GB2033393A

GB2033393A - New Daunorubicin Derivatives

Info

Publication number: GB2033393A
Application number: GB7937184A
Authority: GB
Original assignee: Rhone Poulenc Industries SA
Current assignee: Rhone Poulenc Industries SA
Priority date: 1978-10-27
Filing date: 1979-10-26
Publication date: 1980-05-21
Also published as: DE2943438A1; FR2439788A1

Abstract

Daunorubicin derivatives of the general formula: <IMAGE> wherein the symbols R1 have the same or different significances and each represents a hydrogen atom or a methyl or ethyl radical, and the symbol R2 represents a hydrogen atom, a straight- or branched-chain alkyl radical containing 1 to 4 carbon atoms, or a phenyl or benzyl radical, are new therapeutically useful compounds possessing anti-tumoral- properties.

Description

SPECIFICATION New Daunorubicin Derivatives This invention relates to new derivatives of daunorubicin, to a process for their preparation and pharmaceutical compositions containing them.

The new daunorubicin derivatives of the present invention are those compounds of the general formula:

wherein the symbols R1 have the same or different significances and each represents a hydrogen atom or a methyl or ethyl radical, and the symbol R2 represents a hydrogen atom, a straight- or branchedchain alkyl radical containing 1 to 4 carbon atoms, or a phenyl or benzyl radical, and acid addition salts, preferably hydrochlorides, thereof.

It is to be understood that the above-mentioned daunorubicin derivatives of general formula I can be derived, as required, from the D, Land DL forms of the amino-acid of the general formula:

wherein R2 is as hereinbefore defined.

According to a feature of the invention, the compounds of general formula I are prepared by the process which comprises reacting a reactive derivative of an amino-acid of general formula II, in which the amino radical has been protected beforehand, with.a daunorubicin derivative of the general formula:

(wherein each of the symbols R', represents a hydrogen atom, a methyl or ethyl radical, or a radical protecting the acid group, preferably the tert.-butyl radical), or an acid addition salt (e.g. hydrochloride) thereof, and subsequently removing by methods known per se from the resulting product the group protecting the amino radical of the amino-acid reactant of general formula II and, when appropriate, any protective group(s) R'1.By the term "methods known per se" as used in this specification is meant methods heretofore used or described in the chemical literature.

The amino radical of the amino-acid of general formula II can be protected by any method known per se for blocking the amino radical of an amino-acid, and of which the blocking and unblocking conditions are compatible with the stability of the molecule.

The acid group of the amino-acid of general formula II can be activated either in the form of a mixed anhydride or in the form of a reactive ester.

(a) The acid group of the amino-acid of general formula II can be activated by the formation of a mixed an hydroxide by reacting the amino-acid with, for example, ethyl, isobutyl or sec.-butyl chloroformate.

When the amino-acid of general formula II is used in the form of a mixed anhydride of this kind (which can be prepared in situ), the amino radical is preferably protected beforehand by means of a [2 (biphenyl-4-yl)-prop-2-yl]oxycarbonyl radical which can be introduced in accordance with the method described by P. Sieber and B. Iselin, Helv. Chim. Acta., 51 622 (1968), and which can be readily removed in a dilute acid medium, in particular in the presence of hydrochloric acid.

The condensation of the mixed anhydride of the amino-acid of general formula II with the daunorubicin derivative of the general formula Ill is then advantageously carried out in an anhydrous medium in an organic solvent, such as methylene chloride or tetrahydrofuran, in the presence of a nitrogen-containing base, such as triethylamine or N-methylmorpholine, at a temperature between --200 and OOC. The reaction is preferably carried out under an inert gas (e.g. nitrogen or argon).

The removal of the group protecting the amino radical is preferably carried out in the presence of hydrochloric acid in an organic solvent, such as methylene chloride, at a temperature between 40 and 250C. When possible, removal of a protective group R'1 is effected simultaneously.

(b) The acid group of the amino-acid of general formula II can also be activated by esterification with hydroxylic compounds such as N-hydroxysuccinimide. The activated ester can be prepared in situ.

When the reaction is carried out in accordance with this variant, the amino radical of the aminoacid of general formula II can be protected by means of a group, such as the trityl radical, which can be removed in a dilute acid medium.

In these circumstances, the condensation reaction of an activated ester with a compound of general formula Ill is advantageously carried out in an organic solvent, such as ethyl acetate or dimethylformamide, in the presence of a carbodiimide, such as N,N'-dicyclohexylcarbodiimide, at a temperature between 150 and 250C, optionally in the presence of an organic base such as triethylamine.

The starting materials of general formula III, wherein R'1 is as hereinbefore defined, can be prepared by reacting a quaternary ammonium phosphate of the general formula:

(wherein the symbols R'1 are as hereinbefore defined, and the symbols R3 R4 and Rs have the same or different significances and each represents an alkyl radical containing 1 to 4 carbon atoms) with 14bromodaunorubicin of the formula::

or an acid addition salt thereof, after which, when R', in general formula IV represents a protective radical, removing-if so desired-by methods known per se the protective radical in order to obtain a compound of general formula Ill in which at least one of the radicals R'1 is a hydrogen atom, or an acid addition salt thereof.

The reaction between the compounds of general formulae IV and V is generally carried out in an organic solvent such as a ketone (e.g. acetone), a nitrile (e.g. acetonitrile) or an amide (e.g.

dimethylformamide), at a temperature between 200 and 600C. The 1 4-bromodaunorubicin is preferably reacted in tlsss form of the hydrochloride with a tetramethylammonium phosphate conforming to general formula IV.

When R'1 in the reactants of general formulae Ill and IVis a protective radical (preferably the tert.butyl radical), it is generally removed-when so desired-by the action of hydrochloric acid and the reaction is carried out in a chlorine-containing solvent (e.g. methylene chloride) or in acetonitrile, at a temperature between 0 and 200 C. It is not obligatory to purify the product obtained before removing the protective radical.

The compounds of general formula IV can be prepared in accordance with the process described by R. Hazard et a/., C. R. Acad. Sci., 244, 1556 (1957), by P. Chabrier et awl., C. R. Acad. Sci., 244, 2730 (1957), by J. Cheymol et a/., C. R. Acad. Sci., 247, 1014 (1958) or by A. Zwierzak et a/., Tetrahedron, 27,3163(1971).

1 4-Bromodaunorubicin of formula V and its preparation have been described in French Patent Application 2331351.

The daunorubicin derivatives of general formula I obtained by the aforedescribed process can optionally be purified by physical methods such as crystallisation or chromatography; or by chemical methods such as the formation of acid addition salts, crystallisation of the salts and decomposition of them in an alkaline medium.

The daunorubicin derivatives of general formula I can be converted by methods known per se into acid addition salts, for example by reaction of the basic compounds with acids in appropriate solvents, for example alcohols, ethers, ketones or chlorinated hydrocarbons. The salt which is formed is precipitated if necessary after concentration of its solution, and is isolated by filtration or decantation.

The new daunorubicin derivatives of general formula I, and their acid addition salts, possess valuable anti-tumoral properties coupled with a low toxicity. Preferred compounds are those wherein R2 represents an alkyl radical containing 1 to 4 carbon atoms, and especially those such compounds wherein the symbols R1 both represent the ethyl radical, for example N-(L-leucyl)-14diethoxyphosphoryloxydaunorubicin and acid addition salts thereof.

They have proved particularly active against graftable tumours in mice at doses of between 0.4 and 1 5 mg/kg animal body weight, administered intraperitoneally, against leukaemia L 1210.

Their toxicity to mice, expressed as their 50% lethal dose (LD,,), is between 10 and 30 mg/kg animal body weight administered intraperitoneally.

For therapeutic purposes the daunorubicin derivatives of general formula I may be employed as such or in the form of non-toxic acid addition salts, i.e. salts containing anions which are relatively innocuous to the animal organism in therapeutic doses of the salts (such as hydrochlorides, sulphates, nitrates, phosphates, acetates, propionates, succinates, benzoates, fumarates, maleates, tartrates, theophyllineacetates, sa licylates, phenolphtha linates and methylene-bis-p-hydroxynaphthoates) so that the beneficial physiological properties inherent in the bases are not vitiated by side-effects ascribable to the anions.

The following Example illustrates the preparation of daunorubicin derivatives of general formula I by the process hereinbefore described for their preparation.

Example 1 In a dry three-necked round-bottomed flask L-N-! [2-(biphenyl-4-yI)prop-2-yljoxycarbonyl I- leucine (1.33 g) is dissolved in tetrahydrofuran (30 cc), under an argon atmosphere, and triethylamine (0.5 cc), which has been kept over potassium hydroxide pellets, is then added. The mixture is cooled to -200C and isobutyl chloroformate (0.43 cc) is added rapidly.The mixture is stirred for 30 minutes at 200 C. A solution of 1 4-diethoxy-phosphoryloxydaunorubicin hydrochloride (2.2 g) in anhydrous methylene chloride (50 cc) containing triethylamine (0.42 cc), which has been kept over potassium hydroxide pellets, is then added in the course of 5 minutes and whilst keeping the temperature of the reaction mixture at --200C. The mixture is stirred at -200C for one hour.

The reaction mixture is then poured into a stirred mixture of ice-cooled water (300 cc) and methylene chloride (300 cc); after decantation, the organic phase is washed with water (2x250 cc), 0.05 N hydrochloric acid (2x250 cc) and finally with water (2x250 cc).

The organic phase is dried over sodium sulphate. It is filtered and the filtrate is concentrated to dryness under reduced pressure (20 mm Hg; 2.7 kPa) without exceeding 300C. The residue is dissolved in the minimum amount of methylene chloride and this solution is poured onto a column containing silica gel (50 g) in methylene chloride. Elution is carried out with methylene chloride (2500 cc) in order to remove the less polar impurities, and then with methylene chloride (4000 cc) containing methanol (19/0 by volume).The second eluate is concentrated to dryness under reduced pressure (20 mm Hg; 2.7 kPa) at a temperature of about 30"C. After drying at 200C under reduced pressure (1 mm Hg; 0.13 kPa), N-[N-( [2-(biphenyl-4-yI)prop-2-yl]oxycarbonyl }-L-leucylj-1 4diethoxyphosphoryloxydaunorubicin (1.30 g) is obtained in the form of a red powder.

N-[N-i [2-( Biphenyl-4-yl)prnp-2-yljoxycarbonyl }-L-Ieucyl]- 1 4-diethoxyphosphoryloxydaunorubicin (1.30 g) is dissolved in anhydrous methylene chloride (20 cc). The solution is cooled in a bath of icecooled water, and a 0.77 N solution of dry hydrogen chloride in dioxan (2.68 cc) is then added rapidly.

After 30 minutes, ethyl acetate (200 cc) and diethyl ether (300 cc) are added. The red precipitate thus obtained is filtered off, washed with diethyl ether and dried at 400C under reduced pressure (1 mm Hg; 0.133 kPa). N-(L-leucyl)-1 4-diethoxyphosphoryloxydaunorubicin (0.620 g) is obtained in the form of the hydrochloride.

Rf: 0.41 [silica gel, methylene chioride/methanol/formic acid/water (88/15/2/1 by volume)] Analysis % calculated C 53.59 H 6.08 CI 4.27 N 3.39 P 3.73 % found C 52.8 H 5.9 Cl 4.5 N 3.4 P 3.6 The L-N-j[2-(biphenyl-4-yl)prop-2-yl]oxycarbonyl}-leucine can be obtained in accordance with the method described by P. Sieber and B. Iselin, Helv. Chim. Acta, 51, 622 (1968).

The 1 4-diethoxyphosphoryloxydaunorubicin hydrochloride can be prepared in the following manner.

A mixture of 1 4-bromodaunorubicin hydrochloride (15.8 g) and tetramethylammonium diethylphosphate (12.25 g) in dimethylformamide (1 litre) is stirred for 20 hours at 200C and in an anhydrous atmosphere.

An insoluble material is filtered off and washed with chloroform (3x30 cc) and the filtrate and the chloroform phases are then concentrated to dryness under reduced pressure (1 mm Hg) without exceeding 400 C.

The evaporation residue is taken up in chloroform (250 cc) and an insoluble material is filtered off.

The filtrate is concentrated to dryness under reduced pressure (20 mm Hg) at 300 C.

The pasty residue is taken up in a chloroform/ethyl acetate mixture (1/1 by volume) (700 cc) and extraction is then carried out with 0.2 N hydrochloric acid (4x400 cc).

The acid solutions are washed with ethyl acetate (4x700 cc) and then extracted with butanol (2 x 1500 cc).

The butanol extract is concentrated to dryness under reduced pressure (1 mm Hg) without exceeding 400C.

The solid thus obtained is dissolved in chloroform (300 cc); the solution is dried and filtered and the filtrate is evaporated under reduced pressure (20 mm Hg), at 400C.

The evaporation residue is taken up in ethyl acetate (1 litre), the mixture is filtered and the insoluble material is washed with diethyl ether.

After drying, 1 4-diethoxyphosphoryloxydaunorubicin hydrochloride (7 g) is obtained, which has the following characteristics: Rf: 0.30 [silica gel, methylene chloride/methanol/formic acid/water (88/15/2/1 by volume)] Analysis % calculated C 52.00 H 5.49 Cl 4.95 N 1.96 P 4.32 % found C 51.7 H 5.9 Cl 5.3 N 2.1 P 4.8 The present invention includes within its scope pharmaceutical compositions which comprise, as active ingredient, at least one daunorubicin derivative of general formula I, or a non-toxic acid addition salt thereof, in association with a compatible pharmaceutical carrier, which may be inert or physiologically active. The compositions may be in any of the forms appropriate for the envisaged method of administration. Parenteral administration, especially intravenous administration, is the preferred method.

The compositions according to the invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. These compositions can also contain adjuvants such as preserving, wetting, emulsifying and dispersing agents. They may be sterilized by, for example, filtration through a bacteriaretaining filter, by incorporation in the compositions of sterilizing agents or by irradiation. They may also be manufactured in the form of sterile solid compositions which can be dissolved or dispersed, at the time of use, in sterile water or any other injectable sterile medium.

The daunorubicin derivatives of general formula I and their non-toxic acid addition salts are, more particularly, useful in the treatment of acute lymphoblastic and myeloblastic leukaemias, lymphosarcoma, Hodgkin's disease and solid tumours (lung cancer, breast cancer, cancer of the digestive tract and metastases) at doses which are generally between 5 and 12 mg/kg body weight per day, administered intravenously, in the case of an adult.

The following Example illustrates pharmaceutical compositions according to the invention.

Example 2 A solution containing N-(L-leucyl)-1 4-diethoxyphosphoryloxydaunorubicin hydrochloride (27.8 mg/cc) is prepared by dissolving this product (4.178 g) in a sufficient amount of an apyrogenic physiological solvent to give 1 50 cc. The resulting solution is divided, under aseptic conditions, into ampoules at an amount of 3 cc per ampoule. The ampoules are sealed and each contains 80 mg of N (L-leucyl)- 1 4-diethoxyphosphoryloxydaunoru bicin (base).

Claims

1. Daunorubicin derivatives of the general formula:

wherein the symbols R1 have the same or different significances and each represents a hydrogen atom or a methyl or ethyl radical, and the symbol R2 represents a hydrogen atom, a straight- or branched- chain alkyl radical containing 1 to 4 carbon atoms, or a phenyl or benzyl radical, and acid addition salts thereof.

2. Daunorubicin derivatives according to claim 1 wherein R2 represents a straight- or branchedchain alkyl radical containing 1 to 4 carbon atoms, and acid addition salts thereof.

3. Daunorubicin derivatives according to claim 1 or 2 wherein the symbols R, both represent the ethyl radical, and acid addition salts thereof.

4. N-(L-Leucyl)- 1 4-diethoxyphosphoryloxydaunorubicin and acid addition salts thereof.

5. The hydrochloride of a daunorubicin derivative claimed in any one of claims 1 to 4.

6. Process for the preparation of a daunorubicin derivative as claimed in claim 1 which comprises reacting a reactive derivative of an amino-acid of the general formula:

(wherein R2 is as defined in claim 1), in which the amino radical has been protected before hand, with a daunorubicin derivative of the general formula:

(wherein each of the symbols R'1 represents a hydrogen atom, a methyl or ethyl radical, or a radical protecting the acid group), or an acid addition salt thereof, and subsequently removing by methods known per se from the resulting product the group protecting the amino radical of the amino-acid reactant of general formula II and, when appropriate, any protective group(s) R',.

7. A process according to claim 6 in which the reactive derivative of the amino-acid is a mixed anhydride.

8. A process according to claim 6 in which the reactive derivative of the amino-acid is an ester.

9. A process according to claim 7 in which the amino radical of the reactive derivative of the amino-acid is protected by the [2-(biphenyl-4-yl)-prop-2-yl]oxycarbonyl radical.

10. A process according to claim 8 in which the amino radical of the reactive derivative of the amino-acid is protected by the trityl radical.

11. A process according to any one of claims 6 to 10 followed by the step of converting a daunorubicin derivative of general formula I depicted in claim 1 thus obtained in the form of a free base into an acid addition salt.

12. A process for the preparation of daunorubicin derivatives of the general formula depicted in claim 1, and acid addition salts thereof, substantially as hereinbefore described with especial reference to Example 1.

13. A daunorubicin derivative of the general formula depicted in claim 1 and acid addition salts thereof when prepared by the process claimed in any one of claims 6 to 12.

14. Pharmaceutical compositions which comprise, as active ingredient, a daunorubicin derivative as claimed in any one of claims 1 to 4, or a non-toxic acid addition salt thereof, in association with a compatible pharmaceutical carrier which may be inert or physiologically active.

1 5. Pharmaceutical compositions according to claim 14 in a form suitable for parenteral administration.

16. Pharmaceutical compositions according to claim 14 substantially as hereinbefore described with especial reference to Example 2.

1 7. A duanorubicin derivative as claimed in any one of claims 1 to 4, or a non-toxic acid addition salt thereof, when used as a medicament and, more particularly, as an anti-tumour agent.