, 1501, 1M60, 1180 cm ^-1 .

Example 15

'· · ·. '·' ... 1 4 'Deoxy-vinblastinoyl-23-ethyltryptophanate-4-O-hemisuccinate and coupling with bovine serum albumin (RSA) (RSA-succ-deoxy-; VTTrpE)

, , - · '' ''

MOO mg (0.419 mmol) De oxy VTr pE, 126 mg (1.25 mmol) Succinylanhy.drid and 20 ml toluene: dioxane 1: 1 are placed in a flask. The solution is refluxed for three hours. The solvents are evaporated and the residue taken up in 20 ml of dioxane.

Subsequently, the solution is immersed in ¹ an ice bath. Add 1.26 mmol (126 mg) of triethylamine, 1.25 mmol of isobutyl chloroformate and 5 ml of dioxane, and stir for one hour. Independently, a solution of 3.times.0 mg of RSA in 65 ml of water is prepared and 28 ml of dioxane are added. The pH is adjusted to -8.5 by 1N sodium carbonate. The solution is cooled to 5 ° C. After one hour, the activated ester is added to the RSA solution. The reading is stirred lh hours at 5 ° C, wherein the pH value is kept by addition of 1 N NaOH to 8.5. The precipitation of the conjugated compound and its purification are carried out in the manner described above. '.. V.' , '''->

Example 16.

      .. - / '' ... ,

Coupling of ^ -O-pesacetyl - ^ - O-Hemisuccinate-Deoxy-Vindesine with Ethyl Tryptophanate _{; 7}

Following the procedure described in Example 12, but replacing the pyrrolidine by an equimolar amount of ethyl L-tryptophanate gives k7% of the desired compound.

NMR spectrum. (CDCI ₃ , 360 MHz):

8.45 (IH, bs), 8.02 (IH, bs), 7.50 (2H, m), 7.32 (IH, m), 7.23-7.06 (6H "m), 7.02 (lH, m), 6.50 (IH, s). 6.10 (1H, s), 5.86 (1H, m), 5.50 (iH, s), 5.45 (ΐΗ, πι), 5.35 (1H, s). 2.86 (3H, s), 2.67 (IH, s), 2.46 (4H, m), 1.30 (3H.t), 0.90 (3H, t), 0.75 (3H , t).

IR spectrum (CHCl ₃ , 5% v: v): 3480, 3400, 3010, 2978, 2882,

1732, 1690, 1615, 1505, 1460, 1298 cm " ¹ '

         . - 21 - '' '

Example 17

· '..''<''

Coupling of N'-Cii-O-desacetyl - ^ - O-hemisuccinate-vinblastinoyl-23) -ethyl thyltryptophanate with pyrrolidine

After / IEM described in Example 14. (4-0-deacetyl-Vinbp.astinoyl-23) -Äthyltryptophanät after chromatographic purification over SiO _n and trituration in ethanol, 518 mg (64%) of the desired, starting from 700 mg N Connection.' .... ''. ' .,

NMR spectrum · (GDCI ₃ , 360 MHz):

8, OU (IH, bs), 7.46-7.63 (3H, m), 7.33 (1H, m), 7.20-7.03 (6H, m),

6.63 (1H, s), 6.08 (1H, s), 5.83 (1H, m), 5.55 (IH, s), 5.33 (IH, m), 5.06 (1H , m), 4.13 (2H, m), 3.96 (1H, m), 3 _# 76 (3H, s), 3.61 (3H, s), 3.46 (UH, m), 3 40 (2H), 2.81 (2H, m), 2.73 (3H, s), 2.65 (1H, s), -1.85 cm), 1.20 (t, 3H), 0 , 89 (t, 3H), 0.71 (t, 3H).

IR spectrum (CHCl ₃ 5%, v: v): 347 *; 3002, 2977, 2882, 1730,

1678, 1617. 1500, 1460, 1173 cm " ¹

Example 18

Coupling of U-O-desacetyl ^ -O-hemiglutarate-vinblastine with pyrrolidine

*

A solution of 4-O-deacetyl-vinblastine (700 mg, 0.911 mmol) and gliiaric anhydride (145 mg, y> 7 mmol) in 25 mL of anhydrous dichloromethane is stirred under exclusion of light for 70 hours. After cooling to 0 ° C and the sequential addition of a solution of N-methyl-piperidine (163 mg, 1.64 mmol) _; in 7 ml of dichloromethane and a solution of isobutyl chloroformate

 '. '..' '·) (223 mg, 1.6 mmol 4 mmol) in 7 ml of dichloromethane becomes the mixture

stirred for an hour. Then a solution of pyrrolidine (194 mg, 2.73 mmol) in 7 mL of dichloromethane is added at p ° C. After reaching room temperature, the solution is stirred for five hours. It is then partitioned between 60 ml of dichloromethane and 60 ml of a 5% aqueous potassium carbonate solution. The phases are separated and the aqueous phase extracted twice with 30 ml of dichloromethane. The organic extracts are combined, washed with water and a saturated aqueous NaCl solution, dried over magnesium sulfate and evaporated under reduced pressure. The residue is purified by SiQp chromatography (elution methylene chloride: methanol 95: 5). and trituration in a mixture of ethyl acetate:; heptane purified. Yield: 605 mg (71%) of the desired compound.

NMR spectrum. / (CDCI ₃ , 360 MHz): 7.95 (IH, bs), 7, ¹ »5 (iH, m), 7.16-7.00 (3H, m), 6.55 (1H, s) , 6.02 (1H, s), 5.76 (1H, m), 5, MO (VH, s), 5.20 (1H, m), 3.88 (1H ^ m), 3.71 (6H, 2s), 3.53 (3H, s), 3.38 (UH, m), 2.62 (3H, s), 2.58 (1H, s), 1, 92 (4H, m), 1, 8O (2H, m), 0.80 (3H, t), 0.73 (3H, t).

IR spectrum (CHCl ₃ , 5% v: v): 3473, 3003, 2977, 2884, 1738, 1620, 1500, V460, 1301 era " ¹

Compounds of the invention were tested in% BDF.- or DBA ₂ mice implanted with intraperitoneal or intravenous leukemia P388, the antitumor effect being measured by the percentage of ILS (Increased Life Span, increase in survival time ¹ ). Thus, those with, Vindesin, with N-C'l-O-deacetyl-^ -de-imathoxycarbonyl) -vinblastinoyl-23) -A'thyltryptophariat (see European Patent Application No. kl 935, Omnichem) and with other C 4-derivatives of amino acids coupled via the 3-carbon with fetuin or bovine serum albumin in a ratio of 2 to 6 such that these conjugated compounds are not very effective on the Tumqre P388. (See Table) , ,

The conjugated compound was administered by intraperitoneal or intravenous route, each in the same manner as the tumor injection.

in contrast, surprisingly, these same substances in which the coupling has occurred via C-4 show a significant effect in carrying out similar studies. Survival increases of more than $ 70 were observed. The improved efficacy of the conjugated protein compounds of the invention has also been demonstrated on L1210 cells in vitro in RPMI medium containing 10 $ fetal calf serum. The chosen method after incubation is to measure the content of cell proteins, which is determined by the Lowry method, so that after 3 days at a concentration of 10 to 50 micrograms / ml RSA (S) - ^:! C-4 VDS inhibited cell growth, while non-treated cell culture multiplied by a factor of 2.5. '''

Hepatomes Hep.G2 (300,000 cells) were incubated for 8 days in the presence of AHgal-VDS-C-4 and AHgal-VDS-C-3 at varying concentrations of 1.3 to 21,700 ng VDS / ml. After 8 days, the extent of cell proteins is determined by the Lowry method. The results show that the $ 50 cell-killing concentration in the case of AHgal-VDS-C-3 is> 1,000 ng / ml, while in the case of AHgal-VDS-C-4 it is 215 Hg / ml lies.

TABLE

EFFECTS OF VINBLASTlN DEHFVATES ON TUMORS BEJ MICE

PkODIIKTE

RSA TRPV-C-3

Fetus-VDS-C-3 fetus (S) -VDS-C-3 fetal-Ile-VDSC3 RSA-VDS-C-U

RSA (S) -VDS-C- ^ RSA (S) -VI le-C-4

RSA Succ-VDS

RSA- (S) -SuCC-VDS RSA- (S) -SuCc-VDS

(via guanidine) RSA-Succ-VDS

RSA Succ-VDS

(about animal charcoal) (about guanidine) '

IgC unspecified .- $ _UC c VDS A Hgal-Succ VDS RSA succ-deoxyVtrpE

2 2 6 2 2

2.7 2.5 2.4 1.3

18 18

32 32

32 3.4 -19 16.6 17

24 21.6

10

6.3

15

15

10

423 261 298 298 294 398 500 332 27.5 968 165 550 771

173 371

97 208

208 196 351 403 393

643 356 146 284

DBA ₂ 1 (T P388 iv 9.4

DBA ₂ 10 ⁴ P388 iv -8

BDF ₁ 10 ⁶ P388 ip 4.1

BDF ₁ 10 ⁶ P388 ip 8.2

10 ⁶ P388 ip 2.6

10 ⁶ P388 ip

10 ⁶ P388 ip i »8

10 ⁶ P388 ip

10 ⁶ P388 ip

10 ⁶ P388 ip

VO ⁶ P388 ip

10 ⁶ P388 ip

₁₀ 6 P388 ip

10 ⁶ P388 ip

BDF ₁ BDF ₁ BDF ₁ BDF ₁ BDF ₁ BDF ₁ BDF ₁ BDF

BDF. BDFj

BDF.

BDF lo | j P388 ip

BDF ₁ 10 .P388 ip

10 ⁶ P388 ip

OK ⁶ P388 ip

IO ⁶ P388 ip

IO ⁶ P388 ip

10 ⁶ P388 ip

IO ⁶ P388 ip

BDF ₁ 1Ö ⁶ P388 ip

₁ 10 ⁶ P388 ip BDF IO ⁶ P388 ip

TABLE (continued)

(D)

VDS-chloroacetate

VILE-chloroacetate DAVLB-Pyrrolidlrv

10 12

15 4

8 12 16

6.25 12.5 25 50

DBA ₂ FR DBA ₂ FR

DBA ₂ FR DBA ₂ FR DBA ₂ US

DBA ₂

DBA ₂ DBA ₂ DBA ₃ DBA. DBA. DBA. DBA.

10 "10 ⁵

¹⁰ 10 ^£ 10 ⁵

ro

io ⁶ io ⁶ io ⁶

ro ⁵ ίο ⁵ io ⁵

P388 iv P388 iv

38

P388 iv P388 iv P388 iv

P388 Ip> 60 P388 Ip. ":

P388 ip

P388 ip

P388 ip -29

P388 iv

P388 iv

P388 iv

P388 iv

(a) Vinca molar ratio: protein Cb-). Dosage mg Vinca / kg

(c) dosage mg protein / kg

(d) mouse strain ^; , [ (e) Ah, the injected cells

(f) type of tumor injected _; \,

(g) Injeltiönsart

(h) Percent increase in survivor survival compared to untreated mice (ILS)

The compounds obtained by the process according to the invention have extremely interesting anti-tumor properties and can be used in human therapy. This vinblastine derivatives can in particular for loading \ _v '' -handlung of leukemias, gliomas, lymphosarcomas or other malignant tumors, including so-called "solid" tumors ,, be used.

In human therapy, therefore, they are used to treat Hodgkin's disease and other tumors for which treatment. with vintolastin, vincristine or vindesine is used. .

For their therapeutic use, the compounds obtained according to the invention, if appropriate in lyophilised form, preferably in a parenteric manner, in a pharmaceutical. . dissolved solvent administered. Of the% addition salts, preference is given to non-toxic, pharmaceutically acceptable salts, such as the salts of mineral acids, e.g. of hydrochloric, phosphoric and sulfuric acids or of organic acids, such as acetic, propionic, succinic, tartaric, oxyalic, methane,.

... sulfonic or benzenesulfonic acid. Physiological saline or other phosphate buffered saline solutions are suitable solvents. / ·· '' .- '

: D, he drug is usually in a dosage v.erab- ranges, which can vary from 50 mg to several grams. These compounds may also be used in combination with other activating agents.

Claims (11)

63 939 18 Request claims ... ι A process for preparing conjugated compounds of vinblastine or derivatives of vinblastine with proteins, protein fragments, amino acids or simple amines, characterized in that the coupling is via an ester group derived from the hydroxyl group of the 4-carbon of the vinblastine skeleton. '. ''. '''. _t 2. Method according to item 1, characterized in that the protein, the protein fragment or the amine is coupled via a bridge to the vinblastine compound which has been prepared by prior condensation of the vinblastine derivative with an optionally activated bifunctional organic compound. 3. The method according to item 2, characterized in that the bifunctional compound d s is chloroacetic acid chloride, the chloroacetic anhydride, the succinic anhydride, or the glutaric anhydride. 4. A method according to any one of items 1 to 3, characterized in that a protein or a protein fragment is coupled via ₍ a Sster bond to the C-4 hydroxy group of vinblastine, vindesine or a 23-vinblastinoyl ester derivative of an amino acid , Process according to items 1 to 4, characterized in that the derivative of vinblastine is 4-O-deacetylvinblastine. 6. Method according to items 1 to 4, characterized in that the derivative of vinblastine is 4-0-desacetyl-4'-deoxy-vinblastine Process according to any one of items 1 to 6, characterized in that a compound of the formula O-A-iL in which A is a "bridge" such as -COOH ₂ - or wherein η varies from 1 to 5, R 1 represents an optionally modified protein residue, R _{0 represents} a methoxy group, an amino group or an alpha-amino acid residue represented by an amide bond is bonded and whose ester group contains 1 to 6 carbon atoms, and R 1 represents a hydrogen atom or a hydroxyl group, which may be present in each of the two possible configurations, as well as their addition salts with a mineral acid or an organic acid. 8. The method according to any of items 1 to 7t, characterized in that the protein residue is a derivative of Pötuin or albumin. Method according to any one of items 1 to 7, characterized in that the protein residue is a derivative of an immunoglobulin » 10. A method according to any one of items 1 to 9 »characterized in that the protein residue is a derivative of a monoclonal antibody. 11. A method according to any one of items 4 to 7t, characterized in that the amino acid ester is the Athyltryptophan. 12. A method according to any one of items 4 to 7, characterized in that the amino acid ester is the Ethylisoleucinat. 13. The method according to item 7 », characterized in that R _{2 represents} a Metoxy ^ - or amino group. 14. Method according to item 7 », characterized in that E represents a hydrogen atom with the configuration of the 4 ¹ -deoxyvinblastine-B. Method according to any one of the funects 7 to 10, characterized in that the group of minerals is a group MJJ. or an amino acid linked via the amino substituent, wherein K and R, - represent alkyl groups having one to three carbon atoms or together an AlKandiylgruppe having three to run carbon atoms. 16. A process according to any one of items 1 to 15, characterized in that, in a first stage, the chloroacetic acid chloride or an anhydride is mixed with the. Hydroxyl in the C-4 position of 4-0 ~ I) esacetylvinulastine or a derivative of the type 4-0-besacetylvinblastine ~ 3 ~ carbox * - amide and then condensing the derivatives thus obtained with the protein, the amino acid or the simple amine in a solvent in which these compounds are soluble, Process according to item 16, characterized in that the solvent used in the second condensation stage consists of a mixture of water and dioxane at a corresponding pH value held by a buffer 18. The method according to item 16 or 17, characterized in that isolated by precipitation from the reaction medium conjugated compound and centrifuged, washed, lyophilized and purified by gel filtration and optionally performs a classical Succinylierung. Process according to any one of items 16 to 18, characterized in that the proteins used are treated for their selective modification » 20. The method according to any of items 16 to 19, characterized in that one uses as protein Hinderserum- or human serum albumin, fetuin or immunoglobulins, which were optionally obtained by the process of monoclonal antibodies, preferably monoclonal human üntikörper.