DD254009A5 - CONNECTION TO THE PRODUCTION OF NEW CONJUGATED COMPOUNDS OF VINBLASTINE AND ITS DERIVATIVES - Google Patents

Abstract

The invention relates to a process for the preparation of novel conjugated compounds of vinblastine and certain known derivatives thereof with proteins, protein fragments. According to the invention, compounds of the general formula are prepared in which, for example: A is a bridge such as NH-RCOCH (CH 2) n CO u. a., n is 1 to 5, R is hydrogen, an acyl group, and the like. al .; R1 is an optionally modified protein residue; R2 is a methoxy group u. al .; R3 is hydrogen or a hydroxyl group. formula

Description

Field of application of the invention

The present invention relates to a process for preparing novel conjugated compounds of vinblastine and certain known derivatives thereof with proteins, protein fragments, amino acids or amines used as antitumor agents.

Characteristic of the known state of the art

The vinblastine and certain of its derivatives, in particular vincristine or vindesine, have already been coupled with proteins, for example 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. Clough et V. Marks, Br. J.CIin. Pharmac. 4.169 to 172.1977

- C.H.J. Ford, C.E. Newmann, J.R. Johnson, C.S. Woodhouse, T.A. Reeder, G.F. Rowland, R.G.Simmonds, Br.J. Cancer 47, 35-42, 1983

M.J. Embleton, G.F.Rowland, R.G.Simmonds, E.Jacobs, CH.Marsden, R.W. Balwin, 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. Simmons, 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.Gerzon, G.A.Poore J.Med. Chem.22,391,1979

It is also referred to the exclusion patent DD-PS 219771 referred to the coupling of bis-indole derivatives with proteins, protein fragments or amines via a bridge of the type-COCH ₂ -, - CO (CH ₂ I _n -CO-is described, wherein η can vary from 1 to 5 and the bridge of the type mentioned can be substituted by an alkyl or amino group.

The coupling of these bis-indole derivatives has been undertaken not only for the purpose of developing new immunologically reactive substances, but also in particular for the production of antitumour agents that are more active, more selective and less toxic.

For the latter purpose, numerous conjugated protein compounds have been used with other antitumour agents heretofore.

educated. This is especially true for conjugated compounds of an antibody and a ricin fragment or for conjugated compounds of albumin and methylotrexate (French Patent Application No. 2437213, C M Industries; B.C.F. Chu.

Howell J. of Pharmacology and Exp. Therapeutics, 219 [2], 389-393, 1981).

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

Finally, it is emphasized that the use and evaluation of 1: 1 complexes of bis-indole alkaloids with TubuMn has been described in Belgian Pat. No. 854053. In certain cases this may result in lower toxicity and greater chemotherapeutic efficacy than for the corresponding free alkaloids.

Object of the invention

The aim of the invention is to provide the compounds with greater anti-tumor activity and lower toxicity.

Explanation of the essence of the invention

The invention has for its object to find new compounds with the desired properties and processes for their preparation.

According to the invention, there is provided a process for the preparation of novel products which are conjugated compounds of vinblastine or derivatives of vinblastine with proteins or protein fragments, characterized in that the coupling is effected via an ester group derived from the hydroxyl group of the 4-carbon atom of the Vinblastine skeletons is derived. In a preferred embodiment, the method of the invention is characterized in that the protein or protein fragment is coupled to the vinblastine compound via a bridge prepared by previously condensing the vinblastin derivative with a bifunctional organic compound which is optionally activated.

The 4-0-deacetylvinblastine derivative may be, for example, the vindesine, 4-O-deacetylvinblastine coupled to an amino acid at 3-C, or 4-O-deacetyl-deoxy-vinblastine or 4-O-deacetyl-4 '-deoxy-vinblastine coupled to an amino acid at 3-C.

In particular, the process makes it possible to prepare compounds which have the following general formula:

3 '

CH ₃ OOC

CH ₃ O

-A -R

OH

CO

in which A a "bridge" like

NH-R I

- COCH- (CH ₀ ) CO - or 2. η

NH-R

- CO- (CH ₀ ) -CHCO-z η

wherein η is from 1 to 5, R represents a hydrogen atom, an acyl group such as acetyl, trifluoroacetyl or carbobenzyloxy, R _{1 represents} an optionally modified protein residue, R _{2 represents} a methoxy group, an amino group or an alpha-amino acid residue represented by a Amide bond is bound and whose ester group contains one to six carbon atoms, and R _{3 represents} a hydrogen atom or a hydroxyl group, which may each be in the two possible configurations, as well as of addition salts thereof with a mineral or organic acid.

The therapeutic effect of the compound according to the invention is higher than difi of the compounds described so far,

and their toxicity is noticeably lower.

According to the invention, the derivatives in a first stage by condensation of an anhydride, for example the aspartic acid, glutamic anhydride or a higher homologue to the 4-C-hydroxyl of 4-0-desacetylvinblastine or 4-0-deacetyldesoxyvinblastine or one of Derivatives such as the 4-O-desacetyl-deoxyvinblastine-3-carboxamide or the 4-O-desacetyl-deoxyvinblastine-3-carboxamide prepared.

The resulting hemiaspartate or hemiglutamate derivatives or higher homologues are then condensed with the protein, the protein fragment, in a solvent in which these compounds are soluble. 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 (tritiated, for example) may be used to facilitate

Use marking.

From a chemical point of view, condensation with the proteins can be explained by obtaining covalent bonds formed by the reaction of the amino groups of the protein lysine with the activated ester group of the hemiaspartate or

Hemiglutamate derivatives arise.

Bovine serum albumin, for example, has 56 aminated lysine residues. The number of vinblastine molecules per protein will vary depending on the working conditions, but will generally be between 1 and 34.

Activation can be carried out in a conventional manner by treatment with an alkyl chloroformate, preferably with ethyl or isobutyl chloroformate, in the presence of an amino base, such as N-methyl-piperidine or N-methyl-morpholine

be made.

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 is isolated in the classical manner as is conventional in chemistry or in the case of conjugated protein compounds in biochemistry. The conjugated protein compound is thereby precipitated by the addition of acetone from the reaction medium, then centrifuged, washed, lyophilized and purified by gel filtration.

Optionally, one can subject the resulting derivative to a classical succinylation reaction, thereby avoiding aggregation problems characteristic of certain conjugated or non-conjugated proteins

become.

The proteins which are advantageously used are, in particular, albumin from bovine serum or human serum, the fetuin or immunoglobulins, the latter optionally being obtained by the process of monoclonal antibodies

become.

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

The proteins used can also be treated for selective modification. These modifications make it possible to obtain conjugated protein compounds which, in their therapeutic use, are preferentially enriched in the area of specific tissues, for example in the region of the liver. Thus, it is possible to zugalaktosylieren prior to the condensation of the vinblastin derivative with the protein of the latter. 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 occurs via C-4 via C-3.

According to a variant, a compound of the type AR ₁ , wherein A and Ri have the meanings given above, can also be in a suitable solvent on the 4-C-hydroxyl of 4-O-desacetylvinblastine or 4-O-desacetyldesoxyvinblastine or a of the derivatives such as 4-O-desacetylvinblastine-3-carboxamide or 4-O-desacetyldeoxyvinblastine-3-carboxamide.

The activation is advantageously produced by the action of an alkyl chloroformate, such as ethyl chloroformate or isobutyl chloroformate, in the presence of an amino base, such as N-methyl-piperidine or N-methyl-morpholine or triethylamine.

The following examples describe some conjugated compounds according to the invention wherein the bis-indole derivatives are coupled at the 4-C position with a protein fragment, bridge-type substituted hemiaspartate or substituted hemiglutamate.

The compounds obtained by the process according to the invention have extremely interesting antitumor properties and can be used in human therapy. These vinblastine derivatives can be used in particular for the treatment of leukemias, gliomas, lymphosarcomas and other malignant tumors, including the so-called "solid" tumors.

In human therapy, therefore, they are used for the treatment of Hodgkin's disease and other tumors intended for treatment with vinblastine, vincristine or vindesine.

For their therapeutic application, the. Compounds obtained according to the invention optionally in lyophilised form, preferably in a parenteral manner, dissolved in a pharmaceutically acceptable solvent. Among the addition salts, non-toxic, pharmaceutically acceptable salts, such as the salts of mineral acids, e.g. As the hydrochloric, phosphoric and sulfuric acid or of organic acids such as acetic, propionic, succinic, tartaric, oxalic, methanesulfonic or benzenesulfonic acid. Physiological saline or other phosphate buffered saline solutions are suitable solvents.

The active ingredient is normally administered in a dosage that can vary from 50 mg to several grams. These compounds can also be used in combination with other anti-tumor agents.

embodiment

The invention is explained in more detail below with reference to some examples.

example 1

a) a- and / or 3-isomers of VBL 1 -O-deacetyl-O-L-N-acetyl-hemiasparagic acid ester

To a solution of 700 mg (0.91 mmol) of 4-O-deacetyl-vinblastine in 20 ml of anhydrous dichloromethane is added 250 mg of N-acetyl-L-aspartic anhydride.

The solution is stirred for 20 hours at ambient temperature. After vacuum distillation of the solvent, the residue obtained is purified by chromatography on a silica column (eluent ether / methanol / NH ₄ OH to 25,% 60 / 49.75 / 0.25).

After dry-milling in a petroleum ether / dichloromethane mixture, 650 mg of the above-mentioned compound, in the form of a white powder, are obtained (rdt: 77%).

Product analysis by HPLC (high performance liquid phase chromatography) indicates the presence of the two isomers α and β, in the ratio 85/15.

* IR spectrum (KBr): 3420, 2960, 2880.1, 737.1, 660.1, 613, 1501, 1460, 1430 crrT ¹ .

* Mass Spectrum: DCI (isobutane): 925 (M ⁺ ), 939 (M ⁺ + 14), 857, 769, 693, 635.

b) a- and / or 3-isomers of the methyl ester of VBL-4-OLN-acetyl-hemi-aspartic acid

A solution of 500 mg (0.540 mmoles) of the α- and / or 3-isomers of VBL-4-N-acetyl-L-hemiasparagic acid ester in 10 ml of absolute methanol saturated with dry HCl is stirred for 20 hours at ambient temperature. After distilling off the solvent under vacuum, the residue is taken up in a mixture of 15 ml of distilled water and 15 ml of dichloromethane. The mixture is alkalized by NH ₄ OH addition. The aqueous phase is extracted with 3 parts of 20 ml of dichloromethane. The organic extracts are combined, washed successively with 40 ml of water and 40 ml of an aqueous solution saturated with NaCl, dried on magnesium sulfate and evaporated under reduced pressure. The residue is purified by chromatography on silica (elution by CH ₂ Cl ₂ : CH ₃ OH

95: 5). -

Thus, 410 mg of the α- and / or 3-isomers of the above-mentioned compound are obtained.

Portions of the various separated isomers obtained by chromotography have been analyzed.

- main isomer:

* Mass spectrum (DCI, isobutane): 939 (M ⁺ ), 940 (M ⁺ + 1), 953 (M ⁺ + 14).

* IR spectrum (KBr): 2950, 2880, 1740, 1675, 1615, 1503 cm " ¹ "

* NMR spectrum (CDCl ₃ , 360 MHz) δ: 9.65 (OH), 8.02 (NH), 7.52 (H-9 '), 7.16-7.05 (H-10', H-11 ', H-12'), 6,61 (H-9), 6,52 (NH), 6,07

* (H-12), 5.75 (H-14), 5.52 (H-17), 5.17 (H-15), 4.75 (-CH-NH), 3.95 (H-). 17A '), 3.80 (-0Me), 3.77 (-0Me), 3.70 (-0Me), 3.60 (-0Me), 2.80 (H-21A', H-12B ') , 2.67 (NMe), 2.62 (H-21), 2.00 (MeCO), 0.92-0.75 (2Me)

* Rf: 0.51 (CH ₂ Cl ₂ .H ₃ OH 90:10 silicon dioxide).

- minor isomer:

* Mass spectrum (DCI isobutane): 939 (M ⁺ ), 940 (M ⁺ + 1).

* IR Spectrum (KBr): 1 740, 1 675.1 615cm- ¹ .

* NMR spectrum (CDCl ₃ , 360 MHz) δ: 9.25 (OH), 8.02 (NH), 7.50 (H-9 '), 7.16-7.05 (H-10', H-11 /, H-1 T), 6,57 (H-9), 6,52 (NH), 6,08 (H-12), 5,82 (H-14), 5,47 (H -17), 5.28 (H-15), 4.75 (CH-NHAc), 3.93 (H-17A '), 3.77 (2 x OMe), 3.70 (OMe), 3, 60 (OMe), 2.70 (N-Me), 2.02 (MeCO-), 0.95-0.77 (2 Me). ,

*. Rf: 0.39 (CH ₂ Cl ₂ : CH ₃ OH 90:10 silica).

Example 2

a- and / 3-isomers of the VBL ^ -O-desacetyM-OLN-carboxybenzyloxyglutaminsäureesters A solution of 4-0-deacetylvinblastin (300mg, 0,39mmol) and N-carbobenzyloxy-L-glutamic anhydride (144mg, 0,519mmol) in dichloromethane ( 5 ml) is stirred for 20 hours at ambient temperature.

The solvent is evaporated under vacuum. The residue obtained is purified by chromatography on a silica column (elution: ether / methanol / NH ₄ OH 25% 50 / 49.5 / 0.5). In this way, 230 mg of the above-mentioned product are obtained in the form of an a- and y-isomer mixture

HPLC analysis of the product shows the presence of the a and y isomers in the ratio of 60 to 40.

* IR spectrum (KBr): 3450, 2960, 2880.1 730.1 612.1 593.1 501.1459.1 432.1 228cm " ¹ .

* Mass spectrum (DCI-isobutane): 1032 (M ⁺ + 11.984.928.723 cm; ¹

Example 3

Coupling of VBL ^ -O-deacetyl ^ -O-L-N-acetylhemiasparagic acid ester with galactosylated albumin from human serum

a) 80.6 mg of VBL-4-O-deacetyl-4-O-L-N-acetyl-hemiacetate are dissolved in 2 ml of dioxane and the solution is then immersed in an ice-bath. Subsequently, 24.4 / xl triethylamine in 0.5 ml of dioxane and 22.6μΙ Isobutylchloroformiat be added in 0.5 ml of dioxane. It is stirred for one hour. On the other hand, a solution of 200 mg of galactosylated albumin from human serum in 37 ml of water is prepared. The pH is adjusted to 8.5 with sodium hydroxide solution IN.

The solution is cooled to 4 ^° C. After 1 hour, the activated ester is added to the solution of galactosylated albumin from human serum. The solution is stirred for 14 hours at 4 ⁰ C, while the pH is maintained at 8.5 by adding sodium hydroxide solution. The solution is purified by filtration on gel Sephadex G 25, which is kept at equilibrium by a solution of NaCl 9/1000, pH 7.5. The portions containing the conjugated compound are combined, concentrated by ultrafiltration and sterilized. The content of proteins is measured by the Lowry method and the content of alkaloids is determined by radioactivity measurement.

The conjugated compound thus obtained contains 13 moles of vinblastine per galactosylated albumin from human serum. The determination of the composition of the conjugated compound of monomers, dimers and polymers by HPLC gives 82% monomers and 16% dimers.

b) 80.6 mg of VBL ^ -O-desacetyl ^ -O-L-N-acetylhemiasparagic acid ester are dissolved in 2 ml of dioxane. The solution is then immersed in an ice bath. Subsequently, 24.4μ.Ι Isobutylchloroformiat be added in 0.5 ml of dioxane.

On the other hand, a solution of 200 mg of galactosylated albumin from human serum in 37 ml of 0.1 molar phosphate buffer, pH = 8.2, is prepared. The pH is adjusted to 8.5 by means of 1N sodium hydroxide solution.

The solution is cooled to 4 ° C. After 1 hour, the activated ester is added to the solution of galactosylated albumin from human serum. The solution is stirred for 14 hours at 4 ^° C. while the pH is maintained at 8.5 by addition of 1N sodium hydroxide solution. The solution is purified by filtration on gel Sephadex G25, concentrated by ultrafiltration and sterilized. The content of proteins is measured by the Lowry method and the content of alkaloids is determined by the

Measurement of radioactivity determined. ,

The resulting conjugated compound contains 9.3 moles of vinblastine per mole of galactosylated albumin from human serum. The determination of the composition of the conjugated compound of monomers, dimers and polymers by HPLC reveals 91.5% monomers, 7% dimers and 1.5% polymers.

The sensitivity of the conjugated compound of the Example to lysosomal enzymes is by incubating this conjugated compound for 48 hours at 37 ° C in the presence of 5 mM cysteine, 4 mM acetate buffer and lysosomal enzymes. Over time, aliquots are removed and the undissolved proteins are precipitated by adding trichloroacetic acid (40%). After incubation of the samples at 4 ^° C. for ¹ hour, they are centrifuged and the radioactivity of the above float is determined by counting the scintillations of an aliquot.

The soluble radioactivity is a measure of the decomposition of the conjugated compound. The experiment shows that 75% of the conjugated compound is decomposed after 24 hours. Up to 48 hours no further development is detected.

The chemotherapeutic effect of the conjugated compound of the Example was tested by experiments on leukemia P338 implanted intraperitoneally in female BDF ₁ mice = 10 ⁶ tumor cells are administered intraperitoneally on day 0. The conjugated compound is administered on day 1 intraperitoneally. The results show that the conjugated compounds have a very high effect on this experimental model as they lead to an increase in survival time of more than 650% when administered at a dose of 60 mg / kg; the number of survivors on day 60 is 5/5.

Example 4

a- and γ-isomers of ethyl ester of N- (4-O-deacatyl-4-0-LN-acetylhemiaspartat-vinblastinoyl-23) -L-tryptophan acid.

Following the method of Example 1, the ethyl ester of N- (deacetyl-0-4-vinblastinoyl-23) -L-tryptophanoic acid in ethyl ester of N- (4-O-deacetyl-4-O-N-acetyl-hemiaspartate-vinblastinoyl -23) -tryptophanoic acid (mixture of α- and γ-isomers) has been reacted.

The residue obtained is purified by chromatography on silica (elution-ether / methanol / NH ₄ OH to 25% = 50 / 49.75 / 0.25). There are obtained 200 mg of the above product from 314 mg starting material.

* Mass Spectrum: (DCI, acetone): 1126 (M ^{+ 1} ), 1,066, 984,970,951,911.

* IR spectrum: (KBr): 3400, 2960, 2940.1 740m 1 665.1 618cm " ¹

Example 5

α- and / or 3-isomers of the ethyl ester of NKO-desacetyM-OLN-acetyl-hemiaspartet-vinblastinoyl ^ SJ-L-isoleucic acid. Following the procedure described in Example 4, ethyl ester of N-ideacetyl-CM-vinblastinoyl ^ SJ-L-isoleucic acid in ethyl ester of N- (4-O-deacetyl-4-O-LN-acetyl-hemiaspartet-vinblastinoyl-23) L-isoleucine acid has been reacted.

* Mass spectrum: (DCI-acetone): 1051 (M ⁺ ), 1036, 1009, 977, 897, 838, 755, 709, 652.

* IR spectrum (KBr): 3410, 2963, 2929, 2880.1 734.1 676.1 612.1 500.1460.1430.1 372.1 333.1 293 cm ^-1 .

Example 6

α- and γ-isomers of 4-O-deacetyl-4-O-L-N-acetylhemiglutamate-vinblastine.

Following the procedure described in Example 2, 4-O-deacetylvinblastine was treated with N-acetyl-L-glutamic acid anhydride to give 271 mg of CM-deactetyl-OLN-acetyl-hemiglutamate-vinblastine (a-andy isomer mixture) To produce 380 mg of O-4-deacetylvinblastine.

Claims (3)

claims: 1. Process for the preparation of conjugated compounds of vinblastine or derivatives of vinblastine with proteins or protein fragments according to the formula ₍ O - A - R. in which A a "bridge" like NH-R - COCH- ( ) _n CO - or NH-R - CO- (CH ₂ J _n -CH- wherein η varies from 1 to 5 and R represents a hydrogen atom, an acyl group such as acetyl, trifluoroacetyl or carbobenzyloxy, Ri represents an optionally modified protein residue, R _{2 represents} a methoxy group, an amino group or an alpha-amino acid residue represented by an amino bond is bound and whose ester group contains 1 to 6 carbon atoms, and R _{3 represents} a hydrogen atom or a hydroxyl group, which may be present in each of the two configurations, characterized in that an acid anhydride such as NH-R I - HOOC-CH- (CH ₂ J _n condensed on the hydroxyl group in C-4 des Vinblastins or a derivative of vinblastine, and then condensing the derivative thus obtained with the protein residue. 2. The method according to claim 1, characterized in that the protein residue from the albumin from human serum or bovine serum, from the fetuin or immunoglobulins, which is optionally derived by monoclonal antibodies, preferably monoclonal Anitkörper of human origin, preferably galactosylated. 3. The method according to claim 1 or 2, characterized in that for the condensation of an acid anhydride NH-R
t HOOC-CH- (CH ₂ ) _n -CO H - is used on the hydroxyl group in C-4 of VinblastPns or a derivative of vinblastine, a solvent consisting essentially of dichloromethane, and that for the condensation of the product thus obtained with the protein residue, a solvent is used which consists essentially of a Mixture of water and dioxane.