Beta-L-N4 Hydroxycytosine Deoxynucleosides and their use as Pharmaceutical Agents in the Prophylaxis or Therapy of Viral Diseases

The invention relates to ss-L-N4-hydroxycytosine nucleo-sides, pharmaceutical agents comprising same, and to the use of said ss-f31 L-N4-hydroxycytosine nucleosides and pharmaceutical agents in the prophylaxis or therapy of an infection caused by hepatitis B virus (HBV) or human immunodeficiency virus (HIV). The invention also relates to a method for the preparation of said ss-L-nucleoside and analogs.

EXAMPLES

1. Synthesis of 4-hydroxyaminopyrimidin-2(1H)-one β-L-nucleosides from the corresponding uracil or thymine nucleosides

1-(2,3-Di-O-benzoyl-2-deoxy-β-L-ribofuranosyl)uracil (1.3 g, 2.98 mmol) was dissolved in triethylamine (1.8 ml, 12.9 mmol) and anhydrous acetonitrile (70 ml). The solution was cooled to 0° C. in an argon atmosphere and mixed with 2,4,6-triiso-propylbenzenesulfonyl chloride (1.95 g, 6.3 mmol) and 4-dimethylaminopyridine (300 mg, 2 mmol). The reaction mixture was left at room temperature overnight with stirring. Subsequently, hydroxylamine hydrochloride (450 mg, 6.47 mmol) was added and the reaction solution was stirred at room temperature for 24 hours. Thereafter, water (50 ml) and chloroform (75 ml) were added. The organic phase was washed with saturated sodium chloride solution and dried over sodium sulfate. The residue obtained after removing the solvent in vacuum was purified by means of column chromatography on silica gel, using chloroform/methanol (98/2, v/v) as eluent. 1-(2,3-Di-O-benzoyl-β-L-ribofuranosyl)-4-hydroxyamino-pyrimidin-2(1H)-one was isolated from the corresponding fractions as a white amorphous mass (1.7 g).

The above amount of substance was added to ammonia-saturated methanol (20 ml). The reaction solution was left for 24 hours at room temperature and was subsequently concentrated to dryness in vacuum. The residue was purified by means of column chromatography on silica gel, using a chloroform/methanol (9/1, v/v) mobile phase. 1-(2-Deoxy-β-L-ribofuranosyl)-4-hydroxyaminopyrimidin-2(1H)-one was ob-tained from the corresponding fractions and crystallized from methanol/ether (yield: 232 mg, 0.94 mmol, 31.6%).

According to the general synthetic method described above and starting from 1-(3,5-di-O-acetyl-2-deoxy-β-L-ribofura-nosyl)thymine (500 mg, 1.53 mmol), β-L-5-methyl-N4-hydroxy-deoxycytidine was obtained (132 mg, 0.5 mmol, 32%).

β-L-5-Fluoro-2′-deoxyuridine was prepared according to established methods for the synthesis of the corresponding D-derivative (Ozaki et al., Bull Chem Soc Japan 1977, 50: 2197-2198).

A stirred solution of 1-(5-O-acetyl-2-deoxy-β-L-ribo-furanosyl)-5-fluorouracil (288 mg, 1 mmol) in anhydrous acetonitrile (30 ml) under an argon atmosphere was cooled to 0° C. To this solution were successively added 2,4,6-triisopropyl benzenesulphonyl chloride (654 mg, 2.1 mmol) and 4-dimethylaminopyridine (132 mg, 1 mmol). The resulting mixture was stirred for 20 h at room temperature. Solid hydroxylamine hydrochloride (149 mg, 2.1 mmol) was added and the mixture was stirred for an additional 24 h. The mixture was partitioned between water (25 ml) and chloroform (100 ml). The organic layer was washed with a saturated aqueous sodium chloride solution (30 ml), dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting residue was purified by column chromatography on silica gel eluting with a gradient of methanol (0-10%) in chloroform to afford 1-(5-O-acetyl-2-deoxy-β-L-ribofuranosyl)-5-fluoro-4-hydroxyaminopyrimidin-2(1H)-one as a white solid (138 mg, 0.45 mmol). A solution of this compound in methanol saturated with ammonia at 0° C. was kept for 24 h at room temperature. After removing of the solvent under reduced pressure the residue was purified by column chromatography on silica gel with chloroform/methanol (9/1, v/v) as eluent to afford 1-(2-deoxy-β-L-ribofurano-syl)-5-fluoro-4-hydroxyaminopyrimidin-2(1H)-one (94 mg, 036 mmol) as a white solid.

β-L-2′,3′-Didehydro-2,3′-dideoxy-5-fluorouridine was prepared according to established methods described for the synthesis of the corresponding D-derivative (Joshi et al., J Chem Soc Perkin Trans 11992, 2537-2544). This compound was acetylated in the ususal manner with acetanhydride in pyridine and purified by column chromatography. The isolated product was activated with 2,4,6-triisopropyl benzenesulphonyl chloride and 4-dimethylaminopyridine, then reacted with solid hydroxylamine hydrochloride as described in example 1.3. The reaction product was purified by column chromatography to afford the acetylated N4 hydroxycytidine derivative.

A solution of 1-(5-O-acetyl-2,3-dideoxy-β-L-glycero-pent-2-enofuranosyl)-5-fluoro-4-hydroxyaminopyrimidin-2(1H)-one

The product of that reaction was deacetylated by treatment with a solution of ammonia in methanol (saturated at 0° C.) for 24 h. The solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel eluting with chloroform/methanol(95/5,v/v). 1-(2,3-dideoxy-β-L-glyceropentofuranosyl)-5-fluoro-4-hydroxyamino-pyrimidin-2(1H)-one was afforded as a white foam (67 mg, 0.27 mmol).

β-L-2′,3′-Didehydro-2′,3′-dideoxyuridine was prepared according to established methods described for the synthesis of the corresponding D-derivative (Horwitz et al., J Org Chem 1966, 31:205-211).

This solution was stirred for 24 h at room temperature. Hydroxylamine hydrochloride (149 mg, 2.1 mmol) was then added, and the mixture was further stirred for 1 day at room temperature. Water (25 ml) was added, and the product was extracted with chloroform (100 ml). The organic layer was washed with a aqueous solution saturated with sodium chloride (30 ml), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.

The resulting residue was purified by column chromatography on silica gel eluting with a gradient of methanol (0-5%) in chloroform to give 1-(5-O-acetyl-2,3-dideoxy-β-L-glycero-pent-2-enofuranosyl)-4-hydroxyaminopyrimidin-2(1H)-one as a white foam. This compound was concentrated vacuo, the residue was purified by column chromatography on silica gel eluting with chloroform/methanol(95/5, v/v) to afford 1-(2,3-dideoxy-β-L-glycero-pent-2-enofuranosyl)-4-hydroxyaminopy-rimidin-2(1H)-one (79 mg, 0.35 mmol).

Synthesis of 1-(2,3-dideoxy-β-L-glycero-pentofuranosyl)-4-hydroxyaminopyrimidin-2(1H)-one (β-L-2′,3′-dideoxy-N4-hydroxycytidine) β-L-2′,3′-Didehydro-2′,3′-dideoxyuridine was prepared according to established methods described for the synthesis of the corresponding D-derivative (Horwitz et al., J Org Chem 1966, 31:205-211).

This deoxyuridine derivative was acetylated with acetanhydride in pyridine. The reaction product was purified by column chromatography. The isolated derivative was activated with 2,4,6-triisopropyl benzenesulphonyl chloride and 4-dimethylaminopyridine in acetonitrile, then hydroxylamino chloride was added and the reaction mixture was worked up as de-scribed in example 1.3. After evaporation of the solvent the acetylated hydroxycytidine derivative was purified by column chromatography.

A solution of 1-(5-O-acetyl-2,3-dideoxy-β-L-glycero-pent-2-enofuranosyl)-4-hydroxyaminopyrimidin-2(1H)-one (267 mg, 1 mmol) in dioxane containing 125 mg of 10% palladium-charcoal catalyst was shaken with 1 atm. of hydrogen at room temperature. The theoretical uptake of hydrogen was realized in 0.5 h, the catalyst was filtered, and the filtrate was evaporated to dryness.

The residue was treated with methanol/ammonia (25 ml) overnight at room temperature. After removing the solvent the corresponding residue was purified by column chromatography on silica gel with chloroform/methanol (9/1, v/v) as solvent to afford 1-(2,3-dideoxy-β-L-glycero-pentofuranosyl)-4-hydroxyaminopyrimidin-2(1H)-one (105 mg, 0.46 mmol) as a solid.

β-L-2′,3′-Didehydro-2′,3′-dideoxy-5-fluorouridine was prepared according established methods for synthesis of the corresponding D-derivative (Joshi et al., J Chem Soc Perkin Trans 11992, 2537-2544).

In a similar manner as described under example 1.5 using 1-(5-O-acetyl-2,3-dideoxy-β-L-glycero-pent-2-enofuranosyl)-5-fluorouracil (252 mg, 1 mmol) as starting material, the tit-le compound I-(2,3-dideoxy-β-L-glycero-pent-2-enofuranosyl)-5-fluoro-4-hydroxyaminopyrimidin-2(1H)-one was obtained (69 mg, 0.33 mmol).

2′,3′-Didehydro-2′,3′-deoxy-β-L-thymidine (β-L-thymidinene) was prepared according to established methods described for synthesis of corresponding D-derivative (Horwitz et al., J Org Chem 1966, 31: 205-211). β-L-thymidinene was acetylated in the usual manner with acetanhydride in pyridine. 5′-O-acetyl-2′,3′-didehydro-2′,3′-deoxy-β-L-thymidine (266 mg, 1 mmol) was subjected to the same sequence of reaction steps as described in the example 1.5 to afford 1-(2,3-dideoxy-β-L-glycero-pent-2-enofuranosyl)-5-methyl-4-hydroxy-aminopyrimidin-2(1H)-one (132 mg, 0.55 mmol).

2. Synthesis of 4-hydroxyaminopyrimidin-2(1H)-one β-L-nucleosides from the corresponding cytosine nucleosides

β-L-2′,3′-Dideoxy-3′-thiacytidine was synthesized as described (Beach et al., J Org Chem 1992, 57: 2217-2219). 500 mg (2.18 mmol) of it was mixed with a 7 M hydroxylamine hydrochloride solution (25 ml). The reaction solution was kept at room temperature for four days with stirring. Following removal of the solvent in vacuum, the resulting residue was purified by means of column chromatography on silica gel, using the upper phase of the mixture ethyl acetate/isopropanol/water (4/1/2, v/v/v) as eluent.

The solvent of the corresponding fractions was removed in vacuum. β-L-2′,3′-dideoxy-3′-thia-N4-hydroxycytidine was obtained from the methanol solution of the residue (yield: 95 mg, 0.39 mmol, 17.9%).

400 mg (1.76 mmol) of this compound was dissolved in 10 ml of 5 M hydroxylamine hydrochloride which had been adjusted to pH 6.0 with sodium hydroxide. The solution was stirred for 24 h at room temperature, and the solvent was removed under reduced pressure.

The residue was purified by column chromatography on silica gel with chloroform/methanol (9/1, v/v) as eluent to afford 1-(2,3-dideoxy-2-fluoro-β-L-glycero-pent-2-enofuranosyl)-4-hydroxyaminopyridin-2(1H)one (83 mg, 0.34 mmol, yield 19.3%).

78 mg (0.31 mmol) of this compound was shaken for 24 h in 2 ml of aqueous 5 M hydroxylamine hydrochloride (adjusted to pH 6.0).

The solvent was removed in vacuo, and the residue was purified by column chromatography on silica gel eluting with chloroform/methanol (9/1, v/v). From the corresponding fractions β-L-[2-(hydroxymethyl)-1,3-oxa-thiolan-4-yl]-5-fluoro-4-hydroxyaminopyridin-2(1H)-one was isolated as a foam (14 mg, 0.05 mmol, yield 16%).

β-L-3′-Azido-2′,3′dideoxycytidine was prepared according to established methods described for the synthesis of the corresponding D-derivative. 300 mg, (1.2 mmol) of this corn-pound was dissolved in 10 ml aqueous 5 M hydroxylamine hydrochloride (adjusted to pH 6.0) and treated according example 2.2. The title compound was obtained as a white solid (103 mg, 0.38 mmol, yield 31.6%).

β-L-3′-Azido-2′,3′dideoxy-5-fluorocytidine was prepared according to established methods described for the synthesis of the corresponding D-derivative (Sandstrom et al., Drugs 1986, 31: 462-467).

500 mg (1.85 mmol) of this compound were treated as described in example 2.2. The title compound β-L-3′-azido-2′,3′-dideoxy-5-fluoro-N4-hydroxycytidine (121 mg, 0.42 mmol, yield 22.7%) was obtained.

β-L-3′-Azido-2′,3′dideoxy-5-methylcytidine was prepared according to established methods described for the synthesis of the corresponding D-derivative (Lin et al., J Med Chem 1983, 26: 544-551).

This compound (350 mg, 1.52 mmol) gave according to the synthetic method described in example 2.2, 1-(2,3-dideoxy-3-fluoro-β-L-ribofuranosyl)-4-hydroxyaminopyridin-2(1H)-one (137 mg, 0.56 mmol, yield 36.8%) as a solid.

3. Inhibition of HBV-Replication by the Compounds of Inven-tion in HepG2 2.2.15 Cells

The antiviral efficacy of the compounds of the invention was investigated on HepG2 2.2.15 cells, a human hepatoblastoma cell line which has the replication-competent HBV genome stably integrated therein and produces infectious progeny viruses in a productive manner (Sells et al., Proc Natl Acad Sci USA 1987, 84: 1005-1009).

The above cells were cultured under standardized conditions as specified by Korba and Gerin, and the amount of extracellular viral DNA was determined (Korba et al., Antiviral Res 1992, 19: 55-70).

Following passaging, the HepG2 2.2.15 cells were seeded at a density of about 60% in 12-well plates and cultured to confluence in 10% FBS Dulbecco MEM. Thereafter, the medium was changed to 2% FBS, and the cells were cultured for another 24 hours.

After another change of medium, the cells were treated with varying concentrations of compounds according to the invention. Every 24 hours the compounds were re-added together with the medium. On the 6thday of treatment, the cell supernatants were centrifuged off and stored at −20° C. until analysis of the HBV DNA was effected.

Following treatment of the culture supernatants with proteinase K, the extracellular viral DNA was amplified by means of PCR using the following primers (forward: 5′-CTC CAG TTC AGG AAC AGT AAA CCC-3′(SEQ ID NO. 1); reverse: 5′-TTG TGA GCT CAG AAA GGC CTT GTA AGT TGG CG-3′(SEQ ID NO. 2). The PCR products were separated on 1% agarose, stained with ethidium bromide and quantified using a Fluor-S™ Multimager (Biorad).

For calibration of the PCR reaction, serial dilutions of the pUC19 HBV and pTHBV plasmids with known genome equivalents (GE) were used, resulting in a lower detection limit of about 103GE and a linearity between 103and 105GE. Table 1 shows the concentrations of compounds of the invention required for 50% reduction of extracellular HBV DNA (ED50) after 6 days incubation of the HepG2 2.2.15 cells.

Between the new compounds β-L-2′,3′-didehydro-2′,3′-dide-oxy-N4-hydroxycytidine (L-HyddeC), β-L-2′,3′-didehydro-2′,3′-dideoxy-5-fluoro-N4-hydroxycytidine (L-HyddeFC) and β-2′,3′-didehydro-2′,3′-dideoxy-2′-fluoro-N4-hydroxycytidine (L-HyFddeC) were the most effective nucleoside with EC50-values of <0.1 μM.

A third group of compounds of the invention with EC50-values between 3 and 50 μM includes β-L-N4-hydroxydeoxycy-tidine (L-HyCdR), β-L-5-fluoro-N4-hydroxydeoxycytidine (L-HyFCdR), β-L-5-methyl-N4-hydroxydeoxycytidine (L-HyMetCdR), β-L-3′-fluoro-2′,3′-dideoxy-N4-hydroxycytidine (L-FHyCdR), and β-L-3′-azido-2′,3′-dideoxy-N4-hydroxycytidine (L-N3HyCdR).

It can be argued that the N-4-hydroxy-group of the β-L-cy-tidine derivatives could be metabolized inside of cells to the corresponding NH2-group. Such a reaction could suggest a prodrug function of the presented analogues. In this case Hy3TC as the prodrug of 3TC should also display a high efficiency against HIV because 3TC inhibits the HIV-replica-tion at a EC50of 0.002 μM (Schinazi et al., Antimicrob Agents Chemother 1992, 38: 2423-2431).

However, we found that Hy3TC is inactive against HIV replication (EC50>>25 μM) ruling out the possibility that the metabolic conversion of the NHOH-group to the NH2-group could be the reason for its anti-HBV activity.

4. Inhibition of HBV DNA Polymerase by β-L-N4-Hydroxycytosine Nucleoside Triphosphates

Synthesis and purification of the triphosphates of β-L-N4-hydroxycytosine nucle sides were performed according to well-known methods (Yoshikawa et al., Tetradedron Lett 1967, 50:5065-5068; Hoard et Ott, J Am Chem Soc 1965, 87: 1785-1788).

To determine the endogenous HBV DNA polymerase activity, about 100 ml of serum from patients with untreated hepatitis B virus infections from Charité, Berlin, (>107HBV particles/ml), was centrifuged at 3000 rpm. Virus particles of the cleared serum were sedimented in a Beckman SW28 rotor at 25,000 rpm, 60 min. The virus pellet was suspended in 7 ml of TKM buffer (50 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM MgCl2), layered over a step gradient of 10 ml each of 0.3 M, 0.6 M, 0.9 M saccharose in TKM buffer and centrifuged at 25,000 rpm for 20 hours.

The purified virus pellet was suspended in 250 μl TKM buffer, lysed by ultrasound, divided in aliquots and frozen at −80° C. (Davies et al., Antiviral Res 1996, 30: 133-145).

Following a one-hour incubation at 37° C., 20 μl of the assay volume was placed on paper filter, washed 5 times with 5% trichloroacetic acid and 0.1% Na pyrophosphate, and the3H-dCMP incorporated in the HBV DNA was subsequently measured in a Liquid Scintillation Counter.

Using the concentration-dependent inhibition curves of HBV DNA synthesis, the concentration of β-L-N4-hydroxycytosine nucleoside triphosphates resulting in 50% inhibition of the HBV DNA polymerase activity was determined.

Table 2 demonstrates that the HBV DNA polymerase is inhibited strongly by the triphosphates of L-Hy3TC, L-HyddC and L-HyddeC (IC50between 0.15 and 0.65 μM) pointing out that the 4-NHOH-group of the cytosine nucleoside triphosphates is effective at the target and does not require a previous metabolization to the NH2-group.

To this end, established cells of a human myeloid leukemia (HL-60) in RPMI medium, and the above-mentioned HepG2 cells in Dulbecco MEM, respectively, were incubated for two days using varying concentrations of compounds, and the proliferation rate of the cells was subsequently determined. The data were used to determine the concentration of compounds resulting in 50% inhibition of proliferation (CD50). Table 3 shows that the new compounds display no antiproliferative activity on HepG2- and HL-60 cells.

Thus these data further support our suggestion that the NH2-group could not be formed inside of cells from our β-L-N4-hy-droxycytosine nucleoside analogues.

The state of the art may disclose more common empirical formulae, which do not however describe the special, chosen chemical compounds of the doctrine according to the application. Those special, precise compounds of the invention had not yet been made accessible in the form of common terms and conceptions, since it was not possible to generate exactly the compound of the invention only by conducting routine experiments; those compounds show surprising, unobvious characteristics, for example the fact that hitherto all efforts of experts in this matter were in vain, a different approach to the development of scientific technology, the achievement forwards the development, misconceptions about the solution of the according problem (prejudice), technical progress (such as: improvement, increased performance, price-reduction, saving of the time, material, work steps, costs or resources that are difficult to obtain, improved reliability, remedy of defects, improved quality, increased efficiency, augmentation of technical or medical possibilities, provision of another product, spare product, alternatives, enrichment of the pharmaceutical fund), a special choice (since a certain possibility, the result of which was unforeseeable, was chosen among a great number of possibilities).

The precise, claimed chemical compounds of the application have not yet been disclosed in greater fields that are comprised by a common formula. The precisely chosen compounds of the invention are not arbitrarily chosen specimen, but it is rather selective choice that leads to products with the above-mentioned surprising characteristics.