IL31966A - Process for the preparation of adriamycin and adriamycinone - Google Patents

Description

PROCESS FOB THE PREPARATION OF DRIA YCIN A HP DRIAMYCIKQNE n;ns->aynTTtn -ps^ns-niK mart? τ ηη The invention relates to the preparation of the antibiotics adriamycin and its aglycone adriamycinone,, Adriamycin is an antibiotic having a bacteriostatic and anti tumoral activity which has been described and claimed together wi Israel 2 8l3 its derivatives in our Dtl^inu Patent Specif ication-713 ,773'.

Adriamycin and its aglycone have the following structural formula where R is (adriamycinone) .

According to the invention there is provided a process of preparing adriamycin or a non-toxic pharmaceutically acceptable acid addition salt thereof or its aglycone adriamycinone in which daunomycin or a suitably protected derivative thereof or its aglycone daunomycinone is reacted with bromine or iodine, the 14-halogen derivative so obtained is reacted with an alkali metal acetate, the 14-acetoxy derivative so obtained is subjected to alkaline hydrolysis, any protective group is eliminated, and where appropriate the resulting 14-hydroxy derivative is converted into a salt* The starting materials 9 daunomycin and its aglycone daunomycinone, Israel 20230 are described and claimed in our Br-itioh Patent Specification 1,003, 303 They have the general formula I above except that there is a methyl group i the 14-position and R is : NX OH I — CH — CH. being X OH (daunomycin or a derivative thereof), or R is H (daunomycinone).

The initial reaction with bromine or iodine is preferably carried out in the pre sence of an inert organic solvent.

Halogenation with iodine is better carried out in the pre sence of a base such as calcium oxide. Special operating conditions are not called for. The reaction is preferably carried out at room temperature and may be optionally carried out in the warm, preferably under an atmosphere of an inert gas .

When the starting product is daunomycin, it is better to protect the amino group of the amino sugar with a suitable protecting group, particularly when iodine is used as halogenation agent. Among suitable protecting groups are acyl derivatives and Schiff bases, for example trifluoro- acetyl derivative s or yi Schiff base with salic>iildehyde. The trifluoro- acetyl derivative of daunomycin can be prepared by reacting daunomycin, in the ic form of the free base, with trifluoro- acetyi anhydride in the pre sence of an organic solvent . The Schiff base can be yl obtained by reacting daunomycin hydrochloride with sali ^aldehyde.

The reaction with an alkali metal acetate, such as potas sium acetate, is preferably carried out in the pre sence of a polar solvent, such as acetone, in the warm for a short time or in the cold for a protracted reaction time.

The alkaline hydrolysis of the 14-bromo derivative may be carried out by treating the 14- bromo derivative directly with alkali .

When daunomycin having the amino group of the amino sugar protected with a protecting group is used, the said protecting group is generally eliminated before separating the final product.

The elimination of the salicylideiBgroup can be carried out by e acid hydrolysis of N- salicyliden/adr iamycin. The elimination of the trifluoro-acetyl group can be carried out after treating N-trifluoro- acetyl- adriamycin with ethyl orthoformate and . p. toluene- sulphonic acid in order to obtain N-trifluoroacetyladriamy - 9, 14- ethylorthoformate from which the trifluoroacetyl group is subsequently eliminated by alkaline hydrolysis . The a¾amycin 9, 14- ethylorthoformate, so obtained, gives the free adiiamycin on acid hydrolysis . Adriamy cin and adriamy cinone obtained according to the proce s s of the invention can be isolated and purified by extraction and purification. The invention include s a pharmaceutical compos ition which contains adriamycin or a salt chrrivatrre thereof according to the invention in admixture^ with an acceptable diluent or carrier.

The following Examples illustrate the invention.

EXAMPLE 1 ADRIAMYCIN 1.30 g of daunomycin hydrochloride in 30 ml of methyl alcohol and 100 ml of anhydrous dioxan are treated with 3.3 ml of a bromine solution in chloroform (lOg of bromine in 100 ml of chloroform). The reaction mixture is allowed to stand for 4 hours at room temperature, evaporated to dryness under reduced pressure, and the residue is taken up with chloroform ( 5 to 10 ml ) 3 to 5 volumes of diethyl ether are added. Bromo- daunomycin hydrochloride precipitates ( 1.10 g) and on recry stallisation from chloroform-methanol melts at 177° - 178° C A solution of 1.0 g of this 14-bf omo-daunomycin hydrochloride in 150 ml of methyl alcohol is treated with 70 ml of distilled water, mixed with 0.1 N sodium hydroxide to bring it up to pH 10.3 and maintained at such pH for 20 minutes. The whole operation is carried out under a nitrogen atmosphere. The solution is di.ul d with distilled water and extracted many times with chloroform until all the coloured material has passed into the solvent. The combined chloroform extracts are dried over anhydrous sodium sulphate, concentrated under reduced pressure to a small volume, mixed with 1.8 ml of 0.6 N hydrochloric acid in anhydrous methanol and treated with 10 volumes of diethyl ether.

An amorphous precipitate ( 0. 72 g ) is obtained which, crystallized from methanol-propanol, give s 0. 45 g of ¾¾iamycin hydrochloride melting at 205^ - 210 °C (with decomposition).

EXAMPLE 2 ADRIAMYCIN A suspension of 6. 60 g of 14-bromo- daunomycin hydrochloride, prepared according to Example 1 , in 200 ml of anhydrous acetone is mixed with 1. 8 g of melted and pulverized potas sium acetate .

The suspension is refluxed for 45 minutes, filtered and the filtrate is evaporated to dryness under reduced pressure. The residue is taken up with chloroform and treated with diethyl ether. The precipitate ( 0. 45 g ) is purified by filtration through a column of silicic acid using a methylene dichloride: methanol : water ( 100 : 20 : 2 by volume ) solvent system. 150 mg of 14 -acetoxy-daunomycin melting at 196" - 198 ° C are obtained. A solution of 0. 10 g of this 14-acetoxydaunomycin in 30 ml of acetone-methanol ( 2 : 1 ) is mixed with 10 ml of a 5 % sodium bicarbonate solution. The mixture is allowed to stand for three hour s at room temperature and is then repeatedly extracted with chloroform after diluting with water. The chloroformic extracts are combined, dried over anhydrous sodium sulphate, and concentrated to small volume under reduced pres sure. The concentrated solution is mixed with 0. 15 ml of 0. 6 N hydrochloric acid in anhydrous methanol and then with three volumes of diethyl ether. The amorphous precipitate is pas sed through a column filled with 40 g of cellulose powder.

It is eluted with a propanol : ethyl acetate ; water ( 7 : 1 : 2 by volume ) solvent system. The fractions containing adriamycin are combined and evaporated to dryness. 43 mg of adriamycin hydrochloride melting at 204° - 210° C ( with decomposition) are obtained.

EXAMPLE 3 ADRIAMYCINONE 1 g of daunomycinone are dissolved in 100 ml of chloroform and "mixed with 6.75 ml of a bromine solution in chloroform (2 ml of bromine dissolved in 100 ml of chloroform ). After one night at room temperature the crystalline product formed is filtered off and recrystallised from ethyl acetate. 1 g of 14-bromo- daunomycinone melting at 220° - 225° C ( with decomposition ) are obtained ; Γ 1 20 ° = + 165° ( c = 0.1 in dioxan). 0.6 g of 14-bromo-daunomycinone are suspended in 300 ml of anhydrous acetone and mixed with 1.3 g of melted potassium acetate. The mixture is refluxed for 30 minutes, filtered and evaporated to dryness under reduced pressure. The residue is crystallised from ethyl acetate to give 0.5 g of 14-acetoxy-daunomycinone which melts at 242° - 245°C; |^a] 20° = + 192° (c = 0.1 in dioxan ). 0.1 g of this 14-acetoxy-daunomycinone are dissolved in acetone and mixed with 10 ml of a 10% aqueous solution of sodium bicarbonate. After 3 hours at" room temperature the reaction mixture is extracted with chloroform. The chloroformic extract is dried, concentrated to small volume under reduced pressure, chromatographed through a column of silica gel, and eluted with chloroform containing 2 % of ethanol to remove any starting material still pre sent. The. adriamycinone is eluted with chloroform which contains 5 % of ethanol. 50 mg of product, which on crystallisation from ethyl acetate melts at ° 228 ° - 230 ° C, are obtained ; [ « ] = + 1 56 ° ( c = 0. 1 in dioxan ).

D EXAMPLE 4 ADRIAMYCINONE 0. 1 g of the 14-bromo- daunomycinone obtained according to Example 3 by treatment with 10 ml of 0. 1 N soda at 0 ° C for minutes are converted into adriamycinone which is isolated by chromatogr-jphy through a column of silica gel.

EXAMPLE 5 ADRIAMYCIN 4. 6 g of daunomycin free base in 390 ml of chloroform and 155 ml of anhydrous diethyl ether are treated with 9. 3 ml of tr ifluoroacetic anhydride. The reaction mixture is allowed to stand at room temperature for 30 minute s, is extracted with water, and the organic phase is evaporated to dryne s s under vacuum. The re sidue is taken up with 100 ml of anhydrous methanol and is refluxed for 10 minutes . The methanol solution is distilled to drynes s and the re sidue crystallised from tetrahydrofuran-petroleum ether gives 4. 5 g of N-trifluoroacetyl -daunomycin melting at 16° ° - 171 ° C. 0. 6 g of N-trifluoroacetyl- daunomycin in 30 ml of tetrahydrofuran and 20 ml of anhydrous methanol are mixed with 0. 50 g of iodine and with 0. 50 g of pulverized calcium oxide. The mixture is shaken under a nitrogen atmosphere for five hour s, is filtered and the filtrate, diluted with chloroform, is shaken with water containing hydrochloric acid to make it acid to litmus. The organic phase is evaporated to dryne s s under vacuum. The re sidue is taken up with little chloroform and chromatographed through a column by elution with chloroform containing increasing quantities of ethyl acetate . The product is eluted when the concentration of the ethyl acetate is about 10 % . 0. 25 g of 14- iodo- N-trifluoroacetyl - daunomycin melting at 172 ° - 175 ° C are obtained. 0. 8 g of this compound are refluxed for 15 minute s with 60 ml of anhydrous acetone in the pre sence of 1. 50 g of melted and pulverized potas sium acetate . The mixture is filtered and the filtrate is diluted with chloroform and shaken with water made slightly acid to litmus with dilute hydrochloric acid. The organic phase is evaporated almost to dryne ss under vacuum and petroleum ether is added to the residue . 0. 62 g of 14- acetoxy- N-trifluoro-acetyl- daunomycin melting at 150 ° - 153 ° C are obtained by precipitation. 1. 4 g of this product dis solved in a mixture of methanol ( 50 ml ) and acetone ( 100 ml ) are mixed with 100 ml of a 5 % aqueous sodium bicarbonate solution. The mixture is maintained at room temperature and under a nitrogen atmosphere for 3 hours . The reaction mixture is poured into water, acidified with aqueous tartaric acid to a pH of from 5 to 6, and extracted many time s with a total of 350 ml of chloroform. The organic phases combined and evaporated under reduced pressure give a solid re sidue ( 0. 95 g ) which is taken up with chloroform and chromatographed. N-trifluoroacetyl-adriamycin, thus obtained, is eluted with chloroform which contains 15 % of ethyl acetate. The combined and evaporated eluates give 0. 56 g of the desired product which melts at 174 ° - 176 ° C 0. 45 g of this N-trifluoroacetyl-adriamycin in 80 ml of dioxane are mixed with 10 ml of ethyl orthoformate and 3 mg of p. -toluene- sulphonic acid. After 48 hours at room temperature the reaction mixture is diluted with chloroform and washed with water by strongly shaking in a separating funnel. The organic phase is evaporated to a small volume and gives a precipitate ( 35 g ) on addition of petroleum ether. This product is chromatographed through a column of silicic acid ( 7 g ) by elution with chloroform containing 10 % of ethyl acetate. The evaporated eluate gives 80 mg of 9, 14 - N - trifluoroacetyl-adriamycin ethyl-chloroformate which melts at 162 ° - 164° C. 0. 20 g of this product are dis solved in a mixture of acetone ( 10 ml ) with 0. 1 N sodium hydroxide ( 40 ml ) under a pure nitrogen atmosphere. After 20 minutes at room temperature the pH is adjusted to 8. 6 with 1 N hydrochloric acid and the solution is extracted many times with 50 ml of chloroform until the extracts become coloured. The combined chloroformic extracts are evaporated to small volume and pas sed through a column containing 4 g of silicic acid. The elution is performed with a methylene dichloride : methanol : water ( 50 : 10 : 1 by volume ) solvent mixture. By evaporation of the eluate, 0. 1 1 g of adriamycin 9, 14-ethylorthoformate melting at 203 ° - 206 ° C are obtained. 0. 10 g of this product in 9 ml of acetone are mixed with 1 ml 1 N hydrochloric acid. The mixture is allowed to stand at room temperature for 20 hours. The precipitate formed, crystallised from methanol-propanol, give&40 mg of adriamycin hydrochloride melting at 204° - 210 ° C (with decomposition ).

EXAMPLE 6 ADRIAMYCIN 1. 00 g of daunomycin hydrochloride in 30 ml of distilled water are mixed with 0. 1 N sodium hydroxide to a pH of 8. 6 and then are yi treated with 10 ml of a 10 % saliocddehyde solution in methanol.

The precipitate is taken up and washed with water, crystallised e from ethanol and 0. 97 g of salicyliden daunomycin, melting at 168 ° - 170 ° C, are obtained. e 0. 50 g of this salicyliden/daunomycin in 20 ml of tetrahydrofur are mixed with 10 ml of anhydrous methanol, 1. 0 g of iodine and 1. 0 g of calcium oxide. The mixture is refluxed slowly for 40 minutes, filtered and evaporated to dryness under reduced pres sure. The residue is taken up with chloroform ( 22 ml ).

The chloroformic solution is washed with water by shaking, dried, concentrated to a small volume and mixed with petroleum ether to obtain a complete precipitation. 0. 41 g of crude 14- iodo- salicyliden daunomycin are obtained. The product is taken up with ml of anhydrous acetone, treated with 0. 80 g of melted and pulverized potas sium acetate and refluxed for 20 minute s . The suspension is cooled, filtered and the filtrate is evaporated to drynes s under vacuum. The re sidue consisting of 14- acetoxy- e salicyden daunomycin is dissolved in a mixture of equal parts of methanol and acetone and mixed with 18 ml of a 5 % aqueous sodium bicarbonate solution. The reaction mixture is extracted with chloroform after 3 hour s at room temperature. The chloroformic extract is concentrated to about 10 ml and chromatographed through a column of 3 g of silicic acid. The solution is eluted with a methylene dichloride-petroleum ether -methanol ( 100 : 50 : 5 by volume ) solvent mixture. The fractions containing the starting material and impurities are discarded. The later ones are collected and by evaporation give 65 mg of salicyliden-adriamycin, melting at 1 94 °C ( with decomposition ). . 20 g of salicyliden- adriamycin in 20 ml of chloroform are extracted three time s with 10 ml of 0. 1 N of hydrochloric acid each time. The coloured product is all transferred into the aqueous phase which, under a nitrogen atmosphere is made alkaline to pH 8. 6 with 0. 1 N sodium hydroxide and extracted with chloroform until the extracts become colourless . The extracts are combined, dried over anhydrous sodium sulphate, concentrated under reduced pres sure to about 5 ml, and mixed with 0. 5 ml of 0- 6 N hydrochloric acid and 10 volume s of anhydrous diethyl ether. 0. 12 g of adriamycin hydrochloride melting at 205° - 210° C ( with decomposition ) are obtained.

Claims (12)

HAVING NOW particularly described and ascertained the nature of our said invention and in what manner the same is to be performed, we declare that what we claim is

1. A process of preparing adriamycin or a non-toxic pharmaceutically acceptable acid addition salt thereof or its aglycone adriamycinone in which daunomycin or a suitably protected derivative thereof or it&aglycone daunomycinone is reacted with bromine or iodine, the 14-halogen derivative so obtained is reacted with an alkali metal acetate, the 14-acetoxy derivative so obtained is subjected to alkaline hydrolysis, any protective group is eliminated, and where appropriate the resulting 14-hydroxy derivative is converted into a salt.

2. A proces s according to claim 1 in which the reaction with bromine or iodine is carried out in the presence of an inert organic solvent.

3. A process according to claim 1 or claim 2 in which the initial reaction is with iodine and carried out in the pre sence of a base.

4. A proces s according to claim 3 in which the base is calcium oxide.

5. A proces s according to any of the preceding claims in which the reaction with bromine or iodine is carried out under an atmosphere of an inert gas.

6. A proces s according to any of the preceding claims in which daunomycin has the amino group of the sugar protected with an acyl derivative or a Schiff base. 31966/2

7. » A process according to claim 6 in which the acyl derivative is a trifluoro-acetyl derivative or the Sehiff base is formed with salicaldehyde* ,

8. A process according to any of the preceding claims in which the reaction with an alkali metal acetate is carried out in the presence of a polar aiLvent·

9. * A process according to claim 8 in which the solvent is acetone*

10. · A process according to any of the preceding claims in which the alkaline hydrolysis is carried out by treating directly with alkali*

11. * A process as claimed in claim 1 of preparing adrlamycin or adriamycinone eubstantially as hereindescribed in any of the Examples* 12* A driamycin or adriamycinone prepared by a process according to any of the preceding claims*

12. A pharmaceutical composition which contains adriamycin or a salt thereof according to claim 12 in admixture with an acceptable diluent or carrier. COHEN ZEDEI'v & SPISBACH Regd. Patent Attorneys P. O. Box 33116, TEL-AVIV, ISRAEL