DE2558027A1 - AMIDE DERIVATIVES OF VINBLASTIN, LEUROSIDIN, LEUROCRISTIN AND RELATED DIMERIC ALKALOIDS AND THE METHOD OF PRODUCING THEM - Google Patents

Description

X-3754 M.

SCKi - ■

Eli Lilly and Company / Indianapolis, Indiana, V.St.A.

Amide derivatives of Vinblastine, Leurosidine, Leurocristin and related dimeric alkaloids and processes for their manufacture

The invention relates to new amide derivatives of dimeric alkaloids and a process for their preparation. The invention Compounds are either valuable antiviral and antineoplastic agents or intermediates in manufacture such means.

The amide derivatives of vinblastine, leuosidine, leurocristine and related dimeric alkaloids according to the invention have the formula I.

Af

U 4 ^ CHs-CHs

₁₂ .y Xioi \ \ | /; _ _R 4

1 3 ' _χ A 1 6'. 1 8 '· 1' 14 '

14th

> O-CH ₃

1 of 1 /

CH ₃ -CHs

OfilQfNAL

9/0824

255802?

R for NH ₂ , NH-NH ₂ , N (CH ₃ J ₃ , pyrrolidinyl, NH-alkyl-X,

NH- (C ₃ -C ₈ ) -cycloalkyl, NH-alkyl-Am, NH-alkyl- (OH) .j_ ₃ or N ₃ , where alkyl is (C ₃ -C ₆ ) -alkyl and Am is NH ₂ , NHCH ₃ or N (CH ₃ J ₃ and X is hydrogen, cyano, phenyl, carboxyl, carbo (C.-C ₃ ) alkoxy or carboxamide,

R is hydrogen, hydroxy, 0- (C ₁ -C ₃ ) -alkanoyl or O-chloro- (C ₁ -C ₃ ) -alkanoyl and

2 3 4
R, R and R stand for hydrogen or hydroxy,

3 4 with the proviso that the substituents R and R water

are substance, if the substituent R is hydrogen,

and that further at least one of the substituents R or

4 2

R is hydroxy if the substituent R is hydroxy.

The invention also relates to a process for the preparation of amide derivatives of vinblastine, leuosidine, leuurocristine and related dimeric alkaloids of the formula I given above, which consists in that one

(A) a compound of the formula I given above in which the substituent R is O-CH, the substituent R is water

2 3

Substance or acetyl and the substituents R, R and R have the meanings given above for formula I, with a Compound of formula II

NH ₂ R ⁵ II,

wherein the substituent R is hydrogen, methyl or amino, reacts and

809 8 29/08

(b) if appropriate, the compounds obtained in this way in which the substituent R is —NH — NH ₂ , with a nitrosylating agent and with a compound of the formula III

R ⁶

-NH

in which the substituent R is hydrogen or methyl and the substituent R ^{7 is} methyl, alkyl-X, (C ₃ -C ₈ ) -cycloalkyl, alkyl-Am or alkyl- (OH) ^ ₃ , wherein alkyl is a (Cj- C ^ and Am and X have the meanings given for formula I, and the compounds obtained by process (a) or (b), if the substituent R is hydroxy, if desired acylated to compounds in which the substituent R is another Has meaning as hydroxy,

whereupon the compounds obtained in this way by reaction with a non-toxic inorganic or organic If desired, acid is converted into the corresponding acid addition salts.

Several naturally occurring alkaloids obtained from Vinca rosea have been shown to be effective for treatment Experimentally induced diseases in mammals. Such compounds include Leurosin (US Pat. No. 3,370,057), Vinblastine (vincaleukoblastine) (U.S. Patent 3,097,137), leuosidine (vinrosidine) and leuurocristine (vincristine) (each in U.S. Patent No. 3 205 22O). Two of these alkaloids, vinblastine and leucristine, are now in treatment as remedies of diseases, in particular of leukemia and related diseases in humans, in the trade. One of these Compound, namely Leurocristin, is an extremely effective one

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and interesting agent for treating leukemia, it however, it is the least common compound of the antineoplastic alkaloids found in Vinca rosea.

Chemical modification of the vinca alkaloids has been rather limited so far. The first thing to say about this is that the related molecular structures are extremely complex and involve chemical reactions that have a specific function of the Affecting the molecule, making it difficult to develop. Second, it should be mentioned that Vinca rosea fractions contain alkaloids which do not have sufficient chemotherapeutic properties and a structure determination of these compounds led to the conclusion that they are closely related to the active alkaloids. An antineoplastic activity therefore seems to be limited to very specific structures, and the chances of modifying these structures Acquiring effective remedies would appear to be small. To the successful modifications Physiologically active alkaloids include the production of dihydrovinblastine (ÜS-PS 3 352 868) and the replacement of the acetyl group at C-4 (carbon number * 4 of the Vxnblastinringsystem, see formula I) through a higher alkanoyl group or through an unrelated acyl group (U.S. Patent 3,392,173). Several of these derivatives extend the life of mice that vaccinated with P1534 leukemia. One of these derivatives, in which the ani C-4 located acetyl group of vinblastine has been replaced by a chloroacetyl group, is also an interesting intermediate for the preparation of structurally modified ones Vinblastine compounds in which the one located on the C-4 atom The acetyl group of vinblastine is replaced by an N, N-dialkylglycyl group (US Pat. No. 3,387,001). During the chemical conversion to these last-mentioned derivatives also resulted in another intermediate, namely 4-deacetylvinblastine. This intermediate, which does not have an acyl group in position C-4, has one instead Containing non-esterified hydroxy group has been considered. toxic

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Material described in vivo against P1534 leukemia only has a weak chemotherapeutic effect on the mouse (Lloydia 27, 340 (1964).

Preferred compounds of the formula I according to the invention are the amides of vincadioline, leucolombin, 4-deacetoxyvinblastine, 3'-hydroxy-4-deacetoxyvinblastine, deoxyvinblastine and the 4-deacetyl derivatives of the above dimeric alkaloids which have an acetoxy group at carbon atom 4, as well as the pharmaceutically acceptable ones Salts of the above bases with non-toxic acids, with the exception of those compounds in which the substituent R stands for NH-NH ₂ and N ₃ .

Examples of suitable radicals alkyl (OH) _Λ _, alkyl-Am or alkyl-X in the above formula I are methyl, 2-methylpentyl, isohexyl, isopentyl, n-pentyl, n-hexyl, sec-hexyl, ethyl, Isopropyl, η-butyl, sec-butyl, cyanomethyl, cyanoethyl, 2-hydroxy-n-hexyl, 5-cyano-n-pentyl, 2-hydroxyethyl, 3-hydroxypropyl, 2-dimethylaminoethyl, 2-aminoethyl, 2-methylaminoethyl , 2-hydroxypropyl, benzyl, phenethyl, 4-phenylbutyl, 2-aminopropyl, 2-aminohexyl, 2-dimethylaminopropyl, 2,2'-dihydroxyisopropyl, 2,2'-dihydroxy-tert.-butyl or 2,2 ', 2 "-Trihydroxy-tert-butyl.

In the above formula I, (C ₁ -C ₃ ) -alkanoyl or chloro- (C ₁ -C ₃ ) -alkanoyl radicals such as acetyl, chloroacetyl, propionyl, 2-chloropropionyl, 2-chlorobutyryl or butyryl, and this is where it is concerned groups of the formula (C.-C ₃ ) -alkyl-CO, namely alkanoyl groups, or groups of the formula (C ₁ -C ₃ ) -alkyl (Cl) -CO, namely chloroalkanoyl groups.

Examples of NH- (C ₃ -Cg) -cycloalkyl are cyclopropylamino, cyclobutylamino, cyclopentylamino, cyclohexylamines, cycloheptylamino or cyclooctylamino.

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ο -

The term carbo- (C.-C ₃ ) -alkoxy relates, for example, to carbomethoxy, carboethoxy, carboisopropoxy or carbo-n-propoxy.

If the substituent X in an alkyl-X radical is phenyl then the phenyl group can contain the usual aromatic substituents, such as lower alkyl, lower alkoxy, hydroxy, Halogen or nitro, and such phenyl group may have one or more of the above substituents, either are the same or different from the original substituent. Examples of such groups are 4-hydroxyphenyl, 2,4-dichlorophenyl, 2-methyl-4-chlorophenyl, 2,4-dinitrophenyl, 3,5-xylyl, 4-tolyl, 2-tolyl or 3-ethoxyphenyl.

Regarding non-toxic acids which form pharmaceutically acceptable acid addition salts of the amine bases according to the invention suitable include salts of inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, Hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, as well as salts of non-toxic organic acids including aliphatic mono- or dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanedioates of aromatic acids, as well as aliphatic or aromatic acids Sulfonic acids. Examples of such pharmaceutically acceptable salts are sulfates, pyrosulfates, bisulfates, sulfites, Bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, Metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, Formates, isobutyrates, caprates, heptoanates, propiolates, oxalates, Malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioate, hexyne-1,6-dioate, benzoates, chlorobenzoates, Methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, Phthalates, terephthalates, benzenesulfonates. Toluenesulfonates, chlorobenzenesulfonates, xylene sulfonates, Phenylacetate, Pheny! Propionate, Phenylbutyrate, Citrate, Lactates, 2-hydroxybutyrates, glycolates, malates, tartrates, Methanesulfonates, propanesulfonates, naphthalene-1-sulfonates or naphthalin-2-sulfonate.

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The majority of the compounds of the formula I can generally be described as derivatives of vinblastine. Vinblastine is a compound of the formula I in which R is CH ^ O, R ^{1 is} acetoxy, R ^{2 is} β-hydroxyl (the ethyl group present on the carbon atom 4 * is in the alpha position) and the substituents R as well as

4th
R represent hydrogen. A Deoxyvinblastin- you then have

2 3 4

if in formula I all the substituents R, R and R are hydrogen, the substituent R is CH ~ O and the

Substituent R is acetoxy. There are two isomers of deoxyvinblastine, referred to as isomer A and isomer B

2 den. In the case of isomer A, the hydrogen atom R is in the ß-position, while the ^{ethyl group located on the carbon atom 4 1 is} in the alpha position. Isomer B has a β-A "thyl group and has an alpha hydrogen atom ^{on carbon atom 4 1.}

Vincadiolin is the 3'-hydroxy derivative (R = hydroxy) of Vin-

4 blastin. Leurocolurabin is the 2'-hydroxy derivative (R = hydroxy)

of vinblastine.

Vincadiolin has the following physical properties:

Melting point = 218 to 220.5 ⁰ C with decomposition, x-ray diffraction spectrum in powder form using

ο a filtered chrome radiation lambda = 2.2896 A.

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ο
d in A Y / Y ₁ O
d in A 9.55 TOO -1 3.99 8.87 90) ₉ 3.71 8.63 90) ² 3.64 7.78 05 3.44 7.57 60 3.19 7.21 50 - 3.05 6.00 40 2.85 5.88 40 2.78 5.58 7O -3 2.61 5.22 20th 2.44 5.08 20th 2.21 4.70 50 2.07 4.57 40 1.98 4.42 05 1.91 4.31 05

NMR spectrum delta at 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;

Molecular Weight: 826;
Empirical formula: ^C 46 ^H 5q ^N 4 ° io
Mass ions m / e = 826, 170, 371.

Vincadiolin is made according to the following process: leaves of plants that contain raw Vxnca alkaloids, namely Catharanthus roseus (Vinca rosea), are mixed with a water immiscible solvent such as Benzene, extracted. The extract obtained becomes then the benzene is distilled off in the presence of aqueous tartaric acid. Following this, the pH is adjusted obtained aqueous acidic extract by adding a base

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O _

on 6 a. Alternatively, the leaves can also be treated with an aqueous acid at a pH of 3 and the then extract the acidic layer obtained with benzene. The benzene layer is separated and discarded, and the The aqueous layer is adjusted to pH 6 as indicated above. The dimeric alkaloids are then removed from the aqueous Layer extracted into an organic solvent, usually benzene. The extracted alkaloids can be used if necessary subject to a gel exclusion filtration, including, for example, a crosslinked dextran gel (Sephadex G-25F) is used, and a 0.1 molar ammonium citrate buffer of pH 3.0 is used as the mobile phase. The Gelaus Final chromatography is performed at a pressure of about

1.05 kg / cm. The first thing in this procedure is the dimeric alkaloid fraction elutes, the leuurocristine, vinblastine, des-N-methylvinblastine, leuformin, and leurosine Contains leurosidine. The dimeric alkaloids are extracted from the pH 3 buffer by adjusting the pH with a base to pH 7.0 and then treating the resulting aqueous solution with one with water immiscible solvent, preferably again benzene. Evaporation of the benzene gives a residue from which can be crystallized directly after dissolving in ethanol Leurosin. The Leurosin crystals are separated by decanting, whereupon the resulting supernatant liquid is acidified to pH 4.2 with 3 percent ethanolic sulfuric acid. Here, the remaining dimeric alkaloids are converted into their sulfates, which precipitate. The precipitated salts are collected and converted in the usual way into the corresponding free alkaloid bases, which can be achieved, for example, by dissolving the salts in Water, adjusting the resulting solution to pH 8.0 with ammonium hydroxide and then extracting the dimeric alkaloids with a water-immiscible organic solvent, preferably methylene dichloride, can be carried out. By evaporation of the methylene dichloride one obtains the mixed dimeric alkaloids, which one afterwards one with high pressure over aluminum oxide

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(Activity III) using a solvent system of ethyl acetate-methylene dichloride-water (25: 75: 0.4) as the eluent

medium chromatographed.

For the above procedure, working pressures in the range of

2
10.5 to 24.6 kg / cm applied. High pressure chromatography

However, there are also systems for working pressures from 281 to

2
352 kg / cm, and it is also working at pressures

from 527 to 562 kg / cm imaginable. The separation of the individual alkaloids is generally more favorable at higher pressures. High pressure chromatography processes are carried out in a stainless steel device that is pressure tight sealed is.

The elution of the alkaloids takes place in the above chromatographic Procedure in the following order: residual leuosine, vinblastine, des-N-methylvinblastine, leurocristine and Leurosidin. The dimeric alkaloids present in the eluted fraction are identified by conventional methods, for example by thin-layer chromatographic analysis.

After elution of the known alkaloids, several more polar dimeric alkaloids remain on the column. These alkaloids are eluted with methanol, whereupon the whole thing is rechromatographed until vincadiolin is separated Fraction that is practically free from other dimeric alkaloids present in the polar alkaloid fraction are. Leurocolombin has the following characteristic properties:

pKa values 5.05 and 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, 6O, 9.90 and 10.75 microns;

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Ultraviolet absorption maxima at 217 (a _m = 51 O91) and

265 (a = 15,666) millimicrons;
m

Molecular weight 826;
Empirical formula: C ₄₆ H ₅₈ N ₄ O ₁ ,

Ion fragments in the mass spectrum m / e = 795, 767, 749, 667, 649, 282, 170, 156, 154, 152, 144, 143;

NMR proton spectrum: chemical shifts in ppm at 7.51, 7.13, O, 9O, 3.6O, 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.

Leurocolumbin forms a sulfate salt, using its powder a filtered chromium radiation at 2.2896 A gives the following X-ray diffraction spectrum.

din I ³ Jf ₁

17, OO 30

12.5O 100

9.45 50

7.70 10

7.2O 60

6.2O 20

5.70 30

4.95 05

4.65. 20th

Leurocolombin is made in the following way: leaves of plants that contain raw vinca alkaloids, viz Catharanthus roseus (Vinca rosea) are extracted with a water-immiscible solvent such as benzene. The benzene is then evaporated from the extract obtained in the presence of aqueous tartaric acid. Afterward the pH of the acid extract obtained is adjusted by addition adjusted to pH 6 with a base. Alternatively, the leaves can also be treated with an aqueous acid at pH 3 and then extract the acidic layer obtained with benzene. The benzene layer is separated and discarded, and

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the pH of the aqueous layer is adjusted to pH 6 as above. Then the dimeric alkaloids are made from the aqueous layer into an organic solvent, usually benzene. The extracted alkaloids can be used if necessary subject to a gel exclusion filtration, including, for example, a crosslinked dextran gel (Sephadex G-25F) and an O, 1

molar ammonium citrate buffer of pH 3.0 is used. The GeI-

2 exclusion chromatography is carried out at a pressure of about 1.05 kg / cm carried out. The first thing in this process is the dimeric Alkaloid fraction eluted, the Leurocristin, Vinblastin, Des-N-methylvinblastin, Contains Leuroformin, Leurosin and Leurosidin. The extraction of the dimeric alkaloids from the buffer with pH 3 takes place by adjusting the pH with a base to pH 7.0 and then treating the resulting aqueous Solution with a water-immiscible solvent, preferably again benzene. By evaporating the benzene a residue is obtained from which, after dissolving in ethanol, leurosine can be crystallized directly. The Leurosin Crystals are separated by decanting, whereupon the resulting supernatant liquid with 3 percent ethanol Acidified sulfuric acid to pH 4.2. Here, the remaining dimeric alkaloids are converted into their sulfates, which fail. The precipitated salts are collected and in the usual way in the corresponding free alkaloid bases transferred, for example by dissolving the salts in water, adjusting the resulting solution with ammonium hydroxide pH 8.0 and subsequent extraction of the dimeric alkaloids with a water-immiscible organic solvent, preferably methylene dichloride can be carried out. By evaporation of methylene dichloride one obtains the mixed dimeric alkaloids, which one then at high pressure over aluminum oxide (activity III-IV) using a solvent system from ethyl acetate-methylene dichloride-water (25: 75: 0.4) as the eluent chromatographed.

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Working pressures in the range of

2
10.5 to 24.6 kg / cm applied. However, high pressure chromatography systems are also available for working pressures from 281 to

352 kg / cm, and it is also working at pressures

from 527 to 562 kg / cm imaginable. The separation of the individual alkaloids is generally more favorable at higher pressures, High pressure chromatography processes are performed in a stainless steel device that is pressure tight sealed is.

The elution of the alkaloids takes place in the above chromatographic Procedure in the following order: Remaining Leurosine, Vinblastine, Leurocolumbin, Des-N-methylvinblastine, Leurocristin and Leurosidin. The dimeric alkaloids present in the eluted fraction are determined by conventional methods identified, for example by thin-layer chromatographic analysis.

4-Deacetoxyvinblastine has the following physical and chemical properties: Melting point = 183 to 190 ° C (decomposition) after recrystallization from methanol; / alpha / ^ ⁶ = + 95.3 ° (chloroform); Molecular ion M + = 752, according to an empirical formula of C ₄₄ Hc ₆ N ₄ O ₇ .

Analysis for ^C 44 ^H 56 ^N 4 ^O 7

calculated: C, 70.19; H 7.50 N 7.44; 0 14.87; found: C, 69.71; H 7.47; N 7.08; 0 15.00.

4-Desacetoxyvinblastine is made as follows: Leaves of plants that contain raw vinca alkaloids, namely Catharanthus roseus (Vinca rosea), which has previously been moistened with aqueous ammonia, become immiscible with water Solvent such as benzene extracted. From the extract obtained in this way, the benzene becomes more aqueous in the presence of

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Tartaric acid distilled off. The tartaric acid layer is then mixed with a water-immiscible organic solvent extracted and then made basic by adding ammonia. The dimeric alkaloids are then extracted from the alkaline layer into an organic solvent, usually benzene. Obtained by evaporation of the benzene a mixture of amorphous dimeric alkaloids, which is dissolved in benzene, and chromatography over aluminum oxide (CAMAG activity III) he t.

The alkaloids are eluted in the following order: Leurosine, Vinblastine, Des-N-methylvinblastine, Leurocristine and Leurosidin. The identification of the dimeric alkaloids in the eluted fraction is carried out by known methods, for example by thin layer chromatographic analysis. Elution of vinblastine is usually done using a 1: 1 solvent mixture of benzene and chloroform ". This process is described in detail in US Pat. No. 3,225,030.

The vinblastin fractions obtained in this way contain small amounts of a second alkaloid, 4-deacetoxyvinblastine, based on thin-layer chromatographic analysis is identified. This second alkaloid is isolated as follows: The vinblastine fraction is first determined by standard methods converted into the corresponding sulfate salts, whereupon the sulfates are subjected to a pH gradient separation process, for which you dissolve the sulfates in 2 percent aqueous citric acid and the citric acid solution twice with benzene extracted. The pH is then increased by adding of ammonia to pH 5.5 and extracted two more times with benzene. The second fraction obtained is over Chromatographed aluminum oxide (activity III). The development the chromatogram is carried out with benzene. Those fractions that are subject to thin-layer chromatographic analysis

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contain a second alkaloid in addition to vinblastine combined and chromatographed again over aluminum oxide, developing the chromatogram again with benzene. The process is then repeated. Those fractions which then practically only contain the second alkaloid, namely 4-Deacetoxyvinblastine, containing only small amounts of vinblastine, are combined and recrystallized from methanol. The 4-deacetoxyvinblastine obtained in this way is then subjected to preparative thin-layer chromatography on silica using a 3: 2: 4 mixed solvent of diethylamine-chloroform-benzene cleaned.

The S'-hydroxy - ^ - desacetoxyvinblastine has the following Physical Properties:

Maxima in the NMR proton spectrum at delta 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-deacetoxyvinblastine is made as follows: De-greased leaves of plants containing raw vinca alkaloids, namely Catharanthus roseus (Vinca rosea), are made first moistened with aqueous ammonia and then extracted with a water immiscible solvent such as benzene. The benzene is then evaporated from the extract obtained in the presence of aqueous tartaric acid. Afterward the tartaric acid layer is made basic by adding ammonia. The dimeric alkaloids are then extracted from the alkaline layer in an organic solvent, usually benzene. Evaporation of the solvent gives a mixture of amorphous dimeric alkaloids. The dimeric alkaloid fraction is dissolved in ethanol, which is followed by .Addition of ethanolic sulfuric acid and the corresponding sulfate salts

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forms. The crystalline mixed sulfate salts are collected and then converted into the corresponding free bases by dissolving them in water, making the aqueous solution basic and the alkaloids in a water-immiscible organic solvent, usually methyl dichloride, extracted. Evaporation of the solvent gives a mixture of amorphous dimeric alkaloids. This mixture will again dissolved in methylene dichloride, whereupon the solution Chromatographed on aluminum oxide (CAMAG - activity III-IV).

The alkaloids are used in this procedure in the following order eluted: Leurosin, Vinblastin, Des-N-raethylvinblastin, Leurocristin and Leurosidin. The identification of the dimeric alkaloids in the eluate fractions is carried out by known methods, for example by thin-layer chromatographic analysis. The chromatographic process is in a column made of stainless steel with the dimensions 5 cm χ 730 cm for one

Pressure carried out from 14.1 to 28.1 kg / cm. The ratio of aluminum oxide and loading is about 300: 1. The eluate is monitored at 280 m , w , and appropriate fractions are separated off as a function of the maxima in the ultraviolet spectrum. Analysis of these fractions by thin-layer chromatography shows that they contain predominantly leurosine, vinblastine, des-N-methvinblastine and leurocristine. The fractions coming after the des-N-methylvinblastine and before the leucristine are combined, namely those fractions which come after the maximum of the des-N-methylvinblastine fraction and before the maximum of the leuocristine fraction and which contain more than one dimeric alkaloid, and these fractions are then converted into the corresponding sulfate salts by treatment with an excess of Ί percent strength ethanolic sulfuric acid. The sulfate salts thus prepared are then subjected to a pH gradient separation process in which a solution of the sulfate salts in citric acid buffer of pH 3.4 is extracted with benzene. The pH of the citric acid solution increases gradually

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each is increased by 0.5 pH units, and the resulting aqueous layer is extracted with benzene. Analysis by thin layer chromatography shows that 4-deacetoxy-3'-hydroxyvinblastine is present in the extracts at pH 5.4 and 5.9. The sulfates (vinblastine and leurocristine are the main impurities present as dimeric alkaloids based on thin-layer chromatographic analysis) are obtained from the extract at pH 5.4, and are dissolved in 5 ml of water, whereupon the acidity of the aqueous solution is increased by adding ammonium acetate pH 9 adjusts. The free bases of the alkaloids which precipitate are separated off by centrifugation and dissolved in 3 ml of methylene chloride, whereupon the resulting solution is placed under high pressure in a stainless steel column packed with neutral aluminum oxide / Woelm N-18 (18-30 .Xi) J with the Dimensions 8 mm by 6 m chromatographed using a linear gradient of 0 to 5% ethanol in methylene chloride. The column is operated at a pressure of about 77.3 kg / cm and a flow rate of 180 ml per hour. After the occurrence of material in the column effluent as determined in the ultraviolet spectrum, appropriate fractions are collected every three minutes. Fractions 30 to 32 contain 4-deacetoxy-3'-hydroxyvinblastine, as a thin-layer chromatographic analysis over silica gel using a solvent system of ether-diethylamine-toluene-methanol (100: 5: 5: 5) shows.

Examples of typical compounds according to the invention are: 3'-Hydroxy ^ -desacetoxyvinblastine-C-S-N-methylcarboxamide,

3'-Hydroxy ^ -desacetoxyvinblastine-C-S-N-cyclopropylmethylcarboxyamide,

2'-Hydroxy ^ -desacetoxyvinblastine-C-S-N-cyanoäthylamid,

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2'-hydroxy-4-deacetoxyvinblastine-C-3-N- (2-hydroxypropyl) amide,

Deoxyvinblastine-A-C-3-carboxyazid,

Deoxyvinblastine-A-C-3-N- (2-dimethylaminoethyl) carboxamide, Deoxyvinblastine-B-C-3-N- (2,3-dihydroxypentyl) carboxamide,

4-deacetoxyvinblastine-C-3-N- (3-hydroxypropyl) carboxamide,

4-deacetoxyvinblastine-C-3-N- (2-aminoethylaraino) carboxamide,

2'-Hydroxyvinblastine-C-3-N- (2-acetoxyethyl) carboxamide,

2'-Hydroxyvinblastine-C-3-N- (2-phenylethy1) carboxamide,

2'-Hydroxyvinblastine-C-S-N- (3-phenylpropyl) carboxamide and 3'-Hydroxyvinblastine-C-3-carboxhydrazide.

The compounds according to the invention are derivatives in which the carboxymethyl group located on carbon atom 3 of certain known indole dihydroindole alkaloids is converted into a carboxhydrazide group, a carboxazide group, a carboxamide group or a derivative thereof. Not all of these derivatives are usually formed by a single process. Those compounds in which the substituent R of the above formula I stands for NH ₃ , NH-NH ₃ or NH-CH ₃ are prepared as follows: Leurocolumbine, vincadioline, their corresponding 4-deacetyl compounds or deoxyvinblastine are treated with either ammonia, Methylamine or hydrazine, whereby the corresponding amide, N-methylamide or hydrazide is obtained. The product of this reaction with starting materials that have an intact 4-acetyl group is normally

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a mixture of compounds in which the carbomethoxy group on carbon atom 3 is converted into a carboxamide, N-methylcarboxamide or carboxhydrazide group and in which at the same time also the acetyl group located on carbon atom 4 completely or partially removed. For purification, the C-4-deacetyl derivatives prepared in this way are chromatographed separated from each other.

Those compounds according to the invention in which the substituent R is N (CH ₃ J ₃ , NH-alkyl-X, in which X is hydrogen, cyano or phenyl, NH- (C ₃ -Cg) -cycloalkyl, NH-alkyl-Am or NH -Alkyl- (OH), _ and alkyl and Am have the meanings given above, are prepared by the following process: A hydrazide of the formula I prepared by reacting the C-3 carbomethoxy compound with anhydrous hydrazine, in which R is NH-NH ₂ is converted into the corresponding azide by treating the hydrazide in a known manner with a nitrosating agent, such as nitrous acid, nitrosyl chloride, nitrogen tetroxide or amyl nitrite. The C-3 azide produced in this way is then treated with the primary or secondary amines of Formula HN (CH ₃ ) ₂ , NH ₂ -alkyl-X, NH ₂ - (C ₃ -Cg) -cycloalkyl, NH ₃ -AIkYl- (OH) _1-3 or NH ₂ -alkyl-Am reacted, whereby the The above C-3 azide-amine reaction does not affect the acyl group present on carbon atom 4 ht, so that this, if present at all, remains intact during implementation and work-up. The azide-amine transformation given above is carried out according to the method described in HeIv. Chim. Acta 26, 944 (1943), and reference is also made to US Pat. No. 2,090,429 and 2,090,430.

Compounds which have an acetyl group on carbon atom 4, can be made by combining vincadiolin, leucolumbin or deoxyvinblastine directly with ammonia, methylamine

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or hydrazine, which separates the 4-acetyl derivative from the 4-deacetyl derivative, and, in the case of the hydrazide, converts this to the azide and reacts the azide obtained with an amine, whereby the corresponding amides are obtained. Because of the sensitivity of the 4-acetyl group under basic reaction conditions, the abovementioned reaction sequence hydrazine-azide-amide is generally carried out with a 4-t) esacetyl derivative. The 4-deacetylamides of the above formula I can generally be acylated with an aliphatic acid anhydride or acid chloride to give the corresponding C-4 acetate, propionate or butyrate or a chlorine derivative thereof. For this acylation, a (C ₁ -C ₃ ) -alkyl-COCl or ChIOr- (C ₁ -C ₃ ) -alkyl-CO-Cl or a / (C ₁ -C -) - alkyl-CO / = O or / use chlorine- (C ₁ -C ₃ ) -alkyl-CO / ₂ = O as acid anhydride. The preferred acylation process is that described in US Pat. No. 3,392,173 for vinblastine and leurocristine, in which the first reaction product is a diacyl derivative which is then selectively hydrolyzed to a 4-acyl compound. Other methods of selective acylation or multiple acylation with subsequent selective hydrolysis can also be used to prepare the 4-acyl derivatives according to the invention.

However, certain precautions must be observed when selecting a particular acylation process. If the carboxyamide group located on carbon 3 contains an acylable group, for example the hydroxy or amino group, then the C-4 acylation must be carried out before the azide-amine reaction which leads to the desired C-3 carboxamide group. For this purpose, the C-3-carboxhydrazide is preferably acylated by the above-mentioned processes, the hydrazide group being protected first, since it would otherwise be acylated. The preferred hydrazide protecting group is the propylidene group, which is formed by reacting the NH ₂ -TeU of the hydrazide group with acetone. This group can easily be split off by treatment with acid. The propylidene derivative itself is preferably used

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but directly with nitrite to form an azide group (U.S. Patent 3,470,210, Example VII).

Other methods including selective acylation or multiple acylation followed by selective acylation Hydrolysis or selective protection of an acylatable function and subsequent acylation and subsequent Cleavage of the protective group is known to the person skilled in the art.

Compounds of the formula I given above, in which the substituent R is NH-alkyl-X and the symbol X is carboxyl, carboxamido or carbo- (C.-C ₃ ) alkoxy, can be prepared by adding an amino acid Aminoamide or, preferably, an amino ester of the formula

NH, -C-COQ, Z

wherein Q is OH, NH »or O-alkyl and Z is hydrogen or Cj-Cc-alkyl means with the particular dimeric indole dihydroindole azide selected implements. Amino acids suitable for this purpose and falling under the general formula I include for example leucine, isoleucine, valine, glycine, alanine or norleucine. Of course, other amino acids can also be used and use polypeptides for a reaction with, for example, 4-deacetylvinblastine-C-3-carboxazid, so as to be substituted Manufacture C-3 carboxamides that are antitumorally effective are.

Another process for preparing the primary amide (R = NH ₂ ) from the hydrazide (R = NH-NH ₂ ) is described in US Pat. No. 2,756, according to which the hydrazide is subjected to hydrogenolysis with Raney nickel.

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The new derivatives of formula I are only referred to refers to the new group that is formed on a given carbon atom. By replacing the methyl ester function In the case of vinblastine on carbon atom 3, a compound formed by an amide function, for example, therefore becomes simple referred to as vinblastine-C-3-carboxamide, rather than vinblastine-C-S-descarbomethoxy-C-S-carboxamide.

The compounds of formula I are in the form of their free bases including their carboxamides, carboxazides and carboxhydrazides white or tan amorphous solids. If possible, however, the amides are preferably used in the form of their anionic Salts with non-toxic acids isolated and crystallized. These salts represent high melting point white crystalline or amorphous water-soluble solids.

The invention is further illustrated by the following examples. ^l

Example 1 ^ Desacetyldeoxyvinblastine-BCS-carboxhydrazide

In a sealed reaction vessel, 2.55 g Deoxyvinblastin B are reacted with 30 ml of anhydrous hydrazine in anhydrous methanol at a temperature of about 60 ⁰ C over a period of about 18 hours. The reaction vessel is then cooled and opened, whereupon the contents of the vessel are removed and the volatile constituents are evaporated in vacuo. The residue obtained, which contains 4-deacetyldeoxyvinblastine-BC-3-carboxhydrazide, is taken up in methylene chloride, whereupon the methylene chloride solution is washed with water, separated off and dried. Removal of the methylene chloride in vacuo gives an amorphous powder with the following

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physical characteristics: infrared maxiraa at 3440 cm (NH), 1735 cm " ¹ (COO), 1675 cm" ¹ (CON); Molecular ion spectrum m / e = 752 (in accordance with ^C 43 ^H 5g ^N g ° fi) »NMR spectrum delta 3.78 (ArOCH ₃ ), delta 3.58 (C ₁₈ COOCH ₃ ), delta 2.77 (N- CH ₃ ), delta 4.15 (C ₄ -H).

Example 2 4-Deacetoxyvinblastine-C-3-N- (2-hydroxyethyl) carboxamide

According to the process described in Example 1, 4-deacetoxyvinblastine is reacted with anhydrous hydrazine in methanolic solution at a temperature of 46 ° C. over a period of 3 days in a closed reaction tube. The 4-deacetoxyvinblastine-C-3-carboxhydrazide prepared in this way is isolated and purified as described in Example 1. The compound obtained has the following physical properties: pK = 5.61 and 7.38; Ultraviolet spectrum; lambda_ _{= v} =

el * * Iu α. Χ

215 and 267 nm; Infrared spectrum: maximum at 3450 cm (NH), 1725 cm " ¹ (COO), 1680 cm" ¹ (CON); Molecular spectrum m / e = 252, 411, 355, 244, 154; Molecular ion M + = 752 in accordance with the formula C ₄₃ H ₅₆ NgOg. The compound is a tan amorphous powder.

The produced in the above manner, 4-Desacetoxyvinblastin-C-3-carboxhydrazid is converted by reaction with sodium nitrite in a hydrochloric acid solution at a temperature of 0 ⁰ C to the corresponding Carboxazid. The resulting reaction mixture is made basic by adding an excess of cold 5 percent aqueous sodium bicarbonate. The aqueous solution obtained in this way is extracted three times with methylene dichloride.

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5 ml of ethanolamine are added to a solution containing about 1.2 g of 4-deacetoxyvinblastine-C-3-carboxazide. The reaction mixture is then sealed and protected from light. The batch is left to stand for one day at room temperature, whereupon the reaction vessel is opened and the volatile constituents present in the reaction mixture are removed by evaporation in vacuo. The residue obtained, which contains the 4-deacetoxyvinblastine-03-N- (2-hydroxyethyl) carboxamide formed in the above reaction, is dissolved in methylene dichloride, whereupon the methylene dichloride layer is washed several times with water. The methylene dichloride layer is then separated off and dried, whereupon the solvent is removed by evaporation in vacuo. The residue obtained in this way is then chromatographed over silica gel using a 3: 1 solvent mixture of ethyl acetate and ethanol as the eluent. Those fractions which, according to analysis by thin layer chromatography, contain the desired product are combined and evaporated to dryness in vacuo. The 4-deacetoxyvinblastine-C-3- (N- (2-hydroxyethyl) carboxamide produced in this way is a tan-colored amorphous material with the following physical properties: Molecular ion M + = 781 in accordance with the empirical formula ^C 45 ^H 5g ^N 5 ° 7 Infrared spectrum: "Maxima at 3420 cm" ¹ NH), 1735 cm " ¹ (COO), 1665 cm" ¹ (CON). To produce the sulfate salt, this material is treated with ethanolic sulfuric acid, the pH being adjusted to 3.8 to 4.2. The sulfate salt is obtained by evaporating the volatile constituents in vacuo.

Example 3 4-Deacetylvincadioline-C-3-N-methylamide

Vincadiolin is used according to the procedure described in Example 1 with hydrazine to give the corresponding C-3 carboxhydrazide. The hydrazide obtained in this way sets you then according to the procedure described in Example 2 to the corresponding carboxazide, whereupon this carboxazide after the procedure also described in Example 2 with methylamine

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to react. This gives 4-deacetylvincadioline-C-3-N-methylamide with the following physical properties: maxima in the infrared spectrum at 2.9-5 iti / U, 5.75 m, u and 5.97 m, u; NMR spectrum in agreement with the postulated structure with an additional doublet at delta 3.82 (amide methyl hydrogen); Molecular spectrum: Molecular ion M + = 783 in accordance with ^C 44 ^H 57 ^N 5 ° o *

The procedure described above also produces 4-deacetylleurocolombin-C-3-N-methylamide manufactured.

Example 4 Preparation of Salts

Other salts, which also include salts of inorganic anions, such as the chlorides, bromides, phosphates or nitrates, as well as salts organic anions, such as the acetates, chloroacetates, trichloroacetates, Benzoates, alkyl or arysulfonates can be prepared from the amide bases according to the invention by an analogous process as has been described above in Example 1 for the preparation of the sulfate salt by replacing the 1 percent aqueous sulfuric acid used in each case the corresponding acid in a suitable solvent.

Of course, requires the presence of other ester and / or Amide groups in the indole dihydroindole components take special care in the preparation of salts to ensure hydrolysis, esterification or to avoid other reactions that occur at high temperatures and extremely acidic pH values and the like comes.

The compounds according to the invention oppose each other in vitro herpes virus using a tissue culture system in a platelet suppression test similar to that described in Applied Microbiology 9, 66-72 (1961) is similar to the test described, has been shown to be antivirally effective.

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The compounds according to the invention were also found to be effective in vivo against transplanted mouse tumors. from However, the effectiveness of the compounds according to the invention against Ridgeway osteogenic sarcoma (ROS) is of particular interest. and Gardner Lymphosarcoma (GLS). The effectiveness of the invention Compounds to these tumors were identified using a procedure in which the drug was normally intraperitoneally was administered in a predetermined dose 7 to 10 days after inoculating the tumor.

The following table shows the results of several tests in which mice with transplanted tumors were successfully treated with a compound of the invention. In of this table are in column 1 the name of the respective compound, in column 2 the transplanted tumor, in column 3 the Dose or the dosage range and the number of days over which this dose was administered and in column 4 the percentage inhibition of tumor growth indicated. (ROS is an abbreviation for Ridgeway-Osteogensarcom; GLS is an abbreviation for Gardner-Lymphosarcom and CA 755 an abbreviation for an adenocarcinoma.)

The compounds of the invention are similar to Leurocristin and vinblastine toxic to mice at doses in excess of that which would cause 100 percent inhibition of the transplanted Tumor. For reasons that have not yet been clarified, all active ingredients show in a given test including the active ingredients used for comparison, such as vinblastine, a toxicity at doses at which they usually give tumor inhibition without toxicity. The results given in the table are therefore typical Attempts in which the comparative active ingredients to the lead to the expected results and therefore do not represent the mean values of all tests.

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Tabel

Cr; CO

O CD PO

link

4-Deacetoxyvinblastine-C-3-N- (2-hydroxyethyl) carboxamide sulfate

dose Percentage tumor mg / kg χ days Inhibition ROS 0.5 χ 10 44 GLS 0.75 -1.5x6 51-100

K3 CJI

The compounds of the invention are also effective against other transplanted tumors. So, for example, led a 9 day parenteral injection of 0.25 mg / kg vinblastine C-3-N-methylcarboxamide sulfate in Mecca lymphosarcoma to a 54 percent inhibition of growth, and vinblastine-C-3-amide for example, resulted in a 28 percent inhibition of growth. Vinblastine was found to be at the same dosage level itself to be completely ineffective.

In studies of CA 755 adenocarcinoma, 4-deacetylvinblastine-C-3-carboxamide sulfate was also given 67 percent inhibition of tumor growth with 4-deacetylvinblastine-C-3-N-methylcarboxamide sulfate a 61 percent inhibition and with vinblastine-C-r3-carboxamide sulfate a 49 percent inhibition at a dose of 0.25 mg / kg for 8 days and a 72 percent inhibition at a dose of Q.3 mg / kg. At a vinblastine resulted in a similar experiment at 31 percent Inhibition while walking with. Leurocristin received 79 percent inhibition at the slightly lower dose of 0.2 mg / kg, and according to an extremely effective evaluation system. Opposite to L5178Y lymphocyte leukemia survived using Vinblastine C-3 carboxamide sulfate at a dose of 0.25 mg / kg for 10 days in a test with 5 mice 3 test animals for an unlimited period of time during the lifespan of the two sick mice in this experiment was 26% longer than the comparison animals. In the same experiment you got with Vinblastine, being a 36 percent increase in survival time However, there were no permanently surviving animals, so that this active ingredient is only minimally effective.

The amides and hydrazides of the invention can be administered as antineoplastic agents either parenterally or orally. For oral administration, a suitable amount of a pharmaceutically acceptable salt of a base of the formula I, with the exception of those compounds in which the substituent R is NH — NH ₃ or N _3, is mixed with a non-toxic one

609829/08 2 4

Acid with starch or another excipient and then brings the mixture into telescopic gelatine capsules, each containing 7.5 to 50 mg of active ingredient. In a similar way, the antineoplastic salt can also be mixed with starch, a binder and a lubricant and the mixture can then be compressed into tablets, each containing 7.5 to 50 mg of active ingredient. The tablets may be scored if one wishes to use smaller or divided doses. For parenteral administration, the intravenous route is preferred. For this purpose, isotonic solutions are used which contain 1 to 10 mg / ml of a salt of an indole dihydroindolamide of the formula I, with the exception of the hydrazides and azides. The compounds according to the invention are administered once a week in amounts of 0.1 to 1 mg / kg mammalian body weight, which depends both on the effectiveness and on the toxicity of the respective compounds. The free bases of the compounds of the formula I in which the substituent R is NH-NH ₂ or N ₃ are compounded in a similar manner to give suitable dosage forms and administered in similar dosage concentrations.

Most of the compounds according to the invention are suitable as antineoplastic or antiviral medicaments, but two types of derivatives, namely the hydrazides and azides' (compounds of the formula I in which R is NH-NH ₃ or N-), as already mentioned, can also be used use as intermediates, since the hydrazides can be converted into the azides by reaction with a nitrosating agent, such as, for example, treatment with nitrous acid, or into the simple amides by hydrogenolysis. The azide itself can in turn react with primary or secondary amines, whereby the amides according to the invention are obtained.

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Claims (6)

Claims / i \ Process for the production of vinblastine, leuosidine, leucristine and related dine alkaloids of the formula I. 11 'Τ V ⁴ ' Τ CH2-CH3 'φ X * β— D ^ 2iV N ^ -R ^{4 1 3 #} Χ Λ16 'A 1 8' / λ «-j ■ 1 4 / ι ₅ \ Χ ₇ \ / _. 1 ο · [\ | β? I! Il ' ⁵ ΐΓ T ^Τ CHs-CH ₃ R for NH ₂ , NH-NH ₂ , N (CH ₃ J ₂ , pyrrolidinyl, NH-alkyl-X, NH- (C ₃ -C ₈ ) -cycloalkyl, NH-alkyl-Am, NH-alkyl- (OH) ₁ _ ₃ or N ₃ , where alkyl is (C ₃ -Cg) -alkyl and Am is NH ₃ , NHCH ₃ or N (CH ₃ J ₂ and X is hydrogen, cyano, phenyl, carboxyl, carbo (C ₁ -C ₃ ) is alkoxy or carboxamide, R is hydrogen, hydroxy, 0- (C ₁ -C ₃ ) -alkanoyl or O-chloro- (Cj-C ₃ ) -alkanoyl and 609829/0 824 2 3 4
R, R and R stand for hydrogen or hydroxy, 3 4 with the proviso that the substituents R and R water are substance, if the substituent R is hydrogen, 3 and that at least one of the substituents R or 4 2 R is hydroxy if the substituent R is hydroxy. 2. 4-Deacetyldeoxyvinblastine-B-C-3-carboxhydrazine. 4-Deacetoxyvinblastine-C-3-N- (2-hydroxyethyl) carboxamide. 4. 4-Deacetylvincadioline-C-3-N-methylamide. 4-Deacetylleurocolombin-C ~ 3-N-methylamide. 6. Process for the production of Vinblastine, Leurosidine, Leurocristin and related dimeric alkaloids of the in Claim 1 given formula I, characterized in that one (A) a compound of the formula I given above, in which the substituent R is O-CH-. stands, the substituent R ¹ Is hydrogen or acetyl and the substituents R, 3 4
R and R have the meanings given above for formula I. have, with a compound of formula II NH ₂ R ⁵ II, wherein the substituent R is hydrogen, methyl or amino, reacts and 9829/0824 (b) where appropriate, the compounds obtained in this way in which the substituent R is —NH — NH ₂ , with a nitrosylating agent and with a compound of the formula III .R ⁶ -NH III in which the substituent R is hydrogen or methyl and the substituent R ^{7 is} methyl, alkyl-X, (C ₃ -C ₈ ) -cycloalkyl, alkyl-Am or alkyl- (OH) ._ ₃ , in which alkyl is a (C .-C-O-alkyl and Am and X have the meanings given for formula I, and the process (a) or (b) 1 obtained compounds if the substituent R is hydroxy is, if desired acylated to compounds in which i
the substituent R has a different meaning than hydroxy, whereupon the compounds obtained on this by reaction with a non-toxic inorganic or organic If desired, acid is converted into the corresponding acid addition salts. 6 09 8 29/0824