Enantiomerically Pure Beta-D-(-)-Dioxolane-Nucleosides

Europäisches Patentamt *EP001600448A2* (19) European Patent Office

Office européen des brevets (11) EP 1 600 448 A2

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) Int Cl.7: C07D 405/04, A61K 31/506 30.11.2005 Bulletin 2005/48

(21) Application number: 05075365.6

(22) Date of filing: 05.12.1991

(84) Designated Contracting States: (72) Inventors: AT BE CH DE DK ES FR GB GR IT LI LU MC NL SE • Chu, Chung K. Athens, GA 30605 (US) (30) Priority: 05.12.1990 US 622762 • Schinazi, Raymond F. Decatur, GA 30333 (US) (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: (74) Representative: Hallybone, Huw George et al 01203571.3 / 1 164 133 Carpmaels and Ransford, 92902800.9 / 0 562 009 43-45 Bloomsbury Square London WC1A 2RA (GB) (71) Applicants: • UNIVERSITY OF GEORGIA RESEARCH Remarks: FOUNDATION, INC. This application was filed on 14 - 02 - 2005 as a Athens, Georgia 30602 (US) divisional application to the application mentioned • EMORY UNIVERSITY under INID code 62. Atlanta, GA 30322 (US)

(54) Enantiomerically pure beta-D-(-)-Dioxolane-Nucleosides

(57) An asymmetric process for the preparation of enantiomerically pure dioxolane nucleosides is lower enantiomerically pure β-D-(-)-dioxolane-nucleosides. than that of the racemic mixture of the nucleosides, be- The enantiomerically pure dioxolane nucleosides are cause the nonnaturally occurring α-isomer is not includ- active HIV agents, that are significantly more effective ed. The product can be used as a research tool to study than the prior prepared racemic mixtures of the nucleo- the inhibition of HIV in vitro or can be administered in a sides. The anti-viral activity of the compounds is surpris- pharmaceutical composition to inhibit the growth of HIV ing in light of the generally accepted theory that moieties in vivo. in the endo conformation, including these dioxolanes, are not effective antiviral agents. The toxicity of the EP 1 600 448 A2

Printed by Jouve, 75001 PARIS (FR) 1 EP 1 600 448 A2 2

Description and C4'-substituents are on the same side of the carbohydrate plane (i.e., the substituents are cis) is referred [0001] The government has rights in this invention by to as a "β-configuration." A carbohydrate configuration virtue of grants from the Public Health Service of the Na- in which the C1' and C4'-substituents are on the oppo- tional Institute of Allergy and Infectious Diseases and 5 site side of the carbohydrate plane (i.e., the substituents the Department of Veteran's Affairs that partially funded are trans) is referred to as, an "α-configuration". Refer- research leading to this invention. ring to compound 1 of Figure 2, a nucleoside is designated a D-nucleoside if the non-hydrogen substituent at- Background of the Invention tached to the C4'-atom is above the plane of the carbo- 10 hydrate ring. The nucleoside is designated an L-nucle- [0002] This invention is in the area of organic synthe- oside if the non-hydrogen substituent attached to the sis of nucleosides, and in particular relates to a process C4'-atom is below the plane of the carbohydrate ring. for the preparation of enantiomerically pure β-D-(-)-di- [0007] The non-naturally occurring α-isomers of nucl- oxolane nucleosides. eosides (in which the C1' or C4' substituents are on op- [0003] A number of 2',3'-dideoxynucleosides have 15 posite sides of the carbohydrate plane) are rarely bio- been found to be potent antiviral agents against human logically active, and are typically toxic. immunodeficiency virus (HIV), the causative agent of [0008] An analysis of the solid-state conformations of acquired immunodeficiency syndrome (AIDS). The lead six active and two inactive anti-HIV nucleoside agents compound, AZT (Mitsuya, H.; Broder, S. Proc. Natl. was recently performed to attempt to correlate the pres- Acad. Sci. U.S.A., 1986 83, 1911) has been approved 20 ence or absence of certain stereochemical features with by the U.S. Food and Drug Administration for patients high HIV activity. Van Roey, P.,et al., J. Am. Chem. Soc., with AIDS and AIDS-related complex. Several other 2', 1988, 110, 2277; and Van Roey, P., et al., Proc. Natl. 3'-dideoxynucleosides are undergoing various stages of Acad. Sci. U.S.A., 1989, 86, 3929. The x-ray structures clinical trials, including 3'-azido-2',3'-dideoxyuridine indicated that active anti-HIV nucleosides assume the (AZDU or CS-87, see, Chu, C.K.; et al., J. Med. Chem., 25 C3'-exo or similar carbohydrate conformations while in- 1989, 32, 612; and Eriksson, B.F.H.; et al., Antimicrob. active compounds prefer the C3'-endo conformation. Agents Chemother., 1989, 33, 1927), 2',3'-dideoxyino- (Endo and exo refer to the conformations in which the sine (DDI) and 2',3'-dideoxycytidine (DDC) (see Yarcho- atoms are at the same or opposite side of the sugar ring an, R. et. al., Science, 1989, 245, 412), 3'-deoxy-2',3'- in relation to the base). The C3'-exo and C3'-endo con- didehydrothymidine (D4T, Lin, T.S., et al., Biochem. 30 formations place the C5' atom in axial and equitorial po- Pharmacol., 1987, 36, 311; Hamamoto, Y., et al., An- sitions, respectively. The position of the C5' atom affects tinicrob. Agents Chemother., 1987, 31, 907; Balzarini, the location of the 5'-hydroxyl group in relation to the J., et al., Biochem. Biophys. Res. Commun., 1987, 140, base. Since the 5'-hydroxyl group is the site of phospho- 735), and 2'-fluoro-arabinofuranosyl-2'-3'-dideoxycyti- rylation of the nucleoside, its location with respect to the dine (Martin, T.A., et al., J. Med. Chem., 1990, 33, 2137; 35 rest of the nucleoside is important. Watanabe, K.A., et al., J. Med. Chem., 1990, 33, 2145; [0009] There has been recent interest in the synthesis Sterzycki, R.Z., et al., J. Med. Chem., 1990 33, 2150). of nucleoside derivatives in which the 3'-carbon of the [0004] In the 5'-triphosphorylated form, these nucleo- nucleoside has been replaced with a heteroatom. Nor- sides are known to inhibit HIV reverse transcriptase as beck, D.W., et al., in Tet. Lett., 1989, 30, 6263, reported well as cause chain-termination of the growing viral DNA 40 the synthesis of (±)-1-[(2β,4β)-2-(hydroxymethyl)-4-di- chain. Furman, P.A., et al., Proc. Natl. Acad. Sci. U.S. oxolanyl]thymine (referred to below as (±)-dioxolane-T, A., 1986, 83, 8333; Cheng, Y.C., e al., J. Biol. Chem., see Figure 1), that results in a racemic mixture of dias- 1987, 262, 2187; St. Clair, M.H., et al., Antimicrob. tereomers about the C4' atom. The product is a deriva- Agents Chemother., 1987, 31, 1972; and Schinazi, R. tive of 3'-deoxythymidine in which the C3' atom has F., et al., Antimicrob. Agents Chemother., 1989 33, 115. 45 been replaced with an 03' atom. The product was syn- [0005] The stereochemistry of nucleoside derivatives thesized in five steps from benzyloxyaldehyde dimeth- play an important role in their biological activity. The C1' ylacetal and (±)-methyl glycerate to produce a 79% yield position of the ribose in the nucleoside (the carbon of the 1:1 diastereomeric mixture. The X-ray crystallo- bound to the nitrogen of the heterocyclic base) is a chiral graphic analysis of the product revealed that the diox- 50 3 center because the carbon is attached to four different olane ring adopts the T4 conformation commonly ob- moieties. Likewise, there is an optically active center at served in ribonucleosides, with the 03' atom in the endo C4' of the nucleoside (the ring carbon bound to the hy- position. Norbeck reported that the racemic mixture of droxymethyl group that is phosphorylated in nucle- dioxolane-T exhibits an anti-HIV activity of 20 µMin otides). In the naturally occurring nucleosides, both the ATH8 cells, and attributed the low efficacy against the base attached to the C1' atom and the hydroxymethyl 55 virus to an effect of the endo conformation of the 03' group attached to the C4' atom are on the same side of atom. the carbohydrate ring. [0010] Belleau, et al., in the Fifth International Conf. [0006] A carbohydrate configuration in which the C1' on AIDS, Montreal, Canada June 4-9, 1990, paper No.

2 3 EP 1 600 448 A2 4

T.C.O.l., reported a method of synthesis of cytidine nu- HIV in vivo. cleosides that contain oxygen or sulfur in the 3'-position. The dioxolane ring was prepared by the condensation Brief Description of the Figures of RCO2CH2CHO with glycerine. As with the Norbeck synthesis, the Belleau synthesis results in a racemic 5 [0018] mixture of diastereoisomers about the C4' carbon of the nucleoside. Belleau reported that the sulfur analog, re- Figure 1 is an illustration of the chemical structures ferred to as NGBP-21 or (±) BCH-189 (see Figure 1), of (±)-1-[(2β,4β)-2-(hydroxymethyl)-4-dioxolanyl] had high anti-HIV activity. (±) BCH-189 is currently un- thymine (dioxolane-T) and (±)-1-[(2β,4β)-2-(hy- dergoing preclinical toxicology. 10 droxymethyl)-4-(1,3-thioxolane)]thymine (BCH- [0011] To date, no one has reported a method of syn- 189). thesis of a nucleoside analog with an oxygen in the 3'- Figure 2 is an illustration of the method of synthesis position that results in an enantiomerically pure diox- of enantiomerically pure β-D-(-)-dioxolane-thymine. olane nucleoside that has the same stereochemistry as the nucleosides found in nature (the B stereoisomer). 15 Detailed Description of the Invention There is a need for such a synthesis as a research tool to provide more information on the effect of stereochem- [0019] As used herein, the term "protected" refers to istry on the anti-viral activity of nucleoside derivatives, a moiety that has been placed on a functional group of and to provide new anti-HIV agents. a molecule to prevent further reaction of the moiety dur- [0012] It is therefore an object of the present invention 20 ing derivatization of another portion of the molecule. to provide a method of synthesis of enantiomerically Protecting groups, particularly for oxygen and nitrogen, pure dioxolane nucleosides. are well known to those skilled in the art of organic [0013] It is another object of the present invention to chemistry. provide enantiomerically pure dioxolane nucleosides [0020] The term "1,3-dioxolane nucleoside" as used with significant anti-HIV activity. 25 herein refers to a nucleoside derivative as depicted in Figures 1 and 2, wherein a 1,3-dioxolane is attached to Summary of the Invention a heterocyclic base, typically a purine or pyrimidine base, through the oxathiolane C5 carbon (that becomes [0014] The invention as disclosed is an asymmetric the Cl'-carbon in the nucleoside). process for the preparation of enantiomerically pure 30 β-D-(-)-dioxolane-nucleosides. The process involves I. Preparation of Enantiomerically Pure Dioxolane the initial preparation of (2R,4R)- and (2R,4S)-4-ace- Nucleosides toxy-2-(protected-oxymethyl)-dioxolane from 1,6-anhydromannose, a sugar that contains all of the necessary [0021] In preparing enantiomerically pure dioxolane stereochemistry for the enantiomerically pure final prod- 35 nucleosides, care should be taken to avoid strong acidic uct, including the correct diastereomeric configuration conditions that would cleave the dioxolane ring. Reac- about the 1 position of the sugar (that becomes the 4'- tions should be performed, if possible, in basic or neutral position in the later formed nucleoside). conditions, and when acidic conditions are necessary, [0015] The (2R,4R)- and (2R,48)-4-aaetaxy-2-(pro- the time of reaction should be minimized. tected-oxymethyl)-dioxolane is condensed with a de- 40 sired heterocyclic base in the presence of SnCl4, other A. Preparation of Dioxolane Derivative Lewis acid, or trimethylsilyl triflate in an organic solvent such as dichloroethane, acetonitrile, or methylene chlo- [0022] The key starting material for the synthesis of ride, to provide the stereochemically pure dioxolane-nu- enantiomerically pure β-D-(-)-dioxolane-nucleosides is cleoside. 45 1,6-anhydromannose (compound 1, Figure 2). This sug- [0016] The enantiomerically pure dioxolane nucleo- ar contains all of the necessary stereochemistry for the sides are active HIV agents, that are significantly more enantiomerically pure final product (see for example, effective than the prior prepared racemic mixtures of the compound 11, Figure 2), including the correct diastere- compounds. The anti-viral activity of the compounds is omeric configuration about the 1 position of the sugar surprising in light of the generally accepted theory that 50 (that becomes the 4'-position in the later formed nucle- moieties in the endo conformation, including these di- oside). 1,6-Anhydromannose can be prepared accord- oxolanes, are not effective antiviral agents. Further, the ing to procedures described in Knauf, A.E.; Hann, R.M.; enantiomerically pure dioxolane nucleosides are less Hudson, C.S. J. Am. Chem. Soc. , 1941, 63, 1447; and toxic than the racemic mixture of nucleosides because Zottola, M.A.; Alonso, R.; Vite, G.D.; Fraser-Reid, B. J. the nonnaturally occurring isomer has been eliminated. 55 Org. Chem., 1989; 54, 6123. Prior syntheses of diox- [0017] The product can be used as a research tool to olane nucleosides have used racemic mixtures of start- study the inhibition of HIV in vitro or can be administered ing materials for the preparation of the ribose moiety. in a pharmaceutical composition to-inhibit the growth of When the syntheses begin with a racemic mixture of re-

3 5 EP 1 600 448 A2 6 agents, undesirable racemic mixtures of enantiomeric the corresponding key intermediates (2R,4R)- and (2R, nucleoside products have been produced. The mixtures 4S)-4-acetoxy-2-(protected-oxymethyl) dioxolane (see are very difficult to separate and significantly increase compound 8, Figure 2) in good yield. the cost of the final product. Further, the inclusion of nonnaturally occurring isomers increases the toxicity of the 5 B. condensation of a Heterocyclic Base with the product. Dioxolane Derivative [0023] The 1,6-anhydromannose is converted to its isopropylidene derivative with dimethoxypropane and p- [0028] In the next step of this reaction scheme, the toluenesulfonic acid, which, without isolation, is ben- enantiomerically pure dioxolane prepared as described zoylated in the 4-position to compound 2 (see Figure 2). 10 in Section A. is condensed with a protected base in the An acyl group can also be used to protect the 4-position. presence of trimethylsilyl triflate (trimethylsilyl trifluor- The isopropylidene group of compound 2 is then re- omethanesulfonate) or a Lewis acid in a dry organic sol- moved by a catalytic amount of an acid such as sulfuric vent. acid, hydrochloric acid, formic acid, trifluoroacetic acid, [0029] Any compound containing a nitrogen that is ca- sulfamic acid, in 60% aqueous dioxane or other suitable 15 pable of reaction with a center of electron deficiency can organic solvent at a temperature range of approximately be used in the condensation reaction. Purine bases in- 0to50°C to give (-)-1,6-anhydro-4-0-benzoyl-β-D-man- clude adenine, hypoxanthine, N6-alkylpurines, N6-ben- nopyranose in high yield as a white solid. zylpurine, N6-halopurine, and guanine. Pyrimidine bas- [0024] In the next step, the glycol of (-)-1,6-anhydro- es include thymine, cytosine, 6-azapyrimidine, 2-mer- 4-0-benzoyl-β-D-mannopyranose is oxidatively cleaved 20 captopyrimidine, and uracil. A thymine base is preferred 2 by treatment with NaIO4 in H O/EtOH (1:1) for one hour in a condensation reaction carried out with a dioxolane at approximately room temperature to produce to the derivative, and a cytosine base is preferred when the corresponding dialdehyde. Lead tetraacetate can also condensation reaction is carried out with a 1,3-thiox- be used as the oxidizing reagent for this reaction. The olane. dialdehyde is immediately reduced in situ with any suit- 25 [0030] Functional oxygen and nitrogen groups on the able reducing agent, including NaBH4, diisobutylalumi- heterocyclic base should be protected before conden- num hydride (DIBAL-H), lithium borohydride (LiBH4), or sation with the sugar. Protecting groups are well known sodium bis(2-methoxyethoxy)-aluminum hydride (Red- to those skilled in the art, and include trimethylsilyl, Al), at approximately room temperature or below. Under dimethylhexylsilyl, t-butyldimethylsilyl, and t-butyldiphe- the conditions of reaction, compound 4 isomerizes by 30 nylsilyl, tritylmethyl, alkyl groups, acyl groups (lower benzoyl migration from a secondary to a primary posi- alkyl-C(O)) such as acetyl and propionyl, methylsulfo- tion to produce (-)-(2R,4R)-4-(2-benzoxy-1-hydroxye- nyl, and p-toluylsulfonyl. thyl)-2-(hydroxymethyl)-dioxolane (compound 5, Figure [0031] Friedel-Crafts catalysts (Lewis acids) that can 2). be used in the condensation reaction include SnCl4, 35 [0025] The 2-position of the dioxolane is then protect- ZnCl4, TiCl4, AlCl3, FeCl3,BF3-diethylether, and BCl3. ed with a suitable oxygen protecting group, for example, These catalysts require anhydrous conditions because a trisubstituted silyl group such as trimethylsilyl, dimeth- the presence of water reduces their activity. The cata- ylhexylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trit- lysts are also inactivated in the presence of organic sol- yl, alkyl group, acyl groups such as acetyl, propionyl, vents with active hydrogens, such as alcohols and or- 40 benzoyl, p-NO2 benzoyl, or toluyl, methylsulfonyl, or p- ganic acids. The catalysts are typically used in solvents toluylsulfonyl. A preferred protecting group is t-butyld- such as carbon disulfide, methylene chloride, nitrometh- iphenylsilyl. After protecting the 2-position of the dioxane, 1,2-dichloroethane, nitrobenzene, tetrachlo- olane, the benzoyl group is removed from the 2-hydrox- roethane, chlorobenzene, benzene, toluene, dimethyl- yethyl-position with a strong base such as sodium meth- formamide, tetrahydrofuran, dioxane, or acetonitrile. oxide or ammonia in methanol at approximately 0 to 45 Anhydrous aluminum chloride is not soluble in carbon 50°C to produce (-)-(2R,4R)-2-(proteated-O-methyl)- disulfide. Niedballa, et al., J. Org. Chem. 39, 25 (1974). 4-(1,2-dihydroxyethyl)-dioxolane (compound 6, Figure The preferred catalyst is SnCl4. The preferred solvent 2) in high yield. is 1,2-dichloroethane. Trimethylsilyl triflate can be used [0026] In the next step, the 1,2-dihydroxyethyl group under the same conditions described above for the in the 4-position of the dioxolane is converted to a car- 50 Friedel-Crafts catalysts. The reaction proceeds at a boxylic acid with an oxidizing agent such as NaIO4/ temperature range of from -10°C to 200°C. RuO2, or lead tetraacetate, at approximately 0 to 50°C [0032] The choice of catalyst for condensation will af- to produce (+)-(2R,4R)-2-(protected-oxymethyl)-4-car- fect the final product ratio of α to β nucleoside product. boxyldioxolane (see compound 7, Figure 2). For example, condensation of the intermediates (2R, [0027] A modified Hunsdiecker reaction (Dhavale, D.; 55 4R)-and(2R,4S)-4-acetoxy-2-(t-butyldiphenylsily- et al., Tetrahedron Lett., 1988, 29, 6163) is then carried oxymethyl) dioxolane (compound 8, Figure 2) with si- out in ethyl acetate with Pb(OAc)4 to convert (+)-(2R, lylated thymidine in the presence of trimethylsilyl triflate 4R)-2-(protected-oxymethyl)-4-carboxyldioxolane to in CH2Cl2 gave a mixture of (-)-1-[(2R,4R)-2-(t-butyld-

4 7 EP 1 600 448 A2 8 iphenylsilyloxymethyl)-4-dioxolanyl]thymine 9-β (45%) mmole) in 60% aqueous dioxane (820 ml) was added and (+)-1-[(2R,4S)-2-(t-butyldiphenylsilyloxymethyl)- concentrated H2SO4 (3.36 ml). The mixture was stirred 4-dioxolanyl] thymine 10-α (29%). However, the reac- at 70ϳ80° C for 15 hours, and then cooled in an ice bath, tion with SnCl4 produced exclusively β-isomer 9 with neutralized with NaHCO3 and concentrated until half of trace amounts of α-isomer 10 detectable on TLC. 5 the original volume remained. The solution was then ex- [0033] In the final step of this method of preparation tracted with ethyl acetate and the combined organic lay- of enantiomerically pure (-)-β-D-dioxolane-nucleosides, ers washed with saturated NaHCO3 solution and water, the 5'-O-position of the nucleoside is deprotected. Des- dried, and evaporated to give 3 as a white solid. The ilylation can be carried out with a variety of reagents, solid was crystallized from CH2Cl2-n-hexane to yield 3 including acetic acid, trifluoroacetic acid, hydrogen flu- 10 (7.4 g, 85.3%) as white solid: [α]25D-154.7° (c, 0.21 1 oride, n-tetrabutylammonium fluoride, potassium fluo- MeOH); H NMR (DMSO-d6): δ 3.56-4.61 (m, 5H, ride and pyridinium HC1. For example, desilylation of 2,3,5,6-H), 4.82 (d, J=8.1 Hz, 1H, OH D2O exchangea- compounds 9 and 10 with tetrabutylammonium fluoride ble), 5.02 (s, 1H, 4-H), 5.09 (d, J=3.7 Hz, 1H, OH, D2O gave the desired free nucleosides 11 and 12, respec- exchangeable), 5.28 (s, 1H, 1-H), 7.46-8.05 (m, 5H, Ar- 15 1 tively (Figure 2). Acetic acid is preferred for commercial H); IR (KBr) 3410, 1710 cm- ; Anal. Calcd for C13H14O6: scale use because it is inexpensive. Other reagents for C, 58.64; H, 5.31. Found: C, 58.51; H, 5.34. desilylation are known to those skilled in the art. Dea- cylation is accomplished in acid or base. 5-O-Ethers can (-)-(2R,4R)-4-(2-Benzoxy-1-hydroxyethyl)- be cleaved with BCl3 or trimethylsilyl iodide. 2-(hydroxymethyl)dioxalane (5). [0034] The method of preparation of enantiomerically 20 pure (-)-β-D-dioxolane-nucleosides is further illustrated [0039] To a solution of 3 (7.4 g, 27.8 mmole) in 95% in the following working example for the preparation of ethanol (200 ml) was added a solution of NaIO4 (6.54 (-)-1-[(2β,4β)-2-(hydroxymethyl)-4-dioxolanyl]thymine, g, 30.7 mmole) in water (200 ml). The mixture was referred to as (-)-β-D-dioxolane-T. The enumeration of stirred at room temperature for 1 hour. After checking to compounds in the working examples refer to structures 25 insure the complete conversion of diol to dialdehyde by set out in Figure 2. (-)-1,6-Anhydro-2,3-isopropyli- thin layer chromatography, the reaction mixture was dene-4-0-benzoyl-β-D-mannopyranose concentrated to the half of the original volume. Methanol [0035] 1,6-anhydro-β-D-mannopyranose (compound (200 ml) was added to the residue and the mixture was 1) was mixed with acetone (800 ml) and methanol (300 cooled to 50°C. Sodium borohydride (4.2 g, 111.0 ml) and stirred for approximately thirty minutes until only 30 mmole) was added to the mixture portion-wise for 5 min- a free-flowing solid remained. Dimethoxypropane (300 utes and the mixture was stirred at 50°C for 10 minutes, ml), and p-toluenesulfonic acid (5 g) were then added, neutralized with glacial acetic acid and concentrated to and the mixture stirred for 2 hours. yield crude 3 as yellow oil. The oil was purified by column [0036] The reaction mixture was then made basic with chromatography over silica gel to yield pure 3 as color- triethylamine (pH 8), and filtered to remove the white sol- 35 less oil, that was crystallized from diethyl ether/n-hex- id material. The solvents were evaporated, and the res- ane to yield 5 (6.12 g, 82%) as white solid: [α]25D - 18.5° 1 idue taken up in ethyl acetate and then crystallized to (C 0.20, methanol) ; H NMR (DMSO-d6): δ 3.47 (dd, obtain 4 grams of the 2,3-isopropylidenated product as J=5.9, 3.7 Hz, 2H, CH2OH), 3.72-4.14 (m, 4H, 4, 5-H clear needles. and CHOH), 4.27-4.95 (m, 2H, CH2OBz), 4.81-4.95 (m, 40 [0037] To a solution of the 1,6-anhydro-2,3-isopropyli- 2-H and pri OH), 5.43 (d, J=5.5 Hz, 1H, sec OH, D2O dene-β-D-mannopyranose (5.01 g, 0.025 mol) in pyrid- exchangeable), 7.43-8.09 (m, 5H, Ar-H) ; Anal. Calcd ine (40 ml) was added dropwise benzoyl chloride (3.74 for C13H16O6: C, 58.19; H, 6.02. Found: C, 58.09; H, ml, 0.062 mol) at 0°C. The mixture was stirred for 45 6.01. minutes at 0°C. Ice was then added to the reaction mixture to remove excess of benzoyl chloride. The solvent 45 (-)-(2R,4R)-4-(2-Benzoxy-1-hydroxyethyl)-2-(t- was evaporated under vacuum and the residue was dis- butyldipbenylsflyloxy-methyl)-dioxolane. solved in ethyl acetate (200 ml). The organic layer was washed with water, sat. NaHCO3 and brine. The result- [0040] To a solution of 3 (2.8 g, 10.4 mmole) and im- ing material was dried over anhydrous MgSO4, filtered, idazole (2.04 g, 30.0 mmole) in dimethylformamide (40 and then evaporated to give (-)-1,6-anhydro-2,3-isopro- 50 ml) was added t-butyldiphenylsilyl chloride (3 ml, 11.5 pylidene-4-0-benzoyl-p-D-mannopyranose crude prod- mmole). The mixture was stirred at room temperature uct (compound 2, 8.7 g) as yellowish solid. for 2 hours. The reaction mixture was evaporated to yield a yellow oil, that was purified by column chroma- (-)-1,6-Auhydro-4-0-benzoyl-β-D-mannopyranose tography over silica gel to yield 4 (4.48 g, 85%) as a (3). 55 colorless oil; [α]25D - 14.2° (C 0.26, methanol); 1H NMR (DMSO-d6): δ 1.00 (s, 9H, t-Bu), 3.68-3.87 (m, 3H, [0038] To a solution of 1,6-anhydro-4-0-benzoyl- CH2OTBDPS and CHOH), 3.98-4.16 (m, 3H, 4,5-H), 2,3-isopropylidene-β-D-mannopyranose 2 (10.0 g, 32.6 4.20-4.55 (m, 2H, CH2OBz), 5.07 (t, J=3.3 Hz, 1H, 2-H),

5 9 EP 1 600 448 A2 10

5.47 (d, J-5.7 Hz, 1H, OH, D2O exchangeable), centrated and purified by column chromatography over 1 7.40-8.33 (m, 10H, Ar-H); Anal. Calcd for C29H34O6Si: silica gel to yield 8 (0.29 g, 63.5%) as a colorless oil: H C, 68.73; H, 6.79. Found: C, 68.86; H, 6.83. NMR (CDCl3) δ 1.06 and 1.10 (s, 9H, t-Bu), 1.92 and 2.06 (s, 1H, CH3), 3.71-4.24 (m, 4H, 5-H and 5 (-)-(2R, 4R)-2-(t-Butyldiphenylsilyloxymethyl)- CH2OTBDPS), 5.25 and 5.38 (t, J=4.3 and 3.3 Hz each, 4-(1,2-dihydrosyethyl)-dioxolane (6). 1H, 2-H), 6.27-6.41 (m, 1H, 4-H), 7.20-7.72 (m, 10H, Ar- H); IR (KBr) 3400, 1620 cm-1. [0041] To a solution of (-)-(2R,4R)-4-(2-benzoxy- 1-hydroxyethyl)-2-(t-butyldiphenylsilyloxy-methyl)-di- (-)-1-[(2R,4R)-2-(t-Butyldiphenylsilyloxymethyl)- oxolane (2.52 g, 5.0 mmole) in methanol (40 ml) was 10 4-dioxolanyl]thymine (9) and (+)-1-[(2R,4S)-2-(t- added a 0.078 M solution of sodium methoxide (7.3 ml) Butyldipbenylsilyloxymethyl)-4-diozolanyl] in methanol. The mixture stirred at room temperature for thymine (10). two hours. The mixture was neutralized with acetic acid and concentrated. The residue was then portioned be- [0044] To a suspension of thymine (0.15 g, 1.2 tween ethyl acetate and water, and the aqueous layer 15 mmole) in haxamethyldisilazane (10 ml) was added a extracted with ethyl acetate. The combined organic lay- catalytic amount of (NH4)2SO4, and the mixture refluxed ers were washed with a saturated NaHCO3 solution and for 3 . hours. The clear solution obtained was concen- then water, and then dried, evaporated, and purified by trated to yield silylated thymine as a colorless oil. A so- column chromatography over silica gel to yield 6 (1.9 g, lution of 8 (0.24 g, 0.6 mmole) in CH2Cl2 (5 ml) was add- 25 1 20 95%) as colorless oil: [α] D-2° (C 0.25, MeOH); H ed to a solution of silylated thymine in CH2Cl2 (5 ml) and NMR (DMSO-d6) δ 1.00 (s, 9H, t-Bu), 3.40-3.52 (m, 3H, the mixture cooled to 5°C. To the cooled mixture was CH2OH and CHOH), 3.64 (d, J=3.7 Hx, 2H, added trimethylsilyl triflate (0.23 ml, 1.2 mmole), and the CH2OTBDPS), 3.82-3.95 (m, 3H, 4.5-H), 4.49 (t, J-5:3 mixture stirred at room temperature for 1 hour under N2. Hz, 1H, pri on, D2O exchangeable), 4.82 (d, J=5.1 Hz, A saturated NaHCO3 solution (20 ml) was added to the 25 1H, sec OH, D2O exchangeable), 5.01 (t, J-3.7 Hz, 1H, mixture, and the mixture again stirred at room temper- 2-H), 7.36-7.71 (m, 10H, Ar-H) ; Anal. Calcd for ature for 30 minutes. The organic layer was then sepa- C22H33H30O5Si:C, 65.63; H, 7.53. Found: C, 65.72; H, rated and the aqueous layer extracted with CH2Cl2. The 7.52. combined organic layer was washed with a saturated NaHCO3 solution and water, dried, concentrated and (+)-(2R,4R)-2-(t-Butyldiphenylsilyloxymethyl)- 30 separated by column chromatography over silical gel to 4-carboxyldioxolane (7). yield 9 (0.125 g, 44.6%) as white foam and 10 (0.08 g, 28.6%) as white foam: 9(β-form); [α]25D - 6.98° (C 0.43, 1 [0042] To a biphasic solution of 6 (1.6 g, 4.0 mmole) MeOH); H NMR (CDCl3) δ 1.08 (s, 9H, t-Bu), 1.67 (s, in CH3CN (8 ml), CCl4 (8 ml) and H2O (12 ml) was added 3H, CH3), 3.92 (d, J=3.2 Hz, 2H, CH2OTBDPS), 4.14 35 NaIO4 (3.59 g, 16.8 mmole) and RuO2 hydrate (8.5 mg). (d, J=4.0 Hz, 2H, 5-H), 5.06 (t, J=3.2 Hz, 1H, 2-H), 6.36 The mixture was vigorously stirred at room temperature (t, J+4.0 Hz, 1H, 4-H), 7.26-7.75 (m, 10H, Ar-H), 9.51 for 5 hours. Methylene chloride (40 ml) was added to (bnr s, 1H, H=NH): UV (MeOH) λmax 265.0 (pH 2); 264.4 the mixture. The organic layer was separated. The nm (pH 11); Anal. Calcd for C25H30O5N2Si: C, 64.34; H, aqueous layer was extracted with CH2Cl2. The com- 6.49; N, 6.00. Found C, 64.28; H, 6.51; N, 5.98: bined organic layers were washed with water, filtered 40 10 (α-form); [α]25D + 11.3° (C 0.23, MeOH); 1H through celite pad and then concentrated to yield crude NMR (CDCl3) δ 1.08 (s, 9H, t-Bu), 1.94 (d, J=1.2 Hz, 3H, 7 (1.2 g, 77.4%) as black oil, that was used in the next CH3), 3.70 (d, J=3.2 Hz, 2H, CH2OTBDPS), 4.01 (dd, reaction without further purification. For analytical pur- J=9.5, 2.3 Hz, 1H, 5H), 4.35 (dd, J=9.5, 5.3 Hz, 1H, 5-H), poses crude 7 was purified by column chromatography 5.55 (t, J=3.2 Hz, 1H, 2-H), 6.32 (dd, J=5.3, 2.3 Hz, 1H, over silica gel to yield 7 as a white foam: [α]25D + 15.7° 45 4-H), 7.17 (d, J=1.2 Hz, 1H, 6'-H), 7.37-7.74 (m, 10H, 1 (C 0.28, MeOH) ; H NMR (DMSO-d6) δ 0.99 (s, 9H, t- Ar-H), 9.57 (br s, 1H, NH); UV (MeOH)λmax 265.0; (pH Bu), 3.43-4.05 (m, 4H, 5-H and CH2OTBDPS), 4.25 (t, 2) ; 264.5 nm (pH 11); Anal. Calcd for C25H30O5N2Si:C, J=6.8 Hz, 1H, 4-H), 5.04 (dd, J=5.1, 3.7 Hz, 1H, 2-H), 64.34; H, 6.49; N, 6.00. Found C, 64.23; H, 6.51; N, 5.93. 7.38-7.72 (m, 10H, Ar-H). 50 (-)-1-[(2R,4R)-2-(Hydroxymethyl)-4-dioxolanyl] (2R,4R)- and (2R,48)-4-Aaetoxy-2-(t- thymine (11). butyldiphenylsilyoxymethyl) dioxolane (8). [0045] To a solution of 9 (93.3 mg, 0.2 mmole) in tet- [0043] To a solution of 7 (0.46 g, 1.14 mmole) in ethyl rahydrofuran (THF) (3 ml) was added a 1.0 M solution acetate (10 ml) was added pyridine (0.09 ml, 1.25 55 of tetra-n-butylammonium fluoride in THF (0.24 ml, 0.24 mmole) and Pb(OAc)4 (0.66 g, 1.49 mmole). The mix- mmole) and the mixture stirred at room temperature for ture was stirred at room temperature for 15 hours under 1 hour. The mixture was then concentrated and purified N2, and then filtered through celite pad, and then con- by column chromatography over silica ge1 to yield 11

6 11 EP 1 600 448 A2 12

(42 mg, 92.1%) as white solid: [α]25D-18.8° (C o,17, Antiviral and Cytotoxic Assay in Human Peripheral 1 MeOH); H NMR (DMSO-d6) δ 1.75 (d, J=1.2 Hz, 3H, Blood Mononuclear Cells. CH3), 3.63 (dd, J+6.0, 2.6 Hz, 2H, CH2OH), 4.03 (dd, J=9.9, 5.5 Hz, 1H, 5-H), 4.22 (dd, J=9.9, 2.0 Hz, 1H, [0050] 5-H), 4.90 (t, J=2.6 Hz, 1H, 2-H), 5.16 (t, J-t.0 Hz, 1H, 5 OH), 6.21 (dd, J=5.5, 2.0 Hz, 1H, 4-H), 7.67 (d, J=1.2 A. Three-day-old phytohemagglutinin-stimulated 6 Hz, 1H, 6'-H), 11.27 (br s, 1H NH); UV (H2) max 266.0 PBM cells (10 cells/ml) from hepatitis B and HIV-1 (ʦ 10757) ; 266.5 (ʦ 9894) (pH 2); 266.3 (ʦ 8397) (pH seronegative healthy donors were infected with 11); Anal. Calcd for C9H12O5N2: C, 47.36; H, 5.31; N, HIV-1 (strain LAV) at a concentration of about 100 12.28. found: c, 47.28; H, 5.34; N, 12.29. 10 times the 50% tissue culture infectious dose (TICD 50) per ml and cultured in the presence and ab- (+)-1-[(2R.4S)-2-(Hydroxymethyl)-4-dioxolanyl] sence of various concentrations of antiviral com- thymine (12). pounds. B. Approximately 45 minutes after infection, the me- [0046] Deprotection of 10 (60 mg, 0.13 mmole) ac- 15 dium, with the compound to be tested (2 times the cording to same procedure as described above for 11 final concentration in medium) or without com- yielded 12 (26 mg, 87.6%) as a white foam: [α]25D+ pound, was added to the flasks (5ml; final volume 1 10.7° (C 0.15, MeOH); H NMR (DMSO-d6) δ 1.79 (s, 10 ml). AZT was used as a positive control. 3H, CH3), 3.43 (dd, J=6.0, 3.7 Hz, 2H, CH2OH), 4.02 C. The cells were exposed to the virus (about 2 x (dd, J=9.5, 3.3 Hz, 1H, 5-H), 4.28 (dd, J=9.5, 5.6 Hz, 20 105 dpm/ml, as determined by reverse transcriptase 1H, 5-H), 5.00 (t, J=6.0 Hz, 1H, OH), 5.47 (t, J=3.7 Hz, assay) and then placed in a CO2 incubator. HIV-1 1H, 2-H), 6.17 (dd, J=5.6, 3.3 Hz, 1H, 4-H), 7.43 (d, (strain LAV) was obtained from the Center for Dis- J=1.2 Hz, 1H, 6'-H), 11.32 (br s, 1H NH); UV (H2O) λmax ease Control, Atlanta, Georgia. The methods used 266.5 (ʦ 9454) ; 266.5 (ʦ 9199) (pH 2) ; 266.3 (ʦ 6925) for culturing the PHM. cells, harvesting the virus and 25 (pH=11); Anal. Calcd for C9H12O5N2; C, 47.36; H, 5.31; determining the reverse transcriptase activity were N, 12.28. found: C, 47.22; H.5.32; N, 12.16. those described by McDougal et al. (J. Immun. Meth. 76, 171-183, 1985) and Spira et al. (J. Clin. II. Anti-HIV Activity of Dioxolane Nucleosides Meth. 25, 97-99, 1987), except that fungizone was not included in the medium (see Schinazi, et al., An- [0047] In contrast to the previous report that β-D-(±)- 30 timicrob. Agents Chemother. 32, 1784-1787 dioxolane-thymine has low efficacy against HIV in ATH8 (1988)). The reverse transcriptase activity in the vi- cells, the enantiomerically pure β form 11 exhibited a rus-infected control was about 2 x 105 dpm per ml. potent anti-HIV activity (EC50 = 0.3 µM). It was surpris- Blank and uninfected cell control values were about ing to discover that enantiomerically pure β-D-(-)-diox- 300 and 1,000 dpm, respectively. Similar results are olane-T has significantly higher anti-HIV activity than the 35 obtained when Step c is performed before step B. racemic mixture of the compound. This difference might D. on day 6, the cells and supernatant were trans- be explained based on the rate of phosphorylation of 11 ferred to a 15 ml tube and centrifuged at about 900 in these systems. As expected, the α-isomer 12 did not g for 10 minutes. Five ml of supernatant were re- exhibit any significant anti-HIV activity. moved and the virus was concentrated by centrifu- [0048] β-D-(-)-Dioxolane-nucleosides can be used as 40 gation at 40,000 rpm for 30 minutes (Beckman 70.1 research tools to inhibit the growth of HIV in vitro, or can Ti rotor). The solukillized virus pellet was processed be administered pharmaceutically to inhibit the growth for determination of the levels of reverse tran- of HIV in vivo. scriptase. Results are expressed in dpm/ml of sam- [0049] The ability of β-D-(-)-dioxolane-nucleosides to pled supernatant. inhibit HIV can be measured by various experimental 45 techniques. The technique used herein, and described [0051] The median effective (EC50) concentration for in detail below, measures the inhibition of viral replica- (-)-1-[(2β,4β)-2-(hydroxymethyl)-4-dioxolanyl]thymine tion in phytohemagglutinin (PHA) stimulated human pe- was determined by the median effect method (Antimi- ripheral blood mononuclear (PBM) cells infected with crob. Agents Chemother. 30, 491-498 (1986). Briefly, HIV-1 (strain LAV). The amount of virus produced is de- 50 the percent inhibition of virus, as determined from meas- termined by measuring the virus-coded reverse tran- urements of reverse transcriptase, is plotted versus the scriptase enzyme. The amount of enzyme produced is micromolar concentration of compound. The EC50 is the compared to an HIV control. The method is described concentration of compound at which there is a 50% in- in detail below. hibition of viral growth. 55 [0052] The EC50 of (-)-1-[(2β,4β)-2-(hydroxymethyl)- 4-dioxolanyl]thymine in PBM cells was measured as 0.2 µM. This activity compares favorably with 2',3'-dideoxy- adenosine (DDA, EC50 = 0.91 µM), 3'-azido-2',3'-dide-

7 13 EP 1 600 448 A2 14 oxyuridine (AZDU, EC50 = 0.18-0.46 µM), and 3'-dide- are not intended to limit the scope or practice of the oxythymidine (DDT, EC50 = 0.17 µM), which are struc- claimed composition. The active ingredient may be ad- turally similar compounds that are undergoing clinical ministered at once, or may be divided into a number of phase testing in the FDA. smaller doses to be administered at varying intervals of 5 time. III. Toxicity of Dioxolane Nucleosides [0059] A preferred mode of administration of the active compound is oral. Oral compositions will generally [0053] Mitogen-stimulated uninfected human PBM include an inert diluent or an edible carrier. They may cells (3.8 x 105 cells/ml) were cultured in the presence be enclosed in gelatin capsules or compressed into tab- and 2absence of drug under similar conditions as those 10 lets. For the purpose of oral therapeutic administration, used for the antiviral assay described above. The cells the active compound can be incorporated with excipi- were counted after 6 days using a hemacytometer and ents and used in the form of tablets, troches, or cap- the trypan blue exclusion method, as described by Schi- sules. Pharmaceutically compatible binding agents, nazi et al., Antimicrobial Agents and Chemotherapy, 22 and/or adjuvant materials can be included as part of the 15 (3), 499 (1982). The IC50 is the concentration of com- composition. pound which inhibits 50% of normal cell growth. [0060] The tablets, pills, capsules, troches and the [0054] The IC50 of (-)-1-[(2β,4β)-2-(hydroxymethyl)- like can contain any of the following ingredients, or com- 4-dioxolanyl]thymine was measured as over 100 µM, in- pounds of a similar nature: a binder such as microcrys- dicating that the compound was not toxic in uninfected talline cellulose, gum tragacanth or gelatin; an excipient PBM cells evaluated up to 100 µM. 20 such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such IV. Preparation of Pharmaceutical Compositions as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as su- [0055] Humans suffering from diseases caused by crose or saccharin; or a flavoring agent such as pepper- HIV infection can be treated by administering to the pa- 25 mint, methyl salicylate, or orange flavoring. tient an effective amount of β-D-(-)-dioxolane-nucleo- [0061] When the dosage unit form is a capsule, it can sides or their salts in the presence of a pharmaceutically contain, in addition to material of the above type, a liquid acceptable carrier or diluent. The active materials can carrier such as a fatty oil. In addition, dosage unit forms be administered by any appropriate route, for example, can contain various other materials which modify the orally, parenterally, intravenously, intradermally, subcu- 30 physical form of the dosage unit, for example, coatings taneously, or topically, in liquid or solid form. of sugar, shellac, or other enteric agents. [0056] The active compound is included in the phar- [0062] β-D-(-)-Dioxolane-nucleosides or their salts maceutically acceptable carrier or diluent in an amount can be administered as a component of an elixir, sus- sufficient to deliver to a patient a therapeutically effec- pension, syrup, wafer, chewing gum or the like. A syrup tive amount of compound to inhibit HIV replication in vivo 35 may contain, in addition to the active compounds, su- without causing serious toxic effects in the patient treat- crose as a sweetening agent and certain preservatives, ed. By "HIV inhibitory amount" is meant an amount of dyes and colorings and flavors. active ingredient sufficient to exert an HIV inhibitory ef- [0063] β-D-(-)-Dioxolane-nucleosides or their salts fect as measured by, for example, an assay such as the can also be mixed with other active materials that do not ones described herein. 40 impair the desired action, or with materials that supple- [0057] These preparations should produce a serum ment the desired action, such as antibiotics, antifungals, concentration of active ingredient of from about 0.2 to antiinflammatories, or other antivirals, including other 40 µM. A preferred concentration range is from 0.2 to nucleoside anti-HIV compounds. 20 µM and most preferably about 1 to 10 µM. [0064] Solutions or suspensions used for parenteral, [0058] The pharmaceutical compositions should pro- 45 intradermal, subcutaneous, or topical application can in- vide a dosage of from 1 to 60 milligrams of compound clude the following components: a sterile diluent such per kilogram of body weight per day. The concentration as water for injection, saline solution, fixed oils, polyeth- of active compound in the drug composition will depend ylene glycols, glycerine, propylene glycol or other syn- on absorption, inactivation, and excretion rates of the thetic solvents; antibacterial agents such as benzyl al- drug as well as other factors known to those of skill in 50 cohol or methyl parabens; antioxidants such as ascorbic the art. It is to be noted that dosage values will also vary acid or sodium bisulfite; chelating agents such as ethyl- with the severity of the condition to be alleviated. It is to enediaminetetraacetic acid; buffers such as acetates, be further understood that for any particular subject, citrates or phosphates and agents for the adjustment of specific dosage regiment should be adjusted over time tonicity such as sodium chloride or dextrose. The paren- according to the individual need and the professional 55 tal preparation can be enclosed in ampoules, disposa- judgment of the person administering or supervising the ble syringes or multiple dose vials made of glass or plas- administration of the compositions, and that the concen- tic. tration ranges set forth herein are exemplary only and [0065] If administered intravenously, preferred carri-

8 15 EP 1 600 448 A2 16 ers are physiological saline or phosphate buffered sa- manner (e.g., by washing, drying, and crystallizing it). line (PBS). [0070] The triphosphate can be prepared according [0066] In a preferred embodiment, the active com- to the procedure of Hoard et al., J. Am. Chem. Soc., 87 pounds are prepared with carriers that will protect the (8), 1785-1788 (1965). For example, β-D-(-)-dioxolane- compound against rapid elimination from the body, such 5 nucleoside is activated (by making a imidazolide, ac- as a controlled release formulation, including implants cording to methods known to those skilled in the art) and and microencapsulated delivery systems. Biodegrada- treating with tributyl ammonium pyrophosphate in DMF. ble, biocompatible polymers can be used, such as eth- The reaction gives primarily the triphosphate of the nu- ylene vinyl acetate, polyanhydrides, polyglycolic acid, cleoside, with some unreacted monophosphate and collagen, polyorthoesters, and polylactic acid. Methods 10 some diphosphate. Purification by anion exchange for preparation of such formulations will be apparent to chromatography of a DEAE column is followed by iso- those skilled in the art. The materials can also be ob- lation of the triphosphate, e.g., as the tetrasodium salt. tained commercially from Alza Corporation and Nova [0071] This invention has been described with refer- Pharmaceuticals, Inc. Liposomal suspensions (includ- ence to its preferred embodiments. Variations and mod- ing liposomes targeted to infected cells with monoclonal 15 ifications of the invention, enantiomerically pure β-D-(-)- antibodies to viral antigens) are also preferred as phar- dioxolane-nucleosides, will be obvious to those skilled maceutically acceptable carriers. These may be pre- in the art from the foregoing detailed description of the pared according to methods known to those skilled in invention. It is intended that all of these variations and the art, for example, as described in U.S. Patent No. modifications be included within the scope of the ap- 4,522,811 (which is incorporated herein by reference in 20 pended claims. its entirety). For example, liposome formulations may be [0072] Further embodiments of the invention include: prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phos- 1. A process for the preparation of an enantiomeri- phatidyl choline, arachadoyl phosphatidyl choline, and cally pure β-D-dioxolane nucleoside or β-D-[(2β, cholesterol) in an inorganic solvent that is then evapo- 25 4β)-2-hydroxymethyl-4-dioxolanyl] nucleoside from rated, leaving behind a thin film of dried lipid on the sur- 1,6-anhydromannose which comprises the steps of: face of the container. An aqueous solution of the active compound or its monophosphate, diphosphate, and/or (i) converting 1,6-anhydromannose to (-)- triphosphate derivatives are then introduced into the 1,6-anhydro-4-O-benzoyl-β-D-mannopyran- container. The container is then swirled by hand to free 30 ose; lipid material from the sides of the container and to dis- (ii) converting the (-)-1,6-anhydro-4-O-ben- perse lipid aggregates, thereby forming the liposomal zoyl-β-D-mannopyranose from step (i) to (2R, suspension. 4R) and (2R,4S)-4-acetoxy-2-(protected- oxymethyl)-dioxolane; V. Preparation of Phosphate Derivatives of β-D-(-) 35 (iii) condensing the (2R,4R) and (2R,4S)-4-ac- -Dioxoinne-Nucleosides etoxy-2-(protected oxymethyl)-dioxolane from step (ii) with a heterocyclic base in the presence [0067] Mono, di, and triphosphate derivative of of SnCl4, other Lewis acid or trimethylsilyl tri- β-D-(-)-dioxolane-nucleosides can be prepared as de- flate to form an enantiomerically pure β-D-[(2β, scribed below. 40 4β)-2-(hydroxymethyl)-4-dioxolanyl] nucleo- [0068] The monophosphate can be prepared accord- side. ing to the procedure of Imai et al., J. Org. Chem., 34(6), 1547-1550 (June 1969). For example, about 100 mg of 2. The process of embodiment 1 further comprising β-D-(-)-dioxolane-nucleoside and about 280 µl of phos- converting 1,6-anhydromannose in step (i) o its phoryl chloride are reacted with stirring in about 8 ml of 45 (2,3)-isopropylidene derivative and then benzoylat- dry ethyl acetate at about 0°C for about four hours. The ing the 2,3-isopropylidene derivative of 1,6-anhy- reaction is quenched with ice. The aqueous phase is pu- dromannose to (-)-1,6-anhydro-4-O-benzoyl- rified on an activated charcoal column, eluting with 5% 2,3-isopropylidene-β-D-mannopyranose. ammonium hydroxide in a 1:1 mixture of ethanol and water. Evaporation of the eluant gives ammoni- 50 3. The process of embodiment 2, further comprising um-(β-D-(-)-dioxolane-nucleoside)-5'-monophosphate. converting the (-)-1,6-anhydro-4-O-benzoyl- [0069] The diphosphate can be prepared according 2,3-isopropylidene-β-D-mannopyranose to (-)- to the procedure of Davisson et al., J. Org. Chem., 52 1,6-anhydro-4-O-benzoyl-β-D-mannopyranose (9), 1794-1801 (1987). β-D-(-)-Dioxolane-nucleosides which is then oxidised to (-)-(2R,4R)-4-(2-benzoxy- can be prepared from the corresponding tosylate, that 55 1-hydroxyethyl)-2-(hydroxymethyl)-dioxolane. can be prepared, for example, by reacting the nucleoside with tosyl chloride in pyridine at room temperature 4. The process of embodiment 3, further comprising for about 24 hours, working up the product in the usual protecting the 2-hydroxy position of the dioxolane

9 17 EP 1 600 448 A2 18

with an oxygen protecting group. the tri-phosphate derivative.

5. The process of embodiment 4, wherein the oxy- 6. The compound of claim 1 wherein the compound is gen protecting group is selected from the group a pharmaceutically acceptable salt. consisting of trimethylsilyl, dimethylhexylsilyl, t- 5 butyldimethylsilyl, t-butyldiphenylsilyl, trityl, alkyl 7. A pharmaceutical preparation for the treatment of a groups, acyl groups, benzoyl, p-NO2 benzoyl, viral infection comprising an enantiomerically pure toluyl, methylsulfonyl, and p-toluylsulfonyl. β-D-dioxolane thymine nucleoside according to any of claims 1 to 6. 6. The process of embodiment 4, further comprising 10 removing the benzoyl group from the 2-hydroxye- 8. The composition of claim 7 wherein the viral infec- thyl-position to produce (-)-(2R,4R)-2-(protected- tion is HIV. O-methyl)-4-(1,2-dihydroxyethyl)-dioxolane. 9. The composition of claim 7 wherein the composition 7. The process of embodiment 6, further comprising 15 is acceptable for oral administration. oxidizing the (+)-(2R,4R)-2-(protected-oxymethyl)- 4-carboxyldioxolane to form a compound selected 10. The composition of claim 7 wherein the composition from the group consisting of (2R,4R)- and (2R,4S)- is acceptable for parenteral administration. 4-acetoxy-2-(protected-oxymethyl)dioxolane. 20 11. The composition of claim 7 wherein the composition 8. The process of embodiment 7, further comprising is acceptable for intradermal administration. converting (2R,4R)- and (2R,4S)-4-acetoxy-2-(protected-oxymethyl)-dioxolane to a product selected 12. The composition of claim 7 wherein the composition from the group consisting of (2R,4R)- and (2R,4S)- is acceptable for subcutaneous administration. 4-acetoxy-2-(protected-oxymethyl)-dioxolane. 25 13. The composition of claim 7 wherein the composition is acceptable for topical administration. Claims 14. The composition of claim 7 wherein the composition 1. An enantiomerically pure β-D-dioxolane thymine 30 is acceptable for controlled rapid release. nucleoside of the formula: 15. The composition of claim 7 wherein the composition is in the form of a dosage form.

35 16. The composition of claim 15 wherein the dosage form is a capsule.

17. The composition of claim 15 wherein the dosage form is a tablet. 40 18. The composition of claim 7 wherein the nucleoside is in combination with another active material.

19. The composition of claim 18 wherein the other ac- or a pharmaceutically acceptable salt or mono-, di- 45 tive material is an antiviral agent. or tri-phosphate derivative thereof. 20. The composition of claim 18 wherein the other ac- 2. The compound of claim 1 wherein the enantiomer- tive material is an antibiotic agent. ically pure nucleoside is (-)-1-[(2β,4β)-2-(hydroxymethyl)-4-dioxolanyl]thymine. 50 21. The composition of claim 18 wherein the other active material is an antifungal agent. 3. The compound of claim 1 wherein the compound is the mono-phosphate derivative. 22. The composition of claim 18 wherein the other active material is an anti-inflammatory agent. 4. The compound of claim 1 wherein the compound is 55 the di-phosphate derivative. 23. A use of an enantiomerically pure β-D-dioxolane thymine nucleoside according to any of claims 1 to 5. The compound of claim 1 wherein the compound is 6 in the manufacture of a medicament for the treat-

10 19 EP 1 600 448 A2 20

ment of a viral infection in a host.

24. The use of claim 23 wherein the viral infection is HIV. 5 25. The use of claim 23 wherein the medicament is acceptable for oral administration.

26. The use of claim 23 wherein the medicament is acceptable for parenteral administration. 10

27. The use of claim 23 wherein the medicament is acceptable for intradermal administration.

28. The use of claim 23 wherein the medicament is ac- 15 ceptable for subcutaneous administration.

29. The use of claim 23 wherein the medicament is acceptable for topical administration. 20 30. The use of claim 23 wherein the medicament is acceptable for controlled rapid release.

31. The use of claim 23 wherein the medicament is in the form of a dosage form as defined in any of 25 claims 15 to 17.

32. The use of claim 23 wherein the nucleoside is in combination with another active material as defined in any of claims 18 to 22. 30

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