DE1545759C3 - Vincaleukoblastin derivatives and a process for their preparation - Google Patents

Description

3 4

The invention relates to new vincaleukoblastin derivatives of the general formula

4J-O-C-CH, -N

wherein R _{1 is} a hydrogen atom, an alkyl group with 1, 3, 4 or 5 carbon atoms or a cycloalkyl group with 3 to 8 carbon atoms, R _{2 is} an alkyl group with 1,3,4 or 5 carbon atoms or R ₁ and R ₂ together with the nitrogen atom, to which they are bound, a pyrrolidino or morpholino group and R _{3 is} an alkyl group having 1 to 5 carbon atoms, as well as their non-toxic salts with acids and a process for their preparation.

If R ₁ and R _{2 are} alkyl groups having 1, 3, 4 or 5 carbon atoms and R _{3 is} an alkyl group having 1 to 5 carbon atoms, they can, for. B. methyl, ethyl (R ₃ ), isopropyl, n-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-amyl, tert.-

Be amyl, neopentyl, 2-pentyl, 3-pentyl or 3-methyl-2-butyl groups. When R _{1 is} a cycloalkyl group having 3 to 8 carbon atoms, it can e.g. B. be a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl group.

If the radical R _{3 is} a methyl group, the compounds of the formula I belong to the vincaleukoblastine (vinblastine) series and are esters of deacetylvincaleukoblastine, hereinafter referred to as deacetyl-VLB.

The vincaleukoblastin derivatives of the general formula I and their non-toxic salts with acids are prepared by adding a compound of the general formula

CH ₃ OOC

CH ₃ O

C-CH ₇ -Cl

l / -OH R ₃ COOCH ₃

with an excess of an amine of the general formula

R ₁

HN

(III)

In the reaction is used as an inert solvent, for. B. tetrahydrofuran, carbon tetrachloride, ethanol, Ethyl acetate or benzene is used. During this reaction, the amine hydrochloride precipitates and is filtered off. The compound of the general formula 1 thus formed is prepared by the usual methods

ⁿ d isolated, e.g. B by dissolving the desired

² binding in an aqueous acid, extracting the

aqueous acid with a water-immiscible

in the presence of an inert solvent reacts 65 baren solvent, making the aqueous one alkaline and optionally then the obtained pro-phase, extracting the alkaloid with a with duct with an acid into a non-toxic salt over water-immiscible organic solvent, leads. Separating the organic layer and recovering the

desired compound as a residue after evaporation of the solvent. The received new Alkaloid derivative is obtained either by chromatography or by conversion of the free alkaloid base into a mineral acid salt and recrystallizing this salt or by other suitable methods further cleaned. Another way of cleaning the by following the procedures outlined above produced free bases is the fractional extraction with gradually changing pH values.

For the preparation of the non-toxic salts of the compounds of the general formula i come as Acids e.g. B. hydrochloric, sulfuric, phosphoric and phosphorous acid, boron, vinegar, benzoin, amber, Maleic, tartaric and citric acids are possible. Because of the easy hydrolyzability of various groups of the compounds according to the invention, the preparation of the salts must be carried out considerably more carefully than with common organic bases. So z. B. the sulfate one of the compounds according to the invention be prepared by dissolving the free base in aqueous methanol or in ethanol and then with a equivalent amount of a 1% sulfuric acid solution in the same solvent is added. If an aqueous solvent system is used, the dilute acid can be used until it is reached a predetermined pH value can be added. The salt is then removed by evaporation of the solvent isolated in a vacuum, carefully avoiding any overheating.

Starting materials of the general formula II, in which R _{3 is} not a methyl group, are prepared by alkylating desmethyl desacetyl-VLB- (4) -chloroacetate with an appropriate alkylating agent.

So z. B. Dcsmcthyldesacctyl-VLB- (4) -chloracctate with acetaldehyde and hydrogen in the presence of a platinum catalyst to N-Äthyldcsmcthyldesacctyl-VLB- (4) -chloroacetat be reacted. Other conventional alkylation agents can be used to prepare the higher homologues of this compound, e.g. B. Nn-propyl, Nn-butyl. N-isobutyl and N-isoamyl derivatives can be used.
From DT-PS 1 185 615 it is known that

ίο Halogenalkyldcrivatc with amines to the corresponding Allow aminoalkyl compounds to react. In the compounds to be reacted according to the invention however, if there are several carboxylic acid ester groups which could convert to amides, surprisinglyc However, only the reaction of the a-chlorocarboxylic acid group occurs with the formation of substituted ones fi-aminoacyl ester.

The compounds of the invention can, for. B. serve to treat leukemia.

The compounds of the invention significantly extend the life of DBA / 2 mice. those with a lethal dose of p-1534 leukemia cells as can be seen from the following table. The dose is given in column 4 and in column 5 the extension of the lifespan of the treated mice compared to that of a group of control mice, which have only been inoculated with the respective pharmaceutical carrier, which for the Injection of the active substance was used.

The for. the doses used in the tests each represented the maximum permissible dose, with which none was yet Toxicity was observed; at higher doses all compounds are toxic. The preparations were thus administered in equitoxic amounts.

R, CH ₃ R ₂ Rj dose
(mg / k ») renewal
(%) - (CH ₂ J ₄ - CH ₃ CHj 5 5 out of 5 survivors - (CH ₂ J ₂ -O- (CH ₂ J ₂ - CHj 7.5 100% CH ₂ - CH ₂
\ /
CH CH ₃ 15th 127% H CHj CH ₃ 3.75 201% +
4 out of 5 survivors CHj CH ₃ 3 187% +
3 out of 5 survivors

The compound vincaleukoblastin (VLB) was also tested in the manner indicated above. The dose administered to the mice was 0.35 mg / kg and resulted in an increase in lifespan from 25 to 30%. The compound VLB, however, has a very high toxicity, which requires a higher dosage prohibits, as a dose higher than 0.35 mg / kg is lethal. How to get from this and from the values As shown in the table above, the known VLB was inferior to the compounds according to the invention.

The invention is further illustrated by the following exemplary embodiments.

example 1
Desacetyl VLB- (4) -N, N-dimethyl-glycinate

500 mg of deacetyl-VLB- (4) -chloracctate were dissolved in 10 ecm of tetrahydrofuran. 4 ecm of a 25% Solution of dimethylamine in tetrahydrofuran were added and the resulting solution at overnight Left to stand at room temperature. The resulting precipitate of dimethylamine hydrochloride was filtered off, and the filtrate was evaporated to dryness in vacuo. To the residue were Given 50 ecm of water, and the aqueous solution was by adding 14N ammonia in excess made alkaline. Desacetyl-VLB- (4) -N, N-dimethyl-glycinate, which is insoluble in the alkaline layer was extracted with methylene chloride. The methylene chloride layer was separated and dried and the solvent evaporated in vacuo. The residue that consists of deacetyl-VLB- (4) -N, N-dimethyl-glycinate consisted, was recrystallized from ether; Yield: 475 mg (95%).

Crystalline Acetyl-VLB ^ -N.N-dimethyl-glycinate (400 mg) was in a mixture of methanol

and dissolved water. The pH of the solution was brought to about 1.8 with 1% sulfuric acid. The resulting solution was evaporated to dryness in vacuo to give desacetyl VLB- (4) -N, N-dimethyl glycinate sulfate as a residue in practically 100% yield. The residue was obtained by recrystallization from a mixture of methanol and ethanol cleaned.

Desacetyl - VLB - (4) - N - ethyl - N - η - propyl - glycinate was prepared in 85% yield by using N-ethyl-N-n-propylamine in place of dimethylamine was used in the above procedure.

The required raw material was produced as follows:

8 g were dissolved vincaleukoblastine saturated at 0 ⁰ C solution of hydrogen chloride in anhydrous methanol in 1. 1 The mixture was ^{swirled for about 1} _1/2 hours to dissolve the substance. Then, a calcium chloride drying tube was placed on the flask with the solution and the solution was allowed to stand at room temperature overnight. The solution was evaporated in vacuo and the remaining gummy substance was dissolved in water. Pieces of ice were added to the aqueous solution and then made alkaline with aqueous ammonia. The insoluble alkaloid which had precipitated out was extracted with methylene chloride and, after evaporation of the methylene chloride, was recrystallized in vacuo from a methanol-water mixture. The resulting brown-yellow colored crystals were dissolved in 30 ecm of methanol, and water was added to the solution until it became cloudy, and the pH was brought to 3.0 with 1% strength sulfuric acid. The resulting solution was evaporated in vacuo, benzene being added at intervals to azeotropically distill off the water. The deacetyl VLB sulfate obtained was purified by recrystallization from ethanol.

300 mg of deacetyl VLB sulfate were in water dissolved, the mixture was made alkaline with ammonium hydroxide and the precipitated deacetyl-VLB extracted with methylene chloride. The methylene chloride was evaporated in vacuo and there were several portions of 10 ecm benzene each are added and evaporated in vacuo to azeotrope the substance to dry. The amorphous residue was dissolved in 10 ecm of methylene chloride and treated with 300 mg of chloroacetic anhydride offset. The solution was left to stand overnight, whereupon 10 ecm of methanol was added to destroy excess anhydride. The mixture was left to stand for 2 hours and then evaporated in vacuo. The residue of acylated product was dissolved in 20 ecm of methylene chloride dissolved and the solution was quickly washed with cold, dilute aqueous ammonia. The methylene chloride was evaporated in vacuo, and the residue was dissolved in 20 ecm of methanol, the 5 ecm Contained water, dissolved. 1 g of chromatographically pure silica gel was added to the solution and the The mixture was stirred for 6 hours, with all of the 3-chloroacetate that had formed during the acylation to the 3-hydroxy compound was hydrolyzed. The silica gel was filtered off and repeated with warm Methanol washed. The filtrates were combined and vacuumed to a volume of about 5 ecm constricted. The concentrate was made alkaline with dilute aqueous ammonia, and that aqueous-alkaline mixture extracted quickly with methyl chloride. The methylene chloride extract was in Evaporated to dryness in vacuo and the amorphous residue from deacetyl-VLB- (4) -chloracctate in 25 ecm of ether dissolved, whereupon crystalline deacetyl-VLB - ^ - chloroacetate slowly precipitated. The crystalline Substance was filtered off and air-dried.

Be i s ρ i e 1 2

DesacetyI-VLB- (4) -N-methyl-N-cyclopropyI-glycinal

Deacetyl-VLB- (4) -chloroacetate was treated with N-methyl-cyclopropylamine reacted by the method of Example 1, only the reaction mixture was allowed to stand for about 48 hours at room temperature and then heated to 50 ° C for about 1 hour. The product was isolated according to the procedure of Example 1, in benzene on a column of alumina No. 2 and with a benzene-chloroform mixture Chromatographed 9: 1 as eluent. The first fractions were combined and im Evaporated to dryness in vacuo. The yield was 43%. The sulfate of desacetyl-VLB- (4) -N-methyl-N-cyclopropyl-glycinate was made by dissolving deacetyl - VLB - (4) - N - methyl - N - cyclopropylglycinate in ethanol and decomposition of 1% ethanolic sulfuric acid until the pH value is reached 3.4 produced.

After evaporation of the solvents in vacuo, a residue of deacetyl-VLB- (4) -N-methyl-N-cyclopropyl-glycinate sulfate was obtained obtained, which was recrystallized from a mixture of methanol and ethanol. The yield was 60%.

Example 3 Desacetyl-VLB- (4) - «- morpholinoacetate

Following the procedure of Example 1, desacetyl VLB- (4) chloroacetate was obtained reacted with morpholine in tetrahydrofuran. Desacetyl VLB- (4) -r / -morpholinoacetate was isolated and purified according to the procedure of Example 1. The yield was 58%. The corresponding Sulphate was prepared according to the procedure of Example 1.

Example 4 • Desacetyl-VLB- (4) -a-pyrrolidinoacetate

Following the procedure of Example 1, desacetyl VLB- (4) chloroacetate was obtained with pyrrolidine in tetrahydrofuran to give deacetyl-VLB- (4) - «- pyrrolidinoacetate with implemented a yield of 63%, which separated and purified by the method of the same example became. The deacetyl-VLB- (4) - «- pyrrolidinoacetate obtained was recrystallized from benzene. A Crystalline sulfate was prepared according to the procedure of Example 1.

Example 5 Desacetyl VLB- (4) -N-methyl-glycinate

Following the procedure of Example 1, deacetyl VLB- (4) chloroacetate was obtained and N-methylamine in tetrahydrofuran to give deacetyl-VLB- (4) -N-methyl-glycinate implemented, which was isolated and purified by the method of Example 1. The yield at crystalline material was 40%.

The corresponding sulfate was prepared according to the procedure of Example 1.

509 640/30

All compounds obtained according to Examples 1 to 5 were identified by comparing their nuclear magnetic resonance and infrared spectra with those of VBL and other known derivatives of this alkaloid. After a crystalline product (either the free base or the salt) was obtained before the spectral data obtained by thin layer chromatography (mobile phase: ethyl acetate, adsorbent: silicon dioxide, CerdVJ ammonium sulfate development) (1,3), showed that the material was free of alkaloid impurities was. The crystalline solids were dissolved several times in chloroform and the solvent evaporated in a vacuum to remove the crystallization solvents before the substances (i.e. amorphous solids) were subjected to nuclear magnetic resonance analysis. The characteristic absorptions of the introduced acyl or acetylamino groups were compared with the known absorptions the root connection.

The NMR spectra only depend on the hydrogen atoms in the molecule. In the present case a number of hydrogen atoms are the same. Hence the subsurface spectrum is for all connections roughly the same. The following peaks are common to the NMR spectra of all compounds: 2.73, 3.67; 3.83; 5.57; 6.15 and 6.67 ppm; a triplet at 0.91 ppm and a broad doublet at 3.27 ppm.

The characteristic peaks of the NMR spectra (using tetramethylsilane as the standard) for the compounds of the examples are given below. The NMR spectrum of the deacetyl - VLB - (4) - N ₁ N - dimethyl - glycinate (Example 1) is shown in the drawing. The above common peaks are only given where there is a shift.

The NMR spectrum of the deacetyl-VLB-4-N-methyl-N-cyclopropyl-glycinate (Example 2) has a band at 3.45 ppm, a doublet around 0.43 ppm and a peak at 2.57 ppm (this corresponds to the 2.40 ppm peak of the Ν, Ν-dimethyl compound, which is shifted and occurs with less intensity).
The NMR spectrum of the deacetyl VLB-4-u-morpholinoacetate (Example 3) has a band at 2.40 ppm plus a non-specific absorbance at 2.67 and a band at about 6.67 ppm (shifted to 6.62 ppm Band of Ν, Ν-dimethyl compound).

The NMR spectrum of the deacetyl VLB-4-u-pyrrolidinoacetate (Example 4) has a peak at 6.50 ppm (the shifted peak 6.67 ppm of the Ν, Ν-dimethyl compound) and a non-specific absorption in the range from 1.3 to 2.5 ppm, and a band at 2.40 ppm. .

The NMR spectrum of the deacetyl VLB-4-N-methylglycinate (Example 5) has a 2.40 ppm peak of the Ν, Ν-dimethyl compound, but with a lower level Intensity and also a new band at 1.96 ppm.

Microanalysis proved of little value on these compounds, the molecular weights of which were approaching a thousand, and which stubbornly held back the solvents of crystallization.
In summary, it can be stated that the analytical method used was thin-layer chromatography, both to control the reactions and to prove the purity of the products. The products were then identified primarily by nuclear magnetic resonance analysis.

1 sheet of drawings

Claims (2)

Patent claims:
1. Vincaleukoblastin derivatives of the alluemeincn formula wherein R _{1 is} a hydrogen atom, an alkyl group with 1, 3, 4 or 5 carbon atoms or a cycloalkyl group with 3 to 8 carbon atoms, R _{2 is} an alkyl group with 1, 3, 4 or 5 carbon atoms or R ₁ and R, together with the nitrogen atom, to which they are bound, a pyrrolidino or morpholino group and R _{3 is} an alkyl group with 1 to 5 carbon atoms, as well as their non-toxic salts with acids. 2. Process for the preparation of the vincaleukoblastin derivatives according to claim 1, characterized in that that you can connect; the general formula with an excess of an amine of the general formula HN reacted in the presence of an inert solvent and, if appropriate, then the product obtained converted into a non-toxic salt with an acid.