NO120371B - - Google Patents

Description

Process for the production of new, therapeutically active 4<1->demethyl-epipodophyllotoxin glucosides.

The present invention relates to a method for production

of new, therapeutically active lf-demethyl-epipodophyllotoxin glucosides with the general formula (!)■

where R stands for a phenyl, 2-furyl or a 2-thienyl residue, and the distinctive feature of the method according to the invention is that

•+* -Demethyl-epipodophyllotoxin-(3-D-glucoside of formula (II)

in the presence of an acidic catalyst is reacted with an aldehyde of the general formula (III)

where R has the meaning given above. As a catalyst comes e.g. Lewis acids, such as anhydrous zinc chloride, or a dry cation exchanger with sulfonic acid groups in - the form of speech.

It is particularly advantageous to dissolve V-demethyl-epipodophyllotoxin-B-D-glucoside directly in pure aldehyde and add the catalyst. The reaction usually proceeds at room temperature or at a somewhat elevated temperature and is largely completed after 1 to 10 hours, at 20°C most often after 3-6 hours.

To isolate the condensation product, the reaction mixture is treated in a water-immiscible solvent, e.g. chloroform, it is shaken several times with water to remove water-soluble salts and by-products, the organic phase is filtered and evaporated in a vacuum, whereby the main amount of the excess aldehyde is removed. An oily residue is obtained, from which the last remnants of the aldehyde are easily removed by rinsing the condensation product with a suitable solvent, such as e.g. petroleum ether, pentane, hexane or by chromatography on a neutrally reacting adsorbent, such as e.g. silica gel. The condensation product is isolated and purified in a known manner, e.g. by chromatography or recrystallization.

hf - demethyl- epipodophyllotoxin- P- D- glucoside (formula II)

The starting material h'-demethyl-epipodophyllotoxin-B-D-glucoside, which serves for the preparation of the new compounds with the general formula I, is prepared according to one of the following methods: Either one cleaves from tetra-O-acetyl-M-1 -carbo-benzoxy- ^-'-Demethyl-epipodophyllotoxin-B-D-glucoside of formula (IV)

first the carbobenzoxy group hydrogenolytically, and the thus obtained tetra-O-acetyl-^'-dernetyl-epidophyllotoxin-B-D-glucoside of formula (V) is subjected to an alcoholysis in the presence of anhydrous zinc acetate, or one first splits off the acetyl groups in tetra-O-acetyl- U-<1->carbobenzoxy-^1-dernety1-epipodophyllotoxin-B-D-glucoside alcoholytically in the presence of anhydrous zinc acetate or a mixture of anhydrous zinc acetate/sodium acetate, the carbobenzoxy group being partially split off at the same time. From the thus formed mixture of reaction components, the previously unknown V-carbobenzoxy-V-demethyl-epipodophyllotoxin-B-D-€Lucoside of formula (VI)

is isolated, which is further converted by hydrogenolysis of the carbobenzoxy group into h1-demethyl-epipodophyllotoxin-B-D-glucoside.

Tetra-O-acetyl-4-'-carbobenzoxy-U-'-demethyl-epipodophyllotoxin-3-D-glucoside (Formula IV)

The starting material used for the production of h'-demethyl-epipodophyllotoxin-8-D-glucoside, tetra-O-acetyl-^-i-carbobenzoxy-^1-demethyl-epipodophyllotoxin-(3-D-glucoside), can be prepared in the following way:

It was found that turnover of 2,3,^,6-tetra-0-acetyl-(3-D-glucose

in the form of its pure 8-anomer as well as with h'-carbo benzoxy-1*-1-demethyl podophyllotoxin of formula (VIII)

as well as with h<1->carbobenzoxy-H-'-demethyl-epipodophyllotoxin of formula (VII)

i.e. regardless of the stereochemistry of the OH group on 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 conditions leads to tetra-O-acetyl-^-'-carbobenzoxy-H -' -Demethyl-epipodophyllotoxin-B-D-glucoside.

'+'-carbobenzoxy-H-1-demethyl-podophyllotoxin (formula VIII) and ij-'-carbobenzoxy-^-1-demethyl-epipodophyllotoxin (formula VII)

The starting materials used for the production of tetra-O-acetyl-V-carbobenzoxy-^'-demethyl-epipodophyllotoxin-B-D-glucoside V-carbobenzoxy-V'-demethylpodophyllotoxin resp. h'-carbobenzoxy-^-1-demethyl-epipodophyllotoxin is produced using the following method:

1-Demethyl-podophyllotoxin of formula (IX)

respectively ^'-demethyl-epipodophyllotoxin of formula (X)

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

>+'-carbobenzoxy-V'-demethyl-podophyllotoxin or h'-carbobenzoxy-M-'-demethyl-epipodophyllotoxin.

The *+'-demethyl-epipodophyllo-toxin used as starting material can be obtained by epimerization of<>>+'-dernetyl-podophyllo-toxin (see example 1). The production of h1-carbobenzoxy-^f'-demethyl-podophyllotoxin takes place in the same way, starting from V-demethyl-podophyllotoxin, as per mol V-demethyl podophyllotoxin 1 - 1.6 mol carbobenzoxychloride is used.

The new compounds of the general formula I possess in vitro a high cytostatic effect on mastocytoma cells and fibroblast cultures. Furthermore, they are distinguished by their strong activity against experimental tumours, especially/foot mouse leukemia L-1210. The new compounds with the general formula (I) can be used therapeutically to combat pathological processes related to cell proliferation, e.g. malignant neoplasms.

For more specific demonstration of the superiority of the present compounds in relation to the compounds referred to in DAS 1,020,639, the following is cited: Superiority with respect to the inhibitory effect of the following compounds, respectively: V-demethyl-epipodophyllotoxin-B-D-benzylidene-glucoside (1), V-demethyl-epipodophyllotoxin-B-D-tenylidene glucoside (2),

-dernetyl-epipodophyllotoxin-B-D-furfurylidene-glucoside (3)

in relation to podophyllotoxin-benzylidene-B-D-glucoside (<>>+)

(Example 1, DAS 1,020,639) can be easily demonstrated by the action

of these compounds on cultures of mouse stem cell tumor P-815. If the compounds (1), (2), (3) and (h) are added to such a cell culture in different concentrations over the course of h0 hours, the four substances achieve a $0% inhibition of cell proliferation. This concentration or dose is designated as DE^Q.

It appears from the above table that the compounds (1), (2) and (3) in vitro have a significantly stronger cytostatic effect than the compound (h). The aforementioned in-vitro effect thus gives, with the following in-vivo experiment, a hint that the compounds that can be produced according to the invention can also have a clinical effect.

The superior effect of the compound (1) compared to the compound C+ on the proliferation of malignant cells in vivo was tested experimentally on a transplantable mouse tumor, mouse leukemia L-1210. This showed that no appreciable therapeutic effect could be achieved with the compound (h), but that, on the other hand, the compound (1) in a low adequate dosage showed a remarkable effect (the extension of the survival time to 150$ or more).

The injections of the compounds "(1) and (h) were started on the date of tumor inoculation and continued until the animal's ddd. Six mice were used per dose. In the individual experiments, the survival time (OLT)J, i.e. the time from the date of tumor inoculation to death occurred, measured in days.

The dosage of the new compounds with the general formula (I) varies between 1 and 50 mg per day and night.

The new compounds can be used as drugs in themselves or in corresponding drug forms for oral, enteral or parenteral administration. For the production of suitable pharmaceutical forms, the compounds are processed with organic or inorganic, pharmacologically indifferent excipients. 1 the following examples are all temperature indications in degrees Celsius. The melting and splitting points respectively are determined on the Kofler block.

Examples 1-7 illustrate the production of starting materials.

Example 1: \t'-demethyl-epipodophyllotoxin

Dissolve 2 g of demethyl podophyllotoxin in 25 ml of acetone and 15 ml of water and, after adding 5 ml of concentrated hydrochloric acid, heat for 2 hours under reflux. The acid is then neutralized with

solid barium carbonate, it is filtered and the filtrate is freed from acetone in vacuum at <1>+0°C. The precipitated material is taken up in chloroform plus 5% acetone, the solution is dried over sodium sulfate and evaporated in vacuo. The residue is chromatographed on silica gel with chloroform + 1% methanol, whereby first small amounts of impurities are obtained, then pure V-demethyl-epipodophyllotoxin and finally unreacted starting material. Crystallization of the pure fractions of V-demethyl-epipodophyllotoxin takes place from chloroform and methanol. Melting point 228 - 230°C, Æ/jj0 = -69.8° (c = 0.630 in chloroform).

Example 2; h }- carbobenzoxy- H-'- demethyl- epipodophyllotoxin

60 g finely powdered h<*->demethyl-epipodophyllotoxin

suspended in 1000 ml of anhydrous ethylene chloride and cooled after adding 19 ml of abs. pyridine to -10°C. While stirring and exclusion of moisture, a solution of 3.5 g of chloroformic acid benzyl ester in 100 ml of ethylene chloride is added dropwise at -10°C over the course of 2.5 hours and then left to react for a further hour. The reaction solution is then washed with water, the organic phase is dried over sodium sulphate, evaporated in a vacuum and the residue is dried in a high vacuum at 70-8o°C. Crystallization of the crude product from acetone/ether and then 2 times from methanol go h<*->carbobenzoxy-k*-demethyl-epipodophyllotoxin with double melting point 117 - 119/202

- 205°C. By drying in high vacuum first at 95 - 110°C and then at 130°C or crystallization from acetone/ether, the solvent-free form is obtained with melting point 201 - 20k°C, £% f^~ = -<*>+3j9°

(c = 0.535) in CHC13-

Example 3: h'-carbobenzoxy-^'-demethylpodophyllotoxin

15, And h<1->demethyl-podophyllotoxin is dissolved in hjO 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 chloroformate benzyl ester, dissolved in 55 ml of ethylene chloride, are now added dropwise over the course of 1 hour, and then stirred for 1 hour at -5 - -10°C. It is then evaporated in a vacuum at <1>+0°C bath temperature 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 1 N hydrochloric acid and then until neutral with water. After drying over sodium sulphate, it is evaporated in vacuo and the residue is chromatographed on the 20-fold quantity of silica gel. Chloroform with Vfo methanol first elutes small quantities of by-products, then pure *+' -carbobenzoxy-^f'-demethyl-podophyllotoxin. After crystallization from methanol, the compound melts at 113 - 111°C. faj*<1>= -88.3°C (chloroform).

Example h: Tetra-O-acetyl-^'-carbobenzoxy-V-demethyl-epipodophyllotoxin-3-D-glucoside

10.7 g ^! -carbobenzoxy-M-1-demethyl-podophyllotoxin is dissolved while heating in 30 ml of ethylene chloride, the solution is cooled to +15°C and 1H-,0 g of 2,3,k,6-tetra-0-acetyl-3-D- glucose. After 5 minutes of stirring, it is cooled to -15°C while excluding moisture and, over the course of 5 minutes, 7 ml of pre-cooled boron trifluoride ethyl etherate (h8% BF^) is added dropwise to -10°C.

The mixture is then stirred for 1 hour at -15°C, and then a solution is added drop by drop

of 7 ml abs. 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, it is evaporated in a vacuum and the residue is dried in a vacuum at 60°C. The resulting foam is dissolved in 50 ml of hot ethanol, cooled to approx. 50°C, 150 ml of cold water is added and it is stirred for so long that the initially lumpy and tough precipitate is transformed into a sandy powder. The product is suctioned off, washed with 0 ml of 25% ethanol and dried in a vacuum at 60°C. The preparation is then dissolved in 200 ml of hot methane'61, 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-Lt-'-demethyl-epipodophyllotoxin-Ø-D-glucoside obtained in this way is identical to the preparation prepared according to example 3.

Example 5; Tetra-O-acetyl-1f'-demethyl-epipodophyllotoxin-B-D-glucoside

To cleave off the carbobenzoxy residue from tetra-O-acetyl-^1 - carbobenzoxy-^'-demethyl-epipodophyllotoxin-p-D-glucoside, dissolve 13.g of this compound in 100 ml of acetone ethanol (1:2), add 0.5 ml of glacial acetic acid and 2 g of palladium charcoal (with 10% Pd) and 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 ethanol, allowed to crystallize and the crystals are dried in vacuum after suction and washing with methanol. Pure tetra-O-acetyl-V -carbobenzox<y>-V -demethyl-epipodophyllotoxin-B-D-glucoside crystallizes in fine needles with melting point 225 - 227°C, ZV^<1>= -6^,^° (c = 1.02^) in chloroform.

Example 6: - demethyl-epipodophyllotoxin-B-D-glucoside

25 g of pure tetra-O-acetyl-V-carbobenzoxy-V-demethyl-epipodophyllotoxin-B-D-glucoside, 3.6 g of anhydrous zinc acetate and 1.5 g of anhydrous sodium acetate are heated in 150 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 is added and distilled again. After a total of 18 hours, the cleavage of the acetyl groups is complete and there is a mixture of approx. 60% h1-carbobenzoxy-V-demethyl-epipodophyllotoxin-B-D-glucoside and approx. k0% V-demethyl-epipodophyllotoxin-B-D-glucoside. The reaction is followed in a thin-layer chromatogram on silica gel plates with water-saturated isopropyl acetate/methanol (µl) as eluant. The chromatograms are developed by showering with a 0.2% solution of Ce (IV) sulphate in 50% sulfuric acid and heating to 110 - 130°C.

For processing, 5 ml of glacial acetic acid is added, it is evaporated in a vacuum

at 50°C bath temperature and it is dried for 15 minutes in high vacuum at 50°C. The residue is taken up in 250 ml chloroform/isopropanol (h:1) and 25 ml water, the aqueous phase is separated and the organic phase is further washed with 25 ml water. The two washing waters are extracted with 50 ml of chloroform/isopropanol and the combined organic phases are evaporated in vacuo after drying over sodium sulphate. The residue is distilled 3 times with each time 30 ml of acetone in vacuum and then dried for 2 hours in high vacuum at 75°C.

From the mixture of h.' -carbobenzoxy-^-'-demethyl-epipodophyllotoxin-B-D-glucoside and<1>+'rdemethyl-epipodophyllotoxin-B-D-glucoside can

by crystallization from methanol, >+' -carbobenzoxy-V -

demethyl-epipodophyllotoxin-B-D-glucoside in not completely pure form. Chromatography on the 100-fold amount of silica gel gives

after elution with water-saturated isopropyl acetate/methanol (9:1)

pure !+' -carbobenzoxy-V -demethyl-epipodophyllotoxin-B-D-glucoside, which crystallized from acetone melts at 155-156°C.

Æi/p1 = -92.0° (c = 1.00) in chloroform.

For the cleavage of the carbobenzoxy group, the mixture is dissolved

>+' -carbobenzoxy-Ll-, -demethyl-epipodophyllotoxin-(3-D-glucoside and V-demethyl-epipodophyllotoxin-B-D-glucoside in 200 ml of acetone, the solution is added to a suspension of 3g of palladium charcoal (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-water (M-:1) and the filtrate is evaporated in vacuum to a volume of approx. crystals are filtered off, washed with 25 ml of water and dried in high vacuum at 70° C. Crystallization from methanol gives pure h'-demethyl-epipodophyllotoxin-B-D-glucoside with melting point 225-227° C, / U^ 1 = -88.6° (c = 1.05) in methanol Another modification shows mp 262-26<*>4-°C.

Example 7: - dernetyl-epipodophyllotoxin-B-D-glucoside

2.0 g of the tetra-O-acetyl-4-'-demethyl-epipodophyllotoxin-B-D-glucoside obtained in Example 7 and 1 g of anhydrous zinc acetate are heated in 30 ml of absolute methanol for 25 hours under reflux. The white precipitate formed is then dissolved by adding a few ml of glacial acetic acid and gently heating, the solvent is removed in vacuo at h0°C and the residue is taken up in 50 ml of chloroform/butanol (*f:l). The organic phase is washed twice with 10 ml of water each time, it is evaporated in vacuo after drying over sodium sulphate and the residue is chromatographed on silica gel. Water-saturated isopropyl acetate/methanol (9:1) first elutes non-polar portions, then pure h'-demethyl-epipodophyllotoxin-B-D-glucoside. The individual fractions are examined in thin-layer chromatograms on silica gel

plates with water-saturated isopropyl acetate/methanol (8:1), as eluent, and the glucoside fractions are combined and crystallized twice from methanol, ^f1 -demethyl-epipodophyllotoxin-8-D-glucoside melts at 222-230°C, another modification at 262- 26M-°C. frj2^=_88o (c=0?507) in methanol. Example 8: i+ l- dernetyl- epipodophyllotoxin- B- D- benzylidene- glucoside

1 g tdr.t ^'-demethyl-epipodophyllotoxin-B-D-glucoside is dissolved in 20 ml

pure benzaldehyde and, after adding 0.5 g of anhydrous zinc chloride, shake in a shaking machine while excluding moisture for 5-6 hours

at 20°C. The course of the reaction is followed using thin-layer chromatography. For this, silica gel plates and chloroform with 6% methanol as a flow agent are suitable. To make the substances visible, the plates are showered with a 1% solution of Ce (IV) ammonium nitrate in 50% sulfuric acid and then heated at 100 to 120°C. To work up the condensation product, chloroform is added to the clear red-brown colored reaction solution and shaken with water. The aqueous phase is extracted twice with chloroform. All chloroform phases are combined, washed twice with water, dried over sodium sulphate and the solvent evaporated in vacuo. The oily residue obtained is triturated with pentane to remove the still adhering benzaldehyde 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 a quantity of 100 ml of pentane, whereby a pale yellow to white precipitate is obtained. The crude product can also be purified by chromatography on silica gel columns. For this, a solution as concentrated as possible of 1 g of the crude benzylidene derivative in a mixture of chloroform plus 2% methanol is filtered on 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. h<1->demethyl-epipodophyllotoxin-B-D-benzylidene-glucoside is obtained as a white powder with a melting point of 182-185°C. Recrystallization from abs. ethanol gave crystals with melting point 2k5-2k6°C. The optical degrees of rotation are: /h7^ - -99°

-in methanol and -lO^-0 in chloroform.

Example 9: - dernetyl-epipodophyllotoxin-B-D-thenylidene-glucoside

0.5 g of dry V-demethyl-epipodophyllotoxin-B-D-glucoside is added to 10 ml of pure thiophene-2-aldehyde and 0.25 g of anhydrous zinc chloride and the mixture is shaken while excluding moisture at 20°C on a shaking machine, whereby a eventually clear solution.

The course of the condensation is followed as described above by

using thin layer chromatography. After 3 tllh hours of reaction, the solution is diluted with chloroform and shaken out with water. The chloroform phase is washed twice more with a little water and then dried over sodium sulphate and evaporated. The residue obtained is dissolved in a little acetone to remove excess thiophene-2-aldehyde and reprecipitated by adding pentane. The reprecipitation from acetone-pentane is repeated until the condensation product precipitates out in a crumbly form. For further purification, the crude product is chromatographed on silica gel. The thin-layer chromatographically uniform fractions are combined and give crystals from absolute alcohol. Pure V-demethyl-epipodophyllotoxin-B-D-tenylidene-glucoside has a melting point of 2<1>+2-2<1>+6<0>C (last residues at 255°C) and has in chloroform-methanol (9: 1) the optical rotation £hJ- q -107 •

Example 10: -demethyl-epipodophyllotoxin-B-D-furfurylidene-

glucoside

0.5 g of dry V-demethyl-epipodophyllotoxin-B-D-glucoside is added to 10 ml of pure furfurql and after the addition of 0.25 g of anhydrous zinc chloride is shaken on a shaking machine while excluding moisture in

3 to h hours at 20°C. The course of the condensation reaction is checked by thin-layer chromatography. The thin-layer chromatography can be carried out on silica gel plates with chloroform with 6% methanol as eluent. To make the substances visible, they are showered with a 1% solution of Ce(IV)-ammonium nitrate in 50% sulfuric acid and then heated at 100-120°C. After completion of condensation, chloroform is added to the green-coloured reaction solution and then shaken with water. The aqueous phase is extracted twice with chloroform. All chloroform phases are combined, washing twice with a little

water, dried over sodium sulphate and the solvent evaporated

vacuum at 60°C. The oily residue (approx. 10 ml) is dropped into 150 ml of pentane with stirring to remove adhering furfurol, whereby a tough precipitate is obtained. After decanting the overlying pentane, the precipitate is taken up in a little acetone and a quantity of 150 ml of pentane is added dropwise to this solution. The separated, now flocculated condensation product can be purified by chromatography on silica gel. For chromatography, a column with 100 g of silica gel is supplied

concentrated solution of the crude product in chloroform plus 2% methanol and it is eluted with the same solvent mixture. The thin-layer chromatographically uniform first fractions are combined and reprecipitated from acetone-pentane. h*-demet<y>l-e<p>i<p>odof<y>llotoxin-B-D-furfurylidene glucoside is obtained as a white powder with a melting point of 188-191 C.

From abs. methanol, crystals with a melting point of 267-269°C are obtained. The optical rotation value in chloroform is / qjj^ = -102°.

Example 11: V-dernety1-epipodophy11otoxin-B-D-benzylidene-glucoside

1.0 g of finely powdered -demethyl-epipodophyllotoxin-B-D-glucoside is dissolved in 20 mg of pure benzaldehyde and 2 g of dry "Dowex" 50 WX 2" powder is added. The air in the flask is displaced with nitrogen and the reaction mixture is stirred while excluding moisture by means of The course of the reaction is followed continuously by means of thin-layer chromatography.

/Øystein a) chloroform plus 6% methanol,

b) chloroform methanol water- (70 :'25: 517 •

After 1 hour, the reaction is complete.

For processing from, the catalyst is filtered from the yellowish solution and washed thoroughly with chloroform. The solvent is removed at 50°C in a vacuum. An oil is obtained which is purified on 60 g of silica gel "Merck" (grain size 0.05 to 0.2 mm). After separation of the benzaldehyde with chloroform extraction, the substance is eluted with chloroform plus 2% methanol. This chromatographic purification gives amorphous<>>+'-demethyl-epipodophyllbtoxin-B-D-benzylidene-glucoside which is completely uniform in a thin-layer chromatogram.. For .characterization, the preparation is dissolved in 5 ml of acetone and added dropwise to 60 ral of pentane while stirring. k-1-Demethyl-epipodophyllotoxin-B-D-benzylidene-glucoside is obtained as a barrel-dissolved amorphous powder with a melting point of 182-181+°C. From abs. ethanol, crystals with melting point 21f5-21+60C are obtained.

fvJv°=-1Qlf0 (c = 0.766 in chloroform).

Claims (1)

1. Process for the production of new, therapeutically active h'-demethyl-epipodophyllotoxin glucosides with the usual formula (I) in which R stands for a phenyl, 2-furyl or 2-thienyl residue, characterized in that k <*-> demethyl-epipodophyllo-toxin-B-D-glucoside of formula (II) in the presence of an acidic catalyst is reacted with an aldehyde of the general formula (III) where R has the meaning given above.