CS199615B2 - Process for preparing derivatives of alkaloids - Google Patents

Abstract

Novel amide derivatives of dimeric vinblastin alkaloids of formula <IMAGE> wherein the substituents are defined in Claim 1, are prepared. These compounds are obtained by reacting corresponding compounds in which R denotes methoxy and R<1> represents H, OH or acetoxy, with ammonia, methylamine or hydrazine. Resulting compounds in which R denotes an NH-NH2 group can be converted by reaction with a nitrosylating agent into the azide, which can subsequently be converted by means of primary or secondary amines into further amide derivatives. The novel compounds may be used as antiviral or antineoplastic agents.

Description

R is NH2, NH-NH2, N (CH3) 2, -pyrrolidinyl, NH-alk-X, NH-cycloalkyl of 3 to 8 carbon atoms, NH-alk-Am, NH-al.k- [ OH) ₁ _ ₃ or N3 where alk is alkyl of 2-6 carbon atoms,

Am represents NH2, NHCH3 or N (CH3) 2a

X is hydrogen, cyano, phenyl, -carboxyl, carbalkoxy of 1 to 3 carbon atoms in the alkoxy-group or carboxamide,

R ¹ is hydrogen, - hydroxy, O-alkanoyl of 1-3 carbon atoms or O-chloralkanoyl of 1 to 3 carbon atoms,

R 2, R 3 and R ⁴ are hydrogen or hydroxyl, provided that when R 2 is -hydrogen, R ³ and R 4 are also hydrogen, when R 2 is hydroxyl is at least one of R 3 or R ⁴ also has a hydroxyl group.

The process according to the invention consists in:

a) reacting a compound of formula I in which R is O-CH 3,

R1 is hydrogen or acetyl; and R2, o

R ³ and R ⁴ are as defined above with the compound of formula II

NH ² R ⁵ (III, where

R ⁵ represents a hydrogen atom, a methyl group or an amino group, and optionally, if desired

(b) when R is NH-NH2, the compound thus obtained is reacted with a nitrosating agent and a compound of the formula

R ⁶

OF

NH \

R ⁷ where

R ⁶ is hydrogen or methyl;

R ⁷ represents methyl, -alk-X, cycloalkyl of 3 to 8 carbon atoms, -alk-Am or -alk -; (OH) 1-3, wherein alk represents an alkyl group of 1 to 6 carbon atoms, and

Am and X are as defined in formula I, whereupon the compound obtained in step (a) or (b) is optionally acylated where R ¹ is hydroxyl to form a compound in which R ¹ is different from the hydroxyl group and optionally reacting any of the products obtained with a non-toxic, inorganic or organic acid to form an acid addition salt of the resulting product.

It has been found that several naturally occurring alkaloids obtainable from Vinca rosea have activity against experimental cancers in animals. These are in particular · leurosine U.S. Patent No. 3,370,057, vinblastine i [vincaleukoblastine] U.S. Patent No. 3,097,137, leurosidine (vinrosidine) and leurocristine (vincristine) according to U.S. Patent No. 3,205,220. Two of these alkaloids, vinblastine and leurocristine are now on the market as drugs against malignant nations, especially against leukemias and related diseases in humans. Of these derivatives, leurocristine is the most active for the treatment of leukemias, but this derivative is present in the smallest amount among the alkaloids of Vinca rosea.

Chemical modifications of these alkaloids are more or less limited. First of all, the structure of these substances is very complex and therefore it is difficult to design clinical reactions that could affect specific functions in the molecule. In addition, fractions that are completely ineffective with respect to chemotherapeutic properties are often obtained in this manner, the chemical composition of which is very similar to that of the active substances. Thus, the antitumor activity is apparently limited to very specific structures and there is little chance of obtaining a more active substance by chemical changes.

One successful modification of physiologically active alkaloids was the production of dihydrovinblastine, described in U.S. Patent No. 3,352,868 and replacement of the acetyl group at the C-4 position of the vinblaistine ring in Formula I with a higher alkanoyl or other acyl group, as described in U.S. Patent No. 3 Some of these derivatives extend the life of mice immunized with P1534 leukemia.

One of these derivatives in which the chloroacetyl group has replaced the acetyl group at the 4-position of vinblastine is also a useful intermediate for the production of structurally modified vinblastine compounds in which the acetyl group of the vinblastine at the 4-position is replaced by Ν, Ν-dialkylglycyl. In the course of the production of said derivatives, a further intermediate, 4-desacetylvinblastine, was obtained. This intermediate in which the acyl group at the 4-position, where the non-esterified hydroxyl group is present instead, is toxic and has very low chemotherapeutic activity against rat P1534 in leukemia as described in Hargrove, Lloydia, 27, 340 (1964).

A preferred group of compounds of formula (I) consists of the amides of vinadadioline, leurocolombine, 4-desacetoxyvinblastine, 3'-hydroxy-4-desacetoxyvinblastine, deoxyvinblastine and 4-desacetylderivative, any of the above dimeric alkaloids having an acetoxy group at the 4-position, as well as pharmaceutically acceptable salts of the above compounds except those wherein R is NH-NH 2 and N 3 with non-toxic acids.

Examples of the groups alk- (OH) _1-3 , alk-Am and alk-X in the above formula are:

2-methylpentyl, isohexyl, isopentyl, n-pentyl, n-hexyl, sec.hexyl,

2-hydroxy-n-hexyl,

5-cyano-n-pentyl,

2-hydroxyethyl, ethyl, isopropyl, n-butyl, sec-butyl, cyanomethyl, cyanoethyl,

3-hydroxypropyl,

2-dimethylaminoethyl,

2-aminoethyl,

2-methylaminoethyl,

2-hydroxypropyl, benzyl, phenethyl,

4-fenblbytyl,

2-aminoprypyl,

2-aminohexyl,

2-dimethylaminopropyl, 2,2 &apos; -dihydroxyisopropyl, 2,2 &apos; -dihydroxy-t-butyl, 2,2 &apos;, 2 &apos; -trihydroxy-t-butyl and the like.

In formula (I), the terms alkanoyl of 1 to 3 carbon atoms and chloralkanoyl of 1 to 3 carbon atoms are groups such as acetyl, chloroacetyl, propionyl, 2-chloropropionyl, 2-chlorobutyryl and butyryl, which may also be represented by CO Alkyl having 1 to 3 carbon atoms in the alkyl moiety, alkanoyl or CO- (C1) alkyl having 1 to 3 carbon atoms in the alkyl moiety when it is a chloroalkanoyl group. The term NH-cycloalkyl of 3 to 8. carbon atoms include cyclopropylamino, cyclobutylamino, cyclopentylamino, cyclohexylamino, cycloheptylamino, cyclooctylamino. The term C1-C3 carboalkoxy in the alkoxy moiety includes carbomethoxy, carbethoxy, carbisopropoxy and carb-n-propoxy.

When the radical X in the group alk-X · is phenyl, this phenyl group may carry conventional substituents which are usually found on aromatic nuclei such as lower alkyl, lower alkoxy, hydroxy, halogen, nitro, etc., and one, the phenyl group may carry more than one of these substituents, which may be the same or different and may differ from the original substituents. An example of such groups is

4-hydroxyphenyl,

2,4-dichlorophenyl, 2-methyl-C-chlorophenyl,

2.4- dinitrophenyl,

3,5-xylyl, 4-tolyl,

2-tolyl,

3-ethoxyphenyl and the like

Non-toxic acids which are suitable for the formation of pharmaceutically acceptable amine addition salts are e.g. inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, nitrous, phosphorous and the like as well as non-toxic organic acids, especially aliphatic mono- and dicarboxylates, alkanoates, phenyl substituted, hydroxyalkanoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Thus, pharmaceutically acceptable acids are sulfates, pyrosulfates, acid sulfates, sulfites, acid sulfites, nitrates, phosphates, middle phosphates, acid phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, iodides, decanoates, caprylates, acrylates, formates, isobutyrates, capronates, heptanoates, propionates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleate butin-1,4-dioates, hexin-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybene , phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, 2-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, N2F talef ^{ι-l-sulfofáty,} naphthalene-2- sulfonates and the like

Most of the compounds of formula (I) may be described as derivatives of vinblastine. Vinblastine is a compound of formula I wherein R is CH 2 O, R ¹ is acetoxy, R ² is β-hydroxyl (the C-4'-ethyl group is in the a position) and R 3 and R 4 are hydrogen atoms. Desoxyvinblastine is a compound of formula I wherein R 2, R 3 and R 4 are hydrogen, R is. CH 3 O and R 1 are acetoxy. There are two isomers of desoxyvinblastine, · A and · Β. In the case of the A isomers, the hydrogen atom of R2 is in the β position, i.e. upward, and the ethyl group in the 4 'position is α, i.e. downward. Isomer B has an ethyl group and a hydrogen atom in the a 'to 4' position.

Vincadioline is a 3‘-hydroxy derivative of vinblastine in which R 3 is hydroxy.

Leurocombin is a 2'-hydroxy derivative of vinblastine in which R ⁴ is, for example, a hydroxyl group.

Vincadioline has the following properties: mp 218-220.5 ° C with decomposition. X-ray diffraction using radiation filtered by chromium λ = 2.2986 A.

d in A I / Ii d in A I / Ii

9.55 100—1 3.99 60 8.87 90) 3.71 20 May 8.63 90) 3.64 15 Dec 7.78 05 / 3.44 1OB 7.57 60 3.19 20 May 7.21 50 3.05 05 / 6.00 40 2.85 20 May 5.88 40 2.78 10 5.58 70—3 2.61 10 5.22 20 May 2.44 15 B 5.08 20 May 2.21 05 B 4.70 50 2.07 05 / 4.57 40 1.98 15 Dec 4.42 05 / 1.91 05 / 4.31 05 /

NMR δ 7.13, 7.53, 8.04, 3.60, 6.61, 6.09, 3.79, 2.70, 9.77, 5.47, 2.09, 0 , 80, 5.85, 5.29, 5.63, 3.84, 0.91.

Infrared absorption maxima at 3480, 1745 and 1725 cm @ -1.

Molecular weight 826, empirical formula C 18 H 18 N 2 O 10 and m / e = 826, 170, 371.

Wavadioline is produced as follows: leaves and stems containing a whole group of alkaloids, for example Catharanthus roseus (Vinca rosea), are extracted with a water-immiscible solvent such as benzene. The benzene is distilled from the extract in the presence of an aqueous solution of tartaric acid and the pH of the resulting aqueous acid extract is adjusted to pH 6 by addition of a base. The sheets may also be contacted with an aqueous acid solution at pH 3 and the resulting acidified layer extracted with benzene. The benzene layer was separated and discarded and the pH of the aqueous layer was adjusted to 6 as above. The dimeric alkaloids are then extracted from the aqueous layer into an organic solvent, usually benzene, or gel filtration of the alkaloid extract using a cross-linking dextran gel, for example Sephadex G-25F, with the mobile phase at pH 3.0 with citrate buffer with an ammonium concentration of 0.1 Μ. A pressure of about 105 kPa is used during gel filtration. First, those fractions of dimeric alkaloids containing leurocristine, vinblastine, des-N-methylvinhlastine, leurophormine, leurosine and leurosidine are eluted. The dimeric alkaloids are extracted from a buffer of pH 3 by adjusting the pH of the solution to 7.0 with a base and then mixing the resulting aqueous solution with a water-immiscible solvent, preferably again with benzene. Evaporation of benzene gave a residue which was dissolved in ethanol, directly crystallizing leurosine. The crystals were separated by decantation, the supernatant was acidified to pH 4.2 with a 3% ethanolic sulfuric acid solution to convert the remaining dimeric alkaloids into sulfates which precipitated. The precipitated salts are separated and converted to the corresponding free alkaloids in a standard manner, for example by dissolving in water, adjusting the pH to 8.0 with ammonium hydroxide and extracting the dimeric alkaloids with a water-immiscible solvent, preferably methylene dichloride. Evaporation of methylene dichloride gave a mixture of dimeric alkaloids which was then subjected to high pressure chromatography on alumina of activity III using a 25: 75: 0.4 mixture of ethyl acetate, methylene dichloride and water as the solvent system.

An elevated pressure of between 1 and 50 bar is used. It goes without saying that the high-pressure chromatography apparatus allows processes to be carried out at a pressure of from 28 to 35 MPa, and can withstand a pressure in the range of 52.5 to 56 MPa. The separation of alkaloids is usually more effective at elevated pressure. The device used is of the usual type, mostly of stainless steel, provided with a seal which resists high pressure.

In this case, the alkaloids are eluted in the following order: residual leurosine, vinblastine, de-s-N-imethylvinylblastine, leurocristine and leurosidine. The identification of dimeric alkaloids in the eluate is carried out by standard methods, for example thin layer chromatography.

After the elution of known alkaloids, several higher polarity dimeric alkaloids remain in the column. These alkaloids are eluted with methanol and subjected to further chromatography until vincadioline is obtained as a separate fraction, substantially free of the other dimeric alkaloids that were present in the mixture.

Leurokolombin has the following properties:

pKa at 5.05, 6.3, infrared absorption maxima at 2.80, 2.88, 3.35, 5.74, 6.18, 6.65, 6.83, 6.95, 7 25, 7.50, 8.11, 9.60, 9.90, and 10.75 microns, maximum in ultraviolet light at 217 (? _M = 51.091) and 265 (? _M = 15.666) nm, molecular weight. = 826, empirical formula C46H58N4O10, ion fragments in mass spectroscopy m / e 795, 767, 749, 667, 649, 282, 170, 156,

154, 152, 144, 143

9 6-15 proton NMR spectra with chemical shifts in ppm at 7.51, 7.13, 0.90, 3.60, 3.75, 7.01, 3.84, 6.15, 5.29, 5.85, 5.48, 0.78, 2.68, 3.79, 2.70, 2.10 and 4.16.

Sulfate has the following diffraction spectrum using an X-ray beam and a chrome filter at 2.2896 A.

d in A I / I2 17.00 30 12.50 100 ALIGN! 9.45 50 7.70 10 7.20 60 6.20 20 May 5.70 30 4.95 05 / 4.65 20 May

The leurocombinabs are prepared as follows. Leaves of plants containing crude alkaloids, namely Oatharanthus roseus (Vinca rosea), are extracted with a water-immiscible solvent such as benzene. The benzene was distilled off from the extract in the presence of an aqueous solution of tartaric acid, the pH of the resulting acidified aqueous extract was adjusted to 6 by addition of a base. Alternatively, the leaves are contacted with an aqueous acid solution at pH 3 and the resulting acidic layer is extracted with benzene. The benzene layer is separated and discarded and the pH of the aqueous layer is brought to pH 6 as above. The dimeric alkaloids are then extracted from the aqueous layer with an organic solvent, usually benzene. Optionally, gel filtration of the extracted alkaloids is performed using a cross-linking dextran gel, such as Sephadex G-25F, with the mobile phase at pH 3.0 being buffered with 0.1 M ammonium citrate. approximately 15 psi. In this process, the fraction of ‘dimeric alkaloids containing leurocristine, vinblastine, des-N-methylvinblastine, leurophormine, leurosine and leurosidine are first eluted. The dimeric alkaloids are extracted from the pH 3 buffer by adjusting the pH to 7.0 with a base and extracting the aqueous solution with a water-immiscible solvent, for example benzene again.

Evaporation of benzene gives a residue which can be dissolved in ethanol, directly crystallizing leurosine. These crystals were separated by decantation, the supernatant was acidified to pH 4.2 with a 3% ethanolic sulfuric acid solution to convert the remaining dimeric alkaloids into sulfates which precipitated.

The precipitated salts are separated and converted to the corresponding free alkaloids in a standard manner, for example by dissolving in water, adjusting the pH to 8.0 with ammonium hydroxide and extracting the dimeric alkaloids with a water-immiscible solvent, preferably methylene dichloride. Evaporation of methylene dichloride gave a mixture of dimeric alkaloids which was then subjected to high pressure chromatography on alumina of activity III to IV using a mixture of ethyl acetate, methylene dichloride and water in a volume ratio of 27: 75: 0.4 as the solvent system.

An increased pressure in the range of 1.05 to 2.45 MPa is used. It goes without saying that the high-pressure chromatography apparatus allows processes to be carried out at a pressure of from 28 to 35 MPa, and can withstand a pressure in the range of 52.5 to 56 MPa. The separation of alkaloids is usually more effective at elevated pressure. The equipment used is of the conventional type, mostly of stainless steel, provided with a high pressure seal.

In this case, the alkaloids are eluted in the following order: residual leurosine, vinblastine, des-N-methylvinblastine,. leurocristine and leurosidine. The identification of dimeric alkaloids in the eluate is carried out by standard methods, for example by thin layer chromatography.

4-Desacetoyyvin: Blainine has the following physical and chemical properties:

mp 183-190 ° C dec. after recrystallization from methanol, [α] _D 26 = 4-9-5 · 3 ° (chloroform), molecular ion M + = 752, empirical formula C44H56N4O7

Analysis for C44H56N4O7 calculated:

% C, 70.19;% H, 7.50;

14,87% H found:

% C, 69.71;% H, 7.47;% N, 7.08;% O-10-deacetylblastine is prepared as follows. Leaves of plants containing crude alkaloids, i.e., anthus roseus (Vinca rosea) previously moistened with an aqueous ammonia solution, are extracted with a water-immiscible solvent such as benzene. The benzene is distilled off from the extract in the presence of an aqueous solution of tartaric acid. This layer was extracted with a water-immiscible organic solvent and then basified by the addition of ammonia. The dimeric alkaloids are then extracted from the alkaline layer with an organic solvent, usually benzene. Evaporation of benzene yields a mixture of amorphous dimeric alkaloids which are dissolved in benzene and chromatographed on alumina (CAMAG activity III).

The alkaloids are eluted in the following order: leurosine, vinblastine, des-N-methylvinblastine, leurocristine and leurosidine. The identification of dimeric alkaloids in the fractions eluted in 1996615 is carried out by conventional standard methods, for example thin layer chromatography. Vinblastine is usually eluted with a 1: 1 v / v mixture of benzene and chloroform. The method for obtaining a VLB fraction is described in more detail in US Patent No. 3,225,030.

The vinblastine fractions thus obtained still had small amounts of additional alkaloid, which was identified as 4-desacetoxyvinblastine by thin layer chromatography analysis. This second alkaloid can be isolated as follows. The vinblastine fraction was converted to the corresponding sulfate in a conventional manner, and the sulfate was subjected to separation by dissolving it in a 2% aqueous citric acid solution and extracting the solution twice with benzene. The pH was then adjusted to 5.5 by addition of ammonia and the solution was extracted twice more with benzene. The second fraction is chromatographed on alumina with activity III and the chromatogram is developed with benzene. Fractions containing a second alkaloid in addition to vinblastine by thin layer chromatography analysis are pooled and re-chromatographed on alumina, whereupon the chromatogram is again developed with benzene. This procedure was repeated, the fractions containing predominantly the second alkaloid, 4-desacetoxyvinblastine containing only a small amount of vinblastine, were combined and recrystallized from methanol. The recrystallized 4-desacetoxytinblastine is then further purified by preparative thin-layer silica chromatography using a 3: 2: 4 mixture of diethylamine, chloroform and benzene as solvent mixtures.

The 3‘-Hydroxy-4-desacetoxymembrane has the following physical properties: the proton NMR spectrum has peaks at δ 4.075 (5),

5.85 (15),

5.46 to 5.78 (broad multiplet), mass spectrum ions at m / e 768, 411, 371, 224, 170, 102.

3'-Hydroxy-4-desacetoxyvinblastine is produced as follows: Defatted plant leaves containing crude alkaloids, i.e. Catharanthus roseuis (Vinca rosea), pre-moistened with aqueous ammonia solution, are extracted with a water-immiscible solvent such as benzene. in the presence of an aqueous solution of tartaric acid, the solution in tartaric acid is then made alkaline by addition of ammonia.

Dimeric alkaloids are extracted from the alkali layers with an organic solvent, usually benzene. Evaporation of the solvent gave a mixture of amorphous dimeric alkaloids. The fraction of dimeric alkaloids is dissolved in ethanol and the corresponding sulfates are formed by adding an ethanolic solution of sulfuric acid. The mixed sulfates in crystalline form are separated and then converted to the corresponding free bases by dissolving in water, alkalizing the aqueous solution and extracting the alkaloids with an organic solvent immiscible with water, usually methylene dichloride. Evaporation of the solvent yields a mixture of amorphous dimeric alkaloids, which are redissolved in methylene dichloride and chromatographed on alumina. (SAMAG - activity III to IV).

The alkaloids are eluted in the following order: leurosine, vinblastine, des-N-methylvinblastine, leurocristine and leurosidine. The identification of dimeric alkaloids in the eluate is carried out in a conventional manner, for example by thin layer chromatography. Chromatography is carried out in a 5 x 7 730 stainless steel column; cm at a pressure of 1.4 to 2.8 MPa. The ratio of alumina to the chromatographed material is approximately 300 to 1.

The eluate is monitored at 280 nm and the fractions are separated according to the peaks observed in ultraviolet light at this wavelength. The fractions were then identified by leurosine, vinblastine, des-N-methylvinblastine and leurocristine content by thin layer chromatography. The fractions of which. Columns appear after des-N-methylvinblastine and collected before leurocristin. These are fractions containing more than one dimeric alkaloid, and these fractions are then converted to the corresponding sulfates by adding an excess of a 1% ethanolic sulfuric acid solution. The sulfates are then separated at different pHs by first extracting the solution of these sulfates in citric acid at pH 3.4 with benzene, then increasing the pH of this solution by 0.5 pH units, and extracting the resulting aqueous layer with benzene each time. 4-desacetoxy-3‘-hydroxyvinblastine is present according to monitoring by thin-layer chromatography in extracts obtained at pH 5.4-5.9. The sulphates obtained at pH 5.4, containing mainly vinblastine and leurocristine, are dissolved in 5 ml of water and the pH is adjusted to 9 by addition of ammonium acetate. The precipitated free alkaloids are separated by centrifugation, dissolved in 3 ml of methylene chloride and chromatographed under high pressure in a 5/16 inch X 6-inch stainless steel column with a neutral · · Woelm N-18 aluminum oxide particle size of 18 to 30 microns) at using an increasing amount of ethanol in methylene chloride of 0.5%. The column is subjected to a pressure of 7.7 MPa at a flow rate of 180 ml per hour. Fractions were collected every 3 minutes, and the eluate was monitored in ultraviolet light. Fractions 30-32 contained 4-desacetoxy-3‘-hydroxyvinblastine as shown by thin-layer chromatography on silica gel, the solvent being a mixture of ether, diethylamine, toluene and methanol in a volume ratio of 100-: 5: 5: 5.

Typical compounds are:

3‘-hydroxy-4-desacetoxyvinblastine C-3 N-methylcarboxamide,

^{3-hydroxy-4->} desacetoxyvinblastin C-3 N-cyklopropylmethylkarboxamid,

2‘-hydroxy-4-desacetoxyvinblastine

C-3 N-cyanoethylamide, 2'-hydroxy-4-desacetoxyvinblastine C-3 N - '(2-hydroxypropyl) amide, deoxyvinblastine "A" C-3 carboxamide, deoxyvinblastine "A" C-3 N- (2-dimethylaminoethyl) ) carboxamide, deoxyvinblastine ,, B 'C-3 N- (2,3-dihydroxypentyl) carboxamide,

4-desacetoxyvinblastine C-3 N- (3-hydroxypropyl) carboxamide

4-desacetoxyvinblastine C-3 N- (2-aminoethylamino) carboxamide

2‘-hydroxyvinblastine C-3 N- (2-acetoxyethyl) carboxamide

2‘-hydroxyvinblastine C-3 N- (2-phenylethyl) carboxamide

2‘-hydroxyvinblastine C-3 N- (3-phenylpropyl) carboxamide

3‘-hydroxyvinbla'Stln C-3 carboxhydrazide and the like.

The compounds of the invention are derivatives in which the carboxymethyl group at the 3-position of known indole dihydroindole alkaloids is converted to a carboxyhydrazido group, a carboxazide group, a carboxamide group, or a derivative thereof. Not all of these derivatives can be produced in one way. Those in which R in formula I is NH 2, N-NH 2 or NH-CH 3 can be prepared as follows.

Leurocolumbin, vincadioline, their 4-deacetylated derivatives or desoxyvinblastine are treated with ammonia, methylamine or hydrazine to give the corresponding amide, N-methylamine or hydrazide. Usually the product of this reaction using intact 4-acetyl starting materials is a mixture of compounds in which the carbomethoxy group at the 3-position is changed to a carboxamide, N-methylcarboxamide or carboxhydrazide group, but the mixture also contains those where the acetyl group is in position 4 completely or partially removed. For purification, the 4-deacetylderivatives thus produced are separated by chromatography.

Compounds in which R is М (СНз) 2, NH-alk-X, wherein X is hydrogen, cyano or phenyl, NH-cycloalkyl of 3 to 8 carbon atoms, NH-alk-Am or NH-alk- (OH) 13, wherein alk and Am are as defined above, may be prepared as follows. The hydrazide, i.e. the compound of formula I in which R is NH-NH2, produced by reaction with a carbomethoxy-containing compound in the 3-position with anhydrous hydrazine is converted to the corresponding azld by treatment with nitrous acid, nitrosyl chloride, nitrogen dioxide, amyl nitrite or the like . The thus prepared compound having an azide group at the 3-position is then reacted with a primary or secondary amine, N 2 (NH 2) 2, NH 2 -alk-X, N.H 2 -cycloalkyl of 3 to 8 carbon atoms, NH 2 -alk- or This reaction does not affect the acyl group at the 4-position, which, if present, remains in the original state during the reaction and further processing. The reaction is carried out as described for the first time in Stoli and Huffman, Helv. Chlm. Acta, 26, 944 (1943) and in U.S. Pat. Nos. 2,090,042 and 2,090,430.

Compounds in which the acetyl group is in the 4-position can be prepared by reacting vincadioline, leurocolumbin or desoxyvinblastine directly with ammonia, methylamine or hydrazine followed by separation of the 4-acetyl derivative from the 4-deacetyl derivative and in the case of hydrazide subsequent reaction of the azide with an amine to form an amide.

Typically, however, due to the unstable nature of the 4-acetyl group, the procedure is carried out by reacting the hydrazine via the azide to the amide with the 4-deacetyl derivative. Generally, the amides of the 4-position of the desacetyl group of formula I can be acylated with an aliphatic anhydride or acid chloride to give the corresponding 4-acetate, 4-propionate or 4-butyrate or a chlorinated derivative thereof. The reaction may be carried out using a C 1 -C 3 -alkyl chloride of the alkyl moiety, or a C 1 -C 3 chloroalkyl of the alkyl moiety or an acid anhydride (alkyl-COH) 2 -O s 1 to 3 carbon atoms in the alkyl moiety or C-alkyl-CO); and = O with 1 to 3 carbon atoms in the alkyl moiety. A preferred embodiment of acylation is described in U.S. Pat. No. 3,392,173 for vinblastine or leurocristine, wherein the diacylated derivative of vat is the first product of the reaction, and the derivative is then selectively subjected to hydrolysis to form a 4-acyl compound. Other processes, including selective acylation or multiple acylation followed by selective hydrolysis, can also be used to produce the 4-acyl derivatives of the invention.

However, some possible precautions should be taken during acylation. If the carboxaline 6-position of the 3-position contains an acylatable group, for example a hydroxy group or an amino group, it must be carried out. acylation at the 4-position prior to the reaction of the azide to the amine to give the resulting carboxamide group at the -3-position. In this case, it is preferable to acylate the compound having the carboxhydrazide group in the -3 position as described above, whereby it is possible first to protect the hydrazide group, which would otherwise be acylated. A suitable protecting group for this purpose is the propylidene group formed by the reaction of the NH4 group of the hydrazide group with acetone. This group can be easily removed by acid treatment or the propylidene derivative can be introduced directly with the nitrite to form an azide group as described in Example 7 of U.S. Patent 3,470,210.

Other methods, including selective acylation or multiple acylation followed by selective hydrolysis, can be used, or the acylatable group can be selectively protected and removed after acylation.

Compounds of formula I wherein R is -NH — alk — X, wherein X is carboxyl, carboxamido, aralkoxy, wherein the alkoxy group contains 1 to 3 carbon atoms, can be prepared by reacting an amino acid, an aminoamide , preferably an amyl ester of the formula

NH2-C-COQ

Where ·

Q represents OH, NH 2 or -O-alk and

Z- represents a hydrogen atom or an alkyl of 1 to 5 carbon atoms, with a selected dimeric indole dihydroindolazide. An amino group suitable for use in the process and falling within the above formula is, for example, leucine, isoleucine, valine, glycine, alanine, norleucine, and the like. As will be appreciated, others may be used in the reaction. amino acids and polypeptides that are reacted with, for example, 4-desacetylvinyl-3-carboxazide to form a substituted carboxamide at the 3-position with antitumor activity.

Another possible method for producing a primary amine wherein R is NH 2 from a hydrazide, wherein R is NH-NH 2, is the method disclosed by Ainsworth in U.S. Patent No. 2,756,235, in which hydrazide is hydrogenolyzed by Raney nickel treatment.

The new derivatives will be hereinafter referred to only according to the new - groups that are formed on said - carbon atom. For example, a compound formed by replacing the methyl ester function of vinblastine at the 3-position with an amide function would simply be called vinblastine C-3 carboxamide and not vinblastine C-3 decarbomethoxy C-3 carboxamide. .

The free base compounds including carboxamide, carboxazide and carboxhydrazide are white or off-white solids. However, it is preferred, if possible, to isolate and crystallize amides in the form of anionic salts with non-toxic acids. These salts are white, crystalline or amorphous water-soluble solids with a high melting point.

The invention will be illustrated by the following examples:

Example 1

4-Decacetyldesoxyvinblastine "B" C-3 carboxhydrazide

2.55 g of desoxyvrnblastine, B, is reacted with 3θ ml of anhydrous hydrazine in anhydrous methanol in a sealed reaction vessel at 60 ° C for 18 hours. The reaction vessel is then cooled, - open, - the contents are removed and - the volatile components. is evaporated under vacuum. The residue containing 4-desacetyldesoxyvinblastine B C-3 hydrazide is mixed with methylene chloride, the methylene chloride solution is washed with water, separated and dehydrated and the methylene chloride is evaporated in vacuo to give an amorphous powder having the following properties:

maximum in infrared: 3440 cm -1 (Ν-H), 1735 cm -1 (COO), 1675 cm -1 (CON), molecular ion m / e = 752, corresponds to formula C43H56N6O6

NMR spectrum: δ 3.78 (ArOCH 3), δ 3.58 (C 18 COOCH 3), δ 2.77 (N-CH 3), δ 4.15 (Cd-H).

Example 2.

4-Desacetoxyvinblastine C-3 N- (2-hydroxyethyl) carboxamide

By the method of Example-1, 4-desacetoxyvinblastine was reacted with anhydrous. hydrazine in -methanol solution in a sealed tube at 46 ° C for 3 days. The 4-desacetoxyvinblastine-C-3-carboxhydrazide thus produced is isolated and purified in the same manner as in Example 1. The substance obtained has the following physical properties:

pKa = 5.61 and 7.38, ultraviolet spectrum λ max = = 215 -a 267 nm, infrared spectrum has maxima at

3450 cm -1 (Ν-H), 1725 cm ^-1 (COO), 1680 cm ^-1 (CON), molecular spectrum m / e = 252, 411, 355, 244, 154, molecular ion M + - 752 corresponds to empirical VZOrCi C46H56N6O6.

The compound is an off-white amorphous powder.

4-Deeacetoxyvinblastine C-3-carboxhydrazld is converted to the corresponding carboxazide in a solution of hydrochloric acid at 0 ° C by treatment with sodium nitrite. The reaction mixture was then basified by adding an excess of cold 5% aqueous sodium bicarbonate solution. The aqueous solution was extracted 3X with methylene dichloride.

To a solution containing 1.2 g of 4-desacetoxyvinblastine O 3 carboxazide was added 5 ml of ethanolamine. The reaction mixture was sealed and protected from light. After allowing the mixture to stand at room temperature for 1 day, the reaction vessel was opened and the volatiles removed from the reaction mixture by evaporation in vacuo. The resulting residue, which contains 4-desacetoxyvinblastine O 3 N- (2-hydroxyethyl) carboxamide, formed during the above reaction, is dissolved in methylene chloride and the methylene dichloride solution is washed several times with water. The methylene dichloride solution was then separated and dehydrated and the solvent was evaporated in vacuo. The residue is chromatographed on silica gel, eluting with a 3: 1 mixture of ethyl acetate and ethanol. The fractions were monitored by thin layer chromatography, the fractions containing the desired product were combined and evaporated to dryness in vacuo. The 4-desacetoxyvinblastine C-3 thus obtained N- (2-hydroxyethyl) carboxamide is an off-white solid with the following physical properties.

molecular ion M + = 781, corresponds to the empirical formula C45H59N5O7, the infrared spectrum has maxima at

3420 cm -1 (NH), 1735 cm -1 (COO), 1665 cm -1 (CON).

The sulphate of the resulting product was produced by treating the product with an ethanolic sulfuric acid solution followed by pH adjustment in the region of 3.8 to 4.2. The sulphate was then isolated by evaporating the volatile components of the reaction mixture in vacuo.

Example 3

4-Desacetyl-vincadioline C-3 N-methylamide

By the method of Example 1, vincadioline is reacted with hydrazine to give the corresponding C-3 carboxhydrazide. This hydrazide is converted to the corresponding carboxazide and the azide is then reacted with methylamine.

The product of this reaction, 4-desacetyl-vincadioline C-3 N-methylamide, has the following physical properties:

the infrared spectrum has a maximum at 3.95 nm, 5.75 nm and 5.97 nm,

NMR-Spectrum corresponding to the predicted structure -with doublet at δ 3.82 (amidomethyl-hydrogen atoms), molecular spectrum, molecular ion M = 783 corresponds to the empirical formula C44H57N5O8.

Described above is also prepared možno- ^4-de, sacetylleurokolumbin C-3 N-methylamide.

Example 4

Preparation of salts

Other salts can be prepared from the amides of the invention, including salts with inorganic anions such as chloride, bromide, phosphate, nitrate and the like, as well as salts with organic anions such as acetates, chloroacetates, trichloroacetates, benzoates, alkylsulfonates or arylsulfonates in a manner analogous to was given in Example 1 for the production of sulphate by replacing the sulfuric acid with the appropriate acid and carrying out the reaction in a suitable solvent.

It will be appreciated that the presence of additional ester or amide groups in the indole dihydroindole component requires special precautions in salt production to avoid hydrolysis, transesterification and other reactions that may occur at higher temperatures at too acidic pH and the like.

The compounds of the invention are active against certain viruses, for example against herpes virus, as shown in tissue cultures as described in Simonoff, Applied Microbiology, 9, 66-72 (1961).

In addition, the compounds of the invention are effective against transplanted mouse nations in vivo. Of particular interest is the efficacy of the compounds of the invention against Ridgeway osteogenic sarcoma (ROS and Gardner lymphosarcoma (GLS). In demonstrating the efficacy of the compounds of the invention against these tumors, they were administered intraperitoneally at the indicated dose for 7 to 10 days after tumor inoculation.

Table 1 shows the results of experiments on mice transplanted with tumors successfully treated with the compounds of the invention. In column 1 the name of the substance, in column 2 the name of the tumor, in column 3 the dose and number of days in which it was administered, and in column 4 the inhibition of tumor growth in%. In addition to the abbreviations above, CA 755 stands for adenocarcinoma.

Compounds of the leurocristine and vinblastine type are already toxic to mice at doses lower than 100% of the tumor hibernation. In addition, however, toxicity at lower doses sometimes results from unknown causes. In Table . Typical results are shown in which the compounds gave the expected results, excluding untypical experiments, so that the results are not an average of all experiments.

Table 1

Compound Tumor Dose mg / kg X X days Inhibition v - ¹ % 4-desacetoxy vinblastine ROS 0,5 X 10 44 C-3- (2-hydroxyethyl) - GLS 0.75—1.5 X 6 51—100 carboxamidsulphate

The compounds of the invention are effective against other transplanted tumors. For example, in soft-type lymphosarcoma, a parenteral injection of 0.25 mg / kg vinblastine C-3 N-methylcarboxamide over a period of 9 days results in 54% inhibition of tumor growth and 28% inhibition in tumor growth for the same dose of vinblastine C-3 amide. At the same dose, vinblastine alone was completely ineffective.

In experiments with adenocarcinoma CA 755, 67% inhibition of tumor growth was achieved with 4-deacetylvinblastine C-3 carboxamide sulphate, with 61% inhibition of tumor growth with 4-desacetylvinblastine C-3 carboxyamide sulphate and with vinblastine C-3 carboxamide sulphate at 491% tumor growth at 0.25 mg / kg for 8 days and 72% inhibition at 0.3 mg / kg. In a similar experiment, vinblastine was able to achieve a 31% inhibition of tumor growth, while administration of leurocristine at a somewhat lower dose of 0.2 mg / kg resulted in a 79% inhibition of tumor growth with long-term effect. When used against lymphocytic leukemia L5178Y, administration of vinblastine C-3 carboxamide sulphate at a dose of 0.25 mg / kg for 10 days using a group of mice resulted in the survival of 3 mice with indeterminate improvement. On follow-up, the survival time in 2 of these mice was prolonged by 26% compared to control mice. In the same experiment, an increase in survival time of 36% could be achieved with vinblastine, but no longer survival could be achieved.

When using new amides and hydrazides against tumor growth parenterally or orally, different doses may be used. For oral use, an effective amount of a pharmaceutically acceptable salt of a base of Formula I can also be used, except for those compounds wherein R is NH-NH 2 - or N 3, formed by reaction of a compound of the invention with a nontoxic acid. the carrier and the mixture are dispensed into telescopically gelatin capsules containing 7.5 to 50 mg of active ingredient.

It is also possible to mix the active salt with starch, binder and glidant and compress the mixture into tablets containing 7.5 to 50 mg of active ingredient. The tablets may be provided with scoring lines. For parenteral administration, intravenous administration should be preferred.

Isotonic solutions with a salt content of 1 to 10 mg / ml are used, with the exception of hydrazides and azides. The compounds are administered at doses of 0.1 to 1 mg / kg body weight once a week depending on the efficacy and toxicity of the compound used. The free bases of formula I wherein R is NH-NH 2 or N 5 may also be formulated in appropriate dosage forms and administered in a similar manner and at similar dosages.

Thus, most of these compounds are useful for suppressing tumor growth and against viral infections, in addition, two derivatives can be used, namely hydrazides and the azide of I, wherein R is NH-NH2 or N3 also as intermediates, since hydrazides can be converted to azides by nitrosation, for example nitrous acid - or to amides by hydrogenolysis. Amides can be obtained from the azide by reaction with a primary or secondary amine.

Claims (4)

OBJECT OF THE INVENTION A process for the preparation of alkaloid derivatives of the general formula I wherein R is —NH2, —NH — NH2, —N (CH3) 2, pyrrolidinyl, —NH — alk — X, —NH — cycloalkyl of 3 to 8 carbon atoms, —NH — alk — Am, - NH-alk- (OH) m or N3, in which alk represents an alkyl of 2 to 6 carbon atoms, Am represents - - 2, —NHCH 3 or - Ni (CH 3) 2 a X represents a hydrogen atom, a cyano group, a phenyl group, a carboxyl group, a carbalkoxy group having 1 to 3 carbon atoms in an alkoxy group or a carboxamide group, R1 is hydrogen, hydroxyl, O-alkanoyl of 1 to 3 carbon atoms or O-chloralkanoyl of 1 to 3 carbon atoms, R 2, R 3 and R ⁴ are hydrogen or hydroxyl, provided that when R 2 is hydrogen, R 3 and R 4 are also hydrogen, when R 2 is hydroxyl, at least one of R 3 or R ^{4 is} also hydroxyl A group comprising reacting a compound of formula I in which R is -O-CH 3, R 1 is hydrogen or acetyl and R ² , R 3 and R ⁴ are as defined above with a compound of the formula of formula II NH ² R ⁵ (IIJ, where R5 represents a hydrogen atom, a methyl group or an amino group; and optionally, if desired when R is NH-NH 2, the compound so obtained is reacted with a nitrosating agent and a compound of the formula R® OF NH \ R7 where R ⁶ represents a hydrogen atom or methyl a R ⁷ is methyl, -alk-X, cycloalkyl of 3 to 8 carbon atoms, -alk-Am, or -alk- (OH) 13, wherein alk is an alkyl group of 1 to 6 carbon atoms, and Am and X are as defined in formula I, whereupon the compound obtained in any of the above steps is optionally acylated when R 1 is hydroxyl to form a compound wherein R 1 is different from the hydroxyl group and optionally reacting with a non-toxic, inorganic or organic acid to form an acid addition salt of the resulting product. 2. A process according to claim 1, for the preparation of 4-desyldesoxyvinblastine B C-3 carboxhydrazide, characterized in that it is reacted with desoxyvinblastine B with anhydrous hydrazine in anhydrous methanol. 3. The process of item 1 for producing 4-desacetoxyvinblastine C-3 N- (2-hydroxyethyl) carboxamide, which comprises reacting 4-desacetoxyvinblastine with anhydrous hydrazine in anhydrous methanol to give 4-desacetoxyvinblastine C. The carboxhydrazide and the intermediate carboxhydrazide are reacted with ethanolamine. 4. A process according to claim 1 for the preparation of 4-deacetylvincadioline C-3 N-methylamide, which comprises reacting vincadioline with anhydrous hydrazine in anhydrous methanol to form vincadioline C-3 · carboxyhydrazide, which is reacted with methylamine.