HK1038189B - A method for preparation of a pharmaceutical composition comprising 1, 3-oxathiolane nucleoside analogues - Google Patents

Description

Process for preparing pharmaceutical composition containing 1, 3-oxathiolane nucleoside analogue

The invention relates to nucleoside analogues and their use in medicine. More particularly, the invention relates to 1, 3-oxathiolane nucleoside analogues, pharmaceutical compositions thereof and their use in the treatment of viral infections.

The only currently approved compound for the treatment of HIV-induced disorders is 3 '-azido-3' -deoxythymidine (AZT, zidovudine, BW 509U). However, this compound has a significant side effect, and therefore the compound is either unusable or the compound has to be withdrawn from many patients once it is used, with the result that there is a continuing need to provide compounds that are both effective against HIV and have significantly better therapeutic indices.

A compound of formula (I)

Is a racemic mixture of two enantiomers as (I-1) and (I-2):

we have unexpectedly found that the (-) enantiomer of the compound of formula (I) is much more active than the (+) enantiomer, although both enantiomers show unexpectedly low cytotoxicity. It is therefore a first aspect of the present invention to provide an enantiomer of a compound of formula (I) (-) (or levorotatory) and pharmaceutically acceptable derivatives thereof.

The chemical name of the (one) -enantiomer is (one) -4-amino-5-fluoro 1- (2-hydroxymethyl-1, 3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one (hereinafter referred to as compound (A)). The enantiomer has an absolute stereochemistry as shown in formula (I-1).

Preferred compounds (A) are provided which are substantially free of the corresponding (+) -enantiomer. That is, no more than about 5% (W/W), more preferably no more than about 2%, and most preferably less than about 1% (W/W) of the (+) -enantiomer is present.

"a pharmaceutically acceptable derivative" is any pharmaceutically acceptable salt, ester, or salt of the ester of compound (a) or of any other compound which when administered to a recipient has the ability to provide (directly or indirectly) compound (a) or a metabolite or residue thereof having an antiviral activity.

One skilled in the art will recognize that compound (a) may be modified to provide a pharmaceutically acceptable derivative thereof at a site such as a base moiety or a functional group such as a hydroxymethyl group on the oxathiolane ring. All such modifications on functional groups are included within the scope of the present invention. Of particular interest, however, are pharmaceutically acceptable derivatives obtained by modification of the 2-hydroxymethyl group of the oxathiolane ring.

Preferred esters of the compounds (A) include those in which the hydrogen of the 2-hydroxymethyl group is replaced by an acyl function(iii) a substitution wherein the non-carbonyl moiety R in the ester is selected from the group consisting of: hydrogen, straight or branched chain alkyl (e.g., methyl, ethyl, n-propyl, t-butyl, n-butyl), alkoxyalkyl (e.g., methoxymethyl), arylalkyl (e.g., benzyl), aryloxyalkyl (e.g., phenoxymethyl), aryl (e.g., optionally substituted with halogen, C)_1—4Alkyl or C_1—4Alkoxy-substituted phenyl); sulfonates such as alkyl or aralkylsulfonyl (e.g., methanesulfonyl); amino acid esters (e.g., L-valyl or L-isoleucyl) and mono-, di-, or tri-phosphate esters.

Unless otherwise specified, in the esters described above, any alkyl moiety advantageously contains from 1 to 16 carbon atoms, in particular from 1 to 4 carbon atoms. Any aryl moiety advantageously includes a phenyl group.

In particular the ester may be C_1—16Alkyl esters, unsubstituted benzyl esters or substituted by at least one halogen (bromo, chloro, fluoro or iodo), C_1—6Alkyl radical, C_1—6Alkoxy, nitro or trifluoromethyl substituted benzyl esters.

Pharmaceutically acceptable salts of compound (a) include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, p-toluenesulfonic, tartaric, acetic, citric, methanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic and benzenesulfonic acids. Other acids, such as oxalic, while not pharmaceutically acceptable by themselves, may be useful intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts.

Salts derived from suitable bases include gold hydroxideMetal (e.g., sodium) salts, alkaline earth metal (e.g., magnesium) salts, ammonium salts and NR₄ ⁺Quaternary ammonium salts (wherein R is C_1—4Alkyl groups).

The compounds according to the invention mentioned hereinafter include both compound (a) and its pharmaceutically acceptable derivatives.

The compounds of the invention have antiviral activity themselves and/or are metabolisable to give such compounds. In particular, these compounds are effective in inhibiting the replication of retroviruses, including human retroviruses such as the HIV-causing virus, human immunodeficiency virus (HIV' S).

The compounds of the present invention are also useful in the treatment of animals, including humans, infected with Hepatitis B Virus (HBV).

Thus a second aspect of the present invention provides compounds (a) or pharmaceutically acceptable derivatives thereof, for example, for use as an effective therapeutic agent, especially as an antiviral agent, in the treatment of retroviral infections or Hepatitis B Virus (HBV) infections.

In a further or alternative aspect, the present invention provides a method of treatment of viral infections, particularly infections caused by HBV or retroviruses such as HIV in mammals including humans, which comprises administering an effective amount of compound (a) or a pharmaceutically acceptable derivative of said compound.

Further, the present invention provides the use of compound (a) or one of its pharmaceutically acceptable derivatives for the manufacture of a medicament for the treatment of viral infections.

The compounds of the present invention are also useful in the treatment of AIDS-related disorders such as AIDS-related complex (ARC), progressive diffuse lymphadenopathy (PGL), AIDS-related neurological disorders such as dementia or tropical paraparesis, anti-HIV antibody-positive and HIV-positive disorders. It is also useful in the treatment of Kaposi's sarcoma, thrombocytopenic purpura, and opportunistic-related infections such as Pneumocystis carinii.

The compounds of the invention are also useful in preventing the rise of clinical conditions in individuals who are positive for anti-HIV antibodies or HIV-antigens in the prevention of exposure to HIV.

The compound (a) or a pharmaceutically acceptable derivative thereof can also be used for the in vitro prevention of viral infection of physiological body fluids such as blood or semen.

The compounds of the present invention are also useful in the treatment of animals, including humans, infected with hepatitis b virus.

One skilled in the art will recognize that the treatment referred to herein may be extended to prophylaxis as well as treatment of established infections and conditions.

It will further be appreciated that the amount of a compound of the invention to be used in therapy will vary not only with the compound selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will ultimately be determined by the attending physician or veterinarian. However, generally, a suitable dosage range is from about 0.1 to about 750mg/kg body weight per day, preferably from 0.5 to 60 mg/kg/day, and most preferably from 1 to 20 mg/kg/day.

The required dose may conveniently be presented daily at appropriate intervals in a single dose or in divided doses, for example two, three, four or more small doses.

The compounds may conveniently be administered in unit dosage form, for example containing from 10 to 1500mg, conveniently from 20 to 1000mg, preferably from 50 to 700mg, of active ingredient per unit dosage form.

Ideally, the active ingredient should be administered to achieve peak plasma concentrations of the active compound of about 1 to about 75 μ M, preferably about 2 to 50 μ M, and most preferably about 3 to 30 μ M. This can be obtained by the following method: solutions of 0.1 to 5% active ingredient are injected intravenously, as the case may be, in saline solution, or in boluses containing about 1 to 100mg of active ingredient are administered orally. The desired blood concentration is maintained by continuous infusion to provide about 0.01 to about 5.0 mg/kg/hour or by intermittent infusion containing about 0.4 to about 15mg/kg of the active ingredient.

Although the compounds of the invention may be crude chemicals for use in therapy, it is desirable that the active ingredient is present in the form of a pharmaceutical composition.

The present invention therefore further provides a pharmaceutical composition comprising compound (a) or a pharmaceutically acceptable derivative thereof together with one or more pharmaceutically acceptable carriers and optionally other therapeutic and/or prophylactic ingredients. A carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

Pharmaceutical compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation. The compositions may conveniently be presented in discrete dosage units where appropriate, and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the active compound with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.

Pharmaceutical compositions adapted for oral administration may conveniently be presented in discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; or making into powder or granule; making into solution, suspension or emulsion, or making into pill, medicated candy or paste. Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, disintegrants or wetting agents. The tablets may be coated according to methods well known in the art. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may contain edible oils), or preservatives.

The compounds according to the invention may also be formulated for parenteral administration (e.g. by injection, e.g. bolus injection or continuous infusion) and may be presented in unit dosage form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or emulsifying agents. Alternatively, the active ingredient may be powdered by aseptic separation from solution or by aseptic solidification or lyophilization, and formulated with a stable carrier liquid such as sterile, pyrogen-free water prior to use.

For topical administration to the epidermis the compounds according to the invention may be formulated as ointments, creams or lotions or as transdermal patches. Ointments or creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.

Compositions suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; a mouthwash comprising the active ingredient in a suitable liquid carrier.

Suitable carriers for rectal administration are solid pharmaceutical compositions, most preferably in the form of unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art, suppositories may be conveniently prepared by mixing the active compound with the softened or melted carrier and then freezing and moulding in moulds.

Compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be suitable.

The compounds of the invention for intranasal administration may be used as liquid sprays or dispersible powders or in the form of drops.

Drops may be formulated as aqueous or non-aqueous solutions and may also contain one or more dispersing, solubilising or suspending agents. Liquid sprays are conveniently released from pressurised containers.

For administration by inhalation, the compounds according to the invention may conveniently be delivered from an insufflator, nebulizer or pressurised container or other convenient means of delivering an aerosol spray. The pressurized container may contain a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by a valve which releases a metered amount.

Alternatively, for administration by inhalation or insufflation, the compounds according to the invention may take the form of a dry powder composition, for example a powder mix consisting of the compound and a suitable powder base such as lactose or starch. The powder compositions may be presented in unit dosage form in, for example, capsules or cartridges, or in, for example, gelatin or blister containers, from which the powder may be administered by means of an inhaler or insufflator.

When necessary, the above-mentioned composition suitable for sustained release of the active ingredient can be used.

The pharmaceutical compositions according to the invention may also contain other active ingredients such as antimicrobial agents or preservatives.

The compounds of the present invention may also be used in combination with other therapeutic agents such as other anti-infective agents. In particular, the compounds of the present invention may be used with known antiviral agents.

Thus in a further aspect the present invention provides a combination comprising compound (a) or a physiologically acceptable derivative thereof and another therapeutically active agent, particularly an antiviral agent.

The above-mentioned conjugates may conveniently be presented for use in the form of a pharmaceutical composition, and thus a pharmaceutical composition comprising a conjugate as defined above together with a pharmaceutically acceptable carrier therefor forms a further aspect of the invention.

Therapeutic agents suitable for use in such conjugates include acyclic nucleosides such as acyclovir or ganeiclovir, interferons such as alpha, beta, gamma-interferon, renal excretion inhibitors such as 4- (dipropylsulfamoyl) benzoic acid, nucleoside transport inhibitors such as dipyridamole, 2 ', 3' -dideoxynucleosides such as AZT, 2 ', 3' -dideoxycytidine, 2 ', 3' -dideoxyadenosine, 2 ', 3' -dideoxyinosine, 2 ', 3' -dideoxythymidine, 2 ', 3' -dideoxy-2 ', 3' -dideoxythymidine and 2 ', 3' -dideoxy-2 ', 3' -dideoxycytidine, immunomodulators such as interleukin-2 (IL-2) and granulocyte macrophage colony stimulating factor (GM-CSF), erythropoietin, amploigen, thymomodulin, thymopentin, phosphothioate, trinitrosine and HIV bind to CD4 receptors such as CD4, CD4, inhibitors of glycosylation such as 2-deoxy-D-glucose, castanospermine and 1-deoxynojirimycin on CD4 hybrid molecules.

The individual components of such conjugates may be administered sequentially or simultaneously, separately or in combination with a pharmaceutical composition.

The dose of each compound when compound (a) or one of its pharmaceutically acceptable derivatives is used in combination with a second therapeutically active agent effective against the same virus may be the same or different from the dose when it is used alone. Suitable dosages are readily determined by those skilled in the art.

Compound (A) and pharmaceutically acceptable derivatives thereof can be prepared by any method known in the art for the preparation of compounds having a similar structure, for example, as described in European patent publication No. 0382526A 2.

Those skilled in the art will recognize that the stereochemistry of compound (a) required for certain of the processes described below can either be obtained starting from optically pure starting materials or by resolving the racemic mixture at any convenient step in the synthesis. The optically pure products required in all processes can be obtained by resolution of the final product of each reaction.

In one such process a 1, 3-oxathiolane of the formula (VIII)

Wherein the anomeric group L is a substitutable substituent, with a suitable base. Suitable L groups include-OR wherein R is an alkyl group, e.g., a C_1—6Alkyl radicals such as methyl or R is an acyl radical, e.g. a C_1—6Alkyl groups such as acetyl or halogen, for example iodine, bromine or chlorine.

The compound of formula (VIII) may conveniently be reacted with 5-fluoro-cytosine or one of its suitable pyrimidine base precursors (precursor previously silylated with a silylating agent such as hexamethyldisilazane) in a compatible solvent such as dichloromethane with a Lewis acid such as titanium tetrachloride, trimethylsilyltrifluoromethanesulfonic acid, Trimethylsilyliodide (TMSI) or a tin (IV) compound such as SnCl₄Catalytically.

1, 3-oxathiolanes of the formula (VIII) can be prepared by reacting aldehydes of the formula (VII) with mercaptoacetals of the formula (VI) in a compatible organic solvent such as toluene in the presence of an acid catalyst such as a Lewis acid like zinc chloride.

HSCH₂CH(OC₂H₅)₂　　　　　　(VI)

C₆H₅CO₂CH₂CHO　　　　　　　(VII)

Mercaptoacetals of formula (VI) can be prepared by methods known in the art, for example G.Hesse and I.Jorder, Chem Ber 85, pp.924-932 (1952).

The aldehydes of the formula (VII) Can be prepared by methods known in the art, for example E.G. Halloquist and H.Hibbert, Can J Research, 8, pp.129-136 (1933). The crude aldehyde (VII) can be conveniently purified by conversion to a crystalline bisulfite addition adduct and then to the free aldehyde.

In the second method compound (A) is obtained by the interconversion of the base of a compound of formula (IX),

wherein B is a base which is converted to 5-fluoro-cytosine. Such interconversion can be achieved either by simple chemical conversion (e.g. conversion of uracil bases to cytosines) or by enzymatic conversion using a deoxyribosyltransferase. Such methods and conditions for base interconversion are well known in the art of nucleoside chemistry.

In a third method a compound of formula (XI) can be prepared by converting anomeric NH₂The base is converted to the compound (A) by converting it to a 5-fluoro-cytosine base by methods well known in the art of nucleoside chemistry.

Many of the reactions described above have been widely reported in articles relating to nucleoside synthesis, see, for example, nucleoside analogsChemical, biological and medical applications are described,pages 165-192 and T.Ueda in R.T.Walkeret al, Plenum Press, New York (1979),the chemistry of nucleosides and nucleotides, and the use of,vol I, L.B.Townsend Ed.plenum Press New York (1988), the disclosure of which is incorporated herein by reference.

It will also be seen that the above reaction requires the use of starting materials having protected functional groups and therefore deprotection may be required in the intermediates or in the desired compounds produced in the final step. The protection and deprotection of the functional groups can be carried out by conventional methods. Thus, for example, an amino group may be protected by a group selected from aralkyl (e.g., benzyl), acyl, aryl (e.g., 2, 4-dinitrophenyl), or silyl; the protecting group is then removed, if necessary, by appropriate hydrolysis or hydrogenolysis under standard conditions. The hydroxy group may be protected with any conventional hydroxy protecting group, e.g., as inProtective Groups in Organic ChemistryMcOmie, Ed. plenum Press, New York (1973) or T.W.Greene,Protected Groups in Or ganic Synthesisjohn Wiley and Sons, New York (1981). Examples of suitable hydroxy protecting groups include groups selected from alkyl (e.g. methyl, tert-butyl or methoxymethyl), aralkyl (e.g. benzyl, diphenylmethyl or triphenylmethyl), heterocyclyl such as tetrahydropyranyl, acyl (e.g. acetyl or benzoylsilyl) and silyl such as trialkylsilyl (e.g. tert-butyldimethylsilyl). The protecting group for a hydroxyl group can be removed by a conventional method. Thus, for example, alkyl, silyl, acyl and heterocyclyl groups may be removed by solvolysis, e.g. hydrolysis under acidic or basic conditions. Aralkyl groups such as triphenylmethyl can be similarly removed by solvolysis, e.g., hydrolysis under acidic conditions. Aralkyl radicals such as benzyl can be prepared, for example, by reaction with BF₃The etherate and acetic anhydride treatments are then removed by removing the formed acetoxy groups in a suitable step in the synthesis. The silyl groups may also be conveniently removed using a fluoride ion source such as tetra-n-butylammonium fluoride.

In the above process, the compound (A) is usually obtained in the form of a mixture of cis and trans isomers, of which the cis isomer is the compound of interest.

These isomers can be resolved by physical methods, for example by chromatography on silica gel or by fractional crystallization, directly or as a suitable derivative thereof, for example an acetate salt (for example prepared with acetic anhydride), and then converted after isolation into the parent product (for example deacetylation with methanolic ammonia).

Pharmaceutically acceptable salts of the compounds of this invention may be prepared as described in U.S. patent 4,383,114, which is hereby incorporated by reference for all purposes. Thus, for example, when it is desired to prepare an acid addition salt of compound (A), the product of any of the above processes may be converted to a salt by treating the resulting free base with a suitable acid using conventional procedures. Pharmaceutically acceptable acid addition salts may optionally be prepared by reacting the free base with the appropriate acid in the presence of an appropriate solvent such as an ester (e.g. ethyl acetate) or an alcohol (e.g. methanol, ethanol or isopropanol). Salts of inorganic bases may be prepared by reacting the parent compound with a suitable base, such as an alcohol (e.g. ethanol). Pharmaceutically acceptable salts may be prepared by conventional methods from other salts, including other pharmaceutically acceptable salts of compound (a).

By reaction of compound (A) with a phosphating agent such as POCl₃Or a suitable esterifying agent such as an acid halide or anhydride may be converted to a pharmaceutically acceptable phosphate ester or other ester by appropriate reaction. The ester or salt of compound (a) may be converted to the parent compound, for example by hydrolysis.

The resolution of the final product, or its intermediates or its starting materials, may be carried out by any suitable method known in the art: see, e.g., e.l.eliel,Stereochemistry of Carbon Compounds，McGraw Hill(1962)and S.H。Wilen，Tables of Resolving Agents。

thus, for example, compound (a) may be obtained by chiral HPLC using a suitable stationary phase such as acetylated β -cyclodextrin or cellulose triacetate and a suitable solvent such as an aqueous solution of an alcohol, such as ethanol, or, for example, triethylammonium acetate. Alternatively, the compounds can be resolved by enzyme-mediated enantioselective catabolism using a selective enzymatic degradation by a suitable enzyme, such as cytidine deaminase or a suitable 5' -nucleotidase derivative. When the resolution is carried out enzymatically, the enzyme may be used either in solution or more conveniently in immobilized form. The enzyme may be immobilized by any method known in the art, for example by adsorption onto a resin such as Eupergit C.

The invention will be further described by the following examples which are not intended to limit the invention in any way, all temperatures being in degrees centigrade.

Intermediate (1)

(+/-) -cis-2-hydroxymethyl-5- (5 '-fluorocytosin-1' -yl) 1, 3-oxathiolanes

(i)2-benzoyloxymethyl-5-acetoxy-1, 3-oxathiolane

Benzoyloxyacetaldehyde (216.33g, 1.32mol) was dissolved in pyridine (373ml, 4.61mol), and 1, 4-dithiane-2, 5-diol (100.31g, 0.66mol) was added to the solution. The heterocyclic mixture is stirred for 1 hour at 60-65 ℃ under nitrogen, and a complete solution is obtained after reaction. Dichloromethane (650ml) was added to the reaction mixture which was then cooled to 0 ℃ using an ice-salt bath, and acetyl chloride (281ml, 3.95mol) was added dropwise to the solution at 0-5 ℃ over 1.5-2 hours. The reaction mixture was stirred at 0-5 ℃ for 30 minutes, then carefully poured into a cold (0 ℃) saturated solution of sodium bicarbonate, the organic layer separated, the aqueous layer extracted with dichloromethane (3X 200ml), the combined organic layers washed with saturated sodium bicarbonate solution (3X 200ml) and then with brine (200 ml). The solution was dried over sodium sulfate and concentrated in vacuo and a small amount of pyridine was removed by azeotropic distillation with benzene to give 320.79g of crude product, which was purified by Kugelrohr distillation or by filtration through a short silica gel column [ solvent system: hexane/ethyl acetate (3/1).

(ii)Cis-and trans-2-benzoyloxymethyl-5- (N) ₄ ' -acetyl group -5 '-fluoro-cytosin-1' -yl) -1, 3-oxathiolane

5-fluorocytosine (4.30g, 33.3mmol), hexamethyldisilazane (25ml) and ammonium sulfate (120mg) were boiled under reflux until the cytosine dissolved (3 hours), followed by further reflux for 2 hours. Hexamethyldisilazane was evaporated under vacuum and toluene (100ml) was added to the residue to co-volatilize the solvent. The resulting solution of bis (trimethylsilyl) -fluorocytosine in dichloromethane (40ml) was added under argon to a solution of 2-benzoyloxymethyl-5-acetoxy-1, 3-oxathiolane (8.537 g, 30.3 mmol) in dichloromethane (100ml) and molecular sieves (4) preformed under argon (4 g)2g) and cooled at 0 ℃ for 20 minutes. [ (trifluoromethanesulfonyl) oxy ] trimethylsilane (6ml, 31mmol) was added to the above mixture at 0 ℃ and the resulting solution was stirred at room temperature for 2 hours, and the filtrate was shaken twice with 300ml of brine and once with distilled water. The organic layer was dried over magnesium sulfate, filtered and evaporated to dryness to give the crude 5-fluoro-cytosine derivative (10.1 g). R_f＝0.57(EtOAC：MeOH9：1)。

This residue was acetylated in the next step without further purification. The crude product was dissolved in dry dichloromethane (120ml) and the process was carried out in a 500ml round bottom flask under argon. To this solution was added triethylamine (12.7 ml, 91.1 mmol) and dimethylaminopyridine (111mg, 0.9mmol), then the flask was immersed in an ice bath for 1 hour under argon, acetic anhydride (4.3ml, 45mmol) distilled under sodium acetate was injected into the cooled flask, the mixture was stirred overnight, then carefully decanted into an erlenmeyer flask containing a saturated sodium bicarbonate solution. The product was washed with distilled water and then with brine solution. The dichloromethane fraction was dried and evaporated to dryness under high vacuum to give an acetylated α/β mixture as a colourless foam, weighing 9.6g after drying. This was washed with ethyl acetate: flash chromatography of methanol (9:1) gave 3.1g, 7.8mmol (46%) of pure trans and 3.5 g, 8.9mmol (30%) of the pure cis form of the title compound.

Trans isomer: r_f0.65 (ethyl acetate: methanol 9: 1).

U.V.：(MeOH)Lambda max：309nm

¹H-NMR delta (ppm in CDCL)₃Middle)

8.77(b，1H；C₄′-NH-Ac)

8.06(m, 2H; aromatic)

7.70(d，1H；C₆′-H，J_CF＝6.3Hz)

7.62(m, 1H; aromatic)

7.49(m, 2H; aromatic)

6.51(dd，1H；C₅-H)

5.91(dd，1H；C₂-H)

4.48(dd，2H；C₂-CH ₂OCOC₆H₅)

3.66(dd，1H；C₄-H)

3.34(dd，1H；C₄-H)

2.56(s，3H；NH-COCH ₃)

Cis-isomer: r_f0. 58 (ethyl acetate: methanol 9: 1).

U.V.：(MeOH)λ max：309nm

¹H-NMR delta (ppm in CDCl)₃Middle)

8.72(b，1H；C₄′-NH-Ac)

8.06(m, 2H; aromatic)

7.87(d，1H；C₆′-H，J_CF＝6.2Hz)

7.60(m, 1H; aromatic)

7.49(m, 2H; aromatic)

6.32(dd，1H；C₅-H)

5.47(dd，1H；C₂-H)

4.73(dd，2H；C₂-CH ₂OCOC₆H₅)

3.62(dd，1H；C₄-H)

3.19(dd，1H；C₄-H)

2.55(s，3H；NH-COCH ₃)

(iii)(+ -) -cis-2-hydroxymethyl-5- (5 '-fluorocytosin-1' -yl) -l 1, 3-oxathiolanes

1.2g (3.05 mmol) of cis-2-benzoyloxymethyl-5- (N)₄' -acetyl-5 ' -fluorocytosin-1 ' -yl) -1, 3-oxathiolane is stirred in 30ml of methanolic ammonia at 0 ℃ for 1 hour and then at room temperature overnight. The mixture was evaporated under reduced pressure and the residue was triturated with dry ether twice (2X 30 ml). The solid residue was recrystallized from absolute ethanol to give 655mg (2.64mmol, 87%) of pure cis title product: melting point 204-; rf is 0.21 [ ethyl acetate: methanol (9:1) ]. The desired compound is prepared by¹H，¹³C-NMR and U.V.. lamda.max (H)₂O)280.9 nm.

Cis-isomer:

¹H-NMR delta (ppm in DMSO-d)₆Middle)

8.22(d，1H；C₆′-H，J_CF＝7.26Hz)

7.84(d，2H；C₄′-NH ₂)

6.16(t，1H；C₅-H)

5.43(t，1H；C₂-CH₂-OH)

5.19(t，1H；C₂-H)

3.77(m，2H；C₂-CH ₂OH)

3.35(dd，1H；C₄-H

¹³C-NMR(DMSO-d₆)

C₆′ C₂′ C₄′ C₅′

153.46 158.14 134.63 126.32

(²J_CF＝14.0Hz) (J_CF＝24.1Hz) (J_CF＝32.5Hz)

C₅ C₄ C₂ CH₂OH

86.82 36.80 86.77 62.32

Example 1

(mono) -4-amino-5-fluoro-1- (2-hydroxymethyl-1, 3-oxa-sulfide Heterocyclopent-5-yl) - (1H) -pyrimidin-2-ones

(i)(+/-) cis-2-hydroxymethyl-5- (5' -fluorocytosine-1-) Yl) -1, 3-oxathiolane monophosphates

To a stirred mixture of intermediate 1(500mg, 2.024mmol) cooled to 0 ℃ in dry trimethyl phosphate (10ml) was added dropwise phosphorus oxychlorideChlorine (1.22ml, 13.1 mmol). The reaction mixture was stirred at 0 ℃ for 1 hour and then quenched in ice water. The pH of the cold mixture was adjusted to 3 by addition of 1N aqueous sodium hydroxide solution and then applied to an activated carbon column (5g, on DARCO, which was eluted with water and then with ethanol and ammonia in the ratio (10: 1.) the fractions containing the crude monophosphates were combined and evaporated, then added to a column containing 15g of DEAE Sephadex A25 (HCO)₃ ^-Type) was applied to a column, eluting with water (300ml), 0.1M-NH₄HCO₃(300ml) and 0.2MN H₄HCO₃(100ml) was performed in a gradient and the appropriate fractions evaporated after dilution with water (30ml) to yield (. + -.) cis-2-hydroxymethyl-5- (5 '-fluorocytosin-1' -yl) -1, 3-oxathiolane monophosphate as a white solid Rf ═ 0.5 (n.proH: NH)₄OH 6: 4) yield 612mg, 1.77mmol, 87.9%.

¹H NMR delta (ppm in D)₂O in) 8.27(d, 1H, C'₆-H，J_H-F＝6.47Hz)，6.33(dd，1H，C₅-H)，5.47(t，1H，C₂-H)，4.84(m，2H，C₂-CH ₂OH)，3.63(dd，1H，C₄H)，3.30(dd，1H，C₄H).HPLC>99％.

(ii)(+) -cis-2-hydroxymethyl-5- (5' -fluorocytosine- 1' -yl) -1, 3-oxathiolanes

To a solution of (. + -.) -cis-2-hydroxymethyl-5- (5 ' -fluorocytosin-1 ' -yl) -1, 3-oxathiolane monophosphate (100mg, 0.29mmol) in 3ml of glycine buffer (glycine (52.6mg) and magnesium chloride (19mg) dissolved in 10ml of water) was added a portion of 5 ' -nucleotidase [ Sigma, 3.5mg, 29 units/mg ]. The resulting mixture was incubated at 37 ℃ with shaking. The reaction was monitored by HPLC (activated carbon column for alpha-Acid Glycoprotein (AGP) using 0.2M sodium phosphate as eluent at pH7 at 0.15 ml/min) at various time intervals. Only the (+) -enantiomer was observed after 2.5 hours. More enzyme (2mg) was added and incubation was further continued for 3 hours. HPLC analysis clearly shows the selectivity and complete hydrolysis of the (+) -enantiomer. Adding the obtained mixtureTo DEAE Sephadex A-25 (HCO)₃Type) on a column. Eluted with water (155ml) followed by 0.1 and 0.2MNH₄HCO₃Elution was carried out (100ml each). The appropriate fractions containing the nucleoside eluted in the first step were combined and concentrated, and the remaining solid was purified using a short silica gel column with ethyl acetate, methanol (4.5:0.5) as eluent, and then separated by HPLC (using the conditions mentioned above). Pure (+) -cis-2-hydroxymethyl-5- (5 '-fluorocytosin-1' -yl) -1, 3-oxathiolane (23mg, 0.093mmol, 32%) was obtained as a white solid.

(α)²¹ _δ+123⁰[C，1.00，MeOH]Its melting point 185 ℃ NMR delta (ppm in DMSO) 8.26(d, 1H, C'₆-H，J_H-F＝5.22Hz)，7.87(s，1HNH₂，D₂O exchangeable) 7.63(s, 1H, NH)₂，D₂O interchangeable), 6.20(dd, 1H, C)_5-H)，5.48(t，1H，C₂H)，5.24(t，1H，CH₂-OH，D₂O-exchangeable), 3.84(m, 2H, C)₂-CH ₂OH)，3.50(dd，1H，C₄H)，3.37(dd，1H，C₄H).

(iii)(mono) -cis-2-hydroxymethyl-5- (5' -fluorocytosine- 1' -yl) -1, 3-oxathiolanes

The appropriate fractions containing the second eluted nucleoside from the sephadex column described in step (ii) were combined and evaporated under reduced pressure, the residue was dissolved in 2ml water and treated with alkaline phosphatase (Sigma, 1ml, 60 units/ml) followed by incubation at 37 ℃ for 1.5 h, the solvent was evaporated, the residue was purified by chromatography on a silica gel column using ethyl acetate: methanol (4: 1) as eluent followed by HPLC purification (using the same conditions as mentioned above for isolation) to give pure (mono) -cis-2-hydroxymethyl-5- (5 '-fluorocytosin-1' -yl) -1, 3-oxathiolane (20mg, 0.081mmol, 28%), melting point 190 ℃ (decomposition).

(d)rf＝0.21，EtOAc:MeOH(4:1)。U.V.：(H₂O)max：279.1nm.¹H NMR delta (ppm in DMSO-d)₆Medium 8.25(d, 1H, C'₆-H，J_HF＝7.26Hz)，7.88(b，1H，C′₄-NH₂，D₂O interchangeable), 7.85(b, 1H, C'₄-NH₂:D₂O interchangeable), 5.24(t, 1H, C)₂-H)，3.83(m，2H，C₂-CH ₂-OH)，3.19(dd，1H，C₄-H)，3.15(dd，1H，C₄-H).

Intermediate 2 and example 2 describe an alternative process for the preparation of the compound (a).

Intermediate 2

(1′R，2′S，5′R)- radical-5R- (5 '-fluorocytosine-1' -radical) -1, 3-oxathiolane-2S-carboxylate

5-fluorocytosine (155mg, 1.2mmol) in CH at room temperature under argon protection₂Cl₂To the suspension in (1ml) was added 2, 4, 6-collidine (0.317ml, 2.4mmol) and tert-butyldimethylsilyl triflate (0.551ml, 2.4mmol) successively. The resulting mixture was stirred for 15 minutes to give a clear solution. Introduction of (1 ' R, 2 ' S, 5 ' R) -5R-acetoxy-1, 3-oxathiolane-2S-carboxylate (330mg, 1mmol) in CH₂Cl₂(0.5ml) followed by iodotrimethylsilane (0.156ml, 1.1 mmol). Stirring was continued for 3 hours and the mixture was taken up in CH₂Cl₂(20ml) diluted and successively saturated NaHSO₃Aqueous solution, water, brine, then concentrated. The residue was taken up in ether-hexane (1:1, 10ml) and saturated NaHCO₃The aqueous solution was dissolved and stirred at room temperature for 15 minutes. The aqueous layer was removed and the organic phase was centrifuged to give a white solid which was washed with hexane (3X 5ml) and dried in vacuo. The product thus obtained (1' R)，2′S，5′R)-The radical-5R- (5 "-fluorocytosin-1" -yl) -1, 3-oxathiolane-2S-carboxylate (350mg, 88%) contained about 6% (1 ' R, 2 ' S, 5 ' R) -The radical-5S- (5 "-fluorocytosin-1" -yl) -1, 3-oxathiolane-2S-carboxylate (NMR). The product can be obtained from MeOH/CH₂Cl₂Recrystallization from benzene to give crystalline product:

[α]_D ²⁶+22°(c，0.19，MeOH)；m.p.216-218℃，¹H NMR(CDCl₃) δ 0.78(d, 3H, J ═ 7Hz), 0.91(t, 6H, J ═ 7.3Hz), 1.00(m, 2H), 1.39-2.04(m, 7H), 3.12(dd, 1H, J ═ 6.6Hz, 6.1Hz), 3.52(dd, 1H, J ═ 4.7Hz, 6.1Hz), 4.79(dt, 1H, J ═ 4.4Hz, 4.3Hz), 5.46(S, 1H), 5.75(bs, 1H, exchangeable), 6.42(5t, 1H, J ═ 5.0Hz), 8.10(bs, 1H, exchangeable, 8.48(d, 1H, J ═ 6.6 Hz);¹³C NMR(CDCl₃-DMSO-d₆)：δ 16.7，21.2，22.4，23.7，26.6，31.8，34.4，36.6，40.5，47.2，77.1，79.1，90.8，126.3(d，J＝33Hz)，137.1(d，J＝244Hz)，154.2，158.3(d，J＝15Hz)，170.1.

example 2

2S-hydroxymethyl-5R- (5 '-fluoro-pyrimidin-1' -yl) -1, 3 Oxathiolanes

Slowly adding (1 ' R, 2 ' S, 5 ' R) -A solution of the radical-5R- (5 "-fluorocytosin-1" -yl) -1, 3-oxathiolane (54mg, 0.135mmol) in THF (2ml) was added, and the reaction mixture was stirred for 30 minutes, followed by quenching with excess methanol (2 ml). Then silica gel (3g) was added and the resulting slurry was transferred to a silica gelColumn chromatography (EtOAc-hexane-MeOH, 1: 1: 1) afforded a sticky solid which was azeotropically dried with toluene to give 20.7 mg (63%) of the product as a white solid:

[α]_D ²⁶+114°(c，0.12，MeOH)；¹h NMR (DMSO-d6) δ 3.14(dd, 1H, J ═ 4.3, 11.9Hz), 3.42(dd, 1H J ═ 5.3, 11.9Hz), 3.76(m, 2H), 5.18(m, 1H), 5.42(t, 1H, J ═ 4.8Hz), 6.14(m, 1H), 7.59(br m, 1H, interchangeable), 7.83(br m, 1H interchangeable), 8.20(d, 1H, J ═ 7.66 Hz).

Example 3

Biological activity

(i)Antiviral activity

The antiviral activity of the compound of example 1 against HIV-1 was determined in the following cell lines.

C8166 cells, a human T-lymphoblastoid cell line, were infected with HIV-1 strain RF.

MT-4 cells, a human T-cell leukemia cell line, were infected with HIV-1 strain RF.

Antiviral activity was determined by inhibiting the formation of syncytia in C8166 cells (Tochikura et al Virology, 164542-546), and by inhibiting A in MT-4 cellsTo determine (Ba baet al.Biochem Biophys Res Commun.，142，pp128—134(1987)；Mossman，J.Immun Meth65, pp.55-57 (1983). Antiviral activity can also be determined by assaying the amount of synthetic HIV P24 antigen in the presence and absence of enantiomers.

The results are shown in table 1 and table 2 below:

TABLE 1

50% antiviral Activity (μ g/ml)

Measurement of First of all Inhibition of formation of syncytia cells

Cell MT-4C 8166

Virus (HIV-1) HIV-1 RF

(+) -enantiomer > 10.04

(-) -enantiomer 0.140.0018

Intermediate 10.0650.013

AZT 0.0038

TABLE 2

50% inhibition of HIV P24 Synthesis (μ g/ml)

Cell C8166

Virus RF

(+) -enantiomer 0.1

(-) -enantiomer 0.0022

Intermediate 10.011

AZT 0.017

(ii)Cytotoxicity

Cytotoxicity of the compound of example 1 and the racemic compound (intermediate 1) in two CD4 cell lines: h9 and CEM.

The compounds for testing were serially diluted from 100. mu.g/ml to 0.3mg/ml (final concentration) in 96-well microtiter plates. 3.6X 10 inoculate each well in the plate⁴The number of surviving cells, including no drug control, was determined by removing cell suspension samples and counting cells without trypan blue with a hemocytometer after 5 days of incubation at 37 ℃.

The results are shown in Table 3.

TABLE 3

50% cytotoxicity (μ g/ml)

Compound (I) CEM cells H9 cell

(+) -enantiomer 217334

(-) -enantiomer 148296

Intermediate 1173232

Claims (5)

1. A process for preparing a pharmaceutical composition comprising admixing as an active ingredient the (-) enantiomer of cis-4-amino-5-fluoro-1- (2-hydroxymethyl-1, 3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one substantially free of the corresponding (+) enantiomer or a pharmaceutically acceptable salt or ester thereof, one or more pharmaceutically acceptable carriers, and an acyclic nucleoside.

2. The method according to claim 1, wherein said pharmaceutical composition further comprises the (+) enantiomer of cis-4-amino-5-fluoro-1- (2-hydroxymethyl-1, 3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one and is present in an amount of no more than 5% w/w.

3. A process according to claim 2, wherein the (+) -enantiomer is present in an amount of no more than 2% w/w.

4. A process according to claim 3, wherein the (+) -enantiomer is present in an amount of no more than 1% w/w.

5. A method according to any of claims 1-4 wherein said acyclic nucleoside is acyclovir or 9- [1, 3-dihydroxy-2-propoxymethyl ] guanine.