Synthesis of 7-trifluoromethyl-7-deazapurine ribonucleoside analogs and their monophosphate prodrugs

Abstract

Novel 7-trifluoromethyl-7-deazapurine ribonucleoside analogs (13a-c) and their Protides (15a-c) were successfully synthesized from ribolactol or 1-α-bromo-ribose derivatives using Silyl-Hilbert-Johnson or nucleobase-anion substitution reactions followed by key aromatic trifluoromethyl substitution. Newly prepared compounds were evaluated against a panel of RNA viruses, including HCV, Ebola or Zika viruses.

Introduction

Nucleoside analogs played a pivotal role in the development of antiviral and anti-cancer agents. Since the discovery by William Prussoff in 1962¹ of idoxuridine, the first antiviral drug approved for the treatment of herpes virus, more than 25 nucleoside derivatives have been approved to treat human infectious diseases. 7-Deazapurine nucleoside derivatives are an extremely interesting class of analogs due to their innate biological activity.² Over the past 50 years, more than twenty 7-deazapurine derivatives have been discovered from terrestrial and marine sources.³ Among them, naturally occurring tubercidin (1a),⁴ toyocamycin (1b),⁵ and sangivamycin (1c)⁶ display a broad spectrum of activity and have driven extensive SAR studies towards the development of new antiviral and anticancer agents. Thus, 7-deazaadenosine derivatives of 6-hetaryl-7-deazapurine (2a) and 7-fluoro-7-deazapurine ribonucleosides (2b-c)⁷ as well as 7-hetaryl-7-dazaapurine ribonucleoside (3a)⁸ displayed potent cytostatic activity in multiple cancer cell lines. Recently, a series of 6-substituted 7-furyl or ethynyl-7-deazapurine ribonucleoside analogs (3b-c)⁹ showed significant cytostatic, antimicrobial and promising anti-HCV activity. Furthermore, 7-fluoro-7-deazapurine ribonucleoside (4a)¹⁰ showed improved anti-cancer activity and reduced cytotoxicity when compared with its parent tubercidin (1a). 7-Fluoro-7-deazapurine-2’-C-methyl and 2’-deoxy-2’-fluoro-2’-C-methyl ribonucleosides (4b)¹¹ and (4c)¹² were highly potent inhibitors of HCV replication. Interestingly, 7-deazapurine-2’-C-methylribonucleoside (5a) has been reported to inhibit both HCV¹³ and Zika virus replication.¹⁴ Additionally, 7-deazapurine-2’-C-ethynylribonucleoside (5b, NITD008) potentially inhibited dengue and Zika viruses.¹⁵ Furthermore, 7-vinyl-7-deazapurine-2’-deoxy-2’-fluoro-2’-C-methyl ribonucleoside (5c) exhibited promising anti-HCV activity as well as weak anti-HIV activity.¹⁶ The 1,2,4-oxadiazole-7-deazapurine-2’-C-methylribonucleoside (5d) showed high antiviral potency against HCV replication cells.¹⁷ 7-Deazaneplanocin A analog (6) exhibited potent antiviral activity against cowpox and vaccinia viruses.¹⁸

If a large variety of groups has been introduced at the 7-position of 7-deazapurine nucleosides (Figure 1), the effect of a CF₃ group at this position remains underexplored. Herein, we wish to report the synthesis and antiviral evaluation of novel ribo, β−2’-methyl- or α−2’-fluoro-β−2’-methyl-D-ribofuranoses 7-deazapurine derivatives with a trifluoromethyl group at the 7-position using a key aromatic trifluoromethyl substitution (Figure 2).

Figure 1.

Biologically relevant 7-deazapurine nucleoside analogs

Figure 2.

Target 7-trifluoromethyl-7-deazapurine analogs

Results and discussion

6-Chloro-7-iodo-7-deazapurine (8), prepared from commercially available 6-chloro-7-deazapurine (7) by treatment with N-iodosuccinimide (NIS) in DMF¹⁹, was reacted with 1-α-/β-O-acetyl-2,3,5-tri-O-benzoyl-D-ribose (9a) and 1-α-/β-O-benzoyl-2,3,5-tri-O-benzoyl-2’-C-methyl-D-ribose (9b) under classical Vorbrüggen glycosylation conditions^20–22 to give β- 6-chloro-7-iodo-7-deazapurine ribonucleosides 10a and 10b. Next, aromatic trifluoromethylation reaction^23,24 of compound 10a and 10b was carried out by treatment with methyl fluorosulfonyldifuoroacetate (MFSDA) and CuI in a mixture hexamethylphosphoramide (HMPA) and N,N-dimethylformamide (DMF) to give 7-trifluoromethyl-7-deazapurine nucleoside derivatives 11a and 11b in 90–93% yield.²⁵ Subsequent treatment with a saturated solution of ammonia in MeOH gave 7-trifluoromethyl-7-deazapurine nucleosides 12a and 12b in good yields. Finally, since the monophosphorylation of a nucleoside analog²⁶ can be the limiting step to the formation of the active triphosphate form, nucleosides 12a and 12b were converted into their corresponding phosphoramidate prodrug 14a and 14b by reaction with chlorophosphoramidate derivative 13 in presence of t-BuMgCl.²⁷ Prodrugs 14a and 14b were obtained as 1/1 Rp/Sp-mixtures, as determined by ¹H-NMR and ³¹P-NMR. It is noteworthy that when N-methylimidazole (NMI) was employed instead of t-BuMgCl, prodrugs 14a and 14b were obtained in poor yields due to incomplete reactions and formation of 3’,5’-bis-phosphoramidate by-products.

2’-C-Me,2’-F nucleoside analog 12c and its phosphoramidate prodrug 14c were prepared according to the chemistry described in Scheme 2. 1-α-Bromo-3,5-di-O-benzoyl-2’-deoxy-2’-fluoro-2’-C-methylribose (9c), synthesized from commercially available 3,5-di-O-benzoyl-2’-deoxy-2’-fluoro-2’-C-methyl ribolactone (15),^28,29 was coupled with 7-deaza purine 8 in presence of KOH and tris-[2-(2-methoxyethoxy)]ethylamine (TDA-1)³⁰ to give 3,5-di-O-benzoyl-2’-deoxy-2’-fluoro-2’-C-methyl-6-chloro-7-iodo-7-deazapurine ribonucleoside (10c) in 68% yield. Subsequent reaction with MFSDA and CuI in a mixture of DMF and HMPA afforded 3,5-di-O-benzoyl-2’-deoxy-2’-fluoro-2’-C-methyl-6-chloro-7-tri-fluoromethyl-7-deazapurine ribonucleoside (11c) in 97% yield. Finally, treatment of 11c with a saturated solution of ammonia in MeOH provided 2’-deoxy-2’-fluoro-2’-C-methyl-7-trifluoromethyl-7-deazapurine ribonucleoside (12c) in 85% yield. Phosphoramidate prodrug 14c was prepared by reaction of nucleoside 12c with chlorophosphoramidate 13 in presence of t-BuMgCl (Rp/Sp = 1/1 ratio).

Scheme 2.

Synthesis of compound 12c and 14c

Reagents and reaction conditions: a) i. LiAl(t-BuO)₃H, THF, −20 °C, 4 h: ii. AcCl, DMAP, Et₃N, CH₂Cl₂, 0 °C, 3 h; b) 33% HBr in AcOH, CH₂Cl₂, 0 °C then rt 4 h; c) TDA-1, KOH, 4 Å MS, CH₃CN, rt, 2 h; d) FSO₂CF₂CO₂Me, CuI, HMPA, DMF, 70 °C, 12 h; e) sat. NH₃/MeOH, 90 °C, 12 h, steel bomb; e) 13, t-BuMgCl, THF, −78 °C, 6 h.

Nucleosides 12a-c and their corresponding phosphoramidate prodrugs 14a-c were evaluated against a panel of RNA viruses including HCV³¹ (clone B replicon), Ebola³² (Zaire ebola virus replicon) and Zika³³ (cytopathic effect reduction assay) viruses. In addition, cytotoxicity was determined in primary human peripheral blood mononuclear (PBM) cells, human lymphoblastoid CEM, African Green monkey Vero cells and HepG2 cells.³⁴ Unfortunately, none of these derivatives exhibited significant activity at concentration up to 20 μM against these viruses nor toxicities in PBM, CEM and Vero cells at concentration up to 100 μM.

In conclusion, several novel 7-trifluoromethyl-7-deazapurine ribonucleoside analogs (12a-c) were synthesized by using a key aromatic trifluoromethylation with MFSDA and CuI using HMPA and DMF as co-solvents. Unfortunately, none of the synthesized compounds (12a-c and 14a-c) showed marked activity when tested against HCV, Ebola or Zika viruses.

Experimental

Nuclear magnetic resonance (NMR) spectra (¹H, ¹³C, ¹⁹F and ³¹P) were recorded on a Varian Unity Plus 400MHz and a Bruker Ascend™ 400MHz Fourier transform spectrometer at RT, with tetramethylsilane (TMS) as an internal standard. Chemical shifts (δ) are reported in parts per million (ppm), and signals are quoted as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad), dd (doublet of doublets), or ddd (doublet of doublets of doublets). The phosphoramidates are an approximate 50:50 mixture of diastereomers (R_P/S_P) and the ¹³C NMR data is reported as observed, that is, some carbon signals overlap. High-resolution mass spectra (HRMS) were recorded on a Micromass Autospec high-resolution mass spectrometer with electrospray ionization (ESI). Thin-layer chromatography (TLC) was performed on 0.25mm silica gel. Purifications were carried out on silica gel column chromatography (60 Å, 63–200 μm, or 40–75 μm).

Compound 8

To a solution of 6-chloro-7-deazapurine (1.0 g, 6.51 mmol) in 20 mL of anhydrous DMF was added N-iodosuccinimide (1.60 g, 7.16 mmol). The reaction mixture was stirred for 2 h at room temperature and the solvent removed under vacuum. The residue was purified by silica gel column chromatography (hexanes:EtOAc = 1:1 to 3;1v/v) to give compound 8 in quantitative yield. ¹H NMR (400 MHz, CD₃OD) δ 8.55 (s, 1H), 7.72 (s, 1H); ¹³C NMR (100 MHz, CD₃OD) δ 151.7, 151.6, 150.2, 133.5, 116.3, 50.1; MS-ESI⁺ m/z 280 (M+H⁺).

(2R,3R,4R,5R)-2-((Benzoyloxy)methyl)-5-(4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3,4-diyl dibenzoate (10a)

To a solution of 6-chloro-7-iodo-7-deazapurine 8 (0.55 g, 1.96 mmol) in 10 mL of anhydrous CH₃CN was added N,O-bis(trimethylsilyl)acetamide (0.50 g, 2.45 mmol) at room temperature under N₂ atmosphere. After 30 min, a solution of 1-O-Ac-2,3,5-tri-O-Bz-ribose 9a (0.90 g, 1.79 mmol) in 10 mL of anhydrous CH₃CN and TMSOTf (0.56 g, 2.45 mmol) was added to the reaction mixture at 0 °C. The reaction mixture was then heated to 80 °C over 1 h and stirred for 12 h at this temperature e. The solution was cooled to room temperature and diluted with EtOAc (50 mL). The organic layer was washed with a saturated NHCO₃ aqueous solution (20 mL), cold water (20 mL) and brine (20 mL). The organic layer was then dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexanes:EtOAc = 5:1 to 2:1 v/v) to give compound 10a (0.83 g, 1.15 mmol) in 64% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.58 (s, 1H), 8.21 (d, J = 7.2 Hz, 2H), 7.99 (d, J = 6.8 Hz, 2H), 7.92 (d, J = 8.4 Hz, 2H), 7.64–7.49 (m, 6H), 7.43–7.35 (m, 4H), 6.67 (d, J = 5.6 Hz, 1H), 6.15 (t, J = 5.6 Hz, 1H), 6.11 (dd, J = 5.6, 4.4 Hz, 1H), 4.90 (dd, J = 12.4, 3.2 Hz, 1H), 4.80 (q, J = 3.6 Hz, 1H), 4.68 (dd, J = 12.4, 3.6 Hz, 1H), ¹³C NMR (100 MHz, CDCl₃) δ 166.3, 165.8, 165.3, 153.3, 151.5, 151.2, 134.1, 134.0, 133.8, 132.2, 130.1, 129.9, 129.5, 129.1, 128.9, 128.8, 128.7, 128.6, 118.0, 87.0, 80.9, 74.4, 71.6, 63.7, 53.9; MS-ESI⁺ m/z 724 (M+H⁺); HRMS-ESI⁺: m/z calcd for C₃₂H₂₄ClIN₃O₇ (M+H⁺) 724.0347, found 724.0331.

(2R,3R,4R,5R)-2-((Benzoyloxy)methyl)-5-(4-chloro-5-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3,4-diyl dibenzoate (11a)

To a solution of compound 10a (0.51 g, 0.71 mmol) in 20 mL of anhydrous DMF and hexamethylphosphoric triamide (HMPA, 8.50 mL) was added FSO₂CF₂CO₂Me (170.0 mg, 0.88 mmol) and CuI (160.0 mg, 0.85 mmol) under argon atmosphere. The reaction mixture was stirred for 13 h at 70 °C and then cooled down to room temperature before addition of a saturated aqueous solution of NH₄Cl (20 mL). The mixture was extracted with a mixture of hexanes and EtOAc (50 mL x 2, 1:2 v/v). The combined organic layers were washed with a saturated aqueous solution of NaHCO₃ (20 mL), water (20 mL), brine (20 mL) then dried over Na₂SO₄ and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (hexanes: EtOAc = 10:1 to 5:1 v/v) to give compound 11a (0.44 g, 0.66 mmol) in 93% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.66 (s, 1H), 8.11–8.08 (m, 2H), 8.02–8.00 (m, 2H), 7.93–7.91 (m, 2H), 7.86 (s, 1H), 7.62–7.34 (m, 9H), 6.68 (d, J = 5.6 Hz, 1H), 6.22 (t, J = 5.6 Hz, 1H), 6.15 (dd, J = 5.6, 4.4 Hz, 1H), 4.92 (dd, J = 9.2, 3.2 Hz, 1H), 4.85 (q, J = 3.6 Hz, 1H), 4.72 (dd, J = 12.4, 3.6 Hz, 1H), ¹³C NMR (100 MHz, CDCl₃) δ 166.2, 165.5, 165.2, 152.7, 152.4, 152.1, 134.0, 133.9, 130.0, 129.8, 128.9, 128.8, 128.7, 87.6, 81.1, 74.4, 71.6, 63.5; ¹⁹F NMR (376 MHz, CDCl₃) δ −56.52; MS-ESI⁺ m/z 666 (M+H⁺); HRMS-ESI⁺: m/z calcd for C₃₃H₂₄ClF₃N₃O₇ (M+H⁺) 666.1255, found 666.1245.

(2R,3R,4S,5R)-2-(4-Amino-5-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (12a)

In a steel bomb at 0 °C, NH₃ was bubbled through a solution of compound 11a (0.16 g, 0.24 mmol) in 30 mL of MeOH for 20 min. The reaction was heated at 90 °C for 12 h before being cooled down to 0 °C. After evaporation of the volatiles under reduced pressure, the residue was purified by silica gel column chromatography (CH₂Cl₂: MeOH = 20:1 to 10:1 v/v) to give compound 12a (58.60 mg, 0.18 mmol) in 83% yield. ¹H NMR (400 MHz, CD₃OD) δ 8.20 (s, 1H), 8.03 (s, 1H), 6.11 (d, J = 6.0 Hz, 1H), 4.59 (t, J = 5.6 Hz, 1H), 4.30 (dd, J = 4.8, 3.2 Hz, 1H), 4.13 (q, J = 3.2 Hz, 1H), 3.87 (dd, J = 12.4, 2.4 Hz, 1H), 3.76 (dd, J = 12.4, 2.8 Hz, 1H); ¹³C NMR (100 MHz, CD₃OD) δ 158.1, 153.9, 152.7, 152.2, 129.9, 126.3, 123.7, 105.8, 100.7, 91.2, 87.5, 76.0, 72.3, 63.2; ¹⁹F NMR (376 MHz, CD₃OD) δ −57.14; MS-ESI⁺ m/z 335 (M+H⁺); HRMS-ESI⁺: m/z calcd for C₁₂H₁₄F₃N₄O₄ (M+H⁺) 335.0967, found 335.0953.

Ethyl ((((2R,3S,4R,5R)-5-(4-amino-5-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (14a)

To a solution of compound 12a (0.05 g, 0.15 mmol) in 3.0 mL of anhydrous THF was added t-BuMgCl (0.53 mL, 1.0 M in THF) at −78 0 °C under N₂ atmosphere. After 30 minutes at this temperature and an additional 10 min at room temperature, a solution of phosphoramidate chloride 13 (67.0 mg, 0.23 mmol) in 2.0 mL of anhydrous THF was added to the reaction at −78 °C. The reaction mixture was stirred for 6 h at room temperature and cooled down to 0 °C before addition of a saturated aqueous solution of NH₄Cl (0.25 mL). The resulting solution was poured into EtOAc (50 mL) and the organic layer was washed with a saturated aqueous solution of NH₄Cl (20 mL) and brine (20 mL). The organic layer was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (CH₂Cl₂: MeOH = 20:1 to 10:1 v/v) to give compound 14a (53.0 mg, 0.09 mmol) as a mixture of Rp-/Sp-isomers (~1:1) in 60% yield. ¹H NMR (400 MHz, CD₃OD) δ 8.23 (s, 1H), 7.91–7.89 (m, 1H), 7.35–7.31 (m, 2H), 7.23–7.15 (m, 3H), 6.25–6.22 (m, 1H), 4.48–4.35 (m, 4H), 4.33–4.29 (m, 2H), 4.28–4.20 (m, 2H), 4.14–4.03 (m, 3H), 3.92–3.84 (m, 1H), 1.38–1.16 (m, 11H); ³¹P NMR (162 MHz, CD₃OD) δ 5.06, 4.83; ¹⁹F NMR (376 MHz, CD₃OD) δ −56.99, −57.03; MS-ESI⁺ m/z 590 (M+H⁺); HRMS-ESI⁺: m/z calcd for C₂₃H₂₈F₃N₅O₈P (M+H⁺) 590.1627, found 590.1609.

(2S/R,3R,4R,5R)-5-((Benzoyloxy)methyl)-3-methyltetrahydrofuran-2,3,4-triyl tribenzoate (9b)

To a solution of 2-C-methyl-, 2,3,5-tribenzoyl ribolactone (5.0 g, 10.54 mmol) in 50 mL of anhydrous THF was added LiAlH(Ot-Bu)₃ (1.1 M in THF, 15 mL) over 10 min at 0 °C under N₂ atmosphere. After stirring for 1 h, the reaction was warmed up to room temperature and stirred for 2 h. The reaction was then quenched with a saturated solution of NH₄Cl (10 mL), poured into cold water (50 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column (hexanes:EtOAc = 10:1 to 2:1 v/v) to give a 2-C-methyl-, 2,3,5-tribenzoyl ribolactol in quantitative yield. To a solution of the lactol (2.08 g, 10.25 mmol) in 100 mL of anhydrous CH₂Cl₂ was added benzoyl chloride (2.16 g, 15.38 mmol) and Et₃N (1.25 g, 20.50 mmol) at 0 °C under N₂ atmosphere. After being stirred for 12 h at room temperature, the reaction mixture was quenched with MeOH (10 mL) at 0 °C, stirred for 1 h at room temperature, and then poured into cold water (50 mL). The organic layer was separated and washed with 0.1 N HCl aqueous solution (30 mL), water (30 mL), brine (30 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (CH₂Cl₂ to CH₂Cl₂: MeOH = 50:1 v/v) to give compound 9b (5.81 g, 10.01 mmol) in 95% yield.

¹H NMR (400 MHz, CDCl₃) δ 8.13–8.10 (m, 4H), 8.07–8.05 (m, 2H), 7.88 (dd, J = 8.4, 1.2 Hz, 2H), 7.64–7.60 (m, 3H), 7.51–7.40 (m, 7H), 7.15 (t, J = 8.0 Hz, 2H), 7.06 (s, 1H), 5.95 (d, J = 8.0 Hz, 1H), 4.80–4.77 (m, 1H), 4.68 (dd, J = 12.4, 4.0 Hz, 1H), 4.54 (dd, J = 12.4, 4.8 Hz, 1H), 1.95 (s, 3H); MS-ESI⁺ m/z 581 (M+H⁺).

(2R,3R,4R,5R)-5-((Benzoyloxy)methyl)-2-(4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3-methyltetrahydrofuran-3,4-diyl dibenzoate (10b)

To a solution of 6-chloro-7-iodo-7-deazapurine 8 (0.50 g, 1.79 mmol) in 20 mL of anhydrous CH₃CN was added N,O-bis(trimethylsilyl)acetamide (0.44 g, 2.16 mmol) at room temperature under N₂ atmosphere. After 20 min, 1,2,3,5-tetra-O-Bz-2-methylribose 9b (1.04 g, 1.80 mmol) and then TMSOTf (0.49 g, 2.16 mmol) were added to the reaction mixture at 0 °C under N₂ atmosphere. The mixture was warmed up to room temperature for 30 min and then heated at 80 °C for additional 8 h. The solution was cooled down to room temperature and diluted with EtOAc (50 mL). The organic layer was washed with a saturated solution of NHCO₃ (30 mL), cold water (30 mL), and brine (20 mL). The organic layer was then dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexanes:EtOAc = 10:1 to 3:1 v/v) to give compound 10b (0.97 g, 1.31 mmol) in 73% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.75 (s, 1H), 8.12–8.09 (m, 4H), 7.96 (d, J = 7.6 Hz, 2H), 7.69 (s, 1H), 7.63–7.53 (m, 3H), 7.48–7.44 (m, 4H), 7.34 (t, J = 7.6 Hz, 2H), 6.95 (s, 1H), 6.04 (d, J = 6.0 Hz, 1H), 4.96 (dd, J = 12.4, 3.6 Hz, 1H), 4.86 (dd, J = 12.4, 5.6 Hz, 1H), 4.74–4.70 (m, 1H), 1.59 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 166.5, 165.5, 165.3, 153.3, 151.4, 150.9, 133.9, 133.8, 133.6, 133.2, 130.1, 130.0, 129.9, 129.8, 129.7, 128.9, 128.8, 128.7, 117.9, 89.2, 85.1, 80.2, 75.8, 63.5, 52.9, 29.9, 18.1; MS-ESI⁺ m/z 738 (M+H⁺); HRMS-ESI⁺: m/z calcd for C₃₃H₂₆ClIN₃O₇ (M+H⁺) 738.0504, found 738.0488.

(2R,3R,4R,5R)-5-((Benzoyloxy)methyl)-2-(4-chloro-5-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3-methyltetrahydrofuran-3,4-diyl dibenzoate (11b)

To a solution of compound 10b (0.24 g, 0.32 mmol) in 10 mL of anhydrous DMF and 7.0 mL of HMPA was added FSO₂CF₂CO₂Me (80.0 mg, 0.40 mmol) and CuI (73.0 mg, 0.38 mmol) under argon atmosphere. After being stirred for 12 h at 70 °C, the reaction mixture was cooled down to temperature and treated with a saturated solution of NH₄Cl (10 mL) and then extracted with a mixture of hexanes and EtOAc (30 mL x 2, 1:2 v/v). The combined organic layers were washed with a saturated solution of NaHCO₃ (10 mL), water (10 mL), brine (10 mL), dried over Na₂SO₄ and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (hexanes: EtOAc = 10:1 to 5:1 v/v) to give compound 11b (0.20 g, 0.29 mmol) in 90% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.85 (s, 1H), 8.13–8.08 (m, 4H), 7.97–7.94 (m, 3H), 7.64–7.52 (m, 3H), 7.48–7.43 (m, 4H), 7.33 (t, J = 7.6 Hz, 2H), 6.96 (s, 1H), 6.03 (d, J = 5.2 Hz, 1H), 4.97–4.92 (m, 2H), 4.76–4.72 (m, 1H), 1.60 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 166.6, 165.5, 165.3, 152.9, 152.4, 151.9, 134.90, 133.7, 130.2, 130.0, 129.9, 129.7, 129.6, 128.8, 128.7, 89.5, 84.8, 80.7, 75.9, 63.4, 18.2; ¹⁹F NMR (376 MHz, CDCl₃) δ −56.29; MS-ESI⁺ m/z 680 (M+H⁺); HRMS-ESI⁺: m/z calcd for C₃₄H₂₆ClF₃N₃O₇ (M+H⁺) 680.1411, found 680.1400.

(2R,3S,4R,5R)-2-(4-Amino-5-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3,4-diol (12b)

In a steel bomb at 0 °C, NH₃ was bubbled through a solution of compound 11b (0.20 g, 0.26 mmol) in 30 mL of MeOH for 20 min. The reaction was then heated at 90 °C for 12 h and then cooled down to 0 °C. The volatiles were concentrated under reduced pressure and the residue was purified by silica gel column chromatography (CH₂Cl₂: MeOH = 20:1 to 10:1 v/v) to give compound 12b (77.60 mg, 0.22 mmol) in 87% yield. ¹H NMR (400 MHz, CD₃OD) δ 8.32 (dd, J = 1.2 Hz, 1H), 8.23 (s, 1H), 6.31 (s, 1H), 4.13 (d, J = 9.2 Hz, 1H), 4.06–4.01 (m, 2H), 3.85 (dd, J = 12.4, 2.0 Hz, 1H), 0.85 (s, 3H); ¹³C NMR (100 MHz, CD₃OD) δ 158.1, 154.1, 152.3, 151.5, 126.5, 125.2, 125.1, 123.8, 106.2, 105.8, 100.0, 92.9, 84.2, 80.6, 73.3, 60.7, 20.2; ¹⁹F NMR (376 MHz, CD₃OD) δ −57.04; MS-ESI⁺ m/z 349 (M+H⁺); HRMS-ESI⁺: m/z calcd for C₁₃H₁₆F₃N₄O₄ (M+H⁺) 349.1123, found 349.1113.

Ethyl ((((2R,3R,4S,5R)-5-(4-amino-5-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (14b)

To a solution of compound 12b (40.0 mg, 0.12 mmol) in 2.0 mL of anhydrous THF was added t-BuMgCl (0.42 mL, 1.0 M in THF) at −78 0 °C under N₂ atmosphere. After being stirred for 30 min at this temperature and for 10 min at room temperature, a solution of phosphoramidate chloride 13 (49.60 mg, 0.17 mmol) in 2.0 mL of anhydrous THF was added to the solution −78 °C. The reaction mixture was stirred for 12 h at room temperature and then cooled down to 0 °C before addition of saturated aqueous solution of NH₄Cl (0.10 mL). The resulting solution was poured into EtOAc (30 mL) and washed with saturated aqueous NH₄Cl (10 mL) and brine (10 mL). The organic layer was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (CH₂Cl₂: MeOH = 15:1 to 10:1 v/v) to give compound 14b (42.0 mg, 0.07 mmol) as a mixture of Rp-/Sp-isomers (~1:1) in 58% yield. ¹H NMR (400 MHz, CD₃OD) δ 8.25 (s, 1H), 7.88–7.85 (m, 1H), 7.36–7.32 (m, 2H), 7.26–7.23 (m, 2H), 7.19–7.16 (m, 1H), 6.35–6.33 (s, 1H), 4.89–4.42 (m, 2H), 4.33–4.29 (m, 2H), 4.25–4.18 (m, 1H), 4.13–3.99 (m, 3H), 3.97–3.90 (m, 1H), 1.32–1.27 (m, 4H), 1.22–1.14 (m, 3H), 0.88–0.86 (s, 3H); ³¹P NMR (162 MHz, CD₃OD) δ 5.28, 4.94; ¹⁹F NMR (376 MHz, CD₃OD) δ −56.68, −56.78; MS-ESI⁺ m/z 604 (M+H⁺); HRMS-ESI⁺: m/z calcd for C₂₄H₃₀F₃N₅O₈P (M+H⁺) 604.1784.1627, found 604.1771.

((2R,3R,4R,5R)-3-(Benzoyloxy)-5-bromo-4-fluoro-4-methyltetrahydrofuran-2-yl)methyl benzoate (9c, α-Isomer)

To a solution of 3,5-O-di-Bz-2-F-2-Me-ribolactone (3.0 g, 8.10 mmol) in 40 mL of anhydrous THF was added LiAlH(Ot-Bu)₃ (1.1 M in THF, 8.80 mL) over 10 min at 0 °C under N₂ atmosphere. After being stirred for 2 h at this temperature, the reaction was warmed up to room temperature and stirred for an additional 1 h. The reaction was then quenched with a saturated solution of NH₄Cl (5.0 mL), poured into cold water (30 mL) and extracted with EtOAc (40 mL x 3). The combined organic layers were dried over Na₂SO₄ and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column (hexanes:EtOAc = 10:1 to 2:1 v/v) to give 3,5-O-di-Bz-ribolactol in quantitative yield. To a solution of the lactol (3.0 g, 8.01 mmol) in 50 mL of anhydrous CH₂Cl₂ was added acetyl chloride (0.79 g, 10.02 mmol) and Et₃N (1.22 g, 12.02 mmol) at 0 °C under N₂ atmosphere. After being stirred for 3 h, the reaction mixture was quenched with MeOH (10 mL) at 0 °C and poured into cold water (50 mL). The organic layer was separated and washed with 0.1 N-HCl (20 mL), water (20 mL), brine (20 mL) and then dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (CH₂Cl₂ to CH₂Cl₂: MeOH = 50:1 v/v) to give 1-O-Ac-3,5-O-di-Bz-2-F-2-Me-ribose (3.20 g, 7.69 mmol) as an α-/β-isomers (1:1) in 95% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.13–8.10 (m, 2H), 8.06–8.03 (m, 2H), 7.66–7.62 (m, 1H), 7.57–7.53 (m, 1H), 7.52–7.48 (m, 2H), 7.41–7.37 (m, 2H), 6.25 (d, J = 8.7 Hz, 1H), 5.70 (dd, J = 23.6, 8.2 Hz, 1H), 4.74 (dd, J = 12.0, 4.0 Hz, 1H), 4.69–4.66 (m, 1H), 4.46 (dd, J = 12.0, 4.4 Hz, 1H), 2.00 (s, 3H), 1.55 (d, J = 22.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 168.9, 165.9, 165.7, 133.9, 133.2, 130.1, 129.7, 129.6, 128.6, 128.3, 100.4, 98.4 (d, J = 34.8 Hz), 78.7, 73.7 (d, J = 15.1 Hz), 63.6, 20.9, 16.5 (d, J = 23.0 Hz); ¹⁹F NMR (376 MHz, CDCl₃) δ −170.72; MS-ESI⁺ m/z 417 (M+H⁺). To a solution of 1-O-Ac-3,5-O-di-Bz-2-F-2-Me-ribose (2.0 g, 4.80 mmol) in 20 mL of CH₂Cl₂ was added 2.5 mL of 33% HBr in acetic acid solution at room temperature. The solution was stirred for 16 h at room temperature and then concentrated under reduced pressure. The residue was dissolved in 50 mL of CH₂Cl₂ and then washed with cold water (20 mL) and saturated aqueous NaHCO₃. The organic layer was dried over anhydrous MgSO₄ and filtered. The filtrate was concentrated and purified by silica gel column chromatography to give α-isomer (9c) (1.80 g, 4.13 mmol) in 86% yield and β-Isomer (0.21 g, 0.48 mmol) in 10% of yield.

α-Isomer (bottom spot on TLC): ¹H NMR (400 MHz, CDCl₃) δ 8.17–8.14 (m, 2H), 8.06–8.04 (m, 2H), 7.66–7.59 (m, 2H), 7.52–7.45 (m, 4H), 6.37 (s, 1H), 5.31 (dd, J = 5.2, 2.8 Hz, 1H), 4.92–4.88 (m, 1H), 4.80 (dd, J = 12.4, 3.6 Hz, 1H), 4.65 (dd, J = 12.4, 4.4 Hz, 1H), 1.75 (d, J = 21.6 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 166.0, 165.8, 133.7, 133.4, 130.1, 129.7, 129.3, 128.8, 128.6, 128.5, 95.6, 93.6, 92.0 (d, J = 23.6 Hz), 81.9 (d, J = 1.9 Hz), 73.4 (d, J = 15.4 Hz), 62.4, 22.9 (d, J = 26.3 Hz); ¹⁹F NMR (376 MHz, CDCl₃) δ −151.36; MS-ESI⁺ m/z 437 (M+H⁺).

β-Isomer (upper spot on TLC): ¹H NMR (400 MHz, CDCl₃) δ 8.12–8.10 (m, 2H), 8.08–8.06 (m, 2H), 7.66–7.62 (m, 1H), 7.56–7.48 (m, 3H), 7.40–7.36 (m, 2H), 6.45 (d, J = 11.4 Hz, 1H), 6.07 (dd, J = 23.0, 7.7 Hz, 1H), 4.81 (m, 2H), 4.64 (dd, J = 13.2, 6.6 Hz, 1H), 1.79 (d, J = 22.4 Hz, 3H); ¹⁹F NMR (376 MHz, CDCl₃) δ −160.06. OK

((2R,3R,4R,5R)-3-(Benzoyloxy)-5-(4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4-fluoro-4-methyltetrahydrofuran-2-yl)methyl benzoate (10c)

A solution of powdered KOH (0.23 g, 3.52 mmol, 85%) and TDA-1 (0.03 g, 0.09 mmol) in 10 mL of anhydrous CH₃CN was stirred for 15 min at room temperature under N₂ atmosphere. 6-Chloro-7-iodo-7-deazapurine 8 (0.41g, 1.47 mmol) was then added to the solution. After stirring for 20 min, a solution of compound 9c (0.77 g, 1.76 mmol) in 10 mL of anhydrous CH₃CN was added to the mixture at once. The reaction mixture was stirred to 40 °C for 48 h and then quenched with a saturated solution of NH₄Cl (30 mL) and extracted with EtOAc (50 mL x 2). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexanes:EtOAc = 10:1 to 5:1 v/v) to give compound 10c (0.76 g, 1.20 mmol) in 68% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.65 (s, 1H), 8.12–8.08 (m, 4H), 7.65–7.58 (m, 3H), 7.52–7.46 (m, 4H), 6.64 (d, J = 18.0 Hz, 1H), 5.90 (dd, J = 22.0, 5.6 Hz, 1H), 4.92 (dd, J = 12.8, 2.4 Hz, 1H), 4.78–4.74 (m, 1H), 4.62 (dd, J = 12.8, 2.4 Hz, 1H), 1.20 (d, J = 22.8 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 166.3, 165.7, 153.5, 151.6, 150.7, 134.2, 133.6, 131.4, 130.3, 129.9, 129.1, 128.9, 128.6, 117.7, 101.3, 99.5, 89.8 (d, J = 39.4 Hz), 72.5 (d, J = 16.2 Hz), 62.3, 53.8, 17.4 (d, J = 24.7 Hz); ¹⁹F NMR (376 MHz, CDCl₃) δ −158.15; MS-ESI⁺ m/z 636 (M+H⁺); HRMS-ESI⁺: m/z calcd for C₂₆H₂₁ClFIN₃O₅ (M+H⁺) 636.0198, found 636.0186.

((2R,3R,4R,5R)-3-(Benzoyloxy)-5-(4-chloro-5-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4-fluoro-4-methyltetrahydrofuran-2-yl)methyl benzoate (11c)

To a solution of compound 10c (0.25 g, 0.39 mmol) in anhydrous DMF (20 mL) and 8.50 mL of hexamethylphosphoric triamide was added FSO₂CF₂CO₂Me (63.0 mg, 0.49 mmol) and CuI (90.0 mg, 0.47 mmol) under argon atmosphere. The reaction mixture was stirred for 24 h at 70 °C under argon atmosphere. The solution was then cooled down to room temperature and treated with a saturated aqueous solution of NH₄Cl (20 mL) and then extracted with a mixture of hexanes and EtOAc (40 mL x 2, 1:2 v/v). The combined organic layers were washed with a saturated solution of NaHCO₃ (20 mL), water (20 mL), brine (20 mL), dried over Na₂SO₄ and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (hexanes: EtOAc = 10:1 to 5:1 v/v) to give compound 11c (0.22 g, 0.38 mmol) in 98% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.76 (s, 1H), 8.12–8.09 (m, 2H), 8.06–8.04 (m, 2H), 7.87 (s, 1H), 7.65–7.56 (m, 2H), 7.50–7.43 (m, 4H), 6.66 (d, J = 18.0 Hz, 1H), 5.92 (dd, J = 21.6, 9.2 Hz, 1H), 4.90 (dd, J = 12.8, 2.8 Hz, 1H), 4.81–4.77 (m, 1H), 4.67 (dd, J = 12.8, 4.0 Hz, 1H), 1.24 (d, J = 22.0 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 166.4, 165.6, 153.0, 152.7, 151.7, 134.2, 133.7, 130.3, 129.9, 129.8, 129.4, 128.8, 128.5, 127.2, 123.2, 120.5, 114.1, 107.3 (q, J = 38.5 Hz), 101.2, 99.3, 90.4 (d, J = 40.1 Hz), 77.7, 72.6 (d, J = 16.2 Hz), 62.2, 29.6, 17.6 (d, J = 24.7 Hz); ¹⁹F NMR (376 MHz, CDCl₃) δ −56.40, −157.73; MS-ESI⁺ m/z 578 (M+H⁺); HRMS-ESI⁺: m/z calcd for C₂₇H₂₁ClF₄N₃O₅ (M+H⁺) 578.1106, found 578.1126.

(2R,3R,4S,5R)-5-(4-Amino-5-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol (12c)

In a steel bomb at 0 °C, NH₃ was bubbled through a solution of compound 11c (0.12 g, 0.21 mmol) in 20 mL of MeOH for 20 min and heated to 80 °C for 12 h. The volatiles were concentrated under reduced pressure and the residue was purified by silica gel column chromatography (CH₂Cl₂: MeOH = 20:1 to 10:1 v/v) to give compound 12c (63.10 mg, 0.18 mmol) in 85% yield. ¹H NMR (400 MHz, CD₃OD) δ 8.28 (d, J = 1.2 Hz, 1H), 8.24 (s, 1H), 6.48 (d, J = 17.2 Hz, 1H), 4.59 (dd, J = 24.8, 9.6Hz, 1H), 4.09–4.03 (m, 2H), 3.86 (dd, J = 12.8, 2.4 Hz, 1H), 1.07 (d, J = 22.0 Hz, 1H); ¹³C NMR (100 MHz, CD₃OD) δ 158.1, 154.4, 152.4, 126.3, 124.8 (d, J = 6.2 Hz), 123.6, 106.7 (q, J = 37.8 Hz), 103.4, 101.6, 100.0, 90.2 (d, J = 38.6 Hz), 83.6, 72.2 (d, J = 17.8 Hz), 60.3, 16.8 (d, J = 25.5 Hz); ¹⁹F NMR (376 MHz, CD₃OD) δ −57.26, −164.93; MS-ESI⁺ m/z 351 (M+H⁺); HRMS-ESI⁺: m/z calcd for C₁₃H₁₅F₄N₄O₃ (M+H⁺) 351.1080, found 351.1069.

Ethyl ((((2R,3R,4S,5R)-5-(4-amino-5-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (14c)

To a solution of compound 12c (45.0 mg, 0.13 mmol) in 5.0 mL of anhydrous THF was added t-BuMgCl (0.39 mL, 1.0 M in THF) at −78 0 °C under N₂ atmosphere. After being stirred for 30 min at this temperature and for 10 min at room temperature, a solution of phosphoramidate chloride 13 (75.0 mg, 0.26 mmol) in 2.0 mL of anhydrous THF was added to the reaction at −78 °C. The reaction mixture was stirred for 6 h at room temperature and cooled down to 0 °C before treatment with a saturated solution of NH₄Cl (0.20 mL). The resulting solution was poured into EtOAc (30 mL) and washed with a saturated solution of NH₄Cl (20 mL) and brine (10 mL). The organic layer was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (CH₂Cl₂: MeOH = 20:1 to 10:1 v/v) to give compound 14c (54.50 mg, 0.09 mmol) as a mixture of Rp-/Sp-isomers (1:1) in 69% yield.

¹H NMR (400 MHz, CD₃OD) δ 8.25 (s, 1H), 7.85 (d, J = 1.6 Hz, 1H), 7.36–7.32 (m, 2H), 7.24–7.22 (m, 2H), 7.20–7.15 (m, 1H), 6.54–6.49 (s, 1H), 4.65–4.60 (m, 1H), 4.52–4.47 (m, 1H), 4.26–4.22 (m, 2H), 4.19–4.08 (m, 2H), 4.92–3.88 (m, 1H), 1.29–1.26 (m, 5H), 1.21 (t, J = 7.2 Hz, 3H), 1.12–1.06 (s, 3H); ¹³C NMR (100 MHz, CD₃OD) δ 175.1, 158.1, 154.6, 152.6, 152.3, 130.9, 126.3, 121.6 (d, J = 4.6 Hz), 112.5, 103.0, 101.2, 100.1, 90.8, 81.4, 73.0, 65.8, 62.5, 51.8, 20.4 (d, J = 7.0 Hz), 17.0, 16.7,14.6; ³¹P NMR (162 MHz, CD₃OD) δ 5.23; ¹⁹F NMR (376 MHz, CD₃OD) δ −56.99, −57.04, −163.59; MS-ESI⁺ m/z 606 (M+H⁺); HRMS-ESI⁺: m/z calcd for C₂₄H₂₉F₄N₅O₇P (M+H⁺) 606.1740, found 606.1723. OK

Scheme 1.

Synthesis of compound 12a-b and 14a-b

Reagents and reaction conditions: a) NIS, DMF, rt, 12h; b) BSA, TMSOTf, , 0 °C → 80 °C, 12h for 10a; 8h for 10b; c) FSO₂CF₂CO₂Me, CuI, HMPA, DMF, 70 °C, 12h; d) sat. NH₃/MeOH, 90 °C, 12h, steel bomb; e) 13, t-BuMgCl, THF, −78 °C → rt, 8h for 14a; 12h for 14b