LU83034A1 - N- (VINBLASTINOYL-23) DERIVATIVES OF AMINO ACIDS AND PEPTIDES, THEIR PREPARATION AND THEIR THERAPEUTIC APPLICATION - Google Patents

Description

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- 1 -NU

J * 1 DERIVATIVES N-CVINHLAST1NOYL-23) OF AMINO ACIDS AND PEPTIDES,

THEIR PREPARATION AND THERAPEUTIC APPLICATION

5

The present invention relates to novel bis-indole alkaloids. More particularly, the invention relates to new amino acid coupling derivatives with Vinblastine, including polypeptide derivatives, to their process of preparation, to their application as anti-tumor agents and to the pharmaceutical compositions containing them. .

These new Vinblastine derivatives can be used 1S for the treatment of leukemias and malignant tumors in humans.

The alkaloids bis indoi i c of the type of vir.rlastine are "well known compounds possessing the carbon skeleton of i 20 ^ la ^ ° rmU ^ e ^ n ^ rUjC SU * Vante: Γ i • · ν * 'ν' . ftLJ'ji.ï - 2 - "ch3û2c 1 / \ (I) 5 j l op I 1 4 1 ° H0 # \ 2 3 f = 0 R, 15

Examples include vincaleukoblastine (US patent 3,097,137), leurocristine or vincristine and leurosidine (US patent 3,205,220), vinglycinate (Belgian patent 659. 1 12jet vindesine (Belgian patent 813,168).

The latter compound was obtained by chemical transformation of natural Vinblastine (1, R ^ OCH ^, R7 = C0CHj), which itself is obtained by extraction from the leaves of Catharanthus roseus.

Vinblastme, vincristine and vindêsine are marketed for their use in human therapy, more particularly for the treatment of leukemias and certain solid tumors.

ύ 30 These drugs nevertheless have unfavorable side effects. Vincristine is neurotoxic and Vinblastine is highly toxic in the hematopoetic tissue.

% * —....... '""' .'η - 3 - »1 Their mechanism of action is similar to that which has been postulated for the antimitotic action of colchicine.

These drugs would act by inhibiting the polymerization h of tubulin into microtubules and subsequent arrest of cell division in metaphase.

3 The use of 1: 1 complexes of bisindolic antitumor 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.

Other derivatives obtained by chemical modification of Vinblastine have been tested. Thus vinglycinate sulfate (i, sulfate, Rj ^ 0CH3, R ^ COCll ^ N (Cil ^) 2 (Cancer Research 1 967, 2 7, 221-2271 has been studied in clinical experimentation but has generally not been revealed superior to Vinblastine and vincristine.

20

Belgian patent 813.108 describes vindesine (1, R ^ = NH7, R2 = H) and other C-3 carboxamide derivatives of Vinblastine.

Subsequent publications have confirmed the therapeutic value of vindesin or carboxamide-3 deacetyl-0-4 Vinblastine, but this compound has nonetheless proved inactive for the treatment of L 1210 tumors transplanted in mice (CJ Barnett et al. ., J. Med. Chem., 2J [, 88, 1978).

Vindesine is currently being introduced to the European market (particularly in France and Germany) and the I registration procedure in the United States is underway (NDA-18.348).

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- 4 - 1 Amino acid coupling derivatives with Vinblastine t and other analogous bisindolic alkaloids were generally proposed in the Belgian patent 813,168 * mentioned above.

5 However, no example of a specific amino acid derivative has been disclosed and consequently, no physico-chemical data has been provided. No specific preparation method and no particular pharmacological result was mentioned.

It can therefore be deduced therefrom that these derivatives have not actually been synthesized and / or have not been tested for their anti-tumor activity.

On page 3, line 10 et seq., It is further mentioned that "anti-neoplastic activity seems to be limited to very specific structures and the chances of obtaining more active drugs by modifying these structures also seem low".

It has now been found that the new compounds described in the present invention are capable of effectively delaying the death of mice to which P 388 and L 1210 tumors have been transplanted intravenously.

While Vinca alkaloids administered i.v.

M

25 generally have no activity on L 1210 leukemia, the results obtained show a very significant and unexpected activity for the new derivatives.

They also have other important and unexpected advantages over Vinblastine and its known analogs, particularly vindesine. Numerous complete remissions have been observed.

i - 5 -

V

\ "1 The new compounds of the invention respond more particularly to the general formula 11

Or / rL -! N -A ^ C02ChJ (II), 0 i! oo

ririV

1 ς CH 0 v N R

15 i '| JS. j 3 O

CH3 OH "-Sf - (- NH-CH-C-) n 0KA 0 20 wherein R2 represents a hydrogen atom or a C1-C9 acyl group, preferably an acetyl group.

is a hydrogen atom, an alkyl, hydroxy-carboxy-, amido-, amino-, guanadino-, thio-C 1 -C 8 alkyl, cysteinyl methyl, methyl-thio ethyl, benzyl, hydroxy benzyl chain; a - ph - 7d. at

H

-f "!

J

I “- 6 - 1 or represents with the carbon atom to which it is attached and the nitrogen of the amide group, an azole ring; n varies from 1 to 6; and represents a linear or branched alkyl group having from 1 to 7 carbon atoms, a benzyl group or an OH radical. The group -OR ^ can also be replaced by a group -N ^.

„COOR ^ preferably represents a carboethoxy or carbomethoxy group.

3 10 More particularly, the structural fragment of the

formula II R, O

i! -NH-CH-COR4 represents an ester of any of the natural amino acids and their isomeric optics of configuration D, in particular: glycine aspartic acid cystine alanine glutamic acid methionine valine asparagine phenylalanine leucine glutamine tyrosine 20 isoleucine arginine tryptophan serine lysine proline or threonine cysteine histidine

These compounds can be considered as derivatives of descarbomethoxy-3 deacetyl-0-4 Vinblastine carboxamide-3. However, it will be preferred to designate them as N- (vinblastinoyl-23) derivatives of amino acids.

The preferred compounds of the invention correspond to formula II in which R £ represents a hydrogen atom and the amino acid part is derived from one of the following amino acids: L or D tryptophan, / leucine, isoleucine or phenylalanine.

% * 4 - 7 -

V

3 d, 1 Among the polypeptide ester derivatives, the preferred compounds correspond to formula II in which n = 2 and the amino acids are chosen from the list mentioned above, according to any sequence.

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With regard to pharmacological properties, it is foreseeable that slight changes in the structure of the polypeptide backbone will provide compounds of comparable activity.

10 In particular, the presence of an unnatural α-amino acid (for example an N-monosubstituted α-amino acid φ or an α, a -dialkyl amino acid) or an fj-amino acid would provide active compounds similar against a number of tumors.

Such compounds therefore fall within the scope of the present invention.

The compounds of formula II can be obtained using various synthetic methods.

20 According to one of these methods, these derivatives are obtained from Vinblastine by hydrazinolysis, nitrosation | then coupling with the amino acid or poly- ester! appropriate peptide.

These compounds can also advantageously be obtained by saponification of Vinblastine, followed by coupling of vinblastinoic acid, optionally protected beforehand by acetylation, with the amino acid or peptide ester. In this case, a coupling agent such as dicyclo is preferably used. hexylcarbodiimide.

t /% Λ Λ% «- - 8 - 1 When the amino acid or the peptide contains functional groups in the side chain R3, it can be like lysine or cysteine, necessary to protect these functional groups by using the 5 methods well known to those skilled in the art.

This protection can be carried out, depending on the nature of the group to be protected, by grafting a benzyl, t-butyl, benzyloxycarbonyl, t -butoxycarbonyl or trityl group. Other agents well known in peptide chemistry can be advantageously used.

According to the first process mentioned, the compounds according to the invention can be obtained from the

Vinblastine, in a conventional manner, by hydrazinolysis followed by nitrosation then coupling with the amino acid ester or of polypeptide according to reaction scheme I

20

r ^ l νΛ _ γ-Γ "! n.no2 ^ J4 I

C -a A CH-OH ^ 1 i HCl

v4x5coch, 3 \ ηβρ / T ÛH

4 3 ^ OH 0 ° C 4 \ OH 'W \ HO' or cooch „ho c-nh-nh., C-n, 25 3 <3 2 b 3" "acid ester

5 amino or N

polypeptide _ ^ pvj j Scheme I

CH-C1 H

30 Z Z <A

\ - VÜH

\ ^ H ^ 3

nn '“| J

Æ- -, c- <k-9-CO) „or4

/ OH

/ η = 1 to 6! '- ^ iffie ^ · * -ζψ * · - *. - - -— * -; .., '- · ^ ι ·' -ί ^ .ί> - + - .. _. ·. H | T, ^. _. - ____ ”t *“ • * *.

- 9 - 1 The first step consists in adding an excess of anhydrous hydrazine to a solution of Vinblastine base in anhydrous methanol. The reaction mixture is then heated under an inert atmosphere for 12 to 30 ± 5 hours at a temperature between 30 and 70 ° C.

Preferably, the temperature will be maintained at around 60 ° C., the reaction time then being 24 hours.

The deacetyl-4-vinblastine hydrazide is then isolated by addition of water, extraction with dichloromethane and concentration. The compound can be further purified by preparative chromatography (neutral silica).

In the second step, the hydrazide function of the modified Vinblastine is transformed into acid azide. This transformation is carried out in a known manner, by adding sodium nitrite to the hydrazide in solution in a water-methanol-acid mixture.

The acid used can be hydrochloric acid.

The reaction temperature is maintained between 0 and 5 ° C. After extraction with an aprotic solvent which is not soluble in water, preferably methylene chloride, the organic phase is partially concentrated.

The acid azide itself is preferably not isolated, but directly brought into contact with the amino acid ester or polypeptide or, where appropriate, the corresponding protected derivative, in solution in chloride. methylene.

-10-.

"1 The amount of amino acid used is one to four equivalents of Vinblastine carboxazide.

The reaction medium is maintained between - 3 and + 5 ° C λ for about 15 hours.

The reaction is easily followed by chromatography = on a thin layer. After concentration, the compound of invention II is isolated which can be transformed into sulfate or other mineral or organic acid salt by crystallization from a methanolic solution of the corresponding acid.

The compounds of the invention are purified using the well known techniques of chromatography and recrystallization.

15

If necessary, the deacety1-0-4 Vinblastine derivative thus obtained can be reacetylated either directly to give the Vinblastine II derivative in which R2 = COCH3 (J. Med. Chem. 22, 391, 1979) or by 20 preliminary formation of the 3,4-diacetoxy derivative followed by selective hydrolysis of the acetoxy group in position 3.

The hydroxyl group may, in a manner known per se, be esterified by other activated derivatives of acids containing from 1 to 9 carbon atoms. It has moreover been demonstrated that the derivatives of the invention can also be advantageously obtained at starting from Vinblastine, by saponification of this alkaloid, optionally partial or complete protection of the free hydroxyl groups by treatment with acetic anhydride according to the conventional mode 30 then condensation of vinblastinoic acid-23, thus obtained with an acid ester amino or J polypeptide.

Tt i * '-11- 1 The saponification of Vinblastine provides, after acidification, deacetyl-0-4 vinblastinoic-23 acid. : This derivative was first described by

Hargrove (Lloydia, 27, 340, 1964).

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The coupling is carried out by adding the acid derivative

AT

vinblastinoïque to a coupling agent usually used in peptide chemistry (see for example: Methoden der Organischen Chemie, Houben-Weyl, vol.XV 10 parts 1 and 2, 1974.)

A particularly preferred coupling agent is N, N’-carbonyldiimidazole (Staab H.A., Angew. Chemie, 82, 4596, 1960). In this case, the condensation is carried out by prior addition to the vinblastinoic acid of one or more equivalents of N, N’-carbonyl-diimidazole, followed by the addition of the amino acid ester or peptide.

The reaction takes place in tetrahydrofuran, dimethylformamide or any other solvent inert under the reaction conditions, at a temperature which can vary between -15 ° C and + 40 ° C.

The reaction time then varies from one hour to twenty hours.

25

The product thus obtained, isolated in a conventional manner, can then be deacetylated in an acid medium to provide compounds of formula II in which R7 = H. Optionally controlled deacetylation can provide the compounds of formula II in which ji R, = C0CH ,.

i - 3 * r r i t / -12- * 1 The present invention also extends to industrial and in particular pharmaceutical applications of new bisindolic compounds.

The compounds of the invention have in particular particularly advantageous anti-tumor properties which can be used in human therapy.

These amino acid derivatives can, in particular, be used for the treatment of leukemias of the L 1210, P 388 type, of gliomas, lymphosarcomas and other leukemias or malignant tumors.

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

These compounds can also be used in veterinary medicine for the treatment of tumors in animals.

Other interesting therapeutic applications can be envisaged for these new derivatives of bisindolic alkaloids. It has in fact been noted 25 that Vinblastine could be used in the treatment of certain arthritis (United States Patent No. 4,208,411) and Vincristine for the treatment of psoriasis (United States Patent No. 3,749. 784).

For the treatment of malignant tumors in animals, the chemotherapeutic activities were tested using the sulfate salts of the compounds jL of the invention.

l · f [ji -13- 1 DBA mice? Charles River France females ^. 4 were inoculated intravenously with 10 leukemia cells from seven-day P 388 or L 1210 leukemia ascites. Day 0 is the day of inoculation of the tumor cells. The compound of the invention (in sulfate form) is then injected intravenously in solution in physiological water (NaCl 9/1000) according to the scheme in one injection (day 1) or the scheme in three injections (days 1, 2 and 3).

10 We then calculate the MST (Medium Survival Time), the day when half of the animals died.

This calculation is performed on the thirtieth day.

The percentage of ILS (Increased Life Span) or · percentage increase in survival is calculated using the following formula: MST product% ILS = (- x 100) - 100 MST controls

The number of mice surviving the thirtieth and sixtieth days is also indicated.

At too toxic doses, the percentage of ILS becomes negative, that is to say that the untreated mice survive longer than those having been injected with the anti-tumor substance.

In certain cases, a significant variability in the results is observed depending on the origin of the mice (DBA? France or the United States) or, more rarely, the batch of product used.

The results obtained are to be compared with those observed with Vinblastine (VLB), desacetyl-0-4 Vinblastine (DAVLB) and vindesine (VDS) collated in Table I. The pharmacological superiority of the compounds of the invention is thus demonstrated in Tables * 35 i II to XII.

-14- '1 Table II provides the results obtained with some derivatives of natural leucine.

The following derivatives were thus tested: 5 VLE: N- (deacetyl-0-4 vinblastinoyl-23) -L-ethyl leucinate VLM: N- (deacetyl-0-4 vinblastinoyl-23) -L-leucinate i methyl VLn-But: N- (deacetyl-0-4 vinblastinoyl-23) -L-leucinate n-butyl VL-octyl: N- (deacetyl-0-4 vinblastinoyl-23) -L-octyl leucinate VL -amide: N- (deacetyl-0-4 vinblastinoyl-23) -L-leucin-amide 15

Table III collates the results obtained with a derivative of D-leucine, in this case N- (deacetyl-0-4 vinblastinoyl-23) ethyl D-leucinate (VDLE).

The results obtained with the following L-tryptophan derivatives are shown in Table IV: V-Trypt E: N- (deacetyl-0-4 vinblastinoyl-23) -L-ethyl tryptophanate, MST calculated at 30 and at bO days.

Methyl V-Trypt M: N- (deacetyl-0-4 vinblastinoyl-23) -L- tryptophanate.

V-Trypt Ac: N- (deacetyl-ü-4 vinblastinoy1-23) -L-tryptophan.

Table V presents the activities observed with the ethyl tryptophanate derivative of absolute configuration D (VD Trypt E).

V

/ V

/

P

t; .1% i * Λ -15- 1 Tables VI to XII show the results obtained with the following compounds: - VAE: N- (deacetyl-0-4 vinblastinoyl-23) -L-ethyl alaninate 5 - VPE: N- (deacetyl-0-4 vinblastinoyl-23) -L-ethyl phenyl-alaninate (table VII) - VILE: N- (deacetyl-0-4 vinblastinoyl-23) -L-iso ethyl leucinate (table VIII) 10 - VILM: N- (deacetyl-0-4 vinblastinoyl-23) -L-iso methyl leucinate (Table VIII) - VVE: N- (desacetyl-0-4 vinblastinoyl-23) -L-valinate ethyl (Table IX) - V-Tyr E: N- (deacetyl-ü-4 vinblastinoyl-23) -L- ethyl tyrosinate (Table X)

- V-t-TPA lysine E: N- (deacetyl-0-4 vinblastinoyl-23) -C

J ethyl trifluoroacetyl-L-lysinate. - V-Val-Trypt-E: N- (deacetyl-0-4 vinblastinoyl-23) - f ................. * ......... ...

TABLE I: -1b- • ...........

A. VINBLASTINE (VLB) - P388: dü8e diagram nowbre of MST ILS> 30 J.> 60 J.

„Mg / kg / d animals max 30J. Z

----- I - -------- 1 _ I I

4 1 10 14.6 36.4 '0 0 6 1 10 15.6 45.8' 0 0 8 1 9 18.5 72.9 0 0 - - - · - - # - L J2J0: 6 1 10 10 , 6 24.7 0 0 B. DESACETYL-0-4-VINBLASTINK (DAVLB) - P_388: dose, number of STDs ILS> 30 J.> 60 J.

mg / kg / d SC ema animals max 30J. % 2 1 10 13.5 26.2 0 0 3 1 10 lb 49.5 2 1 4 1 20 18 58 3 0 5 1 10 6.5 -44 1 1 6 1 10 5.6 -52 0 ü - L_I2I0 ; 3 1 10 11.8 38.8 0 0 -17- TAELEAIJ I (continued): C. VINDESINE (VDS) - P_388: dosa 8 diagram number of STDs ILS> 30 J.> 60 J.

mg / kg / d animals max 30J. Z

2 1 10 13.6 27.1 0 0 .3 1 10 14 30.8 ^ 0 0 4 1 10 14.8 38.3 0 0 5 1 10 13.8 30.2 0 0 6 1 10 14 32 0 0! - L_! 2J0: i I "- 3 1 10 14.8 38.3 Q 0 i-18-

TABLE II: DERIVATIVES OF NATURAL LEUCIN

A. ELV

- P_388: düße diagram nümbre of MST ILS> 30 J ·> 60 J-

mg / kg / d animals max 3ÜJ. Z

20 1 10 16 52.4 2 1 • 22 1 10 20 90.5, 4 1 24 1 20 18 65 2 0 26 1 10 17.5 53.5 1 0 28 1 10 19.6 71.9 1 1 30 1 10 21 84.2 2 1 34 1 10 6 -48 2 1 36 1 10 5.4 -53 1 1 5 1.2.3 9 14.5 25 0 0 6 1.2.3 10 15 29.3 0 0 7 1.2.3 10 14.2 24.6 0 0 9 1.2.3 10 5-56 1 1 12_1, 2,3_9_4,8 -59_0_0 - L_J. 210: 30 1 10 5.6 - 26 0 0 32 1 10 6 -21 0 0; > _ * TABLE IX (continued) -19-

B. VLM

- P_388: dose - number of STD ILS> 30 J.> 60 J.

mg / kg / d 1 ma animals max 30J. % λ - -----— I - ... 1. I - 1 - - _ f_ _ - I. _. _ -.

10] 20 16.7 Al 2 1 I. 0.5 1 10 16.4 53.3, 0 0 Π 1 19 17.7 51 2 1 II, 5 1 10 16.8 57 0 0 12 1 20 17, 5 49 32 ^ 12.5 1 30 17.3 49 8 3 13 1 20 20.5 74 7 5 15 1 10 18 55.2 4 1 - L_] 2J0: 10.5 1 10 10.6 24.7 0 0 11.5 1 10 10.8 27 0 0 12.5 1 10 10 17.6 0 0 f ............... "i · Ί

U

; ι il -20- J TABLE II (continued): C. VL-n butyl - P_388: dose schema nunlb, re d * MST ILS> 30 J.> 60 J.

mg / kg / d animals max 30J. % '____J___ 55 1] 0 15 23 0 0 60 1 10 15.4 26.0 0 65 1 10 13.2 13.8 0 0 70 1 10 14.7 26.7 0 0 80 1 10 13.4 27 , 6 0 0 1 ---- - - ____, —1 D. VL Octyl - P_388:

Idose scheme number of STD ILS> 30 J.> 60 J. d mg / kg / d animals max 3ÜJ. % 40 1 10 11.5-0.9 0 0 60 1 10 12.5 7.8 0 0 | 80 1 8 12.7 9.5 0 0 E. VL Friend of i i - P_388: dose, ^ number of STD ILS> 30 J.> 60 J.

1 regimen mg / kg / d animals max 30J. %; 51 10 11.6 1.7 0 0. '15 1 10 12.4 8.7 0 0 20 1 10 11.6 1.7 1 1 30 1 10 11.6 1.7 0 0 40 1 10 12.4 19 0 0 J 50 1 10 13.4 15.5 0 0

TABLE III

-21-

A. VDLE

- P_388: düsô. diagram number of STDs ILS> 30 J.> 60 J.

mg / kg / d animals max 30J. % 6 1 10 14.7 26.7 0 0 '8 1 10 17 46.5 * 2 1 10 1 20 28.5 146 9 2

11 1 10 16 49.5 1 O

12.5 1 10 30 145.9 6 3 - L_I2J0; 9 1 10 10.8 27 Û 0 10 1 10 11 29.4 0 0 -22-

TABLE IV

\ '' Y 'j. ·

A, V Trypt E

- P_388: düSÊ diagram noDlbre of MST ILS> 30 J.> 60 J.

mg / kg / d animals max 30J. % 20 1 10 11.4 43.9 0 0 40 1 10 17 58.8 5 4 50 1 20 24.5 117 9 5 55 1 40 30 171 28 16 60 1 90 30 204 52 24 I 65 1 29 22, 75 114 13 9 70 1 20 6, '5 -39 8 5 - - - -_____- -___. .

- L_1_2J0: 55 1 30 11.8 56 O 0 60 1 30 12.5 65 2 2 65 1 30 12 62 0 0 70 1 10 12.3 61.8 1 1 “E_lbb_i®a: x_623j.). : dose diagram number of STD ILS> 30 J ·> 60 J.

mg / kg / d animals max 60J. Z

55 1 30 60 457 28 1b 60 1 60 36 224 52 24 i-- TABLE IV (continued) -23-

B. V ïrypt M

- P_388: düSe diagram I1Uïbre of MST ILS> 30 J.> 60 J.

rog / kg / d animals max 30J. Z

30 1 10 15 33.9 0 O

40 1 10 16 42.8, 0 0 50 I 10 23.5 199.8 1 0 60 1 20 26 140 7 2 70 1 10 26 145.3 5 C. V Trypt Acid - P_388: dose number of STDs ILS > 30 J. ^ 60 J.

mg / kg / d animals max 30J. Z

20 1 10 11.4 1.8 0 0 25 1 9 11.8 6.3 0 0 30 1 8 12 8.1 0 0 40 1 10 1.6-85.7 0 0 60 1 10 1.4 - 87.5 0 0 .80 1 10 1.4 -87.5 0 0 1 ............—- t

NOT

J

0 ..., - - - * - - "r. - »**« I * X 'Λ. * - * .- * <- * i ·' “'fc ** * I« 4 d -24-

TABLE V

VD Trypt E

- P_388: dose 8 diagram number of * STD ILS> 30 J.> 60 J.

mg / kg / d animals max 30J. Z

20 1 10 13.3 18.7 0 30 1 10 15.8 41.4 ^ 1 40 1 20 16.5 52 1 50 I 10 22 107.5 1 60 1 10 4.4 -60.7 1

TABLE VI VAE

- P_388: dose number of 1 ILS STD> 30 J.> 60 J.

C Γ * ΠΡΤΤ1Λ r r mg / kg / d animals max 30J. % 1 1 10 14.8 27.6 0 0 10 1 10 16.3 40.5 0 0 12.5 1 19 16.2 44 1 1 1 15 1 20 17 46 5 4 25 1 10 19 63.8 2 1 - L_] 2] 0: μ 12.5 1 20 10.4 48 00 -¾ -25-

TABLE VIII A. VPE

- P_388: d ° Diagram number of STD ILS> 30 J.> 60 J.

mg / kg / d animals max 30J. Z

10 1 10 13 12.1 0 0 20 1 10 14.4 24, 0 '0 0 40 1 10 30 158.6 7 7 50 1 30 13.6 26 10 7 55 1 10 14.6 36.4 1 0 60 1 60 13.7 18 6 6 70 1 10 6 -48.3 2 0 75 1 59 14.5 27 0 0 80 1 20 15 38 0 0 - L__m0: 55 1 10 8.5 0 0 0 60 1 10 9 13.9 0 0 65 1 10 8.8 3.5 0 0 70 1 20 8.7 13 0 0 75 1 10 9.5 25 0 0 Λ -> i iî * *! -2b- TABLE VIII (continued)

B. VILE

- L · 388: düSe diagram noDlbre of MST ILS> 30 J.> 60 J.

mg / kg / d animals max 30J. 7.

»8 1 30 30 172 20 2 • 9 1 20 30 179_12_ - LJ. 21_0: 8 1 20 11.57 58 0 0 | 9 1 10 12.8 68.4 0 0

IC. VILM

- P_388: dose, _ number of STD ILS> 30 J.> 60 J.

i, diagram. _ _ * mg / kg / d animals max 30J. % 1: 1- 1 10 16.4 '41.3 0 0 5 1 29 18.2 68 8 3 6 1 69 23.4 115 25 6 7 1 30 30 177 16 5 d 9 1 10 6 -48.3 4 4 15 1 10 5 -53.3 0 0 20 19 3 -72 0 0 25 1 10 2.5 -76.6 0 0 30 1 10 2.5 -76.6_0_0 - L_J_210: i P 6 I 29 11 , 2 60 0 0 -27- "

TABLE IX VVE

- P_388: düae diagram nOBlbre of MST ILS> 30 J.> 60 J.

®g / kg / d animals max 30J. Z

* - - - - - f - _ - _ _ 1 _ I

5 1 10 13.6 17.2 0 0 10 1 10 17.8 53.4 0 0 »12.5 1 10 21 82.6 2 0 14 1 20 21 98 3 15 1 39 23.5 107 14 4 17.5 1 9 8.4 -22.2 0 0 25 1 10 6.5, -44 0_0 - L _. [2K): 15 1 19 11.4 61.9 0 0 - - - - ...

PAINTINGS

V Tyr E

- P_388: dose number of STD ILS,> 30 days> 60 days.

mg / kg / d SC ema animals max 30J. Z

20 1 10 13.9 29.9 0 0 40 1 10 16.4 53.3 1 1 50 1 10 16 49.5 1 1 60 1 10 16 49.5 3. 2 70 1 10 5.4 -49.5 0 0 f --------- / dI * "-28-

TABLE XI

V- g, TFA-lysine E - P_388: dose number of STD ILS> 30 J.> 60 J.

diagram . _

mg / kg / d animals max 30J. Z

A ____ ^ - 20 1 10 12.8 19.6 0 0 30 1 10 12.5 13.8, 0 0 40 1 19 13.6 20 0 0 50 1 10 14.8 27.6 0 0 60 1 10 15 , 2 31 0 0

_ _ _________ I

TABLE XII

V-Val-Trypt E - P_388: düSe nchéma number of STD ILS> 30 J.> 60 J.

mg / kg / d animals max 30J. % 15 1 10 26 132.1 3 25 1 10 27.5 145.5 3

S

35 1 19 27.5 154 8 45 1 10 30 183 8 50 1 9 27.5 145.5 4 i1 -: - 1 r -29- 1 The anti-tumor activities collated in Tables I to XII complete, and in some cases modify, the conclusions which could be drawn from the first results presented in the base patent 5 corresponding to the present application.

;'at

Note in particular the exceptional activity of * sulphate of N- (deacetyl-0-4 vinblastinoyl-23) ethyl tryptophanate which provided an ILS calculated at 60 days of 10,457% with half of the mice surviving more long time.

The optimal dose would then be around 60 mg. On the other hand, the amino acid derivatives which have been tested in the amide or acid form exhibit only weak or non-significant activities. These derivatives can however be used as intermediates which are useful for obtaining other very active bis-indole alkaloids.

In general, the compounds of the invention have been found to be much less toxic than the Vinca alkaloids currently used in cancer therapy.

The lethal dose 50 (DL5Q) of the ELV was determined using female Charles River CD ^ mice weighing at least 24 g. The Litchfield and Willcoxon calculation method provides a value of DL, -q of; 32 mg / kg.

The corresponding doses for Vinblastine and | , vindesine are respectively 24 mg / kg and approximately • 11 mg / kg. In particular, unlike the 30 cases of Vinblastine and deacetyl Vinblastine, the absence of hepatic toxicity was noted at doses of 20 to 4 "I mg / kg.

U * · · · - mal 'l .. · * I / # 1 "-30- I For their therapeutic application, the compounds of the invention, optionally in lyophilized form, are preferably administered parenterally, dissolved in a pharmaceutically acceptable solvent, in the form of a base or an addition salt of a pharmaceutically acceptable acid.

Physiological water or other buffered saline solutions, "for example with phosphate, are suitable solvents. Generally, these compounds can be used drawing inspiration from techniques and limitations which are known for therapeutic treatments with other alkaloids of the Vinca class.

* 15 The active substance is generally administered in a dosage which can vary from 0.01 mg to approximately 5 mg / kg, depending on the derivative used and the therapeutic regimen adopted. Total weekly doses will generally range from 0.05 mg to approximately 5 mg / kg.

20

The compounds of the invention can be used alone or in combination with one or more carcinostatic agents including, for example, alkylating agents, antimetabolites such as methotrexate, fluoro-5-uraci1e, inercapto-b purine, thio-b guanine, the arabinosides of cytosine and antibiotics such as actinomycin, daunorubicin, adriamycin and cis-diamino-dichloro-platinum. In particular, these new Vinblastine 30 derivatives can be used in combination with one another.

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

it -31- A '1 General procedure for obtaining N- (d.ésacetyl-ü-4-vinblastinoyl-23) amino acids _3 1 g (1.3 10 M) of descarbomethoxy-3-desacetyl-0- 4 5 Vinblastine carboxhydrazide-3 is dissolved in 23 ml of absolute methanol and 74 ml of 1N hydrochloric acid.

. This solution is then cooled to -10 ° C and 207 mg of sodium nitrite are added to it at once.

The reaction mixture is then left for ten to thirty minutes at 0 ° C. After checking by thin layer chromatography, the reaction medium is basified to pH 8.5 at -10 ° C with a saturated solution of sodium bicarbonate.

The alkaline solution is extracted at 0 ° C with its own volume of methylene chloride cooled to 0 ° C until a negative Meyer reaction on the aqueous phase is obtained. The organic phases are combined, dried over Na2SO 4 and filtered at 0 ° C.

Then added 1.43.10 3M (1.1 equivalent) of the retained amino acid and concentrated in vacuo to a volume of about 4 ml. This solution is then stored for 24 to 48 hours at 4 ° C. The progress of the reaction is followed by thin layer chromatography.

The solution is then drawn to dryness and provides 1.05 g of crude coupling product which is almost unitary with thin layer chromatography.

Puxification

The dry residue obtained above is placed on a column of silica gel and eluted with an ether-methanol mixture saturated with 96% + 4% ammonia.

10 ml fractions are collected and tested by thin layer chromatography. The revelation will be made with ninhydrin (amino acids) and ceric (alkaloids).

i \, -32-! 1 When the amino acids have passed (ninhydrin -), one elutes again with 100 cc of the solvent used before changing it with a 92% - 8 i mixture.

The alkaloid coupled to the amino acid will leave (caeric +) 5 from fractions 55 to 160.

The identical fractions are combined, concentrated to dryness on a rotary evaporator, taken up in methylene chloride, dried over sodium sulfate, filtered and evaporated to dryness. The foam obtained is the dimer alkaloid coupled with the all-pure amino acid, the physicochemical data of which will be made on an aliquot part.

The rest will be directly transformed into sulfate.

The yields based on the various derivatives are given for information only and are likely to improve.

15

Sulphate preparation:

The amorphous base (foam) obtained above is taken up in 20 times its weight in ethanol.

Two equivalents of sulfuric acid from a 2% H2SO4 / 98% anhydrous ethanol solution are then added very slowly and with vigorous stirring to this solution. (0.484 equiva] ent / 1itre). After adding the two equivalents, the mixture is left to stir for half an hour before concentrating under reduced pressure.

By adding anhydrous sulfuric ether, with vigorous stirring, the sulfate of the committed derivative precipitates.

Filtration and drying under vacuum at 10 ° C. provide the sulfate of the coupled derivative ready for use.

The numbering adopted for the description of the nuclear magnetic resonance spectra of the examples yes following i I is inspired by that proposed by Le Men and Taylor for the derivatives of the aspidospermidine type (Experientia 21, 508, 1965).

*. AT, . . .... ”· 4 4 -33-

Preparation of Vinblastine (VLB) base To a solution of 1.5 g of VLB sulfate (1.65.10 ~ 3M) 5 dissolved in 15 cc of distilled water, 15 cc of methylene chloride are successively added with vigorous stirring as well as 1.5 cc of concentrated ammonia.

After five minutes, the mixture is decanted and the aqueous phase extracted with a further 3 × 15 cc of methylene chloride. The combined organic phases are washed with 2 x 40 cc of deionized water, dried over anhydrous sodium sulfate and then evaporated to dryness on a rotary evaporator. 1.32 g of base VLB (99%) are thus collected.

15

Preparation of descarbomethoxy-3-deacetyl -0-4 Vinblastine carboxhydrazide-3 (VLH) To a solution of 1 g of VLB base (1.23 10 ^ M) in 7 ml of anhydrous ethanol, 14 ml of hydrazine are added anhydrous and 7 ml of anhydrous ethanol. The reaction mixture thus formed is kept for twenty hours at 60 ° C.

After cooling, 28 ml of water saturated with salt are added and the mixture is extracted with the same volume of methylene chloride until a negative Meyer reaction is obtained on the acidified aqueous phase.

The organic phases are dried over MgSO 4 and evaporated to dryness under reduced pressure.

The hydrazide obtained with 88% yield (0.760 g) is unitache by thin layer chromatography.

J

-34- JÏ I 4 1 iiËiDEÎ.ë-? :

Synthesis of N- (deacety1-0-4 vinblastinoyl-23) -L-ethyl leucinate (VLB) - -4 5 A solution of 275 mg (3.58 10 M) of 0-4 deacetyl Vinblastine hydrazide in 7 ml of anhydrous methanol and 20.5 ml of IN HCl, cooled beforehand to 0 ° C., 58 mg of sodium nitrite are added.

The mixture is left to stir for 25 minutes before returning to a pH of 8.5 by adding a sodium bicarbonate solution at 5 h. The azide formed is extracted with methylene chloride. The organic phase is dried over MgSO. and filtered. 68.4 mg of ethyl 4 -4 leucinate (4.29.10 M) are then added thereto and the mixture is concentrated under vacuum, until approximately 4 ml are obtained.

This solution is then left for 24 hours in the refrigerator.

After the reaction is complete, 50 ml of methylene chloride are added and the mixture is washed several times with the same volume of demineralized water and then once with a saturated NaCl solution.

The organic phases are dried over MgSü 4, filtered and then evaporated under vacuum.

300 mg of crude product are thus collected to which 0.035 g of I 2 SO 4 is added in solution in 1 ml of anhydrous methanol.

The salt formed is precipitated with ether.

-. The precipitate is washed ten times with 50 cc of anhydrous diethyl ether.

The residue, 183 mg (57%), contains only pur VLB sulfate which is practically pure and free from ethyl leucinate \ L.

-35- 1 After release of the bases, the latter can be chromatographed on silica gel (10 g of SiC 4) and eluted with 50 cc of ether / MeH-NH 4 sat.

(92 * / 8 *) then 250 ml of ether / MeOH-NH3 (851/151).

5 The VLB is obtained pure at the head of the second eluate.

49 l of purified VLB are thus collected, ie 152 mg of base.

Physico-chemical properties of GBV

10 ~; Melting point: 169 ° C i

---— .... ^ I

r i CHC13 c = 0.35: 00 15 Ultraviolet spectrum (MeUll, A lliax, mn, lo ^ »l): 221 (4.62); 267 (4.15); 287 (4.02); 295 (3.99).

Infrared spectrum (KBr, cm "1): 20 3470, 2960, 2880, 1735, 1665, 1610.

Mass spectrum (m / e, 1): 924 (6) M + + 2 8; 910 (56) M ++ 14; 897 (62); 896 (100); 865 (25); 938 (68); 772 (19); 709 (25); DSI (43); 571 (69).

25 * Nuclear magnetic resonance spectrum (111, CDC J,, ppm, 3 ü OMI 1 z): 9.66 (<111, .b s, C10-011); 8.20 (111, s, N 'UH); 7.52 (111, d); 7.15 (311, m); 6.56 (11I, S, C9-11); 6.05 (111, s, C 1 2-U); 5.86 (11l, dd, C 1 4-11, J 14-15 = 12; J1 4 -3 = 3.6; 5.78 (111, d, C 1 5-H); 4.69 (1 H, m, CH (NUR) CÜ-); 30 4, 2 (21l, q, OÇH2 Cllj); 4.18 (1 11, t, C 1 7-11); 3.77 (311, s, UCHj ); i 3.66 (311, s, 0CH,); 3.47 (11I, s, C5-H); 2.77 (311, s, -Cllj); J> P> y2 (12H, m, -CT8H3 + C, 8'll3 + j sopropyl).

b s.

*. -, • - '' i - \ i - '-36- 1 ËîëîîEIë--: N-deacetyl-0-4 vinblastinoyl-23) -L-methyl leucinate (VLM)

By applying the general procedure on page 31, 5 we obtain the VLM with a yield of 68 I.

Melting point: 172 ° C

10 I (¾ CHC13: (.7- υ c = 0.2 7

Ultraviolet spectrum (CH ^ OH, Amax, nm, log l. J: 220 (4.01); 267 (4.15); 287 (4.03); 294 (3.98).

15

Inlra-red spectrum (KBr, cm: 3475; 2960; 2880; 1740; 1080; 11> 1S.

Mass spectrum (in / e, o): 20 910 (25) M + + 2 8; 890 (78) M ++ 14; 883 (20) M ++ 1; 882 (30) M +; 850 (29); 830 (41); 822 (100); 708 (15); 081 (50); 050 (78); 570 (70).

Naked magnetic resonance spectrum J éa i ru (C1H3 1 3, p pm, OR MHz): 25 9.21 (111, s, Ο1 b-ÜH); 8.1 (111, s, N ’u -11); 7.53 (111, m); 7.23 (311, m); 0.63 (111, s, C9-U); 6.13 (11l, s, C1 2-11); 5.80 (2 II, m, C 1 4 - H + C15-H); 3.83 (311, s, -0C113); 3.80 (3H, s, -C00C113); 3.03 (311, s, -0C113); y 2.8 (3H, s, -if-Cll3J; 0.90 (1211, m, C18H3 + C18 H + isopropyl).

Example 4: N- (deacetyl-0-4 vinblastinoyl-23) -L-n-butyl leucinate (VLn-But) 5 13n applying the. general operating mode on page 31, we obtain the VLn-Goal with a yield of 58% ·

Physico-chinuques_de_J_a_VLnn properties * 10

T us i on point: 1 5 8 0 (2

CIICK

H "c.ojo: 7 '18 Ultraviolet spectrum (Lll ^ üll, A niax, um, log t): 220 (4.00); 200 (4.21); 289 (4.08); 290 (4, 03).

Specter in ira-rouge (KBr, cm ^): 20 3470, 2900, 2880, 1740, 1070, 1015.

Mass spectrum (m / c, 'L): 952 (5) M ++ 28; 938 (31) M ++ M; 924 (12) M +; 923 (15) M "1; .11 87 (1 (1 (10) 891 (13); 803 (30); 835 (5); 821 (11); 708 (33); 050 (bl)), 3-25

1st naked magnetic resonance spectrum (CUC 13.ppm> büMll ^ l · 9.0 (lll, s, C16-ÜU); 8.00 (11l, s, Ν 'α-1Ι); 7.5 (111, m); 7.2 (3H, m); 0.03 (1H, s, c'JH); 0.1 · (1 11, s, C1 2-ll); 5.8b (211, m, C1 4-H + C -H); 4.2 (2U, t, -CÜÜ-Cll?); 3.83 (3H, s, -üCH,); 3.65 (3H, s, -OCIU); 30 2.8 (311, s, -N-Cll3); 1 (1511, m, -C 1I3 + C1-113; butyl Cll3; I CHj isopropyl).

4 ij! -Μ- 1 lixein £ le_5; Octyl N- (deacetyl-0-4 vinblastinoyl-23) -L-leucinate * (VL-octyl) 5 By applying the general procedure on page 31, VL-octyl is obtained with a yield of 48 I.

^ _ia l-'Octy 1 10

Melting point: 14 5 0 C

[u] uCtlCJ3: 54 “u c-0.33 15 Ultraviolet spectrum (CH 011, λ max, nm, Jog ^): 8.62 mg / 1,217 (4.69J; 265 (4.14); 289 ( 4.01); 295 (3.95).

Infrared spectrum (KPr, cm ~ ^): 20 3460; 2940; 2920; 2870; 1735; 1bb5; 1610; 1500; 1455.

Naked magnetic resonance spectrum (cl (CI3, ppm, 60MHz): 1 f \ 9.6 (1H, S, C -OH); 8,? (1H, s, N '-11); 7.55; 1 Il, m); 7.23 (311, m); 6.5 (1H, s, C9-H); 6.12 U11, s, C1 2-H); 5.90 i2H, m, CM-H + C15-H); 4.2 (2H, t, -0-CH2-0ct; 1> 1, d, C1 7-11); 3.83 (311, s, -COOCHj); 3.67 (3H, s, -OCH,); 3.56 (1H, b, C5 -H); 2.83 (3H, s, N-CH); 1.33 (1011, ^ 10 'in ^ i o Γ in, solid octyl + CIi2 »Cll2); 1 (15H, in, solid octyl + CHj + 4> Ch! 8 ').

r> / -39- 1 Example_6: φ · “*“ - N- (deacetyl-0-4 vinblastinoyl-23) -D-ethyl leucinate (VDLE) _________ 5 By applying the general procedure on page 31, the VDLE is obtained with a yield of 74%.

φ

Physical and chemical properties of VDLE:

1 ü Melting point: 181 ° C

fa] n CI1C13: 70 ° L U c = 0.28

Ultraviolet spectrum (methane), amax, nm, logt): 15,220 (4.67); 226 (4.20); 288 (4, 1 1); 295 (4.02).

Specter in lia rouge (K8r, cm: r 3460; 2960; 2880; 1735; 1665; 1605.

20 Mass spectrum (m / e l): 924 (14) M + + 2 8; 910 (32); M ++ 14; 897 (38) M ++ 1; 896 (b6) M +; 863 (28); 835 (43); 709 (43); 651 (81); 570 (100).

Spectrum I magnetic resonance nucleus i re (CDC1, ρρηι, 60MHz d: 25 9.6 (1 H, s, C16-0H); 8.1b (1H, s, N 'UH); 7, b6 (1H, m ); 7.2b (31i, ni); 6.7 (11I, s, C9-Il); 6.16 (1 H, s, C1 2-H); 5.90 (2H, m, C14-H + I C15-I1); 4.26 (2Il, q, -COOCH2); 3.83 (3H, s, -OCH3); 3.66 (3H, s, vL -ÜCH,); 2.90 (3H , s, -Nu-CH,); 1.3 (3H, t, - (COOCH?) - CH.,; 0.96 A? (12H, m, CH, + C IL + isopropyl).

»I -40-! 1 (Example_7: N -> (desacetyl-0-4 vinblastinoyl-23) -L-ethyl serenate (VSE) I. 5 By applying the general procedure on page 31, the VSE is obtained with a yield of ' 35%.

.-1¾ VSIj: 10 ï „T CHC13: 05 ° J t). n v c = 0.7

Ultraviolet spectrum (Meütl, »max, um, 1 og. D: 2 15 (4, 7b D; 26b (4.31); 28B (4.20); 297 (4.15 d.

15

Specter in Ira rouge (CUC 1 ^, l iu:; 3 4 b Ü; 3400; 2965; 29 35; 2880; 1730; looh; U.U5.

S]) ectre of esuiiinia · m.igué 1 1 qne iiik lé.ilic (ClU 1 ^, ppin, OUflIL ·): 20 8.13 (1 II, s, N1 '-H); 7, "J (111, d, -011); 7.52 (111, ml; 7.20 (311, m); b, 03 (111, s, C9-11); 0.1 (1 11, s, C 11-H); 5.9 (211 , w, C N-il + C15-llj; 4.3 (211, q, -C00-CH? -J; 3.83 (311, s, -0CIU); 3.00 (311, s, -DC1L ); 2.67 (3H, sf-Nu-CH, J; 1.34 (311, t, (-COü-Cll -) iU ~); 0.95 {bll, m, CI8.1.3 + c18n3j. * Ί- * -41- N- (deacetyl-0-4 vinblastinoyl-23) -L-ethyl glutamate (VGE) 5 By applying the general procedure on page 31, the VGE is obtained with a yield of 55%.

Pbysjco-chemical_de_Ja_VGE properties: U)

Pu i ni of merger: 149 UC

QJ C ,, t: i3: 5 9 c- 2 1 S Ultraviolet spectrum (MetHl, J Οιιιμ / 1, \ max, ma, 1 ou, t.: 219 (4.40); 2o9 (3.94); 288 (3.80); 29b (3.7).

Spectrum i h 1 ~ i a - ronge (Kbr, cm; 2 U 3 4 0 0; 3395; 2900; 2920; 2870; 1 730; 1005; 1010; 1500.

Nuclear magnetic resonance spectrum (GDG 1 3, p pm, OOMlli): 9.03 (lll, s, C1 (3-0il); 8.10 (111, s, Nu'-11); 7.0 (111 , m); 7.10 (3II, ia); 0.04 (1II, s, C 9 - ί I); 0.07 (1 H, s, C1 “-11); 5.83 (211, m , 25 C 1 411 + C15li); 4.23 (211, q, -CÜüGil, -); 4.10 (211, q, -COOC11, -); 3.8 (311.5, - () CII3) ; 3.04 (311, s, -ÜCllj); 3.5 (111, s, C5 -11);, 2.8 (311, .s, -NuClU); 1.33 (311, t, -Cil -,); 1.20 (3! I, t, -Cli-.); '1 (ùll, in, C, 0ll <c10 Il3).

% "* L. Λ * -42- 1 Example_9: N- (deacetyl-0-4 vinblastinoyl-23) -L-ethyl phenyl-alaninate (VPE)

By applying the general procedure on page 31, we obtain the VPE with a yield of 66 5 -Physico-chemical of the VPE: j i φ

Melting point: 154 ° C.

! 1 ° fu] n CilC13: 78 ° U c = 1, 2

Ultraviolet spectrum (Meüll, 9.8 mg / 1, λ max, jim, 1 ug î d: 217.5 (4.67); 2b5 (4.13); 286.5 (3.99); 295.5 (3.95).

15 -1

Infrared spectrum (KBr, cm): 3560; 3460; 3400; 2860; 2830; 1730; 1650; 1610; 1500; 1455. Mass spectrum (rn / e,% j: 20 958 (17); 944 (41); 930 (35); 871 (64); 651 (41); 588 (41); 571 (53); 401 (100).

Nuclear magnetic resonance spectrum (Cl (Cl ^, ppmgjOQMIIz): 9.48 (1H, bs, ~ C.16-OH); 8.1 (1H, s, N * '-H); 7.6 (1H, d); 7.5 (1H, d); 7.3 to 7.02 (7H, m); 6.6 (1H, s, C9-H); 6.1 (1H, s, C] 2-H); 5.87 (2H, m, C14-H + C15-H) (J15‘14 = 12Hz; J14‘3 = 8.6Iiz); 4.9 (1H, m ° ^ CH-); 4.16 (2H, q, COO-CH2); 3.8 (3H, s, -OCHj); 3.56 (3H, s, -OCH3); I 3.43 (1H, s, C5-H); 2.74 (3H, s, Ν ^ -ΟΗ,); 1.20 (3H, t, -CH3 (ester)); jL 0.97 (3H, t, -C18'H3); 0.88 (3H, t, -C '8h.,).

* i ί i -43- 1 Example_10: N- (deacetyl-0-4 vinblastinoyl-23) -L-leuein-amide (VL-amide) 5 By applying the general procedure on page 31, "we obtain the VL -amide with a yield of 39%.

physical-chiaic of VL-amide

10 Melting point: 240.2 ° C

Gfc CHC13: 47.1 ° c = 0.254

Ultraviolet spectrum (CII ^ OH, xmax, nm, lodge): 15,219 (4.76); 263 (4.29); 288 (4.1 ep); 295 (4.03 ep).

-1

Infrared spectrum (KBr, cm): 3460, 3400, 2960, 2940, 2860, 1715, 1665, 1610, 1495, 1455, 1225.

20

Nuclear magnetic resonance spectrum (CDCl ^, 60MHz, ppm): 9.76 (bs, 1H, Cl6-OH); 8.33 (s, 1H, N-H); 7.4c (m, 2H); 7.16 (m, 2H); 6.5 (s, 1H, C9-H); 6.3 (s, 1H, C12-H); 5.66 (m, 2H, C14-H and C15-H); 3.76 (s, 3H, COOCH3); 3.6, 3H, -0CH3); 3.46 (s, 1H, -C5-H); 2.76 (s, 3H, N-CH3).

h ν '' -44- ♦.

1 Exemgle_11: N- (deacetyl-0-4 vinblastinoyl-23) -L ~ methyl isoleucinate (VILM) 5

By applying the general procedure on page 31, the VILM is obtained with a yield of 66%.

Physico-chemical_properties_of_the_VILM

10 [° Jn CHC13: 66.6 ° C = 0.135! Ultraviolet spectrum (MeQH, λmax, nm, log ε): I 225 (4.55); 266 (4.18); 288 (4.05); 295 (4.00).

15

Nuclear magnetic resonance spectrum (CDCl ^, 360MHz): 9.48 (bs, 1H, C16-0H); 8.03 (s, 1H, NH); 7.51 (m, 2H); 7.23- 7.06 (m, 2H); 6.58 (s, 1H, C9-H); 6.20 (s, 1H, 012-H); 5.85 (dd, 1H, C14-H; J15_14 = 12Hz; J1 4-3 = 3.6Hz); 5.78 (-d, 1H, ClS-H); 4.62 (m, 1H h ^ C ^ qur); 4.17 (d, 1H); 3.77 (s, 3H, COOCH3); 3.75 (s, 3H, C00CH,); 3.6 (s, 3H, -OMe-j; 2.73 (s, 3H, N ^ -CH.,); 91 ^ i ο io »^ 2.58 (s, 1H, c -H); 0 , 92 (m, 12H, C 'IL-C 1 0 H).

T

-45-

V *, V

1 £ xample_12 · N- (deacety1-0-4 vinblastinoyl-23) -L-ethyl tyrosinate (V-Tyr E) 5 By applying the general procedure on page 31, "we obtain V-Tyr E with a yield of 48%.

? I2Eïï§ £ ê§ J ^ h ^ sico-chemical de_la_V-Tyr E

(a] D CHClj; 64.4 ° 10 c = 0.1087

Ultraviolet spectrum (CH ^ OH ^ max, nm, loge): 227 (4.73); 266 (4.26); 288 (4.07); 296 (3.94).

15 Infrared spectrum (KBr, cm ~ ^): 3460, 3400, 3040, 2950, 2840, 1715, 1660, 1610, 1500, 1455, 1225.

Nuclear magnetic resonance spectrum (360 MHz): 9.6 (bs, 1H, Cl6-OH); 8.05 (s, 1H, NH); 7.55 (m, 2H); 7.21 - 7.06 (m, 2H); 7.03 (d, 2H, arom tyr J = 7.5); 6.7 (d, 2H, tyr arom. J = 7.5); 6.55 (s, 1H, C9-H); 6.05 (s, 1H, 012-H); 5.83 (dd, 1H, CU-H; J15 "14 = 12Hz; J14 ~ 3 = 3.6 Hz); 5.76 (d, 1H, 25 C15-H); 4.83 (m, 1H, CH); 4.13 (solid 3H, -COOCH2.0Ί7-H); 3.76 (s, 3H, -0CH3-); 3.6 (s, 3H, -COOCiy; 3.45 (s, 1H, C5-H); 2.71 (s, 3H, -N ^ -CHj); 1.21 (t, 3H, -CHj- (CH? OOCjJ; 0.88 (m, 6H, -C18H_-C18'hJ .

f- 4! -46-

• AT

1 Example_13: N- (deacetyl-0-4 vinblastinoyl-23) -L-ethyl tryptophanate (V-Trypt E) 5

By applying the general procedure on page 31 we obtain the V-Trypt E with a yield of 72 I.

_de_l a _V-T rypt JE

Γ Ί CHC1X

10 LaJD ύ: 89.8 0 c = 0.51119

Ultraviolet spectrum (MeOH, λ max, nm, log ε): —J—— —----- - - '- -.-- K, “* · 225 (5,15); 267 (4.65); 280 (ep); 290 (4.55).

15

Mass spectrum (ra / e%): 970, 391, 279, 165, 108, 35;

Infrared spectrum (KBr, cm ~ ^): 20 3460, 3400, 3040, 2960, 2940, 2880, 1725, 1660, 1610, 1500, 1455, 1225, 740.

Nuclear magnetic resonance spectrum (CDC1 ^, 360MHz): 9.5 (1H, s, C16-OH); 8.2 (1H, s, NH, trypt); 8.05 (1H, s, NH); 7.66 (1H, d, trypt; J = 7.2Hz); 7.58 (1H, d, trypt; J = 7.2Hz); 7.51 (1H, d, trypt; J = 7.2Hz); 7.31 (1H, d, trypt; J = 7.2Hz); 7.25-7.04 (5H, m); 6.58 (1H, s, C9-H); 6.06 (1H, s, C12-H); 5.83 (1H, dd, C -H; j = i2Hz; J = 3.6Hz); 5.78 (1H, d, C15-H; J = 12Hz); 4.95 (1H, qf> CH); 3.75 (3H, s, -COOCHj); 3.6 (3H, s, 30 -OCH3); 3.4 (1H, s, C5-H); 2.77 (1H, s, N-CH3); 1.15 (311, t, -cooch2 (CH3)). ^ 4 '-47- 1 Example £ le_14: N- (deacetyl-ü-4 vinblastinoyl-23) -L-methyl tryptophanate (V-Trypt M) 5 By applying the general procedure on page 31, We obtain the V-Trypt M with a yield of 41%.

Physico-chemical properties_of_the_V-Trypt_M: 10 t “] CHC13: 93.9 ° U c = 1.82

Ultraviolet spectrum (MeQH, ima:?;, Nm, cell): 269 (4.48); 280; 289 (4.32).

15 -1

Infrared spectrum (KBr, cm): 1730, 1710, 1660, 1610, 1495, 1455, 1220, 740.

Nuclear magnetic resonance spectrum (360 MHz):

9.5 (1H, bs, C16-OH); 8.06 (1H, s, NH ind); 8.01 (\ -, s, NH

ind); 7.65 (1H, d); 7.58 (1H, d); 7.38-7.06 (7H, r; 6.41 (1H, s, C9-H); 6.05 (1H, s, C12-H); 5.85 (1H, cc, C14-H; J = 12Hz; J '= 3.6Hz); 5.78 (1H, d, C15-H; J = 12Hz); 4.-5 (1H, q, C); 4.2 (1H, m, C17 -H); 3.75 (3H, s, -OCH3); 3, fe (3H, s, 25 -COOCH3); 3.58 (3H, s, -C00CH3); 3.43 (2H, s, CS - H. 2.6 f3H, s, N-CH3).

* 4 -48- t j 1 Example_15:! 'N- (deacetyl-0-4 vinblastinoyl-23) -L-valinate ethyl (VVE) 5

By applying the general procedure on page 31, the VVE is obtained with a yield of 63%.

Physico-chemical properties of VVE

10

Ultraviolet spectrum (MeOH ^ max, nm, loge): 226 (4.95); 267 (4.57); 285 (4.45); 296 (4.40).

Mass spectrum (m / e I): 910 (0.1); 896 (2); 882 (5); 823 (4.5); 822 (6); 708 (5.3); 1,653 (9.2); 651 (8.7); 650 (14.6); 572 (8.7); 571 (23.3); 539 (10.7); 355 (16); 354 (10); 353 (31); 294 (17); 188 (8.5); 156 (100); 155 (11.8); 154 (11.3); 144 (12.2); 141 (12.1); 20,140 (16.4); 136 (13); 135 (20.4); 124 (61); 122 (35.5); 121 (15.3).

Infrared spectrum (KBr, cm ^): 3540, 3470, 3420, 2960, 2930, 2870, 1730, 1720, 1670, 25 1610, 1500, 1455, 1225, 745.

Nuclear magnetic resonance spectrum: 9.48 (1H, bs, C16-OH); 8.03 (1H, bs, NH ind); 7.55 (1H, d; J = 7.2Hz); 7.51 (1H, d, J = 7.2Hz); 7.51 (1H, d); 7.23 to 7.06 30 (3H, m); 6.58 (1H, s, C9-H); 6.06 (1H, s, C12-H); 5.85 (1H, dd, Cl4-H; J14 ”15 = 12Hz; J14'3 = 3.6Hz); 5.78 (1H, d, C15-H; J = 12Hz); 4.56 (1H, dd, C); 4.21 (2H, q, COOCH2); 4.15 (1H, d, C17-H); 3.96 (1H, t); 3.76 (3H, s, OCH3); 3.6 (3H, s,; C00CH3); 3'46 OH, s, C5-H); 2.73 (3H, s, N-CH ^); 1.31 (6H, 35,; m, C00CH7CHL); 0.96 (14H, m); 0.9 (14H, tj.

ί l · 4 'k ί -49- j 1 Example 16: N- (deacetyl-0-4 ethyl vinblastinoyl-23j-L-iso-leucinate (VILE) 5 By applying the general procedure on page 31, - the VILE is obtained with a yield of 58 I.

Physico-chemical properties_of_the_VILE

10 Ultraviolet spectrum (MeOH, Xmax, nm, lodge): 226 (4.94);, 266 (4.56); 285 (4.44); 295 (4.38).

Mass spectrum (m / e%): 15,924 (7); 910 (11); 897 (23); 896 (44.4); 867 (11.3); 837 (18.7); 836 (26.6); 822 (4); 709 (10); 650 (21); 571 (13); 570 (34.7); 366 (21.7); 154 (100); 126 (11).

Infrared spectrum (KBr, cm ^): 20 3460, 3440, 3040, 2960, 2940, 2880, 1730 1665, 1610, 1500, 1455, 1225, 745.

Nuclear magnetic resonance spectrum (CPC13.360MHz): 9.46 (1H, bs, Cl6-OH); 8.03 (1H, bs, NH); 7.55 (1H, d; J = 7.2Hz); 7.51 (1H, d); 7.23 to 7.06 (3H, m); 6.58 (1H, s, C9-H); 6.06 (1H, s, C12-H); 5.85 (1H, dd, C14-H; J = 12Hz; J 1 = 3.6Hz); 5.78 (1H, d, C1S-H; J = 12Hz); 4.61 (1H, q (dd),; 4.21 (2H, q, COO-CH2-); 4.15 (1H, d, C17-H); 3.96 (1H, t); 3, 76 (3H, s, OCH3 ester); 3.6 (3H, s, OCH3); 3.46 (1H, s, C5-H); 2.73 (3H, s, N-CH3); 1, 25 (3H, t, COOCH2CH3).

1 Λ * * * - -50- 1 Example £ le_17: N- (deacetyl-0-4 vinblastinoyl-23) -D-ethyl tryptophanate (VD Trypt E) 5. By applying the general procedure on page 31, we obtain the DV Trypt E with a yield of 69%.

_Les_VD_Try] 3t_E physiological co-chemicals

10 [“3d CDC13: 69.8 ° c-0.3295

Ultraviolet spectrum (MeOH, At max, nm, compartment): 225 (4.77); 269 (4.30); 290 (4.20); 320 (ep).

15 Mass spectrum (m / e%): 970, 391, 279, 165, 108, 35.

Infrared spectrum (KBr, cnT ^): 3460, 3400, 3050, 2960, 2940, 2870, 1735, 1665, 1610, 20 1495, 1455, 1210, 740.

Nuclear magnetic resonance spectrum: 9.53 (1H, bs, Cl6-OH); 8.1 (1H, s, NH ind tryp); 8.02 (1H, s, NH ind); 7.76 (1H, d); 7.65 (1H, d); 7.51 (1H, d); 7.35 (1H,

D); 7.2 to 7.05 (6H, m); 6.53 (1H, s, C9H); 5.98 (1H, dd, C14H

J1 4-1 5 = -] 2Hz, J * l4-3_ 3> 6 Hz d. 5> 73 (1H, d, C15-H; J = 12Hz); 4.86 (1H, q, C-); 4.1 (SH.mjC17-H-COOCH2); 3.75 (3H, s, COOCH,); 3.6 (3H, s, -OCHj); 3.33 (2H, s, C5-H); 2.43 (3H, s, / N-CH3); 1.16 (3H, t, -COOCH2-CH3); 0.93 (8H, m).

ij Λ -51- 1 Example 18: N- (deacetyl-0-4 vinblastinoyl-23) -L-valinyl-L-ethyl tryptophanate (V-Val-Trypt-E) 5 By applying the general procedure of page 31, we obtain the V-Val-Trypt E with a yield of 40%.

? Γ2ΕϊϊΙΐ§5_physico-chimiques_de_la V-yal-Trypt-E

1Q a D CHC13: 36.2 0 c = 0.3589

Ultraviolet spectrum (methano3, A max, nm, cell): 270 (4.42); 290; 312 (4.96).

15 Infrared spectrum (KBr, cm ^): 3480, 3400, 2960, 2940, 2870, 1735, 1725, 1660, 1610, 1500, 1455, 1230.

Nuclear magnetic resonance spectrum (CüCl3,360MHz):

9.5 (1H, bs, C16-OH); 8.18 (1H, s, NH ind); 8.01 (1H, s, NH

ind); 7.6 to 7.06 (10H, m arom); 6.41 (1H, s, C ^ -H); 5.95 (1H, s, C12-H); 5.85 (1H, dd, C14-H; J15-14 = 12Hz; J8'14 = 3.6Hz) 5.75 (1H, d, C15-H; J15_14 = 12Hz); 5.02 (1H, m, C); 4.9 (1H, m, £); 3.73 (3H, s, COOCH3); 3.56 (3H, s, -OCHj); 3.43 (2H, s, C5-H); 2.61 (3H, s, N-CH3); 1.18 (3H, t, -COOCH2-CH3); 1.05-0.86 (+ 16H, m, CH3).

L

if -52-} 1 Exemgle_l_9: N- (deacetyl-0-4 vinblastinoyl-23) -L-tryptophan (V-Trypt Ac) 5

By applying the general procedure on page 31, we obtain the V-Trypt Ac with a yield of 67

Ghysico-chemical_ properties_of_V_ Trypt_Ac ^ ^ Ultraviolet spectrum (MeOH, A max, nm, cell): 270 (4.29); 289 (4.18); 321 (thick)

Infrared spectrum (KBr, cm 1): 15 3400, 2860, 2820, 2760, 1720, 1650, 1605, 1590, 1500, 1455, 1225, 780.

Λ • χ

Claims (12)

1. As new compounds, the Vinblastine derivatives corresponding to the following general formula: CH3 ° 2C Jf! <MJ XCŒ- CH3 CILj Vf> ÜK2 “u 'cR, in which Rj represents an amino acid or peptide ester, optionally protected, coupled to the vinblas-tinoyl-23 fragment by an amide link, the peptide comprising from 1 to 6 different or identical amino acids, and the ester group, branched or not, having from 1 to 8 carbon atoms and, R2 represents a hydrogen atom or an alkyl-carbonyl group comprising from 2 to 9 carbon atoms , as well as its addition salts with acids | minerals or organic; J r I. ....... “i $ i 4’ -54- 2. New compounds of general formula (; H3Ü2C ff "* I i (HJL J XDŒ- 0c" 3> üK2 "V- (NH-ÇH-Ç-fcOR υ R3 0 in which represents a hydrogen atom or a group alkylcarbonyl having from 1 to 9 carbon atoms; represents each fols independently, a hydrogen atom, a branched or unbranched alkyl group having from 1 to 8 carbon atoms, a carboxy-, hydroxy-, amino-, amido- group , guanadino-, thiol-alkyl having from 1 to 8 carbon atoms, methyl cysteinyl, methyl thioethyl, benzyl, hydroxybenzyl, or R ^, together with the carbon to which it is attached and the nitrogen of the amide group forms an azole ring; n varies from 1 to 6 and OR ^ is a group> benzyloxy, alkoxy, branched or not, containing from 1 ί to 8 carbon atoms, as well as its addition salts with mineral or organic acids. 55- » 3. Compounds according to Claims 1 and 2, characterized in that it is ethyl- or methyl N- (deacetyl-0-4 vinblasti-noyl-23) -L-tryptophanate and their addition salts with pharmaceutically acceptable acids. 4. Compounds according to Claims 1 and 2, characterized in that it is ethyl- or methyl-N- (deacety1-0-4 vinblasti-noyl-23) -leucinate and their addition salts with pharmaceutically acceptable acids. 5. Compounds according to Claims 1 and 2, characterized in that it is ethyl N- (deacetyl-0-4 vinblasti- 'noyl-23) -L-valyl-L-tryptophanate and the salts thereof. addition with pharmaceutically acceptable acids. b. Compounds according to Claims 1 to 5, characterized in that they are in the form of their 1: 1 addition salts with sulfuric acid. 7. Ethyl N- (deacetyl-0-4 vinblastinoyl-23) -L-tryptophanate sulfate. 1 Compound according to claim 1 characterized in that it is chosen from the group consisting of the following vinblastine derivatives, optionally in the form of an addition salt with an acid - mineral or organic: - N- (deacetyl -0-4 vinblastinoyl-23) -L-ethyl leucinate - N- (deacetyl-0-4 vinblastinoyl-23) -L-Methyl Jeucinate fj - N- (deacetyl-0-4 vinblast inoyl-23) - L-leucinate \ J / n-butyl Ί 4 '-5b- j - N- (deacetyl-0-4 vinblastinoyl-23j-L-leucinate! Octyl! - N- (deacetyl-0-4 vinblastinoyl-23 ) -L-leucin-amide I- N- (deacety1-0-4 vinblastinoyl-23) -E-ethyl leucinate - N- (deacetyl-0-4 vinblastinoyl-23) -L-ethyl glutamate - N - (deacetyl-0-4 vinblastinoyl-23) -L-ethyl serinate o - N- (deacetyl-0-4 vinblastinoyl-23) -L-ethyl tryptophanate - N- (deacetyl-0-4 vinblastinoyl -23) -Methyl tryptophanate - N- (deacety1-0-4 vinblastinoyl-23) -L-tryptophan - N- (deacety1-0-4 vinblastinoy1-23) -D-ethyl tryptophanate i - N- (deacetyl-0-4 vinblastinoyl-23) -L-ethyl alaninate - N- (deacety1-0-4 vinblastinoyl-23) -L-phenyl ethyl alaninate - N- (deacetyl-û-4 vinblastinoyl-23j-L-isoleucinate) ethyl - N- (deacety1-0-4 vinb1astinoy1 -23j-L-isol methyl eueinate - N- (deacetyy1-0-4 vinblastinoyl-23J-ethyl ethyl valinate - N- (deacety1-0-4 vinblastinoy1 - 23) -L-ethyl tyrosinate v - N- (deacety 1-0-4 vinblastinoyl-23) - £ -trif 1 uoro-; ethyl acetyl-L-lysinate - N- (deacetyl-0-4 vinblastinoyl-23) -L-valinyl-L-tryptophanate ethyl jt-. '\ ". V * Λ ‘-57- 9. Pharmaceutical composition used in human and veterinary medicine for the treatment of neoplastic diseases containing one or more derivatives defined in any one of claims 1 to 8, 10. Use of a pharmaceutical composition according to * claim 9 characterized in that it is j administered to a patient at a dose between 0.05 mg / kg / week and about 5 mg / kg / week. 11. Pharmaceutical composition according to claim 9 characterized in that the active principle is N- (deacetyl-0-4 vinblastinoyl-23) -L-tryptophanate ethyl, optionally in the form of an addition salt with a pharmaceutically acceptable acid. 12. Pharmaceutical composition according to claim 9 characterized in that the active principle is N- (deacetyl-0-4 vinblast inoyl-23) -L-valyl-L-tryptophanate ethyl, optionally in the form of a salt addition with a pharmaceutically acceptable acid. 13. Pharmaceutical composition according to claim 9 characterized in that the active principle is in a pharmaceutically acceptable diluent. 14. Pharmaceutical composition according to claim 13 1 characterized in that the diluent is a sterile buffered aqueous solution. 1 Use of a pharmaceutical composition according to claim 9 for the treatment of leukemias, solid tumors and Hodgkin's disease.