NO170896B - CLOTHING ELEMENT AND PROCEDURE FOR MANUFACTURING SUCH ITEMS - Google Patents

Description

Process for the production of 4'-demethyl-epipodophyllotoxin-S-D-glucoside with therapeutic

effect.

The present invention relates to a method for the production of <l>+<i_>demethyl-epipodophyllotoxin-|3-D-glucoside with therapeutic effect and with formula (I)

and distinctive in the method according to the invention..is that i. tetra-O-acetyl-^'-kdrboDénzoxy-^'-demethyl-Gpipodophyllo-tok .. n-p-D-glucoside with formula (II) is either cleaved off first .the carbobenzoxy group hydroge. nolytically and the thus obtained tetra-O-acetyl-^'-demethyl-epipodophyllotoxin-(3-D-glucoside of formula (III) .......

subjected ' to an alcoholysis in the presence of anhydrous zinc acetate, or

the acetyl groups are first cleaved alcoholically in the presence of anhydrous zinc acetate or a mixture of anhydrous zinc acetate and sodium acetate^ and the carbobenzoxy group is then removed hydrogenolytically.

The individual procedural steps are carried out in the following way:

UTyTlrcge nolysis

Hydrogonolysis is carried out according to methods known per se. !lnr\/e:i is appropriately hydrated without excess pressure and at 20 to maximum.

k0' C i liser weather of palladium catalysts, e.g. palladium on charcoal or py barium sulphate. Alcohols are preferably used as solvents, such as e.g. methanol or ethanol, with the addition of 0.5 to 5 volumes of glacial acetic acid and 10 to 50 volumes of acetone. The amount of catalyst is 1 to 5% by weight, calculated on the substance used.

Alcohololysis

It was not to be expected that from tetra-O-acetyl-^-'-carbobenzoxy--demethyl-epipodophyllotoxin-p-D-glucoside or tetra-O-acetyl-h'-demethyl-epipodophyllotoxin-p-D-glucoside should be able to split off the acetyl group hydrolytically under normal basic and acidic conditions, in order to arrive at the corresponding free glucoside in this way. As is known, lignanglucosides undergo epimerization with bases, while the influence of acids can lead to cleavage during removal of the sugar residue.

It was now surprisingly found that from tetra-O-acetyl-V-carbobenzoxy-V-demethyl-epipodophyllotoxin-f3-D-glucoside or

from tetra-O-acetyl-^f<1->demethyl-epipodophyllotoxin-p-D-glucoside could remove the acetyl residues without simultaneous epimerization of the aglycon at the C-3 atom and without cleavage of the total sugar residue, as these compounds are subjected to an alcoholysis, preferably with methanol, in the presence of anhydrous zinc acetate.

The methanolysis is repeated in anhydrous methanol at reflux temperature. The amount of catalyst is approx. 20-50 weight$ calculated on the tetra-O-acetyl-^1 -deren tyl-epipodophyllotoxin-B-D-glucoside used. The reaction time is between 15 and 30 hours. During the methanolysis of tetra-O-acetyl-^f'-dem±yl-epipodophyllotoxin-|3-D-glucoside, a precipitate is formed with zinc acetate as catalyst in the course of the reaction.

For processing, this precipitate is dissolved by the addition of acetic acid and gentle heating.

After evaporation of the solvent/t, the residue is dissolved in a mixture of chloroform/butanol and the zinc salt is removed by shaking with water.

From the organic phase, the crude V-demethyl-epipodophyllotoxin-β-D-glucoside is isolated after evaporation, which is purified in a known manner, e.g. by chromatography and/or crystallization.

As indicated above, however, when removing the protective groups, one can also proceed in the reverse order, firstly tetra-O-acetyl-β1-carbobenzoxy-1*'-dernetyl-epipodophyllotoxin-β-D-glucoside in the presence of anhydrous zinc acetate and possible addition of Anhydrous sodium acetate in methanol is boiled under reflux for so long that the cleavage of the acetyl group is completed, whereby at the same time the carbobenzoxy group is also split off in parts. From the thus formed mixture of the reaction components, the hitherto unknown V-carbobenzoky-V-demethyl-epipodophyllotoxin-B-D-glucoside of formula (IV)

is isolated, which is further converted by hydrogenolysis of the carbobenzoxy group into -dernetyl-epipodophyllotoxin-S-D-glucoside.

The starting material used for the production of V-demethyl-epipodophyllotoxin-B-D-glucoside tetra-O-acetyl-M-'-carbobenzoxy-V-demethyl-epipodophyllotoxin-B-D-glucoside is prepared according to the following method:

It was found that the turnover of 2,3,^,6-tetra-O-acetyl-D-glucose

in the form of its pure B-anomer as well as with K<*->carbobenzpxy-V-demethyl podophyllotoxin of formula (V)

as well as with -carbobenzoxy-V-dernetyl-epipodophyllotoxin of formula (VI)

i.e. regardless of the stereochemistry of the OH group at the C-1 carbon atom in the aglycone, in the presence of boron trifluoride ethyl etherate at a temperature below 0°C in a solvent inert under the reaction conditions1 leads to tetra-O-acetyl-V-carbobenzoxy-^ 1 - demethyl-epipodophyllotoxin-β-D-glucoside in high yield.

The starting materials used for the production of tetra-O-acetyl-V-carbobenzoxy-V-dernetyl-epipodophyllotoxin-B-D-glucoside h<1->carbobenzoxy-V-demethyl-podophylltotoxin hechv. V-carbobenzoxy-V-demethyl-epipodophyllotoxin is produced according to the following procedure:

k1-demethyl podophyll toxin' of formula (VII)

respectively V-demethyl-epipodophyllitoxin of formula (VIII)

is reacted in an anhydrous, under the reaction conditions inert organic solvent at temperatures between -20 and -5°C in the presence of a tertiary organic base with chloroformic acid benzyl ester to V-carbobenzoxy-V-demethyl-epipodophyllotoxin resp.

V-carbobenzox<y>-^-'-denretyl podophyllotoxin.

The V-demethyl-epipodophyllotoxin used as starting material can

is obtained by epimerization of h<*->demethyl podophyllotoxin

(see ex. 1). The production of k'-carbobenzoxy-V-demethyl-podophyllotoxin takes place in a known manner, starting from -demethyl-podophyllotoxin, as per mol V-demethyl-podophyllotoxin 1.0 - 1.6 mol carbobenzoxychloride is used.

V-demethyl-epipodophyllotoxin-B-D-glucoside in addition to a high cytostatic effect on mastocytoma cells and fibroblast cultures in vitro. Furthermore, it is distinguished by its strong activity against experimental tumors, especially against mouse leukemia L-1210. h<1->demethyl-epipodophyllotoxin-B-D-glucoside can be used therapeutically to combat pathological processes related to cell proliferation, e.g. malignant neoplasms. The dosage of -demethyl-epipodophyllotoxin-6-D-glucoside is varied between approximately 1 and 50 mg per day.

As mentioned, the produced V-demethyl-epipodophyllotoxin-8-D-glucoside is distinguished by interesting therapeutic properties. In a specific comparison of h'-demethyl-epipodophyllotoxin-B-D-glucoside with the previously known podophyllotoxin-8-D-glucoside in German patent document no. 959,192 and Norwegian patent document no. 93,077, it turns out that according to the present process-producible compound has a significantly more pronounced selective inhibitory effect on the division process in the cell nuclei. The said compound was pi5vet in vi tro the culture of mast cell tumor P 81 5 in mice. In the following table, DE^-q denotes the concentration which inhibits cell growth for the mast cells by 50$.

V-demethyl-epipodophyllotoxin-8-D-glucoside may find application

as a drug in itself or in equivalent drug forms for oral, enteral or parenteral administration. For the production of suitable medicinal forms, the compound is processed with organic or inorganic, pharmacologically indifferent excipients. 1 the following examples are all temperature indications in °C. The melting or fission points are determined on the Kofler block.

Ex. 1 - 4 illustrate the production of starting materials.

EXAMPLE 1: V-demethyl-epipodophyllotoxin

2 g of h<1->demethyl-podophyllotoxin are dissolved in 25 ml of acetone and 15 ml of water and heated after adding 5 ml of concentrated sodium chloride for 2 hours under reflux. The acid is then neutralized with solid barium carbonate, it is filtered and the filtrate is freed of acetone in a vacuum at <1>+0°C. The precipitated material is taken up in chloroform plus 5% acetone, the solution is dried over sodium sulphate and evaporated in vacuo. The residue is chromatographed on silica gel with chloroform plus 1% methanol, whereby small amounts of impurities and then pure V-demethyl-epipodophyllotoxin and finally unreacted starting material are obtained. Crystallization of the pure fractions of V-demethyl-epipodophyllotoxin takes place from chloroform and methanol.

Temp. 228-230°C, C<tJ^ ° = -69.8° (c = 0.630 in chloroform).

EXAMPLE 2: V- carbobenzoxy- V- demethyl- epipodophyllotoxin

60 g of finely powdered V-demethyl-epipodophyllotoxin are suspended in 1000 ml of anhydrous ethylene chloride and after the addition of 19 nil abs. pyridine is cooled to -10°C. While stirring and excluding moisture, a solution of 3^ g of benzyl chloroformic acid in 100 ml of ethylene chloride is added dropwise at -10°C over the course of 2 hours and then a further -g:t is reacted. The reaction solution is then washed with water, the organic phase is dried over sodium sulphate, evaporated in a vacuum and the residue dried in a high vacuum at 70-80°C. Crystallization of the crude product from acetone/ether and then twice from methanol gives V-carbobenzoxy-V-demethyl-epipodophyllotoxin with double melting point 117-119/202-205°C. By drying in a high vacuum solid at 95-110°C and then at 130°C or crystallization from acetone/ether, the solvent-free form is obtained with melting point 201-20<>>+OC, Z«7D21 = -^3.9° (c = 0.535) in CHCl^.

EXAMPLE 3: V-carbobenzoxy-V-demethylpodophyllotoxin

Dissolve 1 5j0 g of hx -demethyl-podophyllotoxin in <4>-50 ml of warm abs. ethylene chloride-tetrahydrofuran mixture 1:1, add 6.0 ml abs. pyridine and cooled to -10°C. While stirring and cooling, 8 ml of chloroformic acid benzyl ester dissolved in 55 nil of ethylene chloride are now added dropwise in the course of 1 g per hour, and then stirred for 1 hour at -5 to -10°C.

It is then evaporated at h0 C bath temperature in a vacuum to a volume

of 200 ml, it is diluted with 250 ml of ethylene chloride and the solution is washed once with 100 ml of 1N hydrochloric acid, then neutralized with water. After drying over sodium sulphate, it is evaporated in vacuo and the residue is chromatographed on the 20-fold amount of silica gel. Chloroform with \% methanol first elutes small amounts of by-products, then pure h<*->carbobenzoxy-V-demethyl-podophyllotoxin. After crystallization from methanol, the compound melts with 113-H<1>+<0>C. Δs7D<21>= -88.3° (chloroform).

EXAMPLE <l>+: tetra- O- acetyl- V - carbobenzoxy- V - demethyl-epipodophyllotoxin- B- D- glucoside

10.7 g of V-carbobenzoxy-V-demethyl-podophyllotoxin are dissolved while heating in 30 ml of ethylene chloride, the solution is cooled to 15°C and 1^.0 g of 2,3,^,6-tetra-O-acetyl-B-D is added -glucose. After 5 min. rowing is cooled to the exclusion of moisture

-15°C>and within 5 min. 7 ml to -10°C pre-cooled boron trifluoride ethyl etherate (h8% BF-.) is added dropwise. It is then stirred for 1 hour at -15 C, and then a solution of 7 ml abs is added drop by drop. pyridine in 20 ml of ethylene chloride. After dilution with 100 ml of chloroform, wash 5 times with 50 ml of water each time. The organic phase is dried over sodium sulphate, evaporated in a vacuum and the residue is dried at 60°C in a vacuum. The resulting foam is dissolved in 50 ml of hot ethanol. It is cooled to approx. 50°C, 150 ml of cold water is added and stirred until the initially lumpy and tough sediment has been transformed into a sandy powder. The product is filtered, washed with ^0 ml of 25% ethanol and dried in vacuum at 60°C. The preparation is then dissolved in 200 ml of hot methanol, filtered clearly, the filtrate is evaporated in a vacuum and the residue is dried in a high vacuum at 80°C to constant weight. The tetra-O-acetyl-V-carbobenzoxy-V-demethyl-epipodophyllotoxin-B-D-glucoside thus obtained melts at 167-169°C, fqj^ 20 <=><->^6.6° (CHC13).

EXAMPLE 5: tetra- O- acetyl- V - detiEbyl- epipodophyllotoxin- g- D- glucoside

To cleave off the carbobenzoxy residue from tetra-O-acetyl-^'-carbobenzoxy-V-demethyl-epipodophyllotoxin-B-D-glucoside, dissolve 13A g of this compound in 100 ml of acetone/ethanol (1:2), add 0.5 ml of glacial acetic acid and 2 g palladium/charcoal (with 10$ Pd) and it is hydrated at 20°C. The catalyst is then filtered off, this is washed with a hot acetone-methanol mixture and the filtrate is evaporated in a vacuum. The residue is poured over with 100 ml of boiling hot ethanol, allowed to crystallize and the crystals are dried after suction and washing with methanol in a vacuum. Pure tetra-0-acetyl-^-<1->demethyl-epipodophyllotoxin-B-D-glucoside crystallizes in fine needles with melting point 225-227°C ^7D<21><=> - 6h, k° (c = 1.02 »f) in chloroform.

EXAMPLE 6: *+'- dernethyl- epipodophyllotoxin- B- D- glucoside

25 g of pure tetra-O-acetyl-V-carbobenzoxy-^<1->demethyl-epipodophyllotoxin-B-D-glucoside, 3.6 g of anhydrous zinc acetate and 1.*f5 g of anhydrous sodium acetate are heated in 1 50 ml of methanol under reflux and stirring . After 2 and h hours, 12.5 ml of methanol are distilled off each time. Then, after every 2 hours, 12.5 ml of methanol are added and distilled again. After a total of 18 hours, the cleavage of the acetyl group is complete and there is a mixture of approx. 60% k<1->carbobenzoxy-V-demethyl-epipodophyllotoxin-B-D-glucoside and approx. h0% h<*->demethyl-epipodophyllotoxin-8-D-glucoside.

The reaction takes place in a thin-layer chromatogram on silica gel plates with the eluent, water-saturated isopropylaceta-methanol (<*>f:1). Development by showering with a 0.2% solution of Ce (IV)-sulphate i

50% sulfuric acid and heating to 110-130°C.

For the preparation, 5 ml of glacial acetic acid is added, it is evaporated in a vacuum at 50°C bath temperature and dried for 15 minutes in a high vacuum tank at 50°C. The residue is taken up in 250 ml of chloroform/isopropanol (*f:1) and 25 ml of water, the aqueous phase is separated and the organic phase is further washed with 25 ml of water. The two washing waters are extracted with 50 ml of chloroform/isopropanol and the combined organic phases are evaporated after drying over sodium sulphate in vacuum. The residue is distilled 3 times with each time 30 ml of acetone in vacuum and then dried for two hours in high vacuum at 75°C.

From the mixture of V-carbobenzoxy-V-demethyl-epipodophyllotoxin-B-D-glucoside and k'-demethyl-epipodophyllotoxin-8-D-glucoside, V-carbobenzoxy-V-demethyl-epipodophyllotoxin-B-D-glucoside can be prepared by crystallization from methanol in not quite pure form. Chromatography on the 100-fold amount of silica gel gives, eluting with isopropylacetalzmethanol (9:1), water-saturated, pure V -carbobenzoxy-M-1-demethyl-epipodophyllotoxin-B-D-glucoside, which melts at 155-156°C after crystallization from acetone .

/"ci7D21 = -92.0°C (c = 1.00) in chloroform.

For the cleavage of the carbobenzoxy group, the mixture of V-carbobenzoxy-V'-demethyl-epipodophyll toxin-B-D-glucoside and

V-demethyl-epipodophyllotoxin-B-D-glucoside in 200 ml of acetone, the solution is added to a suspension of 3 g of palladium metal (10$ Pd) in 50 ml of water and it is hydrated until the removal of the protecting group is complete (1.5 hours). Control of the reaction by thin-layer chromatogram as above. The catalyst is then filtered off, this is washed with 100 ml of acetone (Vi) and the filtrate is evaporated in vacuum to a volume of approx. M-0-V5 ml, as V-demethyl-epipodophyllotoxin-B-D-glucoside already begins to crystallize. Allow another 20 min for complete crystallization. in an ice bath, the precipitated crystals are filtered off, washed with 25 ml of water and dried in high vacuum at 70°C. Crystallization from methanol gives pure V-demethyl-epipodophyllotoxin-8-D-glucoside with a melting point of 225-227°C, /"«7D<21> = -88.6° (c = 1.05) in methanol. A other modification has melting point 262-26Lt-°C.

EXAMPLE 7: V-demethyl-epipodophyllotoxin-3-D-glucoside

2.0 g of the tetra-O-acetyl-V-demethyl-epipodophyllotoxin-8-D-glucoside obtained in example 5 and 1 g of anhydrous zinc acetate are heated in 30 ml of abs. methanol for 25 hours under reflux.

The white precipitate formed is then dissolved by the addition of a few ml of glacial acetic acid and gentle heating, the solvent is removed in vacuo at 1+0°C and the residue is taken up in 50 ml of chloroform butanol (Vi). The organic phase is washed twice with 10 ml of water each time, after drying over sodium sulphate it is evaporated in vacuo and the residue is chromatographed on silica gel. Isopropyl acetate^ethanol (9:1), water saturated, elutes first

nonpolar portions, then pure V-dernetyl-epipodophyllotoxin-B-D^glucoside. The individual fractions are submitted to a thin-layer chromatogram on silica gel plates with the eluant isopropylacetaminoethanol (8:1), saturated with water, and the glucoside fraternoids are combined and crystallized twice from methanol. V-<de>rne tyl-epipodophyllotoxin-B-D-glucoside melts at 222-230° C, another modification at 262-26V°C, Z"d7D<21> = -88° (c = 0.507) in methanol.

Claims (1)

Process for the production of -demethyl-epipodophyllotoxin- 6-D-glucoside with therapeutic effect and of formula (I) characterized in that from tetra-O-acetyl-V-carbobenzoxy-V-demethyl-epipodophyllotoxin-B-D-glucoside of formula (II ) either the carbobenzoxy group is first split off hydrogenolytically and • the thus obtained tetra-O-acetyl-V-demethyl-epipodophylloxyri- 6-D-glucoside of formula (III) is subjected to an alcoholysis in the presence of anhydrous zinc acetate, or the acetyl groups are first cleaved off alcoholically in the presence of anhydrous zinc acetate or a mixture of anhydrous zinc acetate and r^italumaetate, and the carbobenzoxy group is then removed hydrogenolytically-