PH26447A - New conjugates or vinblastine and its derivatives a process for their preparation and pharmaceutical compositions containing the same - Google Patents

Description

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COMPOSITIONS. CONTAINL G THE . ¥ The present invention relates to new con- jugates of vinblastine and of some its known derivatives with proteins or fragments of proteins usable as anti-tumour agent. The invention also relates to a process for their preparation and to pharmaceutical compositions containing these conjugates.

Vinblastine and some of its derivatives, in particular vincristine or vindesine, have already been coupled to proteins, for example ~. albumin or various immunoglobulins. Coupling products or compounds called "conjugates" result.

The following literature references may be re- ferred to in particular : J.D. Teale, Jacqueline

M. Clough and V. Marks, Br.J.Clin.Pharmac. 4, 169-172, 1977; C.H.J. Ford, C.E. Newman, J.&.

Johnson, C.3. Woodhouse, 7.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,

C.H. Marsden, H.W. Baldwin, Br.J. Cancer 47, 43-39, 1983; J.id. Johnson, C.H.J. ford, C.E. Newman,

C.S. Woodhouse, G.Y. Rowland, R.G. Simmonds, Br.J. cancer 44, 472-475, 1981; E11 Lilly Eur. Pat. Applic., 3 v

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Publ. No. 56,322, 21.07.82; and R.A. Conrad, G.J.

Cullinan, K. Gergon, G.A. Poore, J. Med. Chen. 22, 391, 1979.

May also be mentioned the European Patent

Application No. 84 870057.1 which contains a description of the coupling of bls-indole deri- vatives to e¢ protein, a fragment of protein or an amine by means of an arm such as ~COCH,-, -CO(CH,) -Co, a varying from 1 to 5, which may be substituted 3 by an alkyl or amino group.

Coupling of these bis~indole derivatives has been effected not only with the aim of develop- ing new immunological reagents, but in particular with the aim of preparing anti-tumour substances which are more active, more selective and less toxic.

In this last respe:t, numerous conjugates of proteins with other anti-tumor agents are currently being studied, This applies, in part- icular, to conjugates of antibodies and a fragment of ricin ¢r conjugates of albumin and methotrexate (French Patent Application 2,437,213, CM

Industries; and B.C.r. Chu, 5.b, ilowell, J. of

Fharmacology and kxp. Therapeutics, 219 (2), 3b9y=- 393, 1981). a 4 ' ( IEEE aD ORIGINAL 9)

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Monoclonal antibodies, in particular those of human origin, coupled to known anti-tumour medicaments are more particularly the subject of various studies.

Rinally, reference is made to the fact that the use and evaluation of 1:1 complexes of bis-indole alkaloids with tubulin have been . described in Belgian Patent 854,053. In some cases, a lower toxicity and a more significant chemotherapeutic activity than from the corres- ponding free alkaloids may result.

The present invention is particularly directed to new products which consist of con- jugates of vinblastine or of derivatives of vin- blastine with proteins or fragments of proteins, characterised in that the coupling is effected through an ester group which derives from the hydroxyl group at the carbon 4 of the skeletons : of vinblastine.

These conjugates according to the invent- ion are characterised in that the protein or frag- ment of protein is coupled to the compound of the vinblastine t,pe by means of an arm, by pre- vious condensation of the derivative of vinblas- tine with a bifunctional organic derivative, optionally activated, according to a preferred embodiment. 5 ‘BAD ORIGINAL J

The derivative of 4-0O-diacetylvinblastine may be, for example, vindesine, 4-0 deatetylvin- blastine coupled at C-3 with an aminoacid, 4-0- deacetyl-deoxy-4'~-vinblastine or 4-0-deacetyl- deoxy-4'-vinblastine coupled at C-3 with an aminoacid.

More particularly, the compounds accord- ing to the invention correspond to the following ; general formula I pz TTR

IR

NN u - H NU R

AVE Bs , \ >H.,, 00

CH50 C

Se 3 ( pre NL ~~ re

A er

LT

Kd ~~. I - : N -, ad “=~. 0O- A ~R “~~

CH50 | [ ~ OH .

CH

3 co 23

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V—

in which A represents an "arm" such as

NH-R NH-R co- (CH) -CHcO- or COCH= (CH) _CO- in which n varies from 1 to 5 and R represents a hydrogen atom, an acyl group such as acetyl, trifluoroacetyl or carbobengyloxy, Ry represents an optionally modified protein radical, R, re- presents a methoxy group, 8n amino group or an alpha-aminoacid ester radical which is bonded by a bond of the amide type and ln which the ] 10 ester group contains 1 to 6 carbon atoms, and

Ry represents a hydrogen atom or a hydroxyl ” group, in each case in the two phesible config- urations, as well as their addition salts with an toorganic or organic acid.

The therapeutic activity of the compounds according to the jnvention is higher than that of the compounds which have been described up to now and their toxicity 18 substantially lower.

The derivatives according to the present invention are obtained, in a first stage, bY condensation of an anhydride, for example aspartic or glutamic anhydride or a higher homologue, optionally substituted on the amine group, onto the hydroxyl at c-4 of 4-O-deacetyl-vinblaatine or of 4-0O-deacetyldeoxyvinblastine or of one of al the derivatives of the 4-O-deacetyldeoxyvinblast- ine-3-carboxamide or 4-~0O-deacetyldeoxyvinblastine- 3~-carboxamide type.

T'ne hemiaspartate or hemiglutamate or homologue derivatives thus obtained are then con- densed with the protein or the fragment of pro- tein in a solvent in which these compounds are soluble. For this purpose, a water/dioxane mixture at a suitable pd maintained by a buffer, for example a phosphate buffer, can be used. The condensation is confirmed by chromatography or electrophoresis. 1n the later case, radioactive (for example tritiated) vinblastine may be used in order to facilitate the characterisation,

From the chemical point of view, the condensation with the proteins can be explained by the production of covalent bonds resulting from the reaction of the amine groups of the pro- . tein lysine with the activated ester group derived from the optionally substituted hemiappartate or hemiglutamate.

Bovine serum albumin, for example, con- tains 56 amine residues of lysine. The humber of molecules of vinblastine per protein varies as a function of the operating conditions, but is ot 8 . BAD ORIGINAL 9

Sh : ee

Tel TN edgy eq 4} generally 1 and 34.

The activation can be effected in a con- ventional manner by treatment with an alkyl chloroformate, preferably ethyl or isobutyl chloroformate, in the:presence! ol an amine base, such as N-methylpiperidine or N-methylmorpholine.

The condensation can be carried out in situ on the reaction mixture containing the activated anhydride. lowever, in most cases, the activated anhydride can also be isolated.

The conjugate obtained is isolated by means of the conventional methods used in chemistry or, in the case of protein conjugates, in biochemistry. The protein conjugate is accordingly precipitated out of the reaction mixture by addition of acetone and is then cen- trifuged off, rinsed, lyophilised and purified by gel filtration. If appropriate, the derivative thus obtained kay be subjected to a conventional succinylation reaction, which enables the aggregation problems which characterise certain conjugates and non-conjugated proteins to be avoided.

The proteins whicao can advantageously be used are, in particular, bovine or human serum i 9 y CTA hs vil I \aro ORIGINAL dP .

albumin, fetuin or immunoglobulins, the latter oo being obtained, if appropriate, by the monoclonal antibody technique. In the latter case, the use o of monoclonal antibodies of human origin which demonstrate a certain specificaty towards human tumours has proved to be ol particular intesist.

The proteins used can also be treated in order to be selectively modified. These modifi- cations enable protein conjugates to be obtained which, during use in therapy, are concentrated preferahtially in certain tissues, for example in the liver. It is thus possible prior to the condensation of the derivative of the vinblastine with the protein, to galactosylate the latter,

The galactosylation is carried out, for example, by applying the method described by G. Wilson in By the Journal of Biochemistry, 253 (7) 2070-2072, 1978.

In vitro experiments carried out with the compounds according to the invention to de- monstrate their anti-tumour activity indicate that the formation of the conjugate by a bond at C-4 may be more advantageous... than if the bond is effected at C-3. 10 - . | \sa0 ORIGINAL 9

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Alternativeli, it is also possible to condense a compound of the -A-R; type, A and Ry having the same meaning as indicated above, in a suitable solvent onto the hydroxyl at C-4 of 4-O-deacetylvinblastine or 4-O-deacetyldeoxy- vinblastine or of one of the derivatives of 4-O-deacetylvinblastine~3-carboxamide or 4-0- deacetyldeoxyvinblastine-3-carboxanide. The activation can advantageously be carried out by treatment with an alkyl chloroformate, such as ethyl or isobutyl chloroformate, in the presence of an amine base such as N-meth,1lpiperidine, N- methylmorpholine or triethylamine. The following examples describe some conjugates according to the invention wherein the bis-indole derivatives are coupled at the position C-4 to a protein or a fragment of protein by means of an arm of the substituted hemiaspartate or the substituted hemiglutamate vype.

The present invention also relates to ” industrial and, in particular, pharmaceutical uses of the new bis-indole compounds, in fact, the compounds according to the invention have particularly useful anti-tumour properties which are capable of being used in human therapy. 11 \ aap ORIGINAL J) vo

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In particular, these vinblastine derivatives can be used for the treatment of leukemies, gliomas, lymphosarcomas or other malignant tumours, includ- ing so-called "solid" tumours.

In human therapy, they are thus used for the treatment of Hodgkin's disease and for other tumours which may benefit from treatment with vin- blastine, vincristine or vindesine.

For use in therapy, the compounds according to the invention, is appropriate in lyophilised torm, are preferably administered parenterally, in olution in a pnarmaceutically acceptable solvent.

Physiological water or other saline solutions which ere buticred, ror example with a phosphate, are suitable solvents.

The active substance is in general admin- istered in a dosage which can vary from 50 mg to several grammes. The compounds according to the invention can also be used in combination with other anti-tumour agenta.

Example 1 a) +4 and b isomers of 4-0 deacetyl-4-O-L-N-acetyl- hemfaspartete vinblastine 250 mg of N-acetyl-L-aspartio anhydride are added to a solution of 700 mg (0.Yl mmol) 4-0O-deacetyl- 12 | ~

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ee vinblastine in 20 wl anhydrous dichloromethane.

The solution is stirred during 20 hours at ambient temperature. After distillation under vacuum of the solvent, the obtained residue is purified by chromatography on silica (elution ether/methanol/

NH,OH 25% 60/49.75/0.25).

After trituration in a mixture of dichloromethane : and petrol ether, 650) mg 4-O-deacetyl-4-0-L-N- acetylhemiaspartate vinblastine are obtained in the state of a white powder (Yield : TT%).

HPLC analysis of the product indicates the presence of both isomers : and °° in a ratio of 85/15. » IR spectrum (Kbr): 3420, 2960, 2880, 1737, 1660, 1613, 1501, 1460, 1430 cm ©. * Magsepectrum: DCI (isobutane): 925 my, 939 (wf 4 14), 857, 769, 693, 635. i vb) '. and #¢ isomers of 4-0-deacetyl=-4-0~L-N- acetylhemiaspartate methyl ester vinblastine.

A solution of 500 mg (0.540 mmoles) + and ° isomers ot 4-O-deacetyl-4-0O-L-k-acetylhemiaspariate vin~- blastine in 10 ml absolute methanol saturated with dry HCl is stirred for 20 hours, at ambient temper- ature. 13

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After distillation under vacuum of the solvent, the residue is recovered in a mixture of 15 ml distilled water and 1% ml dichloromethane. The mixture is rendered alkaline by NH, OH addition. The aqueous phase 18 extracted by three portions of 20 ml di- chloromethane, Ihe organic extracts are combined, successively washed with 40 ml water and 40 ml of an agueous solution saturated with NaCl, dried on magnesium sulfate and evaporated under reduced pressure, The residue is purified by chromatography on silica (elution by CH, Cl, :CH,OH 95:5). Accord- ingly 410 mg = and isomers of 4-0O-deacetyl-4-0- ;

L=N-acetylhemiaspartate methyl ester vinblastine are obtained, Yield: &1%,

Fractions resulting from chromatography including individual isomers are analyzed. - Principal isomer *# Mass spectrum (DCi; isobutane): 939 ry, 940 (nf £1), 953 (Mf 4 14) * IR spectrum (4Br): 2550, 2880, 1740, 1675, 1615, 1503 cmt * NMR spectrum (CDCl 5, 360 MHz) “1: 9.65(0H), &,02 (NH), 7.52 (H-9'), 7.16-7.05 (N-10', H-11', H-12'), 6.61 (H-9), 6.52 (NY), 6.07 (H-12), 5.75 &H-14), 5.52 (H-1¥), 5.17 (H-15), 4.75 (-CH-NH), 3.95 (H-17A'),

Nao SERRE 14 - : ‘oro ORIGINAL J) em

3.80 (-OMe), 3.77 (-OMe), 3.70 (-OMe), 3.60 (-OMe), 2.00 (UH=-21A°, H-21B'), 2.67 (NMe), 2.62 (H-21), 2.00 (MeCO), 0.92-0.75 (2 Me) # Rf: 0.51 (CH,CL, :CHOH 90:10 silica)

Y - Minor isomer # Mass spectrum (pUI-igobutane): 939 (ty, 940 (uf 4 1) # IR Spectrum (KBr): 1740, 167%, 1615 cm™1 *NMR spectrun (CDCly, 5€0 MHz) / : 9.25 (OH), } 10 8.02 (NH), 7.50 (H-9'), 7.16-7.05 (k-10', H-11"',

H-12'), 6.-f(H-9), 6.52 (iH), 6.08 (K-12), 5.82 (H-14), 5.47 (14-17), 5.28 (11-15), 4.75 (CH-NHAc), 3.9% (H=17A'), 3.77 (2 x OMe), 3.60 (OMe), 2.70 (N=Me), 2.02 (iieCcO0-), 0.95-0.77 (2 Me) # pf: 0.39 (CHyUl, :CH5LH 90:10 silica)

Example 2 4 and isomers Of 4-0-deacetyl-4-0-L-N-carbo- benzyloxyhemiglutanate vinblastine

A solution of 4-O-deacetyl vinblastine (300 mg, 0,34 mmole) and N-carbobenzyloxj-L-glutamic an- hydride (144 mg; 0,514 mmole) in dichloromethane (5 ml) is stirred for 20 hours, at ambient temper- ature.

The solvent is evaporated under vacuum. . 15 ‘a0 ORIGINA- 2 ee tq “17

The obtained residue is purified by chromatography on silica (elution: ether/methanol/NH,OH 25% 50/49.5/0.5).

Accordingly, 230 mg oi 4-0 deacetyl-4-0-L-N- carbobenzyloxyhemiglutanate vinblastine are obtained in the state of an 4. and # isomers mixture.

A HPLC analysis of the product indicates the pre- sence of .« and * isomers in a ratio oi 60:40.

IR spectrum (KBr): 345C, 2960, 2880, 1730, 1612, 1593, 1501, 1459, 1432, 1228 emt

Fass spectruu (DLI-isobutane): 1032 (’ £1), 9u4, Y2u, 723 cu . gxample 3

Coupling of 4-0 deacetyl-4-0-h-N-acetylhemiaspar- tatevinblastine with galactosylated albumin of human origin . a) 80.6 g of 4-0-deacetyl-4-0-L-N-acetylheni- aspartate vinblastine are dissolved in 2ml dioxane. The solution is then plunged in an ice bath. 24.4 ul triethylamine in 0.5 ml dioxane and 22.6 nl isobutyl chloroformate in 0.5 ml dioxane are added. The mixture ig stirred for 1 hour.

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Further, a solution of 200 ng galactosylated human albumin in 37 al H,0 is prepared. The pH . is adjusted to 8.5 by NaOH O.1N.

The solution is refrigerated to 4 degrees Cc.

After 1 hour, the activated ester is added to the solution of galactosylated human albumin. The solution is stirred for 14 hours at 4 degrees C, while the pH iso maintained at &.5 by addition of

NaOH 1N. Thereafter, the solution is purified by filtration on a sephadex gel 6 25 dquilibrated by a solution of NaCl 9/1000, pH - 7.5. The fractions containing the conjugate are combined, concentrated by ultrafiltration and sterilized.

The protein content is determined by the Lowry method and the content of alkaloids is estimated by determination of the radioactivity.

Phe obtained conjugate contains 13 moles of vin-~ blastine per mole of galactosylated human albumin. . The HEL. determination of monomers, dimers and polymers of the conjugate composition indicates 82% _monomers and 18x dimers. b) 80,6 mg 4-0-deacetyl-4-0-L-N-acetylhemi- aspartate vintlastine are dissolved in 2 ml dioxane. The golution is then plunged in an z 11

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Led 4] ice bath. 24.4 pl triethylamine in 0.5 ml di- oxane and 22.6 nl isobutyl chloroformate in 0.5 ml dioxane are added.

Further, a aolution of 200 mg galactosylated human albumine in 37 ml phosphate buffer 0,1 M, pH ~ 8.2, is prepared. The pH is adjusted to 8.5 by NaOH 1N,

The solution is refrigerated to 4 degrees C.

After 1 hour, the activated ester is added to the solution of galactosylated human altumin. ihe solution is stireed for 14 hours at 4 degrees

C, while the pil is maintai.ed at 6.5 by an addition of NaOH 1N. The solution is purified by fil- tration on Jephadex gel 429% syuilibrated by a solution of katCl Y/1000, pH - 7.5, ‘Lhe tractions containing the conjugate are combined, concen- trated by ultrafiltration and sterilized. The protein content is measured by the Lowry method and the content of alkaloide is estimated by determination of the radioactivity,

The obtained conjugate contains 9.3 moles vin- blastine per mole galactosylated human albumin.

The HPLC determination of monomers, dimers and polymers of the conjugate couposition indicates 91.5% monomers, 74 dimers and 1.5% polymers. 18 ee,

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The sensibility of the conjugate to lysosomial ensymes has tden studied by incubation during 48 . hours, at 37°¢C, in the presence of 5 mM cystein, 40 mM acetate buffer and lysosomial angzymes,

Aliquot parts are taken and the non decomposed proteins are precipitated by addition of a trichlo- roacetic acid volume (TCA 40 #4). After incuba- ‘ tion at 4°¢, during one hour, said samples are centriiuged and the radioactivity of the super- 16 natant is estimated by counting of scintillations of an aliquote part of the liquid.

The soluble radioactivity is a measure of the digestion of sald conjugate. Practical tests have shown that 75 of the conjugate has been digested after 24 hours. Ko further evolution - has been observed up to 48 hours.

The chemotherapeutic activity of said conjugate has been estimated on FP 348 leukemia with female . BDF, mice : 108 tumoral cells are introperitoneally inoculated at the day 0. The conjugate is intra- peritoneally administrated at the day 1. The test results show that the conjugate presents an import- ant activity on this experimental model, because it provides an increase in life time of more than 650 9 if it is adminiatered at a rate of 60 mg/kg and the number of surviving mice is 5/% after 60 days. 19

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BXAMPLE

( and ¥ isomers of ethyl N-(4-O-deacetyl-4-0- dcetylhémiaspartate-vinblastinoyl-23)-L-tryp- tophanate.

Following the procedure of example 1, ethyl N- (deacetyl-4-0O-vinblastinoyl-23)-L-tryptophanate was converted to ethyl-N-(4-O-deacetyl-4-0-N- acetyl-hemiagpartate-vinblastinoyl-23)-tryp- - tophanate (mixture of isomers { and ¥ ).

The obtained residue is purified by chromato- graphy on silica (elution ether/methanol/NH,Oh 25% 50/49,75/0,25). 200 mg of the product are obtained from 314 mg of starting product. 15% Fass spectrum (DCI, Acetone): 1126 (wl), 1066, y84, 970, 951, Yll.

IR spectrum (ake): 3400, 2960, 2940, 1740, 1665, 1618 cmt, cXAMPLE 5 d& and f isomers of ethyl $N-(4-0-deacetyl—~-4-0- l-N-acetylhemi aspartate-vinplastinoyl=23)-L~- isoleucinate. oo 20 . J —

Fuld}

Following the procedure of example 4, ethyl N- (deacetyl-4-0O-vinblastinoyl-23)-L-1soleucinate was converted to ethyl-N-(4-O-deacetyl-4-0-L-

N-acetyl hemiaspartate-vinblastinoyl-23)-L- isoleucinate. [lage gpectrum (buL-ucetone) : 10951 (ity, 1036, 1009, 477, 8917, ©38, 755, 709, 652.

IR spectrum (Abr) : 3410, 2963, 2929, 2880, 1754, 1676, 1612, 1500, 1460, 1430, 1372, 1333, 1293. cat oXAMPLE 6

A and ¥ isomers of 4-O-deacetyl-4-O-I-i-acetyl- hemiglutamate vinblastine.

Following the procedure of example 20, 4-0O-de- acetylvinblastine was treated with N-acetyl-L- 19° glutamic anhydride to yield 271 mg of 4-0O-de- acetyl-4-0-L-N-acetyl-nemi; lutamate vinblastine (mixture of isomers. and ¥ ) from 380 mg of 4-0- . deacetylvinblastine. ~ £1 pass spectrum (DcI-acetone) : 940 (n"+), 811,

IR spectrum (KBr) : 3470, 2960, 2&80, 1740, 1665, 1617 cmt. 21 \

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Claims (1)

1. Lec 4} ch ATI S l. Conjugate of vinblastine of of a derivative thereot corresponding to the general formula =~ TTT ] XX: N NT H 7 “ Ry

   CH.,00C J . 3 ] ya ~~ ~ ( 3 Y _ ~ Pa 4 LN 3 a CH, 0 3 OH Cig

   [14] 23 Ry in which A represents Ni-R fin ~colti- (CH) C0= or -CO-(CH,) CHCO- . 22 ar . fr Co ar * \ | J BAD OHIGINAL J) J

   Cut in which n may be 1 or 2 and R represents a . hydrogen atom, acetyl or carbobenzyloxy, Ry re- presents an optionally modified protein radical T, Ry represents a methoxy group, and Ry represents a hydrogen atom or a hydroxyl group, in each cage in the two phssible configurations.

   2. Conjugate accerding to claim 1 characterized in that the protein radical is one obtainable by monoclonal antibody technique. 3, Conjugate according to claim 1 characterized in that the protein radical con- gists in monoclonal antibodies which are of human origin.

   4. Congyugate according to claim 1 characterized in that lhe protein radical con- sists in galactosylated monoclonal antibodies, which are of human origin.

   5. Process tor the preparation ot ) conjugates according to claim 1 characterised in that an anh,dride of an acid of the Mi-R 00¢-Lii- (Ciiy) COOK type is condensed onto ithe hydroxyl at C-4 ot the vinblastine derivative in dichloromethane 23 } BAD ORIGINAL 9 : \

   2 19% as solvent and in that the derivative thus obtained is then condensed with the protein radical, in a water/dioxane mixture, as solvent.

   6. Process for the preparation of conjugates according to claim 1 characterised in that a moiety of the type i Io -CJ~ - wu -" -C - - > CO-CH-( ty) COR, or 0 (CH, ) HCOR, in which fy is a optionally modified protein radical T, is condensed onto the hydroxyl at C-4 of the vinblastine derivative and in that an activation of the starting pro- : duct is eilected by treatwent with an alxyl chloroformnate , in the presence of an amine base.

   Ji. Process according to claim 6 characterised in that the alkyl chloroformate is ethylchliorotormate and the amine base in tri- ethylamine.

   ’ 8. Process according to claim 5, characterised in that a centrifugation, a rinsing, a lyophilisation and/or a purification by gel . filtration are furthermore carried out.

   Y. Process according to claim 8 characterised in that further a conventional succinylation reaction is carried out, 24 Ny TLL BAD ORIGINAL 9 L

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   10. irocess according to claim 5, characterised in that the protein radical is de- rived from bovine or human serum albumin, fetuin or immunoglobulins, the latter being obtained, by the monoclonal antibody technique.

   11. Process according to claim 6, chacterised in that a centrifugation, a rinsing, a lyophilisation and/or a purification by gel filtration are furthermore carried out.

   12. Process according to claim 10, characterised in that further a conventional succinylation reaction is carried out. -

   13. Process according to claim 6, characterised in that the protein radical is derived from bovine or human serum aloumin, fetuin or immunoglobulins.

   14. Pharmaceutical composition, character- . ised in that it comprises the conjugates accord- ) ing to claim 1, in combination with a diluent, a solvent, an excipient or a pharmaceutically accept- able support, the active compound being in lyo- philised form or in solution in a buffered solvent, for the treaiment otf leukemias, lymphosarcomas or other malignant tumours, inciuding so-called 1g0lid" tumours and tor the treatment of Hodgkin's disease. 25 — 'aAD ORIGINAL 2