DK153491B - ANALOGY PROCEDURE FOR PREPARING A 4'-O-TETRAHYDROPYRANYL OR 4 ', 14-BIS (0-TETRAHYDROPYRANYL) -THRACYCLING LYCOSIDE OR A POISONOUS ACID ADDITION SALT - Google Patents

Description

DK 153491B

The present invention relates to an analogous process for the preparation of a novel 4'-O-tetrahydropyranyl or 4 1,14-bis (O- * tetrahydropyranyl) anthracycling glycoside of the general formula o OH f 1

AND, 0 OH

Wherein R 3 is a hydrogen atom or a tetrahydropyranyl oxy group, or a non-toxic acid addition salt thereof. These compounds have antibiotic action.

The process according to the invention is characterized by reacting an anthracycling glycoside of the formula O 9 H in 2 R · 4 ·

AND O OH I

O (II) r <^ H ° Eh ^ wherein r3 is a hydrogen atom or a tetrahydropyranyl oxy group, or an acid addition salt thereof with 3,4-dihydro-2H-pyran in an inert organic solvent and in the presence of a acid catalyst and, if desired, convert

H

2

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the formed compound of formula I into a non-toxic acid addition salt thereof or, if necessary, converts a formed acid addition salt of the compound of formula I into the free compound or to another non-toxic acid addition salt 5 thereof.

A number of anthracycling glycosides are described in the literature. Among these are daunomycin (U.S. Patent No. 3,616,242 and GB Patent No. 1,003,383) and adriamycin (U.S. Patent No. 3,590,028 and No. 3,803,124), which are the known compounds having are closely related to the compounds of formula I, which are recovered from the culture broth from the cultivation of certain streptomyces species, a broad antitumor spectrum against various experimental tumors, and are used clinically as potent chemotherapeutic agents. Despite the usefulness of 15 adriamycins and daunomycins as clinical antitumor agents, they are known to have serious side effects such as alopecia, leukopenia and cardiotoxicity.

It has been found that the compounds of formula I have a stronger anticancer activity and lower toxicity than the above known analogous compounds.

The term "non-toxic acid addition salts" encompasses acid addition salts of compounds of formula I with all the organic and inorganic acids commonly used to prepare substantially non-toxic salts of drugs containing amine functions. Such salts can be formed, for example, from acids such as sulfuric acid, phosphoric acid, hydrochloric acid, hygrogen bromide, nitric acid, phosphoric acid, acetic acid, propionic acid, maleic acid, voleic acid, palmitic acid, citric acid, succinic acid, tartaric acid, fumaric acid, glutamic acid, pantothenic acid, pantothenic acid, pantothenic acid,

The compounds of formula I exist as the individual: diastereomers (herein arbitrarily designated as isomer a and isomer b) which have different configuration at the C-2 position of the tetrahydropyranyloxy group or as mixtures of such lp 3

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isomers. The present invention encompasses the preparation of both the individual diastereomers and the diastereomer mixtures.

In the drawing, FIG. 1 shows the infrared absorption spectrum (KBr) of 5 4'-O-tetrahydropyranyldaunomycin (isomer a); 2 shows the infrared absorption spectrum (KBr) of 4'-O-tetrahydropyranyldaunomycin (isomer b); FIG. 3 shows the infrared absorption spectra (KBr) of 4 ·, 14-bis (O-tetrahydropyranyl) adriamycin (isomer a); 4 shows the infrared absorption spectrum (KBr) of 414-bis (O-tetrahydropyranyl) adriamycin (isomer b); and FIG. 5 to 8 proton NMR spectra (100 MHz, CDCl 3) of the compounds in the same order as the aforementioned infrared absorption spectra.

14-O-Tetrahydropyranyl-adriamycin and daunomycin, which are starting materials for the preparation of the compounds of the invention, have the formulas 0 Od 20 0 in li i: Ul2 All JH 13 "1 2ΛΛΛΛκμ3

Il g0H

4T 5 Π 6 [(7

OCH 2 O OH

0 Daunomycin "'Ίτ \" Ab Nla ..

HO ^ —f I 2 «11¾ and.

|| 4

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«·· · Ί O OH i o O» li n 1 J} 2 111 3? 15 g CH.Q- / 2 ΑΛ / Υν% v_>

J | l π I J ° H

^ οΓ ^^ Ιγ

AND ^ O OH

ISLAND

14-O-Tetrahydropyranyl-4-adriamycin h, / CK,

Hov-f 1 2 '. «%.

Daunomycin has two reactive hyiroxyl groups (excluding the two phenolic hydroxyl groups) on C-9 and C-4 ', and 14-O-tetrahydro-pyranyl-adriamycin has two reactive hydroxyl groups (again excluding the tb phenolic groups) on C 9 and C-4 '.

The starting anthracycline glycoside may be a free base or an acid addition salt. Since the acid addition salts of the tetrahydro-pyranyl ether reaction products can be converted by the known methods to the corresponding free bases or to non-toxic acid salts. 10 addition salts, it is not necessary for a starting material to be non-toxic.

Any non-reactive organic solvent can be used for the tetrahydropyranylation reaction. Suitable solvents are, for example, benzene, toluene, xylene, dimethylformamide, acetonitrile and tetrahydrofuran. The reaction solvent may be a single solvent or a mixture of solvents. A preferred solvent is anhydrous dimethylformamide.

* /, y, *

The acidic catalyst can be any organic acid (e.g.

20 formic acid or trifluoroacetic acid) or an inorganic acid lif 13 ________ v '

DK 153491 B

5 (eg hydrochloric or phosphoric acid). A preferred class of acidic catalysts comprises the organic sulfonic acids. Particularly preferred catalysts are aromatic sulfonic acids such as p-toluenesulfonic acid and benzenesulfonic acid. A particularly preferred catalyst is p-toluenesulfonic acid.

The reaction temperature is not critical. Good results in the ethereal reaction have been obtained at room temperature, although higher and lower temperatures can also be used.

The reaction time varies according to the selected reaction conditions, e.g. temperature, catalyst and solvent. Selection of the optimal reaction time can be done by routine experimentation using the thin layer test described below. Generally, reaction times of approx. 20 to approx. 50 hours proved to yield favorable results.

*

While reaction products and reaction yields depend on factors such as the concentration of the starting materials, the ratio of the reactants, etc., the main products using daunomycin as starting material are 4'-O-tetrahydropyranyl-daunomycin (abbreviated as 4'-O-PD) and 9-0. -tetrahydropyranyl-daunomycin (abbreviated as 9-O-PD). These products can be detected in the reaction mixture by thin-layer chromatography on silica gel (Merck Co., 60F2 ^^) using a chloroform-methanol-acetic acid mixture (80: 20: 4 v / v) as the eluent . The products appear at Rf 0.74 (4'-O-PD) and Rf 0.15 (9-O-PD).

The product 4'-0-PD is separated into two components with Rf values of 0.46 and 0.65 by thin layer chromatography on silica gel using a chloroform-methanol mixture (10: 1 v / v).

These components were found to be the diastereomer of 4'-0-PD. The components with Rf 0.46 and Rf 0.65 were arbitrarily designated as 4'-O-PDa (isomer a) and 4'-o-PDb (isomer b), respectively.

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6

Using 14-O-tetrahydropyranyl-adriamycin as the starting material, the main products detected using the above-mentioned thin-layer chromatography on silica gel were two components which are diastereomers of 4 ', 14-di (O-tetrahydropyranyl) adriamycin, ie. 4 ', 14-di (O-tetrahydropyranyl) adriamycin (isomer a), abbreviated as 4', 14-di-O-PAa at Rf 0.55 and 4 ', 14-di (O-tetrahydropyranyl) adriamycin (isomer b), abbreviated as 4 ', 14-di-O-PAb at Rf 0.73. The compound 41,14-di (O-tetrahydropyranyl) -adriamycin may also be called 4 ', 14-bis (O-tetrahydropyranyl) -adriamycin.

The diastereomeric mixture of 4 ', 14-di-O-PAa and 4', 14-di-O-PAb is prepared in high yield by etheration of 14-O-tetrahydropyranyladriamycin or an acid addition salt thereof with 3,4-dihydro-2H -pyran in an inert organic solvent and in the presence of an acidic catalyst.

The products of formula I can be isolated from the reaction mixture by conventional methods. Thus, the products of the tetrahydropyranylation reaction can be recovered by neutralizing the reaction mixture with a basic substance (e.g., an alkali metal carbonate or hydrogen carbonate), extracting the neutralized reaction mixture with a water-immiscible organic solvent (e.g., ethyl acetate, chloroform, methylene chloride or methyl isobutyl ketone), extraction of the organic extract with a dilute aqueous acid solution (organic or inorganic), neutralization of the aqueous acidic layer with a basic substance, extraction of the neutralized aqueous layer with a water-immiscible organic solvent. and concentration of the organic extract to dryness. The dark red dried powder thus obtained can be purified by silica gel column chromatography or, in the case of a smaller amount, by preparative thin layer chromatography.

Products obtained in the form of a mixture of diastereo-. λ 'more (a and b isomers), can be separated as described above i1 · 1 C 1' • j? · 1®,: by silica gel thin layer chromatograph i in the individual a and b -Y ^ V in _________. -. - ·. il

DK 153491 B

7 isomers essentially in pure form.

The products obtained by the above reactions can be recovered in the form of the free base or a non-toxic acid addition salt. The free bases can easily be converted into nontoxic acid addition salts, whose therapeutic activity is substantially equivalent to that of the corresponding free bases. The salts are formed, isolated, purified and formulated by the methods generally used for salt formation of antibiotics, e.g. anthracyclinglycosidantibiotika. Thus, the free base can be reacted with a non-toxic acid in a suitable solvent and the salt is recovered by lyophilization or by precipitation with a precipitant, ie. a solvent in which the desired salt is only slightly soluble.

Products in the form of an acid addition salt can be converted to the corresponding free base by neutralization with a basic substance. Finally, toxic acid addition salts can be converted to non-toxic acid addition salts by neutralization and treatment with a non-toxic acid as described above.

Physicochemical Properties The present compounds exist in solid form as an amorphous or crystalline red powder. As free bases, they are soluble in ethyl acetate, chloroform and ethanol and are highly soluble in e.g. water, n-hexane and petroleum ether. Ethanol solutions and acidic solutions of the compounds are! 25 reds, gives positive ninhydrin reaction and does not reduce

'A

Fehling's fluid. FIG. 1-8 and Table 1 show elemental analysis, melting point (decomposition), specific rotation (c = 0.2 in CHCl 3), UV and visible light absorption spectrum (methanol), infrared absorption spectrum (KBr tablet) and NMR spectrum 30 (100 MHz, CDCl 3).

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DK 153491 B

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DK 153491B

With respect to the structure of compounds 4'-O-PDa, 41-O-PDb, 4 ', 14-di-O-PAa and 4'f14-di-O-PAb, the number of tetrahydropyranyl groups bound to the compounds may , are analyzed to be either one or two out of 5 of the signal intensity of the methine proton in C-2 and the methylene proton in the C-3-, C-4-, C-5- and C-6 positions of the tetrahydropyranyl group. The binding position of the tetrahydropyranyl group can be analyzed by chemical displacement of the C-4 'proton in the daunosamine moiety towards a lower field (compared to that of daunomycin) due to formation of a glycosidic bond on C-4'.

The difference in configuration between 4'-P-PDa and 4'-O-PDb and 4 ', 14-di-O-PAa and 414-di-O-PAb is presumed to be a difference in absolute configuration R and S of C 2 in the tetrahydro-pyranyl group, with chemical shift and coupling constants (J value) of the C-2 and C-3 protons being different from each other. However, the absolute configuration of the a and b isomers is still unknown. Table 2 shows chemical displacement (from Figs. 5-8) of the C-2 proton in the tetrahydropyranyl group and of the C-4 'proton in the daunosamine moiety.

:> - '1 ·. "·> • v · yå

• fM

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Table 2

Proton V-THP * 14-THP * DS4 '** __ (ppm) _ (ppm) _ (ppm) _

Compound VO-PDa 4-, 38 - 3.62 5 4'-O-PDb 4.72 - 3.64 4 ', 14-di-O-PAa 4.38 4.70 3.60 4', 14- di-O-PAb 4.72 4.72 3.66 * THP: Chemical shift in the C-2-methine group in a substituted tetrahydropyranyl group ** DS4 ': Chemical shift in the C-4'-methine group in daunosamine 10 From the above it was determined the structure of the compounds of the invention to be as set forth above.

Antibiotic activity

The compounds of formula I have been found to have antimicrobial activity against many different pathogenic microorganisms.

The minimum inhibitory concentrations (determined by the broth dilution method) of representative compounds prepared by the process of the present invention are shown in Table 3.

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Table 5 Minimum inhibitory concentration (ΜΓΚ, µg / ml)

Tested compound

Test organism_4'-0-PDa 41 -O-PDb

Staph, aureus 6.25 6.25

PDA 209P

5 Staph .. aureus 12.5 5 »12

Smith

Bacillus subtilis 3> 12 1.56 NBBLB-558

Bacillus cereus 6.25 6.25 ATCC 10702

Bacillus megaterium 6.25 3.12

APP

Sarcina lutea 0.39 0.39 PCI 1001 10 Micrococcus flavus 0.78 1.56 PDA 16

Corynebacterium bovis 0.78 0.78 1810

Pseudomonas aeruginosa> 100> 50 A3

Escherichia coli> 100> 100

NIHJ

Mycobacterium smegmatis 6.25 6.25 ATCC 607 15 Candida albicans> 50 25 (1 -

As shown in Table 3, the compounds of the invention are useful as antibiotic agents, especially against gram-positive bacteria.

Antitumor activity

Compounds of formula I show pronounced antitumor activity 20 of low toxicity in standard tests.

. A. The compounds of the invention were found to have a strong J inhibitory effect on the growth and nucleic acid synthesis of '<L1210 leukemia cells in culture.

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12 For example. For example, L122 cells (5x104 cells / ml) are included in RPMI-1640 medium (Roswell Park Memorial Institute 1640) containing 20% calf serum and grown at 37 ° C in the presence of 0.1 and 0.5 µg / ml of the compounds of this invention in a 5 CC 2 incubator. The number of cells is counted periodically and the growth inhibition (%) of control was determined as shown in Table 4.

Table 4 Growth Inhibitory Effect of Tetrahydropyranyl Derivatives on Ll210 Cells in Culture. 10 Concentration Inhibition (%)

Compound 0.1 / 0.5 µg / ml 41-O-PDa 79.2 95.0 4'-0-PDb 74.6 88.8

Daunomycin 68.8 72.7 4 ', 14-di-O-PAa 37.5 80.8 4', 14-di-O-PAb 25.5 72.1

Adriamycin 70.7 84.2

The effect of the subject compounds on nucleic acid synthesis was investigated as follows: 20 1x10 5 cells / ml of L210 cells were suspended in RPM1 medium containing 10% calf serum and first cultured at 37 ° C for 1 to 2 hours in a CC incubator, after which the subject compounds were added to the medium at various concentrations.

14

After 15 minutes of incubation, C-uridine (0.05 µCi / ml) was added 14 o

25 or C-thymidine (0.05 µCi / ml) and incubated at 37 ° C

for 60 minutes. 10% trichloroacetic acid (TCA) was added to the incubation medium to stop the reaction and precipitate the acid-insoluble materials, and then the precipitate was washed three times with 5 to 10% TCA dissolved in formic acid. Radioactivity 30 was measured and expressed as 50% inhibition concentration for incorporation.

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13

Table 5 14 14 50% inhibition concentration of C-thymidine and C-uridine incorporation into L122 cells in culture. 50 inhibition concentration µg / ml

Compound_Uridine_Thymldine 5 4'-O-PDa 0.13 0.28 4'-O-PDb 0.20 0.20

Daunomycin 0.40 0.70 4 ', 14-di-O-PAa 0.23 0.55 4', 14-di-O-PAb 0.24 0.97 10 Adriamycin 0.50 2.1 B. By against various experimental tumors in animals, the compounds of this invention exhibit a marked anti-tumor activity with reduced toxicity to adriamycin and daunomycin. Accordingly, the compounds are therapeutically useful for inhibiting the growth of mammalian tumors.

For example, BDF 1 mice were intraperitoneally inoculated with 6 x 10 10 cells / mice of L122 leukemia cells. After 24 hours of inoculation, the procedures in question were administered. 20 intraperitoneally adhered to the mice once daily for ten consecutive days, and the mice were kept under observation for 45 days. The antitumor activity was shown at the prolongation of life (T / C,%) relative to the life span of control mice injecting physiological saline. The results are shown in Table 6.

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Table 6

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Antitumor activity of tetrahydropyranyl derivatives (T / C,%).

Dose (mg / kq / day) _

Compound_5_2.5_1.25_0.6_0.3_0.15 5 4'-O-PDa> 320> 320 122 115 96 90 41-O-PDb> 320 256 122 115 103 90 4 ', 14-di-O-PAa 154 115 109 96 103 96 4 ', 14-di-O-PAb 161 109 103 103 96 115

Adriamycin Toxic 231 218 230 165 128 Death From the results of toxic deaths and weight loss in the mice in this experiment, it has been shown that the toxicity of the compounds of the present invention is 1/3 to 1/2 lower than that of adriamycin and daunomycin. which are starting materials of the process of the present invention.

C. The pronounced antitumor effects seen by A and B above were confirmed by the stability of the compounds of the invention against inactivation by hepatic NADPH cytochrome P450 reductase. Specifically, NADPH cytochrome P450 reductase isolated from rat liver homogenate and purified was incubated with the subject compounds at 25 ° C for 25 minutes in nitrogen gas phase, and the resulting incubation product, .. · ·; ! 7-deoxyaglycon was determined as shown in Table 7.

Table 7

Stability of tetrahydropyranyl derivatives against rat NADPH cytochrome P4 50 reductase.

Product (nmol / tube) Compound (7-deoxyaglycone) 4'-O-PDa 37.3? ': 4'-O-PDb 46.6 ϊ%> V: Daunomyc in 65.8 m

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i5,. p

Table 7 continued 4 ·, 14-di-O-PAa 13.1 4 ’, 14-di-O-PAb 15.8

Adriamycin 47.2

Composition of the reaction mixture:

NADPH 0.2 mM

Tris-HCl (pH 8.0) 0.1 M

Substrate 0.1 mM

Enzyme 4.6 µg / ml (Tris-HCl = tris- (hydroxymethyl) -aminomethane).

10 Therapeutic use

As mentioned above, the compounds 4'-O-PDa, 4'-O-PDb, 4 ', 14-di-O-PAa and 414-di-O-PAb and their non-toxic acid addition salts are novel antibiotics useful in both human and veterinary medicine, and which have also exhibited 15 inhibitory effects against malignant mammalian tumors, including both solid and ascitic types.

The present compounds may be formulated as a pharmaceutical composition containing a therapeutically effective anti-microbial or tumor-inhibiting amount of 4'-O-PDa, 4'-0-20 PDb, 414-di-O-PAa or 414-di -O-PAb or a mixture thereof or a non-toxic acid addition salt thereof, in combination with a pharmaceutical carrier or diluent.

Such preparations may be prepared in any pharmaceutical form suitable for parenteral administration.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. They may also be prepared in the form of sterile solid preparations which may be dissolved in sterile water, physiological saline or other sterile injectable medium 30 immediately prior to use.

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The preferred dose size depends on the nature of the compound used, on the formulation of the preparation and on the mode of application, on the situs and host, and on the nature of the disease. In general, the compounds are injected intraperitoneally, intravenously, subcutaneously, or locally in mammals other than humans and intravenously or locally in humans. Many factors that modify the effect of the drug must be considered by those skilled in the art, e.g. age, body weight, sex, diet, time of administration, route of administration, rate of excretion, patient's condition, drug combinations, response sensitivities and severity of the disease. Administration may be performed continuously or periodically within the maximum tolerated dose. Optimal application rates for a given set of conditions can be determined by those skilled in the art using conventional dosage determination tests according to the above guidelines.

For use as antimicrobial agents, the compounds are generally administered such that the concentration of the active ingredient is greater than the minimum inhibitory concentration of the treated organism concerned.

The process according to the invention is further illustrated in the following examples.

Example 1

Preparation of 41-o-tetrahydropyranyldaunomycin (4'-O-PDa 25 and 4'-O-PDb)

To a solution of 60 mg of daunomycin hydrochloride in 5 ml of anhydrous dimethylformamide was added 1 ml of 3,4-dihydro-2H-pyran and a catalytic amount of p-toluenesulfonic acid. After standing for 16 hours at room temperature in the dark, the reaction mixture was added to 20 ml of 0.1 N aqueous sodium hydrogen carbonate solution and extracted with 4 x 10 ml of chloroform. After extracting the chloroform extract with 10 x 10 ml of 1% acetic acid, the resulting acidic aqueous layer was neutralized with sodium hydrogen carbonate and then re-extracted with tr-Vpt 17

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10 x 20 ml of chloroform. The chloroform layer was dried over anhydrous sodium sulfate and concentrated to dryness. The 44 mg residue thus obtained was subjected to thin-layer preparative chromatography on silica gel (Merck Co.) and eluted with a chloroform-methanol mixture (10: 1) (v / v).

Silica gel bands corresponding to Rf 0.46 and 0.65 were scraped off the thin-layer plate, eluted with the chloroform-methanol mixture (10: 1) (v / v) and concentrated to dryness. The residues were dissolved separately in methylene chloride, frozen by the addition of t-butanol under cooling and dried under reduced pressure. 10.1 mg of a reddish brown solid of 4'-O-PDa and 10.3 mg of a red solid of 4'-O-PDb of Rf 0.46 and 0.65 fraction respectively were obtained.

4'-O-PDa and 4'-O-PDb are diastereomers of 4'-O-tetrahydro-pyranyldaunomycin and their physicochemical properties are as shown in Table 1.

Example 2

Preparation of 4 ', 14-bis (O-tetrahydropyranyl) adriamycin from 14-O-tetrahydropyranyladriamycin. To a solution of 35 mg of 14-O-PA in 2 ml of anhydrous dimethylformamide was added 0.5 ml of 3, 4-dihydro-2H-pyran and a catalytic amount of p-toluenesulfonic acid. After standing for 40 hours at room temperature in the dark, the reaction mixture was added to! 10 ml of 0.02 N aqueous sodium hydrogen carbonate solution and extracted with 4 x 5 ml of ethyl acetate. After extraction of the ethyl acetate layer with 3 x 10 ml of 1% acetic acid, the acidic aqueous layer was neutralized with sodium hydrogen carbonate and extracted with 5 x 10 ml of chloroform. The chloroform layer was dried over anhydrous sodium sulfate and concentrated to dryness.

The resulting residue was chromatographed using thin layer preparative chromatography on a silica gel thin layer as described below and 8.4 mg of i m 18 were obtained.

DK 153491B

a red solid of 4 ', 14-di-O-PAa and 8.1 mg of a red solid of 4', 14-di-O-PAb from Rf 0.55 and 0.73 fraction respectively. Their physicochemical properties are as listed in Table 1.

Preparation of the starting material 14-o-tetrahydropyranyl-adriamycin (14-O-PA) from adriamycin.

To a solution of 130 mg of adriamycin hydrochloride in 10 ml of anhydrous dimethylformamide was added 2 ml of 3,4-dihydro-2H-pyran and a catalytic amount of p-toluenesulfonic acid. After standing for 10 hours at room temperature in the dark, the reaction mixture was added to 20 ml of 0.1 N aqueous sodium hydrogen carbonate solution and extracted with 5 x 20 ml of ethyl acetate. After extraction of the ethyl acetate layer with 4 x 40 ml of 1% acetic acid, the acidic aqueous layer was neutralized with sodium hydrogen carbonate and extracted with 10 x 20 ml of chloroform. The chloroform layer was dried over anhydrous sodium sulfate and concentrated to dryness. The resulting 160 mg of solid was eluted and purified by preparative thin layer chromatography on silica gel using a chloroform-methanol mixture 20 (10: 1) (v / v). The Rf 0.12 band was scraped off the thin-layer plate and purified by the method of Example 1.

1 35 mg of a red solid of 14-O-PA was obtained

Rf 0.12 fraction.

4 ', 14-di-O-PAa and 4', 14-di-O-PAb are diastereomers of 4 ', 14-25 bis (O-tetrahydropyranyl) adriamycin and their physicochemical properties are as shown in Table 1 .

· / *

Claims (1)

GB 153491 B Analogous process for preparing a 4'-O-tetrahydro-pyranyl or 41,14-bis (O-tetrahydropyranyl) anthracycline glycoside of the general formula i. Wherein I represents a hydrogen atom or a tetrahydropyranyl oxy group, or a non-toxic acid addition salt thereof, characterized by reacting an anthracyclic glycoside of the formula ° ° H '! And wherein OH represents a hydrogen atom or a tetrahydropyranyl oxy group, or an acid addition salt thereof with 3,4-dihydro-2H-pyran in an inert organic solvent and in the presence of an acidic catalyst and, if desired, 15 forms the formed compound of formula I into a non-toxic acid addition salt thereof, or, if necessary, converts a formed acid addition salt of the compound of formula I into the free compound or into another non-toxic acid addition salt thereof.