LU85161A1 - NOVEL VINBLASTIN CONJUGATES, THEIR PREPARATION PROCESS AND THEIR THERAPEUTIC USE - Google Patents

Description

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The present invention relates to new conjugates of Vinblastine and of some of its known derivatives with proteins, their, method of obtaining and their use as an anti-tumor agent.

The invention also includes certain intermediates active in chemotherapy, and amino derivatives thereof.

Vinblastine and some of its derivatives, in particular vincristine or vindesine, have already been coupled to proteins, for example albumin or various immunoglobulins. This results in coupling products or so-called "conjugate" compounds. We note in particular the following references from the literature: - J.D.Teaie, Jacqueline M.Clough and V.Marks, Br. J.Clin.Pharmac. 4, 169-172, 1977 - CHJFord, CENewman, J, R.Johnson, CSWoodhouse, TAReeder, 15 CFRowiand, RGSimmonds, Br.J. Cancer 47. 35-42, 1983 - MJEmbleton, GF Rowland, RGSimmonds, E.Jacobs, CHMarsden, RWBaidwin Br.J. Cancer 47,43-49, 1983 -JRJohnson, CHJFord, CENewman, CSWoodhouse, GFRowland, RGSimmonds Br.J. Cancer, 44, 472-475, 1981 20 - Eli Lfîlv Eur.PEi.ADDiic. , Pub !. nc5 € .322, 21 .07.82 - R.A. Conrad, G.J.Cuiiinan, K.Gerzon, G.A.Poore J.Med.Cnem. 22, 391, 1979

The coupling of these bis-indole derivatives has been undertaken not only for the purpose of developing new immunological reagents but above all for the preparation of more active, more selective and less toxic anti-tumor substances.

In this latter perspective, numerous protein conjugates with other anti-tumor agents are currently being studied. This is in particular the case with conjugates of antibodies and of a ricin fragment or of conjugates of albumin and methotrexate (French patent application 2,437,213, C »M» Industries; BCFChu, SBHowell J . of Pharmacology and Exp. Therapeutics, 219 (2), 389-393, 1981).

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Monoclonal antibodies, in particular those of human origin, coupled with known anti-tumor drugs are more particularly the subject of various studies.

Finally, it will be noted that the use and evaluation of 1: 1 complexes of bis-indole alkaloids with tubulin has been described in Belgian patent 854,053. In some cases this may result in less toxicity and greater chemotherapeutic activity than that of the corresponding free alkaloids.

The present invention is characterized in particular by the preparation and use of vinblastine-protein conjugates in which the covalent link which unites the active molecule to the protein acting as a chemotherapeutic vector, is an ester type link at the level of 15 C-4 hydroxyl function of 0-4 deacetylvinblastin or its derivatives.

The compounds of the invention more particularly correspond to the following general formula 20 ch3ooc / (\ TQï is. - · 'i; - 4 - j: in which A represents an "arm" such as -COCH2 ~,' -COCH2- CH2GO-., COCH2-CH2-CH2CO-, Rj represents an optionally modified protein radical, an amino group NR ^ R ^, a chlorine or ij an amino acid ester group linked via the amine function and R_ represents ji ^: 5 a methoxy group, an amino group or an alpha-amino acid ester radical linked by an amide bond and the ester group of which contains from 1 to 6 carbon atoms, R 1 and R 1 represent alkyl groups comprising from 1 to 3 carbon atoms or »together, ur, alkanediyl group having from 3 to 5 carbon atoms j 'ίο

In the prior art, the binding was carried out via an amide bond at the level of the vinblastinoyl-23 function of the bis-indole derivative. Surprisingly we have found that the anti-tumor activity can be better preserved when the binding is carried out at C-4 rather than at C-23.

The derivatives of the present invention are obtained, in a first step, by condensation of the chloroacetic acid chloride or of an anhydride, for example succinic or glutaric anhydride, on the C-4 hydroxyl of 0- 4 deacetylvinblastine or one of the derivatives of the type 0-4 deacetylvinblastine - carboxamide-3.

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The 4-chloroacetate hemisuccinate or hemiglutarate derivatives thus obtained are then condensed with the protein, amino acid or simple amine in a solvent in which these compounds are soluble. To this end, it is possible to use a water-dioxane mixture at an adequate pH maintained by a buffer, for example a borate buffer. Condensation is confirmed by chromatography or electrophoresis. In the latter case, radioactive Vinblastine (for example tritiated) can be used in order to facilitate characterization.

Obtaining the C-4 chloro-acetate from Vinblastine is described by W.W. Hargrove Lloydia, 27 (4), 342, 1964. It can thus be obtained by the action of chloroacetic anhydride on deacetyl Vinblastine in 35. dichloromethane.

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From a chemical point of view, condensation with proteins can be explained by the obtaining of covalent bonds resulting from the reaction of the amino groups of the protein lysine with the activated chlorine of the chloroacetate function or with the activated ester group derived from the hemisuccinate or 5 hemiglutarate. Activation can be carried out conventionally by treatment with a chloroformate. Bovine serum albumin, for example, comprises 56 amino residues of lysine. The number of Vinblastine molecules per protein will vary depending on the operating conditions but will generally be between 1 and 20.

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The conjugate obtained is isolated using conventional methods used in chemistry or, in the case of protein conjugates, in biochemistry. The protein conjugate is thus precipitated from the reaction medium by addition of acetone, then centrifuged, rinsed, lyophilized and purified by filtration on gel. Optionally, the derivative thus obtained can be subjected to a conventional succinylation reaction which makes it possible to avoid aggregation problems which characterize certain proteins, conjugated or not.

Proteins which can be advantageously used are in particular bovine or human serum albumin, fetuin or immunoglobulins, the latter possibly being obtained by the technique of monoclonal antibodies. In the latter case, the use of monoclonal antibodies of human origin and showing a certain specificity with respect to human tumors proves to be particularly advantageous.

The proteins used can also be processed in order to be selectively modified. These modifications make it possible to obtain protein conjugates which, during their therapeutic use, are preferentially concentrated in certain tissues, by exerpy in the liver. It is for example possible, prior to the condensation of the derivative of Vinblastine with the protein, to galactosyier the latter. Galactosylation is carried out by applying, for example, the procedure described by G. Wilson in The Journal of 35 ^ Biochemistry, 253 (7) 2070-2072, 1978.

* - 6 - I In vitro tests carried out with the compounds of the invention to demonstrate their antitumor activity indicate that the formation of! · Conjugated by a link in C-4 can be more advantageous than when this link is made in 03.

5: Coupling via carbon 3 of Vinblastine or its derivatives | is carried out in a conventional manner by forming, in a first step, the hydrazide in 023 (carboxhydrazide) then the corresponding acid azide by nitrosation. The azide is then directly coupled with the protein, j

This acid azide can however also be condensed in a manner also known with an amino acid ester, for example a methyl ester. The ester function of the amino acid can in turn be subjected to hydrazinolysis and then to nitrosation. The new acid azide from the amino acid-vinblastine coupling product is then condensed with the protein. The amino acid in this case constitutes the link uniting the protein with the Vinblastine molecule.

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There were thus obtained condensation derivatives coupled to C-3 or C-4 and 0-4-deacetylated for example of the vinblastine-fetuin (Fét-VDS-C-3), vinblastine-tetuin succinylated type (Fét (S) - VDS-C-3) 25 vinblastine-isoleucine-fetuin (Fet-lle-VDS-C-3) vinblastine-tryptophan-bovine serum albumin (BSA-TrpV-C-3) vinblastine-tryptophan-bovine serum succinylated { BSA (S) -T rpV-C-3) vinblastine-isoleucine-succinylated bovine serum albumin (BSA (S) -Life 30 C-4) vindesine-bovine serum albumin (BSA-VDS-C-4).

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succinylated bovine serum albumin-albumin (BSA (S) -VDS-C-4).

i galactosilated bovine serum albumin-albumin (BSA (G) -VD5-C-4)

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Using succinic acid as arm was also obtained many protein conjugates, e.g. vindesine-bovine serum albumin.

The compounds of the invention were tested on BDF ^ or DBA2 5 mice in which P388 leukemia was implanted intraperitoneally or intravenously, the anti-tumor activity being measured by the percentage of ILS (Increased Life Span, increased survivorship / total).

Thus, experiments carried out with vindesine, N- (04-deacetyl-3- from (methoxycarbonyl) - vinblastinoyl-23) ethyl tryptophanate (see European patent application Publ. No. 41.935, Omnichem), as well as other amino acid derivatives C-3, coupled via C-3 carbon with fetuin or bovine serum albumin in a ratio varying from 2 to 15 6, demonstrate that these conjugates are not very active on P388 tumors (See table ). The administration of the conjugate was carried out intraperitoneally or intravenously, identical to the mode of injection of the tumor.

On the other hand, these same substances for which the coupling is carried out in C-4 show, unexpectedly, significant activity during similar tests. Increases in survival time of more than 70% have been observed.

The superior activity of the protein conjugates of the present invention has

also demonstrated on L1210 cells in vitro in RPMl medium containing 10% fetal calf serum. After incubation, the technique adopted consists of measuring the cellular protein content, an estimation carried out using the Lowry technique. So after 2 30 days at a concentration of 10-50 micrograms / mL of BSA (S) -C4-VDS

inhibition of cell growth is obtained, while the untreated cell culture has increased by a factor of 2.5.

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BOARD

VINBLASTINE DERIVATIVE ACTIVITIES FOR INDUCED TUMORS IN THE MOUSE.

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I PRODUCTS (a) (b) (c) TUMOR MOUSE THEY

(d) (e) (f) (g) BSA-TrpV-C-3 2 10 423 DBA ^ ΙΟ9 P388 iv 9.4! BSA (S) TrpV-C-3 2 6.3 261 DBA2 ΙΟ4 P388 iv -8 j Fet-VDS-C-3 6 15 298 BDF ^ ΙΟ6 P388 ip 4.1 Fet (S) -VDS-C-3 2 15 298 BDF ^ ΙΟ6 P388 ip 8,2 Fét-! Le-VDSC3 2 10 294 BDF ^ ΙΟ6 P388 ip 2,6 BSA-VDS-C-4 1, 7 8 398 BDF ^ 106 P388 ip 2 2,7 16 500 BDF1 ΙΟ6 P3B8 ip 48 BSA (S) -VDS-C-4 2.5 10 332 BDF1 ΙΟ6 P388 ip 76 2.4 27.5 968 BDF1 106 P388 ip 71 BSA (S) -Vlle-C-4 1, 3 3 165 BDF1 ΙΟ6 P388 ip 18 1, 3 10 550 BDF1 ΙΟ6 P388 ip 20 1, 3 14 771 BDF1 ΙΟ6 P388 ip 32 BSA-Succ-VDS 18 35 173 BDF. 108 P388, p 39, 18 75 371 BDFj 10b P388 ip 79 • r j! t.

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(b) TUMOR MOUSE THEY

VDS-Chioroacét 4 DBA2 FR ΙΟ5 P388 iv 24 8 DBA2 FR ΙΟ5 P388 iv 38 10 DBA2 FR 105 P388 iv 57 14 DBA2 FR ΙΟ5 P388 iv 70 12 DBA2 US 1Û5 P388 iv 63 5 BDF1 106 P388 ip 15 BDF1 ΙΟ6 P388 ip> 60 VILE-Chloroacét 4 DBA2 ΙΟ6 P388 ip 2 £.

8 DBA2 ΙΟ6 P388 ip 52 12 DBA2 ΙΟ6 P388 ip 80 16 DBA2 ΙΟ6 P388 ip -29 DAVLB-Pyrroiidino 6.25 DBA2 10 ^ P388 iv 9 12.5 DBA2 ΙΟ5 P388 iv 20 25 DBA2 ΙΟ5 P388 iv 43 50 DBA2 ΙΟ5 P388 iv 82 ( a) vinca molar ratio: protein (b) mg vinca dose / kg (c) mg protein dose / kg (d) number of cells injected (e) type of tumor injected, (f) injection route (g) percentage increased survival time of survivors compared to untreated mice / - 10 -

Comparison with hepatoma cells in vitro (300,000 cells on hep 3B medium, 8-day incubation, estimation by the! Lowry technique) demonstrates that albumin of human serum coupled with C4 inhibits growth better than when it is coupled to C3 (0.395 5 micrograms / mL of Vinblastine).

The present invention therefore also extends to industrial and in particular pharmaceutical applications of new bisindolic compounds.

The compounds of the invention indeed have particularly advantageous antitumor properties which can be used in human therapy.

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These Vinblastine derivatives can, in particular, be used for the treatment of leukemias, gliomas, lymphosarcomas or other malignant tumors, including so-called "solid" tumors.

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

For their therapeutic application, the compounds of the invention, optionally in lyophilized form, are preferably administered parenterally, dissolved in a pharmaceutically acceptable solvent. Among the addition salts, non-toxic pharmaceutically acceptable salts are preferred, such as the salts of mineral acids such as hydrochloric acid, phosphoric acid and sulfuric acid or organic acids such as acid. acetic, propionic,. ;

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. Physiological water or other buffered saline solutions, for example with phosphate, are suitable solvents.

The active substance is generally administered in a dosage which can vary from 50 mg to several grams. The compounds of the invention can also be used in combination with other anti-tumor agents.

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

Example 1 0-¾ vindesine chloroacetate (VDS-Chloroacét) 15

0.92 M of vindesin, 25 mL of CH2Cl2 and 4.1 mM of chloroacetic anhydride are introduced into a flask. The solution is stirred in the absence of light for 1 h. 25 ml of methanol are added (aicoolysis of the excess anhydride). The mixture is stirred at normal temperature for two hours. The solvent is evaporated in vacuo and the residue dissolved in 500 ml of CH2Cl2, washed with an aqueous solution of NH 4 OH diluted cold. 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 h at normal temperature. The silica is filtered off and washed several times with hot methanol. The filtrate is concentrated in vacuo, basified with a dilute ammonia solution and extracted with CH2C12 · The extracts are combined, dried over Na ^ Q ^ and evaporated to dryness. The purification is carried out on a silica column, eluting with a CH2Cl2: 5% MeOH mixture. Yield 88%.

30 • IR: 3470, 3040, 2970, 2880, 1740, 1690, 1615, 1510, 1500, 1430, 1225, 1190, 1010, 740 cm “1 I Mass spectrum 830 (M + -1), 813, 796, 773, 754, 297, 277, 293, 108 ”1 tsp! - I 2 - i -! ' I NMR: ppm 8 (1H, s, NHind), 7.45 (1H, d), 7.2-7.0 (4H, m), 6.9 (1H, d), '] * 6.5 (1H, s, 'C14-H), 6.05 (1H, s, C17-H), 5.8 (1H, m, C7-H), 5.5 (1H, m, C4-H), 5.28 (1H, d, C6-H), 4.0 (2H, CH2Cl), 3.7 (3H, s, -C02CH3),; 3.55 (3H, s, 0CH3), 2.8 (3H, s, NCH3}, 0.9-0.65 (8H, m).

5

Example 2 a) Coupling in C-4 of O-4-deacetyl-vindesine (BSA-VDS-C-4) [jq Alpha-chloroacetate in C-4 of radioactive 0-4 deacetyl-vindesine (150 mg ), dissolved in 5 mL of dioxane is added dropwise to a solution of 216 mg of BSA (bovine serum albumin, Cal Biochem) in 5 mL of 0.4 M borate buffer pH 9.0. The mixture is added at room temperature for 48 h. The conjugate is precipitated by the addition of 6 volumes of acetone and centrifuged 30 min at 1300 rpm. The precipitate is washed twice in acetone and centrifuged under the same conditions. After these two rinses the precipitate is lyophilized and purified by filtration on a G-25 gel (3 × 90 cm) equilibrated in a solution of 0.1 M NH 4 HCO 3 pH 7.8. The excluded peak is recovered and lyophilized. The protein content is measured by the Lowry technique and the alkaloid content estimated by measuring the radioactivity. The conjugate obtained contains 2.5 moles of vindesine per mole of BSA. By chromatography on agarose gel, it is demonstrated that radioactivity is well associated with proteins.

B) Succinylation (BSA (S) -VDS-C-4)

The conjugate is dissolved in water at a concentration of 100 mg / 5 ml and the pH of the solution brought to 7 with a 0.1 N NaOH solution. 55 mg of succinic anhydride are then added in small portions.

The pH is maintained at 7 by adding 0.1 N NaOH. When the pH is stabilized, another 55 mg of succinic anhydride is added. The solution is stirred for 1 h at room temperature, dialyzed with water at 0 ° C overnight and lyophilized. The protein and alkaloid contents are estimated by the techniques described in a).

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Example 30-4 N (04-deacetyl-decarbomethoxy-3 vinblastin-23-oyl) L-chloroacetate ethyl isoleucinate (BSA (S) -VIle-C-A) 5

By following the procedure of Example 1, from the corresponding derivative j of Vinblastine (see Belgian patent 889,136), the above derivative is obtained with a yield of 85%.

10 IR: 3470, 3410, 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 15 NMR: ppm 9.85 (1H, s, C3-OH), 8.6 (1H, s, NH), 7.55 (1H, d), 7.4 (1H, d], 7.2-7.0 (m, 3H), 6.65 (1H, s, C14H), 6, 1 (1H, s, C17H), 5.85 (1H, m, C7H), j 5.58 (1H, s, C4H), 5.32 (1H, m, C6H), 4.6 ( 1H, q), 4.2 (2H, m, C02CH2-), 1.4 (2H, C02CH2CI), 3.78 (3H, s, -C02CH3), 3.1 (3H, s, -OCH3) , | 2.7 (3H, s, NCH3), 1.28 (3H, t, C02CH2CH3), 1.0-0.8 (14H, m).

20 'Example 4

0-4 hemisuccinate formation of vindesin and coupling with bovine serum albumin (BSA). (BSA-Succ-VDS)

0.125 mM of vindesin, 0.187 mM of succinic anhydride and 4 ml of anhydrous C 1 -C 4 are introduced into a flask. The solution is stirred away from light at room temperature for 14 h.

The solution is then immersed in an ice bath. 0.250 mM of triethylamine, 0.250 mM of ethyl chloroformate and 5 ml of dioxane are added thereto. The mixture is stirred for 1/2 h. On the other hand, a solution of 90 mg of BSA in 17.3 ml of H 2 O and 17.3 ml of dioxane are prepared therein dropwise. The pK is adjusted to 8.5 with 1 35 I N sodium hydroxide solution. The solution is cooled to 5 ° C. After 1/2 h the activated ester is added to the BSA solution. The solution is stirred for 4 h at 5 ° C., while the pH is maintained at 8.5 by addition of IN NaOH. Precipitation. of the conjugate and its characterization are carried out as previously described. The molar ratio is approximately 18 and remains constant after chromatography on sepharose 6B in the presence of 6M guanidine.

Example 5 10 Vinblastine 0-4 hemisuccinate pyrrolidine (DAVLB-Pyrrolidino) To a solution of 500 mg (0.651 mM) of 0-4 deacetylvinblastine in 15 ml of anhydrous dichloromethane, 97.6 mg (0.976 mM) of succinic anhydride is added . The solution is stirred for 20 h at room t. It is then cooled to 0 ° C. in an ice bath and a solution of 131.5 mg (1.303 mM) of triethylamine in 8 ml of dichloromethane and a solution of 141.2 mg (1, 3) are added dropwise successively. 302 mM) of ethyl chloroformate in 8 ml of dichloromethane. Stirred 1 h 30 at 0 ° C and added 92.5 mg (1.302 mM) of pyrrolidine dissolved in 8 mL 2 0 of dichloromethane. The mixture is left to return to room temperature and the mixture is stirred for 2 hours. 50 ml of dichloromethane and 50 ml of a 10% aqueous solution of sodium carbonate are added. The organic phase is stirred, decanted and separated. The organic phase is extracted 3 times with dichloromethane. The combined organic phases are washed with a saturated aqueous NaCl solution and dried over MgSO 4. The residue which “is obtained after evaporation is purified by chromatography on a column of SiO2 (elution dichloromethane / methano! 92: 8). After trituration in an ethyl acetate / cyclohexane mixture, 386 mg of pure product are thus obtained in the form of an amorphous powder. Yield: 64%.

Mass spectrum: DCI (isobutane): 936 (M + 14), 922 (M): - IR (CHCI ,, 5%): 3477, 3015, 2980, 2885, 1741, 1635, 1505, 1450, 1250 / - 1 J cm - 15 -! * NMR (CDCI3, 360 MHz, ppm): 8.05 (NH, 1H), 7.53 (1H), 7.16 (3H), 6.66 (1H), 6.10 fl H), 5, 85 (1H) and 5.45 (1H) (H14, H15), 5.51 (1H, H17), 3.95 (1H, H17'a), 3.78 (3H, OMe), 3.75 ( 3H, OMe). 3.60 (3H, OMe), 3.70 (1H, H2), 3.43 (7H), 3.11 (2H, H5'b + H6'b), 2.78 (2H, H21a '+ H21 , b). 5 2.70 (3H, NMe), 2.61 (1H, H21), 2.25 (1H, H17 ·), 1.90 (2H, CH2-CO), 1.80 (2H, CH2CO), 1 , 73 (2H, beta pyrrolidine), 1.28 (2H, same), 1.45 (1H, H15'a), 1.37 (1H, H14'b), 0.86 (3H, CH3), 0 , 80 (3H, CH3), 0.76 (1H, H14 ').

10

Example 6

Vinblastine 0-4 hemisuccinate-ethyl tryptophanate amide To a solution of 0.310 g (0.403 mM) of 04-deacetylvinblastine in 7 ml of anhydrous dichloromethane, 52 mg (0.524 mM) of succinic anhydride are added. The solution is stirred for 15 hours at room temperature and then cooled to 0 ° C. After the successive addition of a solution of 81 mg (0.806 mM) of triethylamine in 5 ml of dichloromethane and of a solution of 87 mg of ethyl chloroformate in 5 ml of dichloromethane, the reaction mixture is stirred for 1 hour 30 minutes. 0 ° C. Then 187 mg (0.806 mM) of ethyl L-trypfophanate dissolved in 7 ml of dichloromethane is added. The mixture is left to return to room temperature and the mixture is stirred for 15 h. The solution is then treated as in Example 5. The residue obtained is purified by chromatography on a column of SiO 4 (elution ether-methanol saturated with N92: 8). 305 mg of the pure product are thus obtained in the form of a white powder after trituration in isopropanol. Yield 70%.

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-16- t | k Mass spectrum (DCI, isobutane): 1097 (M + 14), 1083 (M) IR (CHCI3): 3460, 2995, 2960, 2870, 1735, 1669, 1613, 1502, 1457, 1331, 1251, 1167, 1010 cm "1 UV (methanol, max, log): 216 (4.87), 265 (4.27), 288 (3.01) 5 d NMR (360 MHz, CDCI3): 8.44 (1H, NH ), 8.08 (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, 5'b, 6'b), 2.80 (H2V), 2.68 ( NCH3), 10.90 (CH3.3H), 0.78 (CH3.3H), 0.74 (H14 *).

Example 7

Vindesin 0-4 hemisuccinate-pyrrolidine amide 15

Starting from vindesine and applying a procedure analogous to that of Example 1, the corresponding conjugate is obtained with a yield of 62%.

20 Mass spectrum (DCI, isobutane): 921 (M + 14), 907 (M) IR (CHCI3. 4%): 3400, 2975, 2882, 1732, 1693, 1632, 1503, 1462, 1380, 1247 cm “ 1 NMR (360 MHz, CDCI3) 8.05 (1H, NH), 7.50 (1H), 7.15 (3H), 6.10 (1H, H12), 5.86 (1H, H14), 5 , 53 (1H, H17), 5.46 (1H, H15), 3.96 (1H, H17'a), 3.80 (3H, OMe), 3.64 (3H, OMe), 3.45 (4CH2-pyrrolidine), 2.88 • (3H, N-CH,), 0.95 (3H.CH-), 0.83 (3H, CH,).

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Claims (19)

1. Vinblastine derivatives of the general formula co- H / iVfOH CH300C / 10/15 'CH30'Iî: î ^ N'Jn4 ^ 0-a-r1 ch3 OH 1 "1 20 in which A represents an" arm " such that -COCH2-, -COCH ^ -Cl ^ CO-, COCH ^ -CHj-CH ^ CO-, represents an optionally modified protein radical, a halogen atom, an amino group NR ^ R ^ or an ester group d amino acid linked via the amine function and R ^ 25 represents a methoxy group, an amino group or an alpha-amino acid ester radical linked by an amide bond and whose ester group contains from 1 to 6 carbon atoms , R ^ and R ^ represent alkyl groups having from 1 to 3 carbon atoms or together an alkanediyl group having from 3 to 5 carbon atoms. * 2. New anti-cancer products characterized in that they consist of so-called "conjugate" compounds in which a protein! or a protein fragment is coupled by an ester bond to the C-4 hydroxyl group of Vinblastine, vindesine or a derivative! ' dj aminobl ester vinblastinoyl-23. 4 * Φ - 18 -9 \ \ 3. A derivative according to claim 1 in which the protein radical is derived from fêtuine or albumin. i * " 4. A derivative according to claims 1 and 2 wherein the protein radical 5 is derived from an immunoglobulin. 5. A derivative according to claims 1,2 and 4 wherein the protein radical is derived from a monoclonal antibody. 10 6. A derivative according to claims 1 to 5 characterized in that the amino acid ester is ethyl tryptophanate. 7. A derivative according to claims 1 to 5 characterized in that the amino acid ester is ethyl isoleucinate. 1 5 8. A derivative according to claims 2 to 4 characterized in that R2 represents a methoxy or amino group. 9. A derivative according to claim 1 in which A represents a group CO-CH ^ and R.j a chlorine atom but R2 does not represent a methoxy group. 10. The derivative according to claim 1 wherein A represents a CO-CH2-CH2 ~ CO- group and Rj is an ethyl pyrrolidine or N-tryptophanate group. 11. A derivative according to claims 1 to 8 in which the group A represents a CO-CH2 or CO-CH2 ~ CH2-CO- group. 12. Process for the preparation of Vinblastine derivatives according to claim 1, characterized in that, in a first step, the chloroacetic acid chloride or an anhydride condenses on the hydroxyl in position C-4 of the 0- 4-deacetyl- 'Vinblastine or one of the derivatives of the type 0-4-deacetylvinblastim V .. -4 / *! tr I - 19 -. carboxamide-3 and in that the derivatives thus obtained are then condensed with the protein, the amino acid or the simple amine in a solvent in which these compounds are soluble. te 5 13. Method according to claim 12, characterized in that the anhydride used in the first condensing step is succinic anhydride or glutaric anhydride. 10 * 14. Method according to one of claims 12 or 13, characterized in that the solvent used in the second! condensation stage consists of a water-dioxane mixture at an adequate pH maintained by a buffer. 15 15. Method according to any one of claims 12 to 14, characterized in that the conjugate obtained is isolated by precipitation from the reaction medium and in that centrifugation, rinsing, lyophilization and further purification by gel filtration, as well as possibly conventional succinylation. 16. Method according to any one of claims 12 to 15, characterized in that the proteins used are treated in order to be selectively modified. 17. Method according to any one of claims 12 to 16, characterized in that, as protein, bovine or human serum albumin, fetuin or immunoglobulins optionally obtained by the technique of • monoclonal antibodies, preferably 3 human monoclonal antibodies. 18. Pharmaceutical composition characterized in that: “35 comprises the derivatives according to one of claims 1 to 11 in combination with a diluent, solvent, excipiant or pharmaceutically acceptable carrier. 0> Λ t I - 20 - 19. Use of the derivatives according to one of claims 1 to 11 for the treatment of leukemias, gliomas, lymphosarcomas or other malignant tumors, including | _ so-called "solid" tumors as well as for the treatment of Hodgkin's disease and other tumors. % \ i i j. t A "[i i s i