WO2003040139A1

WO2003040139A1 - Diastereoselective process for the preparation of the antiviral agent4-amino-1-(2r-hydroxymethyl-[1,3]oxathiolan-5s-yl)-1h-pyrimidin-2-one

Info

Publication number: WO2003040139A1
Application number: PCT/EP2002/012308
Authority: WO
Inventors: Maurizio Botta; Cristina Crescenzi; Daniele Mancinetti; Marco Pompei; Fausto Bonifacio
Original assignee: Recordati S.A.
Priority date: 2001-11-06
Filing date: 2002-11-05
Publication date: 2003-05-15
Also published as: TW200406212A; ITMI20012317A1

Abstract

Process for the synthesis of 4-amino-1-(2R-hydroxymethyl - [1,3]oxathiolan-5S-yl)-1H-pyrimidin-2-one (LAMIVUDINE) comprising the reduction of 2-(R)-(4'-(R)acetonyl)-5-oxo-1,3-oxathiolan to give 2-(R)-(4'-(R)-acetonyl)-5-hydroxy-1,3-oxathiolan.

Description

Diastereoselective process for the preparation of the antiviral agent 4-amino- 1-(2R-hydroxymethyl - [1,3]oxathiolan-5S-yl)-1H-pyrimidin-2-one

FIELD OF THE INVENTION

The present invention is located in the field of the organic synthesis of chiral nucleosidic analogues, which constitute an important class of antiviral agents, and refers to a stereoselective process for the preparation of 4-amino-1-(2R- hydroxymethyl - [1 ,3]oxathiolan-5S-yl)-1 H-pyrimidin-2-one (LAMIVUDINE).

PRIOR ART

A nucleoside is a molecule constituted of a glycidic portion (ribose or one of its analogues) and a purinic or pyrimidinic base.

3' 2'

In 1985, it was reported that the synthetic nucleosidic analogue 3'-azido-3'- deoxythymidine (AZT) inhibited the replication of the human immunodeficiency virus type 1 (HIV-1 virus) {Mitsuya, H. et al., Proc. Natl. Acad. Sci. U8A 82. 7096 (1985), WO 91/17159}.

The replicative cycle of the virion initiates with its attachment to T4 lymphocyte cells through binding to a glycoprotein (CD4) present on the lymphocyte. Once the attachment has been made, the virion fuses with the cellular membrane and penetrates the host cell, releasing its RNA. The viral enzyme, Reverse Transcriptase, directs the transcription process of the RNA into a single strand of DNA. The RNA is then degraded and a second strand of DNA is created. Finally the double stranded DNA is integrated into the genome of the infected T cell.

AZT, analogously to other nucleosidic derivatives later identified, inhibits the enzyme Reverse Transcriptase and blocks the transcription of viral RNA to viral DNA.

Following phosphorylation at the 5' position, the nucleosidic analogues such as 3'- azido-3'-deoxythymidine (AZT), 2'-3'-dideoxycytidine (DDC), 2',3'-didehydro-3'- deoxythymidine (D4T), 2',3'-dideoxyinosine (DDI) are potent inhibitors of the enzyme Reverse Transcriptase of the HIV virus and induce termination of the nascent viral DNA chain.

The stereochemistry of the nucleosidic derivatives plays an important role in their biological activity. The C1 ' carbon atom on the saccharide unit (the atom bound to the nitrogen of the nucleobase) is a chiral centre. Analogously another asymmetric centre is present in position C4' (to which the hydroxymethyl group is bound). In the naturally occurring nucleosides, the base in position C1 ' and the hydroxymethyl group on C4' are on the same side of the sugar ring. The configuration which sees both substituents of the sugar on the same side (cis substituents) is called the " β configuration". The configuration which sees the substituents in position C1' and C4' on opposite sides of the plane of the sugar (trans substituents) is defined " configuration". Still with reference to the figure above, a nucleoside is defined D if the hydroxymethyl substituent is oriented above the plane of the glycidic portion. The nucleoside is, finally, designated L if the hydroxymethyl substituent in position C4' is oriented below the plane of the sugar.

The non-naturally occurring α anomers of the nucleosides are rarely biologically active and typically toxic. X ray analysis of the solid state conformations {Van Roey, P. et al., J. Am. Chem. Soα, 1988, 10, 2277; Van Roey, P. Et al, Proc. Natl. Acad. Sci. USA. 1989, 86, 3929} has revealed that the C3'-exo conformation belongs to the compounds which are biologically active, whilst the inactive compounds prefer a C3'-endo conformation (the designations endo and exo refer to conformations in which the atoms are on the same side or on the opposite side of the sugar ring with respect to the position of the base). The C3'-exo and C3'- endo conformations place, respectively, the C5' atom in axial and equatorial positions. The position of the C5' atom influences the disposition of the 5'- hydroxymethylic group in relation to the base. Because the 5'- hydroxymethyl group is the site of phosphorylation of the nucleoside, it acquires importance in merit of its position with respect to the rest of the molecule.

More recently, different types of atypical nucleosides have been identified as potent antiviral agents against the HIV, HBV and HTLV viruses {WO 89/04662 and

EP 0349242 A2}.

In particular, the molecule 4-amino-1-(2R-hydroxymethyl - [1 ,3]oxathiolan-5S-yl)-

1 H-pyrimidin-2-one which can be represented by the following formula:

(also known as 3TC™ or LAMIVUDINE or (-)BCH-189) and its various pharmaceutically acceptable derivatives, (EP 382526), shows antiviral activity, in particular against the human immunodeficiency virus (HIV) and hepatitis B virus

(HBV) retroviruses.

Both enantiomers with the β configuration have comparable activities towards the

HIV virus, but the (-)-enantiomer has considerably much lower cytotoxicity

(W091/17159). Therefore a stereoselective general synthesis only of the cis nucleoside with L configuration represents an important synthetic target.

Most processes described in the literature for the synthesis of optically active nucleosides, take into consideration structural modifications of natural nucleoside derivatives. {C.K. Chu et al., J. Orq. Chern. 54, 2217-2225 (1989)}, modifying the base or the sugar through reductive processes such as deoxygenation or radicalic reduction.

The processes reported for the synthesis of Lamivudine can be classified in 3 groups:

1. synthesis starting from a "Chiral Pool";

2. synthesis of the racemic product and final resolution;

3. chiral synthesis. The first approach involves the synthesis of the glycidic portion starting from a chiral precursor, which is then reacted with the silylated nucleobase. Examples reported in the literature involve the use of (+)-3-mercaptoacetic acid (Humber D.C. et al. Tetrahedron Lett 33, 4625 (1992) ) and L-gulose (L. S. Jeong et al. J. Med. Chem. 36, 181 (1993), Beach et al. J. Org. Chem: 57, 2217 (1992)). The second approach involves the synthesis of the racemic product which is then later enzymatically resolved (WO 91/17159, EP 382526).

The third way involves the stereospecific synthesis of the desired intermediate using an appropriate chiral helper. The transformations involve numerous steps, with a consequent reduction in the total yield, and must not influence the stereointegrity of the starting nucleoside. Furthermore, it requires optically active natural nucleosides as starting materials which are expensive.

Other procedures, known for the synthesis of optically active nucleosides, are based on conventional glycosylation reactions using an appropriate nucleobase. The stereochemical control in this phase, so as to lead to just the cis stereoisomer, is generally achieved through substituents in position 2' or 3' of the sugar ring. Because the substituents in position 2' or 3' are capable of directing the stereochemical passage only when found in the trans conformation with respect to the leaving group in C1 ', laborious procedures are required to introduce these substituents in the desired configuration. Further steps are therefore necessary to obtain the finally desired nucleoside eliminating the substituents introduced to regulate the stereochemical pathway.{L. Wilson and D. Liotta "A General Method for Controlling Stereochemistry" Tetrahedron Lett. 31 , 1815-1818 (1990)}. Therefore the need was felt for new synthetic processes for the production of nucleosides with high yield, purity and optical purity, avoiding laborious procedures of protection-deprotection and for addition-removal of the substituents in position 2' and 3' on the glycidic portion, with reactions characterised by high yield, which can be easily carried out on industrial scale, with intermediates having high degree purity, through the use of easily commercially available reactants. SUMMARY

A new synthetic process for 4-amino-1-(2R-hydroxymethyl - [1 ,3]oxathiolan-5S- yl)-1 H-pyrimidin-2-one or β 2-((R)-hydroxymethyl)-5-(citosyl-1 '-yl)-1 ,3-oxathiolan (LAMIVUDINE) has been discovered comprising the reduction reaction of the derivatives of 1 ,3-oxathiolan-5-one, capable of overcoming the drawbacks of the processes known in the art. The applicant has unexpectedly and surprisingly found a new synthetic process for 4-amino-1-(2R-hydroxymethyl - [1 ,3]oxathiolan-5S-yl)-1 H-pyrimidin-2-one (LAMIVUDINE) comprising the reduction of 2-(R)-(4"-(R)-acetonyl)-5-oxo-1 ,3- oxathiolan to give 2-(R)-(4"-(R)-acetonyl)-5-hydroxy-1 ,3-oxathiolan. DESCRIPTION OFTHE FIGURES Figure 1 : scheme of the synthesis of 4-amino-1-(2R-hydroxymethyl - [1 ,3]oxathiolan-5S-yl)-1 H-pyrimidin-2-one (LAMIVUDINE) (XI). DETAILED DESCRIPTION OF THE INVENTION

Therefore, a subject of the present invention constitutes a diastereoselective synthetic process for 4-amino-1-(2R-hydroxymethyl - [1 ,3]oxathiolan-5S-yl)-1 H- pyrimidin-2-one (LAMIVUDINE) comprising the reduction reaction, defined as phase a), with reducing agents selected from the group consisting of: disiamylborane, di-isobutylaluminium hydride (DIBAL-H), (bis(2- methoxyethoxy)aluminium hydride) (RED-AL) and sodium borohydride, of 2-(R)- (4"-(R)-acetonyl)-5-oxo-1 ,3-oxathiolan (III) to give 2-(R)-(4"-(R)-acetonyl)-5- hdroxy-1 ,3-oxathiolan (IV) as a mixture of cis and trans stereo isomers. Preferably the reducing agent is di-isobutylaluminium hydride.

The molar ratios between 2-(R)-(4"-(R)-acetonyl)-5-oxo-1 ,3-oxathiolan (III) and the reducing agent, as defined above, is comprised between 1.1/1.0 and 1.0/1.0, preferably it is 1.04/1.0. The reduction reaction of 2-(R)-(4"-(R)-acetonyl)-5-oxo-1 ,3-oxathiolan is carried out in aliphatic and/or aromatic solvents such as for example benzene, xylene or toluene, preferably toluene, at a temperature comprised between -80°C and 20°C, preferably the temperature is comprised between -10° and 10°C. Compound (IV) can be easily converted into lamivudine through reactions which involve about the cytosilation of the oxathiolan ring, the opening of the dioxolan ring, the formation of the methyl substituent in position 2 of the oxathiolan ring and the separation of the α and β isomers, through chemical reaction, of the lamivudine obtained or, following cytosilation of the oxathiolan ring of the formula (IV) compound, the separation of the corresponding and β isomers thus formed, by their chemical reaction, the opening of the dioxolan ring of the desired β isomer and the formation of the methyl substituent to give lamivudine. One of these particularly preferred forms of realisation of the diastereoselective synthetic process of lamivudine according to the present invention, comprises further to the reduction phase a) mentioned above, also the phases: b) conversion reaction of the hydroxyl group in position 5 of the oxathiolan ring, in the cis-trans mixture of the 2-(R)-(4"-(R)-acetonyl)-5-hydroxy-1 ,3-oxathiolan intermediate, into a leaving group -L, in position 5 of the oxathiolan ring, selected from the group consisting of: acyloxy; alkoxy; aryloxy; alkylaryloxy; alkoxy carbonylic groups ; an aliphatic or aromatic aminocarbonyl group; phosphonate groups; halogens; amide groups; azide; isocyanate; substituted or not substituted, saturated or unsaturated thiolate; seleno, selenyl or selenonyl groups substituted, being non- substituted, saturated or unsaturated; c) silylated cytosine glycosylation reaction of the cis-trans mixture originating from phase b) in the presence of a Lewis acid, as a catalyst, to give the corresponding mixture of ,β-2-(R)-(4"-(R)-acetonyl)-5-(cytosyl-1'-yl)-1 ,3-oxathiolan nucleoside isomers (VII). Preferably in the reaction of phase c) the Lewis acid is selected from SnCI₄ and a compound of general formula:

R₈ Si-

(VI)

R₇ wherein: the R₅, R₆ and R substituents are the same or different from each other and are selected from the group consisting of: hydrogen; alkyl groups of one to twenty carbon atoms optionally substituted by fluorine, chlorine, bromine or iodine, alkoxy groups of one to six carbon atoms, aryloxy groups of six to twenty carbon atoms; arylalkyl groups of seven to twenty carbon atoms optionally substituted by halogens, alkyl groups of one to twenty carbon atoms or alkyloxy groups of one to twenty carbon atoms; aryl groups of six to twenty carbon atoms optionally substituted by fluorine, chlorine, bromine, iodine, alkyl groups of one to twenty carbon atoms, alkoxy groups of one to twenty carbon atoms; trialkylsilyl groups and halogen substituents: F, Cl, Br, I, and the R₈ substituent is selected from the group consisting of: fluorine, bromine, chlorine, iodine, sulphonic esters of one to twenty carbon atoms, optionally substituted by fluorine, chlorine, bromine, iodine; alkyl esters of one to twenty carbon atoms, optionally substituted by fluorine, chlorine, bromine, iodine; polyvalent halides; trisubstituted silyl groups with the general formula RsReRySi, in which R₅, R₆ and R have the aforementioned meanings, selenonyl aryl groups, saturated and unsaturated, of six to twenty carbon atoms; arylsulphonyl groups, substituted or non substituted, of six to twenty carbon atoms; alkoxyalkyl groups substituted or non substituted, of six to twenty carbon atoms; and trialkylsiloxy groups,

Preferably in the reaction of phase b) the hydroxyl group is converted into the leaving group -L selected from the group consisting of: halogens; alkoxy -OR groups, where R is selected from the group consisting of: alkyl groups, saturated or unsaturated, of one to twenty carbon atoms optionally substituted by fluorine, chlorine, bromine, iodine, alkoxy groups of one to six carbon atoms, aryloxy groups of six to twenty carbon atoms; aryloxy groups -OAr, where Ar is selected from the group consisting of: aryl groups of six to twenty carbon atoms optionally substituted by fluorine, chlorine, bromine, iodine, alkyl groups of one to twenty carbon atoms, alkoxy groups of one to twenty carbon atoms; alkylaryloxy groups -OR', where R' is selected from the group consisting of: arylalkyl groups of seven to twenty carbon atoms optionally substituted by halogens, alkyl groups of one to twenty carbon atoms or alkyloxy groups of one to twenty carbon atoms; acyloxy groups -OC(0)R" where R" is selected from the group consisting of: alkyl groups, saturated or unsaturated alkyl groups, of one to five carbon atoms, preferably methyl, ethyl, butyl, optionally substituted by amino, carboxyl, hydroxyl, phenyl, lower alkoxyl groups, preferably methoxyl and ethoxyl; phenyl group; phenyl groups substituted with lower alkyls, carboxyl, halogens, preferably chlorine and bromine, sulphate, sulphonyloxy, lower oxyalkyls, preferably carbomethoxy and carboethoxy; amino group; lower mono and di amino alkyl groups, preferably methylamino, dimethylamino. More preferably the leaving group -L is selected from the group consisting of: ethoxycarbonyl, iodine, bromine, chlorine, fluorine, acetate, benzoate, methylcarbonate, phenylcarbonate, diethylphosphonate.

The hydroxyl group conversion reaction in phase b) is carried out using methods well known and described in the literature such as in T.W. Greene, "Protective Groups In Organic Synthesis", pp 50-72, John Wiley & Sons, New York (1981). Preferably the hydroxyl group conversion reaction of the cis-trans mixture of the intermediate 2-(R)-(4"-(R)-acetonyl)-5-hydroxy-1 ,3-oxathiolan in phase b) in the process, subject of the present invention, takes place in methylene chloride in the presence of pyridine for the reaction with acetyl chloride at a temperature comprised between -5° and 5°C to give the cis-trans 2-(R)-(4"-(R)-acetonyl)-5- acetoxy-1 ,3-oxathiolan (V).

Preferably in the Lewis acid of formula (VI) the substituents R₅, R₆ and R₇, the same or different from each other, are selected from the group consisting of: fluorine, chlorine, bromine, iodine, methyl, ethyl, t-butyl, benzyl. When said substituent groups are alkyl and benzyl, they are in turn substituted with F, Cl, Br, I.

Preferably in the Lewis acid of formula (VI) the Rs substituent is selected from the group consisting of F, Cl, Br, I, tri-iodide.

Preferably the Lewis acid of formula (VI) is iodotrimethylsilane (TMSI) or trimethyl triflate (TMSOTf). The Lewis acid can be generated in situ or prepared using any method known in the literature such as for example those described in A.H. Schimidt "Bromotrimethylsilane and Iodotrimethylsilane - Versatile Reagents for Organic Synthesis". Adrichimica Acta 14, 31-38, (1981).

Cytosine, previously silylated, is reacted with the cis and trans isomers originating from phase b) in the presence of Lewis acids to give the corresponding mixture of ,β-2-(R)-(4"-(R)-acetonyl)-5-(cytosyl-1 '-yl)-1 ,3-oxathiolan nucleoside isomers (VII).

The reaction is normally carried out in aliphatic or aromatic organic solvents, preferably acetonitrile, methylene chloride at a temperature comprised of between 0°C and 25°C, preferably between 0°C and 5°C. The most appropriate procedure involves the addition of the cis-trans mixture originating from phase b) to the previously silylated cytosine nucleotide base, and later addition of the Lewis acid.

The nucleobase is silylated using an appropriate silylating agent, for example hexamethyldisilazane (HMDS) or t-butyldimethylsilyltriflate, in aliphatic or aromatic organic solvents such as for example acetonitrile or methylene chloride, in the presence of a sterically hindered base such as for example 2, 4, 6-collidine.

A further preferred embodiment of the lamivudine synthetic process according to the present invention comprises, following on from phases a), b) and c), as above, also the phases: d) conversion reaction of the nucleoside isomer mixture of α,β-2-(R)-(4"-(R)- acetonyI)-5-(cytosiyl-1'-yl)-1 ,3-oxathiolan (VII) in the corresponding mixture of ,β

2-((R)-hydroxymethyl)-5-(cytosyl-1'-yl)-1 ,3-oxathiolan nucleoside isomers (X); e) separation of the β 2-((R)-hydroxymethyl)-5-(cytosyl-1'-yl)-1 ,3-oxathiolan isomer (XI) (lamivudine) by chemical derivatisation of the mixture of ,β 2-((R)- hydroxymethyl)-5-(cytosyl-1'-yl)-1 ,3-oxathiolan nucleoside isomers (X), separation by physical means, of the mixture of derivatised nucleoside isomers, recovery of the β isomer by chemical removal of the derivatising agent, with the isolation of lamivudine (XI).

The conversion reaction in phase d) is carried out through methods well known and described in the literature such as in T.W. Greene, "Protective Groups In Organic Synthesis", pp 50-72, John Wiley & Sons, New York (1981). Preferably the conversion reaction comprises the removal of the chiral helper 4"-(R)- acetonide, in position 2 of the oxathiolan ring, by hydrolysis of said substituent group to give the corresponding mixture of diol isomers: α,β-2-(R)-(2"-(R)-1 ,2- dihydroxyethyl)-5-(cytosyl-1'-yl)-1 ,3-oxathiolan (VIII), oxidation of the diol isomers to give the mixture of ,β 2-((R)-formyl)-5-(cytosyl-1'-yl)-1 ,3-oxathiolan aldehyde isomers (IX) followed by reduction of the aldehyde isomers to give the corresponding mixture of ,β 2-((R)-idrossimetil)-5-(citosil-1'-il)-1 ,3-ossatiolano isomers (X).

The hydrolysis of the acetonide group of the nucleoside isomers: ,β-2-(R)-(4"- (R)-acetonyl)-5-(cytosyl-1 '-yl)-1 ,3-oxathiolan (VII) preferably takes place with strong mineral acid in protic solvents, more preferably in alcoholic solvents of one to four carbon atoms, still more preferably in methanol. Preferably the acid is hydrochloric acid, more preferably a solution of 5% (w/w) HCI in methanol. The oxidation of the mixture of isomers: α,β-2-(R)-(2"-(R)-1 ,2-dihydroxyethyl)-5- (cytosyl-1'-yl)-1 ,3-oxathiolan (VIII) preferably takes place in protic solvents with oxidants, for example sodium metaperiodate (Nal0₄). Preferably the solvent is an alcoholic solvent of one to four carbon atoms or water or mixtures thereof, more preferably a water/methanol mixture. The oxidation takes place at a temperature comprised of between -15° and -10°C.

The reduction reaction of the mixture of the isomers: ,β 2-((R)-formyl)-5-(cytosyl- 1'-yl)-1 ,3-oxathiolan aldehyde (IX) preferably takes place with sodium borohydride (NaBH₄). The reaction solvent is preferably alcoholic or hydroalcoholic, for example water/methanol.

The reaction temperature is comprised of between -25° and 15°C, preferably at 0°C. The separation of the isomer: β 2-((R)-hydroxymethyl)-5-(cytosyl-1'-yl)-1 ,3- oxathiolan (XI) (lamivudine) in phase e) by chemical derivatisation of the mixture of α,β 2-((R)-hydroxymethyl)-5-(cytosyl-1'-yl)-1 ,3-oxathiolan nucleoside isomers (X), followed by separation by physical means such as chromatography or fractional crystallisation, taking place according to techniques well known in the field of the resolution of mixtures of geometric isomers. Preferably the chemical derivatisation takes place by reacting the isomeric mixture with benzoylchloride in methylene chloride, with the formation of corresponding o-benzoyl cis/trans isomers, the physical separation occurs by fractional crystallisation and the β isomer thus isolated is hydrolysed to lamivudine.

A further preferred embodiment of the lamivudine synthetic process according to the present invention comprises, following on from phases a), b) and c), as above, also the phases: d') separation of the β 2-(R)-(4"-((R)-acetonyl)-5-(cytosyl-1'-yl)-1 ,3-oxathiolan isomer (XII) from the mixture of α,β-2-(R)-(4"-(R)-acetonyl)-5-(cytosyl-1'-yl)-1 ,3- oxathiolan nucleoside isomers (VII), by simple separation of the mixture of nucleoside isomers by physical means; e') the conversion reaction of the β 2-(R)-(4"-((R)-acetonyl)-5-(cytosyl-1 '-yl)-1 ,3- oxathiolan isomer (XII) into the corresponding β 2-((R)-hydroxymethyl)-5-(cytosyl- 1'-yl)-1 ,3-oxathiolan isomer (XI), lamivudine;

The separation of the isomer: β 2-(R)-(4"-((R)-acetonyl)-5-(cytosyl-1'-yl)-1 ,3- oxathiolan (XII) in phase d') is carried out according to methods well known in the field of the resolution of mixtures of geometric isomers by physical means such as chromatography or fractional crystallisation.

The conversion reaction in phase e') is carried out using methods well known and described in the literature such as in T.W. Greene, "Protective Groups In Organic Synthesis", pp 50-72, John Wiley & Sons, New York (1981 ). Preferably the conversion reaction comprises the removal of the chiral helper 4"- (R)-acetonide, in position 2 of the oxathiolan ring, through hydrolysis of said substituent group to give the corresponding β-2-(R)-(2"-(R)-1 ,2-dihydroxyethyl)-5- (cytosyl-1 '-yl)-1 ,3-oxathiolan diol (XIII), oxidation of the diol to give the β 2-((R)- formyl)-5-(cytosyl-1 '-yl)-1 ,3-oxathiolan aldehyde (XIV) followed by reduction of the aldehyde to give the corresponding β 2-((R)-hydroxymethyl)-5-(cytosyl-1 '-yl)-1 ,3- oxathiolan (XI), lamivudine.

The hydrolysis of the acetonide group of the β-2-(R)-(4"-(R)-acetonyl)-5-(cytosyl- 1 '-yl)-1 ,3-oxathiolan nucleoside (XII) preferably takes place with strong mineral in acid protic solvents, more preferably in alcoholic solvents of one to four carbon atoms, still more preferably in methanol. Preferably the acid is hydrochloric acid, more preferably a solution of 5% (w/w) HCI in methanol.

The oxidation of the β-2-(R)-(2"-(R)-1 ,2-dihydroxyethyl)-5-(cytosyl-1 '-yl)-1 ,3- oxathiolan nucleoside (XIII) preferably takes place in protic solvents with oxidants, for example sodium metaperiodate (Nal0₄). Preferably the solvent is an alcoholic solvent of one to four carbon atoms or water or mixtures thereof, more preferably a water/methanol mixture. The oxidation takes place at a temperature comprised of between -15° an -10°C. The reduction reaction of the β 2-((R)-formyl)-5-(cytosyl-1'-yl)-1 ,3-oxathiolan aldehyde (XIV) preferably takes place with sodium borohydride (NaBH ). The reaction solvent is preferably alcoholic or hydroalcoholic, for example water/methanol. The reaction temperature is comprised between -25° and 15°C, preferably 0°C. A further advantage of the synthetic process subject of the present invention is the use of 2-(R)-(4"-(R)-acetonyl)-5-oxo-1 ,3-oxathiolan as the starting reagent, which is easily obtainable through various methods known in the literature, as for example described in "Expeditious preparation of (-)-2'-deoxy-3'-thiacydine" Tetrahedron Letters, Vol. 33, No. 32, pp.4625-4628 (1992). More particularly 2- (R)-(4"-(R)-acetonyl)-5-oxo-1 ,3-oxathiolan is obtained through in situ synthesis of the aldehyde (R)-2,2-dimethyl-4-formyl-1 ,3-dioxolan by oxidation in dimethylsulphoxide with anhydrous phosphoric acid and dicyclohexylcarbodiimide (DCC) of the chiral helper (R)-2,2-dimethyl-4-hydroxymethyl-1 ,3-dioxolan and successive condensation with β-mercaptoacetic acid in dichloromethane at a temperature comprised between -10°C and +20°C. In particular the diastereoisomeric mixture of 2-(S)-(4"-(R)-acetonyl)-5-oxo-1 ,3-oxathiolan and 2- (R)-(4"-(R)-acetonyI)-5-oxo-1 ,3-oxathiolan thus obtained, is resolved by simple physical means, preferably by simple crystallisation obtaining 2-(R)-(4"-(R)- acetonyl)-5-oxo-1 ,3-oxathiolan in the form of a crystalline solid. Thanks to the reduction reaction of 2-(R)-(4"-(R)-acetonyl)-5-oxo-1 ,3-oxathiolan according to phase a) of the present invention it is surprisingly possible to obtain the synthetic intermediate of lamivudine: 2-(R)-(4"-(R)-acetonyl)-5-hydroxy-1 ,3- oxathiolan (IV) as a mixture of cis and trans stereoisomers; the intermediate which makes possible a lamivudine synthetic process with high yield, purity and optical specificity.

This reaction has been applied until now but without success ("2- Mercaptoaldehyde dimers and 2,5-dihydrothiophenes from 1 ,3-oxathiolan-5-ones" Can. J. Chem. Vol. 61 , pp. 1872-1875 (1983), and in "Expeditious preparation of (- )-2'-deoxy-3'-thiacydine" Tetrahedron Letters, Vol. 33, No. 32, pp.4625-4628 (1992)) in the case of 1 ,3-oxathiolan-5-one derivatives.

It constitutes therefore, a further subject of the present invention, the intermediate 2-(R)-(4"-(R)-acetonyl)-5-hydroxy-1 ,3-oxathiolan (IV) in the two stereoisomeric forms, cis and trans.

The process, subject of the present invention, is ideal for the synthesis of various lamivudine analogues, intending for "lamivudine analogues" the nucleotides which are formed by reactions of the 1 ,3-oxathiolan intermediate with pyrimidine nucleotide bases substituted, preferably at position 5. The substituents on the pyrimidine nucleotide bases can be: methyl, halogen, alkyl, alkenyl, alkynyl, hydroxyalkyl, carboxyalkyl, thioalkyl, selenoalkyl, phenyl, cycloalkyl, cycloalkenyl, thioaryl and selenoaryl. The products and the reaction intermediates have been characterised through the analytical techniques of HPLC, TLC, GC, GC/MS, ¹H-NMR and polarimetry.

Some examples for illustrative but not limitative purposes of the present invention, are reported as follows.

Example 1 : synthesis of 2-(R)-(4"-(R)-(acetonyl)-5-oxo-1 ,3-oxathiolan (III) A solution of 80.4g (0.82 mol; 0.5 equivalents) of anhydrous crystalline orthophosphoric acid in 120ml of dimethylsulphoxide, is strained into a solution of 942g (4.56 mol; 3 equivalents) of dicyclohexylcarbodiimide (DCC) in 800ml of dimethylsulphoxide. The mixture is maintained at T=0-5°C for 1 hour, thereafter a solution of 201.1g (1.522 mol; 1 equivalent) of (-)-R-Solketal in 50 ml of dichloromethane is added.

The mixture is maintained at T=25°C for 16 hours and subsequently filtered to remove the dicyclohexylurea. The organic solution is cooled to T=0-5°C, then through a dropping funnel, 106 ml (1.522 mol; 1 equivalent) of β-mercaptoacetic acid are slowly added and the mixture maintained under stirring for 4 hours. The reaction mixture is diluted with 600 ml of methylene chloride, and neutralised with a saturated solution of sodium bicarbonate until the effervescence disappears. The organic phase is washed with water, brine and finally dried over sodium sulphate. About 221 g of a crude oily yellow substance are recovered. The separation of the mixture of diastereoisomers is achieved by crystallisation. The mixture of diastereoisomers is dissolved in a warm solution obtained by mixing 884 ml of hexane and 160 ml of ethyl acetate. The clear solution is placed in a thermostated bath at -18°C for 12 hours. The crystallisation is improved by seeding with the desired product, obtained previously. Following filtration a white crystalline solid is recovered which is washed twice with 20 ml of hexane and dried under high vacuum. 108.8 g of lactone of formula (III) is recovered as a crystalline solid with a total yield for the phases herein described of 35%. Example 2: synthesis of cis-trans 2-(R)-(4"-(R)-(acetonyl)-5-acetoxy-1 ,3-oxathiolan (V) 108.8g (0.53 mol; 1 equivalent) of lactone (III) are dissolved in 500 ml of anhydrous toluene under an inert atmosphere. The reaction temperature is adjusted to -10°C, and under stirring, through a dropping funnel, are added over a period of around 20 minutes about 530 ml of a 1 M solution of DIBAL-H in toluene (1 equivalent).

Intense gas formation is initially observed. At the end of the addition the solution appears clear. For the entire course of the reaction, the temperature is maintained below -5°C. Following 2 hours of stirring, the reaction is stopped by the addition of 300ml of methanol, until the effervescence has disappeared. Following 10 minutes of stirring, the temperature is allowed to rise to room temperature, and a white precipitate forms which seems to be finely dispersed after 60 minutes of stirring. It is filtered on celite washing many times with methanol and recovering an oily residue which is dried under high vacuum. Later the crude product is dissolved, under an inert atmosphere, in 400ml of anhydrous methylene chloride and at 0°C are added, in order 430ml (10 equivalents) of anhydrous pyridine and then 113ml (3.5 equivalents) of freshly distilled acetyl chloride. The reaction proceeds for 1 hour at 0°C, and then it is stopped by dilution with 200ml of methylene chloride and then with 100 ml of an aqueous solution of 10% (w/w) citric acid. The recovered organic phase is washed with water, brine and dried over sodium sulphate. 79.3g of the acetylate product are recovered, with a total yield for the phases herein described of 60%. GC/MS analysis shows that the 2 anomers formed are present in a ratio equal to

1 :6.

Example 3: synthesis of α,β-2-(R)-(4"-(R)-acetonyl)-5-(cytosyl-1'-yl)-1 ,3-oxathiolan (VII) 42.6g of cytosine (0.3834 mol - 1.2 equivalents) are suspended in 242.7ml of hexamethyldisilazane (3.6 equivalents) under an inert atmosphere. A catalytic quantity of ammonium sulphate is added and the mixture is refluxed (at about 150°C) for 1.5 hours. The solution, perfectly clear, is cooled and the excess hexamethyldisilazane is removed by azeotropic distillation with anhydrous toluene. The silylated cytosine is dissolved in double distilled anhydrous acetonitrile (300ml) and at 0°C trimethyl triflate (57.8ml - 0.3195 mol, 1 equivalent) is added. Following stirring for 5 minutes at 0°C, the acetylate (V) (79.3g; 0.3195 mol) dissolved in 300 ml of anhydrous acetonitrile is added. The reaction mixture is kept under stirring for 1 hour at room temperature and then refluxed for 20 minutes (T= 100°C).

It is cooled and diluted with 200 ml of ethyl acetate. 500 ml of a saturated solution of sodium bicarbonate are added, and it is kept under stirring for 20 minutes. The organic phase is recovered and washed with water, brine and dried over sodium sulphate. 120 g of an oily residue is obtained. The purification is carried out by flash chromatography on a column (eluent: methylene chloride - methanol in a volume ratio of 9/1 ). 62.2g (0.2076 mol) of nucleoside (VII) are recovered, with a yield of 65%, in a ratio oc/β of about 1 :1. Example 4: synthesis of α,β 2-((R)-hydroxymethyl)-5-(cytosyl-1'-yl)-1 ,3-oxathiolan (X) 62.2g of nucleoside (VII) (0.208 mol, 1 equivalent) are solubilised in 200ml of methanol (d=0.787). 200 ml of a 5% (w/w) methanol/hydrochloric acid solution are added slowly at 0°C.

The reaction is kept under stirring at 0°C for 3 hours, and finally stopped by dilution with 100 ml of methanol. Still at 0°C solid sodium bicarbonate is added until the effervescence has disappeared (pH=7). It is filtered through celite and the residue obtained evaporated to dryness. The crude product obtained, contaminated with salts, is dissolved in 100 ml of methanol.

At -20°C an aqueous solution obtained by dissolving 62.2g of sodium periodate (1.4 equivalents) in 100 ml of water is added dropwise slowly. The addition takes place over a period of about 20 minutes, then the mixture maintained at -20°C under constant stirring for 10 minutes. Subsequently a large stoichiometric excess of sodium borohydride is added, and the mixture obtained is maintained at -20°C for 30 minutes.

Acetone is added until the effervescence has disappeared. Following 30 minutes of stirring, the reaction mixture is concentrated and the residue dried under high vacuum.

The purification is performed by chromatography on a column (eluent: methylene chloride - methanol in a volume ratio of 8.5/1.5).

33.3g (0.1452 mol) of final product (X) are obtained, with a total yield of the phases herein described of 70%, in a ratio α/β of approx. 1 :1 and 7g of unreacted nucleoside (VII).

¹ H-NMR δ(ppm- CD₃OD) - Compound (X) -β anomer

8.11-8.40 ppm (d, 1H, 7.5Hz, C'6);

5.97-5.93 ppm (d, 1H, 7.6Hz, C'5);

6.29 ppm (t,1H, 4.9Hz, C5); 5.29 ppm (t,1H, 3.9Hz, C2);

3.98-3.83 ppm(m, 2H, CH₂OH);

3.62-3.47 ppm (m, 1h, C4);

3.31-3.14 ppm (m, 1h, C4).

¹H-NMR δ(ppm- CD₃OD) - Compound (X) - anomer 7.75-7.71 ppm (d, 1 H, 7.5Hz, C'6);

5.97-5.93 ppm (d, 1H, 7.6Hz, C'5);

6.47-6.44 ppm (dd, 1H, 1.6Hz, 4.9Hz, C5);

5.63 ppm (t,1H, 4.8Hz, C2);

3.98-3.83 ppm (m, 2H, C2-CH₂OH); 3.62-3.47 ppm (m, 1H,C4);

3.31-3.14 ppm (m, 1H, C4).

Example 5: resolution of β 2-((R)-hydroxymethyl)-5-(cytosyl-1'-yl)-1 ,3-oxathiolan (XI) lamivudine

33.3 g of nucleoside /β (X) (1 equivalent) are dissolved in 200 ml of anhydrous methylene chloride. At 0°C are added 35.5g of freshly distilled benzoylchloride. The suspension assumes a red-orange colour. The reaction proceeds at room temperature for 4 hours, then is stopped by dilution with 100 ml of methylene chloride and washed with 200 ml of a solution of 10% (w/w) citric acid. The organic phase, is washed with water, brine and dried over sodium sulphate. The purification has been performed by flash chromatography on a column (eluent: ethyl acetate - methylene chloride in a volume ratio of 8/2). 23 g of nucleoside β (X)-benzoylate (0.1 mmol - 1 equivalent) are recovered which are dissolved in 10 ml of methanol presaturated with gaseous ammonia. The reaction proceeds overnight. The mixture is taken to dryness and taken up in methanol. The purification has been performed through chromatography under gravity on silica gel (eluent: methylene chloride - methanol in a volume ratio of 8.5/1.5).

10g of the lamivudine (XI) final product are recovered with a yield of these last phases of 30%.

¹ H-NMR δ(ppm- CD₃OD) - Compound β (X)-benzoylate 8.64 ppm (b, 1H, NH-Bz);

8.23 ppm (d,1H, 7.2Hz, C6');

6.39-6.35 ppm (dd, 1H, 3Hz, 5.6Hz, C5);

5.53 ppm (t,1H,3Hz,C2); 4.91-4.71 ppm (m, 2H, C2-CH₂OH);

3.72-3.63 ppm (dd, 1H, 5.0Hz, 12.7Hz, C4);

3.31-3.23 ppm (dd, 1H, 2.9Hz, 12.7 Hz, C4).

¹H-NMR δ(ppm- CD₃OD) - Compound (XJ)

8.01 ppm(d, 1H, C6'); 6.25 ppm (t, 1H, 4.8Hz, C5);

5.84 ppm (d, 1H.C5');

5.24 ppm (t,1H, 3.9Hz, C2); 3.95-3.84 ppm (m, 2H, C2-CH₂OH);

3.51-3.43 ppm (dd, 1H, 5.1Hz, 12.0 Hz, C4); 3.13-3.04 ppm (dd, 1H, 4.3Hz, 12.0Hz, C4).

Polarimetric analysis of Lamivudine

17.4 mg of the nucleoside of formula (XI) (lamivudine) have been subjected to polarimetric analysis using a cell with a capacity of 1 ml. The lamivudine had been dissolved in methylene chloride so as to obtain a final solution with a concentration of c=0.0174 g/ml.

The measured value of [α]_Observe_d=⁺2,017^o.

The corresponding value of

observed/c)=+119°.

The optical purity (OP) was thus determined:

[CC]_D

(+1 19°/+121 °)*100=98,3%.

Claims

1. Diastereoselective synthetic process for 4-amino-1-(2R-hydroxymethyl - [1 ,3]oxathiolan-5S-yl)-1 H-pyrimidin-2-one (LAMIVUDINE) comprising the reduction reaction , defined as phase a), 2-(R)-(4"-(R)-acetonyl)-5-oxo-1 ,3- oxathiolan (III) with reducing agents selected from the group consisting of: disiamylborane, di-isobutylaluminium hydride (DIBAL-H), (bis(2- methoxyethoxy)aluminium hydride) (RED-AL) and sodium borohydride, of to give 2-(R)-(4"-(R)-acetonyl)-5-hydroxy-1 ,3-oxathiolan (IV) as a mixture of cis and trans stereoisomers.

2. Process according to claim 1 wherein the reducing agent is di- isobutylaluminium hydride. 3. Process according to claim 1 wherein the molar ratio between 2-(R)-(4"-(R)- acetonyl)-5-oxo-1 ,

3-oxathiolan (III) and the reducing agent is comprised of between 1.1/1.0 and 1.0/1.0.

4. Process according to claim 3 wherein the molar ratio is 1.04/1.0.

5. Process according to claim 1 wherein the reduction reaction of 2-(R)-(4"-(R)- acetonyl)-5-oxo-1 ,3-oxathiolan (III) is carried out in aliphatic and/or aromatic solvents.

6. Process according to claim 5 wherein the solvent is toluene.

7. Process according to claim 1 wherein the reduction reaction of 2-(R)-(4"-(R)- acetonyl)-5-oxo-1 ,3-oxathiolan (III) is carried out at a temperature comprised of between -80°C and 20°C.

8. Process according to claim 7 wherein the temperature is comprised of between -10° and 10°C.

9. Process according to claim 1 comprising also the phases: b) conversion reaction of the hydroxyl group in position 5 of the oxathiolan ring, in the cis-trans mixture of the intermediate 2-(R)-(4"-(R)-acetonyl)-5-hydroxy-1 ,3- oxathiolan (IV), into a leaving group -L, in position 5 of the oxathiolan ring, selected from the group consisting of: acyloxy; alkoxy; aryloxy; alkylaryloxy; alkoxy carbonylic groups ; an aliphatic or aromatic aminocarbonyl group; phosphonate groups ; halogen; amide groups; azide; isocyanate; substituted or non substituted thiolates, saturated or unsaturated; seleno selenyl or selenonyl groups, substituted or non substituted saturated or unsaturated; c) glycosylation reaction, with silylated cytosine, of the cis-trans mixture originating from phase b) in the presence, as a catalyst, of a Lewis acid to give the corresponding mixture of ,β-2-(R)-(4"-(R)-acetonyl)-5-(cytosyl-1 '-yl)-1 ,3- oxathiolan (VII) nucleoside isomers.

10. Process according to claim 9 wherein in the reaction of phase b) the hydroxyl group is converted into the leaving group -L selected from the group consisting of: halogens; alkoxy groups -OR, where R is selected from the group consisting of: saturated or unsaturated alkyl groups, of one to twenty carbon atoms optionally substituted by fluorine, chlorine, bromine, iodine, alkoxy groups of one to six carbon atoms, aryloxy groups of six to twenty carbon atoms; aryloxy groups -OAr, where Ar is selected from the group consisting of: aryl groups of six to twenty carbon atoms optionally substituted by fluorine, chlorine, bromine, iodine, alkyl groups of one to twenty carbon atoms, alkoxy groups of one to twenty carbon atoms; alkylaryloxy groups -OR', where R' is selected from the group consisting of: arylalkyl groups of seven to twenty carbon atoms optionally substituted by halogens, alkyl groups of one to twenty carbon atoms or alkyloxy groups of one to twenty carbon atoms; acyloxy groups -OC(0)R" where R" is selected from the group consisting of: alkyl groups, saturated or unsaturated, of one to five carbon atoms, preferably methyl, ethyl, butyl, optionally substituted by amino, carboxyl, hydroxyl, phenyl, lower alkoxyl groups, preferably methoxyl and ethoxyl; phenyl group; phenyl groups substituted by lower alkyls, carboxyls, halogens, preferably chlorine and bromine, sulphate, sulphonyloxy, lower oxyalkyls, preferably carbomethoxy and carboethoxy; amino group; mono and di amino lower alkyl groups, preferably methylamino, dimethylamino.

11. Process according to claim 10 wherein the leaving group -L is selected from the group consisting of: ethoxycarbonyl, iodine, bromine, chlorine, fluorine, acetate, benzoate, methylcarbonate, phenylcarbonate, diethylphosphonate.

12. Process according to claim 9 wherein the conversion reaction of the hydroxyl group in the cis-trans mixture of the intermediate 2-(R)-(4"-(R)-acetonyl)-5- hydroxy-1 ,3-oxathiolan (IV) at phase b) is carried out in methylene chloride in the presence of pyridine by reaction with acetyl chloride at a temperature comprised of between -5° and 5°C to give cis-trans 2-(R)-(4"-(R)-acetonyl)-5- acetoxy-1 ,3-oxathiolan (V).

13. Process according to claim 9 wherein in the reaction at phase c) the Lewis acid is selected from SnCI₄ and a compound of general formula:

wherein:

^R8 ?' ^R6 (VI)

R₇ the R5, R₆ and R substituents, are the same or different from each other and are selected from the group consisting of: hydrogen; alkyl groups with one to twenty carbon atoms optionally substituted by fluorine, chlorine, bromine, iodine, alkoxy groups of one to six carbon atoms, aryloxy groups of six to twenty carbon atoms; arylalkyl groups of seven to twenty carbon atoms optionally substituted by halogens, alkyl groups of one to twenty carbon atoms or alkyloxy groups of one to twenty carbon atoms; aryl groups of six to twenty carbon atoms optionally substituted by fluorine, chlorine, bromine, iodine, alkyl groups of one to twenty carbon atoms, alkoxy groups of one to twenty carbon atoms; trialkylsilyl groups and halogen substituents: F, Cl, Br, I, and the Rs substituent is selected from the group consisting of: fluorine, bromine, chlorine, iodine, sulphonic esters of one to twenty carbon atoms, optionally substituted by fluorine, chlorine, bromine, iodine; alkyl esters of one to twenty carbon atoms, optionally substituted by fluorine, chlorine, bromine, iodine; polyvalent halides; trisubstituted silyl groups of general formula RsRβRrSi, wherein R₅, R₆ and R₇ have the same meanings as above, saturated and unsaturated selenonyl aryl groups of six to twenty carbon atoms; substituted or non substituted arylsulphonyl groups of six to twenty carbon atoms; alkoxyalkyl groups substituted or non substituted, of six to twenty carbon atoms; and trialkylsiloxy groups.

14. Process according to claim 13 wherein in the Lewis acid of formula (VI) the substituents R₅, R₆ and R , are the same or different from each other and are selected from the group consisting of: fluorine, chlorine, bromine, iodine, methyl, ethyl, t-butyl, benzyl.

15. Process according to claim 14 wherein the alkyl and the benzyl groups are substituted by F, Cl, Br, I.

16. Process according to claim 13 wherein in the Lewis acid of formula (VI) the substituent Rs is selected from the group consisting of F, Cl, Br, I.

17. Process according to claim 13 wherein the Lewis acid of formula (VI) is iodotrimethylsilane (TMSI) or trimethyl triflate (TMSOTf).

18. Process according to claim 13 wherein the Lewis acid of formula (VI) is generated in situ.

19. Process according to claim 9 wherein the glycosylation reaction in phase c) is carried out in aliphatic or aromatic organic solvents.

20. Process according to claim 9 wherein the glycosylation reaction in phase c) proceeds at a temperature comprised of between 0°C and 25°C.

21. Process according to claim 9 wherein the cis-trans mixture originating from phase b) is added to the cytosine nucleotide base, previously silylated, and subsequently followed by the addition of the Lewis acid.

22. Process according to claim 21 wherein the silylated cytosine nucleotide base used is obtained by silylisation of the cytosine with a silylating agent in aliphatic or aromatic organic solvents in the presence of a sterically hindered base.

23. Process according to claim 22 wherein the silylating agent is selected from the group consisting of: hexamethyldisilazane (HMDS), t-butyldimethylsilyltriflate.

24. Process according to claim 22 wherein the solvent is acetonitrile or methylene chloride.

25. Process according to claim 22 wherein the sterically hindered base is 2, 4, 6- collidine,

26. Process according to claim 9 further comprising the phases: d) conversion reaction of the nucleoside isomer mixture of α,β-2-(R)-(4"-(R)- acetonyl)-5-(cytosyl-1'-yl)-1 ,3-oxathiolan (VII) into the corresponding mixture of ,β 2-((R)-hydroxymethyl)-5-(cytosyl-1 '-yl)-1 ,3-oxathiolan nucleoside isomers (X); e) separation of the β 2-((R)-hydroxymethyl)-5-(cytosyl-1'-yl)-1 ,3-oxathiolan isomer (XI) (lamivudine) by chemical derivasation of the mixture of α,β 2-((R)- hydroxymethyl)-5-(cytosyl-1'-yl)-1 ,3-oxathiolan nucleoside isomers (X), separation by physical means, of the mixture of derivatised nucleoside isomers, recovery of the β isomer by chemical removal of the derivatising agent, with the isolation of lamivudine (XI).

27. Process according to claim 26 wherein the conversion reaction of phase d) comprises the removal of the chiral helper 4"-(R)-acetonide, in position 2 of the oxathiolan ring, through hydrolysis of said substituent group to give the corresponding mixture of diol isomers: α,β-2-(R)-(2"-(R)-1 ,2-dihydroxyethyl)-5- (cytosyl-1'-yl)-1 ,3-oxathiolan (VIII), oxidation of the diol isomers to give the mixture of aldehyde isomers α,β 2-((R)-formyl)-5-(cytosyl-1 '-yl)-1 ,3-oxathiolan (IX) followed by reduction of the aldehyde isomers to give the corresponding mixture of isomers ,β 2-((R)-hydroxymethyl)-5-(cytosyl-1 '-yl)-1 ,3-oxathiolan

(X).

28. Process according to claim 27 wherein the hydrolysis of the acetonide group of the nucleoside isomers α,β-2-(R)-(4"-(R)-acetonyl)-5-(cytosyl-1'-yl)-1 ,3- oxathiolan (VII) takes place in strong mineral acid with protic solvents.

29. Process according to claim 27 wherein the oxidant is sodium metaperiodate (Nal0₄).

30. Process according to claim 27 wherein the reducing agent is sodium borohydride (NaBH₄).

31. Process according to claim 26 wherein the separation of the isomer β 2-((R)- hydroxymethyl)-5-(cytosyl-1 '-yl)-1 ,3-oxathiolan (XI) (lamivudine) in phase e) occurs by reacting the mixture of nucleoside isomers ,β 2-((R)- hydroxymethyl)-5-(cytosyl-1 '-yl)-1 ,3-oxathiolan (X) with benzoylchloride in methylene chloride and the physical separation occurs by fractional crystallisation.

32. Process according to claim 9 further comprising the phases: d') separation of the β 2-(R)-(4"-((R)-acetonyl)-5-(cytosyl-1 '-yl)-1 ,3-oxathiolan isomer (XII) from the mixture of ,β-2-(R)-(4"-(R)-acetonyl)-5-(cytosyl-1 '-yl)- 1 ,3-oxathiolan nucleoside isomers (VII), by simple separation by physical means of the mixture of nucleoside isomers; e') conversion reaction of the β 2-(R)-(4"-((R)-acetonyl)-5-(cytosyl-1 '-yl)-1 ,3- oxathiolan isomer (XII) into the corresponding β 2-((R)-hydroxymethyl)-5- (cytosyl-1 '-yl)-1 ,3-oxathiolan isomer (XI), lamivudine.

33. Process according to claim 32 wherein the separation of the β 2-(R)-(4"-((R)- acetonyl)-5-(cytosyl-1 '-yl)-1 ,3-oxathiolan isomer (XII) in phase d') is performed by fractional crystallisation or chromatography.

34. Process according to claim 32 wherein the conversion reaction in phase e') comprises the removal of the chiral helper 4"-(R)-acetonide, in position 2 of the oxathiolan ring, through the hydrolysis of said substituent group to give the corresponding diol β-2-(R)-(2"-(R)-1 ,2-dihydroxyethyl)-5-(cytosyl-1 '-yl)-1 ,3- oxathiolan (XIII), oxidation of the diol to give the aldehyde β 2-((R)-formyl)-5- (cytosyl-1 '-yl)-1 ,3-oxathiolan (XIV) followed by reduction of the aldehyde to give the corresponding β 2-((R)-hydroxymethyl)-5-(cytosyl-1'-yl)-1 ,3-oxathiolan (XI), lamivudine.

35. Process according to claim 34 wherein the hydrolysis of the acetonide group of the β-2-(R)-(4"-(R)-acetonyl)-5-(cytosyl-1'-yl)-1 ,3-oxathiolan nucleoside (XII) occurs with strong mineral acid in protic solvents.

36. Process according to claim 34 wherein the oxidant is sodium metaperiodate (Nal0₄).

37. Process according to claim 34 wherein the reducing agent is sodium borohydride (NaBH₄).

38. The intermediate 2-(R)-(4"-(R)-acetonyl)-5-hydroxy-1 ,3-oxathiolan (IV) in two isomeric forms, cis and trans.

39. Process according to claim 1 wherein 2-(R)-(4"-(R)-acetonyl)-5-oxo-1 ,3- oxathiolan (III) is obtained from the diastereoisomeric mixture of 2-(S)-(4"-(R)- acetonyl)-5-oxo-1 ,3-oxathiolan and 2-(R)-(4"-(R)-acetonyl)-5-oxo-1 ,3- oxathiolan by simple separation by physical means.

40. Process according to claim 39 wherein the separation of 2-(R)-(4"-(R)- acetonyl)-5-oxo-1 ,3-oxathiolan (III) occurs by crystallisation.