DE4111730A1 - NEW CYTARABIN 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

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

AraC (= cytarabine = 4-amino-1-β-D-arabinofuranosyl-2 (1H) -pyrimidinone or 1-β-D-arabinofuranosylcytosine, Merck Index 11th edition, No. 2790) is a A cytostatic that has been proven in cancer chemotherapy. By however, the cytosine deaminase present in the body quickly becomes the AraC deaminated and therefore ineffective. To achieve a therapeutic Effect, it must therefore be applied in high doses for the Patients are associated with uncomfortable side effects. In addition, that Tumor cells that lack kinase activity are not effective in AraC Phosphorylate 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 previously been masked with acyl protective groups (Int. J. Cancer 37, 149 (1986)). However, the N ⁴ -acyl-AraC derivatives obtained in this way, even if they were applied in the form of liposomes, showed no improved cytostatic effect 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.

The attempt was made to counteract the AraC resistance of tumor cells by that AraC or AraC-5'-monophosphate covalently to natural phopholipids was coupled (DE 83 00 2391). One hoped for these AraC prodrugs that they are taken up by AraC-resistant tumor cells and in the cell would be enzymatically split internally into the active AraC-5'-monophosphate. Prodrugs in which AraC or AraC-5′-monophosphate on natural esters coupled phospholipids and applied in the form of liposomes only partially met expectations. A disadvantage of these Prodrugs is that the free amino group of the cytosine residue before the enzyma table deamination is not protected, so that this phospholipid AraC prodrugs too quickly for inactive 1-β-D-arabinofuranosyluracil deri be metabolized.

New cytarabine derivatives have now been found in which the cytarabine is more effectively protected against enzymatic deamination and the 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 to 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 contain 1 to 2 double bonds and may be branched, aliphatic C _2-24 -acyl radicals which may contain 1 or 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 if 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.

Unbranched alkyl and acyl radicals having 1 to 20 carbon atoms are preferred for R ² and R ³ .

The new cytarabine derivatives of the formula I can be prepared by one

• 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 formula III wherein R ¹ , R ² and R ^{3 have} the meaning given, oxidized.

The chlorinated phenyl radical according to a) is particularly easily cleaved good if the compounds II in an aqueous-organic for 5 to 15 h Stand solvent at room temperature with tetrabutylammonium fluoride leaves.

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

The reaction products thus obtained can be purified by chromatography will.  

Leave the compounds of formula II used as the starting material by condensation of compounds of formula IV

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

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 N-acyl compound.

The compounds IV, V and VI are known or can be according to known Prepare method (Chem. Pharm. Bull. 26, 981 (1978), Nucleic Acid Res. Symposium Series No. 18, 189 (1987)).

Furthermore, the number, length, type and location of the respective sub in the AraC derivatives according to the invention have the amphiphilic properties Surprisingly, can be varied within wide limits. The Amphiphilic AraC derivatives are thus soluble in aqueous buffer systems  and / or dispersible in the form of liposomes. Depending on the amphiphilic The property of the AraC derivative is the liposome formation without and / or with other lipid components available. Everyone can contribute to liposome formation known methods of liposome imaging can be used like for example ultrasound, gel chromatography, detergent dialysis. The the lipophilic residues introduced in each case also significantly influence the Size and stability of the liposomes resulting from the respective form amphiphilic AraC derivatives.

The targeted introduction of lipophilic residues does not, however only the amphiphilic character of the AraC derivatives according to the invention is targeted control, but surprisingly also the cytostatic effect of Optimize AraC decisively.

Like the AraC, the new compounds can even be used to combat malignancies Use diseases of the blood-forming cells, especially against acute ones Leukemia and chronic myeloid leukemia in the blast flare.

The cytostatic effect of the amphiphilic AraC derivatives can be seen in so-called immunoliposomes surprisingly for targeted destruction from certain tumor cells. The amphiphilic AraC Derivatives together with other lipid components in the form of liposomes in dispersed physiological buffer systems. On functional groups of the Liposome membrane are immobilized monoclonal antibodies. The so Immunoliposomes obtained are preferred in vitro by the tumor cells recorded that express the antigen corresponding to the antibody. The 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 demonstrated in the following experiment show arrangement:

DBA / 2 mice were injected with L1210 tumor cells intravenously, causing a Leukemia was simulated. On days 3 and 7 after the tumor cell injection different doses of the test substance were given to the tumor-bearing animals or solvent. The overall classification was as follows into the individual test groups:

• - control group (solvent),
• - i.v. Application (2 doses),
• - i.p. Application (2 doses),
• - i.v. AraC as reference (2 doses),
• - i.p. AraC for 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 effect as AraC.

The new compounds are said to be in a dosage of about 40-1000 mg each Patient and day are used.

example 1 Preparation of 4- (1-hexadecylamino) -1-β-D-5′-O- (1,2-di-O-palmitoyl-sn- glycero-3-phospho) arabinofuranosylcytosine a. Production of the starting material

5 g (10.7 mmol) of 4- (1-hexadecylamino) -1-β-D-arabinofuranosylcytosine were together with 6.8 g / 10.7 mmol) 1,2-di-O-palmitoyl-sn-glycero-3-H-phos phonate in 50 ml of anhydrous pyridine with 6.6 ml (53.5 mmol) of pivalic acid chloride added and with exclusion of moisture for 10 min at room temperature door stirred. The solution was then concentrated to the syrup in a vacuum trated, which was then spun down twice with about 40 ml of toluene.

b. 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 the 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 on a pebble gel column fractionated in chloroform / methanol gradient. The factions of the desired product using chloroform / methanol (9/1, v / v) the column were pooled, concentrated to dryness and then crystallized from methanol. Here, 3.7 g of a white powder get that on the silica gel plate in system chloroform / methanol / 7/3, V / V) had an RF value of 0.46.  

Example 2 Preparation of 4- (palmitoyl) -1-β-D-5′-O- (1,2-di-O-palmitoyl-sn-glycero-3- phospho) arabinofuranosylcytosine a) Production of the starting material

8 g (16 mmol) of 4- (palmitoyl) -1-β-D-arabinofuranosylcytosine were combined with 16 g (21 mmol) 1,2 di-O-palmitoyl-sn-glycero-3- (2-chlorophenyl) - phosphate in 50 ml of anhydrous pyridine with 11 g (36 mmol) of 2,4,6-triiso propylbenzenesulfonyl chloride and 9 ml of N-methylimidazole are added and shaken for 1 h at room temperature with exclusion of moisture. The mixture was then concentrated in vacuo to the syrup, which then was spun down twice with about 150 ml of toluene.

The residue was taken up in 200 ml of chloroform and on a pebble gel column fractionated in a chloroform / methanol gradient. The factions of the desired product with chloroform / methanol / 95/5, V / V) the Left column, were pooled, concentrated to dryness and out Methanol crystallizes. They gave 9.5 g of a white powder.

b. Manufacture of the final product

To split off the 2-chlorophenyl residue, the isolated condensation product was treated with an excess of 3 equivalents of tetrabutylammonium fluoride × 3 H ₂ O 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 purification, the reaction mixture was on a silica gel in Chloroform / methanol gradient fractionated. The fractions of the desired th product was concentrated, crystallized from diethyl ether. Man received 5 g of a white powder that was on the silica gel plate in the system Chloroform / methanol (4/1; v / v) had an Rf value of 0.20.  

Example 3 Preparation of 1-β-D-5′-O- (1-octadecyl-sn-glycero-3-phospho) arabino furanosylcytosine a) Production of the starting material

By condensation of 1-O-octadecyl-2-O-acetyl-sn-glycero-3-H-phosphonate (in analogy to Example 1) or of 1-O-octadecyl-2-O-acetyl-sn-glycero- 3 (2-chlorophenyl) phosphate (in analogy to example 2) with 4 (benzoyl) -1-β- D-arabinofuranosylcytosine was initially 4- (benzoyl) -1-β-D-5'-O- (1-O-octa decyl-2-O-acetyl-sn-glycero-3-phospho) arabinofuranosylcytosine syntheti siert and then chromatographed on silica gel.

b) Production of the final product

The condensation product obtained according to a) was with an excess Open methanolic ammonia closed for approx. 24 h at room temperature maintains. Then the solvent was removed in vacuo and the residue fractionated on a silica gel column in a chloroform / methanol gradient. Fractions containing the desired product were concentrated. The residue, which was crystallized from acetone / water, gave a white powder, which has an RF value in the chloroform / methanol system (1/1, V / V) of 0.19.

Example 4 Presentation of liposome preparations

For the preparation of the liposome dispersion, 100 ml soy phosphatidylcholine, 10 mg cholesterol 1 mg α-tocopherol, 7 mg N ² -palmitoyl-N ⁶ -succinoyl-L- were used per ml chloroform-methanol (1: 1; v: v) lysine and 12 mg of 4- (palmitoyl) -1-β-D-5'-O- (1,2-di-O-palmitoyl) -sn-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 ) and sonicated at 40 watts for 30 min using a micro-tip of a disintegrator. An opalescent liposome dispersion was formed, which was used for the following reaction.

Example 5 Representation of the immunoliposomes

1.2 nmol of an antibody were lyophilized with 50 ul of the liposome preparation from Example 3 and 7 mg (27 µmol) N (3-dimethylaminopropyl) -N'- ethyl carbodiimide × HCl (EDC) and by adding 30 ul PBS (pH 1) adjusted to pH 4. At an hourly interval there were two more each 7 mg EDC added. After stirring for about 5 h at room temperature the reaction mixture is applied to a ULTROGEL ACA 22 column and with PBS (pH 7.4) fractionated. Fractions whose absorption ratio with the Values of the liposome preparation used agree, were ver agreed and used for cell targeting

Claims (3)

1. 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 ₁₄ -alkyl radicals which may optionally contain 1 to 2 double bonds and may be branched, aliphatic C ₂ -C ₂₄ -acyl radicals which may have 1 or 2 double bonds contained 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 if R ² and R ^{3 are} acyl radicals. 2. A process for the preparation of the cytarabine derivatives of the 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 formula III wherein R ¹ , R ² and R ^{3 have} the meaning given, oxidized.

3. cytarabine derivatives of formula I according to claim 1 for use in fighting diseases.