DD205906A5 - METHOD OF PREPARING N- (VINBLASTINOYL-23) DERIVATIVES OF AMINOSAURES - Google Patents

Abstract

VINBLASTINE DERIVATIVES OF GENERAL FORMULA 2, WHICH IS AN ESTER OF ANY AMINO ACID PROVIDED, LINKED TO THE VINBLASTINE 23 OYL PART BY AMID BINDING, IN WHICH THE ESTER GROUP, WHICH MAY BE STRAIGHT OR BRANCHED, CONTAINS 2-9 CARBON ATOMS AND R 1 HYDROXY GROUP, R 2 is an hydrogen atom, a FORMYL GROUP or a C 2 -C 9 ACYL GROUP, R 5 is a hydrogen atom or a METHYL GROUP or a FORMYL GROUP, with the proviso that compounds in which R 5 is METHYL AND R 1 A LYR HYDROX GROUP IS EXCEPT; OR THEIR ADDITIONAL SALT WITH MINERAL OR ORGANIC SAUCE. THE COMPOUNDS ARE AS A MEDICAMENT.

Description

(61 765/12)

Process for the preparation of N- (vinblastinoyl-23) -derivaifen of amino acids

Application s field of the invention

The present invention relates to novel bis-indole alkaloids. More specifically, the invention relates to the combination products of an amino acid with derivatives of vinblatine and to a process for their preparation. They are used as antitumoral agents in pharmaceutical compositions.

These new derivatives of vinblastine can be used in the treatment of leukemia and malignant tumors in the melanocytes.

Known technical solution

Bis-indole alkaloids of the vinblastine type are well known · compounds containing the carbon skeleton of the general formula

OO mn ^ nQO. / \ cj

245703 2

have.

As examples of these compounds may be cited: Vincaleukoblastin (U.S. Patent 3,097,137), Leurocristin or Vincristine and Leurosidin (U.S. Patent 3,205,220), Vinucinate (BE-PS 65,912) and Vindesin; (BE-PS 813 168).

The latter compound is by chemical modification of natural vinblastine (I, R ₁ = OCH ₃ / R ₃ = COCH _3), which can be obtained by extraction from Catharanthus leaves roseus- obtained.

Vinblastine, vincristine and vindesine are commercially available for use in human therapy, specifically for the treatment of leukemia and some solid tumors.

However, it has been found that these drugs have unfavorable side effects. Vincristine shows neurotoxic effects and vinblastine a high toxicity, as far as hematopoietic tissues are concerned.

Their mechanism of action is similar to the mechanism established for the antimitotic effect of colchicine. These drugs are expected to inhibit the polymerization of

24 5 7 O 3 2 -> 19.4.1983

AP G 07 D / 245 703 (61 765/12)

Tubulin to microtubule and therefore inhibit cell division in the metaphase ·

The use of 1, 1 complexes of antitumoral T bisindole alkaloids has been described in Belgian Pat. No. 854,053.

In some cases a lower toxicity and stronger chemotherapeutic activity than for the corresponding free alkaloids is described.

Other chemical modifications of vinblastine have been tested. Thus, Vlglyoinate sulfate (I, sulfate, R ₁ m OCH ₃ , R ₂ = C00H ₂ N (CH ₃ ) ₂ - Cancer Research 1967, 2J, 221-227) was also tested in clinical experiments, but it was found that it was generally not superior to vinblastine or vincristine.

BE-PS 813 168 discloses vindesine (I ₁ R ₁ NHNH ₂ , R ₂ H) and other vinblastine-Cj-carboxamide derivatives. The following confirms the therapeutic interest of vindesine or 3-carboxamide-4-O-deaoetyl-vinbletinine, but this compound appears to be inactive for the treatment of Murine 1210 murine leukemia (CJ Barnett et al., J. Med. Chem. 2J, 88, 1978).

Object of the invention

It is an object of the invention to provide improved preparations for the treatment of human leukemia and malignant tumors.

9 / ^ 7 ΓΠ 2 " ⁴ ~ 19.4.1983

£ Lf vJ / yy * J * ~ AP C 07 D / 245

(61 765/12) essence of the invention

The invention has for its object to develop a process for the preparation of N- (vinblastinoyl ~ 23) derivatives of amino acids ·

The novel compounds described in the present invention are capable of substantially delaying the death of mice intravenously inoculated with P 388 and L 1210 leukemia.

The activities on experimental P388 tumors are apparently surprisingly superior to the particular vinca alkaloids. Numerous complete decreases were observed.

The compounds of the invention further show other important and unexpected advantages compared with vinblastine and known analogues, especially vindesin.

2A5 7 03 2

-JZr-

Specifically, in general, the toxicity observed is less than the corresponding toxicity of vindesine or vincristine.

The new compounds according to the present invention correspond in detail to the general formula II

CH ₃ O

(H)

wherein R is an ester of an optionally protected ct-amino acid attached to the vinblastine-2-3-oyl moiety through an amide linkage, wherein the ester moiety, which may be straight or branched, contains from 2 to 9 carbon atoms and R a Is a hydrogen atom or a hydroxy group, R is a hydrogen atom, a formyl group or a C ₂ -Cg acyl group, R c is a hydrogen atom or a methyl group or a formyl group, provided that compounds in which R cj is methyl and R is a β -Hydroxy group is excluded; or their addition salts with mineral or organic acids.

245703 2

The following compounds are particularly preferred:

(III)

OR

wherein R _{1 is} a hydroxy group or a hydrogen atom, E _{2 is} a hydrogen atom, a formyl group or a C ₂ -C ₉ acyl group, R _{3 are} each independently a hydrogen atom, straight-chain or branched C.sub.Co-alkyl, hydroxyC. Cnalkyl, aminohydroxy-C, -Cg-alkyl, carboxy-C, Cg-alkyl, amido-C-Cg-alkyl, guanadino-C, Cg-alkyl, amino-Cj-Cg-alkyl, thiol-C C ₁ -C 6 -alkyl, cysteinylmethyl, methylthioethyl, benzyl, hydroxybenzyl, or a group:

(IV)

or

CH,

(V)

or R ₃ together with the carbon atom to which it is attached and the nitrogen of the amido group form an azole or a hydroxy-azole ring; R_ is a hydrogen atom or a methyl group or a formyl group, and R _{4 is} a straight-chain or branched C.-Cg-alkyl group or a benz'yl group, with the proviso that compounds in which

245 7 03 2 -

R _{1 is} -β-OH and R ₅ -CH ₃ are simultaneously excluded; -COOR _{4 is} preferably the carboethoxy or the carbomethoxy group; or their addition salts with a mineral or organic acid.

In a preferred embodiment, the structural

segment

R ₀ 0 I 3 11

-NH-CH-COR ₄

in general formula III is an ester derived from any of the naturally occurring amino acids and their D-configuration optical isomers, namely glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, glutamic acid, asparagine, glutamine, Arginine, lysine, cysteine, cystine, methionine, phenylalanine, tyrosine, tryptophan, proline, histidine, hydroxy-lysine or hydroxy-proline.

The compounds may be referred to as S-decarbomethoxy-O-1-deacetylvinblastine-3-carboxamide. However, they are preferably referred to as N- (vinblastine-23-oyl) or N- (vincristinoyl-23) derivatives of the following amino acids.

Particularly preferred compounds according to the invention of the general formula II are those in which R 2 is hydrogen and the amino acid parts are derived from one of the following amino acids: L- or D-tryptophan , * leucine, - iso- leucine, -valine, -alanine or -phenylalanine , Among these, L-tryptophan, L- iso- leucine and L-alanine are of particular interest.

The present invention describes single reaction products of amino acids with vincristine, O-4-deacetyl-vincristine, deformyl-O-4-deacetyl-vincristine and deoxy-vinblastine B.

The latter compound can be obtained by hydrogenation of anhydrovinblastine, again by dehydration of vinblastine or by coupling vindoline with catharantin

245 703 2 ^Λ -

is obtained (Potier et al., JACS 9_8, 7017, (1976)).

The most preferred compound of the present invention is ethyl N- (O-4-deacetyl-4'-deoxyvinblastine-23-oyl) tryptophanate.

If the amino acid contains functional groups in the side chain R ₃ (formula III), such as lysine or cysteine, it may be necessary to protect the functional groups according to methods well known to those skilled in the art.

Protection can be achieved depending on the nature of the group to be protected by condensation of a benzyl _; t-butyl, benzyloxycarbonyl · ^ t-butoxy-trifluoroacetyl-carbonyl or trityl. Other well-known protecting groups used in peptide chemistry can also be used to advantage.

The compounds of the present invention can be obtained in a conventional manner from the corresponding vinblastine derivatives by hydrazinolysis followed by nitrosation and reaction with the amino acid ester according to Reaction Sequence I.

; ^N 2 ^H 4,

4 J - »·

CH OH OCOCH-

OH ^ \ ^J COOCH ₃ '

Aminosäureester

CH ₂ Cl ₂

HO C-H- (S-CO- (

" ^0R <θ ' ^H

Reaction sequence I

245703 2

- Χ9Γ -

The first step of this process is to add anhydrous hydrazine in excess to a solution of vinblastine base in anhydrous methanol. The solution is heated in an inert atmosphere for 12 hours to 12 days at a temperature between about 30 ⁰ C and 7O ⁰ C. Particularly preferably, the temperature is maintained at about 60 ⁰ C.

The hydrazide of the bis-indole derivative 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 case of vincristine, the product obtained is the 0,4-deacetyl-deformyl-vincristine-carboxyhydrazide.

During the second step, the hydrazide function of the modified vinblastine is converted to an acyl azide. This conversion is achieved in a known manner by adding sodium nitrite to the dissolved hydrazide in a water-methanol acid mixture.

The acid that is used can be hydrochloric acid.

The reaction temperature is maintained between 0 ⁰ C and 5 ⁰ C. After extraction with a water-immiscible aprotic solvent, preferably methylene chloride, the organic phase is partially concentrated.

The appropriate acyl azide is preferably not isolated but added directly to the amino acid ester or optionally a protected derivative thereof dissolved in methylene chloride.

245703 2

The amount of amino acid to be used is about 1 to 5 equivalents of the modified vinblastine carboxazide.

The reaction mixture is maintained between about -3 ° C and +10 ⁰ C for 8 to 72 hours and generally for about 15 hours. Monitoring of the reaction is easily accomplished by thin layer chromatography. After concentration, the compound of formula II of the present invention may be converted to a sulfate salt or other salt derived from a mineral or organic acid by crystallization from a methanolic solution of the corresponding acid.

The compounds of the invention can be purified by conventional chromatography and recrystallization techniques.

If desired, the 4-O-deacetyl-vinblastine derivative thus obtained may be re-cyclized, either directly to obtain the vinblastine derivative III, wherein R _{2 is} COCH ₃ (J. Med. Chem. 22 , 391, 1979), or by preliminary formation of the 3,4-diacetoxy derivative, followed by selective hydrolysis of the 3-acetoxy group in the 3-position. The hydroxy group in C can usually be esterified by other activated acid derivatives containing 2-9 C atoms. The O-4-formyl derivatives are obtained by formylation (formic acid-acetic anhydride) of the corresponding O-4-deacetyl compounds (see BE-PS 660 843).

₇ The present invention also relates to the industrial and pharmaceutical applications in particular, the new bis-indole-alkaloids.

In particular, the compounds according to the invention exert very valuable antitumoral properties which can be used in human therapy.

245 703 2

These amino acid derivatives can be used for the treatment of leukemia of the L 1210, P 388 type, gliomas, lymphomas and other leukemias or malignant tumors. In human medicine, they can used to treat Hodgkin ¹ see disease and be used for other solid tumors treatable with vinblastine, vincristine or vindesine. These compounds are also valuable in veterinary medicine for the treatment of tumors of the animals.

Other therapeutic uses of interest may also be given for the novel derivatives of bisindole alkaloids. It has been described that vinblastine can be used for the treatment of certain forms of arthritis (US Pat. No. 4,208,411) and that vincristine can be used for the treatment of psoriasis (US Pat. No. 3,749,784).

For their therapeutic use, the compounds of the invention are optionally administered in lyophilized form, preferably via the parenteral route, dissolved in a pharmaceutically acceptable solvent in the form of a base or a pharmaceutically acceptable acid addition salt. Among the acid addition salts are the non-toxic and pharmaceutically acceptable salts, such as inorganic acid salts, such as hydrochloric, phosphoric and sulfuric salts, or organic acid salts, such as acetic, propionic, succinic, tartaric, oxalic, methanesulfonic and benzene acid salts. sulfonic acid salts are preferred.

Physiological saline and other saline solutions that are buffered, e.g. with a phosphate are suitable solvents. In general, the compounds can be used in human therapy in a manner analogous to the technique and limitations of using other vinca-1 type alkaloids.

24 5 7 03 2

The active substance is generally employed at a posology varying between 0.01 mg to about 5 mg / kg depending on the derivative and the therapeutic regimen used. Total weekly doses will generally vary between 0.1 and about 35 mg / kg.

The compositions according to the invention are prepared as unit doses of 2 to 900 mg.

The compounds of the present invention may be used alone or in combination with other carcinostatic agents, including, for example, the alkylating agents, the antimetabolites such as methotrexate, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, the cystosin-arabinosides, and antibiotics such as actinomycin D, daunorubicin, adriamycin and cis-diamino-dichloro-platinum. In particular, the novel vinblastin derivatives may be used in combination.

The following specific examples, by way of non-limiting example, illustrate the process for obtaining the compounds of this invention.

EXAMPLE 1

1. Preparation of O-4-deacetyl-N-deformyl-vincristine monohydrazide A.

958 mg of vincristine base are dissolved in a mixture of 10 ml of anhydrous hydrazine and 10 ml of methanol. The reaction mixture is kept at 60 ^° C. with stirring for 22 hours. The reaction mixture is then treated with water and then with NaCl saturated water.

2/15703 2

The aqueous solution is extracted eight times with 15 ml of dichloromethane. The collected organic phases are treated with 20 ml of water, then 75 ml of NaCl-saturated water, and finally dried over Na ₂ SO ₄ . After filtration and vacuum concentration, 900 mg of O-4-deacetyl-vincristine monohydrazide (purity over 90%) were obtained.

NMR spectrum (CDCl ,, J6o MHz)

9.76 (s, IH), 8.37 (s, 1H), 8.06 (s, 1H), 7.53 (d, 1H), 7 _; 24 -UHd ^ ₁ 07 (m, 3H), 6.82 (s, 1H), 6.22 (s, 1), 5 ₍ 87 (dd, lh), 5.67 (d, 1H), 5, 43 (s, lH, 4 _; 03 (s, lH), 3.94 (s, 3H), 3.86 (s, IH), 3.73 (s, 3H), 3.52 (s, 3H) , 2 _; 82 (s, 2H), 2.51 (s, l! I),

0.93 (2t, 2 x 3H)

Mass spectrum (electronic ionization bombardment)

154, 294, 355, 413, 524, 555, 695, 710, 723, 736, 739, 755 (M + 1) - 768, 769, 782 calculated for

^C 42 ^H 54 ^N 6 ° 7 ⁷⁵⁴ ' ⁹⁴² 2. Preparation of Acid Acid from Modified Vincristine.

A solution of 850 mg of vincristine monohydrazide A in 20 ml of methanol is treated with 63 ml of HCl 1 N and 175.5 mg of sodium nitrite at 0 ^° C. for 15 minutes.

The solution, kept at 0 ⁰ C and is then neutralized with NaHCO-, at 0 ⁰ C then extracted three times with dichloromethane. The collected organic phases are dried over Na ₂ SO 4, filtered and concentrated in vacuo without heating until a solution of about 10 ml is obtained.

5. Reaction with ethyl tryptophanate.

500 mg of ethyl tryptophanate are then added with stirring. about 4 ⁰ C was added and the reaction was checked by TLC. After 10 days, the reaction product is purified by chromatographic separation (4o g of silica gel βθ, eluent solution).

24 5 7 03 2

medium ether / NEW saturated methanol 96: 4 volume / volume). Fractions of 15 ml are collected, which are checked by TLC (thin layer chromatography). Unreacted amino acid is collected first (Fraction 30 to 4o). The eluent is changed (ether / NH-, - saturated methanol 86:14), and the reacted derivatives are collected as 5 fractions: fractions 44 and 45: 50 mg; Fractions 46-53: 257 mg; Fractions 54-60: 115 mg).

The second part was ethyl N- (0,4-deacetyl-deformyl-virin-β-23-oyl) -tryptophanate Ia, which was homogeneous by TLC.

Mass Spectrum : 984, 970, 956 (M ⁺ + 1) 913, 912, 899 (DCJ I Sobutan.), Calcd. For C55H66N609: 955.181.

UV spectrum (CH ₃ OH):

max: 282 (4j20) - 290 (4 _t 18) - 322 (3.96) min 254 - 287-305

IR spectrum _: (cm " ¹ )

3400-2920-1720-1660-1610-1485-1450-1370-1345

1330 - 1290 - 1220 - 1165 - 1130 - 1025 - 1005 - 920 - 885 830 - 740

NMR spectrum (CDCl3) 3 60 MH, (ppm)

8.50 (s, IH)

8.03 (s, 2H)

7 _f 77 (d, IH)

7.60 (d, IH)

7.57 (d, IH)

6 _f 95 (s, IH)

5 7 03

6, 35 (s, IH)

5, 89 (d.d., IH)

5, 78 (d, IH)

4, 75 (q, IH)

3.88 (s, 3H)

3, 67 (s, 3H)

l _f 23 (t, 3H)

0, 98 (t, 3H)

0, 89 (t, 3H)

Ethyl N- (O _> 4-deacetyl-vincristine-25-oyl) -tryptophanate IB. The deformylated derivative I a (257 mg) is reformylated using the method described in BE-PS 811,110.

The crude product thus obtained is purified by chromatographic separation using a 20 cm column (silica gel, JO g) and successively 3 eluent: Ä the r / NH- * -saturated MeOH 92: 8; 88: 12, 86: Ik cleaned up. The effluent is collected as 15 ml fractions. The fractions kk - 5 ^ (54.1 mg) contained the N-reformylated derivative Ib.

MaS spectrum of fisobutane DCI): 1011 ^ 997, 983 (M ⁺ + 1), 970,

982, 996, 998, 999, 1012 calculated for C56H66N6010 = 983.192

UV spectrum (CH ₃ OH) max: 270 (4, 18) - 290 (4,11) plate 280 (4jl2) shoulder (4 03)

245703 2 IR spectrum (KBr )

Volumes at 3400 - 3040 - 2960 - 2920 - 2880 - 2850 - 1735 1725 - 1680 - 1670 - 1665 - 1610 - 1490 - 1450 - 1225 - 745.

Example ₂

Ethyl N - (0-4 deacetyl-deoxy-4'-vinblastine-23-oyl-B) tryptophanate .Ic.

The O-4-deacetyl-deoxy-vinblastine hydrazide is prepared by the method described in US Pat. No. 4,203,898, column 24, line 51 ff.

500 mg of the hydrazide in 12 ml of methanol are then treated with 37 ml of HCl IN and 104 mg of NaNO ₂ at ⁰ ° C. for 15 minutes.

The solution is then neutralized with NaHCO 3 and extracted 6 times with 15 ml of dichloromethane. The organic phases are dried over Na ₂ SOu and concentrated until a volume of about 10 ml is obtained.

The azide in solution is then contacted with Äthyltryptophanat (300 mg) was reacted with stirring at 4 ⁰ C. The reaction is checked by TLC (ether, MeOH). After 6 days, the reaction is complete and the reaction product is purified by column chromatography over silica gel (18 cm, 30 g), elution through 15 ml fractions.

The following eluents were used sequentially: Ether 96 NH, sat., MeOH 4 fractions 1-49 92 8 fractions 50-57

86 14 fractions 71-90

Fractions 50-57 contain the desired reaction product Ic (108 mg).

24 5.70 3 2

The sulfate is prepared by ether precipitation (2 % H ₂ SO ₄ / ethanol).

UV spectrum of sulfate (CE, 0H)

max: 224 (4,63) - 272 (4,32) min. : 247 (4, 03) Shoulder -: 286 (4,22) - 312 (3,72)

IR spectrum (KBr) of the sulfate ,

3420 - 3060 - 2960 - 2930 - 2880 - 2600 (weak) - 1725

1660 - 1615 - 1500 - 1455 - 1430 - 1375 - 1230 - 1105 -

1060 - 1015 - 920 - 745 cm " ¹ .

NMR spectrum (CDCl ₃ ) 360 MKz of the base in ppm .

9,46 SlH 7,53 d 1 H 4,94 q IH

8.34 slH 7.30 d 1 H 4 _; 18 d IH

7,92 SlH 6,51 s 1 H 4 _; 05 q 2H

7.67 d 1 H 6.04 s 1 H 3.77 s 3H

7.59 d 1 H m. around s. centered on 5 81

245703

3.58 SRH 3 _T 47 s IH 2.82 s 2H 2.77 s 3H

2.59 s IH 1.14 t 3H 0.95 t 3H Of 87 t 3H

Mass spectrum of the base

OCI isobutane

M + 1 ⁺ beL954 M + 14 + l ⁺ teL 968 M + 28 + i ⁺ at 982

Ions at 153-155-157-171-172-185-186-199

213 - 223 - 227 - 255 - 257 - 279 - 281 - 2S3

285 - 349 - 398 - 459 - 516 - 569 - 952 - 953

955 - 967 - 969 - 981 -

calculated for C ₅₆ H ₅₈ N _g O ₈ : 953.208

24 5 7 03. 2

Example 3 Ethyl-N- (0-4-deacetyl-4'-deoxy-vinblastln-23-oyl-B) -

isoleucinate

A solution of O-4-deacetyl-4'-deoxyvinblastine-β-hydrazide (0.6g, 0.79mM - see U.S. Patent 4,203,898, col. 24, line 51) in a mixture of 15ml CH-. OH and 45 ml of HCl 1N were cooled to -7 ⁰ C. NanOp was added to the solution (0.15 g), and stirring was continued for 13 minutes at -7 ⁰ C. After adding an aqueous saturated solution of NaHCO 3 until a pH of 9 is reached, the resulting solution is extracted four times with CH ₂ Cl ₂ . The combined organic phases were washed with saturated aqueous NaCl solution and dried over MgSO4.

The solution was concentrated to a volume of 15 ml and ethyl isoleucinate (1.25 mM) was added. The reaction mixture was allowed to stand in a refrigerator for 4 days. The solvent was evaporated under reduced pressure. The residue was purified by column chromatography (SiO ₂ , 60 g) and eluted with ether / CHgOH / NH, (96: 4). 0.25 g of amorphous product (yield 35 %) was thus obtained.

Mass spectrum : (Molec. Ionization isobutane)

880 (M ⁺ ), 894, 836, 849, 832, 445; 391, 355 cm " ¹

^C 51 ^H 69 ^N 5 ° 8 ^{= 880} * ¹ Melting point 18O-19O ° C

a _D = 104 ° (c = 0.154)

IR (CHCl ₃ ) 3470, 2970, 1728, 1655, 1612, 1502 cm ^-1

UV (CH ₃ OH) 289, 268, 216 nm

245703 2

HO

NMR (CDCLj) 7 _f 9 (NH), 654 and 6.06 H _g , H ₁₃ , 5.81 (H ₁₄ -H ₁₅ )

s 4.60 (CHCO ₂ (NH)) 3.76 CO ₂ CH ₃ -, 3.58 CH ₃ -O, 3.33 and 2 _; 85

(H ₃ A and H ₃ B), 2.73 (N-CH ₃ ), 0.69 (H-15 ¹ ).

Acute toxicity determination of ₀

The acute toxicity of the compound of Example 2: ethyl N- (O-4-deacetyl-deoxy-4'-vinblastine-23-oyl-B) -tryptophanate was determined on female NMRI mice. Rising doses of the drug were injected intravenously in a single injection. The mortality percentage is determined as a function of time. The LDr - ^ values for the

pO

referenced compound (deoxy-VTrpE) and a reference product: deoxy-vinblastine (deoxy-VBL) are given below.

Drug _n Deoxy-VBL 20

Deoxy-VTrpE (namely compound Ic) 91.5

The compound deoxy-VTrpE is as little toxic as the reference compound.

Antitumoral activity

The chemotherapeutic activity of the sulfate salt of deoxy-VTrpE was studied on lymphoblastic leukemia L 1210 and P 588.

1. L 1210 ·

Female DBA ₂ mice were inoculated with 10 leukemic cells. The product was administered intravenously on the day following vaccination at a 50 mg / kg dose. The mortality of the animals is given as a puncture of time. The table below gives the results obtained.

24 5 7 03 2.

Hi

Start with the compound of Example 2 (Ic). At the dose tested, the compound is more active than vinblastine. This superiority was confirmed in comparison with deoxy-vinblastine.

2. P 388

Female DBA ^ mice were inoculated intravenously with 10 leukemia cells. The products were i.v. administered the day after inoculation. The mortality of the animals is given as a function of time. The results are listed in the table. Deoxy-VTrpE is clearly more active than the reference compound and indicates a higher percentage of surviving animals for a long time.

TABLE

Tumor product dose Number of animals MST 50 days ILS Percentage of surviving animals at day 50 Percentage of surviving animals at day 6o L 1210 VBL 8th 10 10.4 57.6 O 0 VBL 19 10 5.8 - 21 _f 6 0 0 Deoxy VTrpE 50 10 11.2 70 0 0 P 388 VBL 4 5 15.6 56 ' 0 0 Deoxy VBL 8th 5 - 42 0 0 Deoxt VTrpE 30 10 16.5 58.7 0 0 40 15 30 191 67 53 50 15 30 191 87 73.3 55 10 30 212 80 60 60 10 30 212 90 90

ro Ul Q

Female DBA1 mice are inoculated via the i.v. pathway with 10 leukemia cells. The drugs were injected via the i.v. route to the indicated doses.

MST = median survival ILS % =% increase in lifespan

Claims (4)

245703 2 claims 1.) Method of making an anti-tumor Compound of the general formula CO ₂ CH ₃ ^CH 3 ° 245703 2 'Ζ *' 19.4.1983 AP G 07 D / 245 703 (61 765/12) wherein R is an ester of an optionally protected C-amino acid bound to the vinblastine-23-oyl moiety by an amide bond, wherein the ester group, which may be straight-chain or branched, contains from 2 to 9 carbon atoms and R _{1 is} a hydrogen atom or a hydroxy group Rg is a hydrogen atom, a formyl group or a Cp-Cg-acyl group, R, - is a hydrogen atom or a methyl group or a pormyl group, with the proviso that compounds in which R, - methyl and R _{1 is} a β- Hydroxy group is excluded; or their pharmaceutically acceptable salts with mineral or organic acids, characterized in that it comprises the following steps ι Reaction of a compound of the aforementioned formula wherein R is methoxy with anhydrous hydrazine to give the corresponding modified 3-carboxyhydrazide-vinblastine, Reaction of this 3-carboxyhydrazide with a nitrosating agent in an acid medium to obtain the corresponding 3-carboxazide, Reaction of this Öarboxazids with one to five equivalents of an OC-amino acid ester, wherein the ester group, which may be straight or branched, containing 2 to 9 carbon atoms. 2Z5
-2- 19.4.1983. ™ "~" ~ - - AP G 07 E / 245 (61 765/12) 2. Method according to item 1, characterized in that the nitrosating agent is sodium nitrite in the presence of methanolic HCl. Method according to item 1, characterized in that a 1: 1 addition salt is prepared with sulfuric acid 4. Process for the preparation of ethyl N- (O-4-deacetyl-4 ^L -deoxy-vinblastine-23-oyl) -L-tryptophanate characterized by the fact that the azide of O-4-deacetyl-4'- deoxyvinblastine-23-oylic acid is reacted with one to five equivalents of ethyl L-tryptophanate.