HU181874B - Process for preparing amides of deacetyl-leurosine and deacetyl-leuroformine - Google Patents

Abstract

Alkaloids of formula (I) and their pharmaceutically acceptable salts are new: (R1=H, CH3 or CHO. R2=NH2, N3, Nalk, or NHCH2(CH2)nX n=1 or 2. X=OH, Oalk, OCOalk or SY. Y=H, alk, or is a bond joining 2 alkaloid residues via C3 - carboxamide gps. alk = 1-3C alkyl. R3=OH or acetoxy). They are made from corresp. cpds. with R2=NHNH2 by reaction with NaNO2 (to R2=N3) when reaction with NH3, alkylamine etc. (I), excluding those with R1=H or CH3 and R2=N3, are antitumour agents effective against e.g. Gardner lymphosarcoma or leukaemia P 388. (I) with R1=H or CH3 and R2=N3 are intermediates for the others.

Description

Process for the preparation of amides of deacetyl leurosin and deacetyl leurofoimin

The present invention relates to novel carboxamides of 4-deacetylleurosin and 4-deacetylleuroform which are useful as antitumor agents.

The most abundant dimeric indole dihydroindole alkaloids from Vinca rosea can be represented by the general formula (1). If in formula 1

Many naturally occurring alkaloids derived from Vinca rosea have been shown to be effective in the treatment of experimental animal tumors. These include leurosin (U.S. Patent No. 3,370,057); vim kaleukoblastin (vinblastine or VLB) (U.S. Patent No. 3,097,137); leurosidine (vinrozidine) and leurocristine (vincristine or VCR) (both disclosed in U.S. Patent 3,205,220); deoxy-VLB "A" and "B" (Tetrahedron Letters 783 (1968): Desacetyl leurosin hydrazide is also described herein); 4-desacetoxyvinblastine (U.S. Pat. No. 3,954,773); 4-deacetoxy-3'-hydroxyvinblastine (U.S. Patent No. 3,944,554); leurocolombin (U.S. Pat. No. 3,890,325); leuroform (N-formyl leurosin, Belgian Patent 811110) and vincadiolone (U.S. Patent 3,887,565). Two of these alkaloids, VLB and VCR, are already commercially available for use in the treatment of human malignancies, particularly leukemias and related diseases.

X ^{1 is} acetoxy,

X ^{2 is} methyl,

X ^{3 is} hydroxyl,

X ⁴ ethyl groups and

X represents ⁵ hydrogen atoms, the compound is vinblastine. Where X ^{1 is} acetoxy, X ^{2 is} formyl, X ^{3 is} hydroxy, X ^{4 is} ethyl and

X represents ⁵ hydrogen atoms, the compound is vincristine. Where X ^{1 is} acetoxy, X ^{2 is} methyl, X ^{3 is} ethyl,

X ⁴ represents a hydroxyl group, and X ⁵ is H, leurosidine the compound. Where X ^{1 is} acetoxy,

X ² methyl groups,

X ³ and X ^{5 are} hydrogen and X ^{4 is} ethyl; the compound is deoxy-VLB "A". Where X ¹ , X ² and X ⁵ are the same as "Á" of the above deoxy-VLB, but

X ³ ethyl group and

-1181874

4

X represents ⁴ hydrogen atoms, the compound is deoxy-VLB "B"; and wherein X ^{1 is} acetoxy,

X ^{2 is} methyl,

X ^{3 is} ethyl; and

X ⁴ and X ^{5, taken} together, form an alpha-epoxide ring, the compound being leurosin.

The structure of leuroform is identical to that of leurosin, except that X ^{2 is} not a methyl but a formyl. Leurocolombin corresponds to 2'-hydroxy-VLB and vincadiolin to 3'-hydroxy-VLB; The structure of 4-desacetoxy VLB VLB identical structure except that X is hydrogen instead of ¹ is acetoxy; and 3'-hydroxy-4-deacetoxy-VLB may also be referred to as 4-deacetoxy-vincadioline.

The possibility of chemical modification of vinca alkaloids is quite limited. In particular, their chemical structure is rather complex and it is difficult to carry out chemical reactions that would affect only one specific site of the molecule. Secondly, alkaloids that did not exhibit adequate chemotherapeutic efficacy from Vinca rosea were also derived and their structure concluded that they are closely related to the active alkaloids. Therefore, it appears that the antitumor activity is limited to a very specific structure and thus the likelihood of modifying these structures to be more effective is low. Successful modifications of physiologically active alkaloids include the preparation of dihydro-VLB (U.S. Patent 3,352,868) and the replacement of the 4 acetyl groups with higher alkanoyl groups or other acyl groups (U.S. Patent No. 3,392,173). (The 4-position carbon atom of the VLB ring system is represented by the numbering of formula I). Many of these derivatives are capable of prolonging the life of mice vaccinated with P1534 leukemia. One derivative wherein the chloroacetyl group replaces the acetyl group of vinblastine 4 is also a useful intermediate for the preparation of structurally modified VLBs, such as Ν, Ν-dialkyl-. glycyl replaces the acetyl group of VLB 4 (U.S. Pat. No. 3,387,001). Intermediate 4-deacetyl VLB is formed during chemical reactions leading to the latter derivatives. This intermediate, which has no -4 acyl group and contains an esterified hydroxyl group, exhibits low in vivo chemotherapeutic activity in the P1534 mouse leukemia system (Hargrove, Lloydia 27, 340 (1964)). However, recent work has shown that 4-desacetyl-VLB is an effective anticancer agent (Owellen, Fed. Proc. 34, 808 (1975) and Cullinan et al., 9th International Congress of Chemotherapy, London, 1975, extract number SC-19).

One of the recent successful chemical modifications of Vinca-derived dimeric indole dihydroindole alkaloids is the substitution of the ester function 3 with an amide or hydrazide group, which is usually accompanied by the loss of the acetyl group 4 (this group can be replaced). THE

Amides of VLB, leurosidine, VCR, deoxy-VLB A &

Two of these alkaloids, VLB and VCR, are commercially available agents for the treatment of human malignancy, and the other two, leuroform and 4-deacetyl-VLB-3-carbox10 amide (vindezine), are now available in Europe and the United States. is being tested as a potential “anti-cancer” agent.

The present invention provides a process for the preparation of compounds of formula I wherein:

R ^{1 is} methyl,

R ^{2 is} azido, -NH-C ₂ H ₄ OH,

NH ₂ - NH- (1- 3 carbon atoms) -alkyl represents -NH-C ₂ H ₄ group of formula -xy je ^2C below, wherein

Y is hydrogen, (C 1 -C 3) alkyl, or a chemical bond that connects two molecules of formula I through the 3-carboxamido group; or ³⁵ R ^{1 is} formyl and

R ^{2 is} amino.

These compounds are prepared by reacting ³⁰ a) a compound of formula II wherein R ^{4 is} methyl and R 5 is ^a hydrazino group, and optionally 3 is formed of ₃₅ , where R ^{5 is} azido, optionally ³ NH ₂ E - where E is hydrogen, C 1-3 alkyl,

-C ₂ H ₄ OH or -C ₂ H ₄ SY, wherein Y is hydrogen, C ₄ -C 1-3 alkyl or -S- bonding two compounds of formula III; obsession

b) reacting a compound of formula II wherein R ⁴ is as defined above and R ^{5 is} methoxy, with a compound of formula IV 45 NH ₂ E ¹ wherein E ^{1 is} hydrogen or C 1 -C 3 alkyl and optionally R ^{1 is} hydrogen; preparation of formula I compounds having a formyl group is appropriate) sara R is oxidized and, if desired, a resulting free base is converted to a pharmaceutically acceptable acid addition salt of compound ¹ is a methyl group.

Pharmaceutically acceptable acid addition salts of the compounds of the invention include salts with inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitric acid, phosphorous acid and the like, as well as organic acids and aliphatic acids. 2181874, dicarboxylic, phenyl-substituted aliphatic, aliphatic and aromatic, aliphatic, aliphatic, hydroxy, and aliphatic, hydroxy, dicarboxylic, aliphatic, and aromatic sulfonic and the like. Pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, , isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, benzoate, chlorobenzoate, methyl benzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, t , toluene sulphonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, 2-hydroxybutyrate, glycollate, malate, tartrate, methanesulfonate _t sulfonate, propanesulfonate, naphthalene-1-sulfonate , naphthalene-2-sulfonate and the like.

Representative compounds within the scope of the invention include:

4-desacetyl-leuroform-3-N- (beta-methoxyethyl) -carboxamide, leurosin-3-N- (beta-isopropoxy-ethyl) -carboxamide, 4-deacetyl-leuroform-3-N- (beta-methyl- mercaptoethyl) carboxamide, leurosin-3-N- (beta-mercaptoethyl) carboxamide, 4-deacetylleuroform-3-N-methylcarboxamide, 4-deacetylleurosin-3-N- (beta- ethoxyethyl) carboxamide, leuroform-3-N- (beta-hydroxyethyl) carboxamide, and 4-deacetylleurosin-3-N- (beta-n-propoxyethyl) carboxamide.

4-Deacetylleurosine-3-carbohydrazide or 1-desformyl-4-deacetylleuroform-3-carbohydrazide is a suitable starting material for the preparation of the antitumor compounds of formula I of the present invention. If the above hydrazides are converted to the corresponding azide by treatment with nitric acid, then with ammonia or other desired primary amines such as methylamine, beta-hydroxyethylamine, beta- (C 1 -C ₃ alkyl mercapto) ethyl (or - with propylamine), 4-deacetyl-3-carboxamide (compound of formula (I) wherein R ^{1 is} methyl) is obtained. The hydrazide azide amide conversion was performed by Stohl and Huffman, Helv. Chim. Acta, 26, 944 (1943) (see also U.S. Patent Nos. 2,090,429 and 2,090,430).

Compounds wherein R ^{2 is} -NH-C ₂ H ₄ -SY * are prepared by reacting 4-deacetylleurosine-3-carboxamide with beta-mercaptoalkylamines. However, mercaptan is oxidized in an alkaline medium to the corresponding disulfide, where the two parts of formula I are linked by a Y bond. Using standard azide synthesis, the mercaptoalkylamides and their corresponding disulfides are isolated by chromatography, which is easily separable. Mercaptoalkylamide free of accompanying disulfides can be prepared by reacting the carboxazide 3 with NH ₂ -C ₂ H ₄ -S-trityl (trityl = triphenylmethyl). Treatment of 3-N-trityl mercaptoalkylcarboxamide with a heavy metal salt such as mercury (II) acetate followed by removal of the metal salt as an insoluble sulfide affords the desired disulfide-free mercaptoalkylamide. Trisubstituted triphenylmethyl derivatives can also be used as blocking groups. The disulfide is clearly prepared by reacting 2 moles of 4-deacetylleurosine-3-carboxazide with cystamine [(NH ₂ -CH ₂ -CH ₂ -S) ₂ ]. Mercapto-propyl-amide, and the corresponding disulfide _ς similarly obtained.

The preparation of leuroform-3-carboxamides requires further consideration. Reaction of leuroformine with hydrazine not only produces 3 carbohydrazides but also undergoes hydrolysis of 4 acetyl and 1 formyl groups. Thus, the starting material for 4-deacetyl leuroform-3-carboxamide is 4-deacetyl-1-desformyl leurofoarminine-3-carboxyhydrazide. This compound can be converted to the corresponding azide by nitro-wash. The azide reagent ₁₅ gáltatva 3 carboxamide formed with ammonia or a suitable primary amine, which újraformilezünk purpose of 4-desacetyl leuroformine-3-carboxamide Alternatively, the 4-desacetyl leurosine 3-carboxamide -60 ° C acetone / acetic acid is oxidized with CrO ₃ (Jovanovics et al., U.S. Patent No. 3,899,494) to give a 1-formyl derivative (a compound of formula I wherein R ^{1 is} formyl) or a 1 desformyl compound (general formula I). (wherein R ^{1 is} hydrogen) which is then re-formylated. This procedure is not suitable for amides containing oxidizable groups, although routine CrO ₃ -C oxidation at -60 ° C is very slow, if at all.

The alkaloid bases according to the invention or their acid addition salts, in particular sulfates, are white crystalline materials. The final starting material for the preparation of the compounds of the invention, leurosin from Vinca rosea, is prepared according to U.S. Patent Nos. 3,370,057 and 3,525,030. When in the air, leurosin is converted into the 5'-hydroxy derivative by autooxidation. Thus, if leurosin is used as a starting material in a reaction, the leurosin must first be purified. Here are 40 ways to do this:

150 g of crude leurosin containing 50% of 5'-hydroxy-leurosin in 5% ethanol are heated for 1 hour in 4 liters of ethanol. The ethanolic solution. The crystals were dissolved in dichloromethane and chromatographed on 1.5 kg of silica. The chromatogram was developed with a 2: 1: 2 mixture of ethyl acetate-methanol-methylene chloride. TLC elution of the first eluted fractions containing 5'-hydroxy leurosine by TLC showed 24 g of purified 5'-hydroxy leurosine. Subsequent leurosin-containing fractions were combined to give 6 g of leurosin. The intermediate fractions contain a mixture of the two alkaloids (42 g), from which further purified compounds can be obtained.

The preparation of compounds of formula I is illustrated by the following examples:

Example 1

Preparation of 4-desacetyl leurosine-3-carbohydrazide

A mixture of 450 mg leurosin, 15 ml tetrahydrofuran, 8 ml anhydrous hydrazine and 6 ml methanol

-3181874 in a sealed glass bomb for 3 days at 50 ° C. The bomb was opened, the solvent was evaporated in vacuo and the remaining 4-deacetylleurosine-3-carbohydrazide was dissolved in methylene chloride. The methylene chloride solution was washed with water and dried. Removal of the solvent in vacuo afforded 4-deacetyl leurosine-3-carboxyhydrazide, which by TLC contained traces of leurosin and

Contains 4-deacetyl leurosin. Pure 4-deacetylleurosine-3-carboxydydrazide (1 spot) was prepared by chromatography on silica gel using methylene chloride / ethyl acetate / methanol 1: 1: 1 as eluent. TLC analysis indicated that the fractions containing 4-deacetylleurosine-3-carbohydrazide were pooled. Physical characteristics of the compound are as follows: mass spectra: m / e 766 (molecular ion) 649, 427; infrared peaks (in chloroform) at 3480, 3440, 1730 and 1670 cm @ -1; pKa (66% in dimethylformamide) 5.32 and 7.12.

Example 2

Preparation of 4-desacetylleurosine-3-carboxamide

Approximately 800 mg of 4-deacetylleurosine-3-carbohydrazide was dissolved in 120 ml of 1N aqueous hydrochloric acid, previously cooled to 0 ° C. To the reaction mixture was added 180 mg NaNCl 2 and stirred at 0 ° C for 10 minutes. The acidic aqueous solution was basified with aqueous NaHCO ₃ and the basic solution was extracted twice with chloroform. The chloroform extracts were combined and dried. The chloroform was evaporated to give 4-deacetyl leurosine-3-carboxazide as a tan amorphous powder. IR (chloroform): absorption peaks at 2135 and 1725 cm @ ^-1.

Example 3

Preparation of 4-Desacetyl-Lurosin-3-carboxamide

To a solution of 4-deacetylleurosine-3-carboxazide prepared in Example 2 in dichloromethane was added 250 ml of dichloromethane previously saturated with NH ₃ at 10 ° C. The reaction vessel is fitted with a drying tube and protected from light. The reaction mixture was kept at room temperature overnight and the volatiles were removed in vacuo. The rest

4-Deacetylleurosine-3-carboxamide was dissolved in methylene chloride and the methylene chloride solution was washed twice with water and dried. Chromatography on silica gel, eluting with ethyl acetate-ethanol (1: 1), gave 4-deacetylleurosine-3-carboxamide fractions purified by thin layer chromatography. These fractions were combined and the solvent evaporated in vacuo. 4-Deacetyl leurosine-3-carboxamide is obtained in the form of a tan powder having the following physical characteristics: mass spectrum (m / e): molecular ion = 751, peaks at 692, 649 and 412 mass spectra. IR: peaks at 3520, 3485, 3410, 1740 and 1695 cm ^_1, .delta.

Example 4

Preparation of 4-Deacetylleurosin-3-N- (beta-hydroxyethyl) carboxamide

The procedure of Example 3 was followed by reacting 4-deacetylleurosine-3-carboxamide with methylene chloride with ethanolamine to give 4-deacetyl-3-N- (beta-hydroxyethyl) carboxamide.

_l0 The compound was purified by chromatography on silica eluting with ethyl acetate-methanol (1: 1) as eluent. The physical properties of the resulting amorphous powder are as follows: Molecular spectrum (m / e): Molecular ion at 795; infrared spectrum: peaks at 3480, 3410,

1735 and 1670 cm ^_1 .

Example 5

Preparation of 4-desacetylleurosin-3-N-methylcarboxamide

Because 0.25 kg of leurosin sulfate is converted to the free base by dissolving the salt in water and basifying the solution. The free base insoluble in the aqueous base is extracted with chloroform. After evaporation of the chloroform, leurosin remains as the free base.

3θ The leurosin free base (corresponding to about 0.25 g of leurosin sulfate) is dissolved in 100 ml of anhydrous methanol; previously, 21 g of methylamine was added to methanol at -78 ° C. The reaction vessel was sealed and heated at 50 ° C for 5 days. The reaction vessel was opened and the volatiles were evaporated in vacuo. The residue containing 4-deacetylleurosin-3-N-methylcarboxamide was dissolved in methylene chloride. The methylene chloride solution was washed three times with water and dried. His metas

evaporation of the dichloride gave 4-deacetylleurosin-3-N-methylcarboxamide as a powder, which was reconstituted and purified by preparative thin layer chromatography on silica gel plates. The chromatogram was eluted with 1: 1 ethyl acetate / ethanol. THE

The band corresponding to 4-deacetyl leurosin-3-N-methylcarboxamide is scraped off and the alkaloid is dissolved from the silica gel with methanol. Evaporation of the methanol gave 4-deacetylleurosin-3-N-methylcarboxamide as a tan amorphous powder having the following physical characteristics (m / e): 765 (molecular ion), 779 (molecular ion + 14), 607, 426 , 353; infrared spectrum: peaks at 2.84, 5.75 and 5.79 μ.

Example 6

Process variant for the preparation of 4-deacetyl leurosin-3-N-methylcarboxamide

According to the procedure of Example 3, 4-deacetylleurosin-3-N-methylcarboxamide was prepared from methylamine and

From 4-deacetylleurosine-3-carboxazide in methylene chloride. Yield: 190 mg (900 mg of carbohydrazide 65 starting from Example 2).

Example 7

Preparation of 4-Dezace til-leurozi η-3-Ν - (beta-mercapto-e til) carboxamide

According to the procedure of Example 3, 2-mercaptoethylamine ⁵

Reaction with 4-deacetylleurosin-3-carboxazide in dichloromethane. The beta-mercaptoethyl-amine was prepared by reacting the hydrochloride salt was dissolved in 1N aqueous sodium hydroxide solution, the aqueous phase was saturated with sodium _1Q chloride and the aqueous layer was extracted with ethyl acetate, ether and methylene chloride (each solvent extracted twice). The extract 6 was combined, dried, filtered and the solvent was evaporated. The mixture of azide and beta-mercapto-ethyl- _15- amine was heated at reflux for 5 minutes in dichloromethane and then cooled. Pyridine (5 mL) was added and the reaction mixture was stirred at room temperature overnight. A 5% aqueous NaHCO ₃ solution was added and the organic layer and the ₂ θ aqueous layer were separated. The organic layer was washed twice with water and dried. The solvent is evaporated in vacuo, 4-desacetyl leurosine 3-N- (beta-mercaptoethyl) carboxamide and bis [beta- (4-desacetyl leurosine 3-carboxamido) ethyl] disulfide A mixture of ₂ 5 back stays . The residue was dissolved in dichloromethane, the methylene chloride solution was washed three times with water and the solvent was evaporated in vacuo. The residue containing the above mixture was chromatographed on silica gel and developed with dichloromethane / ethyl acetate / methanol (1: 1: 1). The TLC fractions containing 4-desacetyl leurosin-3-N- (beta-mercaptoethyl) carboxamide were combined and the solvent evaporated in vacuo. Yellowish - brown amorphous powder

4-Deacetyl leurosin-3-N- (beta-mercaptoethyl) carboxamide is obtained with the following physical characteristics: Molecular spectrum (m / e): 825, [(Molecular ion + 14) - Transmethylation], 486, 353, 299 152. Infrared spectrum (in chloroform): peaks at 1730 and _{40 at} 1665 cm @ -1. Fractions containing bis [beta (4-deacetylleurosine-3-carboxamido) ethyl] disulfide were combined and the solvent was evaporated. The physical properties of the remaining tan solid were substantially the same as those of Example 9 mercaptan. 45

Example 8

Preparation of 4-desacetylleuroform-3-carboxamide was separated into 50 volumes by silica gel chromatography using dichloromethane-methanol-ethyl acetate (1: 1: 1) as eluent. Two components are shown. The faster moving component is identified as 5'-acetonyl-4-deacetylleuroform-3-carboxamide by nuclear magnetic resonance spectroscopy, and the slower-moving, more polar component is identified as 4-deacetylleuroform-3-carboxamide. Physical properties of 4-deacetyl leuroform are as follows: mass spectrum (m / e); 765 (FD); IR (CHCl₃): 1730, 1680 ^cm-first

Physical characteristics of the other component, 5'-acetonyl-4-deacetyl-leuroform-3-carboxamide: mass spectrum (m / e): 821 (FD); the nuclear magnetic resonance spectrum is similar to that of 4-deacetylleuroformin-3-carboxamide but with a further peak at 62.12 (acetonylmethyl); IR (CHCl₃): 1730, 1680 ^cm-first

Example 9

Preparation of bis [beta - (4-deacetylleurosine-3-carboxamido) ethyl] disulfide By converting 4 g of 4-deacetylleurosine-3-carbohydrazide into 250 ml of 1N hydrochloric acid and 500 mg of the corresponding C-3 carboxazide. using sodium nitrite. The azide was isolated by dissolving in dichloromethane. To the solution was added 450 mg of cystamine ((NH ₂ -CH ₂ -CH ₂ -S) ₂ ) in tetrahydrofuran (20 ml). The reaction mixture was stirred overnight and worked up as in Example 3. The resulting bis [beta (4-deacetylleurosine-3-carboxamido) ethyl] disulfide residue was purified by chromatography on silica gel using dichloromethane / ethyl acetate / methanol as eluent.

The TLC analysis showed that the fractions containing the disulfide were combined and the solvent was evaporated. The residue was converted to the disulfate salt by the method of Example 11 below. Physical characteristics of the free base are as follows: mass spectrum: peaks at 825, 839, 486; infrared spectrum; v = 1730.1670 cm ^{1st 13} C NMR: separate resonance peaks at 37.5 and 39.2 ppm compared to 4-deacetylleurosine-3-carboxamide. Titration: pKa = 5.3 and 7.0. Molecular weight determined by osmotic pressure: 1479.

3.4 g of 4-deacetylleurosine-3-carboxamide are dissolved in a mixture of 40 ml of methylene chloride and 450 ml of acetone. The solution is cooled to -61 ° C and 50 ml glacial acetic acid, then a solution of 4 g CrO _{3 in} 8 ml water and 40 ml glacial acetic acid is added over 20 minutes. After the addition was complete, the solution was stirred at -61 ° C for 1 hour and then allowed to warm to -30 ° C. The reaction mixture was maintained at this temperature for 1 hour and then cooled again to -61 ° C. 14 N ammonium hydroxide solution and water were added. The organic layer was separated and the aqueous layer was washed with dichloromethane. The methylene chloride extracts were combined, washed once with water and dried. The residue obtained after evaporation of the solvent was

Example 10

4-desacetyl leurosine 3-N- (beta-methyl-mercapto

Preparation of ethylcarboxamide g-4-deacetylleurosine-3-carbohydrazide is converted to the azide. To a solution of the azide in methylene chloride (200 mL) was added methyl mercaptoethylamine (60 g), and the solution was stirred overnight at room temperature. The solution was washed once with 5% aqueous sodium bicarbonate solution and twice with water, dried over anhydrous sodium sulfate, filtered and evaporated. The evaporation residue 65 was applied to a column packed with silica gel,

And eluted with dichloromethane-methanol-ethyl acetate (1: 1: 1). The appropriate fractions were combined and the solvent was evaporated. The residue was then crystallized from methanol to give 220 mg of the title compound. The physical characteristics of the product are as follows: Mass spectrograph: Molecular ion = 486, 353, 299 and 152. IR: (chloroform): peaks at 1730 and 1665 cm ^_1.

Example 11

Preparation of salts

The sulfate salts of the above amides are prepared by dissolving the amides in absolute ethanol and adjusting the pH of the solutions to about 4.5 with 2% sulfuric acid in ethanol. Other salts, including salts with inorganic ions such as chloride, bromide, phosphate, nitrate and the like, and salts with organic ions such as benzoate, methanesulfonate, maleate, tartrate, are prepared in a similar manner.

The compounds of formula I are active in vivo against transplanted murine tumors and induce metaphase block in tissue-cultured Chinese hamster ovary cells (Siminoff, Applied Microbiology 2, 66-72 (1961)).

The activity of the compounds of Formula I against transplanted mouse tumors is shown to be generally intraperitoneal, daily

12, 6 and 3 mg / kg are administered for 9 days following tumor transplantation.

The following Table 1 shows the results of experiments in which transplanted mice bearing Gardner's lymphosarcoma were effectively treated with the compounds of the invention. Column 1 of the Table indicates the name of the compound; column 2 shows the dose level and

Column 3 shows the percentage inhibition of tumor growth.

Bis [beta (4-deacetyl-leurosine-3-carboxamide) -ethyl] -disulfide disulfate is effective against P388 mucosal leukemia when administered at a total dose of 15 and 30 mg / kg 3 times on 3 different days. The observed prolongation of survival was 55% and 46%, respectively.

When the compounds of the invention are administered to mammals as antitumor agents, the parenteral route of administration is usually selected. Intravenous administration is preferred, although intraperitoneal administration is also possible in smaller mammals than mice. For intravenous use, isotonic solutions containing a salt of the alkaloid base of formula I in a concentration of 1 to 10 mg / ml are used. The compounds are administered in an amount of 0.01 to 1 mg / kg, preferably 0.1 to 1 mg / kg body weight, to mammals once or twice a week or every two weeks, depending on the potency and toxicity of the agent. Another way to determine the therapeutic dose is based on the body surface area; a dose of 0.1-10 mg / nv body surface area is administered every 7 or 14 days.

Table 1

Effect on Gardner's lymphosarcoma

Compound Dose in mg / kg Inhibition% 4-desacetyl leurosine 3-karbox- amide 12 toxic 6 100 3 23 4-desacetyl leurosine 3-N-methyl- -carboxamide 12 toxic 6 100 3 33 4-desacetyl leurosine 3-N- (beta hydroxyethyl) carboxamide 12 100, 98 6 100, 100 3 50, 44 4-deacetyl to uroform-3- -carboxamide 12 39

Patent claims:

Claims (9)

Patent claims: A process for the preparation of compounds of the formula I wherein R ^{1 is} methyl, and R ^{2 is} amino, azido, -NH-C ₂ H ₄ OH, -NH- (C 1 -C 3) -alkyl or -NH-C ₂ H ₄ -SY, wherein Y is hydrogen, (C 1 -C 3) alkyl, or a chemical bond that connects two molecules of formula I through the 3-carboxamido group; obsession R ^{1 is} formyl, and R ^{2 is} an amino group and pharmaceutically acceptable acid addition salts thereof a) preparation of a compound I wherein R ^l is methyl and R ² are as defined hereinbefore, of a compound of formula II - wherein R ^{4 is} methyl, and ^R5 hydrazino - is reacted with nitrous acid, and if desired the resulting compound of formula II, - wherein R ⁵ represents the group -N - a NH ₂ The compound (III) - wherein E is hydrogen, C 1-3 alkyl, -C ₂ H ₄₀ O, or -C ₂ H ₄ -SY, wherein Y is hydrogen, a C 1-3 alkyl group, or a -S- bond that connects two compounds of Formula III, or -613 (b) for the preparation of compounds of the formula I in which: R ^{1 is} methyl, and R ^{2 is} amino or -NH- (C 1 -C 3) alkyl, a compound of formula II wherein R ^{3 4} is methyl, and ^R5 is methoxy, NH ₂ E ', (IV) compound having the formula wherein E ¹ is hydrogen or C1-3 alkyl, and, if desired, compound of the general formula is a methyl group ¹ wherein R is the preparation of compounds of formula I containing a formyl group, R ¹ is - R ² amino group is oxidized, and if desired, a resulting free base of pharmaceutically to an acceptable acid addition salt thereof. (Priority: Vili 1978. 07.) A process for the preparation of a compound according to claim 1, wherein R ^{1 is} methyl, and R ^{2 is} amino, azido, -NH-C ₂ H ₄ OH, -NH- (C 1 -C 3) alkyl, or -NH-C ₂ H ₄ - SY, wherein Y is hydrogen or C 1-3 alkyl; obsession R ^{1 is} formyl, and R ² amino group - and their pharmaceutically acceptable acid addition salts thereof, characterized in that a) for the preparation of compounds of formula I wherein R ^{1 is} methyl and R ^{2 is} as defined in the foregoing, a compound of formula II - wherein R ^{4 is} methyl, and R ^s represents hydrazino - is reacted with nitrous acid, and optionally the resulting compound of the formula II required - wherein R ^s is an azido group - a NH ₂ The compound (III) - wherein It is hydrogen, C 1-3 alkyl, -C ₂ H ₄ OH, or -C ₂ H ₄ -SY, wherein Y is hydrogen or C 1-3 alkyl powder; obsession b) for the preparation of a compound of formula I wherein R ^{1 is} methyl and R ^{2 is} amino or -NH- (C 1-3) alkyl, a compound of formula II wherein R ⁴ is methyl ⁵ and R ^{5 is} methoxy. reacting a compound of formula NH ₂ E * (IV) wherein E ^{1 is} hydrogen or C 1 -C 3 alkyl and, if desired, to form compounds of formula I wherein R ¹ is formyl 10, wherein R ¹ is methyl; The compound of formula I wherein R ^{2 is} amino is oxidized and, if desired, the resulting free base is converted to its pharmaceutically acceptable acid addition salt. (Priority: 08/08/1977) 3. A process according to claim 1 for the preparation of 4-deacetylleurosin-3-carboxamide, characterized in that 4-deacetylleurosin-3 20-KBrbohydrazide is reacted with nitro-wash. (Priority: 08/08/1977) A process for the preparation of 4-deacetylleurosin-3-carboxamide according to claim 1, characterized in that the 4-deacetylleurosine 25 -3-carbohydrazide was washed with nitric acid and then obtained 4-Deacetylleurosine-3-carboxamide is reacted with ammonia. (Priority: Vili, 08 August 1977.) The process of claim a) according to claim 1, wherein 4-desacetylleurosin-3-N- (5-hydroxyethyl) - 30-carboxamide, characterized in that 4-Deacetylhydrazine-0-carbohydrazide is reacted with nitric acid and the resulting 4-deacetylleurosine-3-carboxazide is reacted with ethanolamine. (Priority: 08/08/1977) 35 6. A process according to claim 1 for the preparation of 4-deacetylleurosin-3-N-methylcarboxamide, characterized in that the 4-deacetylleurosin-3-carbohydrazide is reacted with leurosin-3-carboxazide methyl 40-amine. (Priority: 08/08/1977) 7. A process according to claim 1 for the preparation of 4-deacetylleurosin-3-N-methylcarboxamide comprising reacting leurosin with methylamine. (Priority: 1977. VIII 08/08) 8. A process according to claim 1 for the preparation of 4-deacetylleurosin-3-N- (mercaptoethyl) carboxamide, characterized in that the 4-deacetylleurosine-3-carbohydrazide is reacted with a 50 acids, and the resulting 4-deacetylleurosin-3-carboxazide was reacted with 2-mercaptoethylamine. (Priority: 08/08/1977) The process of claim a) according to claim 1, wherein bis [p- (4-deacetylleurosine-3-carboxyl) 55 amido) ethyl] disulfide, characterized in that 4-deacetyl leurosine-3-carbohydrazide is reacted with nitrosoate and then the resulting 4-deacetyl leurosin-3-carboxazide is reacted with cystamine. (Priority: 1978. VIII. 07.) 3 drawings Responsible for publishing: Director of Economic and Legal Publishing 84.4453 - Zrínyi Printing House, Budapest