DD293498A5 - METHOD FOR PRODUCING A MEDIUM FOR THE TREATMENT OR PROPHYLAXIS OF HEPATITE INFECTIONS IN HUMANS AND ANIMALS - Google Patents

Abstract

The invention relates to a process for the preparation of an agent for the treatment or prophylaxis of hepatitis infections in humans and animals. Field of application of the invention is the human and veterinary medicine or the pharmaceutical industry. According to the invention, substituted nucleosides of the formula I, {hepatitis infections; Treatment; Prophylaxis; deoxynucleosides; 3-substituted porters; Human and veterinary medicine; Industry, pharmaceutical}

Description

Field of application of the invention

The invention relates to a method for producing an agent against hepatitis infections in humans and animals. Field of application of the invention is the human and veterinary medicine or the pharmaceutical industry.

Characteristic of the known state of the art

Hepatitis B is an infectious disease caused by the hepatitis B virus (HBV), which affects about 200 million people worldwide and whose chronic form is associated with an increased risk of primary liver carcinoma, about one million in China alone Tumor new diseases per year leads.

Effective and tolerable antiviral therapy has been lacking. Attack could u.a. be the HBV DNA polymerase, an enzyme whose inhibition could prevent the further intracellular virus multiplication and thus stop its spread. The first clinically proven inhibitors of HBV polymerase, such as. B. adenine arabinoside (araA) and acyclovir (ACV), have also shown only partial or transient ameliorations or are accompanied by side effects in combination with corticosteroids or interferon alpha (Alexander et al., British Medical Journal 292, 91 δ [1986]), so there is a need to develop more effective and more selective drugs. In this direction, WO Patent No. 88/00050, which claims 3'-fluorodeoxynucleosides of adenine, guanine, cytosine and thymine as agents against hepatitis B infections, and WO Patent No.89 / 01776, in which the efficacy of 2'-fluoroarabinosyl-5-ethyluracil against hepatitis infections (woodchuck hepatitis) is proven.

Object of the invention

The object of the invention is to provide agents for the treatment and prophylaxis of hepatitis infections in humans and animals.

Explanation of the essence of the invention

The invention has for its object to develop a process for the preparation of a new agent against hepatitis infections, which is effective, well tolerated and has a low toxicity. According to the invention, new and known substituted nucleosides of the general formula I

R ¹ : F, Cl, Br, I, CH ₃ , CH ₂ OH

R ² : 2,3-dideoxyribofuranosyl, 2,3-dideoxy-3-fluororibofuranosyl, 2,3-dideoxy-3-chlororibofuranosyl, 2,3-dideoxy-3-azidoribofuranosyl, 2,3-dideoxy-3-aminoribofuranosyl, 2 , 3-Didehydro-2,3-didesoxyribofuranosyl, arabinofuranosyl, 3-deoxy-3-fluoroarabinofuranosyl or their 5-0-acetyl, 5-0-palmitoyl, 5-0-alkoxycarbonyl derivatives, or their 5-mono Mean, 5-di- or 5-triphosphates (as free acids or alkali metal, ammonium or alkylammonium salts) or other precursor groups for the 5-hydroxyl group,

with pharmaceutically acceptable carriers used for the treatment and prophylaxis of hepatitis infections, such as those caused by the hepatitis B virus (HBV) ₁ the hepatitis A virus (HAV) and the causative agent of non A, non B hepatitis.

These are preferably the new compounds:

2 ', 3'-dideoxy-5-hydroxymethylcytidine

2 ', 3'-dideoxy-3'-fluoro-5-fluorocytidine

2 ', 3'-dideoxy-3'-chloro-5-methylcytidine

2 ', 3'-dideoxy-3'-chloro-5-bromocytidine

2 ', 3'-dideoxy-3'-chloro-5-chlorocytidine

2 ', 3'-dideoxy-3'-az! Do-5-hydroxymethylcytidine

2 ', 3'-didehydro-2 \ 3'-dideoxy-5-methylcytidine

2 ', 3'-didehydro-2', 3'-dideoxy-5-fluorocytidine.

Other effective compounds are:

2 ', 3'-dideoxy-5-methylcytidine

2 ', 3'-dideoxy-5-bromocytidine

2 ', 3'-dideoxy-3'-fluoro-5-methylcytidine

2 ', 3'-dideoxy-3'-azido-5-methylcytidine

2 ', 3'-dideoxy-3'-azido-5-fluorcyfidin

2 ', 3'-dideoxy-3'-amino-5-methylcytidine

2 ', 3'-Didosoxy-3'-amino-5-jodcy1idin

Arabinofuranosyl-5-methylcytosine.

Compounds of formula I are also alone (without carriers) already effective agents against hepatitis infections. The agents according to the invention contain one or more active components, according to the general formula I together with one or more compatible carriers therefor and optionally with other therapeutic means. The agents are prepared as a unit dose or so that a multiple of the active components is included.

Each carrier must be compatible, compatible with the other ingredients of the agent and must not be harmful to the patient.

The agents include those which may be used for oral, rectal, nasal, topical, vaginal or parenteral (including subcutaneous,

intramuscular, intravenous and intradermal) administration. One or more active components are brought into contact with the carrier, which is composed of one or more accompanying ingredients, and, if necessary, brought into an appropriate form.

The agents for oral administration are prepared in the form of dragees, tablets, capsules, powders or granules so that they each contain a certain amount of the active ingredient. Likewise, they can be prepared as a solution or as a suspension. Optionally, flavorants or other conventional agents are added.

Means for rectal administration are prepared as suppositories with a suitable base.

Means for vaginal administration are manufactured as pass are, tampons, creams, gels, pastes, foams or spray products.

The means for parenteral administration may be provided as a unit dose of the active component or as a multi-dose. For this they can be stored in ampoules, vials or in a freeze-dried state. Immediately prepared injection solutions and suspensions can be obtained from sterile powders, granules and tablets. This is done z. B.

by dissolving the substance in physiological saline, glucose or other for the i. v. Injection or infusion of suitable media. For the treatment of patients with hepatitis B infections is z. B. from physiological saline and 2 ', 3'-dideoxy-3'-fluoro-5-methylcytidine the required amount of a 0.3% solution prepared, which is sterilized and the patient is infused intravenously within one hour. The infusion is repeated every 4-8 hours and at least 8-10

Days continued. The chemical preparation of the compounds of formula I was carried out by the treatment of the corresponding Anhydro compounds with nucleophiles optionally with acid catalysis analogous to that of G. Etzold et al. (Tetrahedron 27,

2463 (1971)). In other cases, a suitable 3'-sulfonyloxy group was nucleophilically exchanged directly (J.P. Horwitz et al., J. Org. Chem. 29, 2076 [1964]). The 2 ', 3'-dideoxynucleosides were characterized by catalytic

Reduction of the respective 3'-iodo compounds according to K. E. Pfitzner and J. G. Moffat (J. Org. Chem. 29, 188 [1964]),

while the 2 ', 3'-dideoxy-2'-ene compounds by elimination of suitable 3'-sulfonyloxy groups analogous to that of

J.P. Horwitz et al. (Tetrahedron Lett 13,1343 [1966]). Furthermore, the 5-halogenated pyrimidine compounds were prepared according to the methods described by J. Duval and J.P. Ebel (Bull. Soc. Chim.

1059 [1964]), or K.Kikugawa et al. (Chem. Pharm. Bull.23, 35 [1975]

Phosphoric acid esters were prepared from the respective nucleosides by means of phosphorus oxychloride in trimethyl phosphate as Solvent obtained (M.Yoshikaw »et al .. Tetrahedron Lett 5065 [1967]). To convert the mono- into the respective Triphosphate has been prepared by W. Feist and F. Cramer (in "Nudele Acid Chemistry", Part 2, edit, by L. B. Townsend and R. S.Tipson, John Wiley, New York, 1978, p.827). The still used methods of esterification, etherification or halogenation, the selective blocking or Activation of OH and other functional groups, as well as the removal of protective groups used correspond

the usual method in nucleoside chemistry.

The effectiveness of some of the compounds of the invention is demonstrated as follows:

a) Inhibition of hepatitis B virus polymerase by deoxynucleoside triphosphate analogues The hepatitis B virus (HBV) has a circular, partially double-stranded DNA, which is replicated after infection of a liver cell by a virus-specific polymerase. However, this process requires the synthesis of an RNA by a cellular RNA polymerase. Only this RNA template - also referred to as embossoma in the context of replication - can be used by the HBV polymerase for the synthesis first of an RNA-DNA hybrid and then of the double-stranded DNA.

Thus, the HBV polymerase has an enzymatic activity similar to that of a reverse η transcriptase, Will et al.

(J. Virology 61,904 [1987]). Initially, the inhibitability of HBV-associated polymerase by the 5'-triphosphates of the compounds of this invention was examined. For this purpose, the method described by Kaplan et al. (J. Virology 12,995 [1973])

Used method for the determination of HBV polymerase and added to the tested triphosphates the batches in various concentrations. The inhibition of the enzyme reaction was determined after 3 hours of incubation at 37 ⁰ C by Veigleich with a control value. Table 1 shows the results obtained with 2 ', 3'-dideoxy-3'-fluoro-5-methylcytidine-5'-triphosphate as a typical representative of the compounds of the invention.

Table 1

Inhibition of HBV polymerase activity by 2 ', 3'-dideoxy-3'-fluoro-5-methylcytidine-5'-triphosphate

Inhibitor concentration% inhibition

0.01 μΜ 28 0.03 μΜ 50 0.06 μΜ 62 0.28 μΜ 86 1.40 μΜ 95

This results in the concentration (ID ₆₀ ) of 0.03μΜ required for a 50% inhibition of the HBV polymerase, which means that 2 ', 3'-dideoxy-3'-fluoro-5-methylcytidine-5'-triphosphate is among the strongest known inhibitors of HBV polymerase heard.

b) Effect of Modified Deoxynucleotide Triphosphates on the Cellular DNA Polymerases alpha and beta

In order to examine the extent to which the described effect on the HBV polymerase is selective, ie the cellular DNA polymerases are excluded from inhibition of the deoxynucleoside triphosphates according to the invention, the cellular DNA polymerases al μ a and beta were included in the investigations. The isolation of the enzymes and their activity determination was carried out according to the method of Matthes et al. (Biomed., Biochim. Acta 44, K63-K73 [1985]). The results with 2 ', 3'-dideoxy-3'-fluoro-5-methylcytidine-5'-triphosphate show that the responsible for the replication of cellular DNA DNA polymerase alpha remains largely unaffected. A 50% inhibition of this enzyme is not reached even at 150μΜ, so that the DNA polymerase alpha compared to this compound is considered to be almost resistant. For the DNA polymerase beta, which is considered to be a repair enzyme of cellular DNA damage, the ID ₆₀ value is 4.8μΜ. Thus, the compound described has a high impact specificity.

c) cytotoxicity of the modified nucleosides

From the point of view of possible application of the compounds to humans, their antiproliferative effects on human cell cultures as described earlier were tested, E. Matthes et al. (Biochem Biophys Res., Commun., 153, 825 [1988]). Dabeizeigtesich.daßz. For example, 2 ', 3'-dideoxy-3-fluoro-5-methylcytidine has an ID ₆₀ of 1δ0μΜ over the T-cell line MT4. In another test investigating possible growth inhibition of murine bone marrow colony-forming cells (CFU-GM), E. Matthes et al. (Biochem Biophys Res. Commun. [1989] in press), this compound also proved to be hardly proliferative inhibiting (ID ₆₀ > 5 χ 10 ' ⁴ M).

embodiments

I.Beispiel

injection

From 2 ', 3'-dideoxy-3'-fluoro-5-methylcytidine and physiological saline prepares the required amount of a 0.3% solution.

2nd example tablets or dragees

Powdered 2 ', 3'-dideoxy-3'-fluoro-5-methylcytidine is mixed with one or more of the usual excipients such as starch, talc, magnesium stearate, K-stearate, stearic acid, paraffin, cetyl alcohol, pectin, sucrose, gum arabic , Dextrin processed into tablets or dragees.

3rd example

Preparation of 2 ', 3'-dideoxy-3'-fluoro-5-fluorocytidine

0.12 mmol of 5'-O-acetyl-2 ', 3'-dideoxy-3', 5-difluorouridine are dissolved in 3 ml of pyridine with 34.5 mg (0.5 mmol) of 1,2,4-triazole and 86 mg (0 , 35 mmol) p-Chlorphenoxyphosphorsäuredichlorid added. The reaction solution remains at room temperature for one week. Subsequently, 15 ml of dioxane and 5 ml of concentrated ammonia solution are added. After 20 hours, the solvent is i.Vak. away. The remaining brown residue is pelöst in 3 ml of water and purified on an ion exchanger (5m DOWEX 5OW χ 8, H ⁺ form, 50-100mesh). First it is eluted with water (420ml) followed by ammonia solution (3%, 400ml). The ammonia-alkaline solution is concentrated to dryness and the residue is separated by column chromatography on 10 g of silica gel 40 (0.063-0.2 mm, MERCK). From the appropriate fractions, 10 mg of the title compound are obtained.

F.219 ° C (methanol).

UV (water) pH7max283nm (8400);

pH 1 max 294 nm (9450); MS: m / z 247 (JVr ₁ C ₉ H _n N ₃ O ₃ F ₂ );

129 (base, C ₄ H ₄ N ₃ OF);

119 (sugar residue, C ₆ H ₈ O ₂ F).

4th example

Preparation of 2 ', 3'-dideoxy-3'-chloro-5-methyl-cytidine

6 mmol of O'-O-acetyl-S'-chloro-S'-deoxy-thymidine are dissolved in 100 ml of pyridine, with 1.57 g (22.7 mmol) of 1,2,4-triazole and 4.4 g (17.9 mmol) The reaction mixture remains at room temperature for one week, then add SO ml of dioxane and 20 ml of concentrated ammonia solution and the reaction solution is allowed to stand for 24 hours

Stored at room temperature. After the solvent has been expelled, the residue is first added to 100 ml DOWEX 50 W χ 8

(H ⁺ form) and then separated by 50 g of silica gel 40 (MERCK) by column chromatography. From the ensprechenden

Fractions 2 ', 3'-dideoxy-3'-chloro-5-methyl-cytidine is obtained as the hydrochloride. F. 203-205 ° C (methanol). MS: m / z 259 (M ⁺ ₁₀ H ₁₄ N ₃ O ₃ Cl);

135 (sugar residue, C ₅ H ₈ O ₂ Cl);

125 (base + H ₁ C ₆ H ₇ N ₃ O)

UV (H ₂ O) pH 7max 278.5nm,

pH 1 max 287.0 nm.

5th example

Preparation of 5-bromo-3'-chloro-2 ', 3'-dideoxy-cytidine From 4mMol ö'-O-acetyl-o-bromo-S'-chloro '' a'-dideoxy-uridine is analogous to the angeg) in the 3rd Example added the procedure Title compound prepared and isolated as the hydrochloride. Yield 185mg (15%). MS: m / z 324 (M ⁺ , C ₉ H _n N ₃ O ₃ BrCl);

148 (base + H, C ₄ H ₄ OBr);

135 (sugar residue, C ₆ H ₈ O ₂ Cl).

6th example

Preparation of 2 ', 3'-didehydro-2', 3'-dideoxy-5-methylcytidine

5.5 g (11.3 mmol) of 4-N-benzoyl-2'-deoxy-3 ', 5'-di-O-mesyl-cytidine are dissolved in 350 ml of ethanol. While stirring, 37.5 ml of 1 N NaOH and 87 ml of water are added. The reaction solution is 2h. heated to boiling. After cooling, it is evaporated to dryness and the resulting residue is extracted with hot acetone (5 × 50 ml). The solvent is i. Vak. away. There remains a yellow oil, which is dissolved in 30 ml of DMSO and treated with 1.2 g (10.8 mmol) of potassium tert-butylate. The mixture is 20h. left at room temperature. Subsequently, the DMSO is largely distilled off, the residue dissolved in water and brought to pH 8 to pH 9 with acetic acid. The residue obtained after the solvent has been removed by evaporation is purified by column chromatography on silica gel 40 (MERCK) with chloroform (10% methanol) as eluent.

UV (H ₂ O) pH 7 max 278 nm; pH1 max 289 nm

MS: m / z 223 (M ⁺ , C ₆ H ₇ N ₃ O).

Claims (26)

claims: 1. A process for the preparation of an agent for the treatment or prophylaxis of hepatitis infections in humans and animals, characterized in that substituted nucleosides of the formula I. in the R ¹ : F, Cl, Br, I, CH ₃ , CH ₂ OH R ² : 2,3-dideoxyribofuranosyl, 2,3-dideoxy-3-fluororibofuranosyl, 2,3-dideoxy-3-chlororibofuranosyl, 2,3-dideoxy-3-azidoribofuranosyl, 2,3-dideoxy-3-aminoribofuranosyl, 2 , 3-didehydro-2,3-didesoxyribofuranosyl, arabinofuranosyl, 3-deoxy-3-fluoroarabinofuranosyl, or their 5-O-acetyl, 5-O-palmitoyl, 5-O-alkoxycarbonyl derivatives, or Mono-, 5-di- or 5-triphosphates, as free acids or alkali metal, ammonium or alkylammonium salts, or other precursor groups for the 5-hydroxyl group mixed with pharmaceutically acceptable carriers. 2. The method according to claim 1, characterized in that one adapts the compounds of general formula I for administration by injection. 3. The method according to claim 1, characterized in that one adapts the compounds of general formula I for administration by means of infusion. 4. The method according to claim 1, characterized in that one adapts the compounds of general formula I for oral administration. 5. Process according to Claims 2-4, characterized in that one converts compounds of general formula I in the form of a dragee, a tablet, a capsule, in the powder or granular form. 6. Process according to Claims 1, 4 and 5, characterized in that, if appropriate, a flavoring agent is added. 7. The method according to claim 1, characterized in that one converts the compounds of general formula I in the form of a suppository. 8. Process according to Claims 1-7, characterized in that is used as the compound of general formula 12 ', 3'-dideoxy-5-fluorocytidine. 9. The method according to claims 1-7, characterized in that is used as the compound of the general formula I 2 ', 3'-Οίαβ8θχν-5-ητΐθ ^ νΙ-ονΜΐη. 10. The method according to claims 1-7, characterized in that is used as the compound of general formula 12 ', 3'-dideoxy-5-hydroxy-methylcytidine. 11. The method according to claims 1-7, characterized in that is used as the compound of general formula 12 ', 3'-dideoxy-3'-fluoro-5-fluorocytidine. 12. The method according to claims 1-7, characterized in that is used as the compound of general formula I 2 ', 3'-dideoxy-3'-fluoro-5-methylcytidine. 13. Process according to Claims 1-7, characterized in that the compound of the general formula 12 ', 3'-dideoxy-3'-fluoro-5-hydroxymethylcytidine is used. 14. The method according to claims 1-7, characterized in that is used as the compound of general formula 12 ', 3'-didecxy-3'-chloro-5-methylcytidine. 15. The method according to claims 1-7, characterized in that is used as the compound of general formula 12 \ 3'-dideoxy-3'-chloro-5-bromocytidine. 16. The method according to claims 1-7, characterized in that is used as the compound of general formula 12 ', 3'-dideoxy-3'-chloro-5-chlorocytidine. 17. The method according to claims 1-7, characterized in that is used as the compound of general formula 12 ', 3'-dideoxy-3'-azido-5-methylcytidine. 18. The method according to claims 1-7, characterized in that is used as the compound of general formula 12 ', 3'-dideoxy-3'-azido-5-hydroxymethylcytidin. 19. The method according to claims 1-7, characterized in that is used as the compound of general formula 12 ', 3'-dideoxy-3'-azido-5-bromocytidine. 20. The method according to claims 1-7, characterized in that is used as the compound of general formula 12 ', 3'-dideoxy-3'-amino-5-methylcytidine. 21. Process according to Claims 1-7, characterized in that 2 ', 3'-didehydro-2', 3'-dideoxy-5-methylcytidine is used as the compound of general formula I. 22. The method according to claims 1-7, characterized in that is used as the compound of the general formula 12 ', 3'-didehydro-2 \ 3'-dideoxy-5-fluorocytidine. 23. The method according to claims 1-7, characterized in that is used as the compound of the general formula I arabinofuranosyl-5-methylcytosine. 24. The method according to claims 1-7, characterized in that is used as the compound of the general formula I arabinofuranosyl-5-iodocytosine. 25. Process according to Claims 1-7, characterized in that 13'-deoxy-3'-fluoroarabino-furanosyl-5-methylcytosine is used as compound of the general formula. 26. The method according to claims 1-7, characterized in that is used as the compound of the general formula 13'-deoxy-3'-fluoroarabino-furanosyl-5-iodocytosine.