FI111723B - (-)-Cis-4-amino-1-(2-hydroxymethyl-1,3-oxathiolanyl)-1H-pyrimidinone - Google Patents

Abstract

(-)-Cis-4-amino-1- (2-hydroxymethyl-1,3 -oxathiolan-5-yl)-(1H)- pyrimidin-2-one (I) and its derivs. are new. (I) contg. max. 5 wt% of (+)-enantiomer (pref. below 1 wt%) is also claimed. (I) is prepd. by sepn. from a racemic mixt., preferably by chiral HPLC. The stationary phase used is preferably beta-cyclodextrin or cellulose triacetate. Alternatively sepn. is by enzyme-mediated enantioselective catabolism, preferably using an immobilised enzyme. The enzyme is preferably cytidine deaminase or a 5-nucleotidase.

Description

111723

The process prepares the (-) - enantiomer of cis-4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one for use as an antiviral agent. - Förfarande för framställning av (- This invention relates to a process for the preparation of a) cis-4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one for use in the preparation of anti-viral medication. substantially pure cis-4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one (-) - enantiomer or a salt, ester or ester salt thereof, for use as an antiviral agent.

Compound of formula (I) nh2

«AA

hoch2 ^ ox \ -7 s ®, also known as BCH-189 or NGPB-21, has been described as having a compound having antiviral activity, particularly against human immunodeficiency viruses (HIV t), which are agents of AIDS (5th Anti-Aids). Conference, Montreal, Canada 5th-9th June 1989: Abstracts TCO1 and MCP63; EP Patent Application Publication No. 0382562). The compound of formula (I) is a racemic mixture of two enantiomers of formula (I-1) and (1-2):. NH, NH 2 s Λ Λ

ASJ 0AnJ

- 1-2 Ό (1-2) (1-1) 2 111723 and described and studied in the form of its racemate. The only currently approved compound for the treatment of conditions caused by HIV is 3'-azido-3'-deoxythymidine (AZT, zidovudine, BW 509U). However, this compound has a significant side effect tendency and either cannot be used or, if tested, has to be discontinued in a significant number of patients. Thus, there is a continuing need for compounds that are effective against HIV, but at the same time have a significantly better therapeutic index.

The present inventors have now found that, surprisingly, the enantiomers of the compound of formula (I) are active against HIV, but that one of the 10 enantiomers [(-) enantiomer] has significantly lower cytotoxicity than the other enantiomer. Thus, according to the invention, there is provided a (-) - (i.e., left-rotating) enantiomer (I) of a compound of formula (I), as well as pharmaceutically acceptable derivatives thereof.

The present invention thus relates to a process for the preparation of the substantially pure (-) enantiomer of cis -4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one. a salt, ester or salt of an ester for use as an antiviral agent, wherein the (+) - enantiomer is present in an amount of up to 5% by weight, which process comprises separating the (-) - enantiomer from a mixture of cis -4-amino-1- (+) - and (-) - enantiomers of (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one or their salts, esters or salts of esters, upon separation. ·. ·. by enzyme-mediated enantioselective catabolism.

The chemical name of the (-) - enantiomer is (-) - ε-4-amino-1- (2-hydroxymethyl-1,3-oxo-thiazolan-5-yl). - (1H) -pyrimidin-2-one (hereinafter compound (A)). It has the absolute space chemistry of the compound of formula (I-:: 1), the compound (1-1) being (2R, cis) -: ': *; 254-amino-1- (2-hydroxymethyl-1,3-) oxathiolan-5-yl) - (lH) -pyrimidin-2-one.

. . The compound (A) prepared according to the invention is substantially free of the corresponding compound; of the (+) - enantiomer, i.e. not more than about 5% by weight of the (+) - enantiomer, preferably not more than about 2% by weight, in particular less than about 1% by weight.

The term "pharmaceutically acceptable derivative" means any pharmaceutically acceptable salt, ester or salt of this ester of Compound (A).

It will be apparent to those skilled in the art that compound (A) may be modified to form pharmaceutically active derivatives thereof, in functional groups both in the base moiety of the oxathiolane ring and in the hydroxymethyl group. Of particular interest are the 3111723 pharmaceutically acceptable derivatives obtained by modification of the 2-hydroxymethyl group of the oxathiolane ring.

Preferred esters of compound (A) are compounds having a 2-hydroxymethyl group

hydrogen has been replaced by the acyl function II

RC-5 wherein the non-carbonyl portion of the ester R is selected from: hydrogen, straight chain or branched alkyl (e.g. methyl, ethyl, n-propyl, t-butyl, n-butyl), alkoxyalkyl (e.g. methoxymethyl), aralkyl (e.g. benzyl), aryloxyalkyl (e.g., phenoxymethyl), aryl (e.g., phenyl optionally substituted with halogen, C 1-4 alkyl or C 1-4 alkoxy); sulfonate esters such as alkyl or aralkylsulfonyl 10 (e.g. methanesulfonyl); amino acid esters (e.g. L-valyl or L-isoleucyl) and mono-, di- or triphosphate esters.

With respect to the esters described above, unless otherwise noted, the alkyl moiety preferably contains from 1 to 16 carbon atoms, particularly from 1 to 4 carbon atoms. Any aryl moiety present in these esters preferably contains a phenyl group.

In particular, the ester may be a C 1-6 alkyl ester, an unsubstituted benzyl ester or a benzyl ester substituted with at least one halogen (bromine, chlorine, fluorine or iodine), C 1-6 alkyl, C 1-6 alkoxy, nitro or trifluoromethyl group. .

The pharmaceutically acceptable salts of Compound (A) are those derived from pharmaceutically acceptable inorganic and organic acids and bases. Example: ": 20 of the suitable acids are hydrochloric, hydrobromic, sulfuric, nitrogenous, perchloric, fuicidal, maleic, phosphorous, glycolic, lactic, salicylic, succinic, toluene- p-sulfonic, tartaric, acetic, citric, methanesulfonic, formic, benzoic, malic, naphthalene-2-sulfonic, and benzenesulfonic acids Other acids, such as oxalic acid, although not themselves pharmaceutically acceptable, can be used as intermediates in the preparation of the compounds of the invention and their pharmaceutically acceptable acid addition salts.

Salts derived from suitable bases include alkali metal (e.g. sodium), alkaline earth metal (e.g. magnesium), ammonium and NR / (where R is C 1-4 alkyl) salts.

'·; References to a compound of the invention hereinafter include both compound (A) and: pharmaceutically acceptable derivatives thereof, i.e. salts, esters, or salts of esters.

The compounds of the invention are either antivirally active themselves and / or are metabolizable to such compounds. In particular, these compounds act by inhibiting 4,117,223 retroviral replication, including human retroviruses such as human immunodeficiency viruses (HIV), which cause AIDS.

The compounds of the invention are useful as an antiviral agent, for example in the treatment of infections caused by retroviruses such as HIV.

The compounds of the invention are also useful in the treatment of AIDS-related conditions such as AIDS-related syndrome (ARC), progressive generalized lymph node disease (PGL), AIDS-related neurological conditions (such as dementia or local bilateral limb muscle weakness), HIV antibody positive and HIV positive conditions, Kaposi's sarcoma, thrombocytopenia purpurae and related opportunistic infections such as Pneumocystis carinii.

The compounds of the invention are also useful in preventing the development of clinical disease in individuals who are anti-HIV antibody or HIV antigen positive and in preventing related exposure to HIV.

Compound (A) or pharmaceutically acceptable derivatives thereof may also be used to prevent viral contamination of physiological fluids such as blood or semen in vitro.

It will be appreciated by those skilled in the art that references herein to treatment include both prevention and treatment of identified infections and symptoms.

• ·

It will further be appreciated that the amount of the compound of the invention required for treatment will vary not only with the compound selected, but also with the mode of administration, the nature of the condition being treated, and the age and condition of the patient and is ultimately up to the treating physician or veterinarian. However, in general, a suitable dose will be in the range of about 0.1 to about: *: *: 750 mg / kg of body weight per day, preferably in the range of 0.5 to 60 mg / kg, most preferably in the range of 1 to 20 mg / kg / day.

The desired dose may conveniently be presented as a single dose or in divided doses given at appropriate intervals, for example two, three, four or more sub-doses: '; doses a day.

* ·; · The compound is conveniently administered in unit dosage form; for example, containing 10-1500 mg, suitably 20-1000 mg, most preferably 50-700 mg of active ingredient, per 30 unit dosage forms.

»·

Ideally, the active ingredient should be administered in such a way as to obtain peak plasma concentrations of the active compound of about 1 to about 75 μΜ, preferably about 2 to 50 μΜ, preferably about 3 to 30 μΜ. This can be achieved, for example, by intravenous injection of 0.1-5% solution of the active ingredient, optionally in saline, or orally as a snack containing from about 1 to about 100 mg of the active ingredient. Desired blood levels may be maintained by continuous drip administration of from about 0.01 to about 5.0 mg / kg / hour, or by intermittent drip containing about 0.4 to about 15 mg / kg of active ingredient.

While it is possible for administration of a compound of the invention as a crude chemical for therapeutic use, it is preferred that the active ingredient be in the form of a pharmaceutical preparation.

Such a pharmaceutical preparation contains compound (A) or a pharmaceutically acceptable derivative thereof together with one or more pharmaceutically acceptable carriers, and optionally other therapeutic and / or prophylactic ingredients. The carrier (s) must be "suitable" in the sense that the preparation is not harmful to its recipient.

The pharmaceutical preparations are those suitable for oral, rectal, nasal, topical (both cheek and sublingual), vaginal or parenteral (both intramuscular, subcutaneous and intravenous) administration, or in a form suitable for administration by inhalation or inhalation. inflated. The formulations may, if appropriate, be administered in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. All methods involve contacting the active compound with liquid or finely divided solid carriers, or both, and if necessary: then, if required, forming the product into the desired preparation.

:, ·. Pharmaceutical preparations suitable for oral administration may conveniently be presented in discrete units such as capsules, starch capsules, or tablets each containing a predetermined amount of the active ingredient; in powder or granules; solutions. . as a suspension, suspension or emulsion. The active ingredient may also be administered in the form of a snack, snack or pasta. Tablets and capsules for oral administration may contain conventional excipients such as binders, fillers, lubricants, disintegrants or wetting agents. The tablets may be coated with those well known in the art; . 3 0 la methods. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be; '' As a dry product to be mixed with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifiers, non-aqueous vehicles (which may be edible oils), or preservatives.

6, 111723

The compounds of the invention may be formulated for parenteral administration (e.g., by injection, e.g., by food injection or continuous drip) and may be presented in unit dosage form as ampoules, prefilled syringes, small volume infusion, or in multi-dose containers with added preservative. The compositions may be in the form of suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation from a sterile solid or by lyophilization from a solid, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

For topical administration to the epidermis, the compounds of the invention may be formulated as ointments, creams or lotions, or as a transdermal patch. For example, creams and creams may be formulated in 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 generally contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.

Formulations suitable for topical administration in the mouth include tablets containing the active ingredient in a flavored base, usually sucrose, and acacia or *. * j2 0 tragacanth; pastilles containing the active ingredient in an inert base such as gelatin and glycerol or sucrose and acacia; and mouthwashes containing the active ingredient in a suitable liquid carrier.

.,; Pharmaceutical preparations in solid form suitable for rectal administration; Are most preferably in unit dose suppositories. Suitable carriers include cocoa butter: '25' and other agents commonly used in the art, and suppositories may be suitably formed by mixing the active compound with a softened or thawed carrier (s) and then chilled in molds.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or sprays containing the active ingredient, in addition to carriers known in the art to be suitable.

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

• »7 111723

The drops may be formulated in an aqueous or non-aqueous base which also contains one or more dispersants, solubilizers or suspending agents. Liquid sprays are conveniently provided in pressure packs.

For administration by inhalation, the compounds of the invention are conveniently provided by a medicament injector, nebulizer, or compressed package, or by any other suitable means for administration of an aerosol spray. The pressure kits 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 administering a metered amount of valve.

Alternatively, for administration by inhalation or nebulization, the compound of the invention may be in the form of a dry powder composition, for example, as a powder mixture of the compound and a suitable powder base such as lactose or starch. The powder composition may be in unit dosage form, for example, in capsules or cartridges or, for example, in gelatin or blister packs, from which the powder may be administered by means of an inhaler or medicament gun.

If desired, the above-described formulations adapted for sustained release of the active ingredient may be used.

The pharmaceutical compositions may also contain other active ingredients such as. V antimicrobial agents or antiseptics.

The compounds of the invention may also be used in combination with other therapeutic agents. for example, with other anti-infective agents. In particular, the invention; · ·; the compounds may be used in combination with known antiviral agents.

Such a combination comprises compound (A) or a physiologically acceptable derivative thereof together with another therapeutically active agent, in particular an antiviral. : With 25 substances.

The combinations referred to above may conveniently be in the form of a pharmaceutical preparation for use, wherein such pharmaceutical preparations include the above. ***. a defined combination in combination with a pharmaceutically acceptable carrier therefor.

Suitable therapeutic agents for use in these combinations include acyclic nucleosides such as acyclovir or ganciclovir, interferons such as interferon α, β or τ, renal secretion inhibitors such as probenecid, nucleoside transport inhibitors such as dipyridamole, , 3'-dideoxynucleosides such as AZT, 2 ', 3'-dideoxycytidine, 2', 3'-dideoxyadenosine, 2 ', 3'-dideoxyinosine, 2', 3'-dideoxy thymidine, 2 ', 3' -dideoxy-2 ', 3'-didehydro-thymidine and 2', 3'-dideoxy-2 ', 3'-didehydro-cytidine, immunomodulators such as interleukin II (IL2) and granulocyte macrophage colony stimulating factor (GM-CSF), erythropoietin, ampligene, thymomodulin, thymopentin, foscamet, ribavirin, and HIV binding inhibitors to CD4 receptors, e.g., soluble CD4, CD4 fragments, CD4 hybrid molecules, glycosylation thio-inhibos, such as 2-deoxy-glucan, and 1-deoxynojirimycin.

The individual components of these combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations.

When compound (A) or a pharmaceutically acceptable derivative thereof is used in combination with another therapeutic agent against the same virus, the dose of each compound may be either the same or different than when the compound is used alone. Appropriate doses are readily apparent to those skilled in the art.

The compound of formula (I) and its pharmaceutically acceptable derivatives can be prepared by any method known in the art for the preparation of structurally analogous compounds, for example, in the European patent application. , No. 0382562.

•: V β 0 In one such process (A), a 1,3-oxathiolane of formula (VIII),

H “TA

> L l: S- (VIII) wherein the anomeric group L is a displaceable group, is reacted appropriately. *. with van base. Suitable groups L are -OR groups wherein R is an alkyl group. e.g. a C 1-6 alkyl group such as methyl, or R is an acyl group e.g. C 1-6 acyl group such as acetyl, or a halogen such as iodine, bromine or chlorine.

• I »

The compound of formula VIII is conveniently reacted with cytosine or a suitable pyrimidine base precursor (previously silylated with a silylating agent such as hexamethyldisilazane) in a compatible solvent such as methylene chloride using a Lewis acid such as titanium tetrachloride, , or trimethylsilyl triflate.

9 111723

The 1,3-oxathiolanes of formula (VIII) may be prepared, for example, by reaction of an aldehyde of formula (VII) in an organic solvent compatible with the mercaptoacetal of formula (VI), such as toluene, in the presence of an acid catalyst such as Lewis acid such as zinc chloride.

5 HSCH2CH (OC2H5) 2 (VI) C6H5CO2CH2CH0 (VII)

The mercaptoacetals of formula (VI) may be prepared by methods known in the art, e.g., G. Hesse and I. Jorder, Clhem Rer 85 924-932 (1952).

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

In another process (B), a compound of formula (I) is obtained by a base interconversion of a compound of formula (IX) with HOCH, *. * .S-f (IX) '· * wherein B is a base which can be converted to a cytosine. Such an interconversion may be achieved either by a simple chemical transformation (e.g., conversion of the uracil base to a cytosine) or by an enzymatic change using the deoxyribosyl reverse transcriptase. These methods and conditions: ':'; 20 base interconversion are well known in the art of nucleoside chemistry.

. In the third method (C), a compound of the formula (XI). Ri ° o r ·; (xi) * * »: '· .. can be converted to the compound of formula (I) by converting the anomeric NH2-: * ·, into a cytosine base by methods well known in the art of nucleoside chemistry.

»I

Many of the reactions described hereinabove have been extensively described in connection with nucleoside synthesis, e.g., in Nucleoside Analogs - Chemistry Riology and Medical 10 111723

Applications R.T. Walker et al., Plenum Press, New York (1979), pp. 165-192; T. Ueda, Chemistry of Nucleosides and Nucleotides, Vol. I, LB Townsend ed., Plenum Press, New York (1988), pages 165-192, the disclosures of which are incorporated herein by reference.

It will be appreciated that the above reactions may require, or may be appropriately used, starting materials having protected functional groups, which may require deprotection as an intermediate or final step to obtain the desired compound. Protection or deprotection of functional groups may be accomplished using conventional means. Thus, for example, amino groups may be protected with 10 groups selected from aralkyl (e.g. benzyl), acyl, aryl (e.g. 2,4-dinitrophenyl) or silyl; subsequent deprotection, if desired by hydrolysis or hydrogenation, suitably using standard conditions. The hydroxyl groups may be protected using any conventional hydroxyl protecting group, for example, as described in "Protective Groups in Organic Chemistry", Ed. J. F. W. McOmie (Plenum Press, 1973) or "Protective Groups in Organic Synthesis", by Theodora W. Greene (John Wiley and Sons, 1981). Examples of suitable hydroxyl protecting groups include groups selected from alkyl (e.g. methyl, t-butyl or methoxymethyl), aralkyl (e.g. benzyl, diphenylmethyl or triphenylmethyl), hetero-20 cyclic groups such as tetrahydropyranyl. , acyl (e.g. acetyl or benzoyl) and. V silyl groups such as trialkylsilyl (e.g. t-butyldimethylsilyl). The hydroxyl: V: protecting groups can be removed by conventional methods. Thus, for example, alkyl: silyl, acyl and heterocyclic groups may be removed by solvolysis, e.g. rolysis under acidic or basic conditions. Similarly, aralkyl groups such as triphenyl • • •: • • 2 · 2 -methyl may be removed by solvolysis, e.g., hydrolysis under acidic conditions. Aralkyl groups such as benzyl can be removed, for example, by treatment with BF 3 / etherate and acetic anhydride, and then formed. . the acetate groups are removed at a convenient step in the synthesis. The silyl groups can also be conveniently removed using a fluoride ion source such as tetra-n-butylammonium fluoride.

* * *> »·

In the above methods, the compound of formula (I) is generally obtained by cis- and •; · A mixture of hms isomers where the cis isomer is the compound of interest.

• · • * »; These isomers may be separated by physical means, e.g. by chromatography on silica gel or by fractional crystallization, either directly or by appropriate derivatives, e.g., 35 acetates (prepared, for example, with acetic anhydride, which is subsequently converted to the by capping with methanolic ammonia).

Pharmaceutically acceptable salts of the compounds of the invention may be prepared as described in U.S. Patent No. 4,383,114, the disclosure of which is incorporated herein by reference. Thus, for example, if the acid addition salt of compound (A) is to be prepared, the product of any of the above processes may be converted to a salt by treatment of the resulting free base with a suitable acid using conventional methods. Pharmaceutically acceptable acid addition salts may be prepared by reacting the free base with a suitable acid, optionally in the presence of a suitable solvent, such as an ester (e.g., ethyl acetate) or an alcohol (e.g., methanol, ethanol or isopropanol). The inorganic basic salts may be prepared by reacting the parent compound with a suitable base such as an alkoxide (e.g. sodium methoxide) optionally in the presence of a solvent such as an alcohol (e.g. methanol). Pharmaceutically acceptable salts may also be prepared from other salts of compound (A), such as other pharmaceutically acceptable salts, using conventional methods.

Compound (A) may be converted into a pharmaceutically acceptable phosphate or other ester by reaction with a phosphorylating agent such as POCL or a suitable esterifying agent such as an acid halide or anhydride, whichever is more suitable. The ester or salt of compound (A) can be converted to the parent compound by, for example, hydrolysis.

Resolution of the final product or its intermediate or starting material may be accomplished by any suitable method known in the art: see, e.g., "Stereochemistry of Carbon Compounds", E.L. Eliel (McGraw Hill, 1962) and! Tables of Resolving Agents, S.H. Wilen.

Thus, according to the invention, compound (A) is obtained by separation of the compounds by enzyme-mediated enantioselective catabolism with a suitable enzyme, such as cytidine deaminase, or by selective enzymatic cleavage of a suitable derivative using 5 'nucleotidase. When the resolution is carried out enzymatically, the enzyme; '. can be used either as a solution or more conveniently immobilized. The enzymes can be immobilized by any method known in the art, for example by adsorbing a stick to a resin such as Eupergit C.

• ·

»M

: '. The invention will be further illustrated by the following examples, which are not intended to limit the invention in any way. All temperatures are in degrees Celsius.

, 2 111723 Intermediate 15 5-Methoxy-1,3-oxathiolane-2-methanol, benzoate

A solution of zinc chloride (1.6 g) in hot methanol (15 mL) was added to a stirred solution of mercaptoacetaldehyde, dimethylacetal (34.2 g) and benzoyl-5-hydroxyacetaldehyde (48.3 g) in toluene (1300 mL), which was then heated in Palau. -cooling temperature under nitrogen for 50 minutes. The cooled mixture was concentrated, diluted with a small amount of toluene and filtered through Kieselguhr. The combined filtrates and toluene were washed with saturated aqueous sodium bicarbonate (x2) and brine, dried (MgSO 4), then evaporated to an oil which was subjected to 10 column chromatography on silica gel (2 kg, Merck 9385) eluting with chloroform to afford the title product as an oil. (about 1: 1), 1 H NMR (DMSO-cL) 3.1-3.3 (4H), 3.42 (6H), 4.4-4.6 (4H), 5.41 (1H ), 5.46 (1H), 5.54 (1H), 5.63 (1H), 7.46 (4H), 7.58 (2H), 8.07 (4H); ymax (CHBr3) 1717 cm @ -1.

Intermediate 2 (+) - cis -1- (2-Benzoyloxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidine-2,4-dione

A mixture of finely ground uracil (9.62 g), hexamethyldisilazane (50 mL) and ammonium sulfate (30 mg) was heated at reflux under nitrogen until a clear solution was obtained. This solution was cooled and then evaporated to a colorless oil, which was dissolved in acetonitrile (100 mL) under a nitrogen atmosphere.

The solution was added to a stirred, ice-cooled solution of 5-methoxy-1,3-oxathiolane-2-methanol, benzoate (Intermediate 1) (19.43 g) in acetonitrile: (600 mL) and trimethylsilyl trifluoromethanesulfonate (14 mL) was added. , 7 mL). The ice bath was removed and the solution was heated at reflux under nitrogen for 45 minutes. ·. After cooling and evaporation, the residue was purified by column chromatography tL '1 kg of silica gel (Merck 9385) eluting with a chloroform / methanol mixture of t * Γ 9: 1. The appropriate fractions were cooled and evaporated to give an impure residue.

!; 1 | This fraction was crystallized with a minimum amount of hot methanol (about 1200 mL) to give the title compound (6.32 g) as white crystals.

I I · »

> I

Intermediate 3 (±) - (cis) -4-Amino-1- (2-benzoyloxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one

Method (a) A suspension of cytosine (20.705 g) and ammonium sulfate (few mg) in hexamethyldisilazane (110 mL) was stirred and refluxed under nitrogen for 2.5 hours. The solvent was removed by evaporation and the resulting solid was dissolved in dry acetonitrile (350 mL). This solution was transferred using a flexible needle method to a stirred, ice-cooled solution of 5-10 methoxy-1,3-oxathiolane-2-methanol, benzoate (Intermediate 1) (43.57 g) in acetonitrile (650 mL) under nitrogen. Trimethylsilyl trifluoromethanesulfonate (33 mL) was added, the solution was allowed to warm to ambient temperature (1.5 h) and then refluxed overnight. The residue mixture was concentrated, diluted with saturated aqueous sodium bicarbonate solution (500 mL), then extracted with ethyl 15 acetate (3 x 500 mL). The combined extracts were washed with water (2 x 250 mL) and brine (250 mL), dried (MgSO 4) and then evaporated to a foam which was subjected to silica gel column chromatography (600 g, Merck 7734) eluting with ethyl acetate / methanol mixtures to give an anomeric mixture (n. 1: 1, 31.59 g). Link. . crystallized from water (45 mL) and ethanol (9.0 mL) to give a solid · · * 20 (10.23 g) which was recrystallized from ethanol (120 mL) and water (30 mL) to The title compound was obtained as a white solid (9.26 g), λmax (MeOH) 229.4 mm (E1610); 272.4 mm (E \ L 293); 1 H NMR (DMSO d 6) δ: 3.14 (1H), 3.50 (1H), 4.07 (2H), 5.52 (1H), 5.66 (1H), 6.28 (1H) , 7.22 (2H), 7.56 (2H), 7.72 (2H), 8.10 (2H).

Method 25 (b)

Phosphorus oxychloride (7.0 mL) was added dropwise to a stirred, ice-cooled suspension of 1,2,4-triazole (11.65 g) in acetonitrile (120 mL) and then, keeping the internal temperature below 15 ° C, triethylamine was added dropwise. (22.7 mL). After 10 minutes, a solution of (±) -cis-1- (2-30 benzoyloxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidine-2,4-dione was slowly added (interm. 2). (6.27 g) in acetonitrile (330 mL). Stirring was then continued at room temperature overnight. The mixture was cooled in an ice bath and triethylamine (30 ml) was slowly added followed by water (21 ml). The resulting solution was evaporated and the residue was partitioned between saturated sodium bicarbonate solution (400 ml) and chloroform (3 x 200 ml). The combined chloroform extracts were dried over magnesium sulfate, filtered and evaporated to give an impure residue (9.7 g). The residue was dissolved in 1,4-dioxane (240 mL) and concentrated aqueous ammonia (specific gravity 0.880, 50 mL) was added. After 1.5 h, the solution was evaporated and the residue was dissolved in methanol. This caused a solid to precipitate which was filtered off. The mother liquors were purified by column chromatography over silica gel (Merck 9385, 600 g). The appropriate fractions were combined and evaporated to give the title compound as a tan solid (2.18 g), identical to that obtained in method (a).

Example 1 (±) - (cis) -4-Amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pynmid N-2-one

A suspension of (cis) -4-amino-1- (2-benzoyloxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one (Intermediate 3) (8.19 g) and Amberlite IRA-400 (OH) 15 resin (8.24 g) in methanol (250 mL) was stirred and refluxed for 1 1/4 h. The solids were removed by filtration and then washed with methanol. The combined filtrates were evaporated. The residue was triturated with ethyl acetate (80 mL). The resulting white solid was collected by filtration to give. . title compound (5.09 g), δ 201 H NMR (DMSO d6) 3.04 (1H), 3.40 (1H), 3.73 (1H), 3.73 (2H), 5.18 ( 1H), 5.29: ··· (1H), 5.73 (1H), 6.21 (1H), 7.19 (2H), 7.81 (1H).

* · *

Example 2 T: (-) - cis -4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2 - one • * * * * ', (2 O) (±) -cis-4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidine; ; · 'Noni, dihydrogen phosphate, ammonium salt

Cold (0 °), stirred suspension of (±) -cis-4-amino-1- (2-hydroxymethyl) -1,3-oxathiolan-5-yl) - (1H) -pyrimidine -2-one (1.00 g) (Example 1) in dry dimethyl phosphate (20 mL) was treated with phosphorus oxychloride (2.44 mL) and the mixture was stirred at 0 ° and then cooled in ice water (60 g). The cold mixture was adjusted to pH 2.5 by addition of aqueous N-sodium hydroxide solution, applied to a charcoal column (10 g, DARCO) which was eluted with water and then with a mixture of ethanol 111723 and aqueous ammonia. Fractions containing the crude monophosphate were combined and concentrated. The resulting solution was applied to a column containing 25 g of DEAE Sephadex A-25 (HC03 form). Elution was performed with a gradient from water (120 mL) to 0.1 M NH 4 HCO 3 (240 mL), then 0.2, 0.3 and 4 M NH 4 HCO 3 (120, 5 240, 400 mL, respectively). Appropriate fractions were combined and concentrated. The residual solution was diluted with water (40 mL) and freeze-dried to give the title product as a white solid (1.37 g); ax (pH 6 buffer), 271.0 mm (KL 190); 1 H NMR (D 2 O) δ 3.23 (1H), 3.55 (1H), 4.0-4.2 (2H), 5.43 (1H), 6.07 (1H), 6.33 ( 1H), 8.08 (1H).

(Ii) (+) - (cis) -4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one and (-) - (cis) -4-Amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one 5'-nucleotidase (obtained from crotalus atrox venom) [EC 3.1.3.5 ] (60 mg at 17 units / mg) was added to a solution of (±) -cis-4-amino- (2-hydroxymethyl-1,3-15-oxathiolan-5-yl) - (1H) -pyrimidin-2-one. onium, 6'-dihydrogenphosphate, ammonium salt (1.35 g) in buffer [30 ml, prepared from glycine (526 mg) and magnesium chloride (190 mg) in water (100 ml)] and the mixture was incubated at 37 ° for 2.5 hours. More enzyme (20 mg) was added and incubation continued for a further 3.5 hours. The resulting mixture was applied to a DEAE Sephadex A-25 column (HC03 form). Elution was carried out with water-20 (160 mL), then with 0.1, 0.2, 0.3 and 0.4 M NH4HCO3 (200 mL each). First, the appropriate fractions containing the eluted component were combined and evaporated, the residue was subjected to silica gel column chromatography (60 g, Merck 7734); · ·; eluting with mixtures of chloroform and methanol. Evaporation of the appropriate fractions with Me · ;; ; of a mixture of tanol and ethyl acetate gave (+) - (cis) -4-amino-1- (2-hydroxymethyl-4'-1,3,5-oxathiolan-5-yl) - (1H) -pyrimidin-2-one as a white solid material (0.30 g): (a) D21 + 137 ° (c.1.04 in MeOH); 1 H NMR (DMSO) δ 3.04 (1H), 3.40 (1H), 3.37 (2H),. ···. 5.18 (1H), 5.29 (1H), 5.73 (1H), 6.21 (1H), 7.19 (2H), 7.81 (1H).

The appropriate fractions from the Sephadex column containing the second eluted component were pooled and evaporated. The residue was dissolved in water (30 ml), treated with alkaline phosphatase (Escherichia colista) [EC 3.1.3.1] (1.5 ml at '416 units / ml), and then incubated at 37 ° for one hour. The solvent was removed by evaporation and the residue was subjected to silica gel column chromatography (60 g, Merck 7734), eluting with chloroform-methanol mixtures. Evaporation of the appropriate fractions from a mixture of methanol and ethyl acetate gave (-) - (cis) -4-amino-1- (2-hydroxy-16,117,223-dimethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidine-2- onion as a white solid (0.32 g); (a) D21 + 132 ° (c. 1.08 in MeOH); 1 H NMR (DMSO) δ 3.04 (1H), 3.40 (1H), 3.37 (2H), 5.18 (1H), 5.29 (1H), 5.73 (1H), δ , 21 (1H), 7.19 (2H), 7.81 (1H).

Example 3 (-) - cis -4-Amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one (i) Three 50 ml flasks of nutrient broth (Oxoid Ltd) were each inoculated with bud-like Escherichia coli (ATCC 23848) scraped from a Nutrient Agar-1 ° plate. The flasks were incubated overnight at 37 ° C with shaking at 250 rev / min and then each flask was used to inoculate 4 L of CDD medium (glutamic acid 3 g / L, MgSO 4 0.2 g / L; K 2 SO 4 2.5 g / L). 1; NaCl 2.3 g / L, Na 2 HPO 4 2H 2 O 1.1 g / L; Na 2 H 2 PO 4 2 H 2 O 0.6 g / L; cytidine 1.2 g / L in a seven liter fermenter. Cultures were fermented at 750 rpm, 37 ° C with aeration of 4 l / min. After 24 hours of growth, cells were harvested by centrifugation (5000 g, 30 minutes) to obtain a wet weight of 72 g. The cell pellet was resuspended in 300 ml of 20 mM Tris-HCl buffer (pH 7.5) and disrupted by sonication (4 x 45 seconds). Cell debris was removed by centrifugation (30,000 g, 30 minutes) and protein in the supernatant. V was precipitated by addition of ammonium sulfate to 75% saturation. The precipitate was collected by centrifugation (30,000 g, 30 minutes) and the pellet resuspended in: ··· 25 ml of HEPES buffer (100 mM, pH 7.0) containing ammonium sulfate ··: · (75% saturation). The enzyme solution was prepared by centrifugation at speed; della 12,000 rpm for 30 minutes. The supernatant was discarded and the pellet was dissolved

• t «I

. : ·. Tris-HCl buffer (pH 7.0; 100 mM) to the original volume.

(N) The product of Example 1 (115 mg) was dissolved in water (100 mL) and stirred. Enzyme solution (0.5 mg) was added and the mixture was kept at constant pH by continuous addition of HCl (25 mM). The change was monitored by chiral HPLC, which indicated that the (+) - enantiomer of the substrate was predominantly deaminated. After 22 hours, the (+) enantiomer of the substrate (RT 12.5 min) was completely removed and the pH of the solution was adjusted to 10.5 with additional 30% concentrated sodium hydroxide.

The product prepared above was eluted through a QAE Sephadex column (A25; Pharmacia; 30 x 1.6 cm), pre-equilibrated to pH 11. The column was washed with water (200 mL) and then HCl (0.1 M). . Fractions (40 mL) were collected and analyzed by reverse phase HPLC. Fractions 5-13 containing the unreacted (-) enantiomer of the substrate 17111723 were pooled and adjusted to pH 7.5 with HCl. Fraction 47 containing the deaminated product was adjusted to pH 7.5 with dilute NaOH. Analysis by Chiral HPLC showed that this material was a mixture of one enantiomer (RT 10.2 min) as the major component together with another enantiomer (RT 8.5 min) as the 5 minor components (approximately 90% error).

(iii) Step (ii) above was repeated on a larger scale. The compound of Example 1 (363 mg) in 250 mL of water was incubated with the enzyme solution (0.5 mL) prepared as in step (i). Additional aliquots (0.5 ml) of enzyme were added after 18 and 47 hours. The reaction mixture was stirred for 70 hours and then left standing for an additional 64 hours. Chiral HPLC analysis showed that the (+) enantiomer of the substrate was completely deaminated and the resulting solution was adjusted to pH 10.5 with NaOH.

The above solution was loaded onto the same QAE column and eluted as in step (i). Fractions 2-6 containing the mixture of residual substrate and deaminated product were combined. Fractions 7-13 containing the residual substrate ((-) - enantiomer) were combined and the pH adjusted to 7.5. Fractions 25-26 containing the deaminated product were combined and neutralized.

The above fractions 2-6 were re-eluted through the same QAE column. Fractions 3-. . 11 of these second columns contained an unreacted substrate ((-) enantiomer).

·. ; * 20 The fraction 70 contained deaminated product.

: * * i (iv) The degraded substrate fractions from step (ii) and (iii) were combined and the pH adjusted to 7.5. The solution was eluted through an XAD-16 column (40 x 2.4 cm) packed in water.

· · · ·: The column was washed with water and then eluted with acetone: water (1: 4 v / v). Fractions,. *: ·! containing the (-) enantiomer from BCH 189 were combined and freeze-dried to give a white powder (190 mg).

:: The HPLC methods used above were as follows: I · I · · · · 18 111723

1. Analytical Reverse Phase HPLC

Columns: Capital Cartridge Spherisorb ODS-2 (5 μΜ) 150 x 4.6 mm

Eluent: Ammonium dihydrogen phosphate (50 mM) + 5% MeCN

Flow rate: 1.5 ml / min

Detection: UV, 270 nm

Retention times: BCH 189 5.5 min: Deaminated BCH-189 8.1 min

2. Chiral analytical HPLC

10 Columns: Cyclobond I Acetyl 250 x 4.6 mm

Eluent: 0.2% triethylammonium acetate (pH 7.2)

Flow rate: 1.0 ml / min

Detection: UV, 270 nm

Retention times: BCH 189 11.0 and 12.5 min 15: deaminated BCH-189 8.5 and 10.2 min (After bioconversion, peak disappearance was observed in 12.5 min and product accumulation in 10.2 min).

Example 4 • · · (-) - cis -4-Amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2 * 2 * 2one: Loop E. coli B cells (ATCC 32848) scraped from a well-grown nutrient plate were used to inoculate two Florence flasks, each containing 250 ml of nutrient broth. The culture was incubated at 37 ° with shaking. . (250 rpm, 5 cm swing) for 18 hours. This was then used to inoculate 40 1 '; / 2 5 CDD media with cytidine in a 70 L fermenter.

•: 'The fermentation conditions were as follows: 40 rpm aeration, 750 rpm agitation at 37 ° C. Three Rushton mixers were attached to the fermenter. · Fermentation was carried out 18 hours prior to harvest using Sharples

; * ·. continuous centrifuge. The cell paste (net weight 150 g) was frozen at -20 ° C

: -.- 30 before cell lysis.

1911723 CDD Medium m L-Glutamic Acid 3 MgSO4 0.2 5 K2SO4 2.5

NaCl 2.3

Na 2 HPO 4 1.1

NaH 2 PO 4 0.6

Made in distilled water. Sterilized at 121 ° C for 30 minutes. Cytidine (1.2 g / L) was filtered sterilized and added before inoculation.

Frozen cell paste (150 g) was thawed and suspended in 750 ml of 100 mM Hepes buffer (N- [2-hydroxyethyl] piperazine-N '- [2-ethanesulfonic acid]) (pH 7.5) containing 1 mM ethylenediaminetetra- acetic acid (sodium salt) and 1 mM dithiothreitol (disintegration buffer). Cells were lysed by passing the suspension through a Manton-15 Gaulin homogenizer operating at a pressure of about 5.2 kPa (7500 psi). This was done three times with cooling of the suspension to approximately 5 ° after each visit in the homogenizer. The homogenate was clarified by centrifugation (1400 g; 60 min). Cytidine deaminase activity was adsorbed onto a Q-Sepharose column. . (bed volume 490 mL) pre-equilibrated with 50 mM Tris (hydroxymethyl) t-methyl methylamine (pH 7.5) containing 1 mM sodium chloride. The combined active fractions (210 mL) were added to a phenyl-Sepharose column (bed volume 490 mL): pre-equilibrated with a buffer sample containing 3.2 M ammonium sulfate. The bound enzyme was eluted with a gradient of falling ammonium sulfate. Fractions containing cytidine deaminase activity were pooled (695 mL) and; The partially purified enzyme was then precipitated with 80% ammonium sulfate. After centrifugation (1400 g; 60 min), the pellet was resuspended in 54 ml of super. : the natant obtained above and stored at 4 ° C.

6.2 ml of this solution were centrifuged (18000 g, 90 min) and the pellet was dissolved in 24 ml of 0.5 M potassium phosphate buffer (pH 7.5). The suspension was dialyzed overnight at 1 M

.''30 potassium phosphate buffer (pH 7.5). The residue (20 ml) was then diluted with an equal volume of distilled water. To 35 ml of this solution were added dry Eupergit C beads: '' (1 g) and the mixture allowed to stand at room temperature for 150-300 hours (determined by measuring the residual cytidine deaminase activity in the solution). The immobilized enzyme was washed with 100 mM Tris / HCl buffer (pH 7.0) containing 1 mM EDTA, 35 1 mM DTT, 0.5 M NaCl, and 500 ppm ethyl p-hydroxybenzoic acid (storage buffer). The immobilized enzyme (wet weight 2.7 g) was stored in this buffer at 4 ° C until needed for biotransformation.

The product of Example 1 (3 g) was dissolved in 500 mL of distilled water in a 1 L flask equipped with a magnetic stirrer. The bioconversion was performed at 32 ° C in the pH-stat phase. The pH 5 was kept constant at 7.0 by the addition of 1 M acetic acid. The wet weight of 1 g of the immobilized enzyme beads was washed with distilled water before the reaction started. The change was monitored by chiral HPLC, which showed that the (+) - enantiomer of the substrate was predominantly deaminated. At the end of the reaction (72 h), the enzyme beads were filtered off and the filtrate was used to isolate the desired (-) - enantiomer.

The pH of the reaction mixture was adjusted to 10.5 using ammonia solution (1 M) and the solution was introduced into a Duolite Al 13 super-resin in an OH cycle (50 mL; 0.4 ped volume per hour). The uridine analog was adsorbed to the resin and the (-) enantiomer was directly migrated. The (-) - enantiomer remaining in the resin was removed by washing with 0.04% ammonia solution (2 bed volumes; flow rate 0.8 bed volumes per hour).

The pH of the solutions and washings (600 ml) used was adjusted to 7.0 with concentrated sulfuric acid and the solution was applied to XAD16 resin (50 ml; flow rate 1.4 ppm volume per hour). The column was washed with distilled water (2.5 bed volume, flow rate 2 bed volume per hour) and the (-) enantiomer eluted with acetone: water. . (1: 3) (flow rate 1.5 cubic feet / hour).

The fraction containing the 20 (-) - enantiomeric mass (4 bed volumes) was concentrated in a Buchi: ··: evaporator to a small volume before filtration through glass sinter # 3.

··· The filtered solution was freeze-dried to give 1.2 g of the title product, which was M t * identical to the product obtained in Example 3.

EXAMPLE 5 Tablet Formulations A. The following formulation is prepared by wet granulating the ingredients with a solution containing providone in water, drying and screening, after which : " *; Magnesium stearate is added and pressed.

21 111723 mg / tablet (a) Active ingredient 250 (b) Lactose B.P. 210 (c) Providoni B.P. 15 (d) Sodium starch glycolate 20 (e) Magnesium stearate 5 500 B. The following formulation is prepared by direct compression; lactose is of direct compressible quality.

10 mg / tablet

Active substance 250

Lactose 145

Avicel 100

Magnesium Stearate 5 15 500 C. (Formulation with Controlled Release). The formulation is prepared by wet granulating the ingredients (below) with a solution of providon in water, drying and sieving followed by the addition of magnesium stearate and a press. . a.

2 0 mg / tablet: ": (a) Active ingredient 500 ··· (b) Hydroxypropylmethylcellulose · · · ·: Lose (Methocel K4M Premium) 112 (c) Lactose BP 53 25 (d) Providone BP 28. ( e) Magnesium Stearate 7,700 <* »: L, Example 6: ...: Capsule Formulation · '' 30 The capsule formulation is prepared by mixing the ingredients below and dividing into a two part hard gelatin capsule.

22 11273 mg / capsule

Active substance 125

Lactose 72.5

Avicel 50 5 Magnesium stearate 2.5 250

Example 7

Injectable formulation

Active ingredient 0.200 g Sodium hydroxide solution, 0.1 M q.s. to a pH of about 11.

Sterile water q.s. To 10 ml

The active ingredient is suspended in a small amount of water (which may be heated) and the volume of the batch is made up to volume and filtered through a sterilized membrane filter into a sterile 10 ml glass ampoule and sealed with sterile closures and closures.

Example 8. . Suppository formulation • '', V mg / suppository: · · * V quickener 250 * •• 20 Hard Fat, B.P. 1770 2020 * * * * 11 * • <· '· * * One-fifth of the hard fat is thawed in a steam jacket at a temperature of not more than 45 ° C. Pass the active substance through a 200 μΐη: η sieve and add thawed

• I

:: to the bottom by mixing using a high shear mixer, '' 't': until a soft dispersion is obtained. While maintaining the temperature at 45 ° C, the remainder is added; hard fat in suspension and mix until a homogeneous mixture is obtained. Pass the entire i i · suspension through a 250 μΐη: η stainless steel sieve and allow to cool to 40 ° C while stirring continuously. At 38-40 ° C, 2.02 g of the mixture is filled into suitable 2 ml plastic molds. The suppositories are allowed to: '1,: 3 0 cool to room temperature.

23 1 1 1723

Example 9 Biological Activity Antiviral Activity

The antiviral activity of the compounds of Example 2 was determined against three HIV-15 strains and one HIV-2 strain in the following cell lines.

JM cells, a semi-mature T cell line obtained from a lymphoblastic leukemia patient infected with HIV-1 strain GB8.

C1866 cells, human T-lymphoblast cell line infected with HIV-1 strain RF.

MT-4 cells Human T cell leukemia cell line infected with HIV-1 strain RF.

10 CEM cells, human T-lymphoblast cell line infected with HIV-1 strains RF and U455 or HIV-2 strain ROD.

Antiviral activity in C8166, JM and CEM was determined as inhibition of cell adhesion formation (Tochikura et al., Virology, 164; 542-546) and in MT-4 cells as inhibition of formazan conversion (Baba et al., 1987) Biochem. Biophys Res Commun., 142, 128-134; Mossman (1983) J. Immun Meth; 65, 55-57).

. . Antiviral activities were also determined by analysis of HIV p24 antigen *. an amount synthesized in the presence and absence of enantiomers.

: ·: The results are given in Tables 1 and 2 below: * · · J '\' Table 1: ·; __ 50% antiviral activity (^ Lg / ml) _

Experiment__Pharmacan__Inhibition of cell adhesion formation_

Solut_MT-4_CEM CEM CEM JM_C8166

Virus flHV-11 HIV-1 RF HTV-2 ROD HIV-1 RF HTV-1 IJ455 HIV-1 GR8 HTV-1 RF

I + l-enantiomers. 0.28_ 0045 0-07 0.006 0.03_005 ... "M-enantiom 0.20__0,055__0.04_ 0.008_0,05__0.01_: λ 20 * *»> · ♦ · 24 111723

Table 2 Inhibition of HIV p24 Synthesis 50% Inhibition of HIV p24 Synthesis tjig / mll_

Solut_C8166 I JM MT-4

Virus_RF_GB8 RF

t + Venantiom. 0.021_ 0.033 0.0008 t-Venantiom. 0.016_ 0.016 0.0004 B. Cytotoxicity The cytotoxicity of the compounds of Example 2, the racemic compound (BCH-189; Example 1) and the 50/50 mixture of the two enantiomers was determined in five CD4 cell lines: H9, JM, CEM, C8166 and U937.

Test compounds were serially diluted from 100 pg / ml in 0.3 μg ιηΐ ^ βη (final concentrations) to 96-well microtiter plates. 3.6x104 cells were inoculated into each well of plate 10 as well as non-drug control plates. After incubation at 37 ° C for 5 days, the number of viable cells was determined by taking a cellular pellet sample and counting the cells outside the trypan blue by hemocytometry. you.

• · • ·

The results are shown in Table 3.

: '1: 15 Table 3;: · Cytotoxicity • · ___50% cytotoxicity (µg / ml) __

Compound CEM cells JM cells H9 cells U937 cells r8i66-snlnf ί + 1-enantiom. 1__LJ_2__4__35_ f-Venantiom. > 100__30_> 100_> 100_> iqq_ BCH-189 3_3J_8_ \ 5_90_ 1: 1 blend__2Ji_ND1_ND_ND_ND_ • · ·: ND = not set 20 • 1 ·

Claims (8)

25 1 1 1723 A process for the preparation of substantially pure (-) enantiomer of cis -4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one or a salt, ester or an ester salt for use as an antiviral, wherein the (+) - enan-5 thiomer is present in an amount of up to 5% by weight, characterized in that the (-) - enantiomer is separated from a mixture of cis-4-amino-1- (2- hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one enantiomers or their salts, esters or salts of esters, by separation by enzyme-mediated enantioselective catabolism. Process according to Claim 1, characterized in that the (+) enantiomer is present in the resulting product up to 2% by weight. Process according to claim 1 or 2, characterized in that the (+) - enantiomer is present in the resulting product up to 1% by weight. Process according to one of Claims 1 to 3, characterized in that the product obtained is pure (-) - cis -4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) pyrimidin-2-one or a salt, ester or ester salt thereof. Method according to one of Claims 1 to 4, characterized in that. . the mixture of compounds to be separated is a racemic mixture. v .; A process according to any one of claims 1 to 5, characterized in that: ** i20 is used in immobilized form. • «·!”! Process according to one of Claims 1 to 6, characterized in that the enzyme is a cytidine deaminase. The method according to any one of claims 1 to 6, characterized in that the enzyme is a 5-nucleotidase. '••• '25 I I | t · »« «« · 1 • · »» 26 1 1 1723