LU82514A1 - N- (VINBLASTINOYL-23) DERIVATIVES OF AMINO ACIDS, THEIR PREPARATION AND THEIR THERAPEUTIC APPLICATION - Google Patents

Description

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PATENT

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\ The company called: OMNIUM CHEMIQUE SOCIETE ANONYME

| t in Louvain-la-Neuve (Belgium) i ---- '' --— - - - - - —— - _ - - -. - --— “---“ "j f 1 1 N- (VINBLASTINOYL-23) DERIVATIVES OF AMINO ACIDS,

THEIR PREPARATION AND THERAPEUTIC APPLICATION

The present invention relates to novel bis-indole alca-5 loids. More particularly, the invention relates to new coupling derivatives; 1 i of amino acids with Vinblastine, their preparation process, their use as anti-tumor agents and pharmaceutical compositions | 10 container.

Ü These new Vinblastine derivatives can be :: ’j. used to treat leukemia and | malignant tumors in humans.

; î [; i i s

Bisindolic alkaloids of the Vinblastine type are well known compounds having the skeleton j | carbon of the following general formula: \ t -% i il

. -I

- 2 - • CH302C (I) 5!

: LiXX

, 6 CH3ik% ^ EHjyf \ .R2 JH0 # \ 23 ^ * 0 R, 15

Examples include vincaleukoblastine (US patent 3,097,137), 1 eurocristine or vincristine and leurosidine (US patent 3,205,220), vinglycinate (Belgian patent 659.1 l2) and vindesine (Belgian patent 813.IoSj.

The latter compound was obtained by chemical transformation of natural Vinblastine (I, R ^ = 0CHj, R? = COCH_j, which itself is obtained by extraction from the leaves of Cutharunthus roseus.

-i Vinblastine, vincristine and vindésine are marketed for their use in human therapy, more particularly for the treatment of leukemias; and some solid tumors.

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

P - 3 -! i; i-j he j f h t i ί j! : i \ s 1 Their mechanism of action is analogous to that which was ji postulated for the antimitotic action of colchicine.

1 / _ These drugs would act by inhibiting the polymerization of tubulin into microtubules and subsequent arrest of cell division into metaphase.

i i; ; The use of 1: 1 complexes of bisindolic / anti-tumor alkaloids with tubulin has been described in patent b e 1 g e 8 5 4.0 5 3.

In some cases, this may result in less toxicity; and a more important chemotherapeutic activity than those! corresponding free alkaloids.

h ·! Other derivatives obtained by chemical modification of!! 15 Vinblastine have been tested. Thus vinglycinate sulfate j (I, sulfate, R ^ -üCH ,, R7-CÛCH7N (Cli ^) ^ (Cancer Research 1yo7, 27, 221 -227) has been studied in clinical experimentation but has not generally been revealed superior to Vinblastine i and vincristine.

2ü

Belgian patent 813,108 describes the indes me (1, R. ^ NH- ,, R0 = Hj "! I- and other C-3 carboxamide derivatives of Vinblastine.

_ Subsequent publications have confirmed the therapeutic value of vindesine or carboxamide-3 Jeacetyl-G-4 at 25 Vinblastine, but this compound has nonetheless proved to be inactive for the treatment of L 1210 tumors transplanted in mice (CJ Barnett et al., J. Med. Chem-, 2JL "88, J978).

! w ‘i ^ - i! f, - 4 - a j I 1 Amino acid coupling derivatives with Vinblastine and other analogous bisindolic alkaloids were generally proposed in the Belgian patent 813,168 mentioned above.

* 5 Nevertheless, no example of derivative of specific amino acid j was not disclosed and consequently, no physico-chemical data was provided. No specific preparation method and no particular pharmacological result was mentioned.

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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 1 and following, it is further mentioned 1S 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" .

2ü 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.

; 2¾ generally show no activity on leukemia i L 1210, the results obtained show for the new ί derivatives a very significant and unexpected activity.

They also have other important and unexpected advantages over Vinblastine and its known analogs, particularly vindesine.

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- 5 - i: ί 1 The new compounds of the invention respond more • 1

particularly to general formula II

I '/ ~~ \! 5) C02ch3î (II) i 1 i io; I i rn 15 ch3o or2 p CH3 üH '· £ —-OK4 20 in which ;; R T represents a hydrogen atom or an acyl group, preferably an acetyl group; if Rj is a hydrogen atom, an alkyl, hydroxy-carboxy-, amido-, no-friend, guanadino-, thio-C 1-CS, 25 cysteinyl methyl, methyl-thio ethyl, benzyl, hydroxy chain benzyl; . ^ 1 ï f] | [j “N - f 2

\ 30 kAJJ or 1J

H

Λ.

£: - 6 - 1 or represents, with the carbon atom to which it is attached and the nitrogen of the amide group, an azole ring; and R4 represents an alkyl group, linear or branched § having from 1 to 8 carbon atoms or a benzyl group.

4 More particularly, the structural fragment of formula II

R3 5 - NH - CH - COR4 10 represents an ester of any of the natural amino acids and their optical isomers of configuration D, in particular glycine aspartic acid cystine 15 alanine glutamic acid methionine va line asparagine phenylalanine leucinc glutamine tyrosine isoleucine arginine tryptophan serine lysine proline or threonine cysteine histidine

The preferred compounds of the invention correspond to formula II in which K- represents a hydrogen atom and the amino acid part is derived from one of the following amino acids: • alanine, val inc, leueine, isoleucine or phenylalanine -

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; j 3 -Τ Ι ί Λ (4; Ί ΐ | 1 Regarding the pharmacological properties, it is | expected that slight changes in the

It amino acid structure will provide it with compounds of comparable activity.

\ i | 5 In particular, the presence of a non-α-amino acid! natural (for example an N-monosubstituted α-amino acid | or an amino acid, a -dialkyle) or a β-amino acid || would provide compounds of similar activity against one; * »I number of tumors. Such compounds are Ί 4- 10 therefore within the scope of the present invention.

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

;! According to one of these methods, these derivatives are obtained at • * i * | Starting from Vinblastine by hydrazinolysis, nitrosation; then coupling with the appropriate amino acid ester.

‘Il w i These compounds can also be advantageously obtained by saponification of vinblastine, followed by coupling of vinblastinoic acid, optionally previously protected by acetylation, with the amino acid ester.

i i j * When the amino acid chosen contains functional groups h 25 in the side chain R-, it can be! · "* Such as lysine or cysteine, necessary to protect * these functional groups using the methods!, Well known to those skilled in the art.

• 3 v - 8 - »1 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.

5 According to the first process mentioned, the compounds according to the invention can be obtained from Vinblastine, in a conventional manner, by hydrazinolysis followed by nitrosation 10 then coupling with the amino acid or polypeptide ester according to reaction scheme I .

..T1! HsNO. h-T ^ | acid ester S a! · 1 U, J j _L ·, V "-1 I amine” Τ ’” * Sc * HO COOCU HO C-HH-NH- Hü C-N, J U L "3

O O

20 ^ W> H

c - HO ÇVN-Ç-CO O R ^ Schema I.

'. G H

25 The first step is to add iivdï az ine annvdrc to a solution Vinblastine base in methane! anhydrous. The reaction mixture is then heated under an inert atmosphere for 12 to 30 hours at a temperature between 30 and 70 ° C. Preferably, the temperature will be maintained at around 6 ° C., the reaction time then being 24 hours.

d - 9 - »j. 1 The deacetyl-4-vinblastine hydrazide is then • isolated by adding water, extracting with dichloromethane and concentrating. The compound can be further purified by preparative chromatography 5 (neutral silica).

During the second step, the hydrazide function of the modified Vinblastine is transformed into acid azide.

; 1 This transformation is carried out in a known manner; 10 by addition of 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 ester | of an appropriate amino acid or, where appropriate, the corresponding protected derivative, in solution in methylene chloride.

| The reaction medium is maintained between -3 and + 5 ° C.

j for about 15 hours. The reaction is easily followed by thin layer chromatography. After concentration, the compound of invention II is isolated, which can be transformed into sulphate or other mineral or organic acid salt by crystallization at; from a methanolic solution of the corresponding j j = acid.

30

The compounds of the invention are purified using 1 the well known techniques of chromatography and recrystallization.

! -ΙΟ Ι j 1 If necessary, the deacetyl derivative 0-4 Vinblastine j thus obtained can be reacetylated either directly to give the derivative of Vinblastine II in which R ^ COCHj (J. Med. Chem. 2_2, 391, 1979 ) or by preliminary formation of the diacetoxy-3,4 derivative followed by! selective hydrolysis of the acetoxy group in position 3.

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

The compounds of the invention have in particular particularly advantageous antitumor properties which are capable of being used in human therapy.

15

These amino acid derivatives can be, in particular, 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 which may benefit from treatment with Vinblastine, vincristine and vindesine.

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

Various results obtained during the pharmacological experiment carried out with the ethyl leucinate derivative (I, R1 = NHCH [CH? CH, r <2 = H; VLEj, methyl leucinate (VLM), D-leucinate ethyl (VDLE), ethyl L-phenylalaninate (VPE) and ethyl L-glutamate (fVGE) are collated in 35 · Tables I and II, respectively with respect to tumors; P 388 and L 1210.

i t i: j -11- j! 1 For the treatment of malignant tumors in animals, the chemotherapeutic activities were tested using the sulfate salts of the compounds of the invention. They were compared with the corresponding sulfates of vindesin.

The superiority of the compounds according to the invention is thus demonstrated.

*! DBA2 mice were inoculated intra-! 10 venous with 10 ^ leukemia cells from a | 7-day leukemic ascites P 388 or L 1210, following the one-injection regimen (day 1) or the three-injection regimen f (days 1,2 and 3). Day 0 is the t day of tumor cell inoculation.

15

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

; 5 The corresponding doses for Vinblastine and! o r »I vinuesine are respectively 24 mg / kg and about | 11 mg / kg. We noted in particular unlike! in the case of Vinblastine and deacetyl Vinblastine, the absence of hepatic toxicity at doses of 20 to 4ü i j mg / kg.

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! i j i s! f (i I TABLE IP 388 Number of animals: 10 I ----- | Product Dose MST ILS Scheme VLE 20 16 52.4 1 VLE 22 20 90.5 1 VLE 24 16 52.4 1 VLE 24 20 75 , 4 1 VLE 26 '1 7.5 53.5 1 VLE 28 19.6 71.9 1 VLE 30 21 84.2 1 f VLM 10 16.5 35 1 VLM 10 16.3 40.5 1 VLM 12, 5 37 203.3 1 VLM 12.5 16.3 40.5 1 VLM 15 18 55.2 1 i VDLE | 6 14.7 26.7 1 VDLE | 8 I 17 46.5 1 VDLE j 10! 33 170 , 5 1 VDLE I 12.5! 32 162 1: II! VPE j 10 s 13 12.1 I 1 VPE j 20 j 14.4 24.1 j 1: VPE; 40! I> 505! 1! VPE! 50; |> 223.8 j 1 VPE | 60; I7132.7 S 1 f! Il

! ! : I

VGE 10! 11.5 d -0.9 d 1 '! VGE 20! 12.8! 10 | 1! i: VGE 40! 15.6 34.5 1 î i

, I

: VDS 6 d 16 1 39.1 1: VDS | 2 | 13.7 | 19.1! 1,2,5! i I! i i

\ V

! f} * t: * ”-13-! VLE 5 14.5 25 1,2.3; / ELV 6 15 29.3 1, 2.3 | VLE 7 14.2 24.6 1.2.3 VDS: Vindesine or deacetyl-0-4 Vinblastine carboxamide-3.

4 | * 10 cells are injected i.v. into female mice DBA2 · MST: "Medium Survival Time", day when half of the animals died.

j% ILS: "Percentage of Increased Life Span", percentage increase in survival.

i TABLE II L 1210 Number of animals: 10 r

Product Dose MST ILS Scheme | it ----------------------- 1 ---------- p -------------- ----- VLE 20 10.6 26.2 1 r 'VLE 22 11 30.9 1 VLE 24 11.6 38.1 1 day VPE 60 14 66.7 1 day VDS 6 9.0 14 1! i VLE 6 9.2 9.5 1.2.3} __λ ____ î 4 i l * 10 L 1210 cells are injected i.v. to i

DBA0 female mice. I

ù {ï MST: "Medium Survival Time", day when half of the [animals died. ! % I LS: "Percentage of Increase Life Span", percentage of increase in survival.

i ^ i! > 14- “1 For their therapeutic application, the compounds of the invention, optionally in lyophilized form,! are preferably administered parenterally, dissolved in a pharmaceutically acceptable solvent, | 5 in the form of a base or of a pharmaceutically acceptable acid addition salt.

| Physiological water or other saline solutions buffered, for example with phosphate, are suitable solvents. Generally speaking, these; , 10 compounds can be used drawing inspiration from techniques and limitations which are known for therapeutic treatments with other alkaloids of the Vinca class.

! 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 treatment regimen ί adopted. Total weekly doses will generally range from 0.05 mg to about 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-uracil, mercapto-6 purine, thio-6; guanine, the arabinosides of cytosine and the antibiotics such as actinomycin b, daunorubiein and adriamycin.

| In particular, these new derivatives of 1 a Vinblastine j 30 can be used in combination with one another.

The following examples do not illustrate | limiting the process leading to the compounds of | The invention.

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-15-% 1 Exemgle_l: j Obtaining the 0-4 deacetyl Vinblastine hydrazide: (I, R.j = NHNH2, R2 = H) i i: 1. ‘] 5 To a solution of 14 ml of anhydrous hydrazine and ü 14 ml of anhydrous ethanol, 1.12 g of sulphate is added

*. _ X

i; Vinblastine (1.23.10 M). The reaction mixture thus formed is kept for 18 hours at 60 ° C.

28 ml of water saturated with sodium chloride are then added and the mixture is extracted with methylene chlorue.

After drying (MgSO 4) and evaporation of the solvent i under vacuum, 930 mg (98%) of the hydrazide are recovered! unitache in thin layer chromatography.

15 Example_2:

Synthesis of 0-4 deacetyl N (ethyl L-leucinate)

Vinblastine (VLE, = ethyl leucinate, R -> = H) -4 To a solution of 275 mg (3.58 10 M) of 0-4 deacetyl 20 Vinblastine hydrazide in 7 ml of anhydrous methanol and 20.5 ml of 1N HCl, cooled beforehand at 58 ° 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 5% sodium bicarbonate solution.

The azide formed is extracted with methylene chloride.

The organic phase is dried over MgSO 4 and filtered.

Then added 68.4 mg of ethyl leucinate -4 (4.29.10 M) and concentrated in vacuo, until a solution of about 4 ml is obtained.

i 'i -16- 1 This solution is then left for 24 hours in the refrigerator.

The reaction being then terminated, 50 ml of methylene chloride are added and washed several times with the same volume of demineralized water and then once with a saturated NaCl solution.

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

300 mg of crude product are thus collected to which 0.035 g of solution in 1 ml of anhydrous inethanol is added.

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 practically pure VLE sulfate free from ethyl leucinate.

After release of the bases, the latter can be chromatographed on silica gel (10 g of Si07) and eluted with 50 cc of ether / MeOB-NH 4 sat. f92% / 8%) then 250 ml of ether / MeOH-NH., (851 / 15¾).

The ELV is obtained pure at the head of the second eluate.

49% of purified ELV is thus collected, ie 152 mg of base.

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1 1 Physico-chemical properties of ELV

he

; | Melting point: 169.4 ° C

| Γ f -i CHC1 i S D “J n · 60? 7 ° i! L c = 0.348 ‘or» - /! I; * i-ï î Ultraviolet spectrum (MeOH, Amax, nm, log ε): Ί l »221 (4.62); 267 (4.15); 287 (4.02); 295 (3.99).

ij 10., S Infrared spectrum (KBr, cm):

J

1 3470, 2960, 2880, 1735, 1665, 1610.

: ®i rl

Mass spectrum (m / e,%):, j 1 15 924 (6) M + + 2 8; 910 (56) M ++ 14; 897 (62); 896 (1Ü Ü); 8.05 f 2 5; ; 938 (68); 772 (19); 709 (25); 651 (43); 571 (69).

; j j Nuclear magnetic resonance spectrum (h \ cDC1 ^, ppm, SoiΊΕζ): I 9.66 (, H, .rg, C16-0H); 8.20 (1H, s, N, uH); 7.52 (1H, d); ", 15 3: .mj; 20 6.56 (1H, s, C ~ -H); 6.05 (ΊΗ, s, 01 2-H j; 5.86 (1H, s, Cl4-H, J 1 - - * = || 9.4; J 14-5 = 2); 5.78 (1H, d, C15-H); 4.69 (1H, m, CH (NHR) CO-); jl 4 , 2 (2H, q, OCH2 CH5); 4.18 (1H, t, C1 '-H); 3.77 (3H, s, 0CH, j; j | 3.66 (3H, s, OCH, ); 3.4 7 (1H, s, N-CH- j; 2.7 7 {^ li, s J-CH7); 0.92 (12H, m, -C H-, + isopropyl J.

u J) i! * = I! ; î ’f! -18- 1 Example_3: 0-4 desacetyl N (methyl L-leucinate) Vinblastine (VLM)

By applying a procedure identical to that of Example 2, the VLM is obtained with a yield of 46¾.

Chemical_physical_properties of_VLM

Melting point: 172.3 ° C

10 r T CHC1,, <η 01} 3: 67.2 υ c = 0.275

Ultraviolet spectrum (CH ^ QH, λ max, nm, log e): 220 (4.61); 267 (4.15); 287 (4.03); 294 (3.98).

15 _ 1

Infrared spectrum (KBr, cm): 3475; 2960; 2880; 1740; 1680; 1615.

Mass spectrum (m / e,%): 20,910 (25) M + + 2 8; 896 (78d M ++ 14; 883 (26d M% 1; 8 8 2 {3 61 M +; 850 (29); 836 (41); 822 (1 00); 708 (15); OS 1 (56d; 650 (78); 570 (70).

Nuclear magnetic resonance spectrum fCDC1 ^, ppm, 60 MHzi: 9.21 (1Η, 5.01Ο-0Η); 8.1 (111, s, N -H!; 7.53 ΠΗ, πΐ); 7.23 (5H, m); Q i ° U i ς 6.63 (1H, s, C -H); 6.13 (1H, s, C -!)); 5.8t. 12H, m, C -H + C -H); 3.83 (3H, s, -OCH-); 3.80 (3H, s, -COÛCH-j; 3, tô i5H, s, -OCH-j;. 3 ίο ^ · | ς t 2, S (3H, s, -CH-j; 0.9t> 11 2H, n :, C H_ + C '“+ i scpropvl}«, f I: -19-. Ι iiii ** i

J

i 1 Exemgle_4: i N (butyl L-leucinate) 0-4 deacetyl Vinblastine | (VL but.E) 5 By applying a procedure identical to that of i ^ example 2, we obtain the VL but.E with a yield of] 58¾.

!

; Physico-chemical properties of_l a_VL_but JB

| ., 10

j Melting point: 158 ° C

! CHC1 r.

[“P c = 0.2622: 74> 46 ° 15 Ultraviolet spectrum (QL OH,> max, nm, log aj: 220 (4.66d; 266 (4.21); 289 (4.08d; 296 (4 , 05J.

Infrared spectrum (KBr, cm: 20 3470, 2960, 2880, 1740, 1670, 1615.

; Mass spectrum (m / e, ΐ,): 952 (5) M ++ 28; 958 (31) M ++ 14; 924 (12) M +; 925 (15) M + -1; 1,891 (13); 863 (36); 835 (5); 821 (11); 708 (35); 650 (81); 570 (100).

25

Bare magnetic resonance spectrum! ÉaireiCDC1 ^, ppm, 60MHzj: 1 16 1 9.6 (1H, s, C -OH); 8.06 (1H, s, N “-H); 7.5 (1H, m); 7.2 (5H, m ,; 9 1 -> '14 15 6.63 (1H, s, C -H); 6.1 (1H, s, C ^ -H 1; 5.86 (2H, ir ., C -H + C -H 1; i 4.2 (211, t, -CÛO-CH-, j; 5.85 (5H, s, -QCH ~ j; 5.63 ί 5H, s, -OCH -j; 2.8 (3H, s, -N-CIL); 1 (15H, m, -C1 5 'il- + C18 H ^; CH- butyl; s CHj isopropyl).

i -20- 1 §îë5} EΧ_§ N (ethyl L-leucinate) û-4 desacetyl vinblastine-carboxamide-3 (VL Oct.E) 5 By applying a procedure identical to that of Example 2 , we obtain the VL Oct-E with a yield of 39 l.

Physical_chemical_properties_of_the_VL_Oct.E 1 c »

Melting point: 145 ° C

Γα J n 3: 5 4.2 0 U c = 0.3319 15 Ultraviolet spectrum (CH-OH, at max, nm, log ε): 8.62 mg / 1,217 (4.69); 265 (4, 14); 289 (4.01); 295 (5.95).

Infrared spectrum (KBr, cm ~ *): 20 3460; 2940; 2920; 2870; 1755; 1665; 1610; 1500; 1455.

Nuclear magnetic resonance spectrum (CDC1 -, ppm, 60 MIiz d: 9.6 (1H, s, C16-OH); 8.1 (1H, s, N'-H); 7.55 (1H, mj; 7 , 25 (5H, mj; 6.5 (1H, S, C9-Hj; 6.12 (1H, s, C ^ 2-Hl; 5.90 (2H, m, C14-H + C1:> - Hj; 25 4.2 (2H, t, -0-CH2-0ct; 11l, d, C1 '' - Hj; 3.83 (3H, s, -COOCH3 j; 3.67 * (3H, s, - OCH-J; 3.56 (1H, s, C "1-Hj; 2.83 (3H, s, N-CH_j; 1.35 (10H, m, massive octy] + Cii ~ + CH-,) ; 1 (15H, m, massive ccty] + CH- + -CH-,).

ί. -21- ι i t j h:! k] j · i, it ^:! i \\ N (ethyl D-leucinate) 0-4 deacetyl Vinblastine; (il carboxamide-3 (VDLE) i »___. _______: j 5 By applying a procedure identical to that of Example 2, the VDLE is obtained with a yield of 42¾.

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Properties_physico-chimlques_de_la_VDLE:

10 Melting point: 181.4 ° C

'r * * 1 CHL1, rr °;. fotJ _ ύ: 70.55 U c = 0.2835

Fmethanol ultraviolet spectrum,> max, nm, log t d: 15,220 (4.67); 226 (4.20); 288 (4.11); 295 (4.02).

_ i

Infrared spectrum (KEr, cm j:; 3460; 2960; 2880; 1755; 1665; 1605.

20 Mass spectrum (m / eij:;! 924 (1 4) M + + 28; 910 (32); M ++ 14; 897 (38) M + * 1; h9b (66) IÎ; L; 863 (28) ; 835 (43); 709 (45i; 651 (81); 570 (1 00).

i; ! ! i .; l · Nuclear magnetic resonance spectrum (CDCl τ, ρριη, ϋΟΜΗι J:! '- 25 9.6 (1 H, s, C16-OH); 8.16 (1 H, s, N' Ü-H j; 7, bt (1H, mj; 7.26 i3H, m); 6.7 (1H, s, C9-H); 6.16 MH, s, C12-H); 5.90; 211, m, ( 214-H + 15 C -H); 4.26 (2H, q, -COOCII -, i; 3.8 3! 3H, s, -uCH ~; 3.66 i 3H, s,: -OCH-) ; 2.90 (3H, s,!; 1.3 (3H, t, - i COOCH -, .- CH-; 0.9o

! -3 ι ό i 18 -O

I (12H, m, C + C + isopropyl).

I, | . 1 i »1 âïÊÎEIÊ-Z: N (ethyl L-serinate) -0-4-deacetyl Vinblastine - carboxamide-3 (VSE) -22- 5 By applying a procedure identical to that of Example 2, one obtains the VSE with a yield of 35%.

* Physical-chemical_properties_of_the_SEV: 10 laln CHCl3: 65> 6 ° c = 0.70 7

Ultraviolet spectrum (MeOH, Àmax, nm, loge): 215 (4.76); 266 (4.31); 288 (4.20); 297 (4, 1 5).

1 5 - 1

Infrared spectrum (CHCl ^, cm): 3460; 5400; 2965; 2955; 2880; 1750; 1665; 1 or 5.

Nuclear magnetic resonance spectrum (CDC 1 -, ppm, büMHcj: 20 8.13 (1H, s, Na —HJ; 7.9 (1H, d, -0H); 7.52 (1H, m ,; 7 , 6.63 (1H, s, C9-H); 6.1 (1H, s, C12-H); 5.9 (2H, m, C14-H + C ^ -Hs; 4.3 (2H , q, -C00-CH 7-); 3, S3 (5H, s, -OCH-); 5.66 (5H, s, -OCH-J; 2.67 (5H, s, -NU-CH- ); 1.34 (3H, t, (- COO-CH, jCH „j; 0.95 (6H, m, c'8'h. ♦ c18h.). 'Ο .5" i! -23- - ji> '| * | 1 Bxemgle_8: il N- (ethyl L-glutamate) 0-4-deacetyl Vinblastine -

Icarboxamide-3 (VGE) 5 By applying a procedure identical to that of | In Example 2, the VGE is obtained with a yield of 55%.

J * Physico-chemical properties of the VGE: -j ~ - - - - ~~ ~~ ‘10

i Melting point: 149.7 ° C

fifln CHC13: 59.12 ° c = 1.99 15 Ultraviolet spectrum (MeOH, 10 mg / l, A max, nm, logt.): 219 (4.46); 269 (3.94); 288 (3.80); 296 (5.76).

j | -1; J Infrared spectrum (KBr, cm): 'd 20 3460; 5395; 2960; 2920; 2870; 1,730; 1öö5; lt1ü; 1,500.

*> I Nuclear magnetic resonance spectrum i CDC1-, ppm, bi) MHc. : if 9.63 (1H, s, C16-0H); 8.10 MH, s, KU '-H); 7, o MH, m); 7.1o il (3H, m); 6.64 (1H, s, CH-H); b, 07 (1H, s, C1 "-H); 5.83 (2H, m, U 14 15 ii _ 25 CI4H + Cl H); 4.23 (2H, q, -COOCIi, -); 4 , 16 (2H, q, -CÜOCH, -); i; · 3.8 f3H, s, -OCH3); 3.64 (3H, s, -OCH-); 3.5 (1H, s, C21- Hj; * "2.8 (3H, s, -NaCHM î 1.55 (3H, t, -CH_j; 1.26 î 3i !, t, -CH-J; io J Ifi * ^ -5 1 | 6H , m, C H. + Cic Hj.

_ ·> I! i: *; Ü î, 1 ëiêiBEiË.? : N- (ethyl L-phenylalanynate) 0-4 desacetyl Vinblastine (VPE) -24-

By applying a procedure identical to that of Example 2, the VPE is obtained with a yield of 60%.

5. _from 1 a_VPE: s Melting point: 154 ° C.

»10 Γα3η CHCl3: 75.206 ° U c = 1.2106

Ultraviolet spectrum (MeOH, 9.8 mg / l, Amax, nm, log): 217.5 (4, '67); 265 (4.1 3); 286.5 (3.99); 295.5 (3.95).

15 -1

Infrared spectrum (KBr, cm): 3560; 3460; 3400; 2860; 2850; 1730; 1050; 1610; 1500; 1455. Mass spectrum (m / e, l): 20 958 (1 7); 944 (41); 930 (35.1; 871 (64); 651 (41d; 588 (41d; 571 (53); 401 (100).

Nuclear magnetic resonance spectrum (CDCl,, ρρπι, 60MHc i: 8.1 (1H, s, Na -H); 7.6 (1H, m); 7.3 (5H, s); 7.2 (3H , mi; 6.6 (1H, s, C9-H); 6.1 (1H, s, 012-Hj; 5.87 (2H, m, C14-H + C15-H); 4.9 (1H, m, CH-); 4.16 (2H, q, -C00-CH, J; 3.8 (5H, s, -OEL); 5.56! 5H, s, -OCH, j; 3 , 56 Γ1 H, s, C ^ 1 -! I j; 3.74 »5H,> *,. \ 'J-CH3J; 1.20 ί 5H, t, -CH, (ester)); 1 (6H , m, -Cl & H_ + C1h 'H _ ;.

"J", f \

Claims (15)

3. Compound according to claim 1 characterized in that it i j. this is ethyl N- (L-leucinate) deacetyl-0-4 i j Vinblastine carboxamide-3. t. j. j *; -27-; l i} 14. A compound according to claim 1 characterized in that J, R represents a methyl-2-propyl group and is i * - * | attached to a carbon having the absolute configuration L, R £ is a hydrogen atom and is an ethyl group; | * · As well as its pharmaceutically acceptable addition salts I. Λ \ \ f 5. Compound according to Claim 1, characterized in 1 * 10, which is chosen from the group consisting of 1 of the following Vinblastine derivatives: i] - N- (ethyl L-isoleucinate) deacetyl- 0-4 Ί d Vinblastine carboxamide-3 15. N- (ethyl L-valinate) deacetyl-0-4 Vinblastine carboxamide-3 - K- (ethyl L-phenylalaninate) deacetyl-0-4 Vinblastine carboxamide-3 - N- (Methyl L-phenylalaninate ) deacetyl-0-4 ί 20 Vinblastine carboxamide-5! - N- (ethyl L-alaninate) deacetyl-0-4 Vinblastine carboxamide-3 -i - N- (ethyl L-leucinate) deacetyl-0-4 Vinblastine i carboxamide-3 25. N- (Ethyl L-leucinate) Vinblastine carboxamide-3 - N- (Ethyl L-serinate) deacetyl-0-4 Vinblastine carboxamide-3 0. Compounds according to Claim 1, characterized in that they are L- (L-phenylalany ethyl ate I deacetyl-0-4 Vinblastine carboxamide-5 and its addition salt with acid sulfuric. 7. A pharmaceutically acceptable acid addition salt of a compound according to any one of claims 1 to 5. -28- ι 8. A salt according to claim 7 characterized in that it is an addition salt with sulfuric acid. 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 10. Use of a pharmaceutical composition according to claim 9 characterized in that it is administered to a patient at a dose between 0.05 mg / hg / week and about 5 mg / kg / week. 15 11. Pharmaceutical composition according to claim 9, characterized in that the active principle is N- (ethyl L-leucinate d-desacetyl-0-4 Vinblastine carboxamide-5. 20 12. Pharmaceutical composition according to claim 9 characterized in that the active principle is N- (ethyl L-phenylalaninate) deacetyl-0-4 Vinblastine carboxamide-5. 15. The pharmaceutical composition according to claim 9, characterized in that the active principle is in a pharmaceutically acceptable diluent. 14. Pharmaceutical composition according to vend i cat ior; 12 characterized in that the diluent is a sterile buffered aqueous solution. ! : F I. -29- ί \! Y ft {, ι y If 115. Use of a pharmaceutical composition according to | claim 9 or 14 for the treatment of 18 leukemias, solid tumors and Hodgkin's disease. 5; j j, 16. Process for obtaining the compounds according to claims 1 to 8, characterized in that it comprises the j; following steps: "1 Λ i s 10 1) Reaction of Vinblastine with hydrazine!’ l anhydrous to obtain deacetyl-0-4 carbox- i *, j ;; hydrazide-3 Vinblastine i ’ 2. Treatment of the hydrazide obtained with a nitrosating agent in an acid medium to obtain the corresponding acid azide. fl; 3) Reaction of the acid azide with one to five equivalents of the appropriate amino acid ester. 2 0 17. The method of claim 1c "characterized in; What hydrazide is treated with NaNO? in a solution of hydrochloric acid diluted at low temperature and the acid azide obtained is added to at least one equivalent of ethyl leucinate. - IB. Process for the transformation of Vinblastine into an amino acid ester derivative according to Claims 30 1 to 6, characterized in that it comprises the following stages: 1. Sapblification of Vinblastine to provide, after acidification, deacety acid 1-0-4 j 35 vinblastinoic-23 ί -30- ι i \ ί 1 2) Treatment of the acid thus obtained with j Ν, Ν'- dicarbonyliimidazole in tetrahydro-! furan or dimethylformamide. | 3) Addition of 1 to 5 equivalents of amino acid ester or of suitable peptide. 10. The method of claim 18 characterized in I 3 that it further comprises the following steps: i J? - prior to the addition of N, N'-carbonyldi-; imidazole acetylation of vinblastinoic acid I obtained with acetic anhydride in the presence of pyridine using standard conditions for the protection of hydroxyl groups 15 - selective hydrolysis by acid treatment of the product obtained in the third step for ί! supply either the derivative of Vinblastine | carboxarnide-3 monoacetylated in position 4 or I completely deacetylated. ! 2 0 ί; 2U. Process according to Claim 18, characterized in that the coupling derivative of Vinblastine with an amino acid ester is N- (ethyl L-leucinate) desacetyl-0-4 carboxamide-3 or 25 N- (L -ethyl phenylalaninate) deacetyl-0-4 carboxamide-3.