DE4304038A1 - Cytarabine derivatives, their preparation and use - Google Patents

Abstract

Cytarabine derivatives of the formula <IMAGE> in which R<1> to R<4> have the meaning stated in the description, and their preparation are described. The compounds are suitable, directly or in the form of immunoliposomes, for controlling diseases.

Description

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

AraC (= cytarabine = 4-amino-1-β-D-arabinofuranosyl-2 (1H) - pyrimidinone or 1-β-D-arabinofuranosylcytosine, Merck index 11th edition, No. 2790) is one in the chemotherapy of Cancer-proven cytostatic. Through those in the body however, the AraC quickly becomes present cytosine deaminase deaminated and therefore ineffective. To achieve a It must therefore have therapeutic effects in high doses to be applied to the patient with unpleasant Side effects are associated.

In order to delay the too rapid enzymatic deamination, the amino group of the cytosine residue was originally masked with acyl protective groups. The therapeutic effect of these araC derivatives was superior to that of araC in the L1210 mouse leukemia model. The best effect was shown by N ⁴ -stearyl-araC, while shorter or longer acyl protective groups slightly or drastically reduced the therapeutic effect of the N ⁴ -acyl-araC derivatives (Cancer Res., 36, 2726 (1976)).

A much clearer improvement in the therapeutic effect of araC was achieved by alkylation of the amino group of the cytosine residue, whereby only N ⁴ -alkyl-araC derivatives in the L1210 model of leukemia of the mouse, whose alkyl chain had at least 14 hydrocarbon residues, were effective. However, the investigated N ⁴ -alkyl-araC derivatives only showed alkyl groups with a maximum of 16 hydrocarbon residues (Int. J. Cancer, 51, 466 (1992); PCT / EP 92/00706). N ⁴ -alkyl-araC derivatives with even longer alkyl chains have not been described and their therapeutic effect is therefore unknown.

New cytarabine derivatives have now been found in which the cytarabine is not only protected against too rapid enzymatic deamination, but which are also more effectively absorbed by the tumor cell due to the optimized N ⁴ alkyl residues. The optimized protective groups enable the araC derivatives according to the invention to be used in more stable pharmaceutical preparations which, surprisingly, are therapeutically active even when administered orally.

The invention relates to cytarabine derivatives Formula I.

wherein
R ^{1 is} a C _17-24 alkyl radical which may contain 1-2 double bonds and may be branched,
and
R ² , R ³ and R ⁴ , which may be the same or different, are hydrogen atoms, an aliphatic C _2-5 acyl radical, a benzoyl group, a phosphate or a carboxy-C _1-3 alkylene carbonyl radical
mean.

As alkyl radicals, straight-chain radicals are preferred for R ¹ , n-docosyl and n-eikosanyl being particularly preferred in addition to n-octadecyl.

The acid residues for R ² , R ³ and R ⁴ include, in particular, the succinic and phosphoric acid residues. Compounds in which 1 or 2 radicals of R ² , R ³ , R ^{4 are} hydrogen are preferred.

The new cytarabine derivatives of the formula I can be prepared by using a compound of formula II

wherein Ac is an acetyl group and R is a chlorine atom or the rest

represent with an amine of formula III

H ₂ NR ′ ′ III

wherein R '' is a C _17-24 alkyl radical, which may optionally contain 1-2 double bonds and may be branched, is reacted and, if appropriate, then subsequently exchanged acetyl radicals for R ² , R ³ or R ⁴ in the compounds thus obtained.

The reaction works well in a polar solvent such as Dioxane or methanol at the boil.

The exchange of acetyl residues for hydrogen atoms or other acyl and phosphate residues can be carried out as follows are: The acetyl residues are by ammonolysis in aqueous or methanolic ammonia by standing at 20 to 60 ° C against hydrogen atoms within 2 to 34 hours exchanged. In the compounds thus obtained, by Reaction with appropriate carboxylic anhydrides, carbon acid chlorides or phosphorylating agents the water then atoms against acyl or phosphate residues exchanged.  

The reaction works well in pyridine or acetonitrile Room temperature or in the cold.

The starting materials required for the implementation are known substances (see J.C.S. Perkin I 1982, 1171; Helvetica Chimica Acta 70, 175 (1987)) or can be according to known Establish process.

The compounds of the invention stand out the araC through better deaminase resistance, cell uptake and application options.

Furthermore, depending on the number, length, type and location of each because of the substituents in the araC derivatives according to the invention vaten surprisingly the solubility of the derivatives in wide limits can be varied. The ara derivatives are thus soluble in aqueous buffer systems and / or in the form of very stable liposomes dispersible. Can for liposome formation all known liposome imaging methods such as ultrasound, Gel chromatography, detergent dialysis and pressure filtration. The each introduced residue also has a significant influence the size and stability of the liposomes that result from each araC derivatives along with others Form lipid components.

The targeted alkylation of the N ⁴ amino group with long-chain residues not only allows the solubility of the araC derivatives according to the invention to be controlled in a targeted manner, but surprisingly also optimizes the cytostatic activity.

Like the AraC, the new connections can be countered use malignant diseases of the blood-forming cells, especially against acute leukemia and chronic myeloid Blast relapse leukemia.  

The cytostatic effect of the araC derivatives can be seen in so-called immunoliposomes surprisingly for targeted Use destruction of certain tumor cells. To do this the araC derivatives along with other functionalized ones Lipid components in the form of liposomes in physiological Buffer system dispersed. On functional groups of the Liposome membrane are immobilized monoclonal antibodies. The immunoliposomes thus obtained are preferred in vitro taken up by the tumor cells that the antibody express appropriate antigen. The consequence of this cellular Targeting is the selective destruction of each target tumor cell in the presence of other cells.

The effect of the new compounds can be shown in the following experimental setup:
BDF1 mice were injected with L1210 tumor cells intravenously (iv), which simulated leukemia. On days 2 and 6 after the tumor cell injection, different doses of the araC derivatives or buffer solution were administered to the tumor-bearing animals. The overall breakdown into the individual test groups was as follows:

• - iv application of N ⁴ -octadecyl-araC in liposomes (2 doses)
• - iv application of N ⁴ -hexadecyl-araC in liposomes as reference (2 doses)
• - i.v. Application of araC in buffer solution for reference (1 dosage)
• - i.v. Application of the buffer solution (control group).

The medians were used as parameters for the success of the therapy Survival time of 6 animals per experimental group determined.  

Comparison of the cytostatic effects of araC and N ⁴ -alkyl-araC derivatives in intravenous therapy on days 2 and 6 days of 6 BDF1 mice each, which were infected on day 0 with 10 ⁵ L1210 leukemia cells.

In these experiments, the compounds according to the invention surprisingly showed a significantly better therapeutic effect compared to araC and N ⁴ -hexadecyl-araC.

When treated with 200 µmol araC in buffer, only 1 animal survived <60 days. When treated with 100 µmol N ⁴ -Hexadecyl araC liposomes, all test animals died in the range of 11-16 days. In contrast, all animals survived <60 days if they were treated with 100 µmol N ⁴ octadecyl-araC liposomes.

The new compounds are said to be in a dosage of about 40  -1000 mg per patient per day.

example 1 Preparation of 4- (n-docosylamino) -1-β-D-arabinofuranosyl- 2 (1H) pyrimidinone

4.85 g (11.5 mmol) of 4- (1,2,4-triazol-1-yl) -1-β-D-2 ′, 3 ′, 5′-tri-O-acetylarabinofuranosyl-2 (1H) -pyrimidinone were dissolved in 40 ml dry dioxane. A solution of 6.2 g (19 mmol) of n-docosylamine, dissolved in a mixture of 40 ml of ethanol and 5 ml of chloroform, was added dropwise to this solution. The mixture was then heated under reflux for 2 h. After cooling, the solvent was removed in vacuo. The remaining syrup was in chloroform / methanol; 4: 1; V: V taken up and shaken out with water. The organic phase was concentrated in vacuo to a syrup, which was dissolved in chloroform and fractionated on a silica gel column in a chloroform / methanol gradient with an increasing proportion of methanol. The product-containing fractions were concentrated to a syrup in vacuo, about 150 ml of methanol saturated with ammonia were added and the mixture was left at room temperature for about 12 hours. Then the solvent was largely spun off and the residue was crystallized from methanol. The precipitate was filtered off and dried at about 60 ° C in a drying cabinet. 6.5 g (11.8 mmol) of 4- (n-docosylamino) -1-β-D-arabinofuranosyl-2 (1H) -pyrimidinone were obtained as a white crystalline product, which on the silica gel plate in the chloroform / methanol system (7: 3; V: V) had an R _f value of 0.69.

Example 2 Preparation of 4- (n-octadecylamino) -1-β-D-arabinofuranosyl- 2 (1H) pyrimidinone

60 g (142 mmol) of 4- (1,2,4-triazol-1-yl) -1-β-D-2 ′, 3 ′, 5′-tri-O-acetylarabinofuranosyl-2 (1H) -pyrimidinone were dissolved in 450 ml dry dioxane. A solution of 55 g (202 mmol) of n-octadecylamine, dissolved in 450 ml of ethanol, was added dropwise to this solution. The mixture was then refluxed for 2 h. The solvent of the cooled reaction mixture was removed in vacuo and the remaining syrup was dissolved in 750 ml of methanol which was saturated with ammonia. The solution was left at room temperature for about 12 hours. The desired product gradually fell out. For complete crystallization, the mixture was kept at -20 ° C. for some time. Then the precipitate was suctioned off, washed with diethyl ether and dried at about 60 ° C. in a drying cabinet. The crude product obtained was recrystallized from 80% aqueous methanol. Up to 70 g of 4- (n-octadecylamino) -1-β-D-arabinofuranosyl-2 (1H) -pyrimidinone were obtained as a white crystalline product, which on the silica gel plate in the system chloroform / methanol (4: 1; V: V) had an R _f value of 0.42.

Example 3 Preparation of 4- (n-octadecylamino) -1-β-D-5'-0-succinoyl arabinofuranosylcytosine

2 g (3.8 mmol) of 4- (n-octadecylamino) -1-β-D-arabinofurano sylcytosine were dissolved in 10 ml of dry pyridine, 0.7 g (5.7 mmol) of 4-dimethylaminopyridine and 0.7 g (7 mmol) of succinic anhydride were added. After stirring for about 12 hours at room temperature, the reaction mixture was chromatographed on a cation exchange column (LEWATIT, pyridinium form) with pyridine / water (1: 4, V: V). The eluate was concentrated in vacuo to the syrup, which was then spun off several times with toluene. The foam obtained was dissolved in a little chloroform and fractionated on a silica gel column in a chloroform / ethanol gradient, the ethanol content being increased from 10% to 100%. The fractions of the desired product, which left the column with a high ethanol content, were combined and concentrated in vacuo to the syrup. The syrup was dissolved in chloroform and precipitated from an excess of an ether / hexane mixture (3: 2, V: V). The 2 ′, 3′-isomers which appeared as by-products could be removed by HPLC on a C ₁₈ reverse phase.

Fractions containing the desired product were concentrated. 4- (n-Octadecylamino) -1-β-D-5'-O-succinoylarabinofuranosylcytosine was obtained as a white powder, which had an R _f value on the silica gel plate in the chloroform / ethanol system (4: 1; V: V) of 0.47.

Example 4 Preparation of 4- (n-octadecylamino) -1-β-D-arabinofurano sylcytosine-5'-phosphate

A solution of 1.2 g (2.3 mmol) of 4- (n-octadecylamino) -1-β-D-arabinofuranosylcytosine in 100 ml of anhydrous acetonitrile was stirred with 350 mg (4.6 mmol) of tetrazole and 850 mg (3 mmol) of phosphorous acid-bis - (2-cyanoethyl ester) -N-diisopropylamide added. After about 30 minutes the solvent was removed in vacuo. The residue was treated with 25 ml of a 0.2 M iodine solution in tetrahydrofuran / pyridine / water; (90: 5: 5; V: V: V) and left for about 20 min at room temperature. Then the oxidation was stopped by adding 75 ml of a 2% NaHSO ₃ solution and the yellowish reaction solution was shaken out with about 75 ml of ethyl acetate. The organic phase was extracted twice with a saturated aqueous sodium chloride solution, filtered through Na ₂ SO ₄ and concentrated in a vacuum in the oil. About 50 ml of methanol, which was saturated with ammonia, was added to the oil, it was left at room temperature for about 12 h and then concentrated again in vacuo. The residue was taken up in a little water and extracted with ether. The aqueous phase was chromatographed on a SEPHADEX G15 column. The product-containing eluate was concentrated in a vacuum to form a syrup. The 2 ′, 3′-isomers which appeared as by-products could be removed by HPLC on a C ₁₈ reverse phase. Fractions containing the desired product were concentrated. 4- (n-Octadecylamino) -1-β-D-arabinofuranosylcytosine-5'-phosphate was obtained as a white powder, which on the silica gel plate in the chloroform / methanol system (1: 3; V: V) had an R _f - Had a value of 0.56.

Example 5 Presentation of liposome preparations

For the preparation of the liposome dispersion, 100 mg 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- (n-octa decylamino) -1-β-D-arabinofuranosyl-2 (1H) pyrimidinone 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. 3 ml of 10 mM PBS (0.9% NaCl and 10 mM NaH ₂ PO ₄ , pH 7.3) were added to the lipid film and sonicated with 40 watts for 30 min using a micro-tip of a disintegrator. An opalescent liposome dispersion was formed, which was used for the following reactions and tests.

Example 6 Representation of the immunoliposomes

1.2. nmol of an antibody were lyophilized with 50 ul of the liposome preparation from Example 5 and 7 mg (27 µmol) N (3- Dimethylaminopropyl) -N'-ethyl-carbodiimide X HCl (EDC) added and adjusted to pH 4 by adding 30 µl PBS (pH 1) set. At an hourly interval were two more each 7 mg EDC added. After stirring for about 5 hours in the room the temperature of the reaction to an ULTROGEL ACA 22 column applied and fractionated with PBS (pH 7.4). Fractions whose absorption ratios with the values of used liposome preparation, were pooled and used for cell targeting.

Claims (4)

1. Cytarabine derivatives of the formula I. wherein
R ^{1 is} a C _17-24 alkyl radical which may contain 1-2 double bonds and may be branched and
R ² , R ³ and R ⁴ , which may be the same or different, are hydrogen atoms, an aliphatic C _2-5 acyl radical, a benzoyl group, a phosphate or a carboxy-C _1-3 alkylene carbonyl radical
mean. 2. A process for the preparation of the cytarabine derivatives of the formula I according to claim 1, characterized in that a compound of the formula II wherein Ac is an acetyl group and R is a chlorine atom or the rest represent, with an amine of the formula IIINH ₂ R '' IIIworin R '' is a C _17-24 alkyl radical, which may contain 1-2 double bonds and may be branched, is reacted and optionally subsequently acetyl radicals against R in the compounds thus obtained ² , R ³ or R ⁴ exchanged. 3. Immunoliposomes containing a cytarabine derivative Formula I according to claim 1. 4. Cytarabine derivatives of the formula I according to claim 1 Use in disease control.