DE1543890B2 - NEW 4'-DIMETHYL-EPIPODOPHYLLOTOXINBETA-D-GLUCOSIDE DERIVATIVES, THEIR PRODUCTION AND MEDICINAL PRODUCTS - Google Patents

Description

wherein R is the phenyl, 2-furyl or 2-thienyl radical stands.

2. A process for the preparation of compounds of the general formula I according to claim 1, characterized in that a compound of the formula II

CH ₂ OH

HO

(H)

CH ₃ OI OCH ₃ OH

with an aldehyde of the general formula

R-CHO

(III)

wherein R has the above meaning, reacts in the presence of an acidic catalyst.

3. Medicinal products with cytostatic activity containing a compound of the formula I according to Claim 1 and customary carriers.

The present invention relates to new compounds of the general formula I.

OCH ₃

in which R is the phenyl, 2-furyl or 2-thienyl 50 one is 4'-demethyl-epipodophyllotoxin-j8-D-glucoside the rest stands. Formula II

The new connections are made by

(II)

CH ₃ O OCH ₃

with an aldehyde of the general l-'omiel
R-CHO

wherein R has the above meaning, reacts in the presence of an acidic catalyst. Cornmen as catalysts for example Lewis acids, such as anhydrous zinc chloride, or a dried cation exchanger with sulfonic acid groups in the H + form in question.

It is particularly advantageous to use 4'-demethyl-epipodophyllotoxin-ß-D-glucoside Dissolve directly in pure aldehyde and add the catalyst. The reaction generally takes place at room temperature or at a slightly elevated temperature and is after 1 to 10 hours, at 20 ° C usually after 3 to 6 hours, largely ended.

To isolate the condensation product, the reaction mixture is not taken in one with water miscible solvents, e.g. B. chloroform, shakes several times to remove water-soluble salts and By-products with water, the organic phase is dried and evaporated in vacuo, the Most of the excess aldehyde is removed. An oily residue is obtained from which the last residues of the aldehyde easily by macerating or digesting the condensation product with a suitable solvents, such as. B. petroleum ether, pentane, hexane or by chromatography on a neutral reactive adsorbents, such as. B. silica gel, are removed. The condensation product will then isolated and purified in a manner known per se, e.g. B. by chromatography, reprecipitation or crystallization.

4'-demethyl-epipodophyllotoxin- / 9-D-glucoside
(Formal)

The starting material used for the production of the new compounds of the general formula I is 4'-demethyl-epipodophyllotoxin-j? -D-glycoside is made by one of the following processes:

Either one cleaves from tetra-O-acetyl-4'carbobenzoxy-4'-demethyl-epipodophyllotoxin - ^ - D-glucoside (Formula IV)

CH, CO—

(IV)

CH ₃ OI OCH ₃

O-CO-CH ₂ QH ₅

first the carbobenzoxy group hydrogenolytically and submits the tetra-0-acetyl-4'-demethyl-epipodophyllotoxin-ß-D-glucoside thus obtained (Formula V)

CH ₇ OAc

Hydrogenolysis

45

(V)

CH ₃ O

OH

OCH ₃

Presence of anhydrous zinc acetate alcoholysis or the acetyl groups are cleaved first des Tetra-O-acetyW-carbobenzoxy ^ '- demethyl-epipodophyllotoxin-0-D-glucoside in the presence of anhydrous zinc acetate or a mixture of anhydrous Zinc acetate-sodium acetate alcoholically and then hydrogenolytically removes the carbobenzoxy group.

The hydrogenolysis takes place according to methods known per se. In this case, it is expediently without excess pressure and at 20 to a maximum of 40 ⁰ C in the presence of palladium catalysts, such as. B. palladium on charcoal or on barium sulfate, hydrogenated. The solvents used are preferably alcohols, such as. B. methanol or ethanol, with the addition of 0.5 to 5 percent by volume of glacial acetic acid and 10 to 50 percent by volume of acetone in question. The amount of catalyst is 1-5 percent by weight, based on the substance used.

Alcoholysis

It was not to be expected that from tetra-O-acetyl-4'-carbobenzoxy ^ '- demethyl-epipodophyllotoxin-ß-D-glucoside and tetra-O-acetyl-4'-demethyl-epipodophyl-iotoxin-j3-D-glucoside the acetyl groups hydrolytically under the usual basic and acidic conditions are cleavable in order to arrive at the corresponding free glucoside in this way. As is well known Lignanglucosides suffer from epimerization of bases, while the action of acids leads to Decomposition, with splitting off of the sugar residue, can lead.

It has now surprisingly been found that tetra-O-acetyl-4'-carbobenzoxy-4'-demethylepipodophyllotoxin-jS-D-glucoside or from Tetra-O-ace-

tyl-4'-demethyl-cpipodop! iylloloxin- / j-D-gkicosicl die Acetyl residues without simultaneous epimerization of the aglycone at the C-3 atom and without splitting off the Remove the entire sugar residue by alcoholysis, preferably with these compounds Methanol, in the presence of anhydrous zinc acetate, subjected.

The methanolysis is carried out in anhydrous methanol at reflux temperature. The amount of catalyst is around 20-50 percent by weight, based on the tetra-O-acetyl ^ '- demethyl-epipodophyllotoxin-ß-D-glucoside used. The reaction time is between 15 and 30 hours. In the methanolysis of tetra-O-acetyl ^ '- demethyl-epipodophyllotoxin-jS-D-glucoside With zinc acetate as a catalyst, a precipitate forms in the course of the reaction. For work-up this precipitate is brought into solution by adding acetic acid with gentle warming.

After evaporation of the solvent, the residue is dissolved in a chloroform-butanol mixture and the zinc salts are removed by shaking with water. One wins from the organic phase evaporation of the crude 4'-demethyl-epipodophyllotoxin-ß-D-glucoside, that in a manner known per se, e.g. B. by chromatography and / or crystallization, purified will.

As stated above, the cleavage of the protecting groups can also be done in reverse order proceed by first adding Tetra-O-acetyl-4'-

carbobenzoxy-4'-demethyl-epipodophyllotoxin-] S-D-glucoside in the presence of anhydrous zinc acetate and optionally addition of anhydrous sodium acetate boiled under reflux in methanol until the cleavage of the acetyl groups has ended, wherein at the same time the carbobenzoxy group is also partially split off. From the resulting mixture The previously unknown 4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin-ß-D-glucoside can be added to the reaction components (Formula VI)

CH ₇ OH

HO

(VI)

CH ₃ OI OCH ₃

0-CO-CH ₂ QH ₅ O

isolate, which further by hydrogenolysis of the carbo benzoxy group in 4'-demethyl-epipodophyllotoxin-j9 D-glucoside is converted.

Tetra-O-acetyl-4'-carbobenzoxy-4'-demethylepipodophyllotoxin-] 3-D-glucoside (Formula IV)

The one used for the production of 4'-demethyl-epipodophyllotoxin-ß-D-glucoside serving starting material Tetra-O-acetyl ^ '- carbobenzoxy ^' - demethyl-epipodo- Phylloloxin- / i-D-glucoside is according to one of the following Process made:

Either 4'-carbobenzoxy-4'-dcmelhyl-epipodophyllotoxin is condensed (Formula VI)

OH

(VIl)

CH ₃ OI OCH,

O C- O C H.2 (

with M-acetobromoglucose (Formula VIII)
CH ₂ OAc

05

AcO

(VIII)

AcO

in an organic solvent which is inert under the reaction conditions and in the presence of Zinc oxide or mercury oxide or one condenses

4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin
(Formula VII) or 4'-carbobenzoxy-4'-demethyl-podophyllotoxin (Formula IX)

OH

40

(IX)

CH ₃ O OCH ₃

OCO CH ₂ CgH ₅
O

with 2,3,4,6-tetra-O-acetyl-yii-D-glucose (formula X)

55

AcO

AcO

in the presence of boron trifluoride ethyl etherate, in an organic - 60 inert under the reaction conditions Solvent at a temperature below 0 ° C.

Condensation with oc-acetate bromoglucose

The preparation of the Tetra-O-acetyl ^ '- carbobenzoxy-4'-demethyl-epipodophyllotoxin- / J-D-glucoside came across because of the steric hindrance caused by the unfavorable position of the secondary OH group on the Ci atom at the aglycon for difficulties. It wasn't like that

possible, this connection z. B. under the conditions of the Koenigs-Knorr synthesis in benzene with the addition of silver salts (such as Ag ₂ O or Ag ₂ CO ₃ ) or in acetonitrile, in the presence of Hg (CN) ₂ , in practically usable yield from 4'-carbobenzoxy -4'-demethyl-epipodophyllotoxin and a-acetobromoglucose.

It has now surprisingly been found that Tetra-O-acetyW-carbobenzoxy ^ '- demethyl-epipodophyllotoxin-ß-D-glucoside Can be obtained in good yield if 4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin with α-acetobromoglucose in an organic which is inert under the reaction conditions Solvent and in the presence of mercury (II) oxide or preferably converts zinc oxide.

A preferred embodiment of the method consists in that 4'-carbobenzoxy-4'-demethylepipodophyllotoxin with a-acetobromoglucose in a suitable solvent, such as. B. ethylene chloride or preferably acetonitrile, at temperatures of 20 to 8O ⁰ C, preferably between 40 and 70 ⁰ C, in the presence of mercury (II) oxide or zinc oxide. The temperature, concentration of the starting

f) The reaction time, depending on the substance and grain size of the metal oxide, is in the range from 0.5 to 12 hours. In order to achieve the highest possible conversion of 4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin during glucosidation, α-acetobromoglucose is used in a 2-4 fold molar excess. The metal oxide is used in the same molar amount as the α-acetobromoglucose. The initial concentration of 4 ^/ '-Carbobenzoxy-4'-demethyl-epipodophyllotoxin in the solvent should be between 5 and 20 percent by weight. The reaction is complete under these conditions after 40 to 60 minutes.

For the isolation of the tetra-O-acetyl-4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin-j3-D-glucoside is filtered off from the excess metal oxide, most of the solvent is distilled off in vacuo and the Residue for removing mercury salts with sodium bromide solution or zinc salts with water-methanol (9: 1) washed. To remove most of the decomposition products from the excess α-Acetobromoglucose becomes the reaction mixture

k. either subjected to pre-chromatography or extracted with 5-30% aqueous ethanol at elevated temperature. For further cleaning the top fractions of the pre-chromatography are subjected to post-chromatography or the Ethanol-insoluble residue chromatographed on silica gel.

The pure glucoside fractions are combined and give crude after drying in a high vacuum Tetra-O-acetyl-4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin-j3-D-glucoside, which is purified by repeated chromatography.

Condensation with
2,3,4,6-tetra-O-acetyl-j3-D-glucose

It has surprisingly been found that the implementation of 2,3,4,6-tetra-O-acetyl-D-glucose in . Form of their pure jS anomer both with 4'-carbobenzoxy-4'-demethyl-podophyllotoxin as well as with 4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin, d. H. regardless of the stereochemistry of the OH group at C-I of the aglycon, in the presence of boron trifluoride ethyl etherate at a temperature below 0 ° C. in a solvent which is inert under the reaction conditions to tetra-O-acetyl-4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin-jS-D-glpcoside leads in high yield, the end product can be isolated easily and in high purity.

Due to the reaction described, it was not possible to foresee on the basis of the previous state of the art that practically only the ß-glucoside compound is formed as the end product. It wasn't closed either expect the sensitive 2,3,4,6-tetra-O-acetyl-ß-D-glucose under the specified reaction conditions, especially in the presence of boron trifluoride ethyl etherate, no or only very little anomerization to the α-anomer suffers.

It was also surprising that the implementation of 2,3,4,6-tetra-O-acetyl-ß-D-glucose in the presence of Boron trifluoride ethyl etherate with both 4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin as well as with 4'-carbobenzoxy-4'-demethyl-podophyllotoxin to the same end product, namely almost exclusively for Tetra-O-acetyl-4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin-j8-D-glucoside, leads. The corresponding glucoside of 4'-carbobenzoxy-4'-demethyl-podophyllotoxins is only formed in traces.

A preferred embodiment of the method consists in that a solution or suspension of 4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin or 4'-carbobenzoxy-4'-demethyl-podophyllotoxin • and 2,3,4,6-Tetra-O-acetyl-j9-D-glucose in one, under the reaction conditions inert solvent, such as

z. B. ethylene chloride, chloroform or methylene chloride, at -10 to -25 ⁰ C with boron trifluoride ethyl etherate. For the most quantitative possible conversion of the valuable aglycones, 1.5 to 3 mol of 2,3,4,6-tetra-O-acetyl-j3-D-glucose and 2 to 4 mol

Boron trifluoride ethyl etherate used. The initial concentration of 4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin or of 4'-carbobenzoxy-4'-demethyl-podophyllotoxin in the solvent should be about 25 to 40% be.

For the isolation of the "Tetra-O-acetyl-4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin-j9-D-glucoside the boron trifluoride is inactivated by adding a tertiary organic base, preferably pyridine, and then the Boron trifluoride-pyridine complex washed out with water. That after evaporation of the organic solvents resulting mixture of 2,3,4,6-tetra-O-acetyl-D-glucose and the condensation product is obtained by precipitation in aqueous ethanol from unreacted tetra-acetylglucose freed. To remove a small amount of a troublesome, sulfur-soluble by-product the precipitate is taken up in hot methanol, the solution filtered and the methanol in vacuo removed. The tetra-O-acetyW-carbobenzoxy-4'-demethyl-epipodophyllotoxin-j3-D-glucoside obtained in this way is sufficiently pure for the subsequent stages. By crystallization from benzene-pentane or benzene-cyclohexane it can be obtained in analytically pure form.

4'-carbobenzoxy-4'-demethyl-podophyllotoxin

(Formula IX) and

4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin (formula VII)

The one used for the preparation of Tetra-O-acetyl-4'-carbobenzoxy ^ '- demethyl-epipodophyllotoxin-jJ-D-glucoside serving starting materials 4'-carbobenzoxy-4'-demethyl-podophyllotoxin and 4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin are according to the following Process made:

609 547/407

4'-Dcmethyl-podophyllotoxin (Formula Xl)

(XI)

CH ₃ OI OCH ₃
OH

or 4'-demethylepipodophyllotoxin (formula XII)

OH

(XII)

CH ₃ O

OCH,

is in an anhydrous organic solvent which is inert under the reaction conditions at temperatures from -20 to -5 ° C in the presence of a tertiary organic base with benzyl chloroformate to 4'-carbobenzoxy-4'-demethyl-podophyllotoxin or 4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin implemented.

It was not previously possible to protect groups such. B. acyl groups or the tetrahydropyranyl radical, selectively on the phenolic OH group in position 4 'of the 4'-demethyl-epipodophyllotoxins. So will for example in the usual acetylation of 4'-demethyl-podophyllotoxin or of 4'-demethylepipodophyllotoxin obtained the 1,4'-diacetyl derivative. In the case of such 1,4'-diesters, a direct one is selective Cleavage of the protective group in position 1 cannot be achieved.

In the reaction of 4'-demethyl-podophyllotoxins or 4'-demethyl-epipodophyllotoxins with dihydropyran a selective etherification succeeds, however, this takes place due to a steric hindrance of the phenolic OH group not at C-4 ', but in position 1 (formation of 1-O-tetrahydropyranyl ether).

It has now surprisingly been found that it is possible to use a derivative of 4'-demethyl-podophyllotoxins or of 4'-demethyl-epipodophyllotoxins with a protected hydroxyl group in position 4 'and more free To obtain hydroxyl group in position 1.

A preferred embodiment for the preparation of 4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin consists in that one finely powdered 4'-DemethyI-epipodophyllotoxin in ethylene chloride, methylene chloride or chloroform at a temperature of -5 to - 15 ° C with carbobenzoxychloride (benzyl chloroformate) in the presence of a tertiary organic base, such as. B. pyridine or dimethylaniline, where Per mole of 4'-demethyl-epipodophyllotoxin 1 to 1.35 moles of carbobenzoxychloride should be used to reduce the Avoid the formation of large amounts of the 1,4'-bis-carbobenzoxy compound. The separation of the 4'-carbobenzoxy-4'-demethyl-epipodophyllotoxins formed from the reaction mixture, which also has not yet reacted 4'-demethyl-epipodophyllotoxin as well small amounts of M'-bis-carbobenzoxy ^ '- demethyi-epipodophyllotoxin contains, is carried out in a manner known per se, e.g. B. by crystallization or chromatography.

The 4'-demethylpipodophyllotoxin used as starting material can be obtained by epimerizing 4'-demethylpodophyllotoxin (see Experiment 1). The preparation of 4'-carbobenzoxy-4'-demethyl-podophyllotoxin is carried out in an analogous manner, starting from 4'-demethyl-podophyllotoxin, 1 to 1.6 moles of carbobenzoxychloride being used per mole of 4'-demethyl-podophyllotoxin. The new compounds of general formula I have a high cytostatic effect in vitro on mastocytoin cells and fibroblast cultures. Furthermore, they are distinguished by a strong effectiveness against experimental tumors, in particular the mouse leukemia L-1210. The new compounds of general formula I can be used therapeutically for combating pathological processes which are associated with Zellvermeh- _r tion, z. B. Malignant neoplasms. '.,

The dosage of the new compounds of general formula I varies approximately between 1 and 50 mg per day.

The new compounds can be used as medicaments alone or in corresponding medicament forms for oral, enteral or parenteral administration. In order to produce suitable dosage forms, these are processed with organic or inorganic, pharmacologically indifferent auxiliaries. As auxiliaries are used z. B.
for tablets and coated tablets:

Lactose, starch, talc, stearic acid, etc.,
for syrups:

Cane sugar, invert sugar,

Glucose solutions, etc.,
for injection preparations:

Water, alcohols, glycerine, vegetable oils

and the same.,
for suppositories:

natural or hydrogenated oils,

Waxes i.a. more. Γ

In addition, the preparations can contain suitable preservatives, Contain stabilizers, wetting agents, solubilizers, sweeteners, dyes, flavorings, etc.

In the following examples, which explain how the process is carried out, all temperature data are given in degrees Celsius. The melting or decomposition points are determined on the Kofler block.

example 1

4'-demethyl-epipodophyllotoxin S-D-benzylidene-glucoside

1 g of dried 4'-demethyl-epidopophyllotoxin - ^ - D-glucoside (preparation see below) is in 20 ml Dissolved pure benzaldehyde and after adding 0.5 g of anhydrous zinc chloride with exclusion of moisture shaken for 5 to 6 hours at 20 ° on the machine. The progression of the reaction will followed continuously by thin layer chromatography. Silica gel plates and chloroform are also suitable for this 6% methanol as a solvent. To make the substances visible, the plates are sprayed with a lproz.

Solution of cerium (IV) ammonium nitrate in 50% Schweifesäure and then heated to 100 to 12O ⁰ C. For working-up of the condensation product is added to the clear, red-brown colored reaction solution with chloroform, and shaken with water. The aqueous phase is extracted twice with chloroform. All the chloroform phases are combined, washed twice with water, dried over sodium sulfate and the solvents evaporated in vacuo. The oily residue obtained is triturated with pentane to remove any benzaldehyde that is still adhering until a powdery product is obtained. For further purification, the benzylidene derivative is taken up in 10 ml of acetone and the acetone solution is added dropwise, with stirring, to 100 ml of pentane, a light yellow to white colored precipitate being obtained. The crude product can also be purified by chromatography on silica gel columns. For this purpose, a solution, as concentrated as possible, of 1 g of the crude benzylidene derivative in a mixture of chloroform + 2% methanol is filtered onto a column of 200 g of silica gel and eluted with the same solvent mixture. The thin-layer chromatographically uniform fractions are combined and reprecipitated from acetone-pentane. 4'-Demethyl-epipodophyllotoxin-ß-D-benzylidene-glucoside is obtained as a white powder of Sm p. Get 182-185 ⁰ C. Recrystallization from abs. Ethanol: crystals with a melting point of 245-246 ° C. The amount optical rotation: [ "] = -99 ° C in methanol and - 104 ⁰ C in chloroform!?.

Example 2

4'-Demethyl-epipodophyllotoxin - /? - D-thenylidene-glucoside

0.5 g of dried 4'-demethyl-epipodophyl otoxin - /? - D-glucoside are aldehyde 2-thiophene with 10 ml of pure and mixed 0.25 g of anhydrous zinc chloride and the mixture exclusion of moisture at 20 ⁰ C on the machine shaken, gradually forming a clear solution. The course of the condensation is - as described above - followed by means of thin layer chromatography. After 3 -4stündiger reaction time the solution is diluted with f: chloroform and shaken with water. The chloroform phase is washed twice more with a little water and then dried over sodium sulfate and evaporated. To remove excess thiophene-2-aldehyde, the residue obtained is dissolved in a little acetone and reprecipitated by adding pentane. The reprecipitation from acetone-pentane is repeated until the condensation product precipitates in flaky form. For further purification, the crude product is chromatographed on silica gel. The fractions which are uniform according to thin-layer chromatography are combined and give crystals from absolute alcohol. Pure 4'-demethyl-epipodophyllotoxin-jS-D-thenylidene-glucoside has a melting point of 242-246 ⁰ C (last residues up to 255 ° C) and has the optical rotation in chloroform-methanol (9: 1) [«]? ? = - 107 ⁰ C.

Example 3

4'-Demethyl-epipodophyllotoxin 9-D-furfurylidene-glucoside

0.5 g of dried 4'-demethyl-epipodophyllotoxin - /? - D-glucoside is mixed with 10 ml of pure furfural and after. Addition of 0.25 g of anhydrous zinc chloride with exclusion of moisture for 3 to 4 hours at 20 ^° C. on the machine. The course of the condensation reaction is checked by thin layer chromatography. Thin layer chromatography can be carried out on silica gel plates using chloroform with 6% methanol as the mobile phase. To make the fabrics visible, spray with a 1 percent. Solution of cerium (IV) ammonium nitrate in

ίο 50 percent Sulfuric acid and then heated to 100 to 120 ^° C. After the condensation has ended, chloroform is added to the green-colored reaction solution and then shaken out with water. The aqueous phase is extracted twice with chloroform. Combine all chloroform phases, nor is washed twice with a little water, dried over sodium sulfate and the solvent The remaining oily residue evaporated in vacuo at 6O ⁰ C. (ca. 10 mL) to remove adhering furfural with stirring in 150 ml of pentane are added dropwise whereby a greasy precipitate is obtained. After decanting off the supernatant pentane, the precipitate is taken up in a little acetone and this solution is added dropwise to 150 ml of initially charged pentane. The separated, now flaky condensation product can be purified by chromatography on silica gel. For chromatography, a column of 100 g of silica gel is charged with a concentrated solution of the crude product in chloroform + 2% methanol and eluted with the same solvent mixture. The peak fractions, which are uniform according to thin-layer chromatography, are combined and reprecipitated from acetone-pentane. 4'-Demethyl-epipodophyllotoxin - /? - D-furfurylidene-glucoside is obtained as a white powder with a melting point.

188-19ΓC received. From abs. In ethanol, crystals with a melting point of 267 ° -269 ° C. are obtained. The optical rotation value in chloroform is [«] ² S = -1 02 ° C.

Example 4

4'-Demethyl-epipodophyllotoxin - /? - D-benzylidene-glucoside

1.0 g of finely powdered ^ '- demethyl-epipodophyllotoxin - /? - D-glucoside is dissolved in 20 mg of pure benzaldehyde dissolved and 2 g of dried Dowex powder 50 WX 2 added. The air in the flask is made with nitrogen displaced and the reaction mixture stirred with the exclusion of moisture using a magnetic stirrer. That Progress of the reaction is ongoing means Thin layer chromatography followed [system a) chloroform + 6% methanol, b) chloroform-methanol-water- (70: 25: 5)]. After an hour there is the reaction completed. The orange-colored solution is used for work-up filtered off from the catalyst and washed thoroughly with chloroform. The solvent is removed at 50 ^° C. in vacuo. An oil is obtained which is purified on 60 g of "Merck" silica gel (particle size 0.05 to 0.2 mm). After separating off the benzaldehyde by chloroform extraction, the substance is eluted with chloroform + 2% methanol. This chromatographic purification yields amorphous 4'-demethyl-epipodophyllotoxin-jS-D-benzylidene-glucoside, which is completely uniform in the thin-layer chromatogram. To the cha Characterization, the preparation is dissolved in 5 ml of acetone and added dropwise to 60 ml of pentane while stirring. 4'-Demethyi-epipodophyllotoxin - /? - D-benzylidene-glucoside is as a colorless amorphous powder with a melting point of 182-184 ° C

obtain. From abs. In ethanol, crystals with a melting point of 245-246 ° C. are obtained. O] ₀ = -104 ⁰ C (c = 0.766 in chloroform).

Attempt 1

(Production of
4'-demethyl-epipodophyllotoxin - /? - D-glucoside)

A) 4'-demethyl-epipodophyllotoxin

2 g of 4'-demethyl-podophyllotoxin are dissolved in 25 ml of acetone and 15 ml and, after adding 5 ml of conc. Hydrochloric acid heated under reflux for 2 hours. The acid is then neutralized with solid barium carbonate, filtered and the filtrate is freed from acetone ^{at 40 ° C. in vacuo.} The precipitated material is taken up in chloroform + 5% acetone, the solution is dried over sodium sulfate and evaporated in vacuo. The remaining residue is chromatographed on silica gel with chloroform + 1% methanol, whereby first small amounts of impurities, then pure 4'-demethyl-epipodophyllotoxin and finally unreacted starting material are obtained. Crystallization of the pure fractions of 4'-demethyl-epipodophyIlotoxin takes place from chloroform and methanol. Mp 228-230 ° C, [a] _D = -69.8 ° C (c = 0.630 in chloroform).

B) 4'-carbobenzoxy-4'-demethylepipodophyllotoxin

60 g of powdered 4'-demethyl-epipodophyllotoxin are suspended in 1000 ml of anhydrous ethylene chloride and, after adding 19 ml of abs. Pyridine cooled to -10 ^0C. While stirring and excluding moisture, a solution of 34 g of benzyl chloroformate in 100 ml of ethylene chloride is added dropwise at -10 ° C. over a period of 2.5 hours and then allowed to react for a further half an hour. Subsequently, the reaction solution is washed with water, the organic phase dried over sodium sulfate, evaporated in vacuo and the residue dried under high vacuum at 70 -80 ⁰ C. Crystallization of the crude product from acetone-ether, and then delivers twice from methanol 4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin the double melting point 117-119 ⁰ C / 202-205 ⁰ C. By drying under a high vacuum first at 95-110 ⁰ C and then at 130 ⁰ C or crystallization from acetone-ether solvent-free form of mp. 201 -204 ⁰ C, [ "] is obtained 2 = ⁰ -43.9 C (c = 0.535) in CHCl _3.

C) Tetra-O-acetyl-4'-carbobenzoxy-

V-demethyl-epipodophyllotoxin-

jS-D-glucoside

26.8 g of 4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin are dissolved in 70 ml of ethylene chloride with heating. The solution is cooled to + 15 ° C. and 26.0 g of 2,3,4,6-tetra-O-acetyl-jS-D-glucose are added with stirring. As soon as the major part of the tetraacetyl-jS-D-glucose has dissolved, it is quickly cooled to -11 to -12 ° C with exclusion of moisture. Regardless of small amounts of undissolved starting compounds are then dropped at - 10 ⁰ C to -12? C internal temperature of 17.5 ml to -10 ⁰ C precooled Äthylätherat boron trifluoride (48% BF3) in the course of 10 minutes and then stirred for a further 40 minutes at -1O ⁰ C. Thereafter, with stirring and cooling a mixture of 17.5 ml abs. Pyridine and 35 ml of ethylene chloride were added dropwise and, after the addition of a further 200 ml of ethylene chloride, extracted four times with 100 ml of water each time. The organic phase is evaporated in vacuo after drying over sodium sulfate and the residue under high vacuum at 70 C ^c dried. The crude product dissolves in 125 ml of hot ethanol, mixed with 375 ml of water while stirring and stirred with external cooling with ice-water until the initially greasy and lumpy precipitate has been converted into a sandy powder. The precipitate is then filtered off with suction, washed with an ethanol-water mixture of 1: 3 and dried

ίο in a high vacuum at 70 ° C. This crude product is dissolved in 300 ml of hot methanol, a little undissolved flakes are filtered off and the filtrate is evaporated in vacuo. After drying the residue in a high vacuum at 70 ° C., tetra-O-acetyl-4'-carbobenzoxy-4'-demethyl-epipodophylloto-
xin - /? - D-glucoside as a white foam. [ä] d ^{0 =} -41.7 ^o C (CHCl ₃ ). For further purification, it can be crystallized from benzene-pentane or a benzene-cyclohexane mixture. Pure tetra-O-acetyl-4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin-jS-D-glucoside melts at 167-169 ° C; [A] ² O ⁰ = -46.6 ° C (CHCl ₃ ).

D) Tetra-O-acetyl-4'-carbobenzoxy-

• i'-demethyl-epipodophyllotoxin-

J3-D-glucoside

8.56 g of 4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin are stirred in 55 ml of acetonitrile at 65 ° C. until dissolved, 3.92 g of dry zinc oxide are added and, after the addition of 20.0 g of a-acetobromoglucose, with exclusion of moisture 65 ° C stirred. After every 10 minutes a sample is taken and examined in a thin-layer chromatogram (silica gel / chloroform + 10% acetone). After complete conversion of the acetobromoglucose (approx. 40 to 60 minutes after the start of the reaction), the mixture is cooled to room temperature, diluted with 50 ml of chloroform, unreacted zinc oxide is filtered off and the filtrate is evaporated in vacuo at 40 ^° C. to a volume of approx. 20 ml a. This concentrate is dissolved in 250 ml of chloroform, the chloroform phase is washed twice with 300 ml of water-methanol (9: 1) each time and, after drying over sodium sulfate, the chloroform layer is evaporated in vacuo. The dried residue is extracted four times with 70 ml of boiling hot ethanol / water 1: 3 mixture each time. The proportion äthanolunlöslichen chromato- f is initialed at the 50-fold amount of silica gel using ^ - Chloroform-acetone (95: 5) as eluent.

The pure Glucosidfraktionen are combined and, after drying under high vacuum at 8O ⁰ C, crude tetra-O-acetyl-4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin-ß-D-glucoside. A highly pure preparation can be obtained by further chromatography which corresponds to the physical data of the end product described in the attempt IC).

E) 4'-carbobenzoxy-4'-demethylpodophyllotoxin

15.5 g of 4'-demethyl-podophyllotoxin are dissolved in 450 ml of warm abs. Ethylene chloride-tetrahydrofuran 1: 1 mixture, mixed with 6.0 ml of abs. Pyridine and cooled to - 10 ⁰ C. While stirring and cooling are now in the course of 1.5 hours, 8 ml of benzyl chloroformate dissolved in 55 ml of ethylene chloride, is added dropwise and after 1 hour at -5 to - 10 ⁰ C stirred. It is then evaporated to a volume of 200 ml at a bath temperature of 40 ° C. in vacuo, diluted with 250 ml of ethylene chloride and the solution is washed once with 100 ml of 1N hydrochloric acid and then with water until neutral

Drying over sodium sulfate is evaporated in vacuo and the residue is chromatographed on 20 times the amount of silica gel. Chloroform with 1% methanol first elutes small amounts of by-products, then pure 4'-carbobenzoxy-4'-demethyl-podophyllotoxin. After crystallization from methanol, the compound melts at 113-114 ° C; [a] ² _D = -88.3 · ° C (chloroform).

F) Tetra-O-acetyl ^ '- carbobenzoxy-

4'-demethyl-epipodophyllotoxin-

ß-D-glucoside

10.7 g of 4'-carbobenzoxy-4'-demethyl-podophenyllotoxin is dissolved under heating in 30 ml ethylene chloride and the solution is cooled to +15 ⁰ C., and 14.0 g 2,3,4,6-tetra-O- acetyl-ß-D-glucose too. After 5 minutes of stirring with exclusion of moisture is cooled to -15 ° C within 5 minutes and 7 ml to -10 ⁰ C precooled Äthylätherat boron trifluoride (48% BF ₃₎ was added dropwise.

The mixture is then stirred at -15 ° C. for 1 hour, then a solution of 7 ml of abs. Pyridine was added dropwise in 20 ml of ethylene chloride. After dilution with 100 ml of chloroform, it is washed five times with 50 ml of water each time. The organic phase is dried over sodium sulfate, evaporated in vacuo and the residue is dried at 6O ⁰ C in vacuo. The foam obtained is dissolved in 50 ml of hot ethanol, cooled to about 50 ° C., 150 ml of cold water are added and the mixture is stirred until the initially lumpy and greasy precipitate has turned into a sandy powder. The product is sucked off, washed with 40 ml of 25% ethanol and dried in vacuo at 6O ⁰ C. Then dissolving the preparation in 200 ml of hot methanol, filtered clear and the filtrate evaporated in vacuo and the residue is dried under high vacuum at 8O ⁰ C to constant weight. The tetra-O-acetyl-4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin-j3-D-glucoside obtained in this way is identical to the preparation produced according to experiment IC).

G) Tetra-O-acetyl-4'-demethylepipodophyllotoxin - /? - D-glucoside

To split off the carbobenzoxy radical from tetra-O-acetyM'-carbobenzoxy ^ '- demethyl-epipodophyllo-toxin-jS-D-glucoside, 13.4 g of this compound are dissolved in 100 ml of acetone-ethanol (1: 2), with 0, 5 ml of glacial acetic acid and 2 g of palladium-carbon (with 10% Pd) are added and the mixture is hydrogenated ^{at 20 ° C.} The catalyst is then filtered off, washed with a warm acetone-methanol mixture and the filtrate is evaporated in vacuo. The residue is poured over 100 ml of boiling-hot ethanol, allowed to crystallize and the crystals are dried in vacuo after suction and washing with methanol. The purest tetra-O-acetyl ^ '- demethyl-epipodophyllotoxin-ß-D-glucoside crystallizes in fine needles with a melting point of 225-227 ° C, [a] ² _D ' = -64.4 ° C (c = 1.024) in Chloroform.

H) 4'-demethyl-epipodophyllotoxin-] 3-D-glucoside

60

25 g of pure tetra-O-acetyl-4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin-] 9-D-glucoside, 3.6 g of anhydrous zinc acetate and 1.45 g of anhydrous sodium acetate are refluxed in 150 ml of methanol with stirring warmed up. After 2 and 4 hours, 12.5 ml of methanol are distilled off. Thereafter, after every 2 hours, 12.5 ml of methanol are added and distilled off again. After a total of 18 hours, the splitting off of the acetyl groups is complete and there is a mixture of approx. 60% 4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin - /? - D-glucoside and approx. 40% 4'-demethyl-epipodophyllotoxinß- D-glucoside before. The reaction is followed in a thin-layer chromatogram on silica gel plates using the mobile phase isopropyl acetate-methanol (4: 1), saturated with water; Development by spraying with a 0.2% solution of cerium (IV) sulfate in 50% sulfuric acid and heating at 110- 13O ⁰ C.

For working up, 5 ml of glacial acetic acid, evaporated in vacuum at 5O ⁰ C bath temperature and dried 15 minutes in high vacuum at 50 ⁰ C. The residue is taken up in 250 ml chloroform-isopropanol (4: 1) and 25 ml of water, the aqueous phase is separated off and the organic phase is washed again with 25 ml of water. The two wash waters are re-extracted with 50 ml of chloroform-isopropanol and the combined organic phases are evaporated in vacuo after drying over sodium sulfate. The residue is drawn up three times with 30 ml of acetone each time in vacuo and then dried for two hours at 75 ° C. in a high vacuum.

4'-Carbobenzoxy-4'-demethyl-epipodophyllotoxin-jS -D-glucoside cannot be obtained in an entirely pure form. Chromatography on 100 times the amount of silica gel, when eluted with isopropyl acetate-methanol (9: 1), water-saturated, gives pure 4'-carbobenzoxy ^ '- demethyl-epipodophyllotoxin-β-D-glucoside, which crystallizes from acetone at 155-156 ^{Melts 0} C, [a] ² J = - 92.0 ° C (c = 1.00) in chloroform.

To split off the carbobenzoxy group, the mixture of 4'-carbobenzoxy-4'-demethyl-epipodophyllotoxin - /? - D-glucoside and 4'-demethyl-epipodophyllotoxin-j9-D-glucoside is dissolved in 200 ml of acetone, the solution is added to a Suspension of 3 g of palladium-carbon (10% Pd) in 50 ml of water and hydrogenated until the protective group has been split off (1.5 hours). Check the reaction in the thin-layer chromatogram as above. The catalyst is then filtered off, washed with 100 ml of acetone-water (4: 1) and the filtrate is evaporated to a volume of approx. 40-45 ml in vacuo, with 4'-demethyl-epipodophyllotoxin- /? -D-glucoside crystallized out. To complete the crystallization, the mixture is left to stand in an ice bath for a further 20 minutes, the precipitated crystals are filtered off with suction, washed with 25 ml of water and dried in a high vacuum at 70 ° C. Crystallization from methanol gives pure 4'-demethyl-epipodophyllotoxin-j9-D-glucoside of mp. 225-227 ⁰ C, [α] 2 = -88.6 ° C (c = l, 05) in methanol. Another modification is the mp. 262-264 ⁰ C.

Attempt 2

Stage H of experiment 1 can also be modified by adding 2.0 g of the tetra-O-acetyl ^ '- demethyl-epipodophyllotoxin-ß-D-glucoside obtained according to experiment IG) and 1 g of anhydrous zinc acetate in 30 ml of absolute methanol 25 Refluxed for hours. The resulting white precipitate is then brought into solution by adding a few ml of glacial acetic acid and gentle heating, the solvent is ^{removed in vacuo at 40 °} C. and the residue is taken up in 50 ml of chloroform-butanol (4: 1). the

609 547/407

The organic phase is washed twice with 10 ml of water each time and, after drying over sodium sulfate, evaporated Vacuum and the residue is chromatographed on silica gel. Isopropyl acetate-methanol (9: 1), saturated with water, first elutes non-polar components, then pure 4'-demethyl-epipodophyllotoxin - ^ - D-glucoside. The single ones Fractions are in thin-layer chromatography

grams on silica gel columns with the mobile phase isopropyl acetal-methanol (8: 1), saturated with water, examined and the glucoside fractions combined and crystallized twice from methanol. 4'-Demethyl-epipodophyllotoxin-ß-D-glucoside melts at 222-230 ⁰ C, another modification at 262-264 ° C, [oc] ^? _D '= -88 ° C (c = 0.507) in methanol.

Claims (1)

Patent claims: 1. Compounds of the general formula 1 H CH ₁ O OH OCH ₃ (I)