WO1992018520A1 - Novel cytarabine derivatives, their production and use - Google Patents

Abstract

The description relates to novel cytarabine derivatives of formula (I) in which R?1, R?2 and R?3 have the meanings given in the description and their production. The compounds are suitable for the treatment of diseases.

Description

 New cytarabirt derivatives, their production and use

The present invention relates to new cytarabine derivatives, their preparation and their use in combating diseases.

AraC (= cytarabine = 4-amino-1-ß-D-arabinofuranosyl-2 (1H) -pyrimidinone or 1-ß-D-arabinofuranosylcytosine, Merck index 11th edition, No. 2790) is a chemotherapy of Cancer-proven cytostatic. Due to the presence of cytosine deaminase in the body, however, the AraC is quickly deaminated and therefore ineffective. To achieve a therapeutic effect, it must therefore be applied in high doses, which are associated with unpleasant side effects for the patient. In addition, tumor cells lacking kinase activity do not phosphorylate AraC to the effective 5'-triphosphate and are therefore resistant to AraC.

In order to delay the too rapid enzymatic deamination, the amino group of the cytosine residue has been masked with acyl protective groups (Int. J. Cancer 37, 149 (1986)). However, the N ⁴ -acylAraC derivatives obtained in this way, even if they were applied in the form of liposomes, showed no improved cytostatic activity compared to underivatized AraC. The N ⁴ acylamide bond of these AraC prodrugs was only able to delay the enzymatic deamination in vivo for a short time.

Apart from this, N ⁴ -acyl-AraC is ineffective with AraC resistance.

Attempts were made to counteract the AraC resistance of tumor cells by covalently coupling AraC or AraC-5'-monophosphate to natural phopholipids (DE 83 00 2391). These AraC prodrugs were expected to be from

AraC-resistant tumor cells would be taken up and enzymatically cleaved inside the active AraC 5'-monophosphate. Prodrugs in which AraC or AraC-5'-monophosphate are present coupled to natural ester phospholipids and which were applied in the form of liposomes only partially met expectations. A disadvantage of these prodrugs is that the natural lipid building blocks used to construct the prodrugs are enzymatically cleaved too quickly, so that these phospholipidAraC prodrugs are metabolized too quickly to inactive derivatives.

New cytarabine derivatives have now been found which are more effectively protected against enzymatic deamination and which are suitable for the therapy of AraC-resistant tumor cells.

The invention relates to cytarabine derivatives of the formula I.

 wherein

R ^{1 is} a hydrogen atom, a C ₁₄ -C ₂₄ -alkyl radical which may optionally contain 1 to 2 double bonds and can be branched, an aliphatic C ₂ -C ₂₄ -acyl radical which may optionally contain 1 or 2 double bonds and can be branched, or the Acyl residue of benzoic acid or anisic acid and

R ² , R ³ , which may be the same or different, hydrogen atoms, C ₁ -C ₂₄ -alkyl radicals, which may be 1 to

Contain 2 double bonds and can be branched, aliphatic C ₂ - ₂₄ acyl radicals which may be 1 to

Contain 2 double bonds and may be branched, but with at most 2 of the radicals R ¹ to R ³

Can be hydrogen atoms and R ¹ ≠ H when R ² and R ^{3 are} acyl radicals. Suitable alkyl radicals for R ^{1 are} in particular hexadecyl and octadecyl.

If R ^{1 is} an acyl radical, the following radicals are preferred:

Palmitoyl, oleoyl, behenoyl.

For R ² and R ³ , unbranched alkyl and acyl radicals having 1 to 20 carbon atoms are preferred. The new cytarabine derivatives of the formula I can be prepared by a) preparing compounds of the formula II,

wherein R ¹ , R ² and R ^{3 have} the meaning given and R ^{4 represents} a 2- or 4-chlorophenyl radical, splits off the chlorinated phenyl radical R ⁴ or b) a compound of the formula III

where Ri, R ² and R ^{3 have} the meaning given,

oxidized. The chlorinated phenyl radical according to a) is cleaved particularly well if the compounds II are left to stand for 5 to 15 hours in an aqueous organic solvent at room temperature with tetrabutylammonium fluoride.

The oxidation of the compounds III is particularly successful at room temperature with iodine in organic aqueous solvents.

The reaction products thus obtained can be purified by chromatography.

The compounds of the formula II used as starting material can be obtained by condensation of compounds of the formula IV

wherein R ² , R ³ and R ^{4 have} the meaning given, with compounds of the formula V (R ¹ ≠ H)

produce with the aid of condensing agents in a manner known per se. The compounds of formula III are obtained by reacting a compound of formula VI

wherein R ² and R ^{3 have} the meaning given, with a compound of the formula V (R ¹ ≠ H) in the presence of an acid chloride and subsequent oxidation in a known manner. If one or two of the radicals R ¹ , R ² and R ³ are to be hydrogen, acyl radicals must be exchanged for hydrogen in a corresponding acyl compound.

The compounds IV, V and VI are known or can be prepared by known processes (Chem. Pharm. Bull. 26, 981 (1978), Nucleic Acid Res. Symposium Series No. 18, 189 (1987)). Furthermore, the amphiphilic properties can surprisingly be varied within wide limits by the number, length, type and position of the respective substituents in the AraC derivatives according to the invention. The aπφhiphilen AraC derivatives are thus soluble in aqueous buffer systems and / or dispersible in the form of liposomes. Depending on the amphiphilic property of the

AraC derivative, liposome formation can be achieved without and / or with other lipid components. All known liposome imaging methods can be used for liposome formation, such as, for example, ultrasound, gel chromatography, detergent dialysis. The lipophilic residues introduced in each case also decisively influence the size and stability of the liposomes which are formed from the respective amphiphilic AraC derivatives. Through the targeted introduction of lipophilic residues, not only can the amphiphilic character of the AraC derivatives according to the invention be specifically controlled, but surprisingly the cytostatic effect of AraC can also be decisively optimized. Like the AraC, the new compounds can even be used against malignant diseases of the hematopoietic cells, in particular against acute leukaemias and chronic myeloid leukemia in the blast episode.

The cytostatic effect of the amphiphilic AraC derivatives can surprisingly be used in so-called immunoliposomes for the targeted destruction of certain tumor cells. For this purpose, the amphiphilic AraC derivatives are dispersed together with other lipid components in the form of liposomes in physiological buffer systems. Monoclonal antibodies are immobilized on functional groups of the liposome membrane. The immunoliposomes thus obtained are preferably taken up in vitro by the tumor cells which express the antigen corresponding to the antibody. The result of this cell targeting is the selective destruction of the respective target tumor cell in a mixture of different cells.

The effect of the new compounds can be shown in the following experimental setup:

DBA / 2 mice were injected with L1210 tumor cells intravenously, simulating leukemia. On days 3 and 7 after the tumor cell injection, different doses of the test substance or solvent were administered to the tumor-bearing animals.

 The overall breakdown into the individual test groups was as follows:

Control group (solvent)

 i.v. Application (2 doses)

- i.p. Application (2 doses)

 i.v. AraC as reference (2 doses)

 i.p. AraC as reference (2 doses)

The median

Survival time of the individual test groups (10 animals per

Group).

In these experiments, the new compounds showed a better activity than AraC. The new compounds are said to be in a dosage of about

 40 - 1000 mg per patient per day.

example 1

Preparation of D, L-4- (1-Hexadecyl ami no) l-β-D-5'-0- (1,2-di-0-palmitoyl-glycero-3-phospho) arabinofuranosylcytosine a. Production of the starting material

5 g (10.7 mmol) of 4- (1-hexadecylamino) -1-ß-D-arabinofuranosylcytosine together with 6.8 g (10.7 mmol) of D, Ll, 2-di-0-palmitoyl-glycerol 3-hydrogenphosphonate in 50 ml of anhydrous pyridine is mixed with 6.6 ml (53.5 mmol) of pivalic acid chloride and stirred for 10 minutes at room temperature with exclusion of moisture.

 The solution was then concentrated in vacuo to the syrup, which was then spun down twice with about 40 ml of toluene.

Manufacture of the final product

The residue obtained according to a) was mixed with 200 ml of a 0.2 M iodine solution (tetrahydrofuran / pyridine / water, 90/5/5, V / V / V) and left for 40 min at room temperature. After

Oxidation, the reaction mixture was shaken with a mixture of 500 ml of chloroform and 500 ml of 2% aqueous NaHSO ₃ solution. The organic phase was dried over Na ₂ SO ₄ and concentrated in vacuo to the syrup.

The syrup was taken up with about 80 ml of chloroform and fractionated on a silica gel column in a chloroform / methanol gradient. The fractions of the desired product which left the column with chloroform / methanol (9/1, v / v) were combined, concentrated to dryness and then from methanol

crystallized. 3.7 g of a white powder were obtained, which had an R _F value of 0.46 on the silica gel plate in the chloroform / methanol system (7/3, v / v). Example 2

Preparation of D-4- (1-hexadecylamino) -1-β-D-5'-0- (1,2-di-0-palmitoyl-glycero-3-phospho) arabinofuranosylcytosine

Example 1 was repeated, but with D-1,2-di-O-palmitoyl-glycero-3-hydrogenphosphonate as the starting material. 4.4 g of product were obtained. Example 3

Preparation of D, L-4- (palmitoyl) -1-β-D-5'-0- (1,2-di-0-palmitoyl-glycero-3-phospho) arabinofuranosylcytosine a) Preparation of the starting material

8 g (16 mmol) of 4- (palmitoyl) -1-ß-D-arabinofuranosylcytosine together with 16 g (21 mmol) of 1,2 D, L-di-O-palmitoyl-glycero3- (2-chlorophenyl) phosphate in 50 ml of anhydrous pyridine are mixed with 11 g (36 mmol) of 2, 4, 6-triisopropylbenzenesulfonyl chloride and 9 ml of N-methylimidazole and shaken for 1 h at room temperature with exclusion of moisture. The mixture was then concentrated in vacuo to the syrup, which was then spun down twice with about 150 ml of toluene.

The residue was taken up in 200 ml of chloroform and fractionated on a silica gel column in a chloroform / methanol gradient. The fractions of the desired product which left the column with chloroform / methanol (95/5, v / v) were combined, concentrated to dryness and crystallized from methanol. They gave 9.5 g of a white powder.

Manufacture of the final product

The cleavage of the 2-chlorophenyl residue was isolated

Condensation product treated with an excess of 3 equivalents of tetrabutylammonium fluoride × 3H2O at room temperature for about 6 hours. A 0.05 M tetrabutylammonium fluoride solution in tetrahydrofuran / pyridine / water (8/1/1, V / V / V) was used for this. For the purification, the reaction mixture was fractionated on a silica gel column in a chloroform / methanol gradient. The fractions of the desired product were concentrated and crystallized from diethyl ether. 5 g of a white powder were obtained, which had an Rf value of 0.20 on the silica gel plate in the chloroform / methanol system (4/1, v / v).

Example 4 Preparation of D-4- (palmitoyl) -1-β-D-5'-0- (1,2-di-0-palmitoyl-glycero-3-phospho) arabinofuranosylcytosine

Example 3 was repeated, but with D-1,2-di-0-palmitoyl-glycero-3- (2-chlorophenyl) phosphate as the starting material. The yield was 5.9 g.

Example 5

Preparation of D, L-1-β-D-5'-0- (1-octadecyl-glycero-3-phospho) -arabinofuranosylcytosine a) Preparation of the starting material

By condensation of D, L-1-0-0ctadecyl-2-0-acetyl-glycero-3-hydrogenphosphonate (in analogy to Example 1) or of

 D, L-1-0-octadecyl-2-0-acetyl-glycero-3 (2-chlorophenyl) phosphate (in analogy to Example 3) with 4- (benzoyl) -1-β-D-arabinofuranosylcytosine was first D, L-4- (Benzoyl) -1-β-D-5'-0- (1-0-octadecyl-2-0-acetyl-glycero-3-phospho) arabinofuranosylcytosine synthesized and then chromatographed on silica gel. b) Manufacture of the final product

The condensation product obtained according to a) was kept closed with an excess of methanolic ammonia for about 24 hours at room temperature. The solvent was then removed in vacuo and the residue was fractionated on a silica gel column in a chloroform / methanol gradient. Fractions that the contained desired product were concentrated. The residue, which was crystallized from acetone / water, gave a white powder which had an R _F value of 0.19 in the chloroform / methanol system (1/1, v / v).

D-1-β-D-5'-0- (1-octadecyl-glycero-3-phospho) arabino-furanosylcytosine is obtained in an analogous manner

Example 6

Presentation of liposome preparations

For the preparation of the liposome dispersion were per ml

 Chloroform-methanol (1/1, v / v); each 100 mg soy phosphatidylcholine, 10 mg cholesterol, 1 mg α-tocopherol, 7 mg

N2-palmitoyl-N6-succinoyl-L-lysine and 12 mg of D, L-4- (palmitoyl) -1-β-D-5'-0- (1,2-di-0-palmitoyl) -glycero-3 -phospho) arabinofuranosylcytosine dissolved. 0.6 ml of this lipid stock solution was transferred in a test tube by blowing with air into a lipid film, which was then dried in vacuo for about 1 h at 50 ° C. The lipid film was mixed with 3 ml of 10 mM PBS (0.9% NaCl and 10 mM NaH ₂ PO ₄ , pH 7.3) and sonicated at 40 watts for 30 min with the aid of a microtip of a disintegrator. An opalescent liposome dispersion was formed, which was used for the following reaction.

Example 7

Representation of the immunoliposomes

1.2 nmol of an antibody was treated as a lyophilisate with 50 μl of the liposome preparation from Example 6 and 7 mg (27 μmol) of N (3-dimethylaminopropyl) -N'-ethyl-carbodiimide X HCl (EDC) and by adding 30 μl PBS (pH 1) adjusted to pH 4, 7 mg EDC were added twice at intervals of one hour. After stirring for about 5 hours at room temperature, the reaction mixture was applied to a ULTROGEL ACA 22 column and fractionated with PBS (pH 7.4). Fractions whose absorption ratios match the values of the liposome preparation used were pooled and used for cell targeting.

Claims

Claims Cytarabine derivatives of the formula I.

 wherein R ^{1 is} a hydrogen atom, a C ₁₄ -C ₂₄ alkyl radical, the optionally contain 1 to 2 double bonds and can be branched, an aliphatic C ₂ -C ₂₄ -acyl residue, which can optionally contain 1 or 2 double bonds and can be branched, or the acyl residue of benzoic acid or anisic acid and R ² , R ³ , which may be the same or different, hydrogen atoms, C ₁ -C ₂₄ -alkyl radicals which may optionally contain 1 to 2 double bonds and may be branched, aliphatic C ₂ -C ₂₄ -acyl radicals which may contain 1 to 2 double bonds and can be branched, mean, but at most 2 of the radicals R ¹ to R ³ can be hydrogen atoms and R ¹ ≠ H when R ² and R ^{3 are} acyl radicals. Process for the preparation of the cytarabine derivatives  Formula I according to claim 1, characterized in that a) from compounds of formula II

wherein R ¹ , R ² and R ^{3 have} the meaning given and R ^{4 represents} a 2- or 4-chlorophenyl radical, splits off the chlorinated phenyl radical R ⁴ or b) a compound of the formula III

wherein R ¹ , R ² and R ^{3 have} the meaning given, oxidized. Cytarabine derivatives of the formula I according to claim 1 for Use in disease control.