MXPA06008722A - Methods to manufacture 1,3-dioxolane nucleosides - Google Patents

Abstract

This aplication provides a process for preparing enantiomerically pure beta-D-dioxolane nucleosides. In particular, a new synthesis of (-)-DAPD, suitable for large scale development, is described. In one embodiment the invention provides a process for preparing a substantially pure beta-D or beta-L-1,3-dioxolane nucleosides comprising a) preparing or obtaining an esterified 2,2-dialkoxy ethanol;b) cyclizing the esterified 2,2-dialkoxy ethanol with glycolic acid to obtain a 1,3-dioxolane lactone;c) resolving the 1,3-dioxolane lactone to obtain a substantially pure D- or L-lactone;d) selectively reducing and activating the D-or L-chiral lactone to obtain a substantially pure D- or L-1,3-dioxolane;e) coupling the D- or L-1,3-dioxolane to an activated and/or protected purine or pyrimidine base;and f) optionally purifiying the nucleoside to obtain a substantially pure protected beta-D or beta-L-1,3-dioxolane nucleoside.

Description

METHODS FOR MANUFACTURING 1, 3-DIOXOLAN NUCOSOS FIELD OF THE INVENTION This application provides a process for preparing ß-D-dioxolane nucleosides. In particular, a novel synthesis of (-) - DAPD is described, suitable for large-scale development.

BACKGROUND OF THE INVENTION AIDS, Acquired Immune Deficiency Syndrome, is a catastrophic disease that has reached global proportions. Currently an estimated 40 million people are living with AIDS, with approximately 5 million new infections each year. The Mortality Index every year still follows over 3 million people worldwide. Another virus that causes a serious health problem in humans is the hepatitis B virus (HBV). HBV is second only to tobacco as a cause of cancer in humans. Some estimates put the number of people globally who have been infected with HBV as high as 2 billion people, up to a third of the world's population, with approximately 400 million chronically infected.

It has been found that a number of 2 ', 3' dideoxynucleotides are potent antiviral agents against HIV and / or hepatitis B virus. After cell phosphorylation at 5'-triphosphate by cellular kinase, these synthetic nucleosides are incorporated into a chain in development of viral DNA, causing chain termination due to the absence of the 3'-hydroxyl group. These can also inhibit the viral enzyme reverse transcriptase. There has also been interest in the synthesis of nucleoside derivatives in which the carbon at the 3 'position of the nucleoside has been replaced with a heteroatom. Both 3TC and its 5-fluorocytosine analog (FTC) present activity against HIV and HBV The discovery that a racemic oxotiolane nucleoside, BCH-189, possesses potent activity against HIV replication prompted Chu et al. to synthesize the chiral products (+) and (-) - BCH-189 (Belleau, et al., 5th International Conference on AIDS, Montreal, Canada, June 4-9, 1989, #TCO 1; Chu, et al. Tetr. Lett., 1991, 32, 3791). The last of these compounds, lamivudine, also known as 3Tc or epivir, is currently used clinically in the treatment of both HIV infection and HBV infection. The (-) - enantiomer of the 5-fluorocytosine-oxathiolane analog (FTC) is particularly active against HIV (Choi, W. et al., J. Am. Chem. Soc., 1991, 113, 9377; Schinazi, RF, et al., Antimic Ag. Chemo, 1992, 2423, U.S. Patent Nos. 5,204,4665, 5,210,085, 5,914, 331, and 5,639,814). The 1, 3-oxothiolane nucleosides described above are prepared by condensation of a purine base or silylated pyrimidine with an intermediate of 1,3-oxathiolane. The patent E.U.A. No. 5,204,466 describes a method for condensing a 1,3-oxathiolane with a silylated pyrimidine using tin chloride as a Lewis acid, which provides virtually β-stereoselectivity. A number of patents of E.U.A. describe processes for the preparation of 1, 3-oxathiolane nucleosides by condensation of a 1,3-oxathiolan-2-carboxylic acid ester with a protected silylated base in the presence of a silicon-based Lewis acid, followed by reduction of the ester to the corresponding hydroxymethyl group to obtain the final product (see US Patent Nos. 5,663,320, 5,693,787, 5,696,254, 5,744,596, 5,756,706 and 5,864,164). In addition, these patents contain generic descriptions for the synthesis of 1, 3-oxathiolane nucleosides in a similar manner using the corresponding 1,3-dioxolane intermediate. The patent E.U.A. No. 5,272,151 describes a process using a 2-O-protected-5-0-acylated 1-3-oxathiolane for the preparation of nucleosides by condensation with a purine base or silylated pyrimidine in the presence of a titanium catalyst. The patent E.U.A. No. 6,215,004 describes a method for producing nucleosides of 1, 3-oxathiolane which includes 2-0-protected-methyl-5-chloro-l, 3-oxathiolane condense with a silylated 5-fluorocytosine without a Lewis acid type catalyst. In these cases, the 1,3-oxathiolane ring is prepared in one of the following ways: (i) reaction of an aldehyde which is obtained from a glyoxylate or glycolic acid with mercaptoacetic acid in toluene in the presence of p-acid toluenesulfonic to obtain 5-oxo-l, 3-oxathiolan-2-carboxylic acid; (ii) cyclization of the anhydrous glyoxylates with diethylacetal of 2-mercaptoacetaldehyde at reflux in toluene to obtain the lactone of 5-ethoxy-1,3-oxathiolane; (iii) condensation of the glyoxylic acid ester with mercaptoacetaldehyde (dimeric form) to obtain the 5-hydroxy-l, 3-oxathiolan-2-carboxylic ester or (iv) coupling of an acyloxyacetaldehyde with 2,5-dihydroxy-1, 4 -ditiana, the dimeric form of 2- ercaptoacetaldehyde, to form a 2- (aryloxy) methyl-5-hydroxy-1,3-oxathiolane. The lactone, the 5-oxo compound, has to be reduced to the corresponding lactol during the procedure. The 2-carboxylic acid or its ester must also be reduced to the corresponding 2-hydroxymethyl derivatives using the borane-methyl sulfide complex. The key intermediate, aldehyde, can be prepared using several methods: (i) oxidation of the compound 1,4-di-0-benzoyl-meso-erythritol, 1,6-di-0-benzoyl-D-mannitol or 1.5 -di-O-benzoyl-D-arabitol with lead tetraacetate; (ii) monoacylated ethylene glycol preparation followed by oxidation to aldehyde; (iii) acylation of the ethylenic chlorohydrin followed by oxidation with dimethyl sulfoxide; (v) oxidation with tetra-acetate of lead; (vi) ozonolysis of the allylic acylate or acylate of 3-methyl-2-buten-1-ol; (vii) and more recently, by acylation of 2-buten-l, 4-diol followed by ozonolysis. In addition, the patent E.U.A. No. 6,215,004 describes a process for preparing the acyloxyacetaldehyde diethylacetal by acylation of 2,2-diethoxyethanol. Norbeck, D.W., et al. (Tet. Lett., 1989, 30.6263) reported the synthesis of (±) -1- (2β, 4β) -2- (hydroxymethyl) -4-dioxolanyl-thymine, which results in a racemic mixture of diastereomers around of the C4A atom The crystallographic analysis of the product with X-rays reveals that the dioxolane ring adopts the 3T4 conformation commonly observed in the ribonucleosides, with the 03 'atom in the endo position, which is completely different from the distorted 3E configurations observed in AZT , AZDU, ddA, ddC, and 3 '-deoxy-3' -fluorothymidine, of which all have potent activity against HIV in vi tro. The anti-viral activity of the dioxolane nucleosides prompted Chu et al. to synthesize a series of analogues in a search for powerful anti-viral and / or anti-cancer agents. For example, it is reported that the compound 9- (β-D-hydroxymethyl-1,3-dioxolanyl) aminopurine (β-D-DAPD), and its metabolite 9- (β-D-hydroxymethyl-1,3-dioxolanyl) -guanine (ß-D-DXG) have potent and selective activity against human immunodeficiency virus (HIV) and hepatitis B virus (HBV) (Rajagopalan et al., Antiviral Chem. Chemother., 1996, 7 (2), 65- 70). (-) - DAPD is a potent and selective inhibitor of HIV in vitro and in vivo and of HBV replication in vitro (Furman, et al., Drugs of the Future 2000, 25 (5), 454-461). Likewise, l- (ß-L-hydroxymethyl-1,3-dioxolanyl) -thimine (Dioxolan-T) (Norbeck et al., Tet.Let., 1989, 30, 6263-66) possesses anti-HIV activity and anti-HBV. It was discovered that 1- (β-L-hydroxymethyl-3-dioxolanyl) -cytidine (β-L-OddC) has potent anti-tumor activity towards renal carcinoma as well as human prostate carcinoma (Kadhi et al., Can. Cancer Res. ., 57 (21), 4803-10, 1997). (-) - (2 'S, 4' R) -1 '- [2' - (hydroxy-methyl) -1 ', 3' -dioxolan-4 '-yl] -5-yodouracil) L-IOddU is currently in pre-clinical or clinical studies to determine its value as an anti-viral or anti-cancer agent (see Kim, et al., J. Med. Chem. 1993, 36, 519-528 and references therein; Corbett , and Rublein, Curr Opin, Research Drugs 2001, 2, 348-353, Gu, et al., Antimicrob Agents Chemother, 1999, 43, 2376-2382; Mewshaw, et al., J. Acquir. Immune Defic. Syndr, 2002, 29, 11-20). U.S. Patent Nos. 5,041,449 and E.U.A. No. 5,270,315 for Belleau et al. describe a generic group of 1, 3-dioxolanes substituted at the 2-position and substituted at the 4-racemic position. Table 1 of the reference shows data for two racemic 1,3-dioxolane nucleosides - a racemic trans (a) 1,3-dioxolane nucleoside with a cytosine base (compound XII) and a cis (β) 1 nucleoside, 3-racemic dioxolane with an adenine base (compound XIV) (see also EP 0 337 713 for IAF Biochem International). In June 1989, Belleau et al. , reported a method of synthesis of cytidine nucleosides containing oxygen or sulfur at the 3 'position (Belleau, B., et al., 5th International Conference on AIDS, Montreal, International Center for Research and Development: Ottawa, Ontario, 1989; TCOI). The dioxolane ring is prepared by the condensation of RC02CH2CHO with glycerin. The synthesis results in a racemic mixture of diastereomers around the C4 'carbon of the nucleoside. The racemic DADP compound is synthesized as shown in reaction scheme 1.

REACTION SCHEME 1 M-chloroperbenzoic acid Ba er-Vüli er RACEMICO (+/-) - DAPD Belleau et al. they reacted glycerol and chloroacetaldehyde to generate a dioxolane intermediate. After displacement of the chlorine with a benzoic acid salt, oxidation of the primary alcohol to a carboxylic acid and transposition of Baeyer-Villiger with m-chloroperbenzoic acid, the corresponding racemic dioxolane benzoate is obtained. This compound is then coupled with 2-amino-6-chloropurine and the resulting nucleoside-like intermediate is reacted with ammonia under pressure to obtain racemic DAPD. (±) -Dioxolane-T is also synthesized in a similar way by Choi et al. (Choi, et al., J. Am. Chem. Soc. 1991, 113, 9377-9378 and patent E.U.A. No. 5,852,027). As discussed earlier, at the end of 1989, Norbeck et al. published an article describing the synthesis of racemic cis-1, 3-dioxolane-thymidine which also has anti-HIV activity in vitro (Norbeck, et al., Tet., 1989, 30 (46), 6263-6266). The product is synthesized in five steps from the benzyloxyaldehyde dimethylacetal and (±) methyl glycerate to produce a 79% yield of the 1: 1 diastereomeric mixture. As with the Belleau synthesis, the Norbeck synthesis results in a racemic mixture of diastereoisomers around the C4 'carbon of the nucleoside. See reaction scheme 2.

REACTION SCHEME 2 Pb (Ac?) 4 RACEMICO DIOXOLAN ACETATE R '= Bn The same racemic acetate dioxolane intermediate is synthesized by Liotta et al. starting from cis-2-buten-l, 4-diol (Choi, et al., J. Am. Chem. Soc. 1991, 113 (24), 9377-0379 and Wilson, et al., Bioorg. Med. Chem. Let. 1993, 3 (2), 169-174). See reaction scheme 3.

REACTION SCHEME 3 RACEMICO R '= butyryl or TBDPhSi The difficulty of these methods, shown in reaction scheme 3, is that they involve the synthesis of an unstable aldehyde and a difficult step or oxidative steps. The patent E.U.A. No. 5,179,104 to Chu and Schinazi, describe a method for nucleoside ß-D-1, 3-dioxolane enantiomerically pure through a stereospecific synthesis (see also US patents 5,925,643 us;. 5,767,122; 5,444,063; 5,684,010; 5,834,474 and 5,830,898).

EP 0,515,156 to BioChem Pharma describes a method for obtaining the enantiomers of nucleosides 1, 3-dioxolane using a stereoselective synthesis that includes condensing an intermediate of 1, 3-dioxolane covalently linked to a chiral auxiliary with a Lewis acid of silyl type (see also related U.S. Patent Nos. 5,753,706 and 5,744,596). Chu et al. , published a stereo-specific synthesis of ß-D-1,3-dioxolane nucleosides from 1,6-anhydromanose (Chu, et al., Tet.Let., 1991, 32, 3791-3794). About the same time, Thomas and Surber published an article describing that (i) an exhaustive search of the literature on chiral chromatography could not reveal any examples of separations nucleoside and (ii) documents appear to be the first separation of the enantiomers of a nucleoside by chiral high performance liquid chromatography. The resolved nucleoside is not a 1,3-dioxolane nucleoside, and four of the five chiral columns attempted did not work (Thomas, et al., J Chromat., 1991, 586, 265-270). Kim et al. (Kim, et al. J. Med. Chem. 1993, 36 (1): 30-37) subsequently published a paper describing an asymmetric synthesis of enantiomers ß-D and -D nucleosides 1, 3-dioxolan-pyrimidine from 1,6-anhydro-D-mannose. The synthesis of (-) - DAPD is described as a thirteen-step process from 1,6-anhydro-D-anose that includes a nine-step conversion of 1,6-anhydro-D-mannose to a chiral acetate ( reaction scheme 4).

REACTION SCHEME 4 9 pa 1,6-anhydro-D-mannose After copying the acetate under conditions of Vorbruggen and several steps of purification and deprotection, you get (-) - DAPD with modest performance. This procedure is laborious, difficult and involves complicated oxidation steps. In 1992, Belleau et al. (Belleau, et al. Tet. Let. 1992, 33, 6949-6952) published a synthesis of stereoisomers of 2 ', 3 r dideoxy-3-oxacitidina enantiomerically pure by a procedure eight steps using L-ascorbic acid as a chiral auxiliary. L-ascorbic acid is used to produce a set of diastereomers that can be separated. The use of lead tetra-acetate makes this procedure unsuitable for scaling. In 1992, Kim, et al. , also published an article describing the way in which (-) -L-ß-dioxolane-C and (+) - L-ß-dioxolane-T can be obtained from 1,6-anhydro-L-β -gulopyranose (Kim, et al., Tet., Let., 1992, 32 (46), 5899-6902). Liotta and Shinazi, in the patent E.U.A. Do not. ,276,151, found that 2-0-protected-5-0-acylated-l, 3-dioxolanes can be copied with purine-type bases or pyrimidine in the presence of a Lewis acid containing titanium to predominantly generate the β- isomers racemic (see also WO 92/14729). Jin et al. (Jin, et al., Tet. Asym, 1993, 4 (2), 211-214), describe that Lewis acid type catalysts play an important role in the preparation of 1,3-dioxolane nucleosides. TiCl4 and SnCl4 promote formation of dioxolane nucleosides with racemization in the coupling of 2'-deoxy-3 '-oxarribósidos enantiomerically pure N-acetylcytosine silylated with. The use of trimethylsilyl Lewis triflate acids, trimethylsilyl iodide and TiCl2 (Oi-Pr) 2 provides enantiomerically pure cytosine-dioxolane nucleosides with low diastereo-selectivity. An asymmetric synthesis of dioxolane nucleosides was reported by Evans, et al. (Tet Asym, 1993, 4, 2319-2322). The reaction of D-mannitol with BnOCH2CH- (OCH3) 2 in the presence of SnCl2 in 1,2-dimethoxyethane followed by oxidation with RuCl3 / NaOCl yields cis and trans-dioxolan-4-carboxylic acid, which is then converted into nucleosides of D- and L-dioxolane by decarboxylation, coupling and deprotection reactions. The document also reports an alternative route for these carboxylic acids by reaction of BnOCH2CH (OCH3) 2 with L-ascorbic acid. The chiral carboxylic acid can also be prepared by reacting the commercially available 2,2, -dimethyl-1,3-dioxolan-4- (S) -carboxylic acid with a protected derivative of hydroxy-acetaldehyde such as benzoyloxyacetaldehyde, under acids (see US Patent Nos. 5,922,867 and 6,358,963). Siddiqui, et al. , describe that the cis-2,6-diaminopurin-dioxolane compound can be deaminated selectively using adenosine deaminase (Siddiqui, et al., Bioorg, Med Chem. Let., 1993, 3 (8), 1543-1546) . Although the synthesis of dioxolane nucleosides using the procedures described in the literature is possible, chemistry is not applicable to the synthesis of (-) - DAPD on a large scale. See Chu, et al. Tet. Let. 1991, 32, 3791-3794; Siddiqui, et al. Bioorg. Med. Chem. Let. 1993, 3, 1543-1546; Kim, et al. Tet. Let. 1992, 46, 6899-6902). The patent E.U.A. No. 5,763,606 to Mansour et al. describes processes for producing predominantly pure 1,3-oxathiolane and 1,3-dioxolane nucleosides by coupling a silylated pyrimidine or purine base with a bicyclic intermediate (see also WO 94/29301). The patent E.U.A. No. 6,215,004 for Painter et al. describes processes for preparing 2- [R -'- C (O) OCH2] -1,3-dioxolanyl-5-one by reacting glycolic acid with an acetal of the formula (R10) 2CHR; a hemiacetal of the formula (R20) (HO) CHR; or a mixture thereof, in which R is - (CH2-0-C (O) R1), and R1 and R2 are independently alkyl, aryl, heteroaryl, heterocyclic, alkaryl, alkylheteroaryl, or alkylheterocyclic, or aralkyl , in the presence of a Lewis acid, such as boron trifluoride diethyl ether (see also WO 00/09494). WO 00/47759 and WO 01/58894 both for BioChem Pharma disclose process for separating β anomers and from an anomeric mixture of dioxolane analogs having a COOR portion at the C4 'position before coupling with a purine base or pyrimidine. The procedure for resolving the dioxolane analogs to obtain dioxolanes having a predominant β-L configuration involves the use of enzymes, in particular hydrolases. WO 03/062229 to Shire BioChem Inc., discloses a single reaction vessel method for producing a dioxolane nucleoside analogue by the addition of a Lewis acid, a silylating agent and a non-silylated pyrimidine or purine base. to a dioxolane. The publication also describes a process for producing a dioxolane compound by reacting a dioxolane compound in a solvent in the presence of DIB e 12, using an appropriate energy source. The stereochemistry of oxa-substituted 2 ', 3'-dideoxynucleoside analogs at the 3' position ("dioxolane nucleoside analogs") may play an important role in their biological activity. The Cl 'position of the ribose in the nucleoside is a chiral center because the carbon is bound to four different portions. Similarly, there is an optically active center in the C4 'of the nucleoside.

As shown below, the substituents on the chiral carbons (the specified purine or pyrimidine base and CH2OH) of the 1,3-dioxolane nucleosides can be either cis (on the same side) or trans (on opposite sides) with respect to the dioxolane ring system. For consistency purposes, the same stereochemical designation is used when the methyloxy portion or the base portion is replaced with another substituent group. The racemates both cis and trans consist of a pair of optical isomers. Therefore, each compound has four individual optical isomers. The four optical isomers are represented by the following configurations (when the dioxolane portion is oriented in a horizontal plane such that the -0-CH2-portion is on the front): (1) cis, with both groups "Nhacia above" , which is the configuration ^ -cis (known as ß-D); (2) cis, with both groups "down", which is the opposite ß-cis configuration (known as ß-L); (3) trans with the substituent at C4 '"upwards" and the substituent at Cl' "downwards"; and (4) trans with substituent C4 '"downwards" and the substituent at Cl' "upwards." The two enantiomers cis together are known as a racemic mixture of β-enantiomers, and the two trans enantiomers are known as a racemic mixture of α-enantiomers In general, it is difficult to separate or in some other way obtain the individual enantiomers of the cis-configuration. stereoisomers of the cis-1,3-dioxolane nucleosides are illustrated ac Next: Trans (a) Because the stereoisomers of dioxolane nucleosides normally have different biological activities and toxicities, obtaining the pure therapeutically active isomer becomes crucial. Frequently, one stereoisomer is considerably more active than the other. Chu et al. developed methods for the asymmetric synthesis of dioxolane nucleosides from D-mannose and L-gulonic lactone for SD- and L-dioxolane nucleosides, respectively (U.S. Patent Nos. 5,767,122, 5,792,473). However, these methods involve many steps and it is necessary to purify most of the intermediates by silica gel column chromatography (see Kim, et al., J. Med. Chem. 1993, 36, 519-528). To prepare a large enough amount of the dioxolane nucleoside drug for clinical trials, chiral 2-acyloxymethyl-5-oxo-l, 3-dioxolane is used as the key intermediate. This is prepared by cyclization of ROCH2CHO or its acetal with glycolic acid in the presence of BF3, followed by column separation with chiral resin or by enzymatic resolution which are expensive and difficult techniques. Therefore, there remains a need for cost-effective and stereoselective procedures for producing biologically active isomers of dioxolane nucleosides. It is an object of the present invention to provide novel and cost effective methods for the synthesis of enantiomerically pure dioxolane nucleosides.

SUMMARY OF THE INVENTION The present invention includes an efficient synthesis route for 1,3-dioxolane nucleosides from inexpensive precursors, with the option to introduce functional groups as needed. The process allows the stereo-selective preparation of the biologically active isomer of these compounds. In one embodiment of the present invention, there is provided a process for preparing a substantially pure β-D or β-L-1,3-dioxolane nucleoside, such as β-D-DAPD, which comprises: a) preparing or obtaining a 2, 2-dialkoxyethanol esterified of the formula (la) or (Ib); (la) (Ib) in which: each R1 is independently alkyl, aryl, heteroaryl, heterocyclic, alkaryl, alkylheteroaryl, or alkylheterocyclic, or aralkyl; and R2 is any suitable removable group such that (1) it can be easily removed at the end of the synthesis, (2) it has a low molecular weight to avoid transporting large masses during the process, (3) this can be achieved commercially and economically, (4) the corresponding ester is stable under the reductive acetylation conditions, (5) the subsequent lactone can be resolved, and (6) after coupling, the corresponding anomers can be easily separated, preferably by crystallization (for example iso -butyryl or p-methoxybenzoyl); and then b) cyclizing the esterified 2, 2-dialkoxyethanol of formula (la) or (Ib) with glycolic acid, preferably in the presence of a Lewis acid, such as BF3-Et20, to obtain a lactone of 1.3. -dioxolane of the formula (II): (H) and then c) resolving the 1,3-dioxolane lactone of the formula (II) to obtain a substantially pure D- or L-lactone; and then d) selectively reducing with a reducing agent, such as LiAlH (OtBu) 3 and activating the substantially pure D- or L-chiral lactone to obtain a substantially pure D- or L-1,3-dioxolane of the formula (III ): (III) in which: L is an appropriate leaving group, such as a 0-acyl, for example OAc, halogen (F, Br, Cl or I), 0-mesylates (OMs) and 0-tolutes (OTs) ), or similar; and then e) coupling the substantially pure D- or L-1, 3-dioxolane of the formula (III) with an activated and / or protected purine or pyrimidine base or its derivative, such as activated 2,6-dichloropurine, to get the mixture a: ß of D- or L-1, 3-dioxolane nucleosides substantially protected cigars of the formula (IV): (IV) in which: B is a purine base or pyrimidine or its derivative; and then, f) purify the mixture at: ß of D- nucleosides or L-1, 3-dioxolane substantially pure protected from the formula (IV) to obtain a protected substantially pure β-D- or β-L-1,3-dioxolane nucleoside; and then g) deprotecting the substantially purified β-D- or β-L-1,3-dioxolane nucleoside, if necessary, to obtain a substantially pure β-D or B-L-1,3-dioxolane nucleoside. In one embodiment of the present invention, the 2, 2-dialkoxyethanol esterified of the formula (la) is hydrolyzed to the corresponding aldehyde of the formula (Ib). In a particular embodiment the esterified 2, 2-dialkoxyethanol of the formula (la) is hydrolyzed to the corresponding aldehyde of the formula (Ib) when R2 is p-methoxybenzoyl. In another embodiment of the present invention, the 2, 2-dialkoxyethanol esterified of the formula (Ia) is not hydrolyzed to the corresponding aldehyde of the formula (Ib) In a particular embodiment, the esterified 2, 2-dialkoxyethanol of the formula (la) is not hydrolyzed to the corresponding aldehyde of the formula (Ib) when R2 is isobutyryl. In one embodiment of the present invention, the resolution of the 1,3-dioxolane lactone of the formula (II) is achieved to obtain a substantially pure D- or L-lactone using chiral chromatography. In an alternative embodiment of the present invention, the resolution of the 1,3-dioxolane lactone of the formula (II) is achieved to obtain a substantially pure D- or L-lactone using enzymatic resolution.

REACTION SCHEME 5 DIOXOLAN LACTONE LACTONE DIOXOLANO RACEMICO QUIRAL NUCLEOSIDE OF ACETATE OF DIOXOLANO DIOXOLANO QUIRAL QUIRAL Therefore, the process of the present invention can be used to prepare the compounds of the formula A to D: A B C D and pharmaceutically acceptable salts or esters thereof, in which: R is independently H, halogen (F, Cl, Br, I), OH, OR ', 0CH3, SH, SR', SCH3, NH2, NHR ', NR'2, lower alkyl of C? -C4, CH3, CH = CH2, N3C = CH2, C02H, C02R' , CONH2, CONHR ', CH2OH, CH2CH2OH, CF3, CH2CH2F, CH = CHC02H, CH = CHC02R', CH = CHC1, CH = CHBr, or CH = CHI; each R 'is independently a lower alkyl of C? -C; Z is either CH or C-X; and Each X and Y are independently H, halogen, (F, Cl, Br, I), OH, OR ', OCH3, SH, SR', SCH3, NH2, NHR ', NR'2, or CH3. In a particular embodiment of the present invention, a process for preparing substantially pure ß-D-DAPD is provided. See reaction scheme 6.

REACTION SCHEME 6 HO R0 Resolution HO- > chiral DIOXOLAN LACTONE LACTONE DIOXOLANO RACEMICO QUIRAL DIOXOLAN ACETATE (-) - DAPD QUIRAL BRIEF DESCRIPTION OF THE FIGURES Figure 1 are typical chromatograms for the compounds of the present invention. Figure IA is a typical chromatogram for the lactone of the butyryl ester (compound 1). Figure IB is a typical chromatogram for butyryl ester acetate (compound 3). Figure 1C is a typical chromatogram for the p-methoxybenzoyl lactone (compound 11). Figure ID is a typical chromatogram for the p-methoxybenzoyl lactone (compound 11) using the optimized SFC method. Figure 1E is a typical chromatogram for benzoyl lactone (compound 12). Figure 1F is a typical chromatogram for benzoyl-lactone (compound 12) using the optimized SFC method. Figures 1G-1J are typical chromatograms for the lactone of the iso-butyryl ester (compound 13). Figure 1K is a typical chromoatogram for the lactone of tert-butyl-diphenylsilyl ether (compound 4). Figure 1L is a typical chromatogram for the lactone of t-butyl diphenylsilyl ether (compound 4) using the optimized method. Figure 1M is a typical chromatogram for DAPD. Figure IN is a typical chromatogram for DAPD using the optimized SFC method. Figure 2 is a graphical representation of a theoretical phase diagram for the lactone of the ether-t-butyl-diphenyl-silyl ether (compound 4). Figure 3 are graphic representations of the selectivity of some microbial enzymes for a particular enantiomer of the compounds of the present invention. Figures 3A and 3B show the results from the evaluation of the lactone of the butyryl ester (compound 1) against several microbial enzymes. Figure 3C shows the results from the evaluation of the lactone of p-methoxybenzoyl (compound 11) against several microbial enzymes. Figure 3D shows the results from the evaluation of benzoyl-lactone (compound 12) against several microbial enzymes. Figure 3E shows the results from the evaluation of the lactone of the isobutyryl ester (compound 13) against several microbial enzymes. Figure 4 is a graphical representation of the microbial resolution of the lactone of the isobutyryl ester (compound 13) against the microbial enzymes CMC 103669 and CMC 103661 using various concentrations, buffer solutions, pH ranges and temperatures. Figure 5 is a spectrum of 1 H NMR at 400 MHz for the lactone of the resolved iso-butyryl ester (compound 13).

DETAILED DESCRIPTION OF THE INVENTION The present invention includes efficient synthetic routes for 1,3-dioxolane nucleosides from inexpensive precursors, with the option of introducing functional groups as needed. These methods allow the stereo-selective preparation of the biologically active isomer of these compounds. In one embodiment of the present invention, there is provided a process for preparing a substantially pure ß-Do β-L-1,3-dioxolane nucleoside, such as β-D-DAPD, comprising: a) preparing or obtaining a , Esterified 2-dialkoxyethanol of the formula (la) or (Ib); (the) (Ib) wherein each R1 is independently alkyl, aryl, heteroaryl, heterocyclic, alkaryl, alkylheteroaryl, or alkylheterocyclic, or aralkyl; and R2 is any suitable removable group such that (1) it can be easily removed at the end of the synthesis, (2) it has a low molecular weight to avoid transporting large masses during the process, (3) this can be achieved commercially and economically, (4) the corresponding ester is stable under the reductive acetylation conditions, (5) the subsequent lactone can be resolved, and (6) after coupling, the corresponding anomers can be easily separated, preferably by crystallization (for example iso -butyryl- or p-methoxybenzoyl); and then b) cyclizing the esterified 2, 2-dialkoxyethanol of formula (la) or (Ib) with glycolic acid, preferably in the presence of a Lewis acid, such as BF3-Et20, to obtain a lactone of 1.3. -dioxolane of the formula (II): (II) and then, c) resolving the 1,3-dioxolane lactone of the formula (II) to obtain a substantially pure D- or L-lactone; and then d) reducing selectively with a reducing agent, such as LiAlH (OtBu) 3 and activating the substantially pure D-lactone or chiral L-lactone to obtain a substantially pure D- or L-1, 3-dioxolane. of the formula (III): (III) wherein: L is an appropriate leaving group, such as a 0-acyl, for example OAc, halogen (F, Br, Cl or I), OMs, OTs, or the like; and then e) coupling the substantially pure D- or L-1, 3-dioxolane of the formula (III) with an activated and / or protected purine or pyrimidine base or its derivative, such as activated 2,6-dichloropurine, to obtain the mixture: β of protected substantially pure D- or L-1, 3-dioxolane nucleosides of the formula (IV): (IV) wherein: B is a purine base or pyrimidine or its derivative; and then, f) purifying the mixture to: ß of D- nucleosides or L-1, substantially pure protected 3-dioxolane of the formula (IV) to obtain a protected substantially pure β-D- or β-L-1,3-dioxolane nucleoside; and then g) deprotecting the substantially purified β-D- or β-L-1,3-dioxolane nucleoside, if necessary, to obtain a substantially pure β-D or B-L-1,3-dioxolane nucleoside. In one embodiment of the present invention, the esterified 2,2-dialkoxyethanol of the formula (la) is hydrolyzed to the corresponding aldehyde of the formula (Ib). In a particular embodiment the esterified 2, 2-dialkoxyethanol of the formula (la) is hydrolyzed to the corresponding aldehyde of the formula (Ib) when R2 is p-methoxybenzoyl. In one embodiment of the present invention, the resolution of the 1,3-dioxolane lactone of the formula (II) to obtain a substantially pure D- or L-lactone is achieved using chiral chromatography. In an alternative embodiment of the present invention, the resolution of the 1,3-dioxolane lactone of the formula (II) to obtain a substantially pure D- or L-lactone is achieved using enzymatic resolution. Therefore, the process of the present invention can be used to prepare the compounds of the formula A to D: A B c D and pharmaceutically acceptable salts or esters thereof, in which: R is independently H, halogen (F, Cl, Br, I), OH, OR ', 0CH3, SH, SR', SCH3, NH2, NHR ' , NR'2, lower alkyl of Ca-Cj, CH3, CH = CH2, N3C = CH2, C02H, C02R ', CONH2, CONHR ', CH20H, CH2CH2OH, CF3, CH2CH2F, CH = CHC02H, CH = CHC02R ', CH = CHC1, CH = CHBr, or CH = CHI; each R 'is independently a lower alkyl of C? -C4; Z is either CH or C-X; and each X and Y are independently H, halogen, (F, Cl, Br, I), OH, OR ', OCH3, SH, SR', SCH3, NH2, NHR ', NR'2, or CH3.

DIOXOLANO DIOXOLANO RACEMICO QUIRAL REACTION SCHEME 5 NUCLEOSID OF DIOXOLAN DIOXOLAN ACETATE QUIRAL QUIRAL Therefore, the process of the present invention can be used to prepare the compounds of the formula A to D: A B C D and pharmaceutically acceptable salts or esters thereof, in which: R is independently H, halogen (F, Cl, Br, I), OH, OR ', 0CH3, SH, SR', SCH3, NH2, NHR ', NR'2, lower alkyl of C1-C4, CH3, CH = CH2, N3C = CH2, C02H, C02R', CONH2, CONHR ', CH2OH, CH2CH2OH, CF3, CH2CH2F, CH = CHC02H, CH = CHC02R', CH = CHC1, CH = CHBr, or CH = CHI; each R 'is independently a lower alkyl of C? ~ C4; Z is either CH or C-X; and each X and Y are independently H, halogen, (F, Cl, Br, I), OH, OR ', OCH3, SH, SR', SCH3, NH2, NHR ', NR'2, or CH3. In a particular embodiment of the present invention, a process for preparing substantially pure ß-D-DAPD is provided. See reaction scheme 6.

REACTION SCHEME 6 HO LACTONADE LACTONA DE DIOXOLANO DIOXOLANO RACEMICO QUIRAL DIOXOLAN (-) - DAPD QUIRAL ACETATE In one embodiment, the invention provides methods for the resolution of compounds that can be intermediates in the synthesis of 1,3-dioxolane nucleosides, such as l-β-D-2, 6- diaminopurin-dioxolane (DAPD). In one embodiment, the inventprovides a biocatalytic method for the synthesis of DAPD intermediates as shown in reactscheme 7.

REACTSCHEME 7 Sample routes for DAPD In one embodiment of the invent the 1,3-dioxolane lactone of the formula (II) is the sec-butanoate ester (compound 13). In one embodiment of the present invent if desired, the (S) -enantiomer of the lactone of the butyrate ester (compound 1 of the following structure), Compound 1 PPL is used for chiral resolut As described in the examples, PPL is identified as the most selective enzyme (22% yield, 98% ee), and is found to result in the (S) isomer of the butyrate ester in a full commercial enzyme selectof all available commercial enzymes and 200 microbial enzymes. Although in general embodiments any effective enzyme can be used, the PS lipase, immobilized lipase from Pseudomonas and Lipase M are identified as selective enzymes that result in the (R) isomer of the butyrate ester, as described in the examples. It is found that PS lipase is an efficient enzyme possessing the required enantio-preference, and yields 22% yield of the (R) -enantiomer of compound 1 of 95% ee. Therefore, in an alternative embodiment of the present invent if an (R) -enantiomer is desired, then PS lipase, immobilized lipase from Pseudomonas or Lipase M is used for chiral resolut In a particular embodiment, the enzyme is PS Lipase. Although the inventis not limited to any particular conditfor resolut in one embodiment, the temperature for resolutis approximately 0 ° C. In another embodiment, the temperature is between -5 ° C and 10 ° C, or -2 ° C and + 5 ° C or about -1 ° C to about 1 ° C. In another embodiment, the amount of substrate is approximately 100 g / l. The amount of substrate may vary, for example, from 5 to 500, 20 to 200, 50 to 150, approximately 60, 70, 80, 90, 100, 110, 120, 130, 140 or more g / l of buffer solutor / l of total reactvolume. In one embodiment, the buffer solutis a 1: 1 mixture of toluene-buffer, which may be about pH 6. In a specific embodiment, the condit for resolutcan be 0 ° C, 100 g / l of substrate in a 1: 1 mixture of toluene-buffer at pH 6. As described in the examples, these condit produce 22%, and % enantiomeric excess of the (R) -enantiomer of compound 1 (E = 5.3). The condit may also vary slightly within the experimental parameters for efficient resolut The microbial strains that resolve the compounds can vary. In one embodiment, the strains come from Acterus Acter species. The microbial selectidentifies 2 strains (Acinetobacter and Acinetobacter junii) which produce (R) -butyrate with 80% ee. All commercially available enzymes and 200 microbial enzymes are evaluated in order to identify an efficient resolutof the butyrate ester. The microbial selectreveals 2 promising strains (CMC 3419, Acinetobacter and CMC 3606, Acinetobacter j unii) which produce (R) -butyrate with 80% ee. In one embodiment, when the resolutof the butyrate ester acetate (compound 3) of the following structure is desired: Compound 3 The enzyme that is used is PS lipase. A complete commercial enzyme selectof compound 3 reveals that most of the selective enzymes resolve in the acetate center and not in the required butyrate center. However, Lipase PS, Lipase MY and Lipase AY have moderate selectivity, and produce the (R) isomers of the ester. These enzymes have similar selectivity to those for compound 1. Further investigatreveals that Lipase PS is the most selective (E = 3.7 effective). In the same way, it is of selectivity comparable with the resolutof compound 1 with Lipase PS. In one embodiment of the invent the resolutstep occurs at an early stage of the synthesis, i.e. the lactone stage. In one embodiment, when the resolutof the (S) enantiomer of p-methoxy-benzoyl lactone, 4-oxo- [1,3] dioxolan-2-ylmethyl 4'-methoxybenzoate (compound 11) is desired Compound 11 Chirazyme-L2 is then used for the resolution of the lactone. A wide range of commercial and microbial enzymes is evaluated in order to identify an efficient resolution of compound 11. Chirazyme-L2 is identified as the most selective enzyme that produces the residual ester with 39% yield, > 98% ee of the enantiomer (S). In another embodiment, when the resolution of the benzoyl lactone is desired, 4-oxo- [1, 3] dioxolan-2-ylmethyl benzoate (compound 12) Compound 12 the enzyme that is used can be Chirazyme-L2. A selection of commercial enzyme (ten enzymes) is made for the benzoyl lactone (compound 12). Chirazyme-L2 and protease-DS acid are identified as potential resolution agents in the selection. Chirazyme-L2 produces comparable selectivity for the resolutions of compounds 11 and 12. A limited selection of commercial enzyme for compound 12 determines that Chirazyme-L2 is again the most efficient enzyme. In another embodiment, when the resolution of the (R) enantiomer of iso-butyryl lactone, 4'-methyl- [1, 3] dioxolan-2-yl-methyl 2'-methylpropanoate (compound 13) is desired.

Compound 13 the enzyme used can be Chirazyme-L2. A complete commercial and microbial enzyme selection is made for compound 13. Again Chirazyme-L2 is identified as the most selective enzyme. The configuration is determined to be the (R) -enantiomer by chemical correlation. Initial studies in MTBE-buffer yield the residual ester with 28% yield, 92% ee. A complete selection of commercial enzyme for compound 13 reveals that Chirazyme-L2, Lipase PS, acid DS protease and Chirazyme-L9 possess moderate selectivity, and produce the (R) -enantiomer as the residual ester. A complete microbial selection identifies strains that are selective for any enantiomer. A microbe, CMC 103869, produces more than 95% ee of the lactone by executing the resolution at 10 ° C. In one embodiment, the substrate is compound 13 (iso-butyryl lactone). In another embodiment, the substrate is one that allows simpler chemistry downstream, for example, simpler purification techniques. Chirazyme-L2 can be the enzyme used for resolution. In one embodiment, Chirazyme-L2 can produce the (R) configuration of the residual ester. The reaction is studied in systems of MTBE-buffer, toluene-buffer and 2-propanol-water. Stability and selectivity problems are encountered with the MTBE and toluene systems. However, the results in 2-propanol-water are excellent, yielding a 38% yield with a 94% enantiomeric excess of compound 13 from a 30 g feed of the racemate. Therefore, in one embodiment, the resolution is carried out in 2-propanol-water. An optimization study is carried out. The procedure is improved using a single-phase alcohol-water system as a solvent and using Kugeirohr distillation to purify the product. Therefore, in one embodiment, the process involves a monophasic alcohol-water system and distillation. The bio-resolution of compound 13 with Chirazyme-L2 is scaled up to 30 g to 200 g / l in 2-propanol: water (9: 1) and produces 2'-methyl-propanoate of 4-oxo- [1, 3] - dioxolan-2-yl-methyl with 38% yield, 94% ee and 90% chemical purity. See reaction scheme 8.

REACTION SCHEME 8 Conditions identified for the bio-resolution of compound 13 and.

In contrast, the lactone of t-butyl diphenyl-silyl ether (compound 4) of the following structure: Compound 4 is determined as a true racemate. The phase diagram is constructed for this compound which reveals that the eutectic is at 77.5% ee. Racemic cis-DAPD is evaluated with several enzymes known for their acylation activity in the presence of vinyl butyrate, but found to produce non-stereo-selective reactions. PeptiCLEC-BL and Lipase AY produce rapid reactions for racemic butyrate and therefore may be useful to place an ester in oxygen in a regioselective and non-aggressive manner. In one mode, PeptiCLEC-BL is used. In a separate modality, Lipase AY is used. l-ß-D-2,6-diaminopurin-dioxolane (DAPE Definitions As used in the present invention, the term "substantially pure", "substantially free of", "substantially in the absence of" or "isolated" refers to a nucleoside composition that includes at least 85, at least 90, at least 95%, or at least 99% to 100% by weight, of the designated enantiomer of said nucleoside. In one embodiment, the process produces compounds that are substantially free of enantiomers of the opposite configuration. The term alkyl, as used in the present invention, unless otherwise specified, refers to a straight, branched chain, or cyclic, primary, secondary, or tertiary saturated hydrocarbon typically from Ci to Cio, and specifically includes methyl, trifluoromethyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3-mephenyl, 2,2-dimethylbutyl , and 2, 3-dimethylbutyl. The term includes both substituted and unsubstituted alkyl groups. The portions with which the alkyl group may be substituted are selected from the group consisting of hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulphonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, and either unprotected, or protected as needed, as is known to those skilled in the art, for example, as taught in Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, second edition, 1991, incorporated in the present invention for reference. The term "lower alkyl", as used in the present invention, and unless otherwise specified, refers to a saturated, straight, branched, C 1 to C 4 alkyl group or, if appropriate, an Ci alkyl group at Cyclic C (for example, cyclopropyl), including both substituted and unsubstituted forms. Unless specifically indicated otherwise in this application, when alkyl is an appropriate portion, lower alkyl is preferred. Similarly, when alkyl or lower alkyl is an appropriate portion, alkyl or unsubstituted lower alkyl is preferred. The term "aryl", as used in the present invention, and unless otherwise specified, refers to phenyl, biphenyl, or naphthyl. The term includes both substituted and unsubstituted portions. The aryl group may be substituted with one or more portions which are selected from the group consisting of bromine, chlorine, fluorine, iodine, hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulphonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as needed, as is known to those skilled in the art, for example, as taught in Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, second edition, 1991. The term "alkaryl" or "alkylaryl" refers to an alkyl group with an aryl substituent. The term "aralkyl" or "arylalkyl" refers to an aryl group with an alkyl substituent. The term "halogen", as used in the present invention, includes bromine, chlorine, fluorine, and iodine. The term "heteroatom", as used in the present invention, refers to oxygen, sulfur, nitrogen, and phosphorus. The term "acyl" refers to a carboxylic acid ester in which the non-carbonyl portion of the ester group is selected from straight, branched, or cyclic alkyl or lower alkyl, alkoxyalkyl including methoxymethyl, aralkyl including benzyl, aryloxyalkyl such as phenoxymethyl, aryl including phenyl optionally substituted with halogen, Ci to C4 alkyl or Ci to C alkoxy, sulfonate esters such as alkyl sulfonyl or aralkylsulfonyl including methanesulfonyl, monophosphate, diphosphate or triphosphate, trityl or monomethoxytrityl esters, substituted benzyl, trialkylsilyl (for example dimethyl-t-butylsilyl) or diphenylmethylsilyl. The aryl groups in the esters optimally comprise a phenyl group. The term "lower acyl" refers to an acyl group in which the non-carbonyl portion is lower alkyl. The term "protected" as used in the present invention and unless otherwise defined refers to a group that is added to an oxygen, nitrogen, or phosphorus atom to prevent further reaction or for other purposes. A wide variety of oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis. The term "purine-type" or "pyrimidine" includes, but is not limited to, adenine, N6-alkyl-purines, N6-acylpurines (in which acyl is C (0) (alkyl, aryl, alkylaryl, or arylalkyl), N6-benzylpurine , N6-halogeno-purine, N6-vinylpurine, N6-acetylene purine, N6-acyl-purine, N6-hydroxy-alkylpurine, N6-thioalkyl-purine, N2-alkylpurines, N2-alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine, 5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2- and / or 4-mercaptopyrimidine, uracil, 5-halogenouracil, including 5-fluorouracil, C5-alkylpyrimidines, C5-benzylpyrimidines, C5-halogeno-pyrimidines , C5-vinyl-pyrimidine, C5-acetylenic pyrimidine, C5-acyl-pyrimidine, C5-hydroxyalkyl-purine, C5-amido-pyrimidine, C5-cyanopyrimidine, C5-nitropyrimidine, C5-amino-pyrimidine, N2-alkylpurines, N2- alkyl-6-thiopurines, 5-aza-cytidinyl, 5-azauracilyl, triazolo-pyridinyl, imidazolo-pyridinyl, pyrrolopyrimidinyl, and pyrazolo-pyrimidinyl. are not limited to, guanine, adenine, hypoxanthine, 2,6-dia inopurine, and 6- (Br, Fl, Cl or I) -purine optionally with a substituent including an amino or carbonyl group in the 6-position, and 6- (Br, Cl or I) - purine optionally with a substituent including an amino or carbonyl group in position 2. The oxygen and nitrogen functional groups in the base can be protected as needed or desired. Suitable protecting groups are well known to those skilled in the art, and include trimethylsilyl, dimethylsilylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trityl, alkyl groups, and acyl groups such as acetyl and propionyl, methanesulfonyl, and phenyl. toluenesulfonyl. The term "heteroaryl" or "heteroaromatic", as used in the present invention, refers to an aromatic compound that includes at least one sulfur, oxygen, nitrogen or phosphorus atom in the aromatic ring. The term "heterocyclic" refers to a non-aromatic cyclic group to obtain which at least one heteroatom is present, such as oxygen, sulfur, nitrogen or phosphorus in the ring. Non-limiting examples of heteroaryl and heterocyclic groups include furyl, furanyl, pyridyl, pyrimidyl, thienyl, isothiazolyl, imidazolyl, tetrazolyl, pyrazinyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, indolyl, isoindolyl, benzimidazolyl, purinyl, carbazolyl, oxazolyl, thiazolyl, isothiazolyl, 1,2,4-thiadiazolyl, iso-oxazolyl, pyrrolyl, quinazolinyl, cinolinyl, phthalazinyl, xanthinyl, hypoxanthinyl, thiophene, furan, pyrrole, isopyrrole, pyrazole, imidazole, 1,2,3- triazole, 1,2,4-triazole, oxazole, isoxazole, thiazole, isothiazole, pyrimidine or pyridazine, and pteridinyl, aziridines, thiazole, isothiazole, 1, 2, 3-oxadiazole, thiazine, pyridine, pyrazine, piperazine, pyrrolidine, oxazines phenazine, phenothiazine, morpholinyl, pyrazolyl, pyridazinyl, pyrazinyl, quinoxalinyl, xanthinyl, hypoxanthinyl, pteridinyl, 5-azacytidinyl, 5-azauracilyl, triazolopyridinyl, imidazolo-pyridinyl, pyrrolopyrimidinyl, pyrazolopyr imidinyl, adenine, N6-alkyl-purines, N6-benzylpurine, N6-halogeno-purine, N6-vinylpurine, N6-acetylene purine, N6-acyl-purine, N6-hydroxy-alkylpurine, N6-thioalkyl-purine, thymine, cytosine , 6-azapyrimidine, 2-mercaptopyrimidine, uracil, N5-alkylpyrimidines, N5-benzylpyrimidines, N5-halopyrimidines, N5-vinylpyrimidine, N5-acetylenic pyrimidine, N5-acyl-pyrimidine, N5-hydroxyalkyl-purine, and N6-thioalkyl-purine, and isoxazolyl. The heteroaromatic group may be optionally substituted as described above for aryl. The heterocyclic or heteroaromatic group may be optionally substituted with one or more substituents that are selected from halogen, haloalkyl, alkyl, alkoxy, hydroxy, carboxyl derivatives, amido, amino, alkylamino, dialkylamino. The heteroaromatic group may be partially or completely hydrogenated as desired. As a non-limiting example, dihydropyridine can be used in place of pyridine. The oxygen and nitrogen functional groups in the heterocyclic or heteroaryl group can be protected as necessary or desired. Suitable protecting groups are well known to those skilled in the art and include trimethylsilyl, dimethylhexylsilyl, t-butyl-dimethylsilyl, and t-butyl-diphenylsilyl, trifly or substituted triflyl, alkyl groups, acyl groups such as acetyl and propionyl, methanesulfonyl, and p-toluenylsulfonyl. These purine or pyrimidine bases, heteroaromatics and heterocycles may be substituted with alkyl groups or aromatic rings, linked through single or double bonds or fused to the heterocyclic ring system. The purine base, pyrimidine base, heteroaromatic base, or heterocycle may be attached to the sugar moiety through any available atom, including the ring nitrogen and the amule carbon (which produces a C-nucleoside). The abbreviations used throughout the description include ee: enantiomeric excess for the material (s) or product (p); c: conversion; and E: enantio-selectivity constant.

Detailed description of procedure steps REACTION SCHEME 9 a, R = -CH- (CH3) 2 b, R =, p-MeO-Ph- 7 a, 24% 6 a 5 a, 61% 7 h, 46% 6 b 5 h, 61% DMF NaN3 8a 9 a, 84% 10 93% from 9a 8b 9h, 88% 90% from 9b TOTAL PERFORMANCE OF 10 FROM: 5a, 11.4% 5b, 22.2% Initially, a 2, 2-dialkoxy-ethanol is esterified (1) with the corresponding acid chloride with quantitative yields. In the case of the iso-butyryl ester (2a), the cyclization to the corresponding dioxolane lactone (3a) is carried out with glycolic acid in the presence of a Lewis acid (BF3? T20) in 80% yield. However, under the same conditions, the p-methoxy-benzoate ester (2b) makes it possible to obtain the corresponding lactone with low yield and purity. Much better results are obtained in a two-step process in which the acetal is first hydrolyzed to the corresponding aldehyde (2c), which is then subjected to similar cyclization conditions, which makes it possible to obtain the corresponding lactone (3b) as a solid with 80% yield. The lactones are resolved by chiral chromatography. The selective reduction of chiral lactones (4a and 4b) with LiAlH (OtBu) 3 and subsequent treatment with acetic anhydride makes it possible to obtain the corresponding acetates (5a and 5b) with reasonable yields, after purification by flash column chromatography. The accumulation with 2,6-dichloropurine under Vobruggen conditions makes it possible to obtain crude mixtures of anomers: β of the corresponding nucleosides (6a and 6b). First the iso-butyrate ester is purified by column chromatography and then crystallized with MeOH to obtain the pure β-anomer (7a) in a yield of 24%. The nucleoside of the ester p-methoxy-benzoate does not require chromatography, and is purified by crystallization with MeOH to obtain the pure β-anomer (7b) in 46% yield. The chlorine atoms are replaced with amino groups in a two step process by treatment with sodium azide and then hydrogenation. The corresponding intermediates (9a and 9b) are isolated and fully described. The removal of the protective groups is achieved with n-butylamine in MeoH under reflux to obtain (-) - DAPD (10) with yield of 93% and 90% respectively. The total general yields from the corresponding chiral lactones (5a and 5b) are: 11.4% for iso-butyrate and 22.2% for p-methoxybenzoate.

EXAMPLES The melting points are determined in open glass capillaries using a Melt-Temp II apparatus with a digital thermometer. The 1 H NMR spectra are recorded at 400 MHz with a Varian XL-400 spectrometer. Evaporations are carried out at reduced pressure using a Buchi rotary evaporator at 40 ° C unless otherwise indicated. The solutions are dried with anhydrous Na2SO4. CCF is carried out on glass plates (0.25 mm) pre-coated with silica gel 60F254 (E. Merck, Darmstad). Flash column chromatography is carried out with silica gel 60 (230-400 mesh, E. Merck, Darmstad). The analysis of elements is carried out by Atlantic Microlab (Atlanta, GA). High resolution mass spectra (HRMS) are performed by Analytical Instrument Group Inc. (Raleigh, NC).

Gas chromatography analysis Samples are prepared by dissolving 1 mg of sample per 1 ml of THF, or another appropriate solvent. An injection volume of 1 μl is used. Samples are separated using a 30-m HP-5 capillary column (PH ME 5% interlaced siloxane), Hewlett Packard, part # 9091J-413. The inlet temperature is adjusted to 200 ° C, and the oven temperature as follows: 35 ° C for 1 minute, it increases up to 250 ° C at 12.5 ° C / minute, it is maintained at 250 ° C for 1.8 minutes . The temperature of the flame ionization detector is set to 250 ° C. The carrier gas is nitrogen adjusted to a nominal flow of 8.0 ml / minute.

HPLC Analysis Method A Column: Chiralpak AD-RH, 150 x 4.6 mm. Mobile phase: Methanol. Gradient: Isocratic Flow rate: 0.5 ml / minute. Running time: 30 minutes. Detection: UV at 280 nm.

Method B Column: Aquasil CIS, 150 x 4.6 mm. Mobile phase: solvent A: acetonitrile, solvent B: NHOac 50 mmole, 0.1% AcOH in water. Gradient: time: 0 minutes, A: 1%, B: 99%; time: 17 minutes, A: 50%, B: 50%, then isocratic. Flow rate: 1.0 ml / minutes. Running time: 30 minutes. Detection: UV at 290 nm.

Method C Column: Chiralpak AD, 250 x 4.6 mm. Mobile phase: Methanol. Gradient: Isocratic Flow rate: 0.8 ml / minute. Run time: 20 minutes. Detection: UV at 254 nm.

EXAMPLE 1 Isobutyric acid 2,2-dimethoxy-ethyl ester (2a) Ester 2, 2-diethoxy-ethyl 4-methoxy-benzoic acid (2b) To a well-stirred solution of 2,2-diethoxy-ethanol or 2,2-dimethoxy-ethanol (1, 100 mmol), DMAP (61 mg) , 0.5 mmol) and Et3N (16 mL, 11.64 g, 115 mmol) in EtOAc or tert-butyl methyl ether (50 mL) at 0 ° C is slowly added the corresponding acid chloride (105 mmol). After stirring for 16 hours at room temperature, the reaction mixture is diluted with EtOAc (50 ml), and successively washed with: (c) NaHCO 3 (2 x 100 ml), brine (2 x 100 ml), it is dried, filtered and evaporated to obtain: Isobutyric acid 2, 2-dimethoxy-ethyl ester (2a, 99%) as a yellow liquid that is used in the next step without any additional purification. GC (R t = 5.24 min., 98%). XH NMR (CDC13) d: 4.57 (1H, t, J = 5.2, (MeO) 2CHCH2-), 4.11 (2H, d, J = 5.2, (MeO) 2CHCH2-), 3.40 (6H, s, CH30-) , 2.60 (1H,, OCOCl (CH3) 2), 1.54 (6H, d, J = 6.8, OCOCH (Cff3) 2). Ester 2, 2-diethoxy-ethyl 4-methoxy-benzoic acid (2b, 100%) as a syrup which is used in the next step without any further purification. GC (Rt = 13. 7 min, 99%). XH NMR (CDCl 3) d: 7.98 (2H, d, J = 9.0, ArH), 6.89 (2H, d, J = 9.0, ArH), 4.79 (1H, t, J = 5.6, (EtO) 2CHCH2-), 4.28 (2H, d, J = 5.6, (EtO) 2CHCl3-), 3.82 (3H, s, CH3O-), 3.73 (2H, m, CH3CH2O-), 3.59 (2H, m, CH3Cif20-), 1.22 (6H , t, J = 6.9, Cff3CH20-).

EXAMPLE 2 Ester 4-oxo- [1,3] -dioxolan-2-yl-methyl isobutyric acid (3a) To a well-stirred solution of the corresponding acetal (2a, 30 mmol) and o-hydroxy-acetic acid (3.42 g, 45 mmol) in acetonitrile (30 ml) at 0 ° C is added slowly BF3 • Et02 (6.38 g, 5.70 ml , 45 mmol). The solution is left at room temperature overnight with stirring. The solution is partitioned between EtOAc (150 ml) and (c) NaHCO 3 (150 ml). The organic solution is washed successively with (c) NaHCO3 (150 ml), brine (2 x 150 ml), dried, filtered and evaporated to obtain: Ester 4-oxo- [1, 3] -dioxolan- 2-yl-methyl isobutyric acid (3a, 80%) as a colorless syrup. GC (R t = 7.89 minutes, 95%). XH NMR (CDC13) d: 5.83 (1H, s, H-2), 4.35-4.20 (4H, m, H-5, H-5 'and -Cff2OCO-), 2.62 (1H, m, (CH3) 2COCOO -), 1.19 (6H, d, J = 7.0, (Ci? 3) 2CHCOO-). Mass calculated for C8H? 305 (M + 1) +: 189.0763. Found: (H.R. F.A.B.M. S.): 189.0763.

EXAMPLE 3 4-Oxo- [1,3] -dioxolan-2-yl-methyl ester of 4-methoxybenzoic acid (3b) A solution of 4-methoxybenzoic acid 2,2-diethoxyethyl ester (2b, 7.5) is treated g, 28 mmol) in C12CH2 (75 ml) with TFA (16.7 g, 11.3 ml, 140 mmol) and water (7.5 g, 7.5 ml, 28 mmol). The homogeneous solution is stirred for 3.5 hours at room temperature until GC analysis shows complete reaction. The solution is concentrated in vacuo at 40 ° C and then diluted with hexane and concentrated in vacuo several times to remove traces of TFA. The product, 2-oxo-ethyl ester of 4-methoxybenzoic acid (2c, 5.9 g, 28 mmol, 100%) is isolated as a white amorphous solid and used in the next step without further purification. GC (R t = 11. 0 min, 95%). XH NMR (CDC13) d: 9.72 (1H, s, HCO-), 8.06 (2H, d, J = 8.8, ArH), 6.95 (2H, d, J = 8.8, ArH), 4.87 (2H, s, HCOCtf20 -), 3.87 (3H, s, OCH3). To a well-stirred solution of the crude aldehyde (2c, 5.9 g, 28 mmol) and a-hydroxy-acetic acid (5.2 g, 68 mmol) in DME (100 ml) at 0 ° C is slowly added BF3-Et02 (12.3 g, 11.0 ml, 85 mmol). The solution is left at room temperature overnight with stirring. The solution is partitioned between EtOAc (150 ml) and (c) NaHCO 3 (150 ml). The organic solution is washed successively with (c) NaHCO3 (150 ml), brine (2 x 150 ml), dried, filtered and evaporated to obtain a syrup. The syrup is treated with DME (15 ml) and a solid precipitates. After stirring for 30 minutes, the solid is filtered to obtain the 4-oxo- [1, 3] -dioxolan-2-yl-methyl ester of 4-methoxybenzoic acid. (3b, 5.7 g, 23 mmol, 80%) as a white granulated solid. GC (R t = 14.7 min, 99%); p.f .: 61-63 ° C. XH NMR (CDC13) d: 7.97 (2H, d, J = 9.2, ArH), 6.91 (2H, d, J = 9.2, ArH), 5.95 (1H, t, J = 3.0, H-2), 4.57 (1H, dd, J = 3.0 and J = 12.6, -CH2OCO-), 4.50 (1H, dd, J = 3. 0 and J = 12.6, -CH2OCO-), 4.41 (1H, d, J = 15.0, H-5), 4.31 (1H, d, J = 15.0, H-5), 3.87 (3H, s, OCH3). Calculated for C? 2H1206: C, 57. 14; H, 4 80 Found: C, 57.40; H, 4.93.

EXAMPLE 4 Ester 4-oxo- [1, 3] -dioxolan-2 (R) -yl-methyl isobutyric acid (4a) The 4-oxo- [1, 3] -dioxolan-2-yl-methyl ester of racemic isobutyric acid (3a) is resolved by chiral chromatography13 to obtain two fractions corresponding to each of the enantiomers. Both fractions are colorless syrups. The first fraction corresponds to the R (4a) enantiomer, 4-oxo- [l, 3] -dioxolan-2 (R) -yl-methyl ester of isobutyric acid); HPLC (Method A, R t = 7.80 minutes, 100%); [] D20 = 20.20 ° (c 0.25, MeOH). The second fraction corresponds to the S-enantiomer (4-oxo- [1, 3] -dioxolan-2 (S) -yl-methyl isobutyric acid ester); HPLC (Method A, Rt = 9.30 minutes, 100%); [a.D20 = -4.40 ° (c 0.25, MeOH).

EXAMPLE 5 4-Oxo- [1,3] -dioxolan-2 (R) -yl-methyl ester of 4-methoxybenzoic acid (4b) The 4-oxo- [1, 3] -dioxolan-2-yl-methyl ester of racemic 4-methoxybenzoic acid (3b) is resolved by chiral chromatography7 to obtain two fractions corresponding to each of the enantiomers. Both fractions are white solid. The first fraction corresponds to the R (4b) enantiomer, 4-oxo- [l, 3] -dioxolan-2 (R) -yl-methyl ester of 4-methoxybenzoic acid); mp .: 76-78 ° C; HPLC (Method A, Rt = 13.59 minutes, 99%); [a] D20 = 12.20 ° (c 0.25, MeOH). The second fraction corresponds to the S-enantiomer (4-oxo- [1, 3] -dioxolan-2 (S) -yl-methyl ester of 4-methoxybenzoic acid); mp .: 76-78 ° C; HPLC (Method A, R t = 20.76 minutes, 99%); [α] D20 = -13.50 ° (c 0.25, MeOH).

EXAMPLE 6 Ester 4-acetoxy- [1,3] -dioxolan-2 (R) -yl-methyl isobutyric acid (5a) Ester 4-acetoxy- [1,3] -dioxolan-2 (R) -yl-methyl of 4-methoxybenzoic acid (5b) To a well stirred solution of the corresponding lactone (4a or 4b, 15 mmol) in dry THF (45 ml) at 10 ° C, a 1.0 M solution of LiAlH (OtBu) 3 in THF (19.5 ml, 19.5 mmol) is slowly added. ) in a period of 20 minutes, maintaining the temperature between -15 ° C and -10 ° C. The reaction is followed by GC and stirred for 30 minutes at room temperature (complete disappearance of the starting material). The solution is cooled again to -10 ° C and DMAP (0.92 g, 7.50 mmol) is added in one portion followed by the dropwise addition of Ac20 (15.3 g, 14.20 ml, 150 mmol). The reaction is further stirred for 1 hour at -15 ° C, and then overnight at room temperature. The solution is cooled to -15 ° C and quenched with MeOH (40 ml). After stirring for 20 minutes at room temperature, the reaction is concentrated in vacuo to a red colored syrup which is purified by flash column chromatography (250 g of silica, hexane: EtOAc 3: 1) to obtain: Ester 4 - Isobutyric acid (1, 3) -dioxolan-2 (R) -yl-methyl acid (5a, 61%) as a yellow syrup. The 1 H NMR analysis (CDC13) shows a nearly 1: 1 mixture of the anomers, d: 6.40 (d, J = 3.8) and 6.37 (d, J = 3.8) corresponding to H-4 and H-4β; 5.41 (t, J = 3.7) and 5.32 (t, J = 3.7) corresponding to H-2a: and H-2β. 4-methoxy- [1, 3] -dioxolan-2 (R) -yl-methyl 4-methoxybenzoic acid ester (5b, 61%) as a yellow jaabe. The analysis of 1H NMR (CDCI3) shows an almost 1: 1 mixture of the anomers, d: 6.44 (dd, J = 2.1 and J = 4.2) and 6.37 (d, J = 4.0) corresponding to H-4a and H -4ß; 5.54 (t, J = 3.6) and 5.45 (t, .J = 4.0) corresponding to H-2a and H-2β.

EXAMPLE 7 Ester 4 (R) - (2,6-dichloro-purin-9-yl) - [1,3] -dioxolan-2 (R) -ylmethyl of isobutyric acid (7a) Ester (R) - ( 2, 6-dichloro-purin-9-yl) - [1, 3] -dioxolan-2 (R) -methyl of 4-methoxybenzoic acid 4 (7b) A suspension of 2,6-dichloropurine (1.27 g, 6. 73 mmol), ammonium sulfate (38 mg, 0.29 mmol) and 1, 1, 1, 3, 3, 3-hexamethyldisilazane (8.45 g, 11. 04 ml, 52.3 mmol) is heated at reflux for 2.5 hours. The resulting solution is cooled to room temperature whereupon a thick solid precipitates. The solids are redissolved by addition of dry dichloromethane (14.9 ml). The solution is then cooled to -10 ° C, and a corresponding acetate solution is added slowly (5a or 5b, 8.6 mmol) in dry dichloromethane (10 ml) in a period of 20 minutes, maintaining the temperature between -10 ° C and -5 ° C. Then, TMSOTf (2.5 g, 2.08 ml, 10.4 mmol) is added slowly over a period of 20 minutes. The reaction is left overnight with stirring at room temperature. The solution is diluted with dichloromethane (120 ml) and quenched with water (150 ml). The organic layer is separated and washed successively with water (150 ml), (c) NaHCO3 (2 x 150 ml), water (2 x 150 ml), dried, filtered and evaporated to a syrup (6a). raw) or a yellow solid (6b crude). The crude product 6a is additionally purified by column chromatography (hexane: AcOEt 4: 1) to obtain 6a as a 1.2: 1 mixture of the a: ß anomers, in accordance with the "" "H NMR analysis. crystallize slowly with MeOH (10 ml) to obtain the ester 4 (R) - (2,6-dichloro-purin-9-yl) - [1, 3] -dioxolan-2 (R) -yl-methyl acid isobutyric (7a, 24%) as a white solid and characterized as the ß-anomer; pf: 145-147 ° C; [a] D20 = -47.67 ° (c 0.25, MeOH) XH NMR (CDC13) d : 8.52 (1H, s, H-8), 6.55 (1H, d, J = 4.9, H-4), 5.34 (1H, t, J = 5.7, H-2), 4.58-4.30 (4H, m, -CH2COO-, H-5 and H-5 '), 2.61 (1H, m, (CH3) 2Ci? COO-), 1.19 (3H, d, J = 6.8, (CH3) 2CHC00-), 1.14 (3H, d, J = 6.8, (CJ? 3) 2CHCOO-) Calculated for C? 3H? 4Cl2N404: C, 43.23; H, 3.91; N, 15.51, Found: C, 43.39; H, 3.91; N, 15. 58 The crude product 6b is first washed with hot hexane to obtain the ester 4- (2,6-dichloro-purin-9-yl) - [1,3] -dioxolan-2 (R) 4-methoxy-benzoic acid-methyl-methyl (6b) as a yellow solid. The aH NMR analysis indicates a 2: 1 mixture of the ocß anomers. This mixture is slowly crystallized with MeOH (100 ml) to obtain the ester 4 (R) - (2,6-dichloro-purin-9-yl) - [1,3] -dioxolan-2 (R) -yl-methyl of 4-methoxybenzoic acid (7b, 46%) as a yellow solid and characterized as the β-anomer; p.f .: 154-156 ° C; HPLC (Method B, Rt = 20.73 minutes); [α] D20 = -53.80 ° (c 0.25, MeOH). XH NMR (C13CD) d: 8.41 (lH, s, H-8), 7.91 (2H, d, J = 8.5, ArH), 6.92 (2H, d, J = 8.5, ArH), 6.53 (1H, d, J = 4.8, H-4), 5.44 (1H, bs, H-2), 4.70-4.30 (4H, m, H-5, H-5 'and -CH2OCO-), 3.85 (3H, s, OCH3) . Mass calculated for C? 7H15Cl2N405 (M + 1) +: 425.0419. Found: (H.R. F.A.B.M. S.): 425.0420.

EXAMPLE 8 Ester 4 (R) - (2,6-diazido-purin-9-yl) - [1,3] -dioxolan-2 (R) -yl-methyl-isobutyl acid co (8a) Ester 4 (R) - (2, 6-diazido-purin-9-yl) - [1,3] -dioxolan-2 (R) -methyl-4-methoxybenzoic acid (8b) To a well-stirred solution of the corresponding dichloropurine nucleoside (7a or 7b, 2.9 mmoles) in dry DMF (13.5 ml) is added NaN3 (390 mg, 6.0 mmoles). The reaction mixture is left at room temperature with stirring for 4 hours. The mixture is filtered through celite to obtain a solution of: Ester 4 (R) - (2,6-diazido-purin-9-yl) - [1, 3] -dioxolan-2 (R) -yl-methyl of isobutyric acid (8a) or ester 4 (R) - (2,6-diazido-purin-9-yl) - [1, 3] -dioxolan-2 (R) -yl-methyl of 4-methoxybenzoic acid (8b) ) in DMF (70 ml) which is used in the next step without any further purification.

EXAMPLE 9 Ester 4 (R) - (2, 6-diamino-purin-9-yl) - [1,3] -dioxolan-2 (R) -ylmethyl of isobutyric acid (9a) Ester 4 (R) - (2,6-diamino-purin-9) -il) - [1,3] -dioxolan-2 (R) -methyl-4-methoxybenzoic acid (9b) The solution coming from the previous reaction (8a or 8b) is hydrogenated (Parr apparatus, 3.515 kg / cm2) at room temperature, in the presence of 10% Pd / C (200 mg) overnight. The mixture is filtered through celite and the resulting filter cake is washed with additional DMF. In the case of 8a, the solution is concentrated to dryness and purified by column chromatography (Cl 3 CH: MeOH 9: 1) to obtain a white solid which is crystallized with iso-propanol to obtain ester 4 (R) - (2,6-diamino-purin-9-yl) - [1,3] - dioxolan-2 (R) -yl-methyl of isobutyric acid (9a, 84% after correction for the presence of a molecule of 2-propanol which crystallizes together) as a white solid; mp .: 143-145 ° C; [] D20 = -56.45 ° (c 0.25, MeOH). t NMR (CDC13) d: 7.84 (1H, s, H-8), 6.33 (1H, dd, J = 1 and J = 5.1, H-4), 5.29 (3H, t, J = 3.2, H-2 and NH2), 4.70 (2H, bs, NH2), 4.50 (1H, dd, J = 1 and J = 9.0, H-5), 4.33 (2H, d, J = 3.2, -CH2COO-), 4.23 (1H, dd, J = 5.1 and J = 9.0, H-5 '), 2.60 (1H, m, ( CH3) 2CHCOO ~), 1.18 (3H, d, J = 6.2, (Cií3) 2CHCOO-), 1.14 (3H, d, J = 7.2, (CH3) 2CHCOO-). Calculated for C? 3H? 8N604 • 2-propanol: C, 50.25; H, 6.85; N, 21.98. Found: C, 50.16; H, 6.84; N, 21.92. In the case of 8b, the solution is concentrated to a final volume of 15 ml under vacuum at 60 ° C. The solution is diluted with water (150 ml) and after a few minutes a solid precipitates. The product is filtered, washed with water, dried overnight at 50 ° C under vacuum a to obtain the ester 4 (R) - (2,6-diamino-purin-9-yl) - [1,3] 4-methoxybenzoic acid (2-R) -yl-methyl-dioxolan (9b, 88%) as an amorphous solid; HPLC (Method B, Rt = 15.41 minutes); [a] D2 ° = -95.75 ° (c 0.25, MeOH). XH NMR (DMSO-de) d: 7.82 (2H, d, J = 8.8, ArH), 7.80 (1H, s, H-8), 7.02 (2H, d, J = 8.8, ArH), 6.77 (2H, bs, NH2), 6.23 (1H, dd, J = 5.5 and J = 1.6, H-4), 5.85 (2H, bs, NH2), 5.36 (1H, t, J = 3.3, H-2), 4.65 ( 1H, dd, J = 9.4 and J = 1.6, H-5), 4.46 (2H, d, J = 3.4, -CH2OCO-), 4.26 (1H, dd, J = 9.4 and J = 5.5, H-5 ' ), 3.84 (3H, s, OCH3). Mass calculated for C? 7H? G 6? 5: 387.1417. Found (H. R. F.A.B.M. S.): 387.1417.

EXAMPLE 10 (R) - [- (2,6-diamino-purin-9-yl) - [1,3] -dioxolan-2 (R) -yl] -methanol, [10, (-) - DAPD] a from 9a A solution of 4 (R) - (2,6-diamino-purin-9-yl) - [1, 3] -dioxolan-2 (R) -yl-methyl ester of isobutyric acid (9a, 100 mg, 0.31 mmol) ) and n-butylamine (0.45 g, 0.62 ml, 6.2 mmol) in MeOH (10 ml) is heated at reflux for 4 hours. The reaction is cooled to room temperature and concentrated in vacuo to obtain a solid which is triturated with tert-butyl methyl ether and filtered to obtain a solid which is crystallized with EtOH: water (1: 4.5 ml) to obtain (- ) -DAPD (10.75 mg, 0.29 mmol, 93%) as a white solid; p.f .: 237-239 ° C (p.f. of literature 2: 236-237 ° C); HPLC (Method C, Rt = 8.7 minutes, an authentic sample of (-) - DAPD shows Rt = 8.7 minutes and a sample of (+) - DAPD shows Rt = 6.0 minutes). DAPD; XH NMR (DMSO-d6) d: 7.80 (1H, s, H-8), 6.74 (2H, bs, NH2), 6.20 (1H, d, J = 5.5, H-4), 5.84 (2H, bs, NH2), 5.16 (1H, t, J = 6.3, OH), 5.03 (1H, t, J = 2.9, H-2), 4.42 (1H, d, J = 9.7, H-5), 4.18 (1H, dd, J = 9.7 and J = . 5, H-5 '), 3.58 (2H, dd, J = 6.3 and J = 2.9, -C # 2OH). Calculated for C9H? 2N603: C, 42. 86; H, 4 80; N, 33. 32. Found: C, 42.88; H, 4.79; N, 33. 32.

EXAMPLE 11 4 (R) - [- (2,6-diamino-purin-9-yl) - [1,3] -dioxolan-2 (R) -yl] -methanol, [10, (-) -DAPD] from 9b A suspension of 4-methoxybenzoic acid (4, 6-diamino-purin-9-yl) - [1, 3] -dioxolan-2 (R) -yl-methyl ester (9b, 1.0 g, 2.6 mmol) and n-butylamine (3.7 g, 5.0 ml, 50 mmol) in MeOH (25 ml) is heated at reflux for 4 hours. The reaction is concentrated in vacuo to obtain a yellow solid which is suspended in C13CH at room temperature and filtered, to obtain (-) - DAPD (10.590 mg, 2.3 mmol, 90%) as a white solid . The physical properties are identical to those of the (-) - DAPD sample obtained above.

EXAMPLE 12 Determination of the enantio-seletivity constant The enantio-selectivity of a simple hydrolysis reaction can be characterized by the E value (C. S Chen, C. J. Sih, Y. Fujimoto and G. Girdaukus, J. Am. Chem. Soc., 1982, 104, 7294). This represents numerically the quotient of the catalyst specificity constants for the two enantiomers. The E value allows direct comparison of the selectivity of the catalyst, even if the reactions have proceeded to different conversions. To calculate the value E, the degree of conversion and the optical purity of the remaining starting material and / or the product of the hydrolysis are needed. If both the enantiomeric excess of the starting material (ees) and the enantiomeric excess of the product (eep) are known, the conversion (c) can be calculated using equation 1. If only ees or eep is known, the " c "determined at the end of the hydrolysis to calculate E (equation 2).

Equation 1 c = ee "ees + eep Equation 2 ln [(l - c) (l - ees)] = ln [l - c (l + ee,)] ln ln [(l - c) (l + ee,)] ln [l - c (l - eep )) Abbreviations ee = enantiomeric excess for the starting material (s) or product (p) c = Conversion E = enantio-selectivity constant EXAMPLE 13 Compound 1 Analytical development for compound 1 A chiral test is required for compound 1. A baseline separation is achieved by chiral GC using the Chirasil DEX CB column. See figure IA.

GC conditions Column: Chirasil DEX CB Dimensions: 25 m x 0.25 mm Temperature program: 140 ° C for 10 minutes then up to 200 ° C at 15 ° C / min Carrier gas: Helium at 1,406 kg / cm2 FID detection at 200 ° C Retention times: 1 - 7.46 min (R) -enantiomer 1 - 7.70 min (S) -enantiomer Enzymatic evaluation for compound 1 Lipases To each scintillation flask is added 50 μl of compound 1, MTBE (5 ml) and 50 mM KH2P04, pH 7 (5 ml), followed by 25 mg of enzyme. The bottles are shaken in an incubator set at 30 ° C. Aliquots are periodically removed and analyzed by TLC (50% EtOAc / heptane) and chiral GC.

Proteases and esterases In the manner described above, except that the reaction solvent is 5 ml of 50 mM KH2P0, pH 7 or 8 without MTBE. The acid protease reactions are carried out at pH 3 using 0.1 M lactic acid solution [pepsin, acid protease A (Newlase A), and acid protease II (Newlase II)]. In some cases 25 mg, 50% by weight of the enzyme produce extremely rapid reactions and therefore 100% conversion is reached before meaningful tests can be performed. In these cases, the reactions are carried out again with the appropriate amount of enzyme to obtain a reasonable reaction rate (normally 5% by weight). Enzymes that require smaller loads are Chirazyme-L2, -El and -E2, all CLEC, choline esterase, Candida esterase (Altus), PPL, PS Lipase and AK Lipase. Two sets of conditions are used for the evaluation of racemic compound 1, depending on whether the enzyme used is a lipase or a protease / esterase. For lipase-catalyzed hydrolysis, the reaction solvent is a 1: 1 mixture of MTBE: 50 mM potassium phosphate buffer, pH 7. For hydrolysis catalyzed by protease and esterase, the reactions are carried out in phosphate buffer of 50 mM potassium, pH 7 or 8 (or pH 3 for acid proteases) without immiscible organic co-solvent. The reactions are carried out in an agitator bath at 30 ° C and followed by TLC, and the values of e is determined by GC analysis (Chirasil Dex-CB column). The alcohol product from the hydrolysis of the butyrate ester appears to be unstable because several new spots occur in TLC during the reactions. Therefore, without chiral test for the possible product, approximate conversions are calculated from the CCF analysis. The results from the evaluations are shown in the following tables. Table 1 shows the enzymes that selectively produce peak 1 in the chiral butyrate test, while Table 2 shows the enzymes that selectively produce peak 2 in the chiral butyrate test. It should be mentioned that there seems to be a very large number of enzymes that produce non-stereo-selective hydrolysis of compound 1, which indicates that the substrate is unstable under the reaction conditions. When a control reaction that does not contain an enzyme is carried out, the hydrolysis continues to occur. Further investigation of the stability of the substrate is described in the present invention.

TABLE 1 Enzymes that selectively produce peak 1 in the butyrate ee test TABLE 1 (cont.) TABLE 2 Enzymes that selectively produce peak 2 in the butyrate ee test a-chymotrypsin does not produce reaction with compound 1 The following enzymes produce non-selective reactions with compound 1 (<10% of ees): Lipase AY, Lipase A "Amano" 6, Lipase A "Amano" 12, Lipase N conc, Lipase AP6, CCL of Sigma, Lipase of wheat germ, Chirazyme-L5, ChiroCLEC-CR, Protease M, Peptidase R, Acid Protease A, Acid Protease II, Acid Protease DS, Protease A-DS, Protease N, Protease A2G, Protease NL, Protease DS, Protease S, Protease P "Amano" 6, Prozyme 6, Proleather, Bromelain -F, Papain W-40 (Amano), Papain (Sigma), Protease X, Protease XXXI, Savinase, Esperasa, Pepsin, C iroCLEC-BL, Ketoprofen Esterase and Chirazyme-E2.

EXAMPLE 14 Resolution scaling by PPL of compound 1 In order to determine which is the (R) isomer, an amount of compound 1 solved is solved using the most promising enzyme from the evaluation indicated above, and is carried through the synthesis sequence to an intermediate in which knows the order of elution of the isomer by chiral test. The most promising enzyme from the evaluation is PPL (porcine pancreatic lipase, Sigma), therefore this reaction is scaled up to 50 g of Dlimentation of racemic substrate (reaction scheme 10).

REACTION SCHEME 10 Large-scale resolution of compound 1 with PPL 68% conversion through base consumption The minimum optimization of the reaction conditions is carried out, in addition to increasing the substrate concentration to 50 g / l (a reaction on 5 g of substrate is successfully carried out as a test). The 50 g reaction is started with 5 wt% PPL at 30 ° C and 3 M NaOH which is added as the reaction proceeds in an attempt to maintain the pH at 7. However, the reaction is extremely rapid and after minutes is cooled to 15 ° C and the pH is maintained at pH 6. In particular, they are added to a reaction vessel of 2 liters with steam jacket at 30 ° C, MTBE (500 ml), 50 mM KH2P04, pH 7 (500 ml) and racemic compound 1 (49.75 g, 0.265 moles, TP-0257/98 / D). To the stirred mixture (pH 7) PPL (2.5 g, 5% by weight) is added, then added 3M NaOH to maintain the pH at pH 6. Due to the speed of the reaction, after 25 minutes the vessel is cooled to 15 ° C and the reaction is run at this temperature. The ees are measured at time intervals by removing aliquots of MTBE, diluting with more MTBE, drying (MgSO4), and analyzing by chiral GC. After 1 hour 10 minutes the ees reaches 98% and the reaction is subjected to treatment. The mixture is filtered through celite and the celite is washed with MTBE (200 ml). The mixture is allowed to separate and the organic layer is preserved. The aqueous layer is then further extracted with MTBE (500 ml). The MTBE extracts are combined, dried (MgSO 4) and evaporated in vacuo to obtain 22 g of a crude yellow oil. The crude product is purified by flash column chromatography with silica (20% EtOAc: 80% heptane) to obtain 9.55 g of the (S) -compound 1 as a very pale yellow oil, 98% ee, purity of 98% by GC-MS. An additional 1.5 g of less pure material is also obtained. Combined performance is 22%. GC-MS (CPSÍ18 CB / MS, 30 x 0.25 mm, 60 ° C for 5 minutes, 10 ° C / minute up to 300 ° C) 13.98 minutes, M = 173 (M + -CH3), 95% pure by peak area . aH NMR (200 MHz, CDC13) d 5.85 (t, 1H, CH-O), 4.35 (m, 4H, 2 x CH20), 2.35 (t, 2H, CH2), 1.7 (sextet, 2H, CH2), 0.95 (t, 3H, CH3). The ees is measured at time intervals (peak 2 in the ee test).

TABLE 3 Resolution by PPL of compound 1 The reaction is subjected to treatment after 1 hour to 10 minutes and the isolated resolved compound 1 is purified by column chromatography with silica gel. This produces 9.55 g, 95% pure, 98% ee of the ester, together with 1.5 g of less pure ester (22% of total yield). The impurity in the main batch of ester (9.55 g) is butyric acid. This material is carried through the entire synthesis sequence to an intermediate in which the order of elution of the isomer is known by chiral test, and determined to be the (S) isomer of compound 1. To obtain the isomer (R ), an enzyme that selectively produces peak 1 in the chiral test of compound 1 is needed.

EXAMPLE 15 Optimization of resolution with commercial enzyme to produce the (R) isomer of compound 1 As can be seen from table 1, the initial selection shows that immobilized lipase SAWA, lipase M, lipase PS and lipase Fl Biocon. are potential candidates to efficiently produce the isomer (R) of the compound 1. The resolutions are scaled to determine their selectivities (the Fl Biocon lipase can no longer be obtained commercially). The enzyme is added to 2-5 g of ester in 15-20 ml each of solvent and buffer (KH2P04 0.2 N). Sodium hydroxide solution is added to control the pH. The treatment procedure is to filter (celite), extract (MTBE), wash with saturated aqueous sodium bicarbonate, dry with (MgSO) and concentrate. The analysis is carried out by chiral GC (Chirasil Dex CB). The variables studied are enzyme, solvent, pH and temperature (see table 4). Initial tests with PS lipase, lipase M and immobilized Pseudomonas lipase SAWA in phosphate buffer at pH 7 produce the remaining substrate with enantiomeric excesses of 90%, but recovery of the (R) isomer of compound 1 is low (< 5%) .

The stability of the butyrate is investigated at pH 7 and pH 6 under standard reaction conditions but without the enzyme present (entries 20 and 21). At pH 7 a significant amount of base is consumed to maintain the pH and after treatment and isolation only 50% of (1) is recovered after 3.5 hours. The stability is significantly better at pH 6. Possibly, the mode of dissociation is opening of the lactone-acetal to obtain an aldehyde and glycolic acid (reaction scheme 11).

REACTION SCHEME 11 Possible route of decomposition of compound 1 TABLE 4 Optimization of the resolution of compound 1 15 (i) enantio-selectivity constant - see example 12 Two other controls show that the starting ester with 98% ee is not racemized by the reaction conditions at pH 6 or pH 7. An additional test reaction shows that after 24 hours at pH 6, 100 g / l of substrate in a 2: 1 mixture of toluene: 1 M buffer solution and 0 ° C, more than 90% yield of the starting material is recovered without loss of enantiomeric excess. Therefore, racemization and stability of the substrate under these reaction conditions are not a problem. Considering these results, all the additional enzyme reactions are carried out at pH 6. The best results of the study are shown in entries 7, 8, 9 and 13. The most promising enzyme resolution is with PS lipase in toluene / buffer solution pH 6 which produces an E value of 4.3. This could give a yield of 17% to 95% of us. The resolution with PS lipase is further investigated in the examples in the present invention. The variables studied for optimization are concentration, solvent volume ratio: regulatory solution, enzyme load, additives and temperature. Optimal conditions are determined as 0 ° C, 100 g / l substrate in toluene: potassium phosphate buffer solution 1: 1 (pH 6) which yields 95% ee, 22% yield of the (R) isomer of the compound 1. At 200 g / l of substrate concentration, 95% ee of the (R) isomer of compound 1 is obtained with 17% yield.

EXAMPLE 16 Evaluation of microbial enzyme From an evaluation of around 200 strains (using the novel 96-well plate method), 7 positives are identified which produce the (R) isomer of compound 1 (peak 1). These are classified as positive insofar as they hydrolyze the ester and produce more than 10% ee of the remaining substrate, thus having the potential to generate an ester with high ee. The evaluation is carried out in Na2HP0 / 0.1 M NaH2P0, pH 6.0 with 20 g / l of the ester at 25 ° C for approximately 16 hours. The results are presented in figures 5A and 5B. The seven positive strains (peak 1) from the primary evaluation are grown in flasks with TSB (tryptone-soy broth) and the cell pastes are harvested. These are resuspended at 10% w / v in Na2HP0 / 0.1 M NaH2P04, pH 6.0 + 20 g / l of the ester and stirred in flasks for scintillation (at 1.0 ml per bottle) for up to 92 hours at 25 ° C. The results are shown in table 5.

TABLE 5 Further investigation of positive microbial strains The results from the control show that the ester is not stable for 24 hours at 25 ° C. There is a reduction of 975 in the size of the peak area between the peak areas at 17 and 92 hours. This clearly explains why all the peak areas of the samples show a dramatic drop. Of the strains that are re-evaluated, CMC 3606 (Acinetobacter junii) and CMC 3419 (Acinetobacter) exhibit adequate dwarf-selectivity (80% ee for peak 1). These two strains are recultivated [CMC 3606 in flasks (because this is originally an unidentified bacteria) and CMC 3419 in fermentation vessels] ready for further evaluation. For CMC 3322 (P. alcaligenes) there is no detectable ester after 17 hours. This can be due to a shift in the pH (with lysis of the cells) or to an elevated esterase activity.

EXAMPLE 17 Compound 3 Analytical development for compound 3 A chiral GC test is developed to separate the four possible diastereoisomers of compound 3. The details of the test conditions are indicated below.

GC conditions: Column: Chiraldex GTA Dimensions: 30 x 0.25 mm Programming of 100 ° C for 20 minutes after temperature: up to 160 ° C at 2 ° C / min. Carrier gas: Helium @ 0.9842 kg / cm2 Detection: FID @ 200 ° C Retention times: 1-41.74 minutes 2-41.96 minutes 3-43.07 minutes 4-43.82 minutes A chiral GC-MS analysis of the starting ester produces 2 peaks at 15.98 and 16.27 minutes (diastereoisomers) in a ratio of 1: 1.4 respectively. See figure IB. The comparison of the four peaks of the GC ee / chiral test and the GC-MS peaks indicate that peaks 1 and 2 are enantiomers and peaks 3 and 4 are enantiomers in the ee / de test.

Enzyme evaluation, compound 3 Lipases To each scintillation flask, 50 μl of the compound 3 (TP-0259/98 / A), MTBE (5 ml) and 50 mM KH2P04, pH 7 (5 ml), followed by 20 mg of enzyme. The flasks are shaken in an incubator set at 30 ° C, aliquoting periodically and these are analyzed by TLC (50% EtOAc / heptane) and chiral GC.

Proteases and esterases In the manner indicated above, except that the reaction solvent is 5 ml, 50 mM KH2P04, pH 7 or 8 without MTBE. The acid protease reactions are carried out at pH 3 using 0.1 M lactic acid solution [pepsin, protease acid A (Newlase A), and protease II acid (Newlase II)] • For some enzymes known to be very active for these substrates, the reactions are carried out with an appropriate amount of enzyme to obtain a reasonable reaction rate, usually 10-15% by weight. The enzymes that require these reduced charges are Chirazyme-L2, Chirazyme-El and Chirazyme-E2, all CLEC, choline esterase and esterase of Candida (Altus). The racemic compound 3 is evaluated for enzymatic hydrolysis with commercially available lipases, proteases and esterases. All lipase reactions are carried out in the presence of 50 μl of the compound 3.5 ml of MTBE, 5 ml of pH 7 phosphate buffer and 50% by weight of the enzyme (except Chirazyme-El, Chirazyme-E2, Chirazyme- L2, esterase of Candida, hill esterasA and CLEC, 10% by weight). Reactions with esterase and protease are carried out without MTBE being present. The reactions are monitored by CCF and chiral GC. The stereochemistry in the acetate center is not so important because it is subsequently established in the synthesis of active DAPD in bulk. It would be desirable, therefore, to resolve with respect to the butyrate center. However, initially it is unknown which peak corresponds to which isomer. The control reactions, which do not contain an enzyme, show that the substrate is stable under the reaction conditions. The results from the evaluation regarding resolution of compound 3 are shown in tables 6-11.

TABLE 6 Enzyme evaluation of compound 3 - selective enzymes for peaks 1/3 TABLE 6 (cont.) These results show that peptidase R, protease M and protease A acid (Newlase A) have excellent selectivity for peaks 1 and 3; while protease B, Chirazyme-L2, and protease A have reasonable selectivity for peaks 1 and 3.

TABLE 7 Evaluation of enzymes for compound 3 - selective enzymes for peaks 1/4 Therefore PLE and Chirazyme-El are reasonably selective for peaks 1 and 4.

TABLE 8 Evaluation of Enzymes for Compound 3 - Selective Enzymes for Peak 1 These results show that papain has very adequate selectivity for peak 1.

TABLE 9 Evaluation of enzymes for compound 3 - selective enzymes for 2/3 peaks TABLE 9 (cont.) These results show that Lipase AK, Lipase MY and Lipase AY, are reasonably selective for peaks 2 and 3.

TABLE 10 Evaluation of Enzymes for Compound 3 - Selective Enzymes for Peak 2 Therefore, no enzyme that is selective for peaks 2 and 4 produces particularly high enantiomeric excesses.

TABLE 11 Evaluation of Enzymes for Compound 3 - Selective Enzymes for Peak 3 These results show that PS lipase is reasonably selective for peak 2. No reaction is observed with a-chymotrypsin, Lipase GC, Lipase PGE, Lipase N conc, Lipase G, Lipase R, Lipasa M, Lipasa A "Amano" 12, Lipasa AU Miles 1988, Lipasa Fl Biocon., Wheat germ lipase, papain W-40, protease S, protease X and pepsin. The enzymes that produce non-selective hydrolysis are protease II acid (Newlase II), Bromelain F, Alcalase, Savinase, Esperase, ChiroCLEC-BL, Trypsin, ChiroCLEC-PC, immobilized Pseudomonas lipase SAWA and lipase F. Candida esterase from Altus produces a reaction that is very quick to collect any data of enantiomeric excess.

After this evaluation is completed, a sample of compound 3 is prepared by reduction of the (S) -enantiomer of compound 1 from a resolution with PPL. This runs in the ee / de test and produces peaks 1 and 4. Because the resolution with PPL produces the (S) enantiomer of compound 1, peaks 2 and 3 are necessary for the resolution of compound 3. results from the evaluation show that most enzymes resolve compound 3 in the acetate center, not in the required butyrate center. However, Lipase AK, Lipase MY, Lipase AY and Lipase PS produce the required peaks with reasonable selectivity. Resolutions of compound 3 are further investigated with Lipase PS and Lipase MY (Lipase AK has been discontinued by Amano). Escalation and isolation of the product is necessary to determine if the resolutions are efficient (see table 12). PS lipase in toluene produces 39% yield of recovered starting material with an effective ee of 58% and effective E of 3.7 (this produces a measure of the amount of correct (i?) -butyrate that is present in the mixture ). These selectivities are not better than those obtained for compound 1 with Lipase PS, E = 5.3. In addition, because in one embodiment, the resolution step occurs early in the reaction sequence, the following examples concentrate on optimizing the resolution of compound 1.

TABLE 12 Additional Investigations of Compound 3 Resolutions Conditions: 20 ° C, 1: 1 mixture of solvent: buffer solution at pH 7 EXAMPLE 18 Compound 11 Analytical development for compound 11 A chiral test is required for compound 11. Baseline separation is achieved by chiral HPLC using the Chiralpak AS column. See figure 1C.

HPLC conditions: Column: Chiralpak AS Dimensions: 250 mm x 4.6 mm Mobile phase 1: 1 IPA: EtOH Flow rate: 0.6 ml / min Detection: ÜV 254 nm Temperature: Ambient Peak Times 1 - 11.96 min. Peak 2 - 13.17 min. retention: Because this compound is subject to microbial evaluation (> 400 samples) as well as a commercial enzyme evaluation, a 5 minute sub-run is required. The previous test is submitted to SFC where it is achieved. See figure ID.

SFC conditions: Column: Chiralpak AS Dimensions: 250 mm x 4.6 mm Mobile phase: 95% of C02: EtOH Speed of 3.0 ml / min, 210.9 kg / cm2 flow: Detection: UV 254 nm Temperature: 35 ° C Peak Times 1 - 2.88 min. Peak 2 - 3.95 min. retention: Enzyme evaluation for compound 11 Lipases In a scintillation test, 40 μl of compound 11, MTBE (5 ml), 50 mM KH2P04, pH 6 (5 ml), and approximately 10 mg of enzyme are agitated in a 30 ° incubator. C.

Aliquots are periodically removed and analyzed by TLC (50% EtOAc / heptane and chiral HPLC).

Proteases and esterases In the same way as indicated above, but without MTBE. Two sets of conditions are used for the evaluation of compound 11, the racemic methoxybenzoate ester, depending on whether the enzyme used is a lipase or a protease / esterase. For lipase-catalyzed hydrolysis, the reaction solvent is a 1: 1 mixture of methyl tert-butyl ether (MTBE): 50 mM potassium phosphate buffer at pH 6. For hydrolyses catalyzed by protease and esterase, Reactions are made in phosphate buffer without immiscible organic co-solvent. The reactions are carried out in scintillation flasks on a shaker at 30 ° C and followed by thin layer chromatography (TLC). The enantiomeric excesses of the residual substrate (e-values) are determined by HPLC analysis (Chiralpak AS). It is known that the alcohol product resulting from the hydrolysis of these esters is unstable from the previous example. Therefore, without a chiral test for the possible product, the conversions can not be calculated, so it is observed if any substrate is still present or not by means of CCF analysis. The results from the evaluations are shown in the following tables. Table 13 shows the results for the enzymes that selectively produce the enantiomer corresponding to peak 1 in the chiral HPLC test, and Table 14 shows the results for the selective enzyme for peak 2.

TABLE 13 Enzymes that selectively produce peak 1 in the HPLC test Enzyme Time eea (%) Comments Lipase M 72 hours 24 Peak 1 2 weeks 29 Substrate present Lipase PS 72 hours 7 Peak 2 2 weeks 22 Substrate present Chirazyme-L9 72 hours 19 Peak 1 2 weeks 56 Substrate present Lipasa MY 72 hours 7 Peak 1 2 weeks 21 Substrate present Lipase DS 72 hours 25 Peak 1 2 weeks 66 Substrate present TABLE 13 (cont.) Enzyme Time eeß (%) Comments Lipasa F-DS 72 hours 52 Peak 1 2 weeks 80 Substrate present Lipase F 72 hours 37 Peak 1 2 weeks 68 Substrate present Chirazyme-L2 18 hours 100 Peak 1 2 weeks 100 Substrate present Lipasa AY 72 hours 31 Peak 1 2 weeks 69 Substrate present Lipase A 72 hours 16 Peak 1"Amano" 12 2 weeks 45 Substrate present Lipasa N cone 72 hours 33 Peak 1 2 weeks 46 Substrate present Lipase AP6 72 hours 17 Peak 1 2 weeks 43 Substrate present Sigma CCL 72 hours 20 Peak 1 2 weeks 62 Substrate present Acid protease 48 hours 24 Peak 1 A 2 weeks 76 Substrate present Acid protease 48 hours 66 Peak 1 DS 2 weeks 92 Substrate present TABLE 14 Enzymes that selectively leave peak 2 in the HPLC Enzyme Time ee < V- Comments Lipase PS 72 hours 7 Peak 2 2 weeks 22 Substrate present The following enzymes are non-selective (< 20% of ees in any peak): immobilized lipase SAWA; Chiroclec-PC; Protease B; Newlase F; Alcalase; Lipase R; PPL; Lipase G; PLE; Chirazyme-El; Chirazyme-L5; ChiroCLEC-CR; Protease M; Peptidase R, Acid Protease A-DS; Protease N; A2G protease; NL protease; NL protease; DS Protease; S protease; Protease P "Amano" 6; Prozyme 6; Proleather; Bromelain-F; Papain W-40 (Amano); Papain (Sigma); Protease X; Protease XXXI; Savinasa; Wait Pepsin; ChiroCLEC-BL, esterase choline; Chirazyme-E2; and a-chymotrypsin.

EXAMPLE 19 Scaling of the resolution with C irazyme-L2 of compound 11 During the initial studies of the resolution of compound 11, the manner in which the enantiomers correlate with the peaks in the chiral HPLC test is unknown. In order to determine the absolute configuration, a sample is carried through the complete synthesis sequence to a DAPD intermediary in which the configuration can be determined. One of the most promising enzymes from the evaluation is Chirazyme-L2; therefore, this reaction is scaled in order to prepare a sample (reaction scheme 12). The substrate concentration is increased to 20 g / l and the enzyme load is reduced, but on the other hand the optimization of the procedure is minimal. With a supply of 1 g of racemate, 5% by weight of Chirazyme-L2 at 30 ° C maintaining the pH between 5 and 6, 0.321 g (32%) of the ester with > 98% ee and adequate purity.

REACTION SCHEME 12 Scaling of the resolution of compound 11 11 O, • o 20 g / l The reaction is further scaled to 10 g; after 3.5 hours, 3.9 g of compound 11 are obtained with 39% yield, > 99% ee as a white solid. It is determined that this is the unwanted (S) enantiomer. Accordingly, the desired (R) enantiomer must correspond to peak 2 in the chiral HPLC test. Therefore, a 500 ml steam jacketed container is charged at 30 ° C with MTBE (250 ml), racemic compound 11 (10.00 g, 39.7 mmole, TP-43/100) and 50 mM KH2P04, pH 6 ( 250 ml). Chirazyme-L2 (500 mg, 5% by weight) is added to the mixture with stirring. The pH is maintained between pH 5 and 6 by the addition of 2N NaOH. After 3.5 hours, the base consumption is stopped, and the mixture is filtered through celite® and separated. The aqueous phase is extracted with MTBE (2 x 200 ml), the organic layers are combined, washed with 2N NaOH (200 ml), sodium sulfite (200 ml), 2N HCl (200 ml) and dried (MgSO4). . Concentration in vacuo affords compound 11 as a white solid. Yield 3.89 g, 39%; ee > 99%; XH NMR (400 MHz, CDC13) consistent with the structure.

EXAMPLE 20 Scaling of the resolution with Lipase PS of compound 11 From the initial evaluation, only the Lipase PS is a candidate to produce the desired (R) enantiomer of compound 11 (see Error! Reference source not found). This resolution is scaled up to 2 g in order to determine its selectivity. At about 50% conversion the enantiomeric excess of the residual ester is only 8%. Clearly, the enzyme is not selective enough for a viable procedure. A 100 ml steam jacketed vessel is charged at 30 ° C with MTBE (25 ml), racemic compound 11 (2.00 g, 7.94 mmole, TP-43/100) and 50 mM KH2P04, pH 6 (25 ml) . Lipase-PS (100 mg, 5% by weight) is added to the mixture with stirring. The pH is maintained between pH 5 and 6 by the addition of 2N NaOH. Approximately 50% conversion (by base consumption), an aliquot is taken and analyzed and is 8% ee. After 24 additional hours no substrate is detected.

EXAMPLE 21 Selection of microbial enzyme General procedure for preparing 96-well culture plates 96-well deep-dish plates are used 2. 2 ml. 1.0 ml of sterile TSB (tryptone-soy broth, Oxoide CM129) is added to the bacteria, or YM (yeast broth, Oxoide CM920B) for yeast, per bottle and inoculated with a culture stock. The plates are shaken ° C during > 48 hours. The cell tablets are harvested by centrifugation (1000 g for 10 minutes at 4 ° C) and the supernatant is removed. The cell tablets are stored at -20 ° C until they are required for evaluation.

Selection of deep 96-well culture plates in compound 11 A stock solution of 200 g / l of compound 11 is prepared in acetone. 450 μl of 0.1 M Tris-HCl + 0.1% of Tween 80, pH 7.0 and 50 μl of stock solution of compound 1 are added per cavity of the 96 deep cavity culture dish. These plates are shaken at 25 ° C during >; 72 hours The samples are extracted with MTBE and analyzed by chiral HPLC. The selection is made against a variety of bacteria and yeasts. These are grown in 96-well deep plates and their cell tablets are collected by centrifugation. This allows the tablets to be resuspended in buffer with substrate for selection. The plates are shaken at 25 ° C for > 72 hours The samples are extracted with MTBE and analyzed by chiral HPLC. The results from the selection are presented in summary form in Figure 3C. Several hits are identified as selective to produce the enantiomer corresponding to peak 1 in the test, however only a few strains produce the desired enantiomer (peak 2) at a low ee. This may be due to the low levels of enzyme expressed in the wild type microorganisms, and therefore a low conversion. It should also be mentioned that the high performance chiral HPLC method used can only produce approximate values. The best results are shown in table 15.

TABLE 15 Microbial selection of compound 11 CMC No. Selectivity Strain ee * substrate (%) 103522 Unidentified 23 Peak 2 103978 Lactobacillus plantarum 12 Peak 2 103947 Comamonas acidovorans 11 Peak 2 103826 Bacillus licheniformis 10 Peak 2 103397 Pseudomonas sp. 10 Peak 2 103308 Phaffia rhodozyma 63 Peak 1 103115 Rhodococcus sp. 61 Peak 1 103188 Pseudomonas putida 58 Peak 1 103126. Enterojactericae sp. 44 Peak 1 * Main peak of the residual ester in the HPLC chromatogram The five strains selectively producing peak 2 and 3 that produce peak 1 are selected for additional tests to confirm the results from the initial selection; the results are shown in Table 16. Only CMC 103522, the best candidate for peak 2 from the evaluation primary, confirms its selectivity to < 10% of ee of the peak 2.

TABLE 16 Re-selection of the strains against compound 1 CMC No. Selectivity Strain ee * substrate (%) 103115 Rhodococcus sp. 5 Peak 1 103126 Enterobacte i cae sp. 2 Peak 1 103188 P. putida 5 Peak 1 103397 Pseudomonas sp. 1 Peak 1 103522 Unidentified 9 Peak 2 103826 B. lichenformis 4 Peak 1 103947 C. acidovorans 2 Peak 1 103978 L. planatarum 2 Peak 1 ^ Peak of the residual ester in the HPLC chromatogram EXAMPLE 22 Compound 12 Analytical development for compound 12 A chiral test for compound 12 is also required. See figure 1E. The details of the test conditions are the following: HPLC conditions: Column: Chiralpak AS Dimensions: 250 mm x 4.6 mm IPA mobile phase 1: 1: EtOH Speed 0.8 ml / min flow: Detection: UV 254 nm Temperature: Ambient Peak Times 1 - 6.91 min. Peak 2 - 7.66 min. retention: Again, this test is taken to SFC to achieve a sub-run time of 5 minutes to allow the largest number of microbial selection samples to be analyzed in as short a time as possible. See figure ÍF.

SFC conditions: Column: Chiralpak AS Dimensions: 250 mm x 4.6 mm Mobile phase 95% C02: EtOH Speed 3.0 ml / min, 210.9 kg / cm2 flow: Detection: UV 254 nm Temperature: 35 ° C Peak times 1 - 3.06 min. Peak 2 - 3.81 min. retention: Enzyme Selection Compound 12 is evaluated against 96-well culture plates. The evaluation for compound 12 is based on that developed for compound 11, but limited to only 10 enzymes. Two sets of conditions are used, depending on whether the enzyme is a lipase or a protease / esterase. For lipase-catalyzed hydrolysis, the reaction solvent is a 1: 1 mixture of MTBE: 50 mM potassium phosphate buffer at pH 6. For hydrolysis catalyzed by protease and esterase, the reactions are carried out in a phosphate buffer without an immiscible organic co-solvent. In all reactions, the amount of substrate used is 40 mg. The reactions are carried out in an agitator bath at 30 ° C and the enantiomeric excess values are determined by HPLC analysis (Chiralpak AS). The three positive results are presented in summary form in Table 17.

TABLE 17 Selection of commercial enzyme. of compound 2 Enzyme Time TGg "6) Comments Chira zyme-L2 20 hours 95 Peak 2 1 week 100 Substrate present Protease Acid 20 hours 47 Peak 2 DS 1 week - Without substrate Chirazyme-L9 20 hours 8 Peak 2 1 week 25 Substrate present The following enzymes produce the least selective resolutions with compound 2 (< 20% ees); Lipase F-DS; Lipase PS; Lipasa DS; Lipasa AY; Lipase F, acid protease A; and Lipasa N Cone. The samples are analyzed by chiral HPLC.

The results are presented in summary form in the 3D figure. As can be seen from Figure 3D, it is identified that the microbial strains are selective for both enantiomers. The conversions, and therefore, the real selectivities are unknown. Although Chirazyme-L2 is identified as an excellent enzyme to produce the enantiomer corresponding to peak 2 in the HPLC test, in this limited selection no enzymes are identified that produce peak 1. It is unknown which peak in the chiral test for the compound 12 corresponds to the required (R) enantiomer.

EXAMPLE 23 Compound 13 Chemical determination of the configuration As a quick way to determine which of the peaks in the GC chromatogram corresponds to the desired enantiomer, it is contemplated that samples can be made from the silyl ether. The enantiomerically enriched silyl ether is heated with 2-methylpropanoyl chloride in tetrahydrofuran (THF) with tetrabutylammonium fluoride (TBAF) (see reaction scheme 13).

REACTION SCHEME 13 Determination of the configuration for compound 13 It is known that it is the configuration Peak 1 in GC (Dex CB) (R) TP-0247/98 / E 95% of ee 75% of ee Peak 1 in GC (Dex CB) 88% ee It is known to be the desired enantiomer The objective is to dissociate the silyl ether and trap the alcohol generated in situ with acid chloride. Although the intermediate alcohol is unstable, if only a small fraction reacts with acid chloride faster than its decomposition, then the ester of known configuration is generated. Because the chiral test is a GC method, the sensitivity is high and minute amounts can be detected. Starting from ether (R) -silyl, peaks are observed in GC chromatograms for the product, with peak 1 increasing in both cases. Seeding the samples with the racemate confirms that the peaks correspond to the desired esters. Slight losses are observed in the enantiomeric excess in both cases, possibly due to the acidity of the medium and the potential instability of the precursor alcohol which is supposed to be an intermediate. It is known that the absolute configuration of the starting silyl ester is the desired (R) configuration. The enantiomer that gives rise to peak 1 in the GC test for the n-butanoate ester is also known to be the required enantiomer. Therefore, the reaction of the ester (R) -silyl with 2-methylpropanoyl chloride can produce the required enantiomer of compound 13, and that this corresponds to peak 1 in the GC test. Because the other enantiomer of the silyl ester is available, for further confirmation, it is also shown that it gives rise to the enantiomer of peak 2 in the reaction with 2-methylpropanoyl chloride.

Experimental method - procedure for preparing esters from silyl ether enantiomerically A mixture of 150 mg of silyl ether, 0.5 ml of acid chloride and 0.4 ml of TBAF (1M in THF) is heated at reflux under nitrogen for 3-4 hours, in 5 ml of THF. The CCF analysis shows a small amount of product formed. An aliquot is taken, passed through a plug of silica and analyzed by chiral GC.

EXAMPLE 24 Analytical development for compound 13 A chiral GC test is developed to separate the enantiomers of compound 13. See figure 1G. The details of the test conditions are the following: GC conditions: Column: Chirasil DEX CB Dimensions: 25 mm x 0.25 mm Program of 140 ° C for 8 minutes afterwards to temperature: 200 ° C to 15 ° C / min Carrier gas: Helium at 1,406 kg / cm2 Detection: FID at 200 ° C Peak times 1 - 6.5 min. Peak 2 - 6.8 min. retention: In addition, an achiral GC test is developed to determine the purity of compound 13. See Figure 1H. The details of the test conditions are the following: GC conditions: Column: J &W Scientific DB5 Dimensions: 15 mm x 0.25 mm Film thickness 0.25 μm Program of 40 ° C for 5 minutes after temperature: up to 200 ° C at 10 ° C / min Carrier gas: Helium at 0.8436 kg / cm2 Detection: FID at 200 ° C Retention times: Compound 3 - 17.80 minutes Enzyme Selection It is determined that the stability of the n-butanoate ester is significantly better at pH 6. Therefore, for the selection of the sec-butanoate ester, compound 13, similar conditions are used. The racemic compound 13 is selected for enzymatic hydrolysis with commercially available lipases, proteases and esterases. All lipase reactions are carried out in the presence of 40 μl of the compound 13, 5 ml of MTBE, 5 ml of phosphate buffer at pH 6 and 20% enzyme. Reactions with esterase and protease are carried out without MTBE being present.

The mixtures are shaken in an incubator at 30 ° C and monitored by CCF and chiral GC. The alcohol product, like the other esters, is unstable as previously described. Therefore, a chiral test for the product is not possible and conversions can not be calculated. For the selection, it is mentioned if any substrate is present or not by means of CCF analysis. The best results from the selections are shown in the following table 18.

TABLE 18 Evaluation of commercial enzyme of compound 13 Enzyme Time e Comments (%) Chirazyme-L2 1 hour 81 Peak 1 5 hours - Not enough substrate Lipase F-DS 1 hour 10 Peak 1 1 week 46 Substrate present Acid protease 24 weeks 49 Peak 1 DS 120 hours - Not enough substrate Lipase PS 5 hours 44 Peak 1 24 hours - Not enough substrate Chirazyme-L9 24 hours 42 Peak 1 120 hours - Not enough substrate Protease acid 24 hours 23 Peak 1 A 120 hours _ Not enough substrate left TABLE 18 Enzyme Time Comments (%) Lipase M 24 hours 28 Peak 1 1 week 30 Substrate present Protease M 5 hours 19 Peak 1 1 week 30 There is not much substrate present Preoteasa P 24 hours 21 Peak 1"Amano" 6 1 week 33 Not much substrate remains The following enzymes produce less reactions selective with compound 13 (< 20% ees); Lipasa DS; Lipasa AY; Lipase F; Lipase N Cone; immobilized lipase SAWA; Chiroclec-PC; - MY Lipase; Protease B; Newlase F; Alcalase; Lipase R; PPL; Lipase G; PLE; Chirazyme-El; Lipase A "Amano" 6; Lipase "Amano" 12, Lipase AP6, CCI of Sigma; Chirazyme-L5; ChiroCLEC-CR; Peptidase R, Acid Protease A-DS; Protease N; A2G protease; NL protease; NL protease; DS Protease; S protease; Prozyme 6; Proleather; Bromelain-F; Papain W-40 (Amano); Papain (Sigma); Protease X; Protease XXXI; Savinasa; Wait Pepsin; ChiroCLEC-BL, Chirazyme-E2; and a-chymotrypsin.

From Table 19 it can be seen that all the enzymes selectively produce peak 1 in the chiral GC test, which fortunately corresponds to the desired enantiomer. None of the enzymes selected are selective for peak 2. Four enzymes are chosen for re-selection with smaller loads, the results of which can be seen in the table. The most promising of these is Chirazyme-L2, which leaves a residual ester with 90% ee after 5.5 hours.

TABLE 19 Re-selection of Selective Enzymes of Peak 1 for Compound 3 Enzyme Time e Comments (%) Chirazyme-L2 '1 hour 21 Peak 1 5.5 hours 90 Substrate present Lipase PS 16 hours 65 Peak 1 24 hours - No substrate Acid protease DS 24 hours 42 Peak 1 48 hours 47 Without much remaining substrate Chirazyme-L9 24 hours 21 Peak 1 72 hours 64 Substrate present EXAMPLE 25 Scaling of the resolution with Chirazyme-L2 of compound 13 The resolution with Chirazyme-L2 is scaled up to 1 g, 30 mg of enzyme and 25 ml of each of phosphate buffer at pH 6 and MTBE. After working up the reaction produces 0.212 g, 21% theoretical of compound 13 as a colorless oil. The 1 H NMR spectrum shows a significant amount of impurities present, which have been carried from the substrate. On a scale of 6 g, using the same conditions, at 20 ° C the yield is 1.52 g, 25% of the theoretical 88% ee solved of compound 3 as a pale yellow oil. Again, XH NMR analysis indicates that some impurities are present. The selectivity is improved at a lower temperature, 0 ° C. Starting from 5.25 g of racemate, after 4.5 hours the resolution produces 1.48 g, 28% of the theoretical of compound 3 with 92% ee. The configuration is confirmed as the required (R) enantiomer. Initially, the bio-resolution is carried out at a moderate concentration, 20 g / l. To study the effect of increasing volume efficiency, the resolution is repeated on a scale of 1 g to 50 g / l, 0 ° C in phosphate buffer at pH 6 and MTBE. After 4.5 hours, the bio-transformation produces 0.44 g, 44% of theory, of material like a pale yellow oil with 92% ee. From this it is concluded that the volume efficiency can be improved without significant losses in selectivity.

The purity of the product obtained from these processes is low. Several impurities are carried from the racemate. For a moderate selectivity bio-resolution, the conversion can optionally be increased to gain a high enantiomeric purity of the residual ester. One of the impurities, the 2-methylpropanoate ester of hydroxyacetaldehyde shown in reaction scheme 14, is present in the racemate. However, it is not clear if this occurs or not in the bio-resolution or not, as indicated below.

REACTION SCHEME 14 Formation of impurities In order to determine if any additional aldehyde is generated during the bio-transformation, the resolution is repeated on the same scale (1 g) using distilled lactone, which does not contain aldehyde. This reaction produces 0.29 g, 29%, of product with 93% enantiomeric excess. The spectrum of "" "H NMR shows that the aldehyde is present, clearly this material is produced during the reaction or the treatment.

The change from solvent to toluene results in a slower but more selective resolution. On a scale of 1 g to 50 g / l, the reaction is treated after 22.5 hours to obtain 0.30 g, 30% yield of ester with > 99% of us The concentration is also increased to 200 g / l using 4 g of racemate, in 10 ml of each phosphate buffer solution pH 6 and toluene, at 0 ° C. The procedure is highly efficient in volume, and no problems are encountered. After 17 hours, the bio-transformation produces 1.11 g, 28%, of product with 95% ee. Several reactions are performed on a slightly larger scale (2-10 g) using different enzyme loads and different reaction times; The results are presented in summary form in Table 10 below. The reaction rate is not completely predictable. Several factors may be responsible for this, including different batches of racemate with different impurity profiles and differences in the mixing of the biphasic system of solvent-enzyme immobilized. Yields can be confusing due to impurities carried over from the racemate. It is found in this scale that thiosulfate, bicarbonate and acid washes do not significantly reduce the amount of the aldehyde impurity. The treatment procedure requires a certain amount of clarification, but at this stage it is expected that an appropriate Kugeirohr wash and distillation combination (film rubbed on scale) after resolution will be effective. This is subsequently investigated on a larger scale. All these bio-transformations use the two-phase system toluene-buffer at 200 g / l.

TABLE 10 Resolution with Chiraz? Me-L2 of compound 3 Input Charge of E: 3cal to Time Performance ee (%) enzyme (% (g) (hours) (%) by weight) 1 1 4 112 19 98 2 2 8 16 22 73 3 2 2 47 29 93 4 2. 5 10 15 20 > 99 5 10 10 4 ND 96 The process is scaled to 30 g using 10% by weight of enzyme at 0 ° C, 200 g / l. After a Kugeirohr distillation 4.2 g of the ester > 95% of us As discussed above, the purity of the racemate is vital for the quality and yield obtainable after bio-transformation. Also, it is difficult to determine the true selectivity of a resolution with exact conversion. This can not be obtained from the data of enantiomeric excess because the product of the bio-resolution can not be isolated; the only available information is the enantiomeric excess of the residual substrate. An idea of the selectivity is obtained from the performance of the ester, but because it is contaminated with impurities, the accuracy is in doubt. When considering the final procedure, it would be convenient to use a crude racemate and purify them after bio-resolution, for example by distillation with rubbed film. However, impurities can inhibit the enzyme. The possibility of one of these procedures using very crude substrate is investigated; a mixture of racemate containing DME, water and other impurities is used, which contains approximately 1 g of substrate in the bio-resolution. During the bio-transformation no base is consumed and therefore additional enzyme is added. An aliquot is removed after 16 hours and found to be racemic. These findings may suggest that the enzyme is being "annihilated" by the impurities present in this mixture. Therefore, in one embodiment, the purification of the substrate is preferably carried out before the bio-resolution.

EXAMPLE 26 Bio-transformation in 2-propanol-water As mentioned above, the stability of the substrate is a potential problem. A background process in which the lactone opens up to glycolic acid and the 2-methylpropanoate ester of hydroxyacetaldehyde can not be excluded (reaction scheme 15).

REACTION SCHEME 15 Lactone ring opening OH The mode of action of the enzyme on compound 13 is also unknown. The only products detected after bio-resolution are the 2-methylpropanoate ester of hydroxyacetaldehyde and glycolic acid. However, the common mode of action for a lipase is to hydrolyze the ester; this could give rise to lactone and 2-methylpropionic acid (reaction scheme 16). The product lactone alcohol is not detected and dissociates to glycolic acid and hydroxyacetaldehyde. If this mechanism is correct, then the esterified aldehyde must come from the reaction of hydroxyacetaldehyde with 2-methylpropionic acid. An alternative mechanism is that the enzyme can act directly to open the lactone without first cutting the ester. This mode of action in which a lactone ring is opened has been observed for hepatic esterases (see for example: E. Fouque and G. Rousseau Synthesis 1989, 661; and P. Barton and MI Page J. Chem. Soc., Perkin Trans. 2, 1993, 2317), but perhaps it is unlikely in this case.

REACTION SCHEME 16 Possible reaction mechanism , H OH HO + HO O O The use of additives in bio-transformations has been widely reported to increase selectivities, but their effects on enantiomeric excesses are usually unpredictable. See for example: T.V. Hansen, V. Waagen, V. Partali, H.W. Anthonsen and T. Anthonsen, Tetrahedron Asymmetry 1995, 6, 499; G. Duan and J.Y. Chen, Biotechnology Letters 1994, 16, 1065; N.W. Boaz and R.L. Zimmerman, Tetrahedron Asymmetry 1994, 5, 153; and K. Faber, G Ottolina and S. Silva, Biocatalysis 1993, 8, 91. The mode of action is also often unclear; The additive may be acting as a phase transfer catalyst, as an enzyme modifier, or as an alternative for water as a nucleophile. In the resolution of (-) -2 ', 3' -dideoxy-5-fluoro-3'-thiacytidine (FTC, also known as Coviracil ™ and emtricitabine), it is found that alcohol: water mixtures are an adequate alternative for normal or mainly aqueous biphasic systems. It is contemplated that this could also be effective in the resolution of compound 13, both with respect to the selectivity of the enzyme and the stability of the substrate. This hypothesis is analyzed using 1 g of compound 13, with 3% by weight of enzyme, at 100 g / l in an 8: 2 solvent system of 2-propanol: water. After 47 hours, the reaction produces 0.47 g, 47% of theory, of material with > 98% of us The initial result in the 2-propanol-water system seems excellent, although the water and the aldehyde may have increased the yield. More significant results are obtained on a larger scale, with distillation to purify the product. The racemate is distilled using rubbed film distillation. The conditions are 30 g of racemate in a 9: 1 mixture of 2-propanol (IPA) -water, at 200 g / l with an enzyme load of 5% by weight. This reaction is filtered after 10 hours and reduced in vacuo. It is determined that the enantiomeric excess is 94%. This material is divided into two lots to investigate the purification methods. The first portion is distilled using a Kugeirohr apparatus at 133 ° C / 1.3 Torr to give two required fractions: Fraction 1: 10.8 g containing 61% aldehyde, 31% of compound 3 and an undetermined amount of glycolic acid. Additional distillation at 80 ° C / 4-5 Torr to remove the aldehyde and a water wash produces 4. 4 g of 90% pure material of compound 3 containing 4% aldehyde. Fraction 2: 2.7 g do not contain aldehyde but a significant amount of glycolic acid and compound 3. After washing with water, 1.8 g of compound 3, 91% pure (by GC) are obtained. The second portion of raw material is washed with water before distilling at 140 ° C / 1.6 Torr followed by removal of the aldehyde at 60 ° C / 1.6 Torr, which produces 5.2 g of material with 89% purity, and that contains 6% aldehyde.

Therefore, a total of 11.4 g of ester 2-methylpropanoate with 90% purity, 94% ee, corresponding to an excellent total yield of 38%, is produced. This procedure clearly performs better than that with toluene: a regulatory solution and with additional optimization could provide an efficient route to the optically pure lactone.

EXAMPLE 27 Experimental procedure - resolution of 30 g of compound 13 with Chirazyme-L2 in 2-propanol: water A 500 ml steam jacketed vessel is charged at 0 ° C with 2-propanol (135 ml), water, (15 ml), and racemic compound 13 (30 g, DB / 1005/85/1). The mixture is stirred and Chirazyme-L2 (1.5 g, 5% by weight) is added to this mixture. The ees is measured at intervals by removing aliquots of the solution, which are extracted with ethyl acetate, dried (MgSO), and analyzed by chiral GC. After 10 hours, the ees is 93%. Shortly thereafter the mixture is filtered through celite® and concentrated in vacuo to obtain 32.1 g of crude compound 3 as a pale yellow oil. The oil is then dissolved in toluene and concentrated (2 x 250 ml) to remove any amount of water in azeotropic form. Crude yield 29.4 g, 98%; 94% of us This oil is divided into two parts. The first is distilled using a Kugeirohr apparatus at 133 ° C / 1.3 Torr, which produces 3 total fractions. Fraction 1 (most volatile) contains 10.8 g of 61% aldehyde, 31% lactone and 8% other impurities. Fraction 2 contains 2.7 g of lactone contaminated with a significant amount of glycolic acid and fraction 3 contains impurities of low yellow volatility. Fraction 2 is dissolved in toluene (200 ml), washed with water (200 ml), dried (magnesium sulfate) and concentrated in vacuo to obtain 1.8 g of compound 13 as a colorless oil with 91% purity by GC (no aldehyde observed). The majority of the aldehyde in fraction 1 is then removed by Kugeirohr distillation at 80 ° C / 4-5 Torr to obtain, as the residue, 6.1 g of lactone containing about 12% aldehyde and an undetermined amount of glycolic acid. This material is then dissolved in toluene (200 ml), washed with water (200 ml), dried (MgSO 4) and concentrated in vacuo to obtain 4.4 g of compound 3 as a colorless oil with 90% purity, 4% strength. aldehyde by GC. The second portion of material is dissolved in toluene (200 ml), washed with water (200 ml), dried (MgSO 4) and concentrated in vacuo. The resulting oil is then distilled using a Kugeirohr apparatus at 140 ° C / 1.6 Torr, to remove impurities of low volatility, and then further distilled at 60 ° C / 1.3 Torr to remove any residual aldehyde. It is determined that the material recovered from this distillation is 5.15 g of 89% purity, 6% of aldehyde by GC. Total yield: 11.4 g, 38%; purity by GC: 90%.

EXAMPLE 28 Selection of microbial enzyme As for compounds 11 and 12, compound 13 is evaluated against 96-well culture plates. The samples are analyzed by chiral GC; It should be noted that the high performance chiral GC method used can only give approximate values. The results of the selection are presented in Figure 3E. A large number of strains shows activity on compound 13, with selectivities for both peaks 1 and 2 observed on the GC chromatograms. Those that produce an enantiomeric excess > 40% of ee are shown in table 21.

TABLE 21 Microbial Evaluation of Compound 13 CMC No. Selectivity Strain ee * substrate (%) 103127 Serratia liquifaciens 42 Peak 1 103869 Unidentified 44 Peak 1 103355 Unidentified 54 Peak 1 103032 Bacillus licheniformis 43 Peak 1 103063 Unidentified 74 Peak 1 103071 Unidentified 75 Peak 1 103095 No car di a sp. 54 Peak 1 103134 Unidentified 48 Peak 1 103146 Pseudomonas sp. 46 Peak 1 103188 Pseudomonas putida 46 Peak 2 103322 Pseudomonas alcaligenes 70 Peak 1 103419 Acinetobacter sp. 81 Peak 1 103422 Unidentified 79 Peak 1 103423 Unidentified 71 Peak 1 103669 Unidentified 51 Peak 2 103777 Unidentified 50 Peak 1 103780 Streptomyces sp. 49 Peak 1 103405 Unidentified 62 Peak 1 103785 Pseudomonas fluorescens 87 Peak 1 103869 Unidentified 92 Peak 2 103331 Candida rugosa 40 Peak 1 103587 Staphylococcus sp. 65 Peak 1 103774 Streptoverticillium 87 Peak 2 cinnamoneus From the results of the primary selection, those strains that show the best selectivity for peak 1 and an excellent peak area are reevaluated. The strains that reconfirm their activities are shown in Table 22.

TABLE 22 Re-evaluation of strains against compound 3 CMC No. Strain of the substrate (%) 103063 Not identified 45 103127 S. liquifaciens 41 103373 Acinetobacter sp. 41 103419 Acinetobacter sp. 14 103552 Unidentified 31 103606 Acinetobacter junii 21 103635 Unidentified 44 103661 S. cerevisiae 47 103777 Unidentified 18 103869 Unidentified 65 EXAMPLE 29 Scaling of bio-transformations for CMC 103869 and 103661 From the selection, two strains are chosen for scaling. These are CMC 103869 (Unidentified) and CMC 103661 (S. cerevisiae). The cultures of both are grown and their collected cell pastes are stored at -20 ° C. The bio-transformations are established on a 30 ml scale, using both pH and temperature control. The initial bio-transformations are carried out in 0.1 M Tris-HCl, pH 7. Very low enantiomeric excesses and substrate stability are observed, possibly due to high pH. The reduction of the pH to 6 partially increases the selectivity, but problems of stability of the substrate still exist. A change in the buffer solution to 50 mM KH2P04, pH 6, also increases the enantiomeric excess. The results for both strains are presented in summarized form in Figure 4. The resolution of compound 13 with CMC 103869 produces the residual ester of 70% ee. The results with CMC 103361 are discouraging and therefore additional studies with this strain are abandoned. For all bio-transformations a residual substrate loss is observed, possibly due either to a second enzyme or to very low stability of the substrate at 25 ° C. Finally, reducing the temperature to 10 ° C increases the selectivity up to > 95% ee, however there is still a loss of the total substrate over time. The conversion and therefore the absolute selectivity are unknown. CMC 103869 is grown in TSB medium and CMC 103661 is grown in YM medium in flasks. Both strains are grown at 25 ° C and the cells are harvested by centrifugation (2000 g for 20 minutes at 4 ° C). The cell pastes are stored at -20 ° C. The bio-transformations are run in 200 ml steam jacketed containers with a magnetic stirrer, with temperature and pH control (with NaOH IN).

The cell pastes are re-suspended at 10% w / v in either 0.1 M Tris-HCl buffer (adjusted to the required pH), or 50 mM KH2P04, pH 6. The substrate, buffer and cell paste are added to the vessel and samples are taken for analysis by chiral GC by dilution in MTBE.

EXAMPLE 30 Compound 4 Analytical development for compound 4 The conditions for the chiral HPLC analysis method are reproduced to obtain the resolution shown in the following chromatogram: HPLC conditions: Column: Chiralcel OJ Dimensions: 250 mm x 4.6 mm Mobile phase: 85% heptane 15% EtOH Flow rate; 1.0 ml / min Detection: UV at 254 nm Retention times: 1 - 747 minutes 2 - 11.16 minutes Under the test conditions, the peak forms of the enantiomers are a little broader (Figure 1K), therefore, an attempt is made to improve this test. This is achieved using supercritical fluid chromatography (SFC), in which both isomers produce sharp peaks that resolve completely within 4 minutes. See figure 1L.

Optimized SFC conditions: Column: Chiralcel OJ (250 x 4.6 mm) Mobile phase: 95% C02, 5% MeOH Flow rate: 3.0 ml / min Pressure: 210.9 kg / cm2 Temperature: 35 ° C Retention times: 1 - 2.73 minutes [(-) -enantiomer] 2 - 3.28 minutes [(+) -enantiomer] Bio-resolution of compound 4 During the study of the bio-transformation of compound 1, it is discovered that the material is susceptible to non-enzymatic hydrolysis. This may be due to the high lability of compound 1 towards the hydrolysis and instability of the postulated lactone-alcohol product, such that a possible route for bio-transformation is the opening of the lactone before the saponification of the butyrate. If this is true, then the enzymatic resolution of compound 4 could be possible. Therefore, the reactivity of compound 4 is analyzed with the PS, PPL, M, MY lipases, and the immobilized SAWA lipase in toluene - buffer solution pH 7 at 20 ° C. No reaction observed (CCF, 24 hours). The enzymatic reaction probably proceeds first by cutting the butyrate and then the possible opening of the lactone.

- Conglomerate studies The IR spectra of the racemate and individual enantiomer of compound 4 are compared and found to be similar but not identical. The melting points of each of these compounds are then determined by differential scanning calorimetry (DSC) and found to produce a melting onset of 91.7 ° C for the racemate and 81.1 ° C for the (-) -enantiomer (98.7 ° C). % of ee). Comparison of these melting points reveals that the racemate has a melting point higher than that of the individual enantiomer. For a compound to be a conglomerate, the IR spectrum of the racemate and the individual enantiomer must be identical and it is required that the melting point of the racemate be the lowest point in the phase diagram. Therefore, this compound is clearly not a conglomerate. Subsequently, a phase diagram for compound 4 is constructed. The position of the eutectic point is determined, above which point the ee of this compound can be increased to enantiomeric purity by crystallization. This phase diagram information is obtained by analyzing the melting points (again by DSC) of samples of different enantiomeric excesses in increments of 10% ee. These samples are initially prepared by dissolving the required amounts of racemate and individual enantiomer in acetone and removing the solvent. This method is successful for the samples of 10% ee, 80% ee and 90% ee, in which the DSC traces contain only one peak. For all other samples, however, two peaks are observed by DSC (possibly due to polymorphs). A number of methods are tried to produce these samples to address this problem. These include fusion of the sample; dissolution in acetone and heating overnight at 65 ° C; melting and heating overnight at 65 ° C and using dichloromethane as the solvent; but the DSC keeps giving two spikes. Therefore, the eutectic phase / point diagram can not be determined by this method. In addition, because the compound is extremely soluble and only 1 g of the racemate and each of the individual enantiomers are available, solubility studies are excluded. However, because individual racemate and enantiomer samples produce only one peak by DSC, the melting points and enthalpy values for these samples can be determined. This allows the phase diagram to be theoretically constructed (J. Jacques, A Collet and S.H. Wilden, Enantiomers, Racemates and Resolutions, New York, Wiley, 1981). The Schroder-Van Laar equation can be used to calculate the liquid phase curve between the pure enantiomer and the eutectic point for a true racemate compound, using the melting point and the enthalpy of fusion of the pure enantiomer: Molar fraction: 00..99 tf = 351.64 K 78.64 ° C 0.8 Tf = 346.60 K 73.60 ° C 0.7 tf = 341.05 K 68.05 ° C 0.6 Tf = 334.86 K 61.86 ° C Then, you can determine the part of the curve below which the solid phase consists of pure racemic compound using the following equation (Prigogine-Defay): Molar fraction: 0.9 Tf 349.26 K 76.26 ° C 0.8 Tf 360.81 K 87.81 ° C 0.7 Tf 366.53 K 93.53 ° C 0.6 f 369.41 K 96.40 ° C In which: x = molar fraction of the most abundant enantiomer (0.5 <x <1) of a mixture whose fusion ends in Tf (K). Hf enthalpy of fusion of the individual enantiomer (J. mol 1). ? HfR = fusion enthalpy of the racemate (J-mol-1) R = 8.31 (J-K_1"mol_1) Tf = melting point of the pure individual enantiomer (K) TfR = melting point of the racemate (K) Both of these curves are then plotted on the same graph to obtain the phase diagram. The point at which the lines cross is the eutectic point, which in this case is 77.5% ee. See figure 4.

EXAMPLE 31 Analytical development for DAPD Chiral tests for cis-DAPD and its butyrate ester are developed in order to analyze an enzymatic esterification evaluation of DAPD. See figure 1M.

SFC conditions for DAPD: Column: Chiralpak AD Dimensions: 250 x 46 mm Mobile phase: 70% of C02, 30% of MeOH with 0.1% of TEA Flow rate: 3.0 ml / min Pressure: 210.9 kg / cm2 Temperature of the 35 ° C column: Detection : ÜV at 254 nm Retention times: 1 - 3.57 minutes 2 - 5.64 minutes In order to monitor the progress of the bio-transformations for the DAPD material, the previous test is modified to include the baseline resolution of both the DAPD and the butyrate. See figure ÍN. The conditions and chromatograms are the following: SFC conditions for DAPD and butyrate ester: Column: Chiralpak AD Dimensions 250 x 4.6 mm Mobile phase '80% C02, 20% MeOH with 0.1% TEA Speed 3.0 ml / min flow: Pressure: 210.9 kg / cm2 Temperature: 35 ° C Detection: UV @ 254 nm Time of 4.49 minutes 2 - 5.90 minutes retention: 3 - 9.18 minutes 4 - 11.0 minutes Enzymatic resolution of DAPD A selection of limited enzyme is carried out aimed at resolving cis-DAPD isomers by trans-esterification using vinyl butyrate. The enzymes are chosen based on their known transesterification activity. The solvents investigated are toluene (insoluble in substrate), DMF and pyridine (soluble in substrate). The reactions are followed by TLC and the enantiomeric excesses are measured using supercritical fluid chromatography (SFC) (Chiralpak AD). As confirmation, enantiomeric excesses are also measured using an HPLC test (100% MeOH, Chiralpak AD) and are in accordance with the results of SFC. For each enzyme: racemic cis-DAPD (TP0041 / 97 / D1, 10 mg, 0.04 mmol) is added with solvent (1 ml) (toluene, DMF or pyridine, see table 13), vinyl butyrate (0.1 ml, 0.8 mmol) ) and then the enzyme (5-10 mg). The flasks are shaken in an incubator at 30 ° C and analyzed periodically by removing aliquots of 50 μl, which are diluted with methanol and analyzed by TLC (10: 0.1 EtOAc: MeOH: H20) and chiral. Table 23 shows the results obtained.

TABLE 23 Enzymatic Selection for Acylation of DAPD The results shown in Table 23 show that although DAPD esterification occurs, this is not an enantio-selective reaction. The CCF analysis shows that in some cases more than one product is formed. However, for PeptiCLEC-BL and LYPSA AY, only the desired butyrate is formed in a rapid reaction, therefore these could be suitable enzymes to place an ester in oxygen in a regio-selective and non-aggressive manner.

Claims (17)

NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and therefore the content of the following is claimed as property: CLAIMS

1. - A process for preparing a substantially pure β-D or β-L-1, 3-dioxolane nucleoside, comprising: a) obtaining an esterified 2, 2-dialkoxyethanol of the formula (Ia) or (Ib); (the) (Ib) wherein: each R1 is independently alkyl, aryl, heteroaryl, heterocyclic, alkaryl, alkylheteroaryl, or alkylheterocyclic, or aralkyl; and R2 is any appropriate removable group; b) cyclizing the esterified 2, 2-dialkoxyethanol of the formula (la) or (Ib) with glycolic acid to obtain a 1,3-dioxolane lactone of the formula (II): (II) c) solving the 1,3-dioxolane lactone of the formula (II) to obtain a substantially pure D- or L-lactone; d) selectively reducing with a reducing agent, and activating the substantially pure chiral D- or L-lactone to obtain a substantially pure D- or L-1,3-dioxolane of the formula (III): (III) wherein L is an appropriate leaving group; e) coupling the substantially pure D- or L-1, 3-dioxolane of the formula (III) with a purine-based or activated and / or protected pyrimidine or its derivative, to obtain the mixture at: ß of D-nucleosides or substantially purified L-1, 3-dioxolane of the formula (IV): (IV) wherein B is a purine base or pyrimidine or its derivative; f) optionally purifying the mixture to: ß of protected substantially pure D- or • L-1, 3-dioxolane nucleosides of the formula (IV) to obtain a β-D- or β-Ll, 3-dioxolane nucleoside substantially pure protected; and g) optionally deprotecting the protected substantially pure ß-D- or β-L-1, 3-dioxolane nucleoside to obtain a substantially pure β-D or β-L-1, 3-dioxolane nucleoside.

2. The process according to claim 1, characterized in that the substantially pure β-D or β-L-1, 3-dioxolane nucleoside is β-D-DAPD.

3. The process according to claim 1, characterized in that step (b) is carried out in the presence of a Lewis acid.

4. The process according to claim 3, characterized in that the Lewis acid is BF3-Et20.

5. The process according to claim 1, characterized in that the reducing agent in step (d) is LiAlH (OtBu) 3.

6. The method according to claim 1, characterized in that the appropriate leaving group is selected. from the group consisting of O-acyl, OAc, halogen, O-mesylates and O-tolutes.

7. The process according to claim 1, characterized in that R2 is isobutyryl or p-methoxybenzoyl.

8. The process according to claim 1, characterized in that the base purine or pyrimidine or its activated and / or protected derivative is activated 2,6-dichloropurine.

9. The process according to claim 1, characterized in that the substantially pure protected β-D- or β-D-L-1,3-dioxolane nucleoside is deprotected.

10. The process according to claim 1, characterized in that the esterified 2,2-dialkoxyethanol of the formula (la) is hydrolyzed to the corresponding aldehyde of the formula (Ib).

11. The process according to claim 1, characterized in that the esterified 2,2-dialkoxyethanol of the formula (la) is hydrolyzed to the corresponding aldehyde of the formula (Ib) when R2 is p-methoxybenzoyl.

12. The process according to claim 1, characterized in that the esterified 2,2-dialkoxyethanol of the formula (Ia) is not hydrolyzed to the corresponding aldehyde of the formula (Ib).

13. The process according to claim 1, characterized in that the esterified 2,2-dialkoxyethanol of the formula (Ia) is not hydrolyzed to the corresponding aldehyde of the formula (Ib) when R2 is isobutyryl.

14. The method according to claim 1, characterized in that the resolution of the 1,3-dioxolane lactone of the formula (II) is achieved using chiral chromatography.

15. The process according to claim 1, characterized in that the resolution of lactone 1,3-dioxolane of the formula (II) is achieved using enzymatic resolution.

16. The process according to claim 1, characterized in that the substantially pure ß-D- or β-L-1, 3-dioxolane nucleosides are selected from the group consisting of the formulas A to D: A B c D and pharmaceutically acceptable salts or esters thereof, in which: R is independently H, halogen, OH, ORA OCH3, SH, SRA SCH3, NH2, NHR ', NR'2, lower alkyl of Cx-C4, CH3 , CH = CH2, N3C = CH2, C02H, C02RA CONH2, CONHRA CH2OH, CH2CH2OH, CF3, CH2CH2F, CH = CHC02H, CH = CHC02RA CH = CHC1, CH = CHBr, or CH = CHI; each R 'is independently a lower alkyl of C? -C; Z can be CH or C-X; and X and X are independently H, halogen, OH, ORA OCH3, SH, SRA SCH3, NH2, NHR 'f NR'2, or CH3.

17. A process for preparing substantially pure ß-D-DAPD, comprising: a) providing a compound of the formula (a): (a) in which each R1 is independently alkyl, aryl, heteroaryl, heterocyclic, alkaryl, alkylheteroaryl, or alkylheterocyclic, or aralkyl; (b) converting the compound of the formula (a) to a compound of the formula (b): in which R is independently H, halogen, OH, OR ', OCH3, SH, SR', SCH3, NH2, NHR ', NR' 2, lower alkyl of Cx-C4, CH3, CH = CH2, N3C = CH2, C02H, C02R ', CONH2, CONHR ', CH2OH, CH2CH2OH, CF3, CH2CH2F, CH = CHC02H, CH = CHC02R ', CH = CHC1, CH = CHBr, or CH = CHI; c) cyclizing the compound of the formula (b) to the racemic 1,3-dioxolane lactone of the formula (c): (c); d) resolving the compound of the formula (c) to a dioxolane chiral lactone of the formula (d); (d) e) acetylating the chiral dioxolane lactone of the formula (d) to a chiral dioxolane acetate of the formula (e); Y (and); f) coupling the compound of the formula (e) with an activated and / or protected adenine base; and g) optionally purifying and deprotecting the compound of step (f) to obtain β-D-2,6-diaminopurin-dioxolane.