AN IMPROVED PROCESS FOR THE MANUFACTURE OF LAMIVUDINE

Field of invention

The present invention relates to an improved process for the Manufacture of Lamivudine.

Background of the invention

Lamivudine (I) (CAS No. 134678-17-4) is chemically known as (-)-[2R,5S]-4 _T amino- 1 - [2-(hydroxymethyl)- 1 ,3 -oxathiolan-5-yl] -2( 1 H)-pyrimidin-2-one.

Formula (I)

Lamivudine is a reverse transcriptase inhibitor used alone or in combination with other classes of Anti-HIV drugs in the treatment of HIV infection. It is available commercially as a pharmaceutical composition under the brand name EPIVIR ^® , marketed by GlaxoSmithKline, and is covered under US 5,047,407.

This molecule has two stereo-centres, thus giving rise to four stereoisomers: (±)- Cis Lamivudine and (±)-Trans Lamivudine. The pharmaceutically active isomer however is the (-)-Cis isomer which has the absolute configuration [2R,5S] as show in Formula (I).

US 5,047,407 discloses the 1,3-oxathiolane derivatives; their geometric (cis/trans) and optical isomers. This patent describes the preparation of Lamivudine as a mixture of cis and trans isomers (shown in scheme I). The diastereomers obtained are converted into N-acetyl derivatives before separation by column chromatography using ethylacetate and methanol (99:1); however, this patent remains silent about further resolution of the cis isomer to the desired (-)-

[2R,5S]-Cis-Lamivudine. Secondly, as the ethoxy group is a poor leaving group, the condensation of cytosine with compound VI gives a poor yield, i.e. 30 - 40%, of compound VII. Thirdly, chromatographic separation that has been achieved only after acetylation requires a further step of de-acetylation of the cis-(±)- isomer. Also, separation of large volumes of a compound by column chromatography makes the process undesirable on a commercial scale.

(+/-) Cis (+/-) Cis Lamivudine (VIII)

Scheme - 1

Efforts have been made in the past to overcome the shortcomings of low yield and enantiomeric enrichment, hi general, there have been two approaches to synthesize (— )-[2R,5S]-Cis-Lamivudine. One approach involves stereoselective synthesis, some examples of which are discussed below.

US 5,248,776 describes an asymmetric process for the synthesis of enantiomerically pure β-L-(-)-l,3-oxathiolone-nucleosides starting from optically pure 1,6-thioanhydro-L-gulose, which in turn can be easily prepared from L- Gulose. The condensation of the 1,3-oxathiolane derivative with the heterocyclic base is carried out in the presence of a Lewis acid, most preferably SnCl _4, to give the [2R,5R] and [2R,5S] diastereomers that are then separated chromatographically.

US 5,756,706 relates a process where compound A is esterified and reduced to compound B. The hydroxy group is then converted to a leaving group (like acetyl) and the cis- and trans-2R-tetrahydrofuran derivatives are treated with a pyrimidine base, like N-acetylcytosine, in the presence trimethylsilyl triflate to give compound C in the diastereomeric ratio 4: 1 of cis and trans isomers.

A B C

Z = S ₅ CH

Dissolving compound C in a mixture of 3:7 ethyl acetate-hexane separates the cis isomer. The product containing predominantly the cis-2R,5S isomer and some trans-2R,5R compound is reduced with NaBH ₄ and subjected to column chromatography (30% MeOH-EtOAc) to yield the below compound.

US 6,175,008 describes the preparation of Lamivudine by reacting mercaptoacetaldehyde dimer with glyoxalate and further with silylated pyrimidine base to give mainly the cis-isomer by using an appropriate Lewis acid, like TMS-

I ₅ TMS-Tf, TiCl ₄ et cetera. However the stereoselectivity is not absolute and although the cis isomer is obtained in excess, this process still requires its separation from the trans isomer. The separation of the diastereomers Js done by acetylation and chromatographic separation followed by deacetylation. Further separation of the enantiomers of the cis-isomer is not mentioned.

US 6,939,965 discloses the glycosylation of 5-fluoro-cytosine with compound F (configuration: 2R and 2S)

. F

The glycosylation is carried out in the presence of TiCl ₃ (OiPr) which is stereoselective and the cis-2R,5S-isomer is obtained in excess over the trans- 2S,5S-isomer. These diastereomers are then separated by fractional crystallization.

US 6,600,044 relates a method for converting the undesired trans-l,3-oxathiolane nucleoside to the desired cis isomer by a method of anomerizatioή or transglycosylation and the separation of the hydroxy-protected form of cis-, trans- (-)-nucleosides by fractional crystallization of their hydrochloride, hydrobromide,

methanesulfonate salts. However, these cis-trans isomers already bear the [R] configuration at C2 and only differ in their configuration at C5; i.e. the isomers are [2R,5R] and [2R,5S]. Hence diastereomeric separation directly yields the desired [2R, 5S] enantiomer of Lamivudine.

In the second approach to prepare enantiomerically pure Lamivudine the resolution of racemic mixtures of nucleosides is carried out. US 5,728,575 provides one such method by using enzyme-mediated enantioselective hydrolysis of esters of the formula

wherein, 'R' is an acyl group and 'Rl ' represents the purine or pyrimidine base.

'R' may be alkyl carboxylic, substituted alkyl carboxylic and preferably an acyl group that is significantly electron-withdrawing, eg. α-haloesters. After selective hydrolysis, the process involves further separation of the unhydrolyzed ester from the ^' enantiomerically pure 1,3-oxathiolane-nucleoside. Three methods are suggested in this patent, which are:

1. Separation of the more lipophilic unhydrolyzed ester by solvent extraction with one of a wide variety of nonpolar organic solvents.

2. Lyophilization followed by extraction into MeOH or EtOH. 3. Using an HPLC column designed for chiral separations.

In another of its aspects, this patent also refers to the use of the enzyme cytidine- deoxycytidine deaminase, which is enantiomer-specific, λo catalyze the deamination of the cytosine moiety and thereby converting it to uridine. Thus, the enantiomer that remains unreacted is still basic and can be extracted by using an acidic solution.

However, the above methods suffer from the following drawbacks, (a) Enzymatic hydrolysis sets down limitations on choice of solvents: alcohol solvents cannot be used as they denature enzymes.

(b) Lyophilization on an industrial scale is tedious, (c) Chiral column chromatographic separations are expensive.

WO 2006/096954 describes the separation of protected or unprotected enantiomers of the cis nucleosides of below formula by using a chiral acid to form diastereomeric salts that are isolated by filtration. Some of the acids used are R-

(-)-Camphorsulfonic acid, L-(-)-Tartaric acid, L-(-)-Malic acid, et cetera.

However, the configuration of these CIS-nucleosides are [2R,4R] and [2S,4S] as the heterocyclic base is attached at the 4 position of the oxathiolane ring and the overall stereo-structure of the molecule changes from that of the 2,5-substituted oxathiolane ring.

Thus various methods are described for the preparation of Lamivudine. However there is no mention in the prior art about the separation of an enantiomeric pair, either cis-(±) or trans-(±), from a mixture containing cis-[2R,5S], [2S,5R] and trans-[2R,5R], [2S,5S] isomers. Further, there also is a need to provide resolution of the cis-(±) isomers to yield the desired enantiomer in high optical purity.

CN 1223262 (Deng et aϊ) teaches the resolution of a certain class of compounds called Prazoles by using chiral host compounds such as dinaphthalenephenols (BINOL), diphenanthrenols or tartaric acid derivatives. The method consists of the formation of a 1:1 complex between the chiral host (BINOL) and one of the enantiomers, the guest molecule. The other enantiomer remains in solution. (S)- Omeprazole, which is pharmaceutically active as a highly potent inhibitor of

gastric acid secretion, has been isolated from its racemic mixture in this manner by using S-BINOL.

BINOL is a versatile chiral ligand that has found its uses in various reactions involving asymmetric synthesis (Noyori, R. Asymmetric Catalysis in Organic

Synthesis) and optical resolution (Cram, D. J. et al J. Org. Chem. 1977, 42, 4173-

4184). Some of these reactions include BINOL-mediated oxidation and reduction reactions, C-C bond formation reactions such as Aldol reaction, Michael addition,

Mannich reaction et cetera (Brunei Chem. Rev. 2005 105, 857-897) and kinetic resolution, resolution by inclusion complexation et cetera.

BINOL, or l,l'-bi-2-Naphthol, being an atropoisomer possesses the property of chiral recognition towards appropriate compounds. One of the uses of BINOL in resolution that is known in literature is in Host-Guest complexation. In one such example, 1,1-binaphthyl derivatives have been successfully incorporated into optically active crown ethers for the enantioselective complexation of amino acid esters and chiral primary ammonium ions (Cram, D. J. Ace. Chem. Res. 1978, 11, 8-14). The chiral 'host' is thus able to discriminate between enantiomeric compounds by the formation of hydrogen bonds between the ether oxygen and the enantiomers. The complex formed with one of the isomers, the 'guest', will be less stable on steric grounds and this forms the basis for its separation.

It is evident from the literature cited that there exists a need to (a) synthesize Lamivudine by a process requiring less expensive, less hazardous and easily available reagents, and (b) achieve good yields with superior quality of product without resorting to column chromatography as a means of separation, thereby making the process of Lamivudine manufacture more acceptable industrially.

Object of the invention

Thus, one object of the present invention is to provide a process for the synthesis of_Lamivudine which is cost effective, uses less hazardous and easily available reagents, yet achieves good yields with superior quality of product without resorting to column chromatography.

A further object of the present invention is to provide an improved process for the synthesis of Lamivudine, by separating the mixture of diastereomers: Cis-[2R,5S], [2S,5R] from Trans-[2R,5R], [2S,5S] and then resolving the Cis isomers using BINOL to obtain (-)-[2R,5S]^Cis-Lamivudine with at least 99% ee.

Summary of the invention

Thus, according to one aspect of the present invention there is provided a process to separate the Cis-Trans diastereomeric mixture of the intermediate IX based on the difference in solubility of the diastereomers or their salts with an acid in a suitable solvent, the process comprising the following steps: a. providing the cis-trans mixture of hydroxy-protected Lamivudine of Formula (IX) as starting material, the different isomers having the configurations Cis-

[2R,5S], [2S,5R] and Trans-[2R,5R], [2S,5S], b. treating the hydroxy-protected intermediate IX, in an organic solvent with an acid to form its corresponding salt, c. filtering the above solution to isolate the cis diastereomer, i.e. salt of hydroxy- protected-Cis-(±)-Lamivudine, d. converting the above salt to the free base, i.e. hydroxy-protected-Cis-(±)- Lamivudine, e. carrying out a deprotection step to get Cis (±)-Lamivudine.

According to another aspect of the invention, there is provided a process for the preparation of an optically pure or optically enriched enantiomer of Lamivudine of Formula (I), the process comprises of -

a. providing a mixture of optical isomers i.e. Cis (±)-Lamivudine, the different enantiomers having the configuration [2R,5S] and [2S,5R], b. reacting the mixture of optical isomers with a chiral host in an organic solvent, c. separating the adduct formed by the enantiomer and the chiral host, d. treating the adduct with an acid and then neutralizing it to get (— )-[2R,5S]-Cis-

Lamivudine, e. optionally purifying it by crystallization from a suitable organic solvent, thereby obtaining the (-)-[2R,5S]-Cis-Lamivudine in a substantially optically pure or optically enriched form.

Thus, the problems associated with chromatographic separations have also been eliminated as the separation of isomers, both diastereomeric and enantiomeric, is done by selective crystallization.

Detailed description of the invention:

The process of the present invention for the manufacture of Lamivudine is as presented in Scheme 2, and comprises reacting compound IV

where, Rl is tert-butyldiphenylsilyl or benzoyl

with sodium periodate to yield compound V. Compound V is then condensed with 2-mercaptoacetaldehyde dimer and subsequently acetylated with acetyl chloride to give the protected 2-hydroxymethyl-l,3-oxathiolan-5-yl acetate (compound

VIII). This 1,3-oxathiolane compound VIII is further condensed with silylated cytosine in the presence of a Lewis acid such as trimethylsilyliodide to get protected 6-amino-3 - {2-hydroxymethyl- 1 ,3 -oxathiolan-5-yl} -3 -hydropyrimidine- 2-one (compound IX).

OH

Cis (±) and Trans (±) racemic mixtures

Lamivudine (-)-[2

Compound (IX) is mixture of following optical isomers

SCHEME 2

The separation of the four-component diastereomeric mixture of isomers bearing the following configuration: trans-[2R,5R], [2S.5S] and cis-[2R,5S], [2S,5R] forms the next step. The separation efficiency of the benzoyl-protected compound

IX diastereomers i.e. cis-(±) from trans-(±) from their solution in methanol or dichloromethane was found to be poor. In the former case, the compound is highly soluble in methanol whereas in the latter, despite getting the cis racemates with a purity of 97.7 %, the yield was found to be only 36.44% of material balance.

In one embodiment of the present invention, the benzoyl-protected compound IX is treated with various achiral and chiral acids like succinic acid, oxalic acid, [S]- (+)-mandelic acid and di-para-toluoyl-D-Tartaric acid in a hydroxylic solvent like methanol to give the corresponding four diastereomer salts. Interestingly, the inventors have found that the Cis-(±)-isomer salts alone precipitate from the solution with a diastereomeric purity in almost all cases [excepting di-para- toluoyl-D-Tartaric acid which has 87%] greater than 98.5% at first pass with an accompanying yield of 40.8%, 28.86%, 50.52% and 27.04% respectively of overall material balance. The Cis-(±)-isomers are obtained exclusively because of the formation of a eutectic, leaving behind in solution the trans-(+)-isomers.

In another embodiment of the present invention the tert-butyldiphenylsilyl- protected compound IX when treated with lS-(+)-camphorsulfonic acid in a hydroxylic solvent like methanol also permits the separation of the Cis-(±)- isomers. The solid product isolated is composed of only the [2S, 5R] and [2R, 5S] i.e. the Cis-(±)-isomers with 88.36% diastereomeric purity and 89 % material balance at first pass. After re-crystallizing the product twice with about 45 v/wt % MeOH, the diastereomeric purity increases to 98.9%.

The same separation using lS-(+)-camphorsulfonic acid has also been attempted with other solvents and solvent systems like ethyl acetate and dichloromethane. It can be seen that the separation efficiency is a function of the solvent and the acid used as the following table indicates.

TABLE 1 : Separation of TBDSi-protected Cis-(±)-isomers using lS-(+)-CSA in different solvents

SOLVENT VAVT % CIS %

Ethyl acetate 5 74.5

Ethyl acetate/DCM 5 84.6

DCM 5 88.04

MeOH 4 88.36

The separation of the cis racemates from the trans racemates was also tried using other acids such as Di-para-toluoyl-D-Tartaric acid in IPA and in ethyl acetate, D-

(-)-Tartaric acid in IPA, L-(+)-Tartaric acid in IPA, Naproxen in ethyl acetate and N-(carbobenzyloxy)-L-Phenyl alanine in ethyl acetate. No solid product enabling the diastereomeric separation was obtained with any of these salts.

Cis-(±)-isomers that are isolated are first converted to their free base and further deprotected to get racemic cis-lamivudine (compound XII) having the configurations [2R,5S] and [2S,5R]. Thus, at this stage in the process, the complete separation of two components from the four-component mixture of Cis- [2R,5S], [2S,5R] and Trans-[2R,5R], [2S,5S] has been successfully accomplished.

Another aspect of the present invention provides a method for the separation of

(-)-[2R,5S]-Cis-Lamivudine from its (+)-enantiomer using optically pure (S)- 2,2'-dihydroxy-l,l '-binaphthyl [(S)-BINOL].

The process is operationally simple and comprises the following steps: a) treating the racemates with (S)-BINOL in the presence of a hydroxylic solvent like methanol, b) isolating the adduct formed by the enantiomer and the chiral host by filtration, c) if desired, crystallizing the adduct, d) separating the guest from the host by using an acid, preferably concentrated HCl, neutralizing the solution to get the free base and purifying it by recrystallization; thereby yielding the desired (-)-[2R,5S] -Lamivudine with an enantiomeric excess greater than 99% .

Thus, reacting the racemates with (S)-BINOL results in the formation of a clathrate of (-)-[2R,5S]-Cis-Lamivudine by Host-Guest complexation. On filtration it affords a clean separation between the enantiomers. In the final step, the (-)-[2R,5S]-Cis-Lamivudine-(S)-BINOL' complex is broken in an acidic medium and then neutralized to obtain the desired product: (-)-[2R,5S]- Lamivudine, in high optical purity of greater than 97.5% before purification. Re- crystallization with isopropanol raises the enantiomeric excess to 99.5%. The IR spectra of racemic Lamivudine, S-BINOL and the Lamivudine - S-BINOL complex are provided in Figures 1, 2, and 3 respectively.. It is to be emphasized that when the same separation was attempted using R-BINOL, no resolution of enantiomers was achieved.

Resolution of the Cis enantiomers of Lamivudine by the ^■ formation of diastereomeric salts of cis-(±) lamivudine with acids like malic acid, mandelic acid, dibenzoyl tartaric acid, 3-bromocamphor-8-sulfonic acid, 10- camphorsulfonic acid, and di-p-toluoyltartaric acid have been attempted before by

Liotta et al, one of the inventors named in US. 5,204,466. However, these attempts were unsuccessful as revealed by a declaration attached with the prosecution history file of US 6,703,396.

Interestingly, the inventors have found that when the salts Cis-(±)-Lamivudine were prepared with R-(-)-CSA or with D-(-)-tartaric acid, no separation of enantiomers was achieved.

The following examples illustrate the practice of the invention without being limiting in any way. Example 1

Preparation of 3-(2,2-dimethyl-l,l-diphenyl-l- silapropoxy)propane-l,2-diol (TBDPSi-glycerol) Compound IV

30Og (1.09 moles) of tert-butyldiphenylsilylchloride was added slowly over a period of 60 to 120 minutes at 0 - 5 ⁰ C to a solution of HOg (1.19 moles) of Glycerol and 329.3g (3.26 moles, 453 mL) of triethylamine in 30OmL of dimethylformamide. After stirring for 6 hours it was added to 1.5 L of cooled (5

⁰ C - IO ⁰ C) DM water. 50OmL of DCM was added to this solution with stirring at room temperature and the layers were separated. To the aqueous layer 500 mL of DCM was added and the layers were separated again. The organic layers were combined and washed with a 25% aqueous solution of NaCl. The organic layer was isolated and the solvent was recovered under vacuum. 40OmL of hexane was added to the reaction mixture which was then cooled to 0 — 5 ⁰ C for 60 to 70 minutes. The solution was filtered to isolate the solid which was then washed with 10OmL of hexane and dried under vacuum at 40- 45 ⁰ C. Yield : 200-22Og (55.7 - 61.3 %) ^' m.p. = 91.7-92.9 ⁰ C MS: M ⁺ -I = 329

¹ H NMR (CDCl ₃ ): δ 1.07 (s, 9H), 3.64-3.71 (m,, 4H), 3.73-3.81 (m, IH), 7.35- 7.68 (m, 10H)

Example 2

Preparation of 2-(2,2-dimethyl-l,l-diphenyl-l-silaρropoxy)ethanal (TBDPSi- aldehyde)

Compound V

To TBDPSi-glycerol (20Og, 0.605 moles) in a solution of 2.7 L acetone and

30OmL DM water, 168g (0.786 moles) of NaIO ₄ was added in portions. The reaction mixture was stirred for 2 hours and then filtered at the end of the reaction. The solid was washed with 40OmL of DCM and the solvent was recovered under vacuum at 40- 45 ⁰ C. 1600 mL DCM was added to the concentrate followed by 1 L of DM water. The organic layer was separated and the solvent recovered under vacuum at 45-50 ⁰ C. Yield : 170-175g (94.4-97.2 %) ¹ HNMR (CDCl ₃ ): δ 1.12 (s, 9H), 4.23 (s, 2H), 7.36-7.70 (m, 10H), 9.73(s, IH)

Example 3

Preparation of [2-(2,2-dimethyl- 1 , 1 -diphenyl- 1 -silapropoxy)methyl] -1,3- oxathiolan-5-yl acetate (TBDPSi-acetoxy oxathiolane)

Compound VIII

17Og (0.57 moles) of TBDPSi-aldehyde was heated to 60-65 ⁰ C with 52g (0.34 moles) of l,4-dithiane-2,5-diol for 15-20 minutes in the presence of 188ml of pyridine and stirred for 3 hours at that temperature. After completion of the reaction, the reaction mixture was cooled to 0 - 5 ⁰ C and then 850 mL of DCM was added to it. After stirring for 10-15 minutes, a mixture of 134.3g (1.71moles, 121.6ml) of acetyl chloride in 34OmL of DCM was added slowly over a period of

60-90 minutes and stirred for 1 hour at 0 - 5 ⁰ C. When the reaction was complete 85OmL of DM water was added to it at 5-10 ⁰ C and the reaction mixture was stirred for 10-15 minutes at 20-25 ⁰ C. The organic layer was separated and washed with 85OmL of 5% aqueous solution Of NaHCO ₃ . The organic layer was separated and the washing was repeated with 850 mL of 25% aqueous NaCl. The

organic layer was separated again and the solvent was recovered under vacuum -at 40-45 ⁰ C. 34OmL of hexane was then added and the solution was vacuum distilled (40-45 ⁰ C) to remove the traces of DCM. 85OmL of hexane and 8.5g of activated charcoal were added to the concentrate. The mixture was stirred and filtered through a celite bed. The bed was washed with 150 niL of hexane and the solvent was recovered under vacuum at 40- 45 ⁰ C. Yield: 210-230 g (88.0-96.6 %) ¹ H NMR (CDCl ₃ ): δ 1.10 (s, 9H), 2.11 (s, 3H), 3.08-3.16 (d, IH), 3.26-3.34 (dd, IH), 3.78-3.97 (m, 2H), 5.26 (t, IH), 6.66 (d, IH), 7.36-7.74 (m, 10H) MS: M ⁺ = 357

Example 4

Preparation of 6-amino-3- {2-[(2,2-dimethyl- 1 , 1 -diphenyl- 1 -silapropoxy)methyl]- 1 ,3 -oxathiolan-5-yl) } -3 -hydropyrimidine-2-one (TBDPSi-Cytosine) Compound IX

A mixture of 72.8g (0.65 moles) of Cytosine, 527.5mL (2.5moles, 403.5g) of

Hexamethyldisilazane and 27.16g (0.25moles, 3ImL) of TMS-Cl was heated to 125-130 ⁰ C and refluxed for 2 hours in a nitrogen atmosphere. The reaction mixture was then cooled and the solvent was completely recovered under vacuum at 100-105 ⁰ C. The residue was cooled to room temperature, dissolved in 2.1 L of

DCM and 21Og (0.5 moles) of TBDPSi-acetoxy oxathiolane was added to it. 14Og (10OmL, 0.7 moles) of TMS-I was added to the reaction mixture slowly over 15- 30 minutes. The reaction mixture was stirred for 4 hours. fl4mL of TMS-I can be added to the reaction mixture if the reaction has not reached completion.) It was then cooled to 0 - 5 ⁰ C and diluted with IL of DM water. After stirring for 15 minutes the organic layer was separated and washed with IL of aqueous NaHCO ₃ solution. The organic layer was separated and the solvent was recovered under vacuum at 40- 45 ⁰ C. Yield: 200-23Og (85.0-93.6 %) MS: M+1 = 468 '

¹ H NMR (CDCl ₃ ): δ 1.05 (s, 9H), 3.15-3.17 (dd, IH) ₅ 3.46-3.55 (dd, IH), 3.92- 3.98 (dd, IH), 4.09-4.11 (dd, IH), 5.21-5.24 (t, IH) ₅ 5.72-5.76 (d, IH), 6.07-6.35 (m, IH)

Example 5

Preparation of 6-amino-3-{2-[(2,2-dimethyl-l ₅ l-diphenyl-l-silapropoxy)methyl]- l ₅ 3-oxathiolan-5-yl)}-3-hydropyrimidine-2-one.CSA salt (TBDPSi-

Cytosine.CSA salt)

Compound X

45OmL of methanol was added to 21Og (0.5moles) of TBDPSi-Cytosine and the solvent was recovered at 40-45 ⁰ C to remove the traces of DCM. The residue was diluted with 840 rnL of MeOH and 105g of (lS)-(+)-10-camphorsulfonic acid was added to it at room temperature. The mixture was stirred at room temperature for 6 hours and then filtered. The residue was washed with 21OmL of MeOH and dried under vacuum at 40-45 ⁰ C for 6 hours. 45 v/w % MeOH was added to the dried mass and was refluxed at 65-70 ⁰ C. The solution was concentrated at atmospheric pressure to 10 v/w% and then filtered at room temperature. The residue was dried under vacuum at 40- 45 ⁰ C for 6 hours. Yield: 121-13Og (38.0- 41.26 %w/w)

HPLC Purity Cis-(±) = 97.3 % m.p. - 190.8 ⁰ C

IR(in KBr, cm ^"1 ): 3284, 3075, 2934, 1678, 1741, 1589, 1548, 1471, 1429, 1391,

1270, 1251, 1065, 1036.

Example 6

Preparation of 6-amino-3 - {2- [(2,2-dimethyl- 1 , 1 -diphenyl- 1 -silapropoxy)methyl]- l,3-oxathiolan-5-yl)}-3-hydropyrimidine-2-one (TBDPSi-Cytosine free base)

Compound XI

12Og (0.17moles) of TBDPSi-Cytosine.CSA salt was treated with 10OmL of ammonia solution in the presence of 1.2 L of DCM and 60OmL of DM water at 30-35 ⁰ C (pH 9-10). The solution was stirred for 10-15 minutes and the layers were separated. The organic layer was washed with 30OmL of DM water, the organic layer was isolated again and the solvent was recovered under vacuum at

40-45 ⁰ C. 20OmL of ethylacetate was added to the residue and the solvent was recovered under vacuum at 40-45 ⁰ C. 40OmL of ethylacetate was added to it at 10- 15 ⁰ C and the solution was filtered. The product was dried under vacuum at 40- 45 ⁰ C for 6 hours. Yield: 70-75g (87.8-93.93 %) m.p. : 200.3-205.1 ⁰ C

¹ H NMR (CDCl ₃ ): δ 1.05 (s, 9H), 3.05-3.12 (dd, IH), 3.44-3.53 (dd, IH), 3.87- 3.95 (dd, IH), 4.06-4.14 (dd, IH), 5.19-5.26 (t, IH), 5.52-5.56 (d, IH), 6.27-6.31 (t, IH), 7.25-7.67 (m, 10H), 7.91-7.95 (d, IH) MS: M+l = 468

Example 7

Preparation of (+/-)- 1 -(2R/S-Cis)-4-amino- 1 - [(2-hydroxymethyl)- 1 ,3-oxathiolan- 5-yl]-2(lH)-pyrimidin-2-one (Lamivudine Cis ±) Compound XII

To 7Og of TBDPSi-Cytosine free base dissolved in 70OmL of THF, 82mL of

TBAF (IM soln in THF) is added dropwise over 35-40 minutes at room temperature. The mixture is stirred for an hour and then filtered. 175mL of ethanol- water mixture (EtOH : H ₂ O is 9:1) is added to the product, stirred for 1 hour and filtered. The residue is washed with 25mL of ethanol- water mixture and dried under vacuum at 40- 45 ⁰ C for 4-5 hours. Yield: 25-28g (72.8-81.63 %) The IR spectrum of Cis-(±)-Lamivudine is given in figure 1. ¹ H NMR (DMSO d ₆ ): δ 2.98-3.07 (dd, IH), 3.35-3.4 (dd, IH), 3.71-3.73 (m, 2H), 5.14-5.18 (t, IH)

5.34 (bs, IH), 5.71-5.75 (d, IH), 6.16-6.21 (t, IH) 7.19-7.26 (d, 2H), 7.80-7.83 (d,

IH)

MS: M+l = 230

Example 8

Preparation of (-)-l-(2R-Cis)-4-amino-l-[(2-hydroxymethyl)-l,3-oxathiolan-5 - yl]-2(lH)-pyrimidin-2-one Binol complex (Lamivudine-BINOL complex)

Compound XIV

To 25g (0.1 moles) of Lamivudine (Cis +/-) dissolved in 25OmL of MeOH at 60-

65 ⁰ C, 37.5g (0.131 moles) of S-(-)-BINOL was added. Once a clear solution was obtained it was allowed to cool to room temperature and was stirred for 6 hours.

The solid was filtered and dried under vacuum at 40- 45 ⁰ C for 4-5 hours. Yield : 20-22g (74.6-82 %w/w) Enantiomeric excess = 97.93% which increases to

99.50% after purification. Purification was carried out by crystallizing the product from IPA containing 0.002 moles of [S]-BINOL.

The IR spectrum of the complex is as given in figure 3.m.p. = 190.1-190.6 ⁰ C

¹ H NMR (DMSO d ₆ ): 1.99-3.08 (dd, IH), 3.36 (dd, IH), 3.73 (m, 2H), 5.14-5.19 (t, IH), 5.72-5.76(d, IH), 6.17-6.23 (t, IH), 6.90-6.94 (d, 2H), 7.12-7.33( ^' m,

8H), 7.82-7.87(m, 6H), 9.24 (bs, 2H)

XRD [2θ] (Cu - K _α i=l .5406OA, K ₀₂ =I.54443 A K _β = 1.39225 A; 4OmA, 45kV):

8.02, 10.52, 12.73, 14.33, 14.51, 16.06, 17.06, 17.80, 20.12, 21.94, 22.36, 23.70,

24.06 and 25.05.

Example 9

Preparation of Lamivudine: (-)-[2R,5S]-4-amino-l-[2-(hydroxymethyl)-l,3- oxathiolan-5 -yl] -2(1 H)-pyrimidin-2-one

Compound I

5mL of cone. HCl was slowly added to a solution of 2Og of Lamivudine-BINOL complex in 100ml of ethylacetate and 10OmL of DM water (pH 2-2.5). The layers. were separated and a 10OmL aliquot of ethylacetate was added to the aqueous layer. The layers were separated again and the aqueous layer was neutralized using 1OmL of 10% aqueous NaOH solution. The solvent was recovered under vacuum at 40-45 ⁰ C, the product obtained was dissolved in 160 mL of methanol, filtered, the filtrate was concentrated and 32 mL of water-ethanol mixture (3:1) was added to this product, heated to get a clear solution, cooled to 5 - 10 ⁰ C and then filtered. The residue was vacuum dried at 45-50 ⁰ C. Yield: 4-5g.

Enantiomeric excess = 99.74 % m.p. = 133-135 °C [<X] _D at 25°C = 98.32° (c = 5 water)

¹ H NMR (DMSO d ₆ ): 2.99-3.07 (dd, IH), 3.35-3.38 (dd, IH), 3.72-3.74 (m, 2H), 5.14-5.18 (t, IH), 5.32-5.38 (t, IH), 5.71-5.75 (d, IH), 6.16-6.21 (t, IH), 7.22-

7.27 (d, 2H), 7.80-7.83 (d, IH)

Moisture content: 1.67%

IR (in KBr, cm ^"1 ): 3551, 3236, 2927, 1614, 1492, 1404, 1336, 1253, 1146, 1052,

967, 786. MS: M+l =230

XRD [2θ] (Cu - K _a1 =I.54060A, K _a2 =1.54443A K _β = 1.39225A; 4OmA, 45kV):

5.08, 9.89, 10.16, 11.40, 11.65, 12.96, 13.23, 15.26, 15.82, 17.74, 18.74, 18.88,

19.67, 20.69, 22.13, 22.88, 23.71, 25.47, 26.07.

Example 10

Preparation of Benzoyloxy acetaldehyde

1 Oa] Preparation of 1 ,2-isopropylidene , glycerol 10Og of glycerol in 40OmL of DCM was treated with 5g of PTSA and 40OmL of acetone. The reaction mixture was refluxed with concomitant azeotropic removal

of water, cooled to room temperature and the solvent was distilled off completely under vacuum (300-400mm of Hg). The product was distilled at 50-55 ⁰ C under 1-2 mm of pressure. Yield: 100-12Og (69-83.6%)

¹ H NMR (CDCl ₃ ): δ 1.33 (s, 3H), 1.4 (s, 3H), 2.54 (bs, IH), 3.50-3.71 (m, 2H), 3.73-3.77 (m, IH), 3.96-4.03 (t, IH), 4.03-4.25 (m, IH)

MS: M+l =133

10b] Preparation of benzoylated 1,2-isopropylidene glycerol

135g (1.02moles) of 1,2-isopropylidene glycerol in 1.35L of DCM and 155g

(1.53moles) of triethylamine was cooled to 5-10 ⁰ C and treated with 150.5g (1.07moles) of benzoyl chloride dissolved in 270 mL of DCM. When the reaction was complete, 70OmL of DM water was added to the reaction mixture, the organic layer was separated and washed with another 70OmL of DM water. DCM was distilled off completely under a vacuum of 300-400mm of Hg. 27OmL of THF was added to the residue and then distilled off at 45-50 ⁰ C, 100-150mm of Hg to remove the traces of triethylamine. Yield: 23Og (95%) <

¹ H NMR (CDCl ₃ ): δ 1.35 (s, 3H), 1.42 (s, 3H), 3.80-3.87 (m, IH), 4.07-4.14 (m, IH), 4.29-4.49 (m, 3H), 7.25-7.55 (m, 3H), 8.0-8.04 (m, 2H) MS: m/z = 179, 150

10c] Preparation of benzoyl glycerol Compound IV

225g (0.95moles) of benzoylated 1,2-isopropylidene glycerol dissolved in 1.125 L of THF was heated to 45-50 ⁰ C with 45mL of 2N HCl. When the reaction was complete, the mixture was cooled to room temperature, neutralized with Hg of

NaHCO ₃ , stirred, and filtered. The THF in the filtrate was distilled under vacuum

(100-150m of Hg). Yield: 187g

Twice stirring it in 1.1 L of 5% ethylacetate/hexane for 30 minutes each time re- crystallized the product.

Yield: 165g (88%) MS: M +1= 197

¹ H NMR (CDCl ₃ ): δ 3.21(bs, 2H), 3.6-3.74 (m, 2H), 4.03-4.10 (m, IH), 4.37-4.39 (d, 2H), 7.25-8.03 (m, 5H)

1Od] Oxidation of Benzoyl glycerol to Benzoyloxy acetaldehyde Compound V To 16Og (0.816moles) of Benzoyl glycerol dissolved in 1.6 L of DCM and 8OmL of DM water, 192g (0.897moles) of NaIO ₄ was added in portions at room temperature. When the reaction was complete, the solid was filtered and the filtrate was concentrated to get the desired product.

MS: M ⁺ +1= 165 ^{' 1} H NMR (CDCl ₃ ): δ 4.88 (s, 2H), 7.25-8.11 (m, 5H), 9.7 (s, IH)

Example 11 Preparation of 2-Benzoyloxymethyl-5-acetoxy- 1 ,3 -oxathiolane

Compound VIII

lOOg (0.609moles) of benzoyloxy acetaldehyde, 55.7g (0.365moles) of 1,4- dithian-2,5-diol and 197.5g of pyridine are heated at 60-65 ⁰ C for 2 hours in a 2.0 L, 4-necked round bottom flask. The reaction mixture is cooled to 0-5 ⁰ C and

50OmL of DCM was added to it.143.6g (1.83moles) of acetyl chloride dissolved in 20OmL of DCM was added dropwise to the reaction mixture over a period of 1- 2 hours. After the addition was complete, the mass was stirred for 1 hour. After completion of the reaction, the reaction mixture was poured slowly into 50OmL of saturated NaHCO ₃ solution. The organic layer was separated and washed with

50OmL of brine. The organic layer was isolated and the solvent was distilled off completely under vacuum (200-250mm of Hg). 50OmL of cyclohexane and 5g of activated carbon was added to the residue, stirred for 10 minutes and filtered

through a celite bed. The cyclohexane was distilled off completely under vacuum (100-125mm of Hg ₅ 40-45 ⁰ C). Yield: 145-155g (84.3-90 % w/w) ¹ H NMR (CDCl ₃ ): δ 2.0 (s, 3H), 3.04-3.30 (m, 2H), 4.36-4.49 (m, 2H) ₃ 5.57-5.62 (m, IH), 6.54-6.64 (dd, IH), 7.31-7.48 (m, 3H), 7.94-7.98 (m, 2H)

Example 12

Preparation of cis- and trans-2-Benzoyloxymethyl-5-cytosin-l'-yl-l,3-oxathiolane

Compound IX

76.8g (0.69moles) of cytosine, 652.4g (842mL, 4.04moles) of HMDS and 7.5g

(0.07moles) of TMSCl were taken in a 3.0L, 4-necked round bottom flask under nitrogen atmosphere. The contents were refluxed at 125-130 ⁰ C for 1-2 hours until a clear solution was obtained. The mass was cooled to 100 ⁰ C and the reagents in excess were distilled off completely under vacuum. 15Og (0.53moles) of ^" benzoyloxymethyl-acetoxy-l^-oxathiolane, 1.5 L of DCM and 165.5g (0.74moles) of TMSTf were added to the residue. (Instead of TMSTf, 148.5g of TMS-I may be used.) The reaction mixture was refluxed and when the reaction was complete, it was cooled to 0 ⁰ C and charged with 75OmL of DM water. (The initial 10OmL were added drop wise.) After stirring for 15 minutes, the organic layer was separated, washed with 1.5L of saturated NaHCO ₃ solution and DCM was distilled off completely under vacuum (200mm of Hg, 30-35 ⁰ C). Yield: 150- 16Og (84.7-90.3%) MS: M ⁺ +1= 334 ¹ H NMR (DMSO d ₆ ): δ 3.10 (dd, IH), 3.48 (dd, IH), 4.64 (d, 2H), 5.50 (t, IH), 5.6 (d, IH) 6.25 (t, IH), 7.27 (d, 2H), 7.50-7.72 (m, 4H), 7.95-7.98 (m, 2H)

Example 13

Separation of Cis/Trans-2-Benzoyloxymethyl-5-cytosin-l '-yl-1 ,3-oxathiolane

a] using S-(+)-Mandelic acid

5g (0.015moles) of cis- and trans-2-Benzoyloxymethyl-5-cytosin-l'-yl-l,3- oxathiolane and 10OmL MeOH were heated till a clear solution was obtained. 2.3 g (0.015moles) of (+)-Mandelic acid was added and the contents were heated to 65-66 ⁰ C for the compound to dissolve. The reaction mixture was stirred overnight at room temperature and the solid was filtered, washed with 5mL of cold MeOH to yield the Cis isomer. Yield: 2g (50.52% yield cis racemate)

b] using succinic acid 5g (0.015moles) of cis- and trans-2-Benzoyloxymethyl-5-cytosin-r-yl-l,3- oxathiolane and 14OmL MeOH were heated till a clear solution was obtained. 1.77 g (0.015moles) of succinic acid was added and the contents were heated to 65-66 ⁰ C for the compound to dissolve. The reaction mixture was stirred overnight at room temperature and the solid was filtered, washed with 5mL of cold MeOH to yield the Cis isomer. Yield: 1.5g (40.8% yield cis racemate)

c] using oxalic acid

5g (0.015moles) of cis- and trans-2-Benzoyloxymethyl-5-cytosin-l'-yl-l,3- oxathiolane and 5OmL MeOH were heated till a clear solution was obtained. 2.83 g (0.022 moles) of oxalic acid was added and the contents were heated to 65-66

⁰ C for the compound to dissolve. The reaction mixture was stirred overnight at room temperature and the solid was filtered, washed with 5mL of cold MeOH to yield the Cis isomer. Yield: 1.Og (28.86% yield cis racemate)

d] using dichloromethane ,

5g of cis- and trans-2-Benzoyloxymethyl-5-cytosin-r-yl-l,3-oxathiolane was dissolved in 25mL of DCM and heated to 65-66 ⁰ C. The solution was stirred overnight at room temperature and the solid was filtered to yield the Cis isomer. Yield: 1.Og (36.44% yield cis racemate)

After separation of isomers, the free base is prepared by a procedure as detailed in Example 6.

Example 14 Debenzoylation (Lamivudine Cis ±)

Compound XII

16g of Cis-(±)-2-Benzoyloxymethyl-5-cytosin-r-yl-l,3-oxathiolane was treated with 32OmL of methanolic ammonia at room temperature. The reaction mixture was stirred for 2 hours and after the reaction was complete the solvent was distilled off completely under vacuum. The residue was charged with 4OmL of ethanol, stirred for 1 hour at room temperature, filtered and dried under vacuum at 40 -45 ⁰ C. Yield: 9.Og (81.81%) Purity by HPLC = 99.13% MS: M+l = 230

¹ H NMR (DMSO d ₆ ): δ 2.98-3.07 (dd, IH), 3.34-3.43(dd, IH), 3.70-3.83 (m, 2H), 5.13-5.17 (t, IH), 5.36 (bs, IH), 5.75-5.79 (d, IH), 6.13-6.18(t, IH), 7.21(d, 2H), 7.83-7.86 (d, IH).