DE19750508A1 - New phospholipid-nucleoside adducts useful for treating and preventing mammary tumors and leukemia - Google Patents

Abstract

Phospholipid-nucleoside adducts (I) are new. Phospholipid-nucleoside adducts of formula (I) are new. R = 4-22C hydrocarbon group which is saturated or unsaturated and may be substituted by one or more halo, OH, alkoxy or CN groups, or R = -CH2-CH(B)-CH2Q or -CH(CH2Q)(CH2B); (F-ara-A) = 9- beta -D-arabinofuranosyl-2-fluoroadenyl-5'-group; n, m = 0 or 1; Q = 5-30C alkoxy or alkenyloxy which are saturated or unsaturated and which may be substituted by one or more halo, OH, alkoxy or CN groups; B has the same meaning as Q or is H, OH, halo, CN, 2,2,2-trifluoroethoxy or 2-22C acyloxy.

Description

The invention relates to new phospholipid adducts of 9-β-D- Arabinofuranosyl-2-fluoradenins (F-ara-A, fludarabine) general formula I including all enantiomeric forms as well as all pharmaceutically usable salts of these Links. The invention also relates to methods for the preparation of the compounds I and their pharmaceutical Use. The field of application of the invention is thus chemical and pharmaceutical industries.

F-ara-A takes a leading place in the treatment of the chronic lymphoblastic leukemia (CLL) as well Monotherapy as well as in combination with others Chemotherapy drugs. To improve solubility, also used the 5'-monophosphate (fludarabine phosphate), the is dephosphorylated after intravenous infusion. Intracellular rephosphorylation occurs to the triphosphate, the actual one cytotoxic metabolite, which is different Inhibits key processes of DNS replication.

In the meantime, several studies have confirmed its effectiveness in acute myeloid leukemia, in non-Hodgkin's lymphoma and in cutaneous T-cell lymphomas (e.g. mycosis fungoides).

Therapy is limited by the strong neurotoxicity as well as the restricted area of application of the Links.  

The scope of a nucleoside, its effective cytostatic component that represents nucleoside triphosphate, is limited when the tumor cells to be treated insufficient activity of phosphorylating enzymes (Kinases). This is the case with kinase-deficient tumor cells therapeutic use of a nucleoside is not possible, there is no or insufficient phosphorylation intracellularly via the monophosphate to the effective nucleoside triphosphate he follows. Direct application of a phosphorylated However, nucleoside stands in the way of the cell membrane natural barrier for this type of ionic compounds represents, d. H. that an infiltration in this form is not is possible. On the other hand, it becomes natural Cytidine diphosphate diacylglycerol (CDP-DAG), which acts as a precursor cellular phospholipids is a natural cell component, incorporated, although it is also a bivalent anion represents. Phospholipid nucleoside analogs of the CDP-DAG type as well as corresponding compounds of adenosine intracellular to the corresponding nucleoside monophosphates split. With suitable "lipid packaging" can be also introduce phosphorylated nucleosides into the cell or into the nucleoside monophosphates, the precursors of the nucleoside triphosphate, convert. It is also known that Structural analogues of the above-mentioned cellular CDP-DAG, which as such cytostatic is ineffective, depending on the Structure of the lipid component also cytostatic activity receive. This shows that the lipid component for the physiological activities of phospholipid-nucleoside adducts is of particular importance.

The aim of the invention was now to develop new cytostatic agents F-ara-A derivatives based on phospholipid nucleoside Adducts to synthesize what antitumor properties and in comparison to the parent substance, an expanded spectrum of action own and thus for pharmaceutical use in Come into consideration.

The goal is solved according to the claims.  

The invention relates to new phospholipid-nucleoside adducts of the general formula I

wherein

• a) R is a straight chain or branched, saturated or a mono- or polyunsaturated, unsubstituted or one or more times by halogen, hydroxy, alkoxy or cyano substituted hydrocarbon residue with 4-22 carbon atoms means or
• b)
  is what
  A is a saturated or mono- or polyunsaturated, unsubstituted or mono- or polysubstituted by halogen, hydroxy, alkoxy or cyano-substituted (C ₅ -C ₃₀ ) alkoxy or alkenoxy,
  B has one of the meanings given for A or represents hydrogen, hydroxy, halogen, cyano, 2,2,2-trifluoroethyloxy and (C ₂ -C ₂₂ ) acyloxy.
• c) F-ara-A the 9-β-D-arabinofuranosyl-2-fluoradenyl-5 'radical means
• d) n and m is 0 or 1.

Preferred connections are:
2-fluoro-ara-adenosine-5'-hexadecyl phosphate (formula I: R = C ₁₆ H ₃₃ , n = 1, m = 0),
2-fluoro-ara-adenosine-5'-diphosphate (formula I: R = C ₁₆ H ₃₃ , n = 1, m = 1),
2-fluoro-ara-adenosine-5'-phosphonophosphate (Formula I: R = C ₁₆ H ₃₃ , n = 0, m = 1).

To produce the phospholipid-nucleoside adducts general formula I in which a phosphate grouping (n = 1, m = 0) is present, one generally proceeds from 2-fluoro-ara-adenosine-5'-phosphate, its 2'- and 3'-OH groups protected by acetylation. Then that will acetylated intermediate in the presence of a Condensing agent, e.g. B. 2,4,6-triisopropylphenyl sulfonyl chloride (TPS), with the reactant R-OH, in the R has the meanings given under formula I, implemented. After removing the protective groups from the nucleoside with the target compound is obtained in methanolic ammonia. It it is also possible to condense the 2-fluoro-ara-adenosine-5'- to make phosphates without protecting the OH groups. In this In case it is appropriate to use another condensing agent, e.g. B. To use DCC.

The synthesis of the phospholipid-nucleoside adducts of the general formula I, in which a phosphonate grouping (n = 0, m = 0) is present, is carried out according to methods known per se by condensation of a phosphonic acid RP (O) (OH) ₂ with the nucleoside in the presence of TPS in pyridine solution. To increase the yields, it may be expedient to start from the nucleoside, the OH groups of which are protected, and to remove the protective groups again after the condensation with the phosphonic acid.

Compounds of the general formula I, in which a phosphonophosphate (n = 0, m = 1) or a diphosphate group (n = 1, m = 1) is present, are obtained by condensation of a 2-fluoro-ara-adenosine-5 'monophosphoamidates, e.g. B. the monophosphomorpholidate, with an alkylphosphonic acid RP (O) (OH) ₂ or a phosphoric acid ester ROP (O) (OH) ₂ obtained. Another variant of the method consists in the implementation of the activated phosphonate or phosphate component, e.g. B. in the form of an amidate with the nucleoside monophosphate. These reactions are generally carried out in a polar, anhydrous solvent, preferably pyridine, at temperatures of 20-50 ° C. After isolation from the reaction mixture, the products are purified, if necessary, by chromatographic methods. The structure and purity of the new compounds are ensured by mass spectrometry (FAB, ESI), ¹ H and ³¹ P NMR and thin layer chromatography.

The compounds of general formula I and their salts are biologically active and have a pronounced Antitumor effectiveness, which u. a. through the results of the In vitro testing on human immortalized breast epithelial cells H184A1N4, on the human breast tumor cells MaTu and MCF7 as well as the leukemia cell lines Daudi and HL60.

Tab. 1 shows the inhibition of the growth of the cells mentioned by selected compounds of general formula I in Comparison to the known parent substance 2-fluoro-ara-adenosine-5'- phosphate (2-F-ara-AMP).

Table 1

IC ₅₀ values (50% inhibition of cell growth in µM)

From Table 1 it can be seen that the new compounds of general formula I in contrast to the reference substance 2-F-ara-A the cell growth of the solid mammary tumor MaTu in strong Inhibit dimensions, so that an advantageous use in the Human medicine for the treatment and prophylaxis of Tumor diseases is given.

The invention is intended to be explained in more detail below by means of exemplary embodiments are explained.

Embodiments example 1

2-fluoro-ara-adenosine 5'-hexadecyl phosphate

A mixture of 2-fluoro-ara-adenosine-5'-phosphate (365 mg, 1.0 mmol), acetic anhydride (10 ml) and pyridine (20 ml) is added for 15 h Room temperature stirred. After adding water (10 ml) Mixture stirred for another hour and then in vacuo rotated in. The residue is dissolved in pyridine (25 ml) to Solution becomes TPS (1.21 g, 4.0 mmol) and after stirring for 30 minutes Hexadecanol (266 mg, 1.0 mmol) was added. After stirring for 20 hours water (10 ml) is added, the mixture is stirred for 1 h and evaporated to dryness. The residue is mixed with diethyl ether treated, the suspension is filtered and the ether solution rotated in. The peracetate obtained is by Column chromatography (silica gel 60 Merck (0.015-0.040 mm, Eluent A: chloroform / methanol / 25%. Ammonia 70: 30: 4) cleaned. After treating the product with 2 N ammonia Methanol (20 ml, 5 h) is again eluted with A and then with eluent B (chloroform / methanol / 25%. Ammonia 80: 20: 4) chromatographed. Yield 206 mg.  

Example 2 2-fluoro-ara-adenosine-5'-hexadecylphosphonophosphate

Hexadecylphosphonic acid (380 mg, 1.24 mmol) and morpholine (420 mg, 4.8 mmol) are in a mixture of water (12 ml) and tert. Butanol (12 ml) dissolved. DCC (1.0 g, 4.8 mmol) in tert. Butanol (15 ml) added and the obtained Mixture heated under reflux for 3.5 h. The mixture is on rotated in a volume of 5 ml and cooled to 0-5 ° C. After suction of the precipitate, the filtrate rotated in. The dried hexadecylphosphonomorpholidate dissolved in pyridine (25 ml). The solution is 2-fluoro-ara adenosine 5'-phosphate (402 mg, 1.1 mmol). The mixture is heated to 38 ° C. with stirring for 16 h. After that, water added and stirred for 1 h at room temperature. After Isolation is like the title compound by chromatography cleaned as described in Example 1. Yield 280 mg.

Example 3 2-fluoro-ara-adenosine-5'-hexadecyidiphosphate

The production takes place in analogy to example 2 under Use of phosphoric acid hexadecyl ester (400 mg, 1.24 mmol), Morpholine (420 mg, 4.8 mmol) and 2-fluoro-ara-adenosine-5'- phosphate (402 mg, 1.1 mmol). Yield 194 mg.

Claims (13)

1. New phospholipid-nucleoside adducts of the general formula I
wherein

• a) R is a straight-chain or branched, saturated or a mono- or polyunsaturated, unsubstituted or mono- or polysubstituted by halogen, hydroxy, alkoxy or cyano substituted hydrocarbon radical having 4-22 carbon atoms or
• b)
  is what
  A is a saturated or mono- or polyunsaturated, unsubstituted or mono- or polysubstituted by halogen, hydroxy, alkoxy or cyano-substituted (C ₅ -C ₃₀ ) alkoxy or alkenoxy,
  B has one of the meanings given for A or denotes hydrogen, hydroxy, halogen, cyano, 2,2,2-trifluoroethyloxy and (C ₂ -C ₂₂ ) acyloxy,
• c) F-ara-A represents the 9-β-D-arabinofuranosyl-2-fluoradenyl-5 'radical,
• d) n and m is 0 or 1.

2. 2-fluoro-ara-adenosine-5'-hexadecyl phosphate (Formula I: R = C ₁₆ H ₃₃ , n = 1, m = 0). 3. 2-fluoro-ara-adenosine-5'-diphosphate (Formula I: R = C ₁₆ H ₃₃ , n = 1, m = 1). 4. 2-fluoro-ara-adenosine-5'-phosphonophosphate (Formula I: R = C ₁₆ H ₃₃ , n = 0, m = 1). 5. Process for the preparation of the phospholipid-nucleoside adducts of the general formula I, in which a phosphate grouping (n = 1, m = 0) is present, characterized that one the 2'- and 3'-OH groups of 2-fluoro-ara-adenosine-5'-phosphate protects by acetylation, then the acetylated Intermediate in the presence of a condensing agent, e.g. B. 2,4,6-triisopropylphenyl sulfonyl chloride (TPS), with which Reaction component R-OH, in which R is those mentioned under formula I. Has meanings, implements and the protective groups of Nucleoside removed with methanolic ammonia. 6. Process for the preparation of the phospholipid-nucleoside adducts of the general formula I, in which a phosphate grouping (n = 1, m = 0) is present, characterized in that the Condensation of the 2-fluoro-ara-adenosine-5'-phosphate with the Reaction component R-OH, in which R is those mentioned under formula I. Has meanings without protection of the OH groups in the presence of the condensing agent DCC. 7. A process for the preparation of the phospholipid-nucleoside adducts of the general formula I in which a phosphonate grouping (n = 0, m = 0) is present, characterized in that a phosphonic acid RP (O) (OH) ₂ with the nucleoside, the OH groups of which are optionally protected, is condensed in the presence of TPS in pyridine solution. 8. A process for the preparation of compounds of general formula I in which a phosphonophosphate (n = 0, m = 1) or a diphosphate grouping (n = 1, m = 1) is present, characterized in that a 2- Fluoro-ara-adenosine-5'-monophospho-amidate, e.g. B. a -monophosphomorpholidate, with an alkylphosphonic acid RP (O) (OH) ₂ or a phosphoric acid ester ROP (O) (OH) _{2 is} condensed. 9. Process for the preparation of compounds of general Formula I in which a phosphonophosphate (n = 0, m = 1) or there is a diphosphate grouping (n = 1, m = 1), characterized in that an activated phosphonate or Phosphate component, e.g. B. in the form of an amidate with Nucleoside monophosphate in a polar, anhydrous Solvents, preferably pyridine, at temperatures of 20-50 ° C is implemented. 10. Use of the compounds according to claims 1-4 as Antitumor agent. 11. Use of the compounds according to claims 1-4 as an agent against breast cancer. 12. Use of the compounds according to claims 1-4 as an agent against leukemia.