A method and reagent for synthesizing nucleosides and their analogs

Abstract

The present invention discloses methods and intermediates for synthesizing nucleosides and nucleoside analogs (NAs). More specifically, the present invention discloses methods for synthesizing nucleosides and NAs using simple achiral materials through a "one-step" proline-catalyzed halogenation reaction of heteroaryl-substituted acetaldehydes, followed by a tandem enantioselective aldol condensation reaction, and then a reduction or organometallic addition and cyclization (cyclization) reaction, including a cyclization halide displacement reaction.

Description

Technical Field

[0001] The present invention relates to the synthesis of nucleosides and their analogs. More specifically, the present invention relates to a method and reagents for synthesizing nucleosides and their analogs. Background Art

[0002] Nucleosides play key roles in various cellular processes from cell signaling to metabolism (1). The prebiotic synthesis of DNA (25) and RNA (26) has been proposed to involve the coupling between nucleobase-type enamine and glyceraldehyde to form a nucleobase iminium ion prior to the furanose by the "ribose end" approach.

[0003] Synthetic nucleoside analogs (NAs), designed to mimic their natural counterparts, are widely used in medicinal chemistry and as tool compounds in chemical biology (2 - 18). NAs have been used in the treatment of cancer (2, 6) and are the largest class of small molecule antiviral drugs (3, 4). Mechanistically, NAs can act as toxic antimetabolites, interfering with nucleic acid synthesis (4). Additionally, after phosphorylation in vivo, the resulting nucleotide analogs can inhibit enzymes involved in cancer cell growth or virus replication (e.g., DNA / RNA polymerases, ribonucleotide reductase, or nucleoside phosphorylase) (2, 4). NAs also show promise as epigenetic regulators, and both decitabine and azacitidine can inhibit DNA methyltransferase and have been approved for cancer treatment (4).

[0004] However, the synthesis process of NAs is often long, not suitable for diversification, and relies on a limited library of chiral carbohydrate starting materials, thus presenting many challenges (e.g., 19 - 24, 27, 33, 42 - 44).

[0005] Locked nucleic acids (LNAs) (39) are conformationally restricted NAs that exhibit better stability, and their binding to antisense oligonucleotides can significantly improve specificity and potency. However, similar to the synthesis of other C4'-modified NAs, the synthesis of LNA is usually long-term. Summary of the Invention

[0006] The present invention relates to the synthesis of nucleosides and their analogs.

[0007] In one aspect, the present invention provides a method for synthesizing a nucleoside or its analog, the method comprising halogenating an aryl- or heteroaryl-substituted acetaldehyde compound by proline catalysis, followed by an enantioselective aldol condensation reaction to generate a halohydrin compound; reducing the halohydrin compound to generate a halohydrin diol compound; and contacting the halohydrin diol compound with a Lewis acid or base in a cyclization halide displacement (AHD) reaction to generate a nucleoside or its analog.

[0008] In some embodiments, the Lewis acid can be InCl3 or Sc(OTf)3.

[0009] In some embodiments, the haloalcohol diol compound can be separated before treatment with a Lewis base.

[0010] In some embodiments, the base can be NaOH.

[0011] In some embodiments, the base-AHD reaction can produce a C3', C5'-protected nucleoside or an analogue thereof.

[0012] In an alternative aspect, the present invention provides a method for preparing an intermediate in the synthesis of a nucleoside or an analogue thereof: by halogenating a heteroaryl-substituted acetaldehyde compound catalyzed by proline, and then performing an enantioselective aldol condensation reaction to generate a haloalcohol compound; reducing the haloalcohol compound to obtain a haloalcohol diol compound to generate an intermediate in the synthesis of a nucleoside or an analogue thereof.

[0013] In an alternative aspect, the present invention provides a method for synthesizing a nucleoside or an analogue thereof: (i) providing a haloalcohol diol compound; (ii) contacting the haloalcohol diol compound with a Lewis acid or a base in a cyclization halide displacement (AHD) reaction to generate a nucleoside or an analogue thereof.

[0014] The summary of the invention does not necessarily describe all features of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] These and other features of the present invention will become more apparent from the following description, which refers to the accompanying drawings including:

[0016] Figure 1 is a schematic diagram of the synthesis of nucleosides and nucleoside analogues (NAs) through a series of reactions, which include an asymmetric α-fluorinated aldol condensation reaction (αFAR), followed by a cyclization (cyclization annulation) reaction including a fluoride displacement (AFD reaction). Het = heteroaryl.

[0017] Figure 2A -C shows the synthesis of pyrazolidinyl NA 17. A: The prebiotic synthesis of nucleosides is thought to involve coupling nucleoside enamines such as 12 with glyceraldehyde by a "ribose end" approach. A method for synthesizing ribose end NAs includes the aldol condensation reaction of iminium ion surrogates 14. B: Examination of the proline-catalyzed α-fluorination and aldol condensation reactions shows that this method is compatible with α-pyrazolaldehyde 15, providing fluoroalcohol 16 in good yield and enantioselectivity. Reduction and cyclization fluoride displacement (AFD) provide a rapid route to NA17. C: Mechanistic studies show that AFD proceeds through stereochemical inversion (S NIt is carried out by a 2 - reaction), followed by epimerization. NFSI = N - fluorobenzenesulfonimide; DMF = dimethylformamide; MeCN = acetonitrile; OTf = trifluoromethanesulfonate.

[0018] Figure 3A -F shows the synthesis of nucleosides and NAs. A: A 4 - step reaction sequence converts readily available starting materials into enantiomerically enriched and naturally configured β - D - NAs. B: NaOH can promote AFD to produce uracil, thymine, pyrazolyl, and 5 - pyrimidinyl nucleosides and NAs. C: AFD to produce trifluoromethyluracil, triazolyl, phthalimide, deazapurine, adenosine nucleosides, and NAs can be promoted by Lewis acids Sc(OTf)3 or InCl3. D: NAs have protective effects on both C3’ and C5’ - alcohol functions. E: Unnatural nucleosides (L - enantiomers) utilize D - proline to catalyze the αFAR reaction. F: C2’ - modified NAs. a TEMPO, BAIB, dioxane (92% from 34). b i) Thiocarbonyldiimidazole, THF; ii) Bu3SnH, azobisisobutyronitrile (2 - step 55% from 35). c i) TEMPO, BAIB, dioxane; ii) MeMgBr, THF, - 78 °C (2 - step 80% from 34). d DAST, CH2Cl2, then HCl, MeOH (53% from 35). TEMPO = 2,2,6,6 - tetramethylpiperidin - 1 - yloxy; BAIB = bis(acetoxy)iodobenzene; THF = tetrahydrofuran; DAST = diethylaminosulfur trifluoride.

[0019] Figure 4A -E shows the rapid synthesis of C4’ - modified and other NAs. A: Adding an organomagnesium reagent to the αFAR product generates a tertiary alcohol, directly performing AFD or Lewis acid / base - promoted AFD to C4’ - modified NAs. B: The large - scale (~380 g) production of fluoroalcohol 55 supports the synthesis of MK - 3682 (an HCV RNA polymerase inhibitor). C: Reductive amination of fluoroalcohol 59 provides a direct route to imino nucleoside 60. D: Utilizing the inherent protection of C3’ and C5’ - OH functions, C4’ - modified C2’ - deoxy NA 62 is prepared. E: The synthesis of two LNAs 65 and 68. a Yield of the keto - fluoroalcohol aldol condensation adduct. b Combined yield of diastereomers. cThe product after heating the crude reaction mixture to 50 °C with CSA and dimethoxyacetone. d The product after treating the crude reaction mixture with aqueous HCl solution. e Starting from a single fluoroalcohol 59. Detailed implementation mode

[0020] Detailed description

[0021] The disclosure part of the present invention provides methods and intermediates for synthesizing nucleosides or their analogs.

[0022] Figure 1 It shows the synthesis of nucleoside analogs (NA) using simple achiral synthetic building blocks through proline-catalyzed α-fluorination and hydroxyaldehyde condensation reactions (α-FAR) and annulative fluorine displacement (AFD). The synthesis includes a one-step method of performing α-fluorination-hydroxyaldehyde condensation reaction on heteroaryl-substituted acetaldehyde 9 through proline catalysis, and then through reduction or organometallic addition and AFD reaction. For example, this process allows for the direct obtaining of C3' / C5'-protected NA 10 (and C2'-modified NA), provides the flexibility of base substitution, provides a direct route to C4'-modified NA, etc.

[0023] In some embodiments, the method includes a complementary (ribose-last) method, which also involves the terminal cyclization of base-imine ions for the synthesis of nucleosides and NA. In a proposed DNA prebiotic synthesis, the coupling between base-type enamine 11 ( Figure 2A ) and glyceraldehyde forms a base imine ion 12 in a "ribose-last" manner before the furanose. As a synthetic equivalent of base imine ion 12, halogenated acyclic NA 13 ( Figure 2A ) is proposed. Without being bound by any specific theory, through the organocatalytic hydroxyaldehyde condensation reaction of dihydroxyacetone derivatives (such as 8)(30) and α-haloaldehyde 14 ( Figure 2A ), the ribonucleoside C2'-C3' bond can be formed and the relative and absolute stereochemistry can be controlled. Therefore, the methods described in the present invention include i) utilizing the reactivity of known unstable haloaldehydes (such as 28, 29, 31, 32, 35) to bind to bases connected at the same position (such as 8) and ii) developing an annulative halide displacement (AHD) reaction in the last step to form the ribose ring.

[0024] In some embodiments, the present invention provides a method for synthesizing nucleosides and NAs, using simple achiral materials, through a short (2-3 step) sequence of reactions, including a "one-step" proline-catalyzed α-halogenation reaction of heteroaryl-substituted acetaldehydes and a tandem enantioselective aldol condensation reaction (αHAR), followed by a reduction or organometallic addition and cyclization (annulation) reaction, including a cyclization (annulation) halide displacement (AHD) reaction.

[0025] More specifically, in some embodiments, the present invention provides a method for synthesizing a nucleoside or an analogue thereof, by:

[0026] (i) halogenating an aryl- or heteroaryl-substituted acetaldehyde compound through proline catalysis to generate an α-haloaldehyde compound, and then coupling with a ketone through proline catalysis to generate a haloalcohol compound.

[0027] (ii) reducing the haloalcohol compound to generate a haloalcohol diol compound; and

[0028] (iii) contacting the haloalcohol diol compound with a Lewis acid or a base in a cyclization halide displacement (AHD) reaction to generate a nucleoside or an analogue thereof.

[0029] In some embodiments, the Lewis acid can be, but is not limited to, a halophilic Lewis acid.

[0030] In some embodiments, the Lewis acid can be, but is not limited to, InCl3 or Sc(OTf)3.

[0031] In some embodiments, Lewis acid-promoted AHD can produce C2', C3'-protected nucleosides or NAs.

[0032] In some embodiments, Lewis acid-promoted AHD may result in the migration of protecting groups, i.e., may produce NAs with a migrated acetonide protecting group.

[0033] In some embodiments, Lewis acid-promoted AHD may result in deprotection.

[0034] In some embodiments, the base can be NaOH.

[0035] In some embodiments, base-promoted AHD can produce C3', C5'-protected NAs.

[0036] In some embodiments, before treatment with a Lewis base, the αHAR reaction product can be reduced and isolated.

[0037] In some embodiments, the present invention provides a method for preparing an intermediate in the synthesis of a nucleoside or a nucleoside analogue, by:

[0038] (i) Halogenating a heteroaryl-substituted acetaldehyde compound through proline catalysis, and then performing an enantioselective aldol condensation reaction to generate a haloalcohol compound;

[0039] (ii) Then reducing the haloalcohol compound to obtain a haloalcohol diol compound, which generates an intermediate when synthesizing a nucleoside or its analog.

[0040] In some embodiments, the present invention provides a method for synthesizing a nucleoside or its analog, by:

[0041] (i) Providing a haloalcohol diol compound; and

[0042] (ii) Contacting the haloalcohol diol compound with a Lewis acid or base in a cyclization halide displacement (AHD) reaction to generate a nucleoside or its analog.

[0043] "Haloalcohol" refers to a compound containing a functional group in which a halogen and a hydroxyl group are attached to adjacent groups. The haloalcohol can have the following general structure, where R 1 , R 2 can be any suitable group, as shown in the present invention, and X is also as shown in the present invention:

[0044]

[0045] In some embodiments, the haloalcohol compound can have the following general structure, where NB and X can be as shown in the present invention:

[0046]

[0047] In some embodiments, the haloalcohol compound can be functionalized with an aryl or heteroaryl group, i.e., NB can be an aryl or heteroaryl group.

[0048] In some embodiments, the haloalcohol diol compound can have the following general structure, where NB and X can be as shown in the present invention:

[0049]

[0050] In some embodiments, the haloalcohol diol compound can be functionalized with an aryl or heteroaryl group, i.e., NB can be an aryl or heteroaryl group.

[0051] In some embodiments, the present invention discloses the following nucleosides or their analogs, including but not limited to their diastereomers, where NB can be as shown in the present invention, and each R can independently be -OH, -OC(CH3)2O-, -(CH2)3-, -CH2SCH2- or -CH2OCH2-:

[0052]

[0053] In some embodiments, the present invention discloses and provides the following compounds or their enantiomers, wherein NB and X can be as shown in the present invention, and each R can independently be -OH, -OC(CH3)2O-, -(CH2)3-, -CH2SCH2-, or -CH2OCH2-, which are intermediates for synthesizing nucleosides or their analogs:

[0054]

[0055] In some embodiments, the present invention provides the following compounds or their enantiomers, wherein NB and X can be as shown in the present invention, Y can be CH2, O, S, NR, where R can be alkyl or aryl, and Z can be a protecting group of ethanol, including but not limited to acetonide, silyl protecting group, alkyl protecting group or aryl protecting group (including cyclic or acyclic groups), which are intermediates for synthesizing nucleosides or their analogs:

[0056]

[0057] In some embodiments, the present invention provides the following compounds or their enantiomers, wherein NB and X can be as shown in the present invention, which are intermediates for synthesizing nucleosides or their analogs:

[0058]

[0059] In some embodiments, the present invention discloses and provides the following compounds or their enantiomers, wherein NB and X can be as shown in the present invention, and Y can be CH2, O, S, NR, where R can be alkyl or aryl, which are intermediates for synthesizing nucleosides or their analogs:

[0060]

[0061] In some embodiments, the method disclosed by the present invention can rapidly obtain intermediates for synthesizing nucleosides or their analogs with good enantioselectivity and / or yield (e.g., greater than 10 g to about 400 g, or any value between 10 g, 15 g, 20 g, 25 g, 50 g, 75 g, 100 g, 125 g, 150 g, 200 g, 250 g, 300 g, 350 g or 400 g). Therefore, the method disclosed by the present invention can be used for the production of nucleosides and / or NAs on an industrial scale.

[0062] In some embodiments, the method disclosed by the present invention can directly obtain C3' / C5'-protected NA3, where R can be alkyl, alkynyl or aryl, NB can be as shown in the present invention (and thus C2'-modified NAs), providing flexibility in base substitution, and / or providing a direct route to C4'-modified NA:

[0063]

[0064] In some embodiments, in the methods disclosed in the present invention, carbonyl reduction followed by cyclization and halide displacement provides β-D-NA in the natural configuration, with both the C3'-OH and C5'-OH functional groups protected.

[0065] In some embodiments, the methods disclosed in the present invention are capable of directly conjugating a wide range of bases and selectively functionalizing the C2' position of the furanose core of natural nucleosides and NAs, including but not limited to C-linked or L-configured NAs.

[0066] In some embodiments, in the methods disclosed in the present invention, replacement of the reducing agent with an organomagnesium reagent provides direct access to arrays of C4'-modified NAs, including but not limited to locked nucleic acids (LNAs).

[0067] In some embodiments, the synthetic methods disclosed in the present invention can be but are not limited to used for the production of D- and L-nucleosides and nucleoside analogs, locked nucleic acids, imino nucleosides, C4'-modified nucleosides, and / or C2'-modified nucleosides.

[0068] In some embodiments, the methods disclosed in the present invention can be used as a tool for drug design.

[0069] In some embodiments, the methods disclosed in the present invention can be used to prepare diversity libraries. For example, the methods described in the present invention can be used to generate larger collections of C4'-modified NAs (e.g., focused screening libraries).

[0070] "Nucleoside" refers to a glycosylamine having a nitrogenous base or "base" or "NB" and a sugar ring (such as ribose or deoxyribose), wherein the anomeric carbon is linked by a glycosidic bond to N9 of a purine (such as adenine or guanine) or N1 of a pyrimidine (such as cytosine, thymine, or uracil). Nucleosides include L- and D-nucleoside isomers. Examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine.

[0071] Nucleoside analogs (NAs) are compounds that are structurally similar to naturally occurring nucleosides. NAs can include, but are not limited to, compounds having modifications at the C1', C2', C3', C4', and / or C5' positions of the sugar ring. In some embodiments, NAs can exist as free triols or can be phosphorylated at C3' and / or C5'. In some embodiments, NAs can include, but are not limited to, compounds having saturated or unsaturated carbocyclic rings. In some embodiments, NAs can include nitrogen in the sugar ring, such as as a replacement for naturally occurring oxygen, and / or may include an N-R group, where R can be, but is not limited to, alkyl, allyl, alkynyl, or benzyl. In some embodiments, NAs that include sulfur in the sugar ring, such as as a replacement for naturally occurring oxygen, are specifically excluded.

[0072] The "NB" or base of an NA can be any aryl or heteroaryl attached to a carbon or nitrogen atom at the C1 position. The NB can also be modified, for example, it can be 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine, 5,5,5-trifluoromethylthymine, 5-fluorouracil, 2-thiouracil, 4-methylbenzimidazole, hypoxanthine, 7-deazaguanine, 7-deazaadenine, indole, imidazole, triazole, pyrrole, pyrazole, etc. It should be understood that the use of D-proline catalysis can produce enantiomers of the aldol condensation product (halohydrin) and can be used to prepare enantiomeric NAs.

[0073] "Aryl" refers to a monocyclic or bicyclic aromatic ring containing only carbon atoms, including, for example, 5 - 14 members, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 members. Examples of aryl include phenyl, biphenyl, naphthyl, indanyl, indenyl, tetrahydronaphthyl, 2,3-dihydrobenzofuranyl, dihydrobenzopyranyl, 1,4-benzodioxanyl, etc. Unless specifically stated herein, the term "aryl" refers to aryl optionally substituted by one or more substituents described in the present invention.

[0074] "Heteroaryl" refers to a single or fused aromatic ring group containing one or more heteroatoms (such as N, O, S) in the ring, including, for example, 5-14 members, such as 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 members. Examples of heteroaryl include furan, thiophene, pyrrole, oxazole, thiazole, imidazole, pyrazole, isoxazole, isothiazole, 1,2,3-oxadiazole, triazole (e.g., 1,2,3-triazole or 1,2,4-triazole), 1,3,4-thiadiazole, tetrazole, pyrazine, pyridine, pyridazine, pyrimidine, 2,6-dichloropyrimidine pyrazine, 1,3,5-triazine, imidazole, benzimidazole, benzoxazole, benzothiazole, indolizine, indole, isoindole, benzofuran, benzothiophene, 1H-indazole, purine, 4H-quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, pteridine, uracil, thymine, deazadenine, phthalimide, adenine, etc. Unless specifically stated otherwise in the present invention, the term "heteroaryl" refers to heteroaryl optionally substituted with one or more substituents described in the present invention.

[0075] Halogen includes bromine, chlorine, fluorine, iodine, etc., and is represented by "X" in the chemical structures disclosed in the present invention. In some embodiments, the halogen may include chlorine or fluorine. Accordingly, "halo" refers to bromine, chlorine, fluorine, iodine, etc. A halide is a halogen atom with a negative charge. The so-called "halogenation" refers to introducing a halogen atom into a compound or molecule.

[0076] "Optional" or "optionally" means that the subsequent described event or situation may or may not occur, and the description includes the situation where the event or situation occurs one or more times and the situation where it does not occur. For example, "optionally substituted alkyl" means that the alkyl may or may not be substituted, and the description includes substituted alkyl and unsubstituted alkyl, and the alkyl may be substituted one or more times. Examples of optionally substituted alkyl include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, etc. Examples of suitable optional substituents include, but are not limited to, H, F, Cl, CH3, OH, OCH3, CF3, CHF2, CH2F, CN, halogen, and C 1-10 alkoxy.

[0077] Unless the context clearly dictates otherwise, the singular forms "a", "and", and "the" as used herein include plural referents. For example, "a compound" refers to one or more of such compounds. In the present invention, the term "compound" or "compounds" refers to the compounds discussed in the present invention, including precursors and derivatives of these compounds. The compounds of the present invention may contain one or more asymmetric centers and can thus occur in racemic and racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. Depending on the nature of the various substituents on the molecule, other asymmetric centers may also exist. Each such asymmetric center will independently give rise to two optical isomers, and it is intended that all possible optical isomers and mixtures of diastereomers, as well as pure or partially purified compounds, be included within the scope of the present invention. Any formula, structure, or name of a compound described in this specification, if no specific stereochemistry is specified, is meant to include any and all of the above-mentioned existing isomers and mixtures thereof in any proportion. When stereochemistry is specified, the present invention is intended to include the specific isomer in pure form or as part of a mixture with other isomers in any proportion. Single enantiomers, i.e., the optically active forms, can be obtained by asymmetric synthesis or by resolution of the racemate. Resolution of the racemate can be accomplished by conventional methods, such as crystallization in the presence of a resolving agent; chromatography using, for example, a chiral high performance liquid chromatography column; or derivatizing the racemic mixture with a resolving agent to produce diastereomers, separating the diastereomers by chromatography, and removing the resolving agent to produce the enantiomerically enriched original compound. These steps can be repeated, if necessary, to increase the enantiomeric purity of the compound. When the compounds described in the present invention contain olefmic double bonds or other geometrically asymmetric centers, these compounds shall include the cis, trans, Z- and E-configurations, unless otherwise specified. Similarly, all isomeric forms are also included.

[0078] The starting materials described in the present invention can be obtained from commercial sources, prepared from commercially available organic compounds, or prepared using known synthetic methods.

[0079] The present invention will be further illustrated in the following examples.

[0080] Examples

[0081] Materials and Methods

[0082] General Considerations

[0083] L- and D-proline (99% purity) were purchased from Alfa Aesar. Unless otherwise stated, all described reactions were carried out at ambient temperature and atmosphere. Column chromatography was performed using 230 - 400 mesh silica gel (E.Merck, Silica Gel 60). Concentration and removal of trace solvents were accomplished using a Buchi rotary evaporator with an acetone - dry ice condenser and a Welch vacuum pump.

[0084] Nuclear magnetic resonance (NMR) spectra were recorded using deuterated chloroform (CDCl3), deuterated methanol (CD3OD), deuterated acetone ((CD3)2CO), deuterated acetonitrile (CD3CN), or deuterated dimethyl sulfoxide (DMSO - d6) as solvents. Signal positions (δ) are given in parts per million relative to tetramethylsilane (δ0), and are measured relative to the signals of the solvents ( 1 1H NMR: CDCl3: δ 7.26; CD3OD: δ 3.31; (CD3)2CO: δ 2.05; CD3CN: δ 1.96; DMSO - d6: δ 2.50; 13 13C NMR: CDCl3: δ 77.16; CD3OD: δ 49.00; (CD3)2CO: δ 29.84; CD3CN: δ 1.32; DMSO - d6: 39.5). Coupling constants (J values) are in hertz (Hz) and are reported to the nearest 0.1 Hz. 1 1H NMR spectral data are listed in the following order: multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; sept, septet; m, multiplet; br, broad), coupling constant, number of protons. NMR spectra were recorded on a Bruker Avance 600 equipped with a QNP or TCI cryoprobe (600 MHz), Bruker 400 (400 MHz), or Bruker 500 (500 MHz). The diastereomeric ratio (dr) is based on the 1 analysis of the crude 1H NMR. 1 The assignment of H is based on the 1 analysis of 1 1H - 1H COSY and nOe spectra. 13 The assignment of C is based on the analysis of HSQC spectra.

[0085] High - performance liquid chromatography (HPLC) analysis was performed on an Agilent 1100 HPLC equipped with a variable - wavelength UV - Vis detector.

[0086] Infrared (IR) spectra were clearly recorded on a Perkin Elmer Spectrum Two FTIR spectrometer. Only selected characteristic absorption data are provided for each compound.

[0087] The optical rotation was measured at 589 nm using a Perkin-Elmer 341 polarimeter.

[0088] General procedure

[0089] General procedure A (one-pot organocatalytic α-fluorination / aldol condensation reaction)

[0090] At 4 °C, a sample of aldehyde (1.5 equiv) was added to a stirred suspension of NFSI (1.5 equiv), L-proline (1.5 equiv), and NaHCO3 (1.5 equiv) in DMF (0.75 M). When complete conversion to α-fluoroaldehyde was observed by 1 1H NMR spectroscopic analysis, a solution of 2,2-dimethyl-1,3-dioxane-5-one (8) (1.0 equiv) in CH2Cl2 or THF or MeCN (1.25× volume of DMF) was then added, and the resulting mixture was warmed to room temperature. After 36 - 72 h, or when complete reaction of 8 was observed by 1H NMR spectroscopic analysis of aliquots of the reaction mixture, the mixture was diluted with CH2Cl2 and the organic layer was washed once with saturated sodium bicarbonate solution and once with water. The organic layer was then dried over MgSO4, concentrated in vacuo, and the crude product was purified by flash chromatography as specified. 1 General procedure B (syn-reduction)

[0091] At -15 °C, tetramethylammonium triacetoxyborohydride (5.0 equiv) and acetic acid (10 equiv) were added to a stirred solution of cis- and trans-fluorohydrins (1.0 equiv) in MeCN (0.10 M). The resulting mixture was stirred for 16 h or until the starting materials had reacted completely (determined by TLC analysis). The reaction mixture was then diluted with a saturated solution of Rochelle salt and washed three times with CH2Cl2. The organic layer was separated, dried over MgSO4, concentrated in vacuo, and the crude product was purified by flash chromatography.

[0092] General procedure C (base-promoted cyclization)

[0093]

[0094] ​To a stirred solution of cis-diol, cis- and trans-fluorohydrin (1.0 equiv) in MeCN (0.10 M) was added 2 M NaOH (2.5 - 10 equiv), and then the reaction mixture was stirred for 5 h or until the starting materials reacted completely (determined by TLC analysis). The reaction mixture was then diluted with CH2Cl2 and washed with saturated ammonium chloride solution. The organic layer was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography.

[0095] General procedure D (Lewis acid promoted cyclization)

[0096] To a stirred solution of cis-diol, cis- and trans-fluorohydrin (1.0 equiv) in MeCN (0.10 M) was added Sc(OTf)3 or InCl3 (0.10 - 2.5 equiv), and then the reaction mixture was stirred for 6 h or until the starting materials reacted completely (determined by TLC analysis). The reaction mixture was then diluted with CH2Cl2 and washed with saturated sodium bicarbonate solution. The organic layer was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography.

[0097] General procedure E (Grignard addition method)

[0098] A stirred solution of fluorohydrin aldol adduct (1 equiv) in CH2Cl2 (0.025 M) was cooled to -78 °C. Then an organomagnesium reagent (2.2 - 5 equiv) was added dropwise, and the resulting reaction mixture was stirred for 5 h. The reaction mixture was then quenched at -78 °C with ammonium chloride:methanol solution (1:1 - saturated ammonium chloride solution:methanol) and warmed to room temperature. The resulting mixture was diluted with CH2Cl2 and washed twice with water. The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure to give the crude product. The crude product was then purified by flash chromatography or used directly for cyclization.

[0099] Preparation and characterization of compounds

[0100] Preparation of S1, aldehyde SM1, aldol adduct A1, diol adducts 18a / 18b, and nucleoside analogs 17, 19, and 34.

[0101] A solution of pyrazole (1.00 g, 14.7 mmol, 1.0 equiv), bromoacetaldehyde diethyl acetal (2.67 mL, 17.6 mmol, 1.2 equiv) and K2CO3 (4.06 g, 29.4 mmol, 2.0 equiv) in DMF (74 mL) was stirred at 90 °C for 36 h. The reaction mixture was then filtered, washed with 40 mL of CH2Cl2 and concentrated under reduced pressure. The crude product S1 was purified by flash chromatography (pentane:ethyl acetate - 7:3) to give SI (2.43 g, yield 90%), which was a colorless oil. A solution of S1 (0.100 g, 0.543, 1.0 equiv) in 0.5 M hydrochloric acid (0.54 mL) was heated at 90 °C for 5 h. After complete conversion to SM1, the reaction mixture was concentrated under reduced pressure and the resulting product SM1 was used for the next reaction without purification.

[0102]

[0103] Data for S1: IR (neat): υ = 2977, 2904, 1516, 1396, 1129, 1063, 751, 621 cm -1 ; 1 H NMR (400 MHz, CDCl3): δ 7.51 (d, J = 1.8 Hz, 1H), 7.46 (d, J = 2.3 Hz, 1H), 6.24 (dd, J = 2.3, 1.8 Hz, 1H), 4.77 (t, J = 5.5 Hz, 2H), 4.22 (d, J = 5.5 Hz, 2H), 3.70 (m, 2H), 3.41 (m, 2H), 1.16 (t, J = 7.1 Hz, 6H); 13 C NMR (125 MHz, CDCl3): δ 139.7, 130.6, 105.6, 101.7, 63.8, 55.2, 15.3 HRMS (EI + ) Calcd for C9H 17 N2O2 [M+H] + 185.1285; found 185.1284

[0104] α-Fluorination / aldol condensation

[0105] According to the conventional procedure A, a solution of SM1 (0.543 mmol), NFSI (0.170 g, 0.543 mmol), L-proline (0.063 g, 0.543 mmol) and NaHCO3 (0.045 g, 0.543 mmol) in DMF (0.72 mL) was stirred at 4 °C for 12 h. Then a solution of 8 (0.043 mL, 0.362 mmol) in MeCN (0.90 mL) was added, and the reaction mixture was stirred at room temperature for 60 h. The crude product fluoroaldehyde A1 (0.060 g, yield 64%, dr 1.4:1) was purified by flash chromatography (pentane:Et2O - 25:75) to afford cis- and trans-fluoroaldehyde A1 as a pale yellow oil.

[0106]

[0107] Data for cis- and trans-fluoroalcohol A1: IR (neat): υ = 2989, 1749, 1446, 1376, 1091, 1042, 764 cm -1 ; 1 1H NMR (600 MHz, CDCl3): δ 7.88, 7.78, 7.63, 6.45, 6.44, 6.39, 6.37, 4.89, 4.50, 4.36, 4.34, 4.31, 4.26, 4.07, 4.04, 1.50, 1.45, 1.45, 1.34; 13 13C NMR (150 MHz, CDCl3): δ 209.0, 207.4, 141.7, 141.4, 131.5, 131.1, 107.7, 107.5, 101.8, 101.4, 95.0, 94.6, 74.3, 72.4, 71.0, 70.2, 67.0, 66.9, 24.0, 23.7, 23.7, 23.4; 19 19F NMR (470 MHz, CDCl3): δ -144.9, -154.1 HRMS (EI + ) Calcd for C 11 H 16 F N2 O4 [M + H] + 259.1089; found 259.1093

[0108] Synchronous reduction of cis- and trans-fluoroalcohol A1

[0109] According to the conventional procedure B, at -15 °C, Me4NHB(OAc)3 (0.968 g, 3.68 mmol) and AcOH (0.442 mL, 7.36 mmol) were added to a stirred solution of A1 (0.190 g, 0.736 mmol) in MeCN (7.36 mL), and the reaction mixture was stirred for 18 h. The crude diols 18a and 18b were purified by flash chromatography (pentane:ethyl acetate - 1:1) to give a mixture of 18a and 18b as a colorless oil (0.151 g, 79% yield, d.r. (cis / trans) = 1:1.2).

[0110]

[0111] Data for cis-diol, cis-fluoroaldehyde 18a: [α] D 20 = +83.2 (c 0.37 in MeCN); IR (neat): υ = 3001, 1442, 1375, 1039, 918, 749 cm -1 ; 1 H NMR (600 MHz, CDCl3): δ 7.68 (d, J = 2.4 Hz, 1H), 7.64 (d, J = 1.5 Hz, 1H), 6.38 (dd, J = 2.4, 1.5 Hz, 1H), 6.18 (d, J = 51.2 Hz, 1H), 4.27 (dd, J = 22.4, 8.8 Hz, 1H), 3.95 (dd, J = 11.1, 5.6 Hz, 1H), 3.93 (dd, J = 9.5, 8.0 Hz, 1H), 3.80 (m, 1H), 3.70 (dd, J = 11.2, 11.0 Hz, 1H), 1.52 (s, 3H), 1.39 (s, 3H); 13 C NMR (150 MHz, CDCl3): δ 141.5, 132.0, 107.2, 99.0, 91.9 (d, J = 211.0 Hz), 72.3 (d, J = 21.8 Hz), 70.6, 67.1, 63.8, 28.7, 19.4; 19 F NMR (470 MHz, CD3CN): δ -150.3 HRMS (EI + ) Calcd for C 11 H 18 FN2O4 [M + H] + 261.1245; found 261.1255

[0112]

[0113] Data for cis-diol, trans-fluoroalcohol 18b: [α] D20 = -10.8 (c 0.91 in MeCN); IR (neat): υ = 3646, 3001, 1443, 1375, 1039, 918 cm -1 ; 1 1H NMR (600 MHz, CDCl3): δ 7.70 (d, J = 0.9 Hz, 1H), 7.65 (d, J = 2.5 Hz, 1H), 6.40 (dd, J = 2.5, 0.9 Hz, 1H), 6.29 (dd, J = 48.4, 2.9 Hz, 1H), 4.41 (ddd, J = 8.0, 4.0, 2.9 Hz, 1H), 3.87 (m, 2H), 3.52 (dd, J = 11.3, 2.7 Hz, 1H), 3.17 (dd, J = 8.8, 8.8 Hz, 1H), 1.34 (s, 3H), 1.16 (s, 3H); 13 13C NMR (150 MHz, CDCl3): δ 142.1, 132.0, 106.9, 98.9, 93.1 (d, J = 207.9 Hz), 76.2 (d, J = 24.7 Hz), 72.2 (d, J = 5.3 Hz), 67.3 (d, J = 4.6 Hz), 63.8, 28.5, 19.3; 19 19F NMR (470 MHz, CD3CN): δ -145.9

[0114] HRMS (EI + ) calculated for C 11 H 18 FN2O4 [M + H] + 261.1245 found 261.1262

[0115] Cyclization of Diols 18a and 18b

[0116]

[0117] Following the conventional procedure C, diols 18a and 18b were cyclized to the same product (17) respectively. The α - anomer generated by the cyclization of 18b's S N 2 cyclized to the thermodynamically more stable β - isomer 17 under the reaction conditions. In addition, taking the 2:1 product mixture (19:17) and following the conventional procedure C, only the β - isomer 17 was obtained. Also note that the e.r. of 17 (95:5) represents the average e.r. of 18a (93:7) and 18b (98:2).

[0118] Following the conventional procedure C, a mixture of 18a and 18b (0.025 g, 0.096 mmol, d.r. (syn / anti) = 1:1) and 2 M NaOH (0.48 mL, 0.962 mmol) was stirred in MeCN (0.96 mL) at 50 °C for 5 h. The crude product 34 was purified by flash chromatography (pentane:ethyl acetate - 65:35) to afford the white solid nucleoside analogue 34 (0.018 g, 76% yield). Sometimes, a product mixture of up to 5:1 (β:α) could be observed.

[0119]

[0120] Data for nucleoside analogue 34: [α] D 20 = -58.9 (c 2.0 in MeCN); IR (neat): υ = 3339, 2926, 1647, 1450, 1397, 1092, 1045, 759 cm -1 ; 1 1H NMR (400 MHz, CD3CN): δ 7.70 (d, J = 2.4 Hz, 1H), 7.56 (d, J = 1.6 Hz, 1H), 6.30 (dd, J = 2.4, 1.6 Hz, 1H), 5.70 (s, 1H), 4.47 (d, J = 4.6 Hz, 1H), 4.12 (dd, J = 9.6, 4.6 Hz, 1H), 4.11 (dd, J = 9.6, 4.6 Hz, 1H), 3.91 (dd, J = 10.3, 9.6 Hz, 1H), 3.83 (dd, J = 9.6, 4.6 Hz, 1H), 3.72 (br s, 1H), 1.54 (s, 3H), 1.43 (s, 3H); 13 13C NMR (100 MHz, CD3CN): δ 141.7, 130.1, 106.7, 101.7, 96.1, 74.7, 74.4, 71.8, 65.9, 29.3, 20.1 HRMS (EI + ) calcd for C 11 H 17 N2O4 [M + H] + 241.1183; found 241.1197

[0121] Deprotection of nucleoside analogue 34

[0122] 34 (0.021 g, 0.088 mmol) was dissolved in MeOD (1.0 mL), and two drops of 1 M HCl were added. Then the solution was left at room temperature for 12 h. Subsequently, the reaction mixture was concentrated under reduced pressure to afford the white solid 17 (0.018 g, 100%).

[0123]

[0124] Data for nucleoside analogue 17: [α] D 20 = +70.4 (c 0.48 in MeOH); IR (neat): υ = 3325, 2944, 2832, 1449, 1022, 631 cm -1 ; 1 H NMR (600 MHz, CD3CN): δ 7.74 (d, J = 2.3 Hz, 1H), 7.58 (d, J = 1.0 Hz, 1H), 6.30 (dd, J = 2.3, 1.0 Hz, 1H), 5.70 (d, J = 4.3 Hz, 1H), 4.51 (m, 1H), 4.33 (m, 1H), 4.08 (br s, 1H), 3.74 (dd, J = 12.3, 2.8 Hz, 1H), 3.67 (d, J = 5.7 Hz, 1H), 3.59 (dd, J = 12.3, 2.5 Hz, 1H), 3.52 (d, J = 4.3 Hz, 1H); 13 C NMR (150 MHz, CD3CN): δ 141.2, 131.1, 106.4, 94.7, 87.2, 76.6, 72.3, 63.4. HRMS (EI + ) Calcd for C8H 13 N2O4 [M + H] + 201.0870; found 201.0870

[0125] Cyclization of diol 18b

[0126] A solution of 18b (0.043 g, 0.165 mmol) and 2 M NaOH (0.21 mL, 0.443 mmol, 2.5 equiv) in MeCN (1.65 mL) was stirred at 50 °C for 3 h. The crude product 19 was purified by flash chromatography (pentane:ethyl acetate - 65:35) to give the white solid nucleoside analogue 19 (0.026 g, 76% yield).

[0127]

[0128] Data for nucleoside analogue 19: [α] D 20 = +72.2 (c 0.98 in MeCN); IR (neat): υ = 3366, 2992, 1306, 1383, 1200, 1076, 754 cm -1 , 11H NMR (600 MHz, CD3CN): δ 7.76 (d, J = 2.3 Hz, 1H), 7.56 (d, J = 1.2 Hz, 1H), 6.35 (d, J = 2.3 Hz, 1H), 5.38 (d, J = 0.9 Hz, 1H), 4.12 (dd, J = 0.9, 2.1 Hz, 1H), 3.94 (d, J = 2.1, 9.7 Hz, 1H), 3.81 (dd, J = 5.0, 10.6 Hz, 1H), 3.59 (m, 2H), 3.37 (m), 1.45 (s, 3H), 1.33 (s, 3H); 13 13C NMR (150 MHz, CDCl3): δ 142.1, 131.0, 108.2, 99.9, 71.8, 65.4, 65.2, 64.7, 59.0, 29.1, 19.9. HRMS (EI + ) Calculated for C 11 H 17 N2O4 [M + H] + 241.1183; found 241.1176

[0129] Determination of the relative stereochemistry of diol 18a

[0130] Diol 18a was converted to the bis-p-nitro-benzoyl ester and recrystallized from ethanol. The relative stereochemistry could then be determined by single X-ray crystallography.

[0131] Determination of the relative stereochemistry of nucleoside analogue 17

[0132] 2D NOESY analysis of nucleoside analogue 17 supported the assigned stereochemistry.

[0133]

[0134] Determination of the relative stereochemistry of nucleoside analogue 19

[0135] 2D NOESY analysis of nucleoside analogue 19 supported the assigned stereochemistry.

[0136]

[0137] Determination of the enantiomeric excess of diol 18a

[0138] The racemate of diol 18a was prepared according to the conventional procedures A and B using a 1:1 mixture of L-:D-proline. Using Enantiomeric diols were separated by chiral HPLC using a 3μm Amylose-1 column at a flow rate of 0.40 mL / min; eluent: hexane-iPrOH 90:10; detection wavelength 210 nm; retention times: (+)-18a at 6.66 min; (-)-18a at 8.10 min. The enantiomeric ratio of optically enriched (+)-18a diol was determined using the same method (93:7 e.r.).

[0139] Determination of enantiomeric excess of diol 18b

[0140] A racemate of diol 18b was prepared using a 1:1 mixture of L-:D-proline according to conventional procedures A and B. Using Enantiomeric diols were separated by chiral HPLC using a 3μm Amylose-1 column at a flow rate of 0.40 mL / min; eluent: hexane-iPrOH 90:10; detection wavelength 210 nm; retention times: (-)-18b at 6.13 min; (+)-18b at 11.72 min. The enantiomeric ratio of optically enriched (-)-18b diol was determined using the same method (98:2 e.r.).

[0141] Determination of enantiomeric excess of nucleoside analogue 34

[0142] A racemate of nucleoside 34 was prepared using a 1:1 mixture of L-:D-proline according to conventional procedures A, B and C. Using Nucleoside enantiomers were separated by chiral HPLC using a 3μm-i-cellulose-5 column at a flow rate of 0.10 mL / min; eluent: hexane-iPrOH 90:10; detection wavelength 254 nm; retention times: (-)-34 at 8.91 min; (+)-34 at 13.32 min. The enantiomeric ratio of optically enriched (-)-34 was determined using the same method (95:5 e.r.).

[0143] Preparation of aldol adduct A2, diol adduct D2 and nucleoside analogues 24, 35 and ent-24

[0144] α-Fluorination / aldol condensation

[0145] Prepare the corresponding starting aldehyde / hydrate SM3 according to the literature procedure (45). According to the conventional procedure A, a solution of aldehyde (1.32 mmol), NFSI (0.416 g, 1.32 mmol), L-proline (0.152 g, 1.32 mmol) and NaHCO3 (0.111 g, 1.32 mmol) was stirred in DMF (1.76 mL) at 4 °C for 12 h. Then a solution of 8 (0.105 mL, 0.880 mmol) in THF (2.64 mL) was added, and the reaction mixture was stirred at 4 °C for 96 h. The crude fluoroaldehyde A2 was purified by flash chromatography (pentane:ethyl acetate - 1:1) to give a mixture of inseparable off-white solids, cis- and trans-fluoroaldehyde A2 (0.159 g, 60% yield, d.r. 1.2:1).

[0146]

[0147] Data for cis- and trans-fluoroaldehyde A2: IR (neat): υ = 3432, 2992, 2900, 1692, 1381, 1079 cm -1 ; 1 1H NMR (600 MHz, CDCl3): δ 8.87, 8.79, 7.74, 7.68, 6.68, 6.67, 5.80, 5.77, 4.53, 4.40, 4.34, 4.33, 4.30, 4.13, 4.11, 4.06, 3.70, 3.48, 1.52, 1.46, 1.44, 1.44; 13 13C NMR (150 MHz, CDCl3): δ 211.3, 208.7, 162.8, 162.6, 150.3, 149.8, 141.7, 141.1, 103.2, 102.6, 102.1, 101.9, 90.7, 90.3, 73.3, 71.4, 70.7, 70.5, 66.6, 66.5, 23.7, 23.6, 23.6, 23.3; 19 19F NMR (470 MHz, CDCl3): δ –162.0, –178.6. HRMS (EI + ) Calcd for C 12 H 16 F N2O6 [M + H] + 303.0987; found 303.0982

[0148] Synchronous reduction of cis- and trans-fluoroalcohol A2

[0149]

[0150] According to conventional step C, diols D2a and D2b are cyclized to the same product (35) respectively. The α-isomer formed by the cyclization of D2b's S N 2 cyclizes to the thermodynamically more stable β-isomer 35.

[0151] According to conventional step B, at -15 °C, Me4NHB(OAc)3 (0.174 g, 0.660 mmol) and AcOH (0.076 mL, 1.32 mmol) were added to a stirred solution of A2 (0.040 g, 0.130 mmol) in MeCN (1.32 mL), and the reaction mixture was stirred for 24 hours. The crude diols D2a and D2b were purified by flash chromatography (pentane:ethyl acetate - 1:3) to give the white solid diols D2a and D2b (0.020 g, 50%, d.r. (cis / trans) = 1.2:1).

[0152]

[0153] Data for cis-diol, cis-fluoroalcohol D2a: 1 H NMR (600 MHz, MeOD): δ 7.76 (d, J = 8.0, 1H), 6.46 (dd, J = 44.4, 4.8 Hz, 1H), 5.73 (d, J = 8.0 Hz, 1H), 4.03 (ddd, J = 18.3, 7.0, 5.0 Hz, 1H), 3.82 (dd, J = 11.4, 5.1 Hz, 1H), 3.71 (m, 2H), 3.60 (dd, J = 11.4, 8.1 Hz, 1H), 1.42 (s, 3H), 1.28 (s, 3H); 13 C NMR (150 MHz, MeOD): δ 165.8, 151.7, 143.1 (d, J = 2.6 Hz), 102.9, 100.1, 94.3 (d, J = 208.4 Hz), 74.6 (d, J = 24.6 Hz), 73.7 (d, J = 4.5 Hz), 67.3, 65.3, 28.3, 19.7. HRMS (EI + ) Calculated for C 12 H 18 FN2O6 [M + H] + 305.1143; found 305.1142

[0154]

[0155] Data for cis-diol, trans-fluoroalcohol D2b: 11H NMR (600 MHz, MeOD): δ 7.90 (d, J = 8.1 Hz, 1H), 6.71 (dd, J = 44.2, 6.1 Hz, 1H), 5.74 (d, J = 8.1 Hz, 1H), 4.32 (m, 1H), 3.81 (m, 3H), 3.60 (m, 1H), 1.43 (s, 3H), 1.32 (s, 3H); 13 13C NMR (150 MHz, MeOD): δ 165.8, 152.2, 143.0, 103.2 100.2, 92.6 (d, J = 204.4), 75.9 (d, J = 2.8 Hz), 71.5 (d, J = 29.1 Hz), 65.7, 64.5 (d, J = 2.2 Hz), 28.6, 19.4. HRMS (EI + ) Calculated for C 12 H 18 FN2O6 [M + H] + 305.1143; found 305.1123

[0156] Cyclization of Diols D2a and D2b

[0157] Following the general procedure C, a solution of D2 (0.022 g, 0.072 mmol, d.r. cis / trans = 1.2:1) and 2 M NaOH (0.36 mL, 0.72 mmol) in MeCN (0.72 mL) was stirred for 24 h. The crude product 35 was purified by flash chromatography (CH2Cl2:MeOH - 92.5:7.5) to afford the white solid nucleoside analogue 35 (0.019 g, 95% yield).

[0158]

[0159] Data for nucleoside analogue 35: [α] D 20 = +48.1 (c 0.90 in MeOH); IR (neat): υ = 2912, 1436, 1407, 1042, 952, 697 cm -1 ; 11H NMR(600MHz,(CD3)2CO):δ7.71(d,J=8.0Hz,1H),5.81(s,1H),5.61(d,J=8.0Hz,1H),4.45(d,J=4.6Hz,1H),4.20(dd,J=9.8,4.7Hz,1H),4.12(dd,J=10.0,10.0Hz,1H),3.90(dd,J=10.0,4.8Hz,1H),3.86(ddd,J=10.0,10.0,4.7Hz,1H),1.56(s,3H),1.42(s,3H); 13 13C NMR(150MHz,(CD3)2CO):δ164.2,151.8,142.4,103.4,102.3,94.5,75.3,74.6,72.5,66.1,33.1,22.8HRMS(EI + )Calculated for C 12 H 17 N2O6[M+H] + 285.1081; found 285.1085

[0160] Deprotection of nucleoside analogue 35

[0161] 35(0.019 g, 0.068 mmol) was dissolved in MeOD(0.68 mL) and two drops of 1M HCl were added, and the solution was left at room temperature for 12 hours. Subsequently, the reaction mixture was concentrated under reduced pressure to give nucleoside 24(0.017 g, 100%) as a white solid. The spectroscopic data was consistent with a previous report(46).

[0162]

[0163] Data for nucleoside 24: [α] D 20 =-23(c = 0.1, MeOH); IR(neat): ν = 3347, 2927, 2857, 1679, 1464, 1381, 1260, 1202, 1104, 1053, 806 cm –1 ; 11H NMR (600 MHz, MeOD): δ 8.03 (d, J = 8.1 Hz, 1H), 5.91 (d, J = 4.7 Hz, 1H), 5.70 (d, J = 8.1 Hz, 1H), 4.18 (dd, J = 4.9, 4.9 Hz, 1H), 4.15 (dd, J = 4.9, 4.9 Hz, 1H), 4.00 - 4.01 (m, 1H), 3.84 (dd, J = 12.2, 2.6 Hz, 1H), 3.74 (dd, J = 12.2, 3.1 Hz, 1H); 13 13C NMR (150 MHz, MeOD): 166.2, 152.5, 142.7, 102.6, 90.6, 86.4, 75.7, 71.3, 62.3 HRMS (EI + ) Calculated for C9H 13 N2O6 [M + H] + 245.0768; found 245.0770

[0164] Determination of the relative stereochemistry of diols D2a and D2b

[0165]

[0166] Based on the J - based configurational analysis of compounds D5a / D5b, D8a / D8b and the XRD analysis of compounds 18a, D7b, D9a, a clear trend was established between the stereochemistry at the fluoromethine center and the chemical shift of the fluoromethine proton (*). In each case, the chemical shift of the cis - fluoroalcohol diol was lower than that of the diastereomeric trans - fluoroalcohol diol. Here, the chemical shift of D2a was 6.46 ppm, while that of the fluoromethine proton of D2b was 6.71 ppm. D2a was assigned as the cis - fluoroalcohol diol and D2b as the trans - fluoroalcohol diol.

[0167] Determination of the relative stereochemistry of nucleoside 35

[0168] The 2D NOESY analysis of nucleoside 35 showed the indicated stereochemistry. In addition, the 1H NMR and 13C NMR of nucleoside 24 were in agreement with the reported data (38). 1 1H NMR and 13 13C NMR were in agreement with the reported data (38).

[0169]

[0170] Determination of the enantiomeric excess of nucleoside enantiomer - 35

[0171] Following the conventional procedures A, B and C and using 1:1 L - :D - proline, a racemate of nucleoside enantiomer - 35 was prepared. Using The 3 μm Amylose-1 column was used to separate nucleoside enantiomers by chiral HPLC; flow rate 0.25 mL / min; eluent: hexane-iPrOH 85:15; detection wavelength 254 nm; retention times: (-)-35 was 19.99 min; (+)-35 was 23.30 min. The enantiomeric ratio of the optically enriched enantiomer-35 was determined using the same method (95:5 e.r.).

[0172] Preparation of the aldol adduct A3, the diol adduct D3 and the nucleoside analogs NA3 and 25

[0173] α-Fluorination / aldol condensation

[0174] The corresponding starting aldehyde / hydrate SM3 was prepared according to the literature procedure (47). A solution of SM3 (0.40 mmol), NFSI (0.126 g, 0.40 mmol), L-proline (0.046 g, 0.40 mmol) and NaHCO3 (0.034 g, 0.40 mmol) in DMF (0.53 mL) was stirred at 4 °C for 14 h according to the conventional procedure A. Then a solution of dioxanone 8 (0.032 mL, 0.27 mmol) in CH2Cl2 (0.67 mL) was added and the reaction mixture was stirred at 4 °C for 96 h. The crude fluoroalcohol A3 (0.072 g, yield 84%, d.r. 1.3:1) was purified by flash chromatography (pentane:ethyl acetate - 3:7) to give the pale white solid fluoroalcohol A3. A mixture of 2 diastereomers and their corresponding isomers (1:1.1:0.65:0.28). Changing the pH of the solution changes the ratio of these products. After reduction, only 2 products (d.r. (syn / anti) = 1.3:1) were present in the crude product.

[0175]

[0176] Data for cis- and trans-fluoroalcohol A3: IR (neat): υ = 2995, 1696, 1451, 1376, 1087, 1049 cm -1 ; 11H NMR (600 MHz, CDCl3): δ 8.65, 8.60, 8.52, 7.57, 7.46, 7.41, 7.23, 6.67, 6.66, 6.64, 6.52, 4.59, 4.54, 4.52, 4.40, 4.39, 4.36, 4.35, 4.35, 4.33, 4.33, 4.32, 4.32, 4.12, 4.11, 4.07, 4.06, 3.67, 3.37, 1.97, 1.95, 1.95, 1.94, 1.52, 1.51, 1.51, 1.49, 1.47, 1.46, 1.45, 1.44; 13 13C NMR (150 MHz, CDCl3): δ 211.4 208.5, 207.9, 206.4, 163.4, 163.2, 163.2, 163.1, 150.8, 150.5, 149.9, 149.9, 137.2, 136.2, 135.7, 134.6, 112.6, 112.0, 111.9, 111.0, 102.1, 102.1, 101.8, 101.7, 91.9, 90.8, 90.7, 90.1, 73.7, 73.0, 71.5, 70.8, 70.6, 70.5, 68.2, 68.0, 67.1, 66.8, 66.6, 66.5, 24.0, 23.9, 23.7, 23.7, 23.7, 23.6, 23.6, 23.4, 12.7, 12.7, 12.7, 12.7; 19 19F NMR (470 MHz, CDCl3): δ –159.9, –161.6, –169.6, –177.8 HRMS (EI + ) Calculated for C 13 H 18 FN2O6 [M + H] + 317.1143; found 317.1142

[0177] Synchronous reduction of cis-fluoroalcohol and trans-fluoroalcohol A3

[0178] According to the conventional procedure B, at -15 °C, Me4NHB(OAc)3 (0.416 g, 1.58 mmol) and AcOH (0.181 mL, 3.16 mmol) were added to a stirred solution of A3 (0.100 g, 0.316 mmol) in MeCN (2.10 mL), and then the reaction mixture was stirred for 18 hours. The crude diol D3a was purified by flash chromatography (pentane:ethyl acetate - 3:7) to give white solids D3a and D3b (0.063 g, yield 63%, d.r. (cis:trans) = 1.3:1).

[0179]

[0180] Data for cis - diol, cis - fluoroalcohol D3a: [α] D 20 = -11.8 (c 1.0 in MeOH); IR (neat): υ = 3363, 2924, 2858, 1674, 1380, 1209, 1075 cm -1 ; 1 1H NMR (600 MHz, CD3CN): δ 7.42 (d, J = 0.90 Hz, 1H), 6.36 (dd, J = 44.9, 5.1 Hz, 1H), 4.04 (ddd, J = 18.1, 6.6, 5.1 Hz, 1H), 3.79 (dd, J = 11.3, 4.5 Hz, 1H), 3.67 (m, 2H), 3.55 (m, 1H), 1.83 (d, J = 0.90 Hz, 3H), 1.39 (s, 3H), 1.24 (s, 3H); 13 13C NMR (150 MHz, CD3CN): δ 164.7, 151.5, 137.9, 111.7, 99.9, 94.0 (d, J = 205.9 Hz), 74.8 (d, J = 25.1 Hz), 73.0 (d, J = 4.3 Hz), 67.1, 65.0, 28.8, 19.9, 12.7; 19 19F NMR (470 MHz, CD3CN): δ –169.1

[0181] For the assignment of relative stereochemistry of cis - diol, cis - fluoroalcohol D3a in MeOD 1 1H NMR is used: 1 1H NMR (600 MHz, MeOD): δ 7.58 (s, 1H), 6.43 (dd, J = 4.1 Hz, 1H), 4.06 (m, 1H), 3.81 (m 1H), 3.71 (m, 2H), 3.59 (m, 1H), 1.89 (s, 3H), 1.41 (s, 3H), 1.26 (s, 3H). HRMS (EI+) calculated for C13H20FN2O6 [M + H]+ 319.1300; found 319.1329

[0182]

[0183] Data for cis - diol, trans - fluoroalcohol D3b: [α] D 20 = +26.2 (c 0.45 in CH3CN); IR (neat): υ = 3360, 2922, 2855, 1670, 1380, 1207, 1078 cm-1 ; 1 1H NMR (600 MHz, MeOD): 7.72 (d, J = 1.1 Hz, 1H), 6.71 (dd, J = 44.3, 6.8 Hz, 1H), 4.32 (m, 1H), 3.82 (m, 3H), 3.60 (m, 1H), 1.90 (d, J = 1.1 Hz, 3H), 1.44 (s, 3H), 1.32 (s, 3H); 13 13C NMR (150 MHz, MeOD): δ 166.1, 152.5, 138.3, 112.0, 100.2, 92.6 (d, J = 204.7 Hz), 75.9, 71.3 (d, J = 29.9 Hz), 65.7, 64.4 (d, J = 2.1 Hz), 28.6, 19.5, 12.4. 19 19F NMR (470 MHz, CD3CN): δ –160.3. HRMS (EI + ) Calculated for C 13 H 20 FN2O6 [M + H] + 319.1300; found 319.1320

[0184] Cyclization of Diols D3a and D3b

[0185]

[0186] According to the conventional procedure C, diols D3a and D3b were cyclized separately to the same product NA3. The α-isomer generated from the S N 2 cyclization of D3b was converted to the thermodynamically more stable β-isomer NA3 after cyclization.

[0187] According to the conventional procedure C, a solution of D3a and D3b (0.100 g, 0.314 mmol, d.r. syn / anti = 1.5:1) and 2 M NaOH (0.236 mL, 0.472 mmol) in MeCN (3.14 mL) was stirred for 10 h. The crude nucleoside NA3 was purified by flash chromatography (ethyl acetate) to give the white solid nucleoside NA3 (0.089 g, 95% yield).

[0188]

[0189] Data for nucleoside NA3: [α] D 20 = +39.4 (c 1.1 in MeCN); IR (neat): ν = 3405, 2993, 1687, 1267, 1138, 845, 734 cm –1 ; 11H NMR (600 MHz, CD3CN): δ 9.04 (br s, 1H), 7.19 (d, J = 1.1 Hz, 1H), 5.67 (s, 1H), 4.22 (dd, J = 4.8, 3.1 Hz, 1H), 4.15 (dd, J = 9.1, 3.5 Hz, 1H), 4.02 (dd, J = 10.1, 9.8 Hz, 1H), 3.70 (m, 2H), 3.55 (m, 1H), 1.85 (d, J = 1.1 Hz, 3H), 1.53 (s, 3H), 1.41 (s, 3H); 13 13C NMR (150 MHz, CD3CN): δ 164.9, 151.6, 137.5, 111.8, 102.3, 93.8, 74.7, 74.1, 72.1, 65.6, 29.6, 20.5, 12.7 HRMS (EI + ) Calculated for C 13 H 19 N2O6 [M + H] + 299.1238; found: 299.1277.

[0190] Deprotection of Nucleoside Analogue NA3

[0191] NA3 (0.010 g, 0.034 mmol) was dissolved in MeOD (0.34 mL) and two drops of 1 M HCl were added, and then the solution was left at room temperature for 12 h. Subsequently, the reaction mixture was concentrated under reduced pressure to give white solid 25 (8.7 mg, 100%). The spectral data was consistent with the previous report (48).

[0192]

[0193] Data of nucleoside analogue 25: [α] D 20 = -33.0 (c = 0.1 in MeOH); IR (neat): ν = 3346, 2928, 2867, 1688, 1466, 1378, 1262, 1200, 1104, 1050, 803 cm –1 ; 1 1H NMR (600 MHz, MeOD): δ 7.86 (d, J = 1.1 Hz, 1H), 5.91 (d, J = 4.6 Hz, 1H), 4.15 - 4.18 (m, 2H), 3.98 - 4.00 (m, 1H), 3.86 (dd, J = 12.2, 2.7 Hz, 1H), 3.75 (dd, J = 12.2, 3.0 Hz, 1H), 1.88 (d, J = 0.9 Hz, 3H); 1313C NMR (150 MHz, MeOD): δ 166.4, 152.7, 138.4, 111.5, 90.3, 86.3, 75.5, 71.3, 62.3, 12.4. HRMS (EI + ) calcd for C 10 H 15 N2O6 [M + H] + 259.0925; found: 259.0923.

[0194] Determination of the relative stereochemistry of diols D3a and D3b

[0195]

[0196] Based on the J-based configurational analysis of compounds D5a / D5b, D8a / D8b and the XRD analysis of compounds 18a, D7b, D9a, a clear trend was established between the stereochemistry at the fluoromethyl center and the chemical shift of the fluoromethyl proton (*). In each case, the chemical shift of the cis-fluoroalcohol diol was lower than that of the diastereomeric trans-fluoroalcohol diol. Here, the chemical shift of D3a was 6.43 ppm, while that of the fluoromethyl proton of D3b was 6.69 ppm. D3a was assigned as the cis-fluoroalcohol diol and D3b as the trans-fluoroalcohol diol.

[0197] Determination of the absolute stereochemistry

[0198] The [α] D 20 value of nucleoside 25 was compared with the literature value, confirming the absolute stereochemistry (49).

[0199] Determination of the enantiomeric excess of nucleoside NA3

[0200] A racemate of nucleoside NA3 was prepared according to the conventional procedures A, B and C using a 1:1 mixture of L-:D-proline. The enantiomeric nucleosides were separated by chiral HPLC using a 3 μm amylose-1 column; flow rate 0.25 mL / min; eluent: hexane-iPrOH 85:15; detection wavelength 254 nm; retention times: (+)-NA3 5.18 min; (-)-NA3 12.61 min. The enantiomeric ratio of optically enriched (+)-NA3 was determined by the same method (91:9 e.r.).

[0201] Preparation of the aldol adduct A4, the diol adducts D4a / D4b and the nucleoside analogue 27

[0202] α-Fluorination / hydroxyalcohol condensation and concurrent reduction of cis- and trans-fluoroalcohols

[0203] According to the conventional procedure A, a solution of 2-(4,6-dichloropyrimidin-5-yl)acetaldehyde (0.250 g, 1.31 mmol, 1 equiv), NFSI (0.413 g, 1.31 mmol, 1 equiv), L-proline (0.151 g, 1.31 mmol, 1 equiv) and sodium bicarbonate (0.110 g, 1.31 mmol, 1 equiv) in DMF (1.19 mL) was stirred at 4 °C for 1 h. Dioxanone 8 (0.521 mL, 4.36 mmol, 3.33 equiv) was added to the reaction mixture and stirred at 4 °C for 24 h. The crude fluoroalcohol A4 was purified by flash chromatography (pentane:ethyl acetate - 3:7) to give fluoroalcohol A4 as an orange oil (0.301 g, 68% yield). According to the conventional procedure B, at -15 °C, Me4NHB(OAc)3 (2.16 g, 8.21 mmol) and AcOH (0.905 mL, 16.4 mmol) were added to a stirred solution of A4 (0.555 g, 1.64 mmol) in MeCN (16.4 mL), and the reaction mixture was stirred for 24 h. The crude diol D4a was purified by flash chromatography (pentane:ethyl acetate - 4:1) to give diol D4a as an off-white solid (0.295 g, 53% yield, d.r., (syn / anti) = 3:1).

[0204]

[0205] Data for cis-diol D4a: [α] D 20 = +26.6 (c 5.0 in MeCN); IR (neat): υ = 3000, 1442, 1375, 1039, 918, cm -1 ; 1 H NMR (600 MHz, CDCl3): δ 8.73 (s, 1H), 6.05 (dd, J = 46.0, 7.9 Hz, 1H), 4.64 (m, 1H), 3.89 (dd, J = 11.5, 5.7 Hz, 1H), 3.80 (m, 1H), 3.73 (dd, J = 9.1, 8.5 Hz, 1H), 3.61 (dd, J = 11.5, 9.5 Hz, 1H), 1.29 (s, 3H), 0.94 (s, 3H); 13 C NMR (150 MHz, CDCl3): δ 161.5, 157.4, 127.8, 98.3, 91.1 (d, J = 179.4 Hz), 75.5 (d, J = 21.3 Hz), 71.7 (d, J = 5.5 Hz), 66.6, 63.3, 28.2, 18.7; 1919F NMR (470 MHz, CDCl3): δ –193.0. HRMS (EI + ) calcd for C 12 H 16 C l2 FN2O4 [M + H] + 341.0466; found 341.0425

[0206] Cyclization of diol D4a

[0207] Following the general procedure C, a solution of D4a (0.014 g, 0.044 mmol, 1 equiv) and 2 M sodium hydroxide (0.11 mL, 0.22 mmol, 5 equiv) in MeCN (0.30 mL) was stirred for 15 min. The crude nucleoside 27 was purified by flash chromatography (ethyl acetate:pentane – 50:50) to afford the white solid nucleoside 27 (6.4 mg, 51% yield).

[0208]

[0209] Data for nucleoside analogue 27: [α] D 20 = +51.2 (c 0.34 in CH2Cl2); IR (neat): υ = 3363, 2927, 1602, 1598, 1571, 1408, 968 cm -1 ; 1 1H NMR (600 MHz, CD3CN): δ 8.66 (s, 1H), 4.19 (dd, J = 10.1, 4.9 Hz, 1H), 3.91 (dd, J = 10.2, 10.1 Hz, 1H), 3.86 (dd, J = 10.1, 4.7 Hz, 1H), 3.30 (ddd, J = 10.2, 10.1, 4.8 Hz, 1H), 1.56 (s, 3H), 1.51 (s, 3H); 13 13C NMR (150 MHz, CD3CN): δ 176.6, 160.8, 158.7, 114.6, 101.7, 82.2, 79.1, 75.6, 69.0, 64.7, 28.9, 19.5. HRMS (EI + ) calcd for C 12 H 14 ClN2O4 [M + H] + 285.0637; found 285.0644

[0210] Determination of the relative stereochemistry of nucleoside 27

[0211]

[0212] 2D NOESY analysis of nucleoside 27 showed the stereochemistry as shown.

[0213] Determination of enantiomeric excess of diol D4a

[0214] A racemate of diol D4a was prepared according to conventional procedures A and B using a 1:1 mixture of L-:D-proline. Using A 3 μm amylose-1 column was used to separate the enantiomeric diols by chiral HPLC; flow rate 0.25 mL / min; eluent: hexane-iPrOH 90:10; detection wavelength 254 nm; retention times: (-)-D4a at 11.81 min; (+)-D4a at 12.68 min. The enantiomeric ratio of (+)-D4a of the optically enriched product was determined by the same method (95:5 e.r.).

[0215] Preparation of S5, hydrate SM5, aldol adduct A5, diol adducts D5a and D5b, and nucleoside analogue 28

[0216] A solution of 1,2,3-triazole (1.00 mL, 17.2 mmol, 1.0 equiv), bromoacetaldehyde diethyl acetal (3.10 mL, 20.7 mmol, 1.2 equiv), and potassium carbonate (4.75 g, 34.4 mmol, 2.0 equiv) in DMF (86 mL) was stirred at 90 °C for 24 h. The reaction mixture was then filtered and washed with 40 mL of dichloromethane and concentrated under reduced pressure. The crude product S5 (pentane:ethyl acetate - 7:3) was purified by flash chromatography to give S5 as a colorless oil (2.90 g, 91% yield). A solution of S5 (0.100 g, 0.54 mmol, 1.0 equiv) in 0.5 M HCl (0.54 mL) was heated to 90 °C for 5 h. When the reaction mixture was completely converted to SM5, it was concentrated under reduced pressure and the resulting product SM5 was used in the reaction without purification.

[0217]

[0218] Data for S5: 1 H NMR (400 MHz, CDCl3): δ 7.68 (d, J = 0.90 Hz, 1H), 7.66 (d, J = 0.90 Hz, 1H), 4.76 (t, J = 5.3 Hz, 1H), 4.48 (d, J = 5.3 Hz, 2H), 3.73 (m, 2H), 3.47 (m, 2H), 1.17 (m, 6H); 13 C NMR (125 MHz, CDCl3): δ 133.8, 124.9, 101.1, 64.0, 52.9, 15.3. HRMS (EI+ )calcd for C8H 16 N3O2 [M+H] + 186.1237;found 186.1233

[0219] α-Fluorination / Hydroxy Aldol Condensation

[0220] According to the conventional procedure A, at 4 °C, a solution of S5 (0.54 mmol), Selectfluor (0.192 g, 0.54 mmol), L-proline (0.063 g, 0.54 mmol) and sodium bicarbonate (0.045 g, 0.54 mmol) in DMF (0.72 mL) was stirred for 12 hours. Then, dioxanone 8 (0.043 mL, 0.36 mmol) in MeCN (0.43 mL) was added, and the reaction mixture was then stirred at room temperature for 72 hours. The crude fluoroalcohol A5 was purified by flash chromatography (Et2O) to give pale yellow oily fluoroalcohol A5 (0.061 g, yield 65%, d.r., 1:1).

[0221]

[0222] Data for cis- and trans-fluoroalcohol A5: IR (neat): υ = 3138, 2990, 1749, 1455, 1379, 1224, 1070, 799 cm -1 ; 1 H NMR (600 MHz, CDCl3): δ 8.24 (1H), 8.12 (1H), 7.79 (1H), 7.77 (1H), 6.89 (1H), 6.86 (1H), 4.74 (1H), 4.49 (1H), 4.33 (2H), 4.26 (1H), 4.14 (1H), 4.06 (1H), 3.89 (1H), 1.55 (3H), 1.48 (3H), 1.44 (3H), 1.31 (3H); 13 C NMR (150 MHz, CDCl3): δ 210.8, 209.4, 134.5, 134.5, 124.4, 124.4, 102.1, 102.0, 94.5, 93.5, 72.1, 71.3, 70.8, 70.1, 66.5, 66.5, 23.8, 23.5, 23.4, 23.4; 19 F NMR (470 MHz, CDCl3): δ -154.6, -163.8. HRMS (EI + ) calcd for C 10 H 15 FN3O4 [M+H] +260.1041; found 260.1044

[0223] Synchronous reduction of cis- and trans-fluoroalcohol A

[0224] According to the conventional procedure B, at -15 °C, Me4NHB(OAc)3 (0.391 g, 1.49 mmol) and AcOH (0.170 mL, 2.98 mmol) were added to a stirred solution of A5 (0.077 g, 0.30 mmol) in MeCN (3.00 mL). The resulting mixture was stirred for 24 h. The crude diols D5a and D5b were purified by flash chromatography (CH2Cl2:MeOH - 96:4) to give the white solid diols D5a and D5b (0.072 g, yield 94%, d.r. (syn / anti) = 1.2:1).

[0225]

[0226] Data for cis-diol, cis-fluoroalcohol D5a: [α] D 20 = +52.4 (c 0.51 in MeCN); IR (neat): υ = 3432, 2997, 2253, 1444, 1375, 1071, 1039 cm -1 ; 1 1H NMR (600 MHz, CD3CN): δ 8.17 (d, J = 1.0 Hz, 1H), 7.78 (d, J = 1.0 Hz, 1H), 6.69 (dd, J = 48.1, 4.7 Hz, 1H), 4.36 (ddd, J = 18.4, 5.0, 5.0 Hz, 1H), 3.79 (dd, J = 11.4, 5.0 Hz, 1H), 3.63 (m, 2H), 3.54 (m, 2H), 1.39 (s, 3H), 1.31 (s, 3H); 13 13C NMR (150 MHz, CD3CN): δ 135.2, 126.2, 100.0, 95.9 (d, J = 206.7 Hz), 74.7 (d, J = 22.7 Hz), 73.1 (d, J = 4.4 Hz), 66.0, 65.2, 28.8, 19.9; 19 19F NMR (470 MHz, CDCl3): δ -156.0 HRMS (EI + ) calcd for C 10 H 17 F N3 O4 [M + H] + 262.1198; found 262.1209.

[0227]

[0228] Data of cis - diol, trans - fluoroalcohol D5b: [α] D 20 = +40.0 (c 0.37 in MeCN); IR (neat): υ = 3000, 1442, 1375, 1039, 918, 740 cm -1 ; 1 1H NMR (600 MHz, CD3CN): δ 8.22 (d, J = 1.0 Hz, 1H), 7.79 (d, J = 1.0 Hz, 1H), 6.78 (dd, J = 46.4, 6.0 Hz, 1H), 4.53 (ddd, J = 10.4, 6.0, 4.7 Hz, 1H), 4.09 (br s, 1H), 3.83 (m, 2H), 3.57 (m, 2H), 3.41 (br s, 1H), 1.35 (s, 3H), 1.34 (s, 3H); 13 13C NMR (150 MHz, CD3CN): δ 135.3, 125.7, 100.0, 96.5 (d, J = 204.3 Hz), 74.2 (d, J = 2.3 Hz), 72.9 (d, J = 27.2 Hz), 65.4, 65.3 (d, J = 2.0 Hz), 28.9, 19.8; 19 19F NMR (470 MHz, CDCl3): δ -151.2 HRMS (EI + ) calcd for C 10 H 17 FN3O4 [M + H] + 262.1198; found 262.1206

[0229] Cyclization of diol D5a

[0230]

[0231] According to the conventional procedure D, diol D5a was cyclized alone to 28, while diol D5b was not cyclized. This indicates that the product formed from the diol mixture is only cyclized from D5a diol via S N 2 cyclization.

[0232] According to the conventional procedure D, a solution of D5a and D5b (0.025 g, 0.096 mmol, 1.0 equiv., d.r. (syn / anti) = 1.2:1) and Sc(OTf)3 (0.118 g, 0.239 mmol, 2.5 equiv.) was stirred in dry MeCN (1.00 mL). After 12 h, pyridine (0.50 mL) and acetic anhydride (0.25 mL) were added, and the reaction mixture was stirred for 3 h. The crude 28 was purified by flash chromatography (pentane:ethyl acetate - 1:3) to afford the nucleoside analogue 28 (0.015 g, 47% yield) as a clear colorless oil.

[0233]

[0234] Data for nucleoside analogue 28: [α] D 20 = +1.3 (c 0.60 in CH2Cl2); IR (neat): υ = 2926, 1747, 1373, 1227, 1064 cm -1 ; 1 H NMR (600 MHz, CDCl3): δ 7.76 (s, 1H), 7.26 (s, 1H), 6.19 (d, J = 3.7 Hz, 1H), 5.85 (dd, J = 5.0, 3.8 Hz, 1H), 5.63 (dd, J = 5.3, 5.0 Hz, 1H), 4.49 (ddd, J = 5.3, 4.3, 3.0 Hz, 1H), 4.41 (dd, J = 12.4, 3.0 Hz, 1H), 4.22 (dd, J = 12.4, 4.3 Hz, 1H), 2.13 (s, 3H), 2.13 (s, 3H), 2.06 (s, 3H); 13 C NMR (150 MHz, CDCl3): δ 170.5, 169.6, 169.5, 134.3, 122.9, 90.0, 81.0, 74.5, 70.8, 62.9, 20.8, 20.6, 20.6; HRMS (EI + ) calcd for C 13 H 18 N3O7 [M + H] + 328.3005; found 328.3000

[0235] Determination of the relative stereochemistry of diol D5a

[0236]

[0237] The relative stereochemistry of diol D5a was determined by J-based configurational analysis. For details, see the J-based configurational analysis section.

[0238] Determination of the relative stereochemistry of diol D5b

[0239]

[0240] The relative stereochemistry of diol D5b was determined using J-based configurational analysis. For details, see the J-based configurational analysis section.

[0241] Determination of the relative stereochemistry of nucleoside 28

[0242]

[0243] The 2D NOESY analysis of nucleoside 28a supported the shown stereochemistry.

[0244] Determination of the enantiomeric excess of diol D5a

[0245] A racemate of diol D5a was prepared using a 1:1 mixture of L-:D-proline according to conventional procedures A and B. Using a 3 μm i-Cellulose-5 column, the enantiomeric diols were separated by chiral HPLC; flow rate 0.20 mL / min; eluent: hexane-iPrOH 90:10; detection wavelength 210 nm; retention time of (+)-D5a was 4.69 min; retention time of (-)-D5a was 5.80 min. The enantiomeric ratio of the optically enriched (+)-D5a diol was determined using the same method (93:7 e.r.).

[0246] Determination of the enantiomeric excess of diol D5b

[0247] A racemate of diol D5b was prepared using a 1:1 mixture of L-:D-proline according to conventional procedures A and B. Using a 3 μm i-Cellulose-5 column, the enantiomeric diols were separated by chiral HPLC; flow rate 0.20 mL / min; eluent: hexane-iPrOH 90:10; detection wavelength 210 nm; retention time: (-)-D5b was 3.94 min; retention time of (+)-D5b was 4.95 min. The enantiomeric ratio of the optically enriched (+)-D5b diol was determined using the same method (96:4 e.r.).

[0248] Determination of the enantiomeric excess of ent-D5a diol

[0249] A racemate of diol ent-D5a was prepared using a 1:1 mixture of L-:D-proline according to conventional procedures A and B. Using The 3 μm i-Cellulose-5 column was used to separate enantiomeric diols by chiral HPLC; flow rate 0.20 mL / min; eluent: hexane-iPrOH 90:10; detection wavelength 210 nm; retention times: (+)-D5a at 4.69 min; (-)-D5a at 5.80 min. The enantiomeric ratio of optically enriched ent-D5a diol was determined by the same method (95:5 e.r.).

[0250] Determination of enantiomeric excess of ent-D5b diol

[0251] According to the conventional procedures A and B, a racemate of diol ent-D5b was prepared using a 1:1 mixture of L-:D-proline. Using The 3 μm i-Cellulose-5 column was used to separate enantiomeric diols by chiral HPLC; flow rate 0.20 mL / min; eluent: hexane-iPrOH 90:10; detection wavelength 210 nm; retention times: (-)-D5b at 3.94 min; (+)-D5b at 4.95 min. The enantiomeric ratio of optically enriched ent-D5b diol was determined by the same method (95:5 e.r.).

[0252] Preparation of S6, hydrate SM6, aldol condensation adduct A6, diol adducts D6a and D6b, and nucleoside analogue 29

[0253] A solution of trifluoromethyluracil (1.00 g, 5.52 mmol, 1.0 equiv), bromoacetaldehyde diethyl acetal (1.66 mL, 11.1 mmol, 2.0 equiv) and potassium carbonate (1.53 g, 11.1 mmol, 2.0 equiv) in DMF (27.6 mL) was stirred at 90 °C for 24 h. The reaction mixture was then filtered and washed with 40 mL of CH2Cl2 and concentrated under reduced pressure. The crude S6 was purified by flash chromatography (pentane:ethyl acetate - 7:3) to give S6 as a colorless oil (0.605 g, 37% yield). A solution of S7 (0.100 g, 0.340 mmol, 1.0 equiv) in 0.5 M HCl (0.34 mL) was heated to 90 °C for 5 h. After complete conversion to the aldehyde / hydrate SM6, the reaction mixture was concentrated under reduced pressure and the resulting aldehyde / hydrate SM6 could be used directly in the reaction without purification.

[0254]

[0255] Data for S6: IR: υ = 3430, 2988, 2800, 1109, 1025 cm -1 ; 11H NMR (600 MHz, CDCl3): δ 8.56 (brs, 1H), 7.82 (s, 1H), 4.61 (t, J = 5.0 Hz), 3.88 (d, J = 5.0 Hz), 3.78 (m, 2H), 3.54 (m, 2H), 1.21 (m, 6H); 13 13C NMR (150 MHz, CDCl3): δ 158.6, 150.0, 147.0 (q, J = 5.8 Hz), 121.9 (q, J = 270.5 Hz), 104.7 (q, J = 33.5 Hz), 100.0, 64.6, 51.0, 15.3. HRMS (EI + ) calcd for C 11 H 16 F3N2O4 [M + H] + 297.1057; found 297.1056

[0256] α-Fluorination / Aldol Condensation

[0257]

[0258] According to the conventional procedure A, at 4 °C, a solution of SM6 (0.340 mmol), NFSI (0.107 g, 0.340 mmol), L-proline (0.039 g, 0.340 mmol), and sodium bicarbonate (0.029 g, 0.340 mmol) in DMF (0.45 mL) was stirred for 12 hours. Then, a solution of dioxanone 8 (0.027 mL, 0.227 mmol) in dichloromethane (0.57 mL) was added, and the reaction mixture was stirred at 4 °C for 96 hours. The crude fluoroalcohol A6 (pentane:ethyl acetate - 65:35) was purified by flash chromatography to give fluoroalcohol A6 as a pale yellow oil (0.050 g, 60% yield).

[0259] Data for cis- and trans-fluoroalcohol A6: IR: υ = 2991, 1699, 1450, 1087, 1049 cm -1 ; 1 1H NMR (600 MHz, CD3CN): δ 9.53, 9.52, 8.15, 8.11, 6.58, 6.46, 4.62, 4.56, 4.55, 4.43, 4.31, 4.29, 3.98, 3.98, 1.43, 1.40, 1.40, 1.38; 1313C NMR (150 MHz, CD3CN): δ 208.4, 207.9, 159.6, 159.5, 150.6, 150.1, 144.0, 144.0, 123.6, 123.5, 106.6, 106.0, 102.4, 102.3, 95.3, 92.4, 76.3, 76.1, 69.9, 69.1, 67.9, 67.8, 24.5, 24.4, 24.2, 23.9; 19 19F NMR (470 MHz, CD3CN): δ –64.1, –64.1, –161.4, –169.1. HRMS (EI + ) calcd for C 13 H 14 F4N2NaO6 [M+Na] + 393.0680; found 393.0682

[0260] Synchronous reduction of cis- and trans-Fluoroalcohol A6

[0261] According to the conventional procedure B, at -15 °C, Me4NHB(OAc)3 (0.355 g, 1.35 mmol) and AcOH (0.155 mL, 2.79 mmol) were added to a stirred solution of A6 (0.100 g, 0.27 mmol, 1 equiv) in MeCN (1.80 mL). Then the reaction mixture was stirred for 24 h. The crude diols D6a and D6b were purified by flash chromatography (pentane:ethyl acetate - 4:1) to give the white solid diol D6a (0.040 g, 40% yield) and D6b (0.019 g, 19% yield).

[0262]

[0263] Data for cis-diol, cis-Fluoroalcohol D6a: [α] D 20 = +18.4 (c 0.50 in CH2Cl2); IR (neat): υ = 3426, 2996, 1702, 1463, 1379, 1070 cm -1 ; 11H NMR (600 MHz, CD3CN): δ 9.42 (br s, 1H), 8.10 (s, 1H), 6.33 (dd, J = 45.1, 5.6 Hz, 1H), 4.28 (dd, J = 14.8, 5.6 Hz, 1H), 3.79 (dd, J = 11.1 5.5 Hz, 1H), 3.70 (m, 2H), 3.60 (dd, J = 9.5, 2.7 Hz, 1H), 3.55 (dd, J = 10.4, 9.5 Hz, 1H), 1.35 (s, 3H), 1.30 (s, 3H); 13 13C NMR (150 MHz, CD3CN): δ 159.5, 150.1, 144.2 (q, J = 6.3 Hz), 123.5 (q, J = 266.4 Hz), 106.3 (q, J = 32.9 Hz), 99.9, 96.3 (d, J = 210.9 Hz), 73.9 (d, J = 3.8 Hz), 70.5 (d, J = 24.5 Hz), 65.4, 63.0, 29.1, 19.8; 19 19F NMR (470 MHz, CD3CN): δ –64.1, –168.0. HRMS (EI + ) calcd for C 13 H 17 F4N2NaO6 [M+Na] + 395.0837; found 395.0836.

[0264]

[0265] Data for cis - diol, trans - fluoroalcohol D6b: [α] D 20 = -37.2 (c 1.1 in CH2Cl2); IR (neat): υ = 3424, 1703, 1466, 1379, 1281, 1138, 1042 cm -1 ; 1 1H NMR (600 MHz, CD3CN): δ 8.26 (s, 1H), 6.67 (dd, J = 43.0, 4.9 Hz, 1H), 4.34 (m, 1H), 3.78 (dd, J = 11.2, 5.1 Hz, 1H), 3.72 (m, 2H), 3.54 (dd, J = 11.2, 8.3 Hz, 1H), 1.39 (s, 3H), 1.26 (s, 3H); 1313C NMR (150 MHz, CD3CN): δ 159.5, 150.6, 144.2, 123.6 (q, J = 272.9 Hz), 105.9 (q, J = 32.5 Hz), 100.0, 92.5 (d, J = 206.1 Hz), 74.2 (d, J = 4.4 Hz), 72.3 (d, J = 27.7 Hz), 65.4, 64.8, 29.0, 19.7; 19 19F NMR (470 MHz, CD3CN): δ –64.1, –161.7. HRMS (EI + ) calcd for C 13 H 17 F4N2NaO6, [M+Na] + 395.0837; found 395.0838.

[0266] Cyclization of Diols D6a and D6b

[0267]

[0268] According to the conventional procedure D, diol D6b was cyclized alone to 29, while diol D6a did not cyclize. This indicates that the product generated from the diol mixture is formed only by the S N 2 cyclization of D6b diol.

[0269] According to the conventional procedure D, a solution of D6a and D6b (0.045 g, 0.121 mmol, d.r. (syn / anti) = 1:2) and Sc(OTf)3 (8.9 mg, 0.018 mmol, 0.15 equiv) in dry MeCN (1.21 mL) was stirred for 24 h. The crude 29 was purified by flash chromatography (pentane:ethyl acetate - 3:7) to give the nucleoside 29 as a colorless oil (0.013 g, 45% yield (from trans - fluoroalcohol D6b)).

[0270]

[0271] Data for nucleoside analogue 29: [α] D 20 = -16.7 (c 0.49 in CH2Cl2); IR (neat): υ = 3405, 2924, 2854, 1702, 1465, 1276 cm -1 ; 1HNMR(600MHz,CD3CN):δ9.33(br s,1H),7.97(q,J=1.2Hz,1H),6.18(d,J=4.1Hz,1H),4.86(m,2H),4.42(dd,J=3.6,2.4Hz,1H),3.67(m,2H),3.21(dd,J=5.6,4.4Hz,1H),1.36(s,3H),1.30(s,3H); 13 C NMR(150MHz,CD3CN):δ159.4,149.9,143.6(q,J=6.0Hz),123.6(q,J=269.7Hz),113.6,103.4(q,J=33.2Hz),87.7,84.7,82.8,80.2,64.0,25.7,24.0; 19 F NMR(470MHz,CD3CN):δ–63.8HRMS(EI + )calcd forC 13 H 16 F3N2O6[M+H] + 353.0955;found 353.0971

[0272] Determination of the relative stereochemistry of nucleoside 29

[0273]

[0274] The 2D NOESY analysis of nucleoside 29 supported the shown stereochemistry.

[0275] Determination of the relative stereochemistry of diols D6a and D6b

[0276]

[0277] Based on the J-based configurational analysis of compounds D5a / D5b, D8a / D8b and the XRD analysis of compounds 18a, D7b, D9a, a clear trend was established between the stereochemistry at the fluoromethyl center and the chemical shift of the fluoromethyl proton (*). In each case, the chemical shift of the cis-fluorohydrin diol was lower than that of the trans-fluorohydrin diol. Here, the chemical shift of D6a was 6.33 ppm, while that of the fluoromethyl proton of D6b was 6.67 ppm. D6a was the cis-fluorohydrin diol and D6b was the trans-fluorohydrin diol.

[0278] Determination of the enantiomeric excess of nucleoside 29

[0279] According to the conventional procedures A, B and C, using a 1:1 mixture of L-:D-proline, a racemate of nucleoside 29 was prepared. Using Enantiomeric nucleosides were separated by chiral HPLC on a 3 μm amylose-1 column; flow rate 0.25 mL / min; eluent: hexane-iPrOH 90:10; detection wavelength 254 nm; retention times: (+)-29 was 9.10 min; (-)-29 retention time was 13.14 min. The enantiomeric ratio of optically enriched (-)-29 nucleoside was determined by the same method (94:6 e.r.).

[0280] Preparation of S7, hydrate SM7, aldol adduct A7, diol adducts D7a and D7b, and nucleoside analogue 30.

[0281] α-Fluorination / Aldol Condensation and Concurrent Reduction of cis- and trans-Fluoroalcohol A

[0282] According to conventional procedure A, a solution of phthalimidoacetaldehyde (0.100 g, 0.529 mmol, 1.5 equiv), NFSI (0.167 g, 0.529 mmol, 1.5 equiv), L-proline (0.061 g, 0.529 mmol, 1.5 equiv), and 2,6-lutidine (0.061 mL, 0.529 mmol, 1.5 equiv) in DMF (0.71 mL) was stirred at 4 °C for 12 h. A solution of dioxanone 8 (0.042 mL, 0.353 mmol, 1 equiv) in dichloromethane (0.88 mL) was added, and the reaction mixture was stirred at room temperature for 48 h. The crude fluoroalcohol A7 was purified by flash chromatography (pentane:ethyl acetate - 1:1) to give fluoroalcohol A7 as a yellow oil (0.069 g, 58% yield, d.r. 2.2:1). According to conventional procedure B, at -15 °C, Me4NHB(OAc)3 (0.776 g, 2.95 mmol) and AcOH (0.337 mL, 5.90 mmol) were added to a stirred solution of A7 (0.200 g, 0.59 mmol) in MeCN (5.90 mL), and the reaction mixture was stirred for 24 h. The crude diols D7a and D7b were purified by flash chromatography (pentane:ethyl acetate - 3:7) to give the white solid diols D7a and D7b (0.094 g, 47% yield, d.r. (syn / anti) = 1.5:1).

[0283]

[0284] Data for cis-diol, cis-fluoroalcohol D7a: [α] D 20 = -11.4 (c 2.0 in CH2Cl2); IR (neat): υ = 3442, 2992, 1785, 1724, 1377, 1074, 721 cm-1 ; 1 1H NMR (600 MHz, CD3CN): δ 7.93 (m, 2H), 7.89 (m, 2H), 6.07 (dd, J = 48.6, 7.9 Hz, 1H), 4.76 (m, 1H), 4.43 (m, 1H), 3.73 (m, 2H), 3.58 (dd, J = 8.8, 6.0 Hz, 1H), 3.47 (m, 1H), 3.41 (m, 1H), 1.21 (s, 3H), 0.92 (s, 3H); 13 13C NMR (150 MHz, CD3CN): δ 167.8 (d, J = 1.5 Hz), 136.0, 132.5, 124.6, 99.1, 91.1 (d, J = 202.0 Hz), 73.3 (d, J = 6.6 Hz), 71.8 (d, J = 25.3 Hz), 65.1, 64.5, 28.1, 19.3; 19 19F NMR (470 MHz, CD3CN): δ –157.8 HRMS (EI + ) calcd for C 16 H 19 FNO6 [M + H] + 340.1191; found 340.1190.

[0285]

[0286] Data of cis - diol, trans - fluoroalcohol D7b: [α] D 20 = -1.0 (c 2.3 in CH2Cl2); IR (neat): υ = 3442, 2992, 1784, 1725, 1375, 1070, 723 cm -1 ; 1 1H NMR (600 MHz, CD3CN): δ 7.94 (m, 2H), 7.89 (m, 2H), 6.34 (dd, J = 46.0, 9.2 Hz, 1H), 4.80 (m, 1H), 3.92 (ddd, J = 9.5, 1.8, 1.4 Hz, 1H), 3.84 (m, 2H), 3.73 (m, 1H), 3.60 (dd, J = 10.8, 8.7 Hz, 1H), 3.30 (m, 1H), 1.47 (s, 3H), 1.35 (s, 3H); 1313C NMR (150 MHz, CD3CN): δ 168.1 (d, J = 1.6 Hz), 136.0, 132.3, 124.6, 99.4, 89.5 (d, J = 202.4 Hz), 75.1, 68.7 (d, J = 31.7 Hz), 65.3, 63.1 (d, J = 3.1 Hz), 28.6, 19.5; 19 19F NMR (470 MHz, CDCl3): δ –159.8. HRMS (EI + ) calcd for C 16 H 19 FNO6 [M + H] + 340.1191; found 340.1172

[0287] Cyclization of Diols D7a and D7b

[0288]

[0289] According to conventional procedure D, diol D7a was cyclized to 30, while diol D7b was cyclized to a mixture of 30 and its corresponding α - isomer. The diol mixture was derived from the cyclization of the diol via S N 2 and also from some isomerization of the diol α - isomers. There have been reports on the heat treatment (emperization) of nucleosides (31).

[0290] According to conventional procedure D, a solution of D7a and D7b (0.033 g, 0.097 mmol, 1.0 equiv., d.r. (syn / anti) = 2:1) and Sc(OTf)3 (0.120 g, 0.243 mmol, 2.5 equiv.) in MeCN (0.65 mL) was stirred. Then 0.25 mL of pyridine and 0.25 mL of acetic anhydride were added and stirred for 1.5 h. The crude product 30 was purified by flash chromatography (pentane:ethyl acetate – 7:3) to give the nucleoside analogue 30 as a colorless oil (0.027 g, 69% yield).

[0291]

[0292] Data for nucleoside analogue 30: [α] D 20 = -9.0 (c 1.96 in CH2Cl2); IR (neat): υ = 2922, 1781, 1744, 1721, 1374, 1222, 1047, 720 cm -1 ; 11H NMR (500 MHz, CDCl3): δ 7.88 (m, 2H), 7.77 (m, 2H), 5.94 (dd, J = 6.0, 4.1 Hz, 1H), 5.87 (d, J = 4.1 Hz, 1H), 5.65 (dd, J = 6.1, 6.0 Hz, 1H), 4.49 (dd, J = 12.1, 3.4 Hz, 1H), 4.29 (ddd, J = 9.5, 5.9, 3.4 Hz, 1H), 4.21 (dd, J = 12.1, 5.9, 1H), 2.12 (s, 3H), 2.11 (s, 3H), 2.09 (s, 3H); 13 13C NMR (150 MHz, CDCl3): δ 170.9, 169.8, 169.7, 166.9, 134.8, 131.7, 124.0, 82.8, 79.2, 72.0, 70.6, 63.2, 20.9, 20.7, 20.7. HRMS (EI + ) calcd for C 19 H 23 N2O9 [M+NH4] + 423.1398; found 423.1378

[0293] Determination of the relative stereochemistry of diol D7b

[0294]

[0295] Recrystallization from ethanol allowed the relative stereochemistry to be assigned using single X-ray crystallography.

[0296] Determination of the relative stereochemistry of nucleoside 30

[0297]

[0298] The 2D NOESY analysis of nucleoside 30 supported the stereochemistry shown.

[0299] Determination of the enantiomeric excess of diol ent-D7a

[0300] A racemate of diol D7a was prepared using a 1:1 mixture of L-:D-proline according to the conventional procedures A and B. Using a 3 μm Amylose-1 column, the enantiomeric nucleosides were separated by chiral HPLC; flow rate 0.25 mL / min; eluent: hexane-iPrOH 90:10; detection wavelength 254 nm; retention times: 9.10 min for (-)-D7a; 13.14 min for (+)-D7a. The enantiomeric ratio of the optically enriched (+)-D7a diol was determined using the same method (95:5 e.r.).

[0301] Preparation of SM8, Aldol Adduct A8, Diol Adduct D8a / D8b and Nucleoside Analogs 32 / 33

[0302] A solution of deazoadenine (0.500 g, 1.79 mmol, 1.0 equiv), bromoacetaldehyde diethyl acetal (0.323 mL, 2.15 mmol, 1.25 equiv) and potassium carbonate (0.491 g, 3.58 mmol, 2.0 equiv) in DMF (9.00 mL) was stirred at 90 °C for 24 h. The reaction mixture was then filtered and washed with 10 mL of dichloromethane and concentrated under reduced pressure. The crude product S8 was purified by flash chromatography (pentane:ethyl acetate - 7:3) to give white solid S8 (0.375 g, 53% yield). Then a solution of S8 (17.0 g, 43.0 mmol, 1.0 equiv) in 2.0 M HCl (129 mL, 258 mmol, 6.0 equiv) was heated to 70 °C for 1 h. The reaction mixture was then cooled to room temperature and stirred for an additional 2 h. The reaction mixture was stored at -20 °C overnight, and the precipitate formed was then filtered and washed with 1:1 dioxane:water (10 mL x 2). The filtrate SM8 was dried under reduced pressure, and the resulting product SM8 (7.88 g, 54% yield) was used in the reaction without purification.

[0303]

[0304] Data for S8: 1H NMR (600 MHz, CDCl3): δ 8.61 (s, 1H), 7.50 (s, 1H), 4.67 (t, J = 5.1 Hz, 1H), 4.35 (d, J = 5.1 Hz, 2H), 3.73 (m, 2H), 3.48 (m, 2H), 1.16 (m, 6H); 13 C NMR (150 MHz, CDCl3): δ 152.7, 151.1, 150.8, 136.3, 116.9, 100.7, 63.9, 50.6, 47.7, 15.3. HRMS (EI + ) calcd for C 12 H 16 ClIN3O2 [M + H] + 395.9970; found 395.9973

[0305] α-Fluorination / Aldol Condensation

[0306] According to the conventional procedure A, a solution of SM8 (2.00 g, 5.86 mmol, 1 equiv), NFSI (1.85 g, 5.86 mmol, 1.0 equiv), L-proline (0.674 g, 5.86 mmol, 1.0 equiv) and sodium bicarbonate (0.984 g, 11.71 mmol, 2.0 equiv) in DMF (10 mL) was stirred at 20 °C for 18 h. Dioxanone 8 (0.762 g, 5.86 mmol, 1.0 equiv) was added to the reaction mixture and stirred at room temperature for 36 h. The crude product A8 was purified by flash chromatography (25 - 75% ethyl acetate in pentane) to give the light yellow solid cis- and trans-fluoroalcohol A8 (1.58 g, yield 57%, d.r 1.2:1).

[0307]

[0308] Data for cis- and trans-fluoroalcohol A8: IR (neat): υ = 3145, 2988, 1747, 1575, 1539, 1444, 1205, 1084, 949, 734 cm -1 ; 1 H NMR (600 MHz, dmso-d6): δ 8.76, 8.74, 8.39, 8.24, 6.89, 6.85, 6.37, 6.12, 4.98, 4.76, 4.61, 4.32, 4.30, 4.05, 3.95, 3.93, 1.40, 1.34, 1.33, 1.31 13 C NMR (150 MHz, dmso-d6): δ 206.3, 206.1, 151.6, 151.5, 151.3, 151.2, 151.0, 134.5, 134.1, 116.8, 116.7, 100.4, 100.1, 91.4, 09.4, 76.1, 74.7, 68.7, 68.0, 66.6, 66.4, 55.3, 55.1, 24.6, 24.1, 22.9, 22.7 19 F NMR (470 MHz, dmso-d6): δ –146.0, –152.6. HRMS (EI + ) calcd for C 14 H 15 ClFIN3O4 [M + H] + 469.9774; found 469.9779

[0309] Synchronous reduction of cis- and trans-fluoroalcohol A8

[0310] According to the conventional procedure B, a solution of NaHB(OAc)3 (0.316 g, 1.49 mmol, 5 equiv) and AcOH (0.171 mL, 2.98 mmol, 10 equiv) was added to a stirred solution of A8 (0.140 g, 0.298 mmol, 1 equiv) in MeCN (2.8 mL) at 0 °C. The reaction mixture was then stirred at room temperature for 2 h. The crude diols D8a and D8b were purified by flash chromatography (pentane:ethyl acetate - 70:30) to afford the white solid diols D8a and D8b (0.141 g, 77% yield, d.r. (syn / anti) = 1.5:1).

[0311]

[0312] Data for cis-diol, cis-fluoroalcohol D8a: [α] D 20 = -19.6 (c 2.0 in CH2Cl2); IR (neat): υ = 3335, 2989, 2890, 1577, 1540, 1445, 1206, 1076, 951 cm -1 ; 1 H NMR (600 MHz, dmso-d6): δ 8.73 (s, 1H), 8.27 (s, 1H), 6.73 (dd, J = 49.4, 7.0 Hz, 1H), 6.08 (br s, 1H), 4.84 (d, J = 4.1 Hz, 1H), 4.59 (m, 1H), 3.59 (m, 1H), 3.44 (m, 1H), 3.42 (m, 1H), 3.33 (m, 1H), 1.16 (s, 3H), 1.13 (s, 3H); 13 C NMR (150 MHz, dmso-d6): δ 151.4, 151.2, 151.1, 134.5, 116.7, 97.8, 92.0 (d, J = 203.3), 73.2 (d, J = 5.7 Hz), 71.0 (d, J = 24.2 Hz), 63.8, 62.5, 54.9, 28.0, 19.1; 19 F NMR (470 MHz, dmso-d6): δ –147.1. HRMS (EI + ) calcd for C 14 H 15 ClFIN3O4 [M + H] + 471.9931; found 471.9940.

[0313] Data for cis-diol, trans-fluoroalcohol D8b: [α] D 20= -11.6 (c 0.38 in CH2Cl2); IR (neat): υ = 3363, 2931, 2890, 1579, 1540, 1444, 1212, 1067, 951 cm -1 ; 1 1H NMR (600 MHz, dmso-d6): δ 8.73 (s, 1H), 8.34 (s, 1H), 6.97 (dd, J = 46.9, 7.9 Hz, 1H), 5.74 (d, J = 5.7 Hz, 1H), 5.22 (d, J = 5.7 Hz, 1H), 4.61 (m, 1H), 3.84 (m, 1H), 3.72 (m, 1H), 3.52 (dd, J = 11.7, 8.7 Hz, 1H), 1.35 (s, 3H), 1.20 (s, 3H); 13 13C NMR (150 MHz, dmso-d6): δ 151.5, 151.4, 151.2, 134.1, 116.6, 97.9, 90.9 (d, J = 203.5 Hz), 74.3, 69.1 (d, J = 30.3 Hz), 64.2, 61.4, 54.8, 28.4, 19.0; 19 19F NMR (470 MHz,, dmso-d6): δ –146.3. HRMS (EI + ) calcd for C 14 H 15 ClFIN3O4 [M + H] + 471.9931; found 471.9940

[0314] Cyclization of diol D8a

[0315]

[0316] According to conventional procedure D, diol D8a cyclizes to 32, while diol D8b cyclizes to 33. This supports S N 2 cyclization without subsequent epimerization.

[0317] According to conventional procedure D, a solution of D8a (0.050 g, 0.106 mmol, 1.0 equiv) and InCl3 (2.3 mg, 0.011 mmol, 0.10 equiv) in dry MeCN (1.00 mL) was stirred for 16 h. The crude nucleoside 32 was purified by flash chromatography (20 - 80% ethyl acetate in pentane) to give the white solid nucleoside 32 (0.029 g, 61% yield).

[0318]

[0319] Data for nucleoside analogue 32: [α]D 20 = -23.9 (c 0.46 in CH2Cl2); IR (neat): υ = 3339, 3113, 2935, 1576, 1539, 1445, 1207, 1108, 951 cm -1 ; 1 H NMR (600 MHz, dmso-d6): δ 8.69 (s, 1H), 8.23 (s, 1H), 6.34 (d, J = 3.1 Hz, 1H), 5.19 (dd, J = 6.3, 3.1 Hz, 1H), 5.14 (br s, 1H), 4.94 (dd, J = 6.3, 2.9 Hz, 1H), 4.20 (m, 1H), 3.56 (m, 2H), 1.54 (s, 3H), 1.31 (s, 3H); 13 C NMR (150 MHz, dmso-d6): δ 151.2, 150.8, 150.4, 133.9, 116.7, 113.2, 89.4, 86.3, 83.9, 80.9, 61.4, 53.7, 27.0, 25.1. HRMS (EI + ) calcd for C 14 H 16 ClIN3O4 [M + H] + 451.9869; found 451.9875

[0320] Cyclization of diol D8b

[0321] According to the conventional procedure D, a solution of D8b (0.050 g, 0.106 mmol, 1.0 equiv) and InCl3 (2.3 mg, 0.011 mmol, 0.10 equiv) in dry MeCN (1.00 mL) was stirred for 16 h. The crude nucleoside 33 was purified by flash chromatography (20 - 80% ethyl acetate in pentane) to give the white solid nucleoside 33 (0.034 g, 70% yield).

[0322]

[0323] Data for nucleoside analogue 33: [α] D 20 = -47.8 (c 0.51 in CHCl3); 11H NMR (600 MHz, dmso-d6): δ 8.66 (s, 1H), 7.81 (s, 1H), 6.73 (d, J = 4.3 Hz, 1H), 5.22 (br s, 1H), 4.91 (m, 2H), 4.41 (dd, J = 3.6, 3.1 Hz, 1H), 3.62 (m, 2H), 1.32 (s, 3H), 1.23 (s, 3H); 13 13C NMR (150 MHz, dmso-d6): δ 151.0, 150.7, 149.8, 134.6, 116.3, 112.3, 85.6, 83.1, 81.9, 79.4, 62.5, 51.9, 25.2, 23.9. HRMS (EI + ) calcd for C 14 11 16 11ClIN3O4 [M + H] + 451.9869; found 451.9888

[0324] Determination of the relative stereochemistry of diol D8a

[0325]

[0326] The relative stereochemistry of diol D8a was determined by J-based configurational analysis. For details, see the J-based configurational analysis section.

[0327] Determination of the relative stereochemistry of diol D8b

[0328]

[0329] The relative stereochemistry of diol D8b was determined by J-based configurational analysis. For details, see the J-based configurational analysis section.

[0330] Determination of the relative stereochemistry of nucleoside 32

[0331]

[0332] The 2D NOESY analysis of nucleoside 32 supported the shown stereochemistry.

[0333] Determination of the relative stereochemistry of nucleoside 33

[0334]

[0335] The 2D NOESY analysis of nucleoside 33 supported the shown stereochemistry.

[0336] Determination of the enantiomeric excess of diol D8a

[0337] The racemate of diol D8a was prepared using a 1:1 mixture of L-:D-proline according to the conventional steps A and B. The enantiomeric diols were separated by chiral HPLC using an IB column; eluent: 90:10 (MeCN:water) to 10:90 (MeCN:water); detection wavelength 230 nm; retention time: (+)-D8a was 12.23 min; (-)-D8a retention time was 13.39 min. The enantiomeric ratio of the optically enriched ent-D8a diol was determined by the same method (90:10 e.r.).

[0338] Determination of enantiomeric excess of diol D8b

[0339] The racemate of diol D8b was prepared using a 1:1 mixture of L-:D-proline according to the conventional steps A and B. The enantiomeric diols were separated by chiral HPLC using an IG column: eluent: 90:10 (MeCN:water) to 10:90 (MeCN:water); detection wavelength 230 nm; retention time: (-)-D8b was 12.35 min; (+)-D8b retention time was 12.56 min. The enantiomeric ratio of the optically enriched ent-D8b diol was determined by the same method (93:7 e.r.).

[0340] Preparation of SM9, aldehyde S9, aldol condensation adduct A9, diol adducts D9a / D9b and nucleoside analogs SI9 / NA9

[0341] A solution of iodouracil (2.50 g, 10.5 mmol, 1.0 equiv), bromoacetaldehyde diethyl acetal (1.91 mL, 12.7 mmol, 1.2 equiv) and potassium carbonate (2.92 g, 21.1 mmol, 2.0 equiv) in DMF (70 mL) was stirred at 90 °C for 16 h. The reaction mixture was filtered and the filtrate was diluted with 200 mL of ethyl acetate. The organic layer was washed 3 times with water, separated, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude S9 was purified by flash chromatography (pentane:ethyl acetate - 75:25) to give white solid S9 (0.301 g, 8% yield). The solution of S9 (0.142 g, 0.401 mmol, 1.0 equiv) in 0.5 M HCl (0.40 mL) was heated at 90 °C for 5 h. After complete conversion to the aldehyde / hydrate SM9, the reaction mixture was concentrated under reduced pressure and the resulting aldehyde / hydrate SM9 was used for the reaction without purification.

[0342]

[0343] Data for S9: IR (neat): υ = 2975, 1686, 1439, 1121, 1059, 1021 cm -1 ; 11H NMR (600 MHz, CDCl3): δ 8.56 (br s, 1H), 7.82 (s, 1H), 4.61 (t, J = 5.0 Hz), 3.88 (d, J = 5.0 Hz), 3.78 (m, 2H), 3.54 (m, 2H), 1.21 (m, 6H); 13 13C NMR (150 MHz, CDCl3): δ 158.6, 150.0, 147.0 (q, J = 5.8 Hz), 121.9 (q, J = 270.5 Hz), 104.7 (q, J = 33.5 Hz), 100.0, 64.6, 51.0, 15.3. HRMS (EI + ) calcd for C 10 H 16 IN2O4 [M + H] + 355.0149; found 355.0145

[0344] α-Fluorination / Aldol Condensation and Concurrent Reduction of cis- and trans-Fluoroalcohol A9

[0345] Following the conventional procedure A, a solution of S9 (0.401 mmol), NFSI (0.126 g, 0.401 mmol), L-proline (0.046 g, 0.401 mmol), and sodium bicarbonate (0.034 g, 0.401 mmol) in DMF (0.53 mL) was stirred at 4 °C for 12 h. Then, 1,4-dioxan-2-one 8 (0.053 mL, 0.270 mmol) in dichloromethane (0.67 mL) was added, and the reaction mixture was stirred at 4 °C for 72 h. The crude fluoroalcohol A9 was purified by flash chromatography (pentane–ethyl acetate 1:1) to afford fluoroalcohol A9 as a yellow oil. Following the conventional procedure B, Me4NHB(OAc)3 (0.066 g, 0.251 mmol) and AcOH (0.030 mL, 0.502 mmol) were added to a stirred solution of A9 (0.021 g, 0.049 mmol) in MeCN (0.49 mL) at −15 °C, and the reaction mixture was stirred for 24 h. Due to challenges in stability and purification, the crude diols D9a and D9b were used directly for cyclization.

[0346] Cyclization of Diols D9a and D9b

[0347] According to the conventional steps C, a solution of D9a and D9b (16.2 mg, 0.038 mmol, 1 equiv) and 2 M sodium hydroxide (0.038 mL, 0.38 mmol, 10 equiv) in MeCN (1.51 mL) was stirred for 18 h. The crude nucleoside SI9 was purified by flash chromatography (CH2Cl2:MeOH - 90:10) to give the white solid nucleoside SI9. SI9 (10.3 mg, 0.025 mmol) was dissolved in MeOD (0.25 mL), and 2 drops of 1 M hydrochloric acid were added dropwise. The resulting solution was left at room temperature for 12 h. Subsequently, the reaction mixture was concentrated under reduced pressure to give the white solid NA9. The spectral data was in agreement with the previous report (37).

[0348]

[0349] Data of nucleoside analogue SI9: 1 H NMR (600 MHz, MeOD): δ 7.99 (s, 1H), 5.58 (s, 1H), 4.35 (d, J = 4.5 Hz, 1H),, 4.19 (dd, J = 10.0, 4.6 Hz, 1H), 4.08 (dd, J = 10.0, 9.7 Hz, 1H), 3.83 (m, 2H), 1.57 (s, 3H), 1.45 (s, 3H); 13 C NMR (150 MHz, MeOD): δ 162.8, 151.7, 147.2, 102.5, 95.7, 74.5, 73.8, 72.5, 68.9, 65.8, 29.3, 20.0

[0350]

[0351] Data of nucleoside NA9: [α] D 20 = -41 (c = 0.1, MeOH); IR (neat): ν = 3353, 2929, 1679, 1447, 1262, 1101, 1023, 799 cm –1 ; 1 H NMR (600 MHz, MeOD): δ 8.61 (s, 1H), 5.86 (d, J = 3.6 Hz, 1H), 4.16 - 4.17 (m, 2H), 4.02 - 4.03 (m, 1H), 3.89 (dd, J = 12.2, 2.6 Hz, 1H), 3.76 (dd, J = 12.1, 2.5 Hz, 1H); 13 C NMR (150 MHz, MeOD): δ 162.8, 152.2, 147.3, 90.9, 86.3, 76.1, 70.9, 68.3, 61.7. HRMS (EI+ )calcd for C9H 12 IN2O6[M+H] + 370.9735;found:370.9739

[0352] Determination of the relative stereochemistry of diol D9a

[0353]

[0354] Recrystallization from ethanol allowed the relative stereochemistry to be assigned using single X-ray crystallography.

[0355] Preparation of nucleoside analogue 36

[0356] To a dry dichloromethane (0.83 mL) solution of nucleoside analogue 17 (0.020 g, 0.083 mmol, 1.0 equiv) was added TEMPO (1.3 mg, 0.008 mmol, 0.10 equiv) and (diacetoxyiodo)benzene (0.067 g, 0.208 mmol, 2.5 equiv). After 18 h or after complete consumption of 17 as monitored by 1H NMR spectroscopy, the reaction mixture was cooled to room temperature and diluted with dichloromethane. The organic layer was then washed with saturated sodium bicarbonate solution, dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give the crude product 36. Purification of the crude nucleoside 36 by flash chromatography (pentane:ethyl acetate - 1:1) gave the white solid nucleoside 36 (0.019 g, 92% yield).

[0357]

[0358] Data for nucleoside analogue 36: [α] D 20 = -115.6 (c 1.0 in MeCN); IR (neat): υ = 3001, 2989, 1694, 1374, 1305, 1088 cm -1 ; 1 1H NMR (600 MHz, CD3CN): δ 7.80 (d, J = 2.4 Hz, 1H), 7.62 (d, J = 1.5 Hz, 1H), 6.36 (dd, J = 2.4, 1.5 Hz, 1H), 5.78 (s, 1H), 4.69 (d, J = 11.1 Hz, 1H), 4.22 (d, J = 10.0, 5.0 Hz, 1H), 4.13 (dd, J = 10.6, 10.6 Hz, 1H), 3.87 (ddd, J = 11.1, 10.0, 5.0 Hz, 1H), 1.56 (s, 3H), 1.45 (s, 3H); 1313C NMR (150 MHz, CD3CN): δ 201.5, 143.3, 133.2, 108.1, 103.5, 86.5, 76.8, 69.4, 66.1, 29.3, 20.0. HRMS (EI + ) calcd for C 11 H 17 N2O5 [M + H] + 257.1132; found 257.1130

[0359] Determination of the relative stereochemistry of nucleoside 36

[0360]

[0361] The 2D NOESY analysis of nucleoside 36 supported the shown stereochemistry.

[0362] Preparation of nucleoside analogue 37

[0363] To a solution of nucleoside analogue 35 (0.100 g, 0.352 mmol, 1 equiv) in THF (3.52 mL) was added 1,1'-thiocarbonyldiimidazole (0.125 g, 0.704 mmol, 2 equiv). The reaction mixture was stirred for 24 h. Subsequently, dichloromethane (10 mL) was added to the reaction mixture, followed by washing with water three times. The organic layer was dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give the crude product S37. The crude product S37 was purified by flash chromatography (ethyl acetate) to give S37 (0.129 g, 96%).

[0364]

[0365] Data for nucleoside analogue S37: [α] D 20 = +25.8 (c 1.2 in MeCN); IR (neat): υ = 3000, 1701, 1443, 1375, 1039, 918, 749 cm -1 ; 11H NMR (600 MHz, CD3CN): δ 9.34 (br s, 1H), 8.38 (s, 1H), 7.73 (s, 1H), 7.43 (d, J = 7.4 Hz, 1H), 7.04 (s, 1H), 6.08 (d, J = 5.2 Hz, 1H), 5.88 (d, J = 5.2 Hz, 1H), 5.69 (d, J = 7.4 Hz, 1H), 4.22 (m, 2H), 4.06 (dd, J = 10.4 Hz, 1H), 3.83 (ddd, J = 10.4, 10.3, 5.0 Hz, 1H), 1.55 (s, 3H), 1.39 (s, 3H); 13 13C NMR (150 MHz, CD3CN): δ 184.8, 164.1, 151.3, 143.3, 138.4, 132.3, 119.8, 103.8, 102.9, 92.4, 82.7, 73.5, 72.8, 65.5, 29.5, 20.4. HRMS (EI + ) calcd for C 16 H 19 N4O6S [M + H] + 395.1020; found 395.1010

[0366] Under nitrogen, tributyltin hydride (0.024 mL, 0.090 mmol, 2 equiv) and AIBN (1.8 mg s, 0.011 mmol, 0.25 equiv) were added to a solution of nucleoside S37 (0.020 g, 0.045 mmol, 1 equiv) in dry toluene (3.0 mL). The resulting reaction mixture was purged with nitrogen for 30 minutes. Subsequently, the reaction mixture was stirred at 90 °C for 16 hours. The reaction mixture was diluted with dichloromethane (10 mL). The organic layer was washed with water, separated, dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give the crude product 37. The crude product 37 was purified by flash chromatography (ethyl acetate) to give nucleoside 37 as a colorless oil (6.8 mg, 57%).

[0367]

[0368] Data for nucleoside analogue 37: [α] D 20 = +7.8 (c 0.32 in MeOH); 11H NMR (600 MHz, CD3CN): δ 8.94 (br s, 1H), 7.50 (d, J = 8.2 Hz, 1H), 6.14 (dd, J = 8.7, 2.1 Hz, 1H), 5.63 (d, J = 8.2 Hz, 1H), 4.10 (dd, J = 10.0, 4.6 Hz, 1H), 4.00 (dd, J = 10.3, 10.0 Hz, 1H), 3.94 (m, 1H), 3.35 (ddd, J = 10.3, 10.0, 4.6 Hz, 1H), 2.27 (m, 1H), 2.17 (m, 1H), 1.52 (s, 3H), 1.37 (s, 3H); 13 13C NMR (150 MHz, CD3CN): δ 164.1, 151.6, 142.6, 103.3, 102.2, 84.4, 76.3, 72.7, 65.6, 36.4, 29.8, 20.5. HRMS (EI + ) calcd for C 12 H 17 N2O5 [M + H] + 269.1132; found 269.1111.

[0369] Preparation of Nucleoside Analogue 38

[0370] At -78 °C, methylmagnesium bromide (0.126 mL, 0.378 mmol, 4.5 equiv) was added to a dry THF (0.84 mL) solution of nucleoside 36 (0.020 g, 0.084 mmol, 1.0 equiv). The resulting reaction mixture was then stirred for 3.5 h. The reaction mixture was quenched at -78 °C with 0.50 mL of ammonium chloride:methanol solution (1:1 - saturated ammonium chloride solution:methanol) and heated to room temperature. The resulting mixture was diluted with 3 mL of dichloromethane and then washed twice with water. The organic layer was dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give the crude product 38. The crude product 38 was purified by flash chromatography (ethyl acetate:pentane - 30:70) to give the white solid nucleoside analogue 38 (19.1 mg, 90%).

[0371]

[0372] Data for nucleoside analogue 38: [α] D 20 = -117.7 (c 0.57 in CH2Cl2); IR (neat): υ = 3425, 2992, 1398, 1384, 1088, 851 cm -1 ; 11H NMR (600 MHz, CD3CN): δ 7.73 (d, J = 2.3 Hz, 1H), 7.60 (d, J = 1.3 Hz, 1H), 6.33 (dd, J = 2.3, 1.3 Hz, 1H), 5.60 (s, 1H), 4.13 (d, J = 10.0 Hz, 1H), 4.06 (dd, J = 9.8, 4.7 Hz, 1H), 3.93 (dd, J = 10.1, 9.8 Hz, 1H), 3.54 (s, 1H), 3.48 (ddd, J = 10.1, 10.0, 4.7 Hz, 1H), 1.53 (s, 3H), 1.41 (s, 3H), 1.36 (s, 3H); 13 13C NMR (150 MHz, CD3CN): 142.1, 132.5, 107.2, 102.2, 95.1, 80.5, 78.4, 71.6, 66.2, 29.7, 20.6, 20.4. HRMS (EI + ) calcd for C 12 H 19 N2O4 [M + H]+ + 255.1339; found 255.1333

[0373] Determination of the relative stereochemistry of nucleoside 38

[0374]

[0375] The 2D NOESY analysis of nucleoside 38 supported the shown stereochemistry.

[0376] Preparation of nucleoside analogue 39

[0377] At 0 °C, diethylaminosulfur trifluoride (0.058 mL, 0.44 mmol, 5 equiv) was added dropwise to a solution of nucleoside analogue 35 (0.025 g, 0.088 mmol, 1 equiv) in dichloromethane (0.45 mL). The reaction mixture was heated to room temperature and stirred for 1 h. Subsequently, ethyl acetate (10 mL) was added, and the organic layer was washed 3 times with saturated sodium bicarbonate solution. Then the organic layer was separated, dried, filtered, and concentrated under reduced pressure. The crude product S39 was purified by flash chromatography (CH2Cl2:MeOH 95:5) to give 2’,2’-anhydrouridine S39 as a white solid (0.012 g, 51% yield). Then 2’,2’-anhydrouridine S39 (0.011 g, 0.039 mmol, 1 equiv) was dissolved in 1 M HCl:MeOH solution (0.20 mL:0.20 mL). The reaction mixture was heated to 50 °C for 24 h and then concentrated under reduced pressure to give nucleoside 39 (9.5 mg, 100% yield). The spectroscopic data were in agreement with the previous report (41).

[0378]

[0379] Data for nucleoside analogue 39: 1 H NMR (600 MHz, dmso-d6): δ 11.28 (d, J = 2.1 Hz, 1H), 7.62 (d, J = 8.1 Hz, 1H) 5.98 (d, J = 4.5 Hz, 1H), 5.56 (dd, J = 8.1, 2.1 Hz, 1H), 3.99 (dd, J = 4.4, 3.2 Hz, 1H), 3.89 (dd, J = 3.6, 3.2 Hz, 1H), 3.73 (ddd, J = 5.6, 4.6, 3.6 Hz, 1H), 3.60 (dd, J = 11.6, 4.6 Hz, 1H), 3.56 (dd, J = 11.6, 5.6 Hz, 1H); 13 C NMR (150 MHz, dmso-d6): δ 163.4, 150.5, 142.3, 100.0, 85.1, 84.7, 75.5, 75.1, 60.7. HRMS (EI + ) calcd for C9H 13 N2O6 [M + H] + 245.0768; found 245.0777

[0380] Preparation of nucleoside analogue 43

[0381] At -78 °C, methylmagnesium chloride (3.0 M in THF, 1.49 mL, 4.47 mmol, 2.1 equiv) was added dropwise to a solution of 41 (cis- / trans-fluoroalcohol = 3:1, 1.00 g, 2.13 mmol, 1.0 equiv) in CH2Cl2 (10 mL). The reaction mixture was then stirred at this temperature for 2 h, then gradually warmed to room temperature and stirred for 12 h. The reaction mixture was then quenched with saturated ammonium chloride solution and diluted with ethyl acetate. The organic layer was separated, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product 43 was purified by flash chromatography (0 - 10% methanol in CH2Cl2) to give the white solid nucleoside 43 (0.418 g, 42%).

[0382]

[0383] Data for nucleoside analogue 43: [α] D 20 = -13.6 (c 0.28 in CH2Cl2); IR (neat): υ = 3443, 2250, 1661, 1053, 1005, 821 cm -1 ; 1 H NMR (600 MHz, CDCl3): δ 8.64 (s, 1H), 7.55 (s, 1H), 6.28 (d, J = 7.6 Hz, 1H), 4.92 (ddd, J = 9.8, 7.5, 4.4 Hz, 1H), 4.21 (d, J = 4.5 Hz, 1H), 3.83 (d, J = 12.6 Hz, 1H), 3.74 (d, J = 12.6 Hz, 1H), 3.40 (d, J = 9.8 Hz, 1H), 1.53 (s, 3H), 1.49 (s, 3H), 1.42 (s, 3H); 13 C NMR (150 MHz, CDCl3): δ 153.2, 151.0, 151.0, 132.6, 118.1, 99.2, 89.9, 79.1, 75.5, 73.9, 66.2, 52.8, 27.4, 23.0, 20.8. HRMS (EI + ) calcd for C 15 H 18 ClIN3O4 [M + H] + 466.0025; found 466.0054

[0384] Determination of the relative stereochemistry of nucleoside 43

[0385]

[0386] 2D NOESY analysis of nucleoside 43 supported the stereochemistry shown.

[0387] Preparation of Nucleoside Analogue 44

[0388] At -78 °C, methylmagnesium chloride (3.0 M in THF, 1.56 mL, 4.68 mmol, 2.2 equiv) was added dropwise to a solution of 41 (cis- / trans-fluoroalcohol = 3:1, 1.00 g, 2.13 mmol, 1.0 equiv) in CH2Cl2 (20.0 mL). The resulting reaction mixture was stirred at -78 °C for 5 h. Then the reaction mixture was quenched with ammonium chloride:methanol solution (1:1 saturated ammonium chloride solution:methanol) and warmed to room temperature. The reaction mixture was diluted with dichloromethane (50 mL), and the organic layer was separated, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product 42b was purified by flash chromatography (pentane:ethyl acetate - 65:35) to give 42b as an off-white solid (0.498 g, 48%).

[0389]

[0390] Data for 42b: [α] D 20 = -17.7 (c 1.8 in CH2Cl2); IR (neat): υ = 3316, 2991, 1206, 1086, 863, 736 cm -1 ; 1 H NMR (600 MHz, dmso-d6): δ 8.76 (s, 1H), 8.28 (s, 1H), 6.92 (dd, J = 45.8, 3.3 Hz, 1H), 6.23 (d, J = 5.0 Hz, 1H), 4.65 (s, 1H), 4.45 (m, 1H), 3.44 (d, J = 11.1 Hz, 1H), 3.28 (d, J = 8.0 Hz, 1H), 3.23 (d, J = 11.1, 1H), 1.28 (s, 3H), 1.13 (s, 3H), 0.75 (s, 3H); 13 C NMR (150 MHz, dmso-d6): δ 151.5, 151.4, 151.2, 134.3, 116.0, 98.3, 90.2 (d, J = 202.7 Hz), 74.1 (d, J = 4.5 Hz), 70.1 (d, J = 25.1 Hz), 70.0, 66.7, 55.2, 28.4, 19.7, 18.1; 19 F NMR (470 MHz, dmso-d6): δ –151.1. HRMS (EI + ) calcd for C 15 H 19 ClFIN3O4 [M + H] +486.0087; found 486.0080

[0391] To a stirred solution of 42b (0.100 g, 0.206 mmol, 1.0 equiv) in dry MeCN (2.0 mL) was added InCl3 (0.046 g, 0.206 mmol, 1.0 equiv). The resulting reaction mixture was heated to 50 °C and maintained for 2 h. Then 2,2-dimethoxypropane (0.214 mg, 2.06 mmol, 10.0 equiv) and camphorsulfonic acid (9.6 mg, 0.041 mmol, 0.20 equiv) were added, and the reaction mixture was stirred at 50 °C for an additional 1 h. The reaction mixture was then concentrated and purified by flash chromatography (0 - 10% methanol in CH2Cl2) to afford the white solid nucleoside 44 (0.049 g, 51%).

[0392]

[0393] Data for nucleoside analogue 44: [α] D 20 = +1.4 (c 0.84 in MeOD); 1 H NMR (600 MHz, CDCl3): δ 8.58 (s, 1H), 7.68 (s, 1H), 6.83 (d, J = 4.5 Hz, 1H), 5.01 (dd, J = 6.0, 4.7 Hz, 1H), 4.77 (d, J = 6.0 Hz, 1H), 3.79 (dd, J = 10.9, 5.2 Hz, 1H), 3.74 (dd, J = 10.9, 3.6 Hz, 1H), 2.02 (dd, J = 5.2, 3.6 Hz, 1H), 1.48 (s, 3H), 1.41 (s, 3H), 1.31 (s, 3H); 13 C NMR (150 MHz, CDCl3): δ 152.6, 150.8, 150.3, 134.5, 117.4, 113.2, 85.1, 85.0, 83.0, 81.1, 69.5, 50.8, 25.6, 24.1, 17.4. HRMS (EI + ) calcd for C 15 H 18 ClIN3O4 [M + H] + 466.0025; found 466.0000

[0394] Determination of the relative stereochemistry of nucleoside 44

[0395]

[0396] The 2D NOESY analysis of nucleoside 44 supported the shown stereochemistry.

[0397] Preparation of nucleoside analogue 45

[0398]

[0399] At -78 °C, ethylmagnesium chloride (0.5 M in THF, 8.94 mL, 4.47 mmol, 2.1 equiv) was added dropwise to a solution of 41 (cis- / trans-fluoroalcohol = 3:1, 1.00 g, 2.13 mmol, 1.0 equiv) in dichloromethane (10 mL). The reaction mixture was stirred at this temperature for 2 h, then gradually warmed to room temperature and stirred for 12 h. The reaction mixture was then quenched with saturated ammonium chloride solution and diluted with ethyl acetate. The organic layer was separated, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product 45 was purified by flash chromatography (0 - 10% methanol in CH2Cl2) to afford the white solid nucleoside 45 (0.415 g, 41%).

[0400] Data for nucleoside analogue 45: [α] D 20 = -29.5 (c 0.58 in MeOH); IR (neat): υ = 3291, 2924, 1446, 1201, 1023, 600 cm -1 ; 1 H NMR (600 MHz, dmso-d6): δ 8.72 (s, 1H), 8.02 (s, 1H), 6.44 (d, J = 8.1 Hz, 1H), 5.05 (dd, J = 8.1, 3.6 Hz, 1H), 4.44 (d, J = 3.6 Hz, 1H), 4.16 (s, 1H), 4.01 (d, J = 13.2 Hz, 1H), 3.82 (d, J = 13.2 Hz, 1H), 3.44 (br s, 1H), 1.49 (s, 3H), 1.43 (s, 3H); 13 C NMR (150 MHz, dmso-d6): δ 151.7, 151.4, 151.1, 132.8, 116.6, 97.5, 86.5, 81.1, 80.5, 75.0, 74.1, 72.3, 64.2, 53.0, 28.5, 18.9. HRMS (EI + ) calcd for C 16 H 16 ClIN3O4 [M + H] + 475.9869; found 475.9849

[0401] Determination of the relative stereochemistry of nucleoside 45

[0402] The relative stereochemistry was determined based on the comparison of the chemical shifts of the heteromeric protons with those of compounds 43 and 46.

[0403] Preparation of nucleoside analogue 46

[0404] At -78 °C, phenylmagnesium chloride (2.0 M in THF, 2.24 mL, 4.47 mmol, 2.1 equiv) was added dropwise to a solution of 41 (cis- / trans-fluoroalcohol = 3:1, 1.00 g, 2.13 mmol, 1.0 equiv) in CH2Cl2 (10 mL). The reaction mixture was stirred at this temperature for 2 h, then gradually warmed to room temperature and stirred for 12 h. The reaction mixture was then quenched with saturated ammonium chloride solution and diluted with ethyl acetate. The organic layer was separated, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product 46 was purified by flash chromatography (0 - 10% methanol in CH2Cl2) to give the white solid nucleoside 46 (0.496 g, 45%).

[0405]

[0406] Data for nucleoside analogue 46: [α] D 20 = -23.6 (c 1.7 in CH2Cl2); IR (neat): υ = 3309, 2990, 2938, 1575, 1538, 1445, 1200 cm -1 ; 1 1H NMR (600 MHz, dmso-d6): δ 8.70 (s, 1H), 7.63 (s, 1H), 7.43 (m, 5H), 6.55 (d, J = 8.3 Hz, 1H), 5.55 (d, J = 6.9 Hz, 1H), 4.77 (d, J = 3.8 Hz,, 1H), 4.67 (ddd, J = 8.3, 6.9, 3.8 Hz, 1H), 3.81 (d, J = 12.9 Hz, 1H), 3.68 (d, J = 12.9 Hz, 1H), 1.62 (s, 3H), 1.50 (s, 3H); 13 13C NMR (150 MHz, dmso-d6): δ 152.0, 151.3, 151.0, 140.4, 133.4, 128.5, 128.0, 125.3, 111.8, 97.4, 86.1, 80.8, 73.9, 72.5, 67.0, 54.3, 28.3, 20.2. HRMS (EI + ) calcd for C 20 H 20 ClIN3O4 [M + H] + 528.0182; found 528.0206.

[0407] Determination of the relative stereochemistry of nucleoside 46

[0408]

[0409] The 2D NOESY analysis of nucleoside 46 supported the shown stereochemistry.

[0410] Preparation of nucleoside analogue 47

[0411] At -78 °C, ethynylmagnesium chloride (0.5 M in THF, 8.94 mL, 4.47 mmol, 2.1 equiv) was added dropwise to a solution of 41 (cis- / trans-fluoroalcohol = 3:1, 1.00 g, 2.13 mmol, 1.0 equiv) in CH2Cl2 (20 mL). The resulting reaction mixture was stirred at -78 °C for 1 h. Then the reaction mixture was quenched with ammonium chloride:methanol solution (1:1 - saturated ammonium chloride solution:methanol) and heated to room temperature. The reaction mixture was diluted with CH2Cl2 (50 mL), the organic layer was separated, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product S47 was purified by flash chromatography (pentane:ethyl acetate - 65:35) to give the off-white solid S47 (0.720 g, 68%, 1:1 mixture of diastereomers).

[0412] To a stirred solution of S47 (0.050 g, 0.101 mmol, 1.0 equiv) in dry MeCN (2.0 mL) was added InCl3 (0.022 g, 0.101 mmol, 1.0 equiv). The resulting reaction mixture was heated to 50 °C and maintained for 2 h. After the addition of 2,2-dimethoxypropane (0.124 mL, 1.01 mmol, 10.0 equiv) and camphorsulfonic acid (4.7 mg, 0.020 mmol, 0.20 equiv), the reaction mixture was further stirred at 50 °C for 1 h. Then the reaction mixture was concentrated and purified by flash chromatography (0 - 10% methanol in CH2Cl2) to give the white solid nucleoside 47 (0.029 g, 60%).

[0413]

[0414] Data for nucleoside analogue 47: [α] D 20 = +6.3 (c 2.0 in CH2Cl2); 11H NMR (600 MHz, CDCl3): δ 8.59 (s, 1H), 7.82 (s, 1H), 6.85 (d, J = 4.6 Hz, 1H), 5.03 (dd, J = 6.0, 4.9 Hz, 1H), 4.98 (d, J = 6.0 Hz, 1H), 3.97 (dd, J = 11.5, 4.4 Hz, 1H), 3.92 (dd, J = 11.5, 3.5 Hz, 1H), 2.82 (s, 1H), 2.18 (dd, J = 4.4, 3.5 Hz, 1H), 1.53 (s, 3H), 1.34 (s, 3H); 13 13C NMR (150 MHz, CDCl3): δ 152.7, 150.9, 1505., 134.6, 117.4, 114.6, 85.3, 83.0, 82.9, 80.6, 78.2, 77.8, 68.7, 51.4, 25.7, 24.5. HRMS (EI + ) calcd for C 16 H 16 ClIN3O4 [M + H] + 475.9869; found 475.9885

[0415] Determination of the relative stereochemistry of nucleoside 47

[0416]

[0417] The 2D NOESY analysis of nucleoside 47 supported the indicated stereochemistry.

[0418] Preparation of nucleoside analogue 48

[0419] At -78 °C, methylmagnesium iodide (3.0 M in THF, 0.39 mL, 1.16 mmol, 3 equiv) was added dropwise to a solution of A5 (0.100 g, 0.388 mmol, 1 equiv) in CH2Cl2. The resulting reaction mixture was gradually heated to -10 °C and then stirred for 2 h. After monitoring the completion of the reaction by thin-layer chromatography analysis, the reaction mixture was quenched with saturated ammonium chloride solution and diluted with CH2Cl2. The organic layer was then washed twice with water and once with brine. Then the organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product S48 was then purified by flash chromatography (pentane:ethyl acetate - 25:75) to give S48 as a yellow transparent oily liquid (0.089 g, 84%).

[0420]

[0421] Data for S48: 11H NMR (600 MHz, CDCl3): δ 8.16, 8.02, 7.76, 7.76, 6.80, 6.58, 4.62, 4.52, 4.40, 4.31, 4.07, 3.81, 3.59, 3.55, 3.45, 3.25, 3.13, 3.10, 1.52, 1.47, 1.45, 1.40, 1.38, 1.17; 13 13C NMR (150 MHz, CDCl3): δ 134.2, 134.1, 124.9, 124.3, 99.8, 99.8, 95.6, 93.4, 72.4, 72.4, 71.9, 71.8, 70.2, 70.0, 67.9, 67.8, 28.8, 28.7, 20.0, 19.8, 19.2, 18.5; 19 19F NMR (470 MHz, CDCl3): δ –157.8, -162.8 HRMS (EI + ) calcd for C 11 H 19 FN3O4 [M+H] + 276.1354; found 276.1366

[0422] To a dry MeCN (2.18 mL) solution of S48 (0.060 g, 0.218 mmol, 1 equiv) was added Sc(OTf)3 (0.268 g, 0.545 mmol, 2.5 equiv). After stirring the reaction mixture for 16 h, 0.50 mL of acetic anhydride and 0.50 mL of pyridine were added to the reaction mixture. The reaction mixture was stirred for an additional 4 h and then diluted with CH2Cl2. The organic layer was washed twice with 1 M HCl, once with water, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product 48. The crude product 48 was purified by flash chromatography (pentane: ethyl acetate - 60:40) to give 48 (0.024 g, yield 32%).

[0423]

[0424] Data for nucleoside analogue 48: [α] D 20 = +18.4 (c 1.46 in CH2Cl2); IR (neat): υ = 2925, 1744, 1374, 1215, 1049 cm -1 ; 11H NMR (600 MHz, CDCl3): δ 7.76 (d, J = 0.60 Hz, 1H), 7.75 (d, J = 0.60 Hz, 1H) 6.19 (d, J = 4.7 Hz, 1H), 6.02 (dd, J = 5.4, 4.7 Hz, 1H), 5.67 (d, J = 5.4 Hz, 1H), 4.17 (d, J = 12.0 Hz, 1H), 4.08 (d, J = 12.0 Hz, 1H), 2.15 (s, 3H), 2.09 (s, 3H), 2.03 (s, 3H), 1.37 (s, 3H); 13 13C NMR (150 MHz, CDCl3): δ 170.3, 169.3, 169.2, 134.4, 122.7, 89.4, 85.6, 75.0, 71.9, 67.9, 20.8, 20.5, 20.5, 19.3. HRMS (EI + ) calcd for C 14 H 20 N3O7 [M + H] + 342.1296; found 342.1312

[0425] Determination of the relative stereochemistry of nucleoside analogue 48

[0426]

[0427] The 2D NOESY analysis of nucleoside 48 supported the shown stereochemistry.

[0428] Preparation of nucleoside analogue 49

[0429] According to the conventional procedure E, at -78 °C, p-tolylmagnesium bromide (1.0 M in THF, 0.712 mL, 0.71 mmol) was added to a solution of 59 (0.050 g, 0.158 mmol) in dichloromethane (6.30 mL). The reaction mixture was stirred for 4.5 h. Without further purification, the crude product S49 was dissolved in MeCN (1.58 mL), and 2 M sodium hydroxide (0.198 mL, 0.395 mmol) was added. Then the reaction mixture was heated to 50 °C and maintained for 4 h. The crude product 49 was purified by flash chromatography (pentane:ethyl acetate - 35:65) to give nucleoside 49 as a colorless oil (0.024 g, 39% yield over two steps).

[0430]

[0431] Data for nucleoside analogue 49: [α] D 20= -56.5 (c 0.4 in MeOH); IR (neat): υ = 3432, 2939, 1700, 1466, 1378, 1129, 1051 cm -1 ; 1 H NMR (600 MHz, CD3CN): δ 8.96 (br s, 1H), 7.38 (d, J = 8.1 Hz, 2H), 7.26 (d, J = 8.1 Hz, 2H), 6.78 (d, J = 0.90 Hz, 1H), 6.24 (d, J = 8.2 Hz, 1H), 4.76 (d, J = 3.8 Hz, 1H), 4.19 (s, 1H), 3.80 (d, J = 13.2 Hz, 1H), 3.73 (d, J = 13.2 Hz, 1H), 3.48 (br s), 2.35 (s, 3H), 1.68 (d, J = 0.90 Hz, 3H), 1.60 (s, 3H), 1.49 (s, 3H); 13 C NMR (150 MHz, CD3CN): δ 164.6, 152.8, 139.6, 138.7, 137.4, 130.6, 126.7, 112.0, 99.2, 88.9, 81.6, 74.9, 74.3, 68.7, 29.0, 21.4, 20.8, 12.8. HRMS (EI + ) calcd for C 20 H 25 N2O6 [M + H] + 389.1707; found 389.1707

[0432] Determination of the relative stereochemistry of nucleoside 49

[0433]

[0434] The 2D NOESY analysis of nucleoside 49 supported the stereochemical results

[0435] Preparation of nucleoside analogue 50

[0436] According to the conventional procedure E, at -78 °C, cyclopropylmagnesium bromide (1.0 M in 2-methyltetrahydrofuran, 0.79 mL, 0.79 mmol, 5 equiv) was added to a solution of 59 (0.050 g, 0.158 mmol, 1 equiv) in CH2Cl2 (6.30 mL). The reaction mixture was then stirred for 5 h. Without further purification, the crude product S50 was directly dissolved in MeCN (1.60 mL), 2 M sodium hydroxide (0.193 mL, 0.395 mmol) was added, and the reaction mixture was then stirred at 50 °C for 4 h. The crude product 50 was purified by flash chromatography (pentane:ethyl acetate - 30:70) to give the nucleoside 50 as an off-white solid (0.021 g, 40% yield).

[0437]

[0438] Data for nucleoside analogue 50: [α] D 20 = -32.6 (c 0.47 in CH2Cl2); IR (neat): υ = 3500, 3251 2997, 2175, 1690, 1088, 888 cm -1 ; 1 H NMR (600 MHz, CDCl3): δ 7.10 (s, 1H), 6.04 (d, J = 7.9 Hz, 1H), 4.25 (dd, J = 7.9, 5.1 Hz, 1H), 4.08 (d, J = 5.1 Hz, 1H), 3.70 (d, J = 11.9 Hz, 1H), 3.63 (d, J = 11.9 Hz, 1H), 3.15 (br s, 1H), 1.93 (s, 3H), 1.44 (s, 3H), 1.43 (s, 3H), 1.21 (m, 1H), 0.63 (m, 1H), 0.55 (m, 1H), 0.46 (m, 1H), 0.42 (m, 1H); 13 C NMR (150 MHz, CDCl3): δ 163.3, 151.0, 134.9, 111.9, 100.1, 87.5, 81.2, 74.0, 72.5, 64.3, 25.9, 25.6, 16.2, 12.9, 1.31, 0.50. HRMS (EI + ) calcd for C 16 H 22 N2O6 [M + H] + 339.1551; found 339.1575

[0439] Determination of the relative stereochemistry of nucleoside 50

[0440]

[0441] The 2D NOESY analysis of nucleoside 50 supports the shown stereochemistry.

[0442] Preparation of nucleoside analogue 51

[0443] Following general procedure E, at -78 °C, p-methoxyphenylmagnesium bromide (0.5 M in THF, 1.58 mL, 0.79 mmol, 5 equiv) was added to a solution of 59 (0.050 g, 0.158 mmol, 1 equiv) in CH2Cl2 (6.30 mL), and the reaction mixture was stirred for 5 h. Without further purification, the crude product S51 was directly dissolved in MeCN (1.60 mL), and 2 M sodium hydroxide (0.193 mL, 0.395 mmol) was added. The reaction mixture was stirred at 50 °C for 4 h. The crude product 51 was purified by flash chromatography (pentane:ethyl acetate - 30:70) to give the white solid nucleoside 51 (0.026 g, 41% yield).

[0444]

[0445] Data for nucleoside analogue 51: [α] D 20 = -52.8 (c 1.0 in CH2Cl2); IR (neat): υ = 3197, 2990, 1693, 1252, 1036, 834 cm -1 ; 1 H NMR (600 MHz, CDCl3): δ 7.38 (d, J = 8.7 Hz, 2H), 6.96 (d, J = 8.7 Hz, 2H), 6.78 (s, 1H), 6.37 (d, J = 7.9 Hz, 1H), 4.75 (d, J = 4.1 Hz, 1H), 4.16 (m, 1H), 3.87 (d, J = 13.1 Hz, 1H), 3.84 (s, 3H), 3.79 (d, J = 13.1, 1H), 2.99 (br s, 1H), 1.63 (s, 3H), 1.56 (s, 3H); 13 C NMR (150 MHz, CDCl3): δ 163.2, 159.9, 151.1, 135.8, 131.8, 126.5, 114.5, 111.7, 98.7, 88.7, 80.6, 74.8, 73.2, 67.7, 55.6, 28.1, 20.4, 12.7. HRMS (EI + ) calcd for C 20 H 25 N2O7 [M + H] +405.1656; found 405.1650

[0446] Determination of the relative stereochemistry of nucleoside analog 51

[0447]

[0448] The 2D NOESY analysis of nucleoside 51 supports the stereochemistry shown.

[0449] Preparation of nucleoside analog 52

[0450]

[0451] According to the conventional procedure E, at -78 °C, p-methoxyphenylmagnesium bromide (0.5 M in THF, 4.66 mL, 2.33 mmol, 3 equiv) was added to a solution of A1 (0.200 g, 0.775 mmol, 1 equiv) in dichloromethane (7.75 mL), and the reaction mixture was stirred for 6 hours. The crude product S52 was purified by flash chromatography (ethyl acetate - pentane - 4:6) to give S52 (0.157 g, 55% yield). S52 (0.155 g, 0.423 mmol, 1 equiv) was dissolved in MeCN (2.82 mL), and 2 M sodium hydroxide (0.53 mL, 1.06 mmol, 2.5 equiv) was added. The resulting reaction mixture was stirred at 50 °C for 5 hours. The crude nucleoside analog 52 was purified by flash chromatography (pentane:ethyl acetate - 40:60) to give 52 as a pale orange oil (0.085 g, 58% yield). Data for nucleoside analog 52: [α] D 20 = -14.8 (c 1.4 in CH2Cl2); IR (neat): υ = 3418, 2991, 1611, 1512, 1250, 1032, 759 cm -1 ; 1 H NMR (600 MHz, CD3CN): δ 7.69 (d, J = 2.7 Hz, 1H), 7.56 (d, J = 1.4 Hz, 1H), 7.39 (d, J = 8.9 Hz, 2H), 6.91 (d, J = 8.9 Hz, 2H), 6.35 (dd, J = 2.7, 1.4 Hz, 1H), 5.99 (d, J = 7.9 Hz, 1H), 4.73 (dd, J = 7.9, 3.7 Hz, 1H), 4.59 (d, J = 3.7 Hz, 1H), 3.92 (d, J = 13.3 Hz, 1H), 3.78 (s, 3H), 3.68 (d, J = 13.3 Hz, 1H), 1.62 (s, 3H), 1.51 (s, 3H); 1313C NMR (150 MHz, CD3CN): δ 160.6, 141.5, 133.7, 132.1, 128.4, 114.8, 107.6, 99.1, 93.9, 82.0, 75.9, 75.1, 68.9, 56.3, 29.0, 21.2. HRMS (EI + ) calcd for C 18 H 23 N2O5 [M + H] + 347.1601; found 347.1610

[0452] Determination of the relative stereochemistry of nucleoside 52

[0453]

[0454] The 2D NOESY analysis of nucleoside 52 supported the shown stereochemistry.

[0455] Preparation of nucleoside analogue 53

[0456] According to the conventional procedure E, at -78 °C, methylmagnesium bromide (3.0 M in THF, 0.258 mL, 0.78 mmol, 4 equiv) was added to a solution of A1 (0.050 g, 0.194 mmol, 1 equiv) in dichloromethane (3.90 mL). The reaction mixture was stirred for 6 h. The crude product S53 was purified by flash chromatography (ethyl acetate - pentane - 6:4) to give S53 (0.026 g, 49% yield). S53 (0.030 g, 0.109 mmol) was dissolved in MeCN (1.09 mL), and 2 M sodium hydroxide (0.545 mL, 1.09 mmol, 10 equiv) was added. The resulting reaction mixture was stirred at 50 °C for 5 h. The crude nucleoside analogue 53 was purified by flash chromatography (pentane:ethyl acetate - 25:75) to give 53 as a pale yellow oil (0.017 g, 61% yield).

[0457]

[0458] Data for nucleoside analogue 53: [α] D 20 = +11.3 (c 0.38 in CH2Cl2); ); IR (neat): υ = 3383, 2992, 2922, 1382, 1199, 1090, 908 cm -111H NMR (600 MHz, CDCl3): δ 7.60 (d, J = 2.4 Hz, 1H), 7.59 (d, J = 1.6 Hz, 1H), 6.35 (dd, J = 2.4, 1.6 Hz, 1H), 5.29 (d, J = 1.3 Hz, 1H), 4.12 (dd, J = 3.0, 1.3 Hz, 1H), 3.98 (d, J = 3.0 Hz, 1H), 3.76 (d, J = 11.3 Hz, 1H), 3.52 (d, J = 11.3 Hz, 1H), 1.47 (s, 3H), 1.44 (s, 3H), 1.41 (s, 3H); 13 13C NMR (150 MHz, CDCl3): δ 141.3, 129.3, 107.4, 99.6, 72.6, 70.3, 67.3, 64.9, 57.0, 28.8, 20.5, 19.0. HRMS (EI + ) calcd for C 12 H 19 N2O4 [M + H] + 255.1339; found 255.1320

[0459] Determination of the relative stereochemistry of nucleoside analogue 53

[0460]

[0461] The 2D NOESY analysis of nucleoside 53 supports the stereochemistry shown.

[0462] Preparation of nucleoside analogue 54

[0463] At 0 °C, p-chlorophenylmagnesium bromide (1.0 M in diethyl ether, 4.32 mL, 4.32 mmol, 3.2 equiv) was added dropwise to a stirred solution of fluoroalcohol aldol condensation adduct A6 (0.500 g, 1.35 mmol, 1 equiv) in THF (10.0 mL). The resulting reaction mixture was then stirred at room temperature for 14 h and then at 40 °C for an additional 8 h. The reaction mixture was then diluted with ethyl acetate (100 mL) and washed once with water (100 mL) and once with brine (50 mL). The organic layer was separated, dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give the crude product 54. The crude nucleoside analogue 54 was purified by flash chromatography (pentane:ethyl acetate – 50:50) to give 54 (0.289 g, 46%).

[0464]

[0465] Data for nucleoside 54: [α] D20 = +10.5 (c 0.8 in CH2Cl2); IR (neat): υ = 3087, 2996, 1699, 1467, 1283, 1129, 1085 cm -1 ; 1 1H NMR (600 MHz, dmso-d6): δ 11.94 (br s, 1H), 8.74 (s, 1H), 7.57 (d, J = 8.7 Hz, 2H), 7.50 (d, J = 8.7 Hz, 2H), 6.13 (d, J = 7.2 Hz, 1H), 5.67 (br s, 1H), 4.66 (d, J = 4.3 Hz, 1H), 4.17 (dd, J = 6.8, 4.3 Hz, 1H), 3.98 (d, J = 13.4 Hz, 1H), 3.88 (d, J = 13.4 Hz, 1H), 1.63 (s, 3H), 1.409 s, 3H); 13 13C NMR (150 MHz, CD3CN): δ 159.9, 150.5, 144.3 (q, J = 5.9 Hz), 138.0, 134.9, 129.9, 128.1, 124.1 (q, J = 269.0 Hz), 104.0 (q, J = 32.0), 99.6, 84.7, 81.6, 73.6, 73.6, 67.7, 28.6, 19.9; 19 19F NMR (470 MHz, CD3CN): δ –62.9. HRMS (EI + ) calcd for C 19 H 19 ClF3N2O6 [M + H] + 463.0878; found 463.0875

[0466] Determination of the relative stereochemistry of nucleoside 54

[0467]

[0468] The 2D NOESY analysis of nucleoside 54 supports the shown stereochemistry.

[0469] Preparation of nucleoside analogue 57

[0470] To a solution of nucleoside 35 (0.285 g, 1.0 mmol, 1.0 equiv) in dry dioxane (20 mL) was added (diacetoxyiodo)benzene (0.805 g, 2.5 mmol, 2.5 equiv) and TEMPO (0.031 g, 0.20 mmol, 0.2 equiv). The reaction mixture was then stirred at room temperature for 24 h until the starting material was completely consumed as detected by thin layer chromatography analysis. The reaction mixture was concentrated to 2 mL and purified by flash chromatography (CH2Cl2:Et2O - 75:25) to afford the white solid ketone 56 (0.265 g, 0.94 mmol, 94% yield). Ketone 56 (0.053 g, 0.19 mmol, 1.0 equiv) was dissolved in methanol (0.94 mL) and 3 drops of AcCl were added. The solution was stirred at room temperature for 12 h until the starting material was completely consumed as detected by thin layer chromatography analysis. The reaction mixture was concentrated under reduced pressure to a white solid S57. The spectral data matched those previously reported (50). The crude product was then dissolved in tetrahydrofuran (4.0 mL) and the resulting solution was cooled to -78 °C. Then methylmagnesium bromide (3.0 M in THF, 0.38 mL, 1.13 mmol, 6.0 equiv) was added. The resulting brown suspension was stirred at -78 °C for 3 h. The reaction mixture was then quenched at -78 °C with a solution of methanol:TFA (10:1) and then concentrated under reduced pressure. The crude product 57 was purified by flash chromatography (CH2Cl2:MeOH - 85:15) to afford the white solid nucleoside analogue (0.024 g, 49% yield). The spectral data were consistent with those previously reported (51).

[0471]

[0472] Data for nucleoside analogue 57: 1 H NMR (600 MHz, MeOD): δ 7.86 (d, J = 8.1 Hz, 1H), 5.96 (s, 1H), 5.64 (d, J = 8.1 Hz, 1H), 3.85 (m, 4H), 1.29 (s, 3H). HRMS (EI + ) calcd for C 10 H 15 N2O6 [M + H] + 259.0925; found 259.0915

[0473] Preparation of nucleoside analogue 60

[0474] To a stirred solution of 59 (0.100 g, 0.316 mmol, 1 equiv) in THF (3.10 mL) was added BnNH2 (0.086 ml, 0.790 mmol, 2.5 equiv) and glacial acetic acid (18.2 μl, 0.316 mmol, 1 equiv), and the resulting mixture was stirred at 20 °C for 1 h. Then NaBH3CN (0.050 g, 0.79 mmol, 2.5 equiv) was added, and the mixture was stirred for an additional hour. The reaction mixture was then diluted to 0.05 M with dichloromethane and then treated with water. The layers were separated, the organic layer was washed with brine, dried over magnesium sulfate, and concentrated under reduced pressure. The resulting product S60 was used without any further purification. To a stirred solution of S60 in MeCN (8.7 mL) was added 2 M sodium hydroxide (0.240 mL, 0.478 mmol, 1.1 equiv). The reaction mixture was stirred at room temperature for 14 h. The reaction mixture was then diluted with dichloromethane and quenched with saturated ammonium chloride solution. The organic layer was washed with saturated ammonium chloride solution and water, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product 60 was purified by flash chromatography (ethyl acetate:pentane - 80:20) to give the nucleoside analogue 60 as a pale yellow oil (0.060 g, 49% yield over two steps).

[0475]

[0476] Data for nucleoside analogue 60: [α] D 20 = -15.5 (c 0.53 in CH2Cl2); IR (neat): υ = 2990, 1670, 1382, 1200, 1078, 701 cm -1 ; 1 H NMR (600 MHz, CDCl3): δ 7.23 - 7.32 (m, 4H), 7.19 (d, J = 7.0 Hz, 2H), 5.07 (s, 1H), 4.11 (d, J = 4.8 Hz, 1H), 3.81 (d, J = 12.9 Hz, 1H), 3.77 (d, J = 12.9 Hz, 1H), 3.72 (dd, J = 10.4, 4.6 Hz, 1H), 3.67 (dd, J = 10.4, 10.2 Hz, 1H), 3.61 (dd, J = 9.8, 4.8 Hz, 1H), 3.11 (ddd, J = 10.2, 9.8, 4.6 Hz, 1H), 1.86 (s, 3H), 1.49 (s, 3H), 1.46 (s, 3H); 1313C NMR (150 MHz, CDCl3): δ 163.5, 150.7, 136.8, 135.9, 129.1, 128.7, 128.2, 110.1, 101.0, 83.1, 74.9, 73.2, 66.6, 58.1, 58.0, 29.2, 19.9, 12.8. HRMS (EI + ) calcd for C 20 H 26 N3O5 [M + H] + 388.1867; found 388.1843.

[0477] Determination of the relative stereochemistry of nucleoside 60

[0478]

[0479] The 2D NOESY analysis of nucleoside 60 supports the stereochemistry shown.

[0480] Preparation of nucleoside analogue 61

[0481] According to the conventional procedure E, at -78 °C, allylmagnesium bromide (1.0 M in diethyl ether, 1.42 mL, 1.42 mmol, 4.5 equiv) was added to a solution of 59 (0.100 g, 0.316 mmol, 1 equiv) in dichloromethane (12.6 mL). Then the reaction mixture was stirred for 5 h. Without further purification, the crude product S61 was dissolved in MeCN (3.16 mL), and 2 M sodium hydroxide (0.395 mL, 0.79 mmol, 2.5 equiv) was added. Then the reaction mixture was stirred at 50 °C for 4 h. The crude product 61 was purified by flash chromatography (CH2Cl2:MeOH - 4:96) to give nucleoside analogue 61 as a dark orange oil (0.050 g, 47% yield).

[0482]

[0483] Data for nucleoside analogue 61: [α] D 20 = -6.0 (c 0.4 in MeOH); IR (neat): υ = 3340, 2992, 1670, 1376, 1044 cm -1 ; 11H NMR (600 MHz, CD3CN): δ 8.95 (br s, 1H), 7.27 (s, 1H), 6.02 (d, J = 8.3 Hz, 1H), 5.87 (m, 1H), 5.22 (d, J = 17.7 Hz, 1H), 5.20 (d, J = 10.1 Hz, 1H), 4.31 (ddd, J = 9.3, 8.3, 4.9 Hz, 1H), 4.11 (d, J = 4.9 Hz, 1H), 3.68 (d, J = 12.2 Hz, 1H), 3.64 (d, J = 12.2 Hz, 1H), 3.41 (d, J = 9.3 Hz, 1H), 2.50 (dd, J = 14.2, 6.7 Hz, 1H), 2.41 (dd, J = 14.2, 8.1 Hz, 1H), 1.85 (s, 3H), 1.40 (s, 3H), 1.39 (s, 3H); 13 13C NMR (150 MHz, CD3CN): δ 164.6, 152.3, 136.8, 133.7, 120.4, 112.3, 100.3, 88.4, 81.7, 73.9, 73.3, 65.3, 41.7, 26.9, 22.4, 12.8. HRMS (EI + ) calcd for C 16 H 22 N2O6 [M + H]+ + 339.1551; found 339.1556

[0484] Determination of the relative stereochemistry of nucleoside 61

[0485]

[0486] The 2D NOESY analysis of nucleoside 61 supported the shown stereochemistry.

[0487] Preparation of nucleoside analogue 62

[0488] To a solution of nucleoside 61 (0.022 g, 0.061 mmol, 1 equiv) in dry THF (0.61 mL) was added 1,1'-thiocarbonyldiimidazole (0.022 g, 0.122 mmol, 2 equiv). The reaction mixture was then stirred for 18 h. Subsequently, CH2Cl2 (5 mL) was added and the mixture was washed with water three times. The organic layer was dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give the crude product S62. The crude product S62 was purified by flash chromatography (pentane:ethyl acetate - 40:60) to give S62 (0.018 g, 66% yield). Under nitrogen, to a solution of nucleoside S62 (0.014 g, 0.031 mmol, 1 equiv) in dry toluene (4.45 mL) was added tributyltin hydride (8.35 μL, 0.031 mmol, 1 equiv) and AIBN (5.1 mg s, 0.031 mmol, 1.0 equiv). The resulting reaction mixture was then purged with nitrogen for 30 min. Subsequently, the reaction mixture was stirred at 90 °C for 16 h. Upon competition, dichloromethane was added to the reaction mixture and the mixture was washed with water. The organic layer was dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give the crude product 62. The crude nucleoside analogue 62 was purified by flash chromatography (ethyl acetate) to give the white solid nucleoside analogue 62 (6.0 mg, 61%).

[0489]

[0490] Data for nucleoside analogue 62: [α] D 20 = +13.3 (c 0.46 in CH2Cl2); IR (neat): υ = 2924, 1690, 1467, 1375, 1263, 1226, 1053 cm -1 ; 1 H NMR (600 MHz, CDCl3): δ 8.26 (s, 1H), 7.31 (d, J = 1.1 Hz, 1H), 6.38 (dd, J = 9.6, 4.8 Hz, 1H), 5.86 (m, 1H), 5.25 - 5.27 (m, 2H), 4.22 (d, J = 5.2 Hz, 1H), 3.69 (d, J = 12.0 Hz, 1H), 3.64 (d, J = 12.0 Hz, 1H), 2.50 (m, 2H), 2.41 (dd, J = 13.5, 4.8 Hz, 1H), 2.00 (dd, J = 13.5, 9.6, 5.2 Hz, 1H), 1.92 (s, 3H), 1.37 (s, 6H); 1313C NMR (150 MHz, CDCl3): δ 163.3, 150.0, 135.0, 131.9, 120.2, 111.1, 99.5, 85.8, 84.0, 73.9, 63.9, 40.9, 37.8, 25.6, 22.5, 12.7 HRMS (EI + ) calcd for C 16 H 23 N2O5 [M + H] + 323.1601; found 323.1580

[0491] Preparation of fluoroalcohols 63 and 64

[0492] According to the conventional procedure E, at -78 °C, ethynylmagnesium chloride (0.5 M in THF, 3.5 mL, 1.75 mmol, 3.5 equiv) was added to a solution of 59 (0.160 g, 0.50 mmol, 1 equiv) in dichloromethane (25.0 mL). The reaction mixture was then stirred for 4 h. The crude products 63 and 64 were purified by flash chromatography (ethyl acetate:hexane - 70:30) to give white solid 63 (0.072 g, 42% yield) and 64 (0.058 g, 34%).

[0493]

[0494] Data for fluoroalcohol 63: [α] D 20 = -60.8 (c 0.4 in MeOH); IR (neat): υ = 3320, 2944, 2832, 1670, 1449, 1022, 638 cm -1 ; 1 1H NMR (600 MHz, dmso - d6): δ 11.47 (br s, 1H), 7.56 (s, 1H), 6.36 (dd, J = 43.7, 4.1 Hz, 1H), 6.21 (d, J = 5.3 Hz, 1H), 5.37 (br s, 1H), 4.14 (m, 1H), 3.71 (d, J = 8.7 Hz, 1H), 3.68 (br s, 1H), 3.42 (s, 1H), 3.16 (d, J = 5.0 Hz, 1H), 1.78 (s, 3H), 1.33 (s, 3H), 1.21 (s, 3H); 1313C NMR (150 MHz, dmso-d6): δ 163.6, 150.0, 136.7, 109.2, 98.8, 92.7 (d, J = 206.6 Hz), 83.7, 76.2, 72.8 (d, J = 2.8 Hz), 71.2 (d, J = 24.6 Hz), 68.2, 65.7, 27.8, 18.7, 12.1; 19 19F NMR (470 MHz, dmso-d6): δ –170.5 HRMS (EI + ) calcd for C 15 H 20 N2O6 [M+H] + 343.1300; found 343.1298.

[0495]

[0496] Data of fluoroalcohol 64: [α] D 20 = -38.0 (c 1.2 in MeOH); IR (neat): υ = IR (neat): υ = 3395, 2994, 1694, 1468, 1381, 1282, 1043 cm -1 ; 1 1H NMR (600 MHz, CD3CN): δ 9.29 (br s, 1H), 7.41 (s, 1H), 6.40 (dd, J = 43.4, 4.6 Hz, 1H), 4.54 (m, 1H), 4.27 (m, 1H), 4.22 (m, 1H), 3.82 (d, J = 9.5 Hz, 1H), 3.79 (d, J = 11.5 Hz, 1H), 3.75 (d, J = 11.5 Hz, 1H), 2.81 (s, 1H), 1.85 (s, 3H), 1.41 (s, 3H), 1.28 (s, 3H); 13 13C NMR (150 MHz, CD3CN): δ 164.8, 151.4, 137.7, 111.5, 100.8, 94.1 (d, J = 206.9 Hz), 84.4, 75.7, 73.6 (d, J = 3.8 Hz), 73.4 (d, J = 24.7 Hz), 69.3, 67.3, 28.8, 19.4, 12.8; 19 19F NMR (470 MHz, CD3CN): δ –175.5 HRMS (EI + ) calcd for C 15 H 20 N2O6 [M+H] + 343.1300; found 343.1305

[0497] Preparation of nucleoside analogue 65

[0498] According to the conventional procedure C, a solution of 63 (0.100 g, 0.292 mmol, 1.0 equivalent) and sodium hydroxide (29.2 mg, 0.73 mmol, 2.5 equivalents) in MeCN (2.0 mL) was heated to 50 °C and maintained for 36 hours. The crude product 65 was purified by flash chromatography (0 - 10% MeOH in dichloromethane) to give the white powder nucleoside analogue 65 (58.6 mg, 62% yield).

[0499]

[0500] Data of nucleoside analogue 65: [α] D 20 = -8.7 (c 0.6 in CH2Cl2); IR (neat): υ = 2994, 1748, 1690, 1270, 1043 cm -1 ; 1 1H NMR (600 MHz, dmso - d6): δ 11.42 (s, 1H), 7.61 (d, J = 1.3 Hz, 1H), 5.46 (s, 1H), 4.86 (s, 1H), 4.63 (d, J = 11.2 Hz, 1H), 4.45 (d, J = 2.6 Hz, 1H), 4.37 (d, J = 2.6 Hz, 1H), 4.23 (d, J = 11.2 Hz, 1H), 3.91 (s, 1H), 1.84 (s, 3H), 1.51 (s, 3H), 1.30 (s, 3H); 13 13C NMR (150 MHz, dmso - d6): δ 163.8, 158.8, 150.0, 135.0, 109.3, 100.4, 87.2, 83.0, 78.4, 76.5, 71.9, 58.5, 28.7, 19.5, 12.0 HRMS (EI + ) calcd for C 15 H 19 N2O6 [M + H] + 323.1238; found 323.1235

[0501] Preparation of nucleoside analogue 68

[0502] According to conventional C, a solution of 64 (0.220 g, 0.64 mmol, 1 equivalent) and 2 M sodium hydroxide (0.640 mL, 1.28 mmol, 2.0 equivalents) in MeCN (6.4 mL) was heated to 50 °C and stirred for 24 h. The crude product 66 was purified by flash chromatography (MeOH:CH2Cl2 - 3:97) to afford the white powder nucleoside analogue 66 (0.144 mg, 70% yield).

[0503]

[0504] Data for nucleoside analogue 66: [α] D 20 = +30.8 (c 1.66 in CH2Cl2); 1 H NMR (600 MHz, CD3CN): δ 9.06 (br s, 1H), 7.48 (s, 1H), 6.16 (d, J = 8.2 Hz, 1H), 4.61 (ddd, J = 8.4, 8.2, 3.7 Hz, 1H), 4.41 (d, J = 3.7 Hz, 1H), 4.06 (d, J = 13.3 Hz, 1H), 3.88 (d, J = 13.3 Hz, 1H), 3.64 (d, J = 8.4 Hz, 1H), 3.29 (s, 1H) 1.86 (s, 3H), 1.48 (s, 3H), 1.43 (s, 3H); 13 C NMR (150 MHz, CD3CN): δ 164.7, 152.5, 136.9, 112.7, 99.3, 89.4, 81.2, 80.8, 76.5, 75.5, 73.9, 65.9, 29.1, 19.7, 13.1. HRMS (EI + ) calcd for C 15 H 19 N2O6 [M + H] + 323.1238; found 323.1245

[0505] Determination of the relative stereochemistry of nucleoside 66

[0506]

[0507] The 2D NOESY analysis of nucleoside 66 supported the shown stereochemistry.

[0508] A solution of 66 (0.050 g, 0.155 mmol, 1 equiv) in dry dichloromethane (0.78 mL) was cooled to 0 °C, and then diethylaminosulfur trifluoride (0.102 mL, 0.776 mmol, 5 equiv) was added dropwise over 5 minutes. The resulting reaction mixture was slowly heated to room temperature over 3 hours. After monitoring the completion of the reaction by thin-layer chromatography, the reaction mixture was diluted with 5 mL of ethyl acetate and washed with 3 mL of water (3×). Subsequently, the organic layer was dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography (ethyl acetate) to give the white solid nucleoside analogue S66 (0.043 g, 91%).

[0509]

[0510] Data for nucleoside analogue S66: [α] D 20 = -47.5 (c 1.1 in MeCN); IR (neat): υ = 3284, 3002, 1626, 1554, 1497, 1134, 1066, 1030 cm -1 ; 1 1H NMR (600 MHz, CD3CN): δ 7.46 (s, 1H), 6.32 (d, J = 5.3 Hz, 1H), 5.13 (d, J = 5.3 Hz, 1H), 4.74 (s, 1H), 4.10 (d, J = 13.7 Hz, 1H), 4.00 (d, J = 13.7 Hz, 1H), 2.87 (s, 1H), 1.87 (s, 3H), 1.47 (s, 3H), 1.34 (s, 3H); 13 13C NMR (150 MHz, CD3CN): δ 173.1, 161.5, 132.3, 119.6, 99.7, 91.8, 87.4, 79.8, 79.1, 77.8, 74.6, 64.9, 29.0, 19.3, 14.4. HRMS (EI + ) calcd for C 15 H 17 N2O5 [M + H] + 305.1132; found 305.1108

[0511] To a solution of S66 (0.042 g, 0.138 mmol, 1 equiv) in wet MeCN (2.76 mL) was added InCl3 (0.122 g, 0.553 mmol, 4 equiv). The resulting reaction mixture was heated to 50 °C and stirred for 16 h or until the reaction was complete as monitored by thin layer chromatography. The reaction mixture was then concentrated under reduced pressure and purified by flash chromatography (MeOH:CH2Cl2 - 7.5:92.5) to afford S68 (0.038 g, 96%). To a solution of S68 (0.038 g, 0.133 mmol, 1 equiv) in DMF (1.73 mL) was added potassium carbonate (0.096 g, 0.69 mmol, 5 equiv). The resulting reaction mixture was heated to 90 °C and stirred for 7 days or until the reaction was complete as monitored by 1 1H NMR. The reaction mixture was then filtered, concentrated under reduced pressure, and the crude product was purified by flash chromatography (MeOH:CH2Cl2 - 10:90) to afford the white solid 68 (0.027 g, 71%).

[0512]

[0513] Data for nucleoside analogue 68: [α] D 20 = +16.9 (c 1.0 in MeOH); IR (neat): υ = 3261, 2988, 1686, 1272, 1203, 1047, 799 cm -1 ; 1 1H NMR (600 MHz, CD3CN): δ 9.43 (br s, 1H), 7.31 (d, J = 1.1 Hz, 1H), 5.48 (s, 1H), 4.27 (s, 1H), 4.15 (s, 1H), 4.03 (d, J = 8.0 Hz, 1H), 3.93 (d, J = 8.0 Hz, 1H), 3.16 (s, 1H), 1.85 (d, J = 1.1 Hz, 3H); 13 13C NMR (150 MHz, CD3CN): δ 165.1, 151.4, 135.6, 111.0, 88.6, 80.9, 80.3, 80.2, 75.8, 75.2, 75.1, 13.0. HRMS (EI + ) calcd for C 12 H 13 N2O5 [M + H] + 265.0819; found 265.0813

[0514] General procedure F (α-fluorination / aldol condensation reaction with cyclohexanone / thiopyranone 35)

[0515] At -10 °C, an aldehyde sample (1.0 equiv) was added to a stirred suspension of NFSI (1.0 equiv), L-proline (1.0 equiv), and sodium bicarbonate (1.0 equiv) in DMF (0.75 M). When complete conversion to the α-fluoro aldehyde was analyzed by NMR, cyclohexanone or thiopyranone 35 (5.0 - 10.0 equiv) was added and the resulting mixture was gradually warmed to room temperature. After a total of 18 h, the reaction mixture was diluted with Et2O, the organic layer was washed twice with water and once with brine. The organic layer was then dried over magnesium sulfate, concentrated in vacuo, and the crude product was purified by flash chromatography.

[0516] Preparation of cis-fluoroalcohol 68a and trans-fluoroalcohol 68b

[0517] Following general procedure F, at room temperature, an aldehyde (2.00 g, 5.86 mmol, 1.0 equiv), NFSI (1.85 g, 5.86 mmol, 1.0 equiv), L-proline (0.674 g, 5.86 mmol, 1.0 equiv) and sodium bicarbonate (0.984 g, 11.71 mmol, 2 equiv) in DMF (10 mL) were stirred for 2 h. Then cyclohexanone (1.15 g, 11.71 mmol) was added and the reaction mixture was stirred for 18 h. The reaction mixture was then diluted with ethyl acetate (100 mL) and water (30 mL). The organic layer was washed with brine (2 × 30 mL), dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude fluoroalcohol 68 was purified by flash chromatography (25 - 75% ethyl acetate in hexanes) to give the white solid cis-fluoroalcohol 68a (0.92 g, 36% yield) and trans-fluoroalcohol 68b (1.21 g, 47% yield).

[0518]

[0519] Data for cis-fluoroalcohol 68a: 1 H NMR (500 MHz, CDCl3): δ 8.73 (s, 1H), 8.27 (s, 1H), 7.02 (dd, J = 50.0, 5.6 Hz, 1H), 5.82 (d, J = 6.9 Hz, 1H), 4.47 (m, 1H), 2.43 (m, 1H), 2.24 (m, 1H), 2.16 (m, 1H), 2.05 (m, 1H), 1.80–1.86 (m, 2H), 1.73 (m, 1H), 1.55–1.60 (m, 2H); 1313C NMR (125 MHz, CDCl3): δ 209.9, 151.5, 151.3, 151.0, 134.0, 116.6, 92.5 (d, J = 205.2 Hz), 69.7 (d, J = 24.4 Hz), 55.3, 51.5, 51.5, 41.5, 29.2, 26.3, 23.5; 19 19F NMR (470 MHz, CDCl3): δ –147.6.

[0520]

[0521] Data for trans-fluoroalcohol 68b: 1 1H NMR (500 MHz, CDCl3): δ 8.75 (s, 1H), 8.34 (s, 1H), 7.05 (dd, J = 47.6, 7.3 Hz, 1H), 5.59 (d, J = 6.7 Hz, 1H), 4.55 (m, 1H), 2.70 (m, 1H), 2.39 (m, 1H), 2.27 (m, 1H), 1.87–1.99 (m, 2H), 1.84 (m, 1H), 1.56–1.76 (m, 3H); 13 13C NMR (125 MHz, CDCl3): 210.1, 151.6, 151.4, 151.3, 133.8, 116.6, 91.5 (d, J = 204.6 Hz), 68.9 (d, J = 30.5 Hz), 55.2, 51.1, 41.7, 29.1, 26.4, 23.5

[0522] Determination of the relative stereochemistry of cis-fluoroalcohol 68a

[0523] Fluoroalcohol 68a was converted to nucleoside 86. NOE analysis of nucleoside 86 confirmed the relative stereochemistry of fluoroalcohol 68a.

[0524] Determination of the enantiomeric excess of fluoroalcohol 68a

[0525] A racemate of fluoroalcohol 68a was prepared using a 1:1 mixture of L-:D-proline. The enantiomeric fluoroalcohols were separated by chiral SFC using Daicel OJ-3; 2900 PSI CO2, 40 °C, 3 ml / min, gradient of 20–30% 25 mM isobutylamine in isopropanol:CO2 for 7 min, retention times = 2.57 min and 2.77 min. The enantiomeric excess of optically enriched fluoroalcohol 68a was determined by the same method (94% ee).

[0526] Determination of the enantiomeric excess of fluoroalcohol 68b

[0527] The racemate of fluoroalcohol 68b was prepared using a 1:1 mixture of L-:D-proline. The enantiomers of fluoroalcohol were separated by chiral SFC using Daicel OJ-3; 2900 PSI CO2, 40 °C, 3 ml / min, gradient of 1 - 20% of 25 mM diethylamine in methanol:CO2 for 5 minutes, retention times = 3.10 min and 3.32 min. The enantiomeric excess of the optically enriched fluoroalcohol 68b was determined by the same method (93% ee).

[0528] Preparation of the aldol adduct 69

[0529] According to the conventional procedure F, a solution of phthalimidoacetaldehyde (0.050 g, 0.265 mmol), NFSI (0.84 g, 0.265 mmol), L-proline (0.031 g, 0.265 mmol), and 2,6-dimethylpyridine (0.031 ml, 0.265 mmol) in DMF (0.35 ml) was stirred at 4 °C for 15 h. Then, thiopyranone 35 (0.307 g, 2.65 mmol) was added and the reaction mixture was stirred for 18 h. The ratio of diastereomers was determined to be 5:1 by 1H NMR spectroscopic analysis of the crude product. Purification by flash chromatography (pentane:EtOAc - 60:40) gave an inseparable mixture of cis- and trans-fluoroalcohols 69 as a white solid (0.075 g, 87% yield, d.r. = 5:1). 1 1H NMR spectroscopic analysis of the crude product was used to determine the ratio of diastereomers to be 5:1. Purification by flash chromatography (pentane:EtOAc - 60:40) gave an inseparable mixture of cis- and trans-fluoroalcohols 69 as a white solid (0.075 g, 87% yield, d.r. = 5:1).

[0530]

[0531] Data for fluoroalcohol 69: 1 1H NMR (600 MHz, CDCl3): δ 7.93, 7.92, 7.79, 7.79, 6.26, 6.11, 5.37, 4.78, 3.44, 3.25, 3.24, 3.16, 3.11, 3.09, 3.03, 2.99, 2.98, 2.85, 2.80, 2.79; 13 13C NMR (150 MHz, CDCl3): δ 212.8, 210.2, 167.1, 167.1, 135.1, 134.9, 131.6, 131.5, 124.3, 124.2, 89.6, 88.3, 70.1, 66.1, 54.6, 53.6, 45.7, 44.9, 34.6, 31.3, 30.7, 30.1; 19 19F NMR (470 MHz, CDCl3): δ –155.5, –158.5 HRMS (EI + ) calcd for [C 15 H 14 FNO4S + NH4]+ 341.0966; observed 341.0938

[0532] Preparation of the aldol condensation adduct 70

[0533] According to the conventional procedure F, at 4 °C, a solution of phthalimidoacetaldehyde (0.050 g, 0.265 mmol), NFSI (0.84 g, 0.265 mmol), L-proline (0.031 g, 0.265 mmol) and 2,6-dimethylpyridine (0.031 mL, 0.265 mmol) in DMF (0.35 mL) was stirred for 16 h. Then cyclohexanone (0.275 mL, 2.65 mmol) was added, and the reaction mixture was stirred for 18 h. By 1 1H NMR spectroscopic analysis of the crude product, the ratio of diastereomers was determined to be 5:1. Purification by flash chromatography (pentane: EtOAc - 60:40) gave a non-separable mixture of cis- and trans-fluoroalcohol 70 as a white solid (0.068 g, yield 84%, d.r. = 5:1).

[0534]

[0535] Data for fluoroalcohol 70: 1 1H NMR (600 MHz, CDCl3): δ 7.92, 7.91, 7.78, 7.78, 6.29, 6.07, 5.37, 4.63, 3.51, 2.93, 2.92, 2.89, 2.80, 2.44, 2.41, 2.30, 2.25, 2.16, 2.01, 1.99, 1.87, 1.78, 1.71; 13 13C NMR (150 MHz, CDCl3): δ 215.9, 213.5, 167.1, 167.1, 134.9, 134.8, 131.7, 131.6, 124.1, 124.1, 89.9, 88.3, 69.9, 65.5, 51.8, 51.0, 43.3, 42.7, 32.4, 28.3, 27.8, 26.1, 25.4, 24.8; 19 19F NMR (470 MHz, CDCl3): δ –156.0, –160.7 HRMS (EI + ) calcd for [C 16 H 17 FNO4] + 306.1136; observed 306.1135

[0536] Preparation of the nucleoside analogue 86

[0537] At 0 °C, acetic acid (131 μL, 2.285 mmol) was added to a suspension of 68a (100 mg, 0.228 mmol) in MeCN (2.0 mL), followed by sodium triacetoxyborohydride (242 mg, 1.142 mmol). The resulting mixture was stirred at room temperature for 16 h, at which point LCMS showed complete conversion to the reduced product with a selectivity of approximately 2.5:1. The reaction mixture was then diluted with water and ethyl acetate. The organic layer was washed with brine, then dried over MgSO4, filtered, and concentrated under reduced pressure. The crude reduced product was then diluted with MeCN (2.0 mL), and indium chloride (50.5 mg, 0.228 mmol) was added. The resulting reaction mixture was stirred at 50 °C overnight. The reaction mixture was then concentrated under reduced pressure and purified by flash chromatography (25 - 100% ethyl acetate in hexane) to afford the white solid nucleoside 86 (43 mg, 45%).

[0538]

[0539] Data for nucleoside analogue 86: [α] D 20 = -15.0 (c 0.17 in MeOH); IR (neat): υ = 3298, 2938, 2852, 1537, 1442, 1204, 1108 cm -1 ; 1 H NMR (600 MHz, CDCl3): δ 8.68 (s, 1H), 7.98 (s, 1H), 6.11 (s, 1H), 5.59 (d, J = 4.7 Hz, 1H), 4.23 (dd, J = 4.7, 4.4 Hz, 1H), 3.64 (ddd, J = 11.1, 11.1, 4.0 Hz, 1H), 2.08 (m, 1H), 1.72–1.82 (4H), 1.49 (m, 1H), 1.19–1.40 (m, 3H); 13 C NMR (150 MHz, CDCl3): δ 151.1, 150.7, 150.1, 133.3, 116.5, 91.0, 80.9, 76.1, 53.4, 47.7, 40.8, 24.8, 23.6, 23.3 HRMS (EI + ) Calcd for C 14 H 16 ClIN3O2 + 419.9970; Found 419.9952.

[0540] Determination of the relative stereochemistry of nucleoside 86

[0541]

[0542] The 2D NOESY analysis of nucleoside 86 supports the depicted stereochemistry.

[0543] Preparation of nucleoside analogue 87

[0544] At -15 °C, to a stirred solution of fluoroalcohol 70 (0.105 g, 0.344 mmol, 1.0 equiv) in MeCN (3.00 mL) was added tetramethylammonium triacetoxyborohydride (0.453 g, 1.72 mmol, 5.0 equiv) and acetic acid (0.190 mL, 3.44 mmol, 10 equiv). The resulting mixture was then stirred for 16 h. The reaction mixture was then diluted with a saturated solution of Rochelle salt and washed three times with CH2Cl2. The organic layer was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The resulting crude product S70 was purified by flash chromatography (EtOAc:pentane - 70:30) to give white solid S70 (0.076 g, 72%).

[0545] To a stirred solution of cis - diol - fluoroalcohol S70 (0.076, 0.248 mmol, 1.0 equiv) in MeCN (2.50 mL) was added InCl3 (0.014 g, 0.062 mmol, 0.25 equiv), and the reaction mixture was stirred for 24 h. The reaction mixture was diluted with CH2Cl2 and washed with saturated sodium bicarbonate solution. The organic layer was separated and dried over MgSO4, filtered, and concentrated under reduced pressure. By 1 1H NMR spectroscopic analysis of the crude product, the ratio of isomers (α:β) was determined to be 2.5:1. The crude product 87 was purified by flash chromatography (EtOAc:pentane - 25:75) to give nucleoside 87 (α - isomer) as a colorless oil (42.7 mg, 60%).

[0546]

[0547] Data for nucleoside analogue 87 (α - isomer): [α] D 20 = +46.6 (c 0.38 in CH2Cl2); IR (neat): υ = 3475, 2935, 1708, 1370, 720 cm -1 ; 11H NMR(600MHz,CDCl3):δ7.88(m,2H),7.77(m,2H),6.13(d,J = 5.0Hz,1H),4.40(ddd,J = 11.8,5.0,4.8Hz,1H),4.03(ddd,J = 10.6,10.6,4.1Hz,1H),3.13(d,J = 11.9Hz,1H),2.22(m,1H),1.94(m,1H),1.85(m,2H),1.62(dddd,J = 11.9,11.9,4.6,3.2Hz,1H),1.51(m,1H),1.23–1.40(3H); 13 13C NMR(150MHz,CDCl3):δ169.1,134.6,132.1,123.8,84.4,81.1,75.3,51.4,31.7,25.4,24.0,24.0HRMS(EI + )calcd for C 16 H 18 NO4[M + H + 288.1230; found 288.1246

[0548] Determination of the relative stereochemistry of nucleoside 87

[0549]

[0550] The 2D NOESY analysis of nucleoside 87 (α - isomer) supports the stereochemistry shown.

[0551] Preparation of nucleoside analogue 88

[0552] At - 15 °C, trimethylammonium triacetoxyborohydride (0.395 g, 1.50 mmol, 5.0 equiv) and acetic acid (0.172 mL, 1.50 mmol, 10 equiv) were added to a stirred solution of fluoroalcohol 69 (0.097 g, 0.30 mmol, 1.0 equiv) in MeCN (3.00 mL). The resulting mixture was stirred for 16 h. The reaction mixture was then diluted with a saturated solution of Rochelle salt and washed three times with CH2Cl2. The organic layer was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product S69 was purified by flash chromatography (EtOAc: pentane - 70:30) to give white solid S69 (0.068 g, 70%).

[0553] To a stirred solution of cis - diol - fluoroalcohol S69 (0.047, 0.143 mmol, 1.0 equiv) in MeCN (1.43 mL) was added InCl3 (7.9 mg, 0.036 mmol, 0.25 equiv), and then the reaction mixture was stirred for 24 h. The reaction mixture was diluted with CH2Cl2 and washed with saturated sodium bicarbonate solution. The organic layer was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. By 1 1H NMR spectroscopic analysis of the crude product, the ratio of isomers (α:β) was determined to be 3:1. The crude product 88 was purified by flash chromatography (EtOAc:pentane - 40:60) to give 88 (α - isomer) as a colorless oil (23.7 mg, 73%).

[0554]

[0555] Data for nucleoside analogue 88 (α - isomer): [α] D 20 = +18.6 (c 2.37 in CH2Cl2); IR (neat): υ = 3475, 2923, 1774, 1709, 1373, 719 cm -1 ; 1 1H NMR (600 MHz, CDCl3): δ 7.88 (m, 2H), 7.77 (m, 2H), 6.13 (d, J = 4.9 Hz, 1H), 4.40 (ddd, J = 11.5, 4.7, 4.7 Hz, 1H), 4.03 (ddd, J = 11.2, 11.2, 3.6 Hz, 1H), 3.35 (d, J = 11.9 Hz, 1H), 2.98 (dd, J = 13.1 11.9 Hz, 1H), 2.82 (m, 2H), 2.69 (m, 1H), 2.50 (m, 1H), 2.10 (m, 1H), 1.74 (m, 1H); 13 13C NMR (150 MHz, CDCl3): δ 169.2, 134.8, 131.9, 124.0, 83.0, 80.2, 75.2, 51.3, 33.5, 27.6, 27.4. HRMS (EI + ) calcd for C 15 H 19 N2O4S [M + NH4 + 323.1060; found 323.1037

[0556] Determination of the relative stereochemistry of nucleoside 88

[0557]

[0558] 2D NOESY analysis of nucleoside 88 (α-isomer) supported the shown stereochemistry.

[0559] J-based configurational analysis (JBCA)

[0560] The fluorine stereochemistry of the following compounds was assigned using NMR J-based configurational analysis and then verified using density functional theory calculations. The other stereocenters were known based on synthesis.

[0561]

[0562] Nuclear magnetic resonance spectroscopy

[0563] NMR samples were prepared by dissolving a few milligrams in 0.75 mL of DMSO-d6. These solutions were then transferred to 5-mm NMR tubes. Proton chemical shifts were referenced to residual DMSO-d5 at 2.50 ppm and carbon chemical shifts were referenced to DMSO-d6 at 39.52 ppm. NMR spectra were acquired on a 600 MHz Bruker AVANCE III HD spectrometer equipped with a 5-mm triple resonance (HCN) helium cryoprobe or a 500 MHz Bruker-AVANCE III HD spectrometer equipped with a 5-mm inverse Prodigy probe. Data were processed using Mnova version 12.0.4. 1 H, 13 C, COSY, HSQC, and HMBC data were acquired to assign proton and carbon chemical shifts. NOESY or ROESY spectra were acquired with a 200-ms mixing time to assist in stereochemical determination.

[0564] DFT calculations

[0565] Density functional theory (DFT) calculations of nuclear magnetic resonance parameters, chemical shifts (δ, ppm), and coupling constants (J, Hz) were performed to verify peak assignments and relative stereochemistry. First, a conformational ensemble was generated using hybrid torsion / low-mode sampling search of the OPLS3e force field, as implemented in MacroModel (52). Then, DFT geometry optimizations and frequency determinations (to verify potential energy minima) were further carried out for conformers with energies less than 5 kcal / mol using the B3LYP / 6-31G(d) model chemistry in Gaussian'16 (53). Then, isotropic magnetic shielding values σ were calculated starting from the optimized geometries using the WP04 / cc-pVDZ or wB97X-D / 6-31G(d,p) gauge (including the gauge including atomic orbitals (GIAO) method for protons and carbons), and implicit solvent corrections were made according to the polarizable continuum model (PCM). The σ values were converted to chemical shifts, δ, in ppm using a linear scaling factor [δ = intercept - σ / -slope]. The scaling factors were previously determined from a large test set of known structures curated by Rablen et al. (54) and Lodewyk et al. (55) ( 1 H: intercept = 31.8465, slope = -0.9976; 13 C: intercept = 198.1218, slope = -0.9816). Coupling constants were calculated using the B3LYP / 6-31G(d) model chemistry. Gibbs free energies were calculated using M06-2X / 6-31+G(d,p) and the SMD solvation model, and chemical shifts and coupling constants were weighted according to the Boltzmann energy distribution.

[0566] Single-crystal X-ray diffraction

[0567] A suitable crystal was suspended in paratone oil, mounted on a MiTeGen Micro Mount, and then transferred to an X-ray diffractometer and set to 150 K using an Oxford Cryosystems Cryostream. Data were collected at 150 K on a Bruker Smart instrument equipped with an APEXII CCD area detector fixed 5.0 cm from the crystal and a CuKα fine-focus sealed tube operating at 1.5 kW (45 kV, 0.65 mA) and filtered with a graphite monochromator. Data were collected and integrated using the Bruker SAINT software package, and absorption effects were corrected using the multi-scan technique (SADABS) (56). The structure was solved using the direct method (SIR92) and subsequent refinement was carried out using SHELXL (57) and ShelXle (58). Hydrogen atoms on carbon atoms were included in geometrically idealized positions (C–H bond distances ), and not refined. The isotropic thermal parameter of the hydrogen atom was fixed at 1.2 times that of the previous carbon atom. Diagrams were drawn using Mercury(59) and POV-RAY(60). Table 1 shows a summary of the XRD analysis.

[0568] Table 1: Summary of XRD analysis

[0569]

[0570]

[0571] Examples of large-scale preparation of αFAR products

[0572] In large-scale synthesis, no additional optimization of reaction conditions was carried out. In most cases, only selected chromatographic fractions were included in the final mass.

[0573] Large-scale preparation of 55

[0574]

[0575] Three reactions were carried out in parallel. DMF (2.1 L) and uracil (300.0 g, 2.68 mol, 1.0 equivalent) were added to a large reactor at 15 - 25 °C. Then, DBU (807 mL, 5.35 mol, 2.0 equivalents) and 2-bromo-1,1-diethoxyethane (483 mL, 3.21 mol, 1.2 equivalents) were added to the reactor respectively. The reaction mixture was heated to 90 °C - 100 °C and maintained for 16 hours. The reaction mixture was cooled to 25 °C, and the three batches of the mixture were combined and concentrated to dryness to obtain a residue. Water (2.5 L) was added to the residue, the pH of the resulting mixture was adjusted to 6 - 7 with 1 M HCl, and the mixture was extracted with EtOAc (2.0 L × 8). The combined organic layers were dried over Na2SO4, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain a residue. The crude residue was triturated with MBTE (3 L) at 20 °C for 60 minutes. The crude residue was purified by silica gel chromatography (petroleum ether:EtOAc:CH2Cl2 = 10:2:1). The alkylated uracil product (738 g, 3.23 mol, 40.3% yield) was isolated as a white solid.

[0576] At 15 - 25 °C, HCl (1 M, 2.89 L, 1.0 equivalent) and the alkylated thymine product (660 g, 2.89 mol, 1.0 equivalent) were added to a large reactor. The reaction mixture was heated to 90 - 100 °C and stirred for 3 hours. After the starting materials were completely consumed, the reaction mixture was cooled to 0 °C and stirred for 30 minutes. The resulting suspension was filtered and dried, and the crude product was used in the next step without further purification. The resulting aldehyde / hydrate (425 g, 2.76 mol, 95.4%) was a pale white solid.

[0577] DMF (2800 mL) and the aldehyde (400 g, 2.60 mol, 1.0 equivalent) were added to a large reactor, and the resulting mixture was cooled to 4 °C. Then, NFSI (818 g, 2.60 mol, 1.0 equivalent), NaHCO3 (218 g, 2.60 mol, 1.0 equivalent), and L - proline (299 g, 2.60 mol, 1.0 equivalent) were added to the reactor respectively. Then the reaction mixture was stirred at 4 °C for 18 hours. HPLC (ET24077 - 13 - P1A) showed that the starting material (RT = 0.34) was completely consumed. At 4 °C, a solution of dioxanone (226 g, 1.74 mol, 0.67 equivalent) in CH2Cl2 (1.3 L) was added dropwise to the reaction mixture. The reaction mixture was stirred at 15 - 25 °C for 18 hours. HPLC (ET24077 - 13 - P1A) showed that the starting material (RT = 1.72 minutes) α - fluoro hydrate was completely consumed. 14.0 L of H2O was added to the reaction mixture, and it was extracted with EtOAc (3.0 L × 8). The organic phase was dried over Na2SO4, then filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (using a 0 - 50% ethyl acetate / petroleum ether gradient as the eluent) to obtain 55 as a yellow oil (380 g, 72% yield, d.r. 1).

[0578] Large - scale preparation of A3

[0579]

[0580] At 15 - 25 °C, DMF (1.7 L) and thymine (85.0 g, 0.674 mol, 1.0 equivalent) were added to a large reactor. Then, DBU (203 mL, 1.35 mol, 2.0 equivalents) and 2 - bromo - 1,1 - diethoxyethane (122 mL, 0.809 mol, 1.2 equivalents) were added to the reactor respectively. The reaction mixture was heated to 90 °C and maintained for 14.5 hours. The reaction mixture was concentrated to dryness to obtain a residue. EtOAc (1.7 L) and water (1.7 L) were added to the residue, the organic layer was separated, and the aqueous phase was extracted with EtOAc (1.7 L × 2). The combined organic phases were washed with brine (500 mL), dried over Na2SO4, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography ( 5000 g Silica flash column, eluent: 30 - 60% ethyl acetate / petroleum ether gradient, @ 800 mL / min). The resulting alkylated thymine product (80.0 g, 301 mmol, yield 22.4%, purity 91.3%) was a pale - white solid.

[0581] At 15 - 25 °C, HCl (1 M, 330 mL, 1.0 equivalent) and the alkylated thymine product (80.0 g, 0.330 mol, 1.0 equivalent) were added to a large reactor. The reaction mixture was heated to 90 - 100 °C and stirred for 15 hours. HPLC (ET17680 - 15 - P1A) showed that the starting material (RT = 2.77) was completely consumed. The mixture was concentrated to dryness, and the crude product was used for the next step without further purification. The resulting aldehyde / hydrate (63.0 g mixture) was a pale - white solid.

[0582] DMF (190 mL) and aldehyde (0.131 mol, 1.0 equiv) were added to a large reactor, and the resulting mixture was cooled to 4 °C. Then, NFSI (41.3 g, 0.131 mol, 1.0 equiv), NaHCO3 (11.0 g, 0.131 mol, 1.0 equiv), and L-proline (15.1 g, 0.131 mol, 1.0 equiv) were added to the reactor respectively. The reaction mixture was stirred at 4 °C for 18.5 h. HPLC (ET17918 - 3 - P1A) showed that the starting material (RT = 1.99) was completely consumed. At 4 °C, a solution of dioxanone (11.4 g, 0.088 mol, 0.67 equiv) in CH2Cl2 (200 mL) was added dropwise to the reaction mixture. Then the reaction mixture was stirred at 15 - 25 °C for 20.5 h. 570 mL of CH2Cl2 was added to the mixture, and the organic phase was washed with water (190 mL×3). The organic phase was dried over Na2SO4, then filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography ( 330 g Silica flash column, eluent: 0 - 100% ethyl acetate / petroleum ether gradient, @200 mL / min) to obtain yellow oil A3 (21.0 g, 76% yield, d.r. 3:1 (cis:trans)).

[0583] Preparation on a 16 g scale of 59

[0584] 39.0 g of A3 was dissolved in 240 mL of ethyl acetate and repurified by prep - HLPC to obtain 18.0 g of the product. Then 18.0 g of the product was dissolved in 240 mL of CH2Cl2 and concentrated under reduced pressure to obtain 17.5 g of 59. Finally, 17.5 g of 59 was lyophilized to obtain 15.8 g of 59 as a white solid (purity 94.3%).

[0585]

[0586] Data of cis - fluoroalcohol 59: [α] D 20 = - 89.4 (c 1.1 in MeOH); IR (neat): υ = 2993, 1694, 1450, 1369, 1082, 1045 cm -1 ; 11H NMR (400 MHz, CDCl3): δ 8.30 (br s, 1H), 7.57 (dd, J = 1.3, 1.2 Hz, 1H), 6.66 (ddd, J = 42.7, 2.3, 1.3 Hz, 1H), 4.40 (dd, J = 8.9, 1.4 Hz, 1H), 4.33 (dd, J = 17.7, 1.4 Hz, 1H), 4.12 (d, J = 17.7 Hz, 1H), 4.10 (ddd, J = 15.4, 3.1, 2.3 Hz, 1H), 3.64 (d, J = 3.0 Hz, 1H), 1.95 (d, J = 1.2 Hz, 3H), 1.52 (s, 3H), 1.46 (s, 3H); 13 13C NMR (100 MHz, CDCl3): δ 211.2, 163.2, 149.9, 137.1 (d, J = 4.0 Hz), 111.0, 102.1, 90.2 (d, J = 207.8 Hz), 71.6 (d, J = 2.3 Hz), 70.9 (d, J = 23.4 Hz), 66.5, 23.8, 23.4, 12.6; 19 19F NMR (470 MHz, CDCl3): δ –177.8 HRMS (EI + ) calcd for C 13 H 18 FN2O6 [M + H] + 317.2929; found 317.1142

[0587] Large-scale preparation of A5

[0588]

[0589] The reaction was carried out without further optimization. The crude product A5 was purified by column chromatography to give 16.5 g of A5 (the impure fractions were discarded).

[0590] Large-scale preparation of A6

[0591]

[0592] The reaction was carried out without further optimization. The reaction was stopped only after 16 h. The crude product A6 was purified by prep-HPLC to give 36.6 g of A6 (the impure fractions were discarded).

[0593] Large-scale preparation of A8

[0594]

[0595] The reaction was carried out without further optimization. The crude product A8 was purified by prep-HPLC to give 47 g of A8 (the impure fractions were discarded).

[0596] Research progress on a short de novo synthesis of NA.

[0597] This invention studied the α-fluorination (33) of α-pyrazole aldehyde 15 ( Figure 2B ), and found that the combination of L-proline and N-fluorobenzenesulfonamide (NFSI) in DMF (34) provided α-fluoro hydrate as the sole product (Table 2).

[0598] Table 2: Optimization of αFAR for α-pyrazole aldehyde

[0599]

[0600]

[0601] a 1.5 equivalents. b The volume of the added solvent was 1.25× the volume of DMF in Step 1. c The volume of the solvent added in the first step was 9× the volume of DMF.

[0602] MeCN of dioxanone 8 was directly added to the reaction mixture to give fluoroalcohols 16a and 16b with good yields and enantioselectivities ( Figure 2B , Entry 2). As shown, fluoroalcohols 16a and 16b formed as an epimeric mixture of ~1.4:1 at the pseudo-anomeric carbon (denoted by *), and did not interconvert under the reaction or separation / purification conditions.

[0603] Reduction of fluoroalcohols 16a and 16b provided a mixture of 1,3-cis-diols, which was then treated with one of several Lewis acids to promote the displacement of fluoride through the distal alcohol functional group, and an AFD reaction using the fluorophilic Sc(OTf)3 (36) was achieved to provide NA 17 as the single β-isomer in 38% yield ( Figure 2B , Entry 4). In addition, this invention found that treatment of the mixture of diols 12a and 12b with a base (NaOH) formed a mixture of α- and β-anomeric NAs whose composition varied with reaction time and base equivalents ( Figure 2B , Entries 5 and 6). Using a large excess of NaOH (10 equivalents, Entry 6), the β-isomer 17 was formed as the sole product in excellent yield (76%). To further study the cyclization mechanism, the intermediate diols 18a and 18b were separated by flash column chromatography and their relative stereochemistry was assigned by J-based configurational analysis and / or X-ray analysis of the derivatives.

[0604] The purified cis-fluoroalcohol 18a was subjected to the AFD reaction (NaOH, CH3CN, Figure 2C ), and clean cyclization was facilitated by the S N 2 process to afford the β-isomer 17. Similarly, the trans-fluoroalcohol 18b was also cyclized via stereochemical transformation to give the α-isomer 19. Under these same reaction conditions, the α-isomer 19 epimerized to the β-isomer 17 of the natural configuration, so that the two fluoroalcohol hydroxyaldehyde products could be converted together to a β-D-NA of the natural configuration. The enantiomeric purity of NA 17 (e.r. = 95:5, Figure 2B , entry 6) represents the average of the enantiomeric purities of the epimeric fluorouracil FAR products 16.

[0605] Preparation of NAs using the αFAR and AFD strategies.

[0606] A series of acetaldehyde derivatives were prepared by alkylation of several heterocycles with bromoacetaldehyde diethyl acetal ( Figure 3A ). Using Selectfluor or NFSI as the electrophilic fluorinating agent (F + ), the resulting aldehydes 21 were subjected to proline-catalyzed αFAR with dioxane 8 to afford a series of fluoro-hydroxyaldehyde condensation products 22 functionalized with one of heterocyclic uracil, thymine, triazole, deazane, pyrazole, phthalimide, adenine, 2,6-dichloropyrimidine or tetrazole. The yields and enantiomeric purities of these fluoroalcohols were generally high, even excellent. Table 3 shows the optimization of αFAR of α-(1,2,3)-triazole aldehyde.

[0607] Table 3: Optimization of αFAR of α-(1,2,3)-triazole aldehyde

[0608]

[0609]

[0610] a 1.5 equivalents. b The volume of solvent added was 1.25× the volume of DMF in the first step. c The volume of solvent added in the first step was 9× the volume of DMF.

[0611] In the case of fluoroalcohols containing adenine, the enantiomeric purity was reduced by the competitive (non-proline) catalysis in αFAR. Each αFAR product was separated as a mixture of epimers at the fluoromethyl center, followed by 1,3-synchronous selective carbonyl reduction and promoted by base (NaOH, Figure 3B ) or Lewis acid ( Figure 3C ) for AFD, as shown. Several heterocycles were compatible with this process (Figure 3B -E), Uracil-, thymine-, or adenine-substituted acetaldehydes can be utilized in the short (4-step total) de novo synthesis of the endogenous ribonucleosides uridine (U:24), 5-methyluridine (m 5 U:25), and adenosine (A:31). In these studies, the Lewis acids promoting the AFD reaction were InCl3 or Sc(OTf)3, and the pyrazole- and uracil-derived fluoroalcohols were cyclized using NaOH. In the present study, with the exception of triazoles 28, trifluoromethyluracil 29, and deazaadenines 32 and 33, the production of NAs was an approximate average of the enantiomeric purity of the single precursor fluoroalcohol epimer 22. Thus, most NAs epimerize after AFD, providing a direct method for converting a mixture of epimeric hydroxyaldehyde products into a single, naturally configured β-D-nucleoside analogue. For trifluoromethyluracil 29 and deazaadenines 32 and 33, the αFAR products (e.g., 22) were reduced, isolated, and treated with Sc(OTf)3 or InCl3, respectively. As Figure 3C shown, for trifluoromethyluracil, only the trans-fluoroalcohol undergoes AFD to form 29, which does not epimerize under the reaction conditions. In the case of deazaadenine, AFD of the cis- and trans-fluoroalcohols provides the β- and α-isomers 32 and 33, respectively, confirming that these reactions proceed via direct fluoride displacement.

[0612] Some αFARs have been demonstrated on scales greater than 10 g (e.g., 25, 28, 29, 30, and 32( Figure 3C )) and the present invention notes that diastereoselectivity is improved when the reaction is conducted on a larger scale. The present invention also found that this series of reactions starting from dichloropyrimidine can be used to prepare C-linked NA 27, further extending the utility of this strategy to another important class of NAs. (37) Here, the major product of αFAR is the trans-fluoroalcohol, which stereoselectively cyclizes to the α-D-nucleoside analogue and undergoes a second cyclization event under the reaction conditions to form tricyclic 27. In addition to naturally configured NAs, by using D-proline in αFAR, this strategy can be readily adapted for the synthesis of enantiomeric (L-configured) nucleosides and NAs ( Figure 3E ). Thus, L-uridine (ent-24) and the L-configured NA ent-28 were obtained in this straightforward manner. Although the crude reaction mixture is typically treated with aqueous acid to remove the acetonide protecting group and enable isolation of the target NA, the present invention can directly isolate the C3' / C5'-protected NAs (e.g., 34 and 35, Figure 3D)。To demonstrate that these acetone-protected NAs can be further derivatized using standard protocols, the present invention prepared several C2'-modified NAs, including C2'-oxo (36), C2'-deoxy (37), C2'-3° alcohol (38), and C2'-epi (39)( Figure 3F )。

[0613] The optimization of the AFD reaction is shown in Tables 4 and 5.

[0614] Table 4: Optimization of the AFD reaction

[0615]

[0616]

[0617]

[0618] a 0.10 M. b 2.5 equivalents. c 10 equivalents.

[0619] Table 5: Optimization of the AFD reaction.

[0620]

[0621]

[0622] a 0.10 M. b 10 equivalents. c 0.15 equivalents. d 1.5 equivalents. e 2.5 equivalents.

[0623] Rapid synthesis of C4'-modified α-L-configured NAs.

[0624] The present invention investigated whether the addition of organometallic reagents (instead of reduction with hydrides) to a series of αFAR products could provide tertiary alcohols, the subsequent AFD of which would directly result in C4'-modified NAs. To this end, the present invention investigated the reaction of the deazapurine-substituted fluoroalcohol 41 with a series of organometallic reagents (e.g., MeMgCl, MeMgBr, Me2Zn, Me3ZnLi, MeLi, Me2Mg, Me3MgLi) in CH2Cl2 or THF at -78 °C, 0 °C, or room temperature ( Figure 4A, illustration). From this panel, the testing of Grignard reagents (e.g., MeMgX) in CH2Cl2 is compatible with highly functionalized fluoroalcohols. The 1,2-addition reaction is carried out at -78 °C because higher temperatures promote 1,2-hydride shift / fluoride displacement as the major degradation pathway. Regarding stereochemistry, the 1,2-addition reaction yields a mixture of tertiary alcohols, preferably adding from the face with the least hindered carbonyl functional group in 33 (the back face). (30) When the reaction is carried out in CH2Cl2 and the crude reaction mixture is heated to room temperature overnight, the intermediate magnesium alkoxide 42a undergoes AFD to directly provide the C4-modified NA 43. Thus, this sequence enables the enantiomerically enriched C4'-modified NAs to be obtained from simple achiral heterocycles and bromoacetaldehyde diethyl acetal in only 3 steps. Alternatively, quenching the mixture of magnesium alkoxides 42a and 42b with ammonium chloride, followed by subsequent Lewis acid-promoted AFD using InCl3, gives the anomeric α-D NA 36. Thus, in this case, each of the magnesium alkoxides 42a and 42b is selectively cycled using complementary base- or Lewis acid-promoted AFD processes to obtain NAs in the α-L and α-D configurations.

[0625] The present invention also examined the reactions of several other organomagnesium reagents with fluoroalcohol aldol adducts containing triazole, deazapurine, thymine, pyrazole, or trifluoromethyluracil functional groups ( Figure 4A ). In this study, the present invention found that the degree of stereoselectivity in the 1,2-addition reaction depends on the solvent and the heterocycle. For example, adding MeMgBr to a ketone fluoroalcohol in THF gave a mixture of tertiary alcohols with a different composition than that produced in CH2Cl2. Adding MeMgBr to a ketone fluoroalcohol substituted with triazole mainly gave the 1,3-cis-diol, which undergoes AFD to produce the naturally configured NA α-D-48.

[0626] Thus, a series of deazapurine-substituted NAs 35-39 are readily accessible in the form of α- and β-isomers. In these studies, base-promoted AFD led to C3',C5'-protected NAs (such as 49-54), while Lewis acid-promoted AFD led to deprotection or protecting group migration (such as 44, 47, and 48). As summarized in Figure 4, a series of highly functionalized C4'-modified NAs can be rapidly obtained from the corresponding ketone fluoroalcohol aldol adducts, including NAs substituted with methyl, cyclopropyl, aryl, and alkynyl groups. The preparation of each C4'-methyl, cyclopropyl, p-methoxyphenyl, p-chlorophenyl, alkynyl NA 43-54 requires only 3 or 4 steps in total.

[0627] The optimization of the 1,2-addition reaction is shown in Table 6.

[0628] Table 6: Optimization of the 1,2-addition reaction

[0629]

[0630]

[0631] a 3 equivalents. b 0.10 M. c by 1 H NMR analysis of the crude reaction mixture. d Independent yields.

[0632] Large-scale αFAR for the synthesis of Uprifosbuvir.

[0633] The present invention investigated the synthesis of D-uridine derivative 56 starting from 900 g of uracil. Without any additional optimization, the present invention was able to generate approximately 380 g of the aldol condensation adduct 55 ( Figure 2B ), which could be converted to the protected uridine 56 by base-promoted AFD in good yield. Oxidation of the C2'-OH functionality, followed by deprotection in tetrahydrofuran and addition of MeMgBr, gave the tertiary alcohol 57. This latter compound was an intermediate (38) for the previously reported large-scale production of MK-3682 (Uprifosbuvir: 58).

[0634] Synthesis of imino nucleosides, deoxynucleosides, and locked nucleic acids.

[0635] The present invention also evaluated the utility of the method for obtaining a class of unusual NAs (termed imino nucleosides or 4'-aza nucleosides), in which the furan oxygen is replaced by a nitrogen atom. Thus, in one embodiment ( Figure 4C ), it was shown that reductive amination of the fluoroalcohol-aldol adduct 59 (isolated as a single diastereomer as shown) with benzylamine, followed by basic treatment, directly gave the imino nucleoside 60 in β-D configuration in good yield.

[0636] To demonstrate the utility of this route for obtaining C2' and C4'-modified NAs, the present invention prepared a C4'-modified, C2'-deoxy NA ( Figure 4D ). Here, the C4'-allyl thymine 61 was prepared in good yield by adding allylmagnesium bromide to the fluoroalcohol aldol condensation adduct 59, followed by base-promoted AFD. Then, the 4'-allyl NA 62 was obtained from thymine in only 6 steps by the Barton-McCombie deoxygenation reaction.

[0637] To demonstrate the utility of this process for NA synthesis, the present invention investigated methods for the preparation of locked nucleic acids (LNAs) by C4'-functionalization. To achieve unified LNA synthesis, the present invention evaluated the addition of alkylmagnesium bromide to the thymine-containing aldol adduct 59 and found that the reaction gave two diastereomeric addition products 63 and 64 in good overall yield. The major product was directly converted to the unusual LNA 67 by reaction with NaOH, which promoted the AFD reaction and subsequent cyclization between the free alcohol functionality and the alkyne in very good overall yield. This four-step total synthesis compares favorably with the 23-step route to the analogous uracil LNA 67 (40) reported previously. We were also able to generate the unusual alkyl-functionalized LNA 68 by simply subjecting the 1,2-addition product 64 to AFD, a scaffold for nucleoside chemistry not previously reported. From here, 2,2'-anhydrothymidine was formed, followed by deprotection and base treatment in warm DMF (41) to afford LNA 68. This unique scaffold provides a basis for further diversification by standard click reactions or Sonagashira coupling reactions.

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[0699] All citations are incorporated herein by reference.

[0700] The present invention has been described with respect to one or more embodiments. However, it will be apparent to those skilled in the art that some variations and modifications can be made without departing from the scope of the invention as defined in the claims. Therefore, although various embodiments of the present invention are disclosed herein, many adjustments and modifications can be made within the scope of the present invention according to the common general knowledge of those skilled in the art. Such modifications include substituting any aspect of the present invention with known equivalents so as to achieve the same result in substantially the same manner. Numerical ranges include the numbers defining the range. In the specification, the term "comprising" is used as an open-ended term, substantially equivalent to the phrase "including but not limited to", and the term "comprises" has a corresponding meaning. However, it should be understood that when using "comprising" or "including" or variants with the same root here, it is also possible to consider varying or modifying it to "consisting of", which excludes any unspecified elements, steps or components, or "consisting essentially of" or "essentially consisting of", which is limited to the specified materials or the steps mentioned, and those materials that do not materially affect the basic and novel features of the claimed invention. In addition, unless the context otherwise requires, any arrangement and combination of all the elements described in the present invention shall be regarded as being disclosed in the description of the present invention. The citation of references herein should not be construed as an admission that these references are prior art to the present invention. All publications are incorporated herein by reference as if each individual publication was specifically and individually indicated to be incorporated by reference herein and as if fully set forth herein. The present invention includes all embodiments and variations substantially as described above and with reference to the embodiments and the drawings.

Claims

1. A method for synthesizing a nucleoside or its analogue, characterized in that, The method comprises: (i) fluorinating an aryl- or heteroaryl-substituted acetaldehyde compound by proline catalysis, and then performing an enantioselective aldol condensation reaction to obtain a fluoroalcohol compound; the aryl or heteroaryl comprises 5-14 members; (ii) reducing the fluoroalcohol compound to generate a fluoroalcohol diol compound; and (iii) contacting the fluoroalcohol diol compound with a Lewis acid or a base in a cyclization halide displacement (AHD) reaction; to generate a nucleoside or an analogue thereof.

2. The method according to claim 1, characterized in that The Lewis acid is InCl3 or Sc(OTf)3.

3. The method according to claim 1 or 2, characterized in that, The fluoroalcohol diol compound is separated before being treated with a Lewis base.

4. The method according to claim 1, wherein The base is NaOH.

5. The method according to claim 1 or 4, characterized in that, The base-AHD reaction produces a C3',C5'-protected nucleoside or an analogue thereof.

6. A method for preparing an intermediate in a synthetic nucleoside or its analogue, characterized in that, The method comprises: (i) fluorinating an aryl- or heteroaryl-substituted acetaldehyde compound by proline catalysis, and then performing an enantioselective aldol condensation reaction to obtain a fluoroalcohol compound; the aryl or heteroaryl comprises 5-14 members; and (ii) reducing the fluoroalcohol compound to obtain a fluoroalcohol diol compound, which generates an intermediate in the synthesis of a nucleoside or an analogue thereof.

7. The method according to claim 6, characterized in that, The fluoroalcohol compound is: wherein NB is an aryl or heteroaryl, comprising 5-14 members, X is F, R is independently -OH, or is a part of a cyclic structure composed of -OC(CH3)2O-, -(CH2)3-, -CH2SCH2-, or -CH2OCH2-.

8. The method according to claim 6, wherein The fluoroalcohol compound is as follows: wherein NB is an aryl or heteroaryl, comprising 5-14 members, X is F, Y is CH2, O, S, NR, wherein R is an alkyl or aryl, and Z is a protecting group for ethanol.

9. The method according to claim 8, wherein The protecting group for ethanol is selected from acetone dimethyl acetal, silyl protecting groups, alkyl protecting groups, and aryl protecting groups.

10. The method according to claim 6, characterized in that, The fluoroalcohol compound is: wherein NB is an aryl or heteroaryl, comprising 5-14 members, X is F.

11. The method according to claim 6, wherein The fluoroalcohol compound is: wherein NB is an aryl or heteroaryl, comprising 5-14 members, X is F, Y is CH2, O, S, NR, wherein R is an alkyl or aryl, comprising 5-14 members.

12. The method according to any one of claims 1-11, characterized in that, The fluoroalcohol compound is: wherein NB is an aryl or heteroaryl, comprising 5-14 members, X is F.

13. A method for synthesizing a nucleoside or its analogue, characterized in that, The method comprises: (i) providing an aryl- or heteroaryl-substituted fluoroalcohol diol compound; the aryl or heteroaryl comprises 5-14 members; and (ii) in a cyclization halide displacement (AHD) reaction, contacting the aryl- or heteroaryl-substituted fluoroalcohol diol compound with a Lewis acid or a base, to obtain a nucleoside or an analogue thereof.

14. The method according to claim 13, wherein The Lewis acid is InCl3 or Sc(OTf)3.

15. The method according to claim 13 or 14, characterized in that, The aryl- or heteroaryl-substituted fluoroalcohol diol compound is separated before being treated with a Lewis base.

16. The method according to claim 13, characterized in that The base is NaOH.

17. The method according to claim 13 or 16, characterized in that, The base-AHD reaction produces a C3',C5'-protected nucleoside or an analogue thereof.

18. The method according to any one of claims 1-17, characterized in that, The fluoroalcohol diol compound is: wherein NB is an aryl or heteroaryl, comprising 5-14 members, X is F.

19. The method according to any one of claims 1-18, characterized in that, The nucleoside or its analogue is a D-nucleoside, an L-nucleoside, a locked nucleic acid, an imino nucleoside, a C4'-modified nucleoside or a C2'-modified nucleoside.

20. The method according to any one of claims 1-18, characterized in that, The nucleoside or its analogue is: wherein, NB is an aryl or heteroaryl, including 5-14 members, and each R is independently -OH, or is part of a cyclic structure composed of -OC(CH3)2O-, -(CH2)3-, -CH2SCH2-, or -CH2OCH2-.

21. The method according to any one of claims 1-19, characterized in that, The aryl or heteroaryl is selected from adenine, guanine, cytosine, thymine, uracil, 5,6-dihydro uracil, 5-methyl cytosine, 5-hydroxymethyl cytosine, 5,5,5-trifluoromethyl thymine, 5-fluorouracil, 2-thiouracil, 4-methyl benzimidazole, hypoxanthine, 7-deazaguanine, 7-deazaadenine, indole, imidazole, triazole, pyrrole, pyrazole, phenyl, biphenyl, naphthyl, dihydroindenyl, indenyl, tetrahydronaphthyl, 2,3-dihydrobenzofuranyl, dihydrobenzopyranyl, 1,4-benzodioxanyl, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, 1,2,3-oxadiazole, 1,3,4-thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, 2,6-dichloropyrimidine pyrazine, 1,3,5-triazine, benzimidazole, benzoxazole, benzothiazole, indolizine, isoindole, benzofuran, benzothiophene, 1H-indazole, purine, 4H-quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, pteridine, deazauracil, or phthalimide.

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