GB2032420A

GB2032420A - New Daunorubicin Derivatives

Info

Publication number: GB2032420A
Application number: GB7923301A
Authority: GB
Original assignee: Rhone Poulenc Industries SA
Current assignee: Rhone Poulenc Industries SA
Priority date: 1978-07-07
Filing date: 1979-07-04
Publication date: 1980-05-08
Also published as: NL7905098A; FR2430425B1; SE7905940L; IT7924158A0; IT1122046B; CH641190A5; GB2032420B; CA1118414A; DE2927452A1; BE877555A; JPS5511593A; FR2430425A1

Abstract

Daunorubicin derivatives of the general formula: <IMAGE> (wherein R1 represents a group of the general formula: <IMAGE> in which (i) X1 and X2 both represent oxygen atoms or both represent sulphur atoms and the symbols R3 each represent a straight- or branched-chain alkyl radical containing 1 to 4 carbon atoms, a phenyl radical or a phenyl radical substituted in the para-position by a methyl, methoxy or methylthio radical, or together form an alkylene radical containing 2 to 4 carbon atoms, or (ii) one of X1 and X2 represents an oxygen atom and the other represents a sulphur atom and the symbols R3 together form an alkylene radical containing 2 to 4 carbon atoms; and R2 represents a group of the general formula: <IMAGE> wherein R4 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 antitumoral properties and can be incorporated into pharmaceutical compositions.

Description

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

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

wherein R, represents a group of the general formula:

in which (i) X1 and X2 both represent oxygen atoms or both represent sulphur atoms and the symbols R3 each represent a straight- or branched-chain alkyl radical containing 1 to 4 carbon atoms, a phenyl radical or a phenyl radical substituted in the para-position by a methyl, methoxy or methylthio radical, or together form an alkylene radical containing 2 to 4 carbon atoms, or (ii) one of X1 and X2 represents an oxygen atom and the other represents a sulphur atom and the symbols R3 together form an alkylene radical containing 2 to 4 carbon atoms, and R2 represents a group of the general formula::

wherein R4 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, preferably hydrochlorides, thereof.

It is understood that the abovementioned daunorubicin derivatives of general formula I can be derived, as required, from the D, L and DL forms of the amino-acid of the general formula:

wherein R4 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 IV, in which the amino radical has been protected beforehand, with a daunorubicin derivative of the general formula:

(wherein R1 is as hereinbefore defined) or an acid addition salt (e.g. hydrochloride) thereof, and then removing the group protecting the amino radical from the resulting product by methods known per se. 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 IV can be protected by any method known per se for biocking 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 IV 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 IV can be activated by the formation of a mixed anhydride by reacting the amino-acid with, for example, ethyl, isobutyl or sec.-butyl chloroformate.

When the amino-acid of general formula IV 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 IV with the daunorubicin derivative of the general formula V 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 nitrogen.

The removal of the group protecting the amine function 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.

(b) The acid group of the amino-acid of general formula IV 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 IV 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 V is advantageously carried out in an organic solvent, such as ethyl acetate or dimethyl-formamide, 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 V can be obtained in accordance with the method described in British Patent 1525180.

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 daunorubicin derivatives of general formula 1, and their acid addition salts, possess valuable antitumoral properties combined with a low toxicity.

They have proved to be particularly active against graftable tumours in mice, in particular at doses between 0.20 and 20 mg/kg animal body weight, administered intraperitoneally, against leukaemia L 1210.

Their maximum tolerated dose, determined on mice, is between 0.5 and 30 mg/kg animal body weight, administered intraperitoneally.

The daunorubicin derivatives of general formula I of particular interest are those wherein R1is a group of general formula II wherein (i) X1 and X2 represent oxygen atoms and the symbols R3 each represent an alkyl radical containing 1 to 4 carbon atoms, or (ii) Xt and X2 have the same or different significances and each represents an oxygen or sulphur atom and the symbols R3 together form an alkylene radical containing 2 to 4 carbon atoms, and R2 represents a leucyl radical.

Amongst such preferred products, N-(L-leucyl) 1 4-diethoxyacetoxydaunorubicin and N-(L leucyl)-1 4-(1 ,3-dithiolan-2- yl)carbonyloxydaunorubicin are especially active.

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, salicylates, phenolphthalinates 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 Examples illustrate the preparation of the daunorubicin derivatives of the present invention.

Example 1 In a dry three-necked round-bottomed flask L N-[2-(biphenyl-4-yl)prop-2-yl]oxycarbonyl- leucine (7.66 g) is dissolved, under a nitrogen atmosphere, in tetrahydrofuran (100 cc), and triethylamine (2.9 cc), which has been kept over potassium hydroxide pellets, is added. The mixture is cooled to -200C and isobutyl chloroformate (2.48 cc) is added all at once. The mixture is stirred at -200C for 30 minutes. A solution of 1 4-diethoxyacetoxydaunorubicin hydrochloride (12.3 g) in dry methylene chloride (200 cc), containing triethylamine (2.43 cc), which has been kept over potassium hydroxide pellets, is added all at once whilst keeping the temperature of the reaction mixture at 200 C.

The mixture is stirred for 1 5 minutes at -200C.

Ice-cooled water (1000 cc) is then added, the mixture is separated by decantation and the organic phase is washed with water (3x200 cc).

The organic phase is dried over sodium sulphate, filtered and the filtrate concentrated to dryness under reduced pressure (20 mm Hg) at 300 C. The residue is dissolved in the minimum amount of methylene chloride and the solution is poured onto a column (diameter 5 cm, height 60 cm) containing silica gel (450 g) in chloroform. Elution is carried out successively with methylene chloride (4000 cc) containing ethyl acetate (10% by volume), and then with methylene chloride (5100 cc) containing ethyl acetate (25% by volume), the eluate being collected in fractions (300 cc).

Fractions 14 to 30 are combined. They are concentrated to dryness under reduced pressure (20 mm Hg) at 300C and the residue is then dried under reduced pressure (1 mm Hg) at 200C.

N-[N-{2-(Biphenyl-4-yl)prop-2-yl]oxycarbonyl I- L-leucyl]-1 4-diethoxyacetoxydaunorubicin (11.3 g) is thus obtained in the form of an amorphous red solid: [o2O=+1 26t 1 60(c=0. 182, chloroform).

N-[N-{ [2-(Biphenyl-4-yl)prop-2-yljoxycarbonyl I- L-leucyl]-1 4-diethoxyacetoxydaunorubicin (11.3 g) is dissolved in methylene chloride (130 cc). The mixture is cooled to +4"C by means of an ice/water bath. A solution (0.77N; 25.7 cc) of dry hydrogen chloride in dioxan is added all at once, whilst stirring. After 10 minutes, ethyl acetate (300 cc) and diethyi ether (500 cc) are added. The resulting red precipitate is filtered off, washed with diethyl ether and dried under reduced pressure (1 mm Hg) at 200C. The red powder is suspended in ethyl acetate (100 cc). The suspension is stirred for 1 5 minutes and filtered.

The resulting red solid is washed with ethyl acetate (3 x 100 cc) and dried under reduced pressure (1 mm Hg) at400C.

N-(L-leucyl)-1 4-diethoxyacetoxydaunorubicin (6.62 g) is obtained in the form of the hydrochloride which is a red powder: [a]020=+255+180 (c=0.216, methanol) Rf=0.66 silica gel; methylene chloride/methanol/formic acid/water (88/1 5/2/1 by volume)] Analysis % Calculated C 56.90 H 6.24 Cl 4.31 N 3.40 % Found C 56.4 H 6.5 Cl 4.4 N 3.5 Example 2 In a dry three-necked round-bottomed flask, L N-[2-(biphenyl-4-yl)prop-2-yl]oxycarbonyl leucine (1.33 g) is dissolved, under an argon at mosphere, in anhydrous tetrahydrofuran (120 cc), and triethylamine (0.505 cc), which has been kept over potassium hydroxide pellets, is then added.

The mixture is cooled to -200C and isobutyl chloroformate (0.426 cc) is added rapidly. The mixture is stirred for 30 minutes at -200C.

Methylene chloride (240 cc), triethylamine (0.42 cc), which has been dried over potassium hydroxide, and 14-(1 ,3-dithiolan-2-yl)- carbonyloxydaunorubicin hydrochloride (2.14 g), in the pulverulent form, are then added to the white suspension. The mixture is stirred at -200C for 45 minutes. The reaction mixture is then poured into ice-cold water (300 cc). The mixture is separated by decantation and the organic phase is washed with hydrochloric acid (0.05N; 2x250 cc) and then with water (3x250 cc). The organic solution is dried over sodium sulphate, filtered and the filtrate is concentrated to dryness under reduced pressure (20 mm Hg) without exceeding 300C.The solid residue is dissolved in a mixture of methylene chloride containing ethyl acetate (25% by volume) and the solution is chromatographed on Merck silica (0.04-0.063 mm; 140 g) under nitrogen (0.5 bar).

Elutiori is followed by thin later chromatography and the eluate corresponding to the pure product is concentrated to dryness under reduced pressure (20 mm Hg) at a temperature of about 300C. After drying at 200C under reduced pressure (1 mm Hg), N-[N-([2-(biphenyl-4-yl)- prop-2-yljoxycarbonyl }-L-leucyl]- 1 4-( 1,3- dithiolan-2-yl)-carbonyloxydaunorubicin (2.44 g) is obtained in the form of a red powder.

N-[N-{ [2-(Biphenyl-4-yl)prop-2- yl]oxycarbonyl}-L-leucyl]- 14-(1,3-dithiolan-2- yl)carbonyloxydaunorubicin (2.44 g) is dissolved in anhydrous methylene chloride (150 cc). The solution is cooled to 50C and a solution (0.77N; 6.88 cc) of dry hydrogen chloride in dioxan is then added rapidly. The mixture is stirred for 10 minutes and the preciptate which has formed is then filtered off. The solid is washed with methylene chloride (100 cc). The insoluble material is dissolved in anhydrous methanol (50 cc) and then reprecipitated by adding diethyl ether (150 cc). After filtration, washing with diethyl ether and drying under reduced pressure (1 mm Hg), N-(L leucyl)-1 4-(1 ,3-dithiolan-2- yl)carbonyloxydaunorubicin hydrochloride (1.3 g) is obtained.

Rf: 0.57 [silica gel; methylene chloride/methanol/formic acid/water (88/15/2/1 by volume)] Analysis % Calculated C 53.84 H 5.50 Cl 4.30 N 3.39 S 7.77 % Found C 53.9 H 5.4 Cl 4.9 N 3.2 S7.6 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 wtih a compatible pharmaceutical carrier, which may be inert or physiologically active. The compositions may be in any of the forms appropriate for the envisaged methods 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 adjuvant such as preserving, wetting, emulsifying and dispersing agents. They may be sterilized by, for example, filtration through a bacteria-retaining 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 some other sterile injectable medium.

The daunorubicin derivatives of general formula I and their non-toxic acid addition salts are, more particularly, used in the treatment of acute lymphoblastic and myeloblastic leukaemia, lymphosarcoma, Hodgkin's disease and solid tumours (lung cancer, breast cancer, cancer of the digestive tract and metastasis) at daily doses which are generally between 1 and 5 mg/kg body weight, administered intravenously, in the case of an adult. In general the physician will decide the posology considered appropriate, taking into account the age, weight and other factors intrinsic to the patient being treated.

The following Example illustrates pharmaceutical composition according to the invention.

Example 3 A solution containing N-(L-leucyl)-1 4- diethoxyacetoxydaunorubicin hydrochloride (27.9 mg/cc) is prepared by dissolving this product (4.185 g) in a sufficient amount of a nonpyrogenic physiological solvent to obtain 1 50 cc.

The resulting solution is divided aseptically between ampoules in an amount of 3 cc per ampoule. The ampoules are sealed and each contains 80 mg of N-(L-leucyl)-14diethoxyacetoxydaunorubicin (base).

Claims

1. Daunorubicin derivatives of the general formula:

wherein R, represents a group of the general formula:

in which (i) X1 and X2 both represent oxygen atoms or both represent sulphur atoms and the symbols R3 each represent a straight- or branched-chain alkyl radical containing 1 to 4 carbon atoms, a phenyl radical or a phenyl radical substituted in the para-position by a methyl, methoxy or methylthio radical, or together form an alkylene radical containing 2 to 4 carbon atoms, or (ii) one of X, and X2 represents an oxygen atom and the other represents a sulphur atom and the symbols R3 together form an alkylene radical containing 2 to 4 carbon atoms, and R2 represents a group of the general formula::

wherein R4 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 R, is a group of general formula II wherein (i) X, and X2 represent oxygen atoms and the symbols R3 each represent an alkyl radical containing 1 to 4 carbon atoms, or (ii) X and X2 have the same or different significances and each represents an oxygen or sulphur atom and the symbols R3 together form an alkylene radical containing 2 to 4 carbon atoms, and R2 represents a leucyl radical, and acid addition salts thereof.

3. N-(L-Leucyl)- 1 4-diethoxyacetoxy- daunorubicin and acid addition salts thereof.

4. N-(L-Leucyl-14-(1,3-dithiolan-2-yl)- carbonyloxydaumorubicin and acid addition salts thereof.

5. A 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 R4 is as defined in claim 1). in which the amino radical has been protected, with a daunorubicin derivative of the general formula:

(wherein R1 is as defined in claim 1) or an acid addition salt thereof, and then removing the group protecting the amino radical from the resulting product by methods known per se.

6. A process according to claim 5 in which the reactive derivative of the amino-acid is a mixed anhydride.

7. A process according to claim 5 in which the reactive derivative of the aminoacid is an ester.

8. A process according to claim 5, 6 or 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 or trityl radical.

9. A process according to any one of claims 5 to 8 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.

10. A process for the preparation of daunorubicin derivatives of the general formula depicted in claim 1, and acid addition salts thereof, substantially as herein before described with especial reference to Example 1 or 2.

11. A daunorubicin derivative of the general formula specified in claim 1 and acid addition salts thereof when prepared by the process claimed in any one of claims 5 to 10.

12. 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.

13. Pharmaceutical compositions according to claim 12 in a form suitable for parenteral administration.

14. Pharmaceutical compositions according to claim 12 substantially as hereinbefore described with especial reference to Example 3.

1 5. A daunorubicin 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.