Synthesis of Cyclopentanyl Carbocyclic 5-Fluorocytosine ((−)-5-Fluoro-Carbodine) Using a Facially Selective Hydrogenation Approach

Abstract

An efficient synthetic route to biologically relevant (−)-5-fluorocarbodine 6 was developed. Direct coupling of N⁶ protected 5-fluorouracil 15 with cyclopentenyl intermediate 13, followed by formation of a macrocycle between the base and the carbocyclic sugar moiety, via ring closing metathesis, allowed for a facial selective hydrogenation of the sugar double bond to give, exclusively, the desired 4’-β stereoisomer.

Carbocyclic nucleosides, in which the furanose ring oxygen atom has been replaced by a carbon atom, are, in general, more chemically and enzymatically stable and some of them have demonstrated interesting biological properties.¹ Thus, naturally occurring (−)-aristeromycin (1)² and (−)-neplanocin A (2)³ manifest cytotoxic and antiviral properties while the drugs abacavir (3)⁴ and entecavir (4)⁵ are Food and Drug Administration (FDA) approved for the treatment of human immunodeficiency virus (HIV) and hepatitis B virus (HBV), respectively (Figure 1.).

Figure 1.

Structures of Biologically Relevant Carbocyclic Nucleosides

The carbocyclic cytosine analogs (−)-carbodine (5) and (−)-5-fluorocarbodine (6) was reported to possess significant antitumor and antiviral activities. For instance, compound (6) showed selective submicromolar IC₅₀’s against a human T cell lymphoblast cell line and human B cell lymphoma cell line but also potent inhibition of HCV RNA replication (ΔCt of 6 at 10 µM in a Huh-7 cell based subgenomic replicon assay).⁶ In addition, compounds (5) and (6) demonstrated selective activity against Venezuela Equine Encephalitis Virus (VEEV)⁷ and various strains of H5N1 influenza.⁸

The most recently reported syntheses of (−)-carbodine (5) and (−)-5-fluorocarbodine (6) used a linear approach (Scheme 1) from known cyclopentenyl intermediate including stepwise construction of the heterocyclic base. The key intermediate 7 was prepared from D-ribose in 13 steps by a sequence that used protecting/deprotection strategies and each step required purification by column chromatography on silica gel. After building the pyrimidine base, intermediate 8 was either converted, after a series of protection/deprotection, to its cytosine counterpart then fluorinated (ref 8), or directly fluorinated and then converted to its cytosine analog (ref 6). Overall, both approaches are lengthy (18 and 19 steps) and resulted in the formation of (6) in an overall yield of 2⁶ and 10%.⁸

Scheme 1.

Linear Approaches for the Synthesis of 5-Fluorocarbodine (6)

Preparation of (−)-carbodine (5) has also been achieved by either enzymatic hydrolysis⁹ or selective hydrogenation of cyclopentenyl cytosine.¹⁰ However, both of these methods resulted in poor overall yields and low selectivities. In the later approach, the authors demonstrated that the use of diimide as a source of hydrogen was not completely diastereoselective and led to a mixture of 5, along with isocarbodine (10) (Scheme 2). Furthermore, separation of the two isomers appeared problematic and purification of enantiomerically pure 5 could only be achieved using semi-preparative HPLC. With this last result in mind, we hypothesized that the introduction of a temporary sugar-base bridging group on compound A (Scheme 2; X = OH) would act as an umbrella, covering the β face of the cyclopentene and allow for selective α face hydrogenation. Furthermore, we envisioned a sequence to form a cyclic eight member linker using a ring-closing metathesis (RCM)¹¹ reaction that would connect the 5’-O of the sugar and 3-N of the uridine base as depicted in Scheme 2 compound B. Facial selectivity of the subsequent hydrogenation could also be rationalized by the calculated¹² increased ring strain of the macrocycle for beta face hydrogenation that would ultimately lead to isocarbodine (10) formation.

Scheme 2.

Facially Selective Hydrogenation

In order to prepare 5-fluorocarbodine (6), the key cyclopentenol derivative 13 was synthesized in 9 steps from d-ribose, using a slightly modified known methodology (Scheme 3).¹³ Thus, chiral intermediate 11 was selectively protected by treatment with TBDPSCl in presence of DMAP to give compound 12. Diene 12 was then cyclized via a ring-closing metathesis reaction in the presence of catalytic amount of 2^nd generation Grubbs’ catalyst D, affording desired cyclopentenol 13. Meanwhile, N3-benzoyl-5-fluorouracil 15 was prepared in 2 steps from 5-fluorouracil 14 (Scheme 4) by benzoylation followed by selective deprotection using K₂CO₃ in dioxane.¹⁴

Scheme 3.

Preparation of Protected Sugar 13

Scheme 4.

Synthesis of 5-Fluorocarbodine 6

Coupling of chiral intermediate 13 with 3-N-Bz-5-F-uracil 15 under Mitsunobu conditions provided the desired N-alkylated derivative as a mixture with its O-alkylated isomer and DIAD byproducts (Scheme 4). The partially column purified mixture was directly deprotected by treatmentwith 7 N NH₃ in MeOH, followed by NH₄F in MeOH, to afford pure carbocyclic 5-fluorouridine derivative 17 along with pure O-alkylated compound 16 (ratio 4:1) in 93% yield over 3 steps. At this stage, introduction of pentenoyl moieties on both 5’-OH and 3-NH positions of compound 17 had to be optimized. While use of pyridine either alone or with DMAP or Et₃N gave the desired compound in low yields (the monoalkylated compound being predominant), the use of Et₃N in dichloromethane lead to the nearly quantitative formation of dialkylated compound 18. With intermediate 18 in hand, a ring closing metathesis reaction to form the desired macrocycle 19 was investigated. Evaluation of solvents, catalyst loading and reaction time was undertaken. The best results were obtained when compound 18 was treated with 2.0 mol% of 2^nd generation Grubbs’ catalyst D (Figure 2) in toluene at 80 °C for 1 h.

As predicted, hydrogenation of unsaturated derivative 19 in the presence of Pd/C in EtOAc lead to complete stereoselective formation of cyclopentane derivative 15 in 85% yield. The absolute stereochemistry of the final product was determined by ¹H-NMR and 1D-NOE experiments on compound 21, which was obtained after removal of the linker using NH₃ in MeOH. The NOE experiment showed an enhancement of the 4’ proton when the 1’ proton was irradiated indicating that they reside on the same face of the sugar moiety. Finally, carbocyclic 5-F-uridine 21 was converted to its cytosine counterpart following a previously reported method.¹⁵ The primary alcohol in compound 21 was first protected using TBSCl. Compound 22 was then being reacted with 2,4,6-triisopropylbenzenesulfonyl chloride followed by NH₄OH to give intermediate 23. Final deprotection was achieved using HCl in MeOH providing the target 5-fluoro-carbodine 6 in 98% isolated yield.

CONCLUSION

In summary, an efficient synthetic route to 5-fluoro-carbodine 6 was developed utilizing a key α-face-selective hydrogenation driven by the introduction of a temporary macrocyclic tether. This approach represents a noteworthy improvement to the existing syntheses of biologically relevant compound 6 and allowed us to prepare 6 in 23% overall yield (compared with 2 to 10% yields from previous approaches) with no major purification problems nor use of special synthetic techniques. We are currently evaluating our umbrella approach’s compatibility with other reactions to modify the olefin functionality and further explore the scope of this innovative methodology.

EXPERIMENTAL SECTION

General Considerations

Nuclear magnetic resonance (NMR) spectra (¹H, ¹³C and ¹⁹F) were recorded on a 400 MHz FT-NMR spectrometer at ambient temperature, 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), dt (doublet of triplets) or ddd (doublet of doublets of doublets). High-resolution mass spectra (HRMS) were recorded on a linear ion trap LTQ-FT mass spectrometer with electrospray ionization (ESI). Thin-layer chromatography (TLC) was performed on 0.25 mm silica gel. Purifications were carried out by silica gel column chromatography (60 Å, 63–200 µm, or 40–75 µm).

(R)-1-((4R,5S)-5-(3-(tert-Butyldiphenylsilyloxy)prop-1-en-2-yl)-2,2-dimethyl-1,3-dioxolan-4-yl)prop-2-en-1-ol (12)

To a solution of compound 11 (4.0 g, 20.5 mmol) in 100 mL of anhydrous CH₂Cl₂ was added DMAP (0.2 g, 1.4 mmol), TBDPSCl (6.3 g, 24.6 mmol) and Et₃N (3.1 g, 30.8 mmol) at 0 °C under N₂ atmosphere. The reaction mixture was stirred for 12 h at room temperature and quenched with 5.0 mL of MeOH. The solution was adsorbed on silica gel and purified by silica gel column chromatography (hexane:EtOAc = 20:1 to 2:1 v/v) to give compound 12 as a white foam (8.7 g, 19.1 mmol) in 93% yield. HRMS-ESI⁺ calcd. for C₂₇H₃₇O₄Si (M+H⁺) 453.2462, found 453.2456; ¹H NMR (400 MHz, CDCl₃) δ 7.71-7.67 (m, 4H), 7.46-7.37 (m, 6H), 6.03-5.95 (m, 1H), 5.47 (dd, J = 13.2, 1.2 Hz, 2H), 5.32 (dt, J = 17.2, 1.2 Hz, 1H), 5.21 (dt, J = 10.4, 1.6 Hz, 1H), 4.65 (d, J = 5.6 Hz, 1H), 4.24 (s, 2H), 4.04-3.99 (m, 1H), 3.94 (dd, J = 8.8, 6.0 Hz, 1H), 2.63 (d, J = 3.2 Hz, 1H), 1.44 (s, 3H), 1.32 (s, 3H), 1.09 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ; 143.2, 138.0, 135.9, 135.8, 132.9, 132.8, 130.2, 130.1, 128.1, 128.0, 116.2, 113.7, 108.2, 80.9, 77.6, 70.3, 66.2, 27.5, 27.0, 25.4, 19.4.

(3aS,4S,6aR)-6-((tert-Butyldiphenylsilyloxy)methyl)-2,2-dimethyl-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ol (13)

To a solution of compound 12 (8.2 g, 18.1 mmol) in 500 mL of anhydrous CH₂Cl₂ was added a solution of 2^nd Grubb’s catalyst (0.1 mol%, 0.02 g, 0.02 mmol) in 100 mL of anhydrous CH₂Cl₂ at room temperature under argon atmosphere. After stirring for 24 h, the reaction mixture was treated with 5.0 mL of DMSO, additionally stirred for 1 h at room temperature and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:EtOAc = 10:1 to 1:1 v/v) to give compound 13 as a white foam (7.40 g, 17.43 mmol) in 96% yield. HRMS-ESI⁺ calcd. for C₂₅H₃₃O₄Si (M+H⁺) 425.2149, found 425.2143; ¹H NMR (400 MHz, CDCl₃) δ 7.69-7.65 (m, 4H), 7.44-7.34 (m, 6H), 5.84 (d, J = 1.6 Hz, 1H), 4.86 (d, J = 5.6 Hz, 1H), 4.74 (t, J = 5.2 Hz, 1H), 4.56 (m, 1H), 4.38 (dd, J = 15.2, 20.0 Hz, 2H), 2.70 (d, J = 10.0 Hz, 1H), 1.35 (s, 3H), 1.33 (s, 3H), 1.07 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 145.5, 135.7, 133.5, 129.9, 129.5, 127.9, 112.7, 83.0, 78.2, 73.5, 61.0, 27.8, 27.0, 26.9, 19.5.

N₃-Benzoyl-5-fluorouracil (15)

To a solution of 5-fluorouracil 14 (3.6 g, 27.3mmol) in 50 mL of anhydrous CH₃CN was added BzCl (9.3 g, 68.2 mmol), pyridine (10.8 g, 136.5mmol) at 0 °C under N₂ atmosphere. The solution was stirred for 24 h at room temperature and concentrated under reduced pressure. The residue was dissolved in 200 mL of CH₂Cl₂ and then washed with cold water (50 mL × 3) and concentrated under reduced pressure. The residue was re-dissolved in 50 mL of 1,4-dioxane and treated with aqueous K₂CO₃ (20 mL, 0.5 M in water solution) at 0 °C. After stirring for 30 min at room temperature, the pH of the solution was lowered to ca. 5.0 by careful addition of glacial acetic acid. The solution was concentrated in vacuo and the residue was treated with a saturated solution of NaHCO₃ (100 mL). After stirring for 1 h, the white solid was filtered and washed with cold water (20 mL × 5) then dried under vacuum for 48 h. The crude product was purified by silica gel column chromatography (hexane:EtOAc = 1:1 to EtOAc) to give compound 15 as a white amorphous solid (5.6 g, 23.7 mmol) in 87% yield. MS-ESI⁺m/z 235 (M+H⁺); ¹H NMR (400 MHz, DMSO-d₆) δ 11.54 (br, 1H), 8.06 (d, J = 6.4 Hz, 1H), 8.01 (d, J = 8.4 Hz, 2H), 7.77 (t, J = 7.6 Hz, 1H), 7.58 (dd, J = 8.4, 7.6 Hz, 2H).

5-Fluoro-2-(((3aS,4R,6aR)-6-(hydroxymethyl)-2,2-dimethyl-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)pyrimidin-4(3H)-one (16) and 5-Fluoro-1-((3aS,4R,6aR)-6-(hydroxymethyl)-2,2-dimethyl-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (17)

To a solution of Ph₃P (11.1 g, 42.4 mmol) in 150 mL of CH₃CN was added DIAD (8.6 g, 42.4 mmol) at 0 °C under N₂ atmosphere. After 30 min, to the reaction mixture was added a solution of compound 13 (7.2 g, 17.0 mmol) in 150 mL of CH₃CN and N-Bz-5-fluorouracil 15 (6.0 g, 25.4 mmol) at −78 °C. The reaction mixture was stirred for 24 h at room temperature and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:EtOAc = 4:1 to 1:1 v/v) to give a non separable mixture of N-/O-alkylated products along with DIAD byproducts. This mixture was dissolved in 100 mL of MeOH and treated with 100 mL of 7 N NH₃ in MeOH at 0 °C. The solution was stirred for 12 h at room temperature and concentrated under reduced pressure. The residue was then directly treated with a solution of ammonium fluoride (1.6 g, 42.4 mmol) in 200 mL of MeOH at room temperature and the solution was stirred at 50 °C for 6 h. After removal of the volatiles under vacuum, the residue was purified by silica gel column chromatography (hexane:EtOAc = 1:5 to EtOAc v/v) to give 16 as an off white gum (0.94 g, 3.2 mmol) and 17 as an off white gum (3.8 g, 12.6 mmol) in 93% yield over 3 steps. Compound 16: MS-ESI⁺m/z 299 (M+H⁺); HRMS-ESI⁺calcd. for C₁₃H₁₆FN₂O₅ (M+H⁺) 299.1039, found 299.1035; ¹H NMR (400 MHz, CD₃OD) δ 7.77 (d, J = 3.2 Hz, 1H), 5.86 (d, J = 1.6 Hz, 1H), 5.72 (s, 1H), 5.19 (d, J = 5.6 Hz, 1H), 4.73 (d, J = 5.6 Hz, 1H), 4.24 (dd, J = 16.0, 10.8 Hz, 2H), 1.38 (s, 3H), 1.34 (s, 3H); ¹³C NMR (100 MHz, CD₃OD) δ 154.7, 149.3, 146.8, 136.0, 123.7, 113.7, 87.3, 85.0, 84.4, 59.00, 27.8, 26.2; Compound 17: MS-ESI⁺m/z 299 (M+H⁺); HRMS-ESI⁺calcd for C₁₃H₁₆FN₂O₅ (M+H⁺) 299.1038, found 299.1036; ¹H NMR (400 MHz, CD₃OD) δ 7.54 (d, J = 6.4 Hz, 1H), 5.60 (m, 1H), 5.36 (s, 1H), 5.24 (d, J = 6.0 Hz, 1H), 4.62 (d, J = 5.6 Hz, 1H), 4.29 (s, 2H), 1.40 (s, 3H), 1.33 (s, 3H); ¹³C NMR (100 MHz, CD₃OD) δ 155.0, 151.5, 143.1, 140.8, 127.8, 122.6, 113.5, 85.6, 84.8, 68.9, 60.0, 27.7, 26.1; ¹⁹F NMR (376 MHz, CD₃OD) δ −169.69 (d, J = 6.0 Hz).

((3aR,6R,6aS)-6-(3-But-3-enoyl-5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-dimethyl-6,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)methyl but-3-enoate (18)

To a solution of compound 17 (1.4 g, 4.7mmol) in 40 mL of anhydrous CH₂Cl₂ was added Et₃N (2.4 g, 23.5 mmol) and 4-pentenoyl chloride (1.7 g, 14.1 mmol) at 0 °C under argon atmosphere. After stirring for 4 h at room temperature, the solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:EtOAc = 4:1 to 1:1 v/v) to give a compound 18 as a white foam (2.1 g, 4.5 mmol) in 97% yield. MS-ESI⁺m/z 463 (M+H⁺); HRMS-ESI⁺calcd for C₂₃H₂₇FN₂O₇Na (M+Na⁺) 485.1705, found 485.1696; ¹H NMR (400 MHz, CDCl₃) δ 7.09 (d, J = 5.6 Hz, 1H), 5.90-5.78 (m, 2H), 5.57 (s, 1H), 5.39 (s, 1H), 5.21 (d, J = 5.6 Hz, 1H), 5.15-5.01 (m, 4H), 4.82 (q, J = 15.2 Hz, 2H), 4.59 (d, J = 5.6 Hz, 1H), 2.97 (t, J = 7.2 Hz, 2H), 2.51 (m, 4H), 2.43 (m, 2H), 1.43 (s, 3H), 1.35 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 173.9, 172.8, 158.7, 149.3, 148.0, 136.6, 135.6, 124.9, 122.9, 116.5, 116.1, 113.4, 94.6, 84.1, 83.5, 68.1, 60.9, 39.8, 33.4, 28.9, 27.5, 27.4, 25.9; ¹⁹F NMR (376 MHz, CDCl₃) δ −165.50 (d, J = 4.5 Hz).

Compound 19

To a solution of compound 18 (1.0 g, 2.16 mmol) in 500 mL of anhydrous toluene was added 2^nd Generation Grubbs’ catalyst D (2.0 mol%, 0.037 g, 0.043 mmol) at room temperature under an argon atmosphere. After stirring for 1 h at 80 °C, the reaction mixture was treated with 2.0 mL of DMSO at room temperature and additionally stirred for 1 h. The solution was concentrated under reduced pressure and then purified by silica gel column chromatography (hexane:EtOAc = 2:1 v/v) to give compound 19 as a white foam (0.85 g, 2.0 mmol) in 90% yield. MS-ESI⁺m/z 435 (M+H⁺); HRMS-ESI⁺calcd for C₂₁H₂₄FN₂O₇ (M+H⁺) 435.1562, found 435.1560; ¹H NMR (400 MHz, CDCl₃) δ 7.29 (d, J = 5.2 Hz, 1H), 5.62 (s, 1H), 5.56-5.40 (m, 3H), 5.25 (d, J = 11.6 Hz, 1H), 4.90 (d, J = 5.6 Hz, 1H), 4.42 (s, 1H), 4.25 (d, J = 11.2 Hz, 1H), 3.07 (ddd, J = 18.0, 8.0, 2.4 Hz, 1H), 2.86 (ddd, J = 18.0, 10.4, 2.4 Hz, 1H), 2.60 (m, 1H), 2.50-2.30 (m, 3H), 2.28-2.15 (m, 2H), 1.40 (s, 3H), 1.36 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 174.5, 172.7, 155.9, 146.9, 141.1, 138.7, 129.8, 129.1, 127.7, 125.5, 112.3, 84.5, 82.7, 74.9, 58.6, 39.4, 32.8, 27.4, 25.8; ¹⁹F NMR (376 MHz, CDCl₃) δ −165.63 (d, J = 6.0 Hz), −166.20 (d, J = 6.0 Hz).

Compound 20

To a solution of compound 19 (2.5 g, 5.8 mmol) in 100 mL of EtOAc was added Pd/C catalyst (10 wt %, 0.25 g). The solution was stirred for 12 h under H₂ atmosphere (1 atm) and then treated with Celite. After stirring for 30 min, the suspension was carefully filtered, the filtrate concentrated under vacuum, and then purified by silica gel column chromatography (hexane:EtOAc = 1:1 v/v) to give compound 20 as a white foam (2.1 g, 4.9 mmol) in 85% yield. MS-ESI⁺m/z 439 (M+H⁺); HRMS-ESI⁻calcd for C₂₁H₂₇FN₂O₇Cl (M+Cl⁻) 473.1496, found 473.1499; ¹H NMR (400 MHz, CDCl₃) δ 7.28 (d, J = 5.6 Hz, 1H), 5.00 (dd, J = 6.4, 2.4 Hz, 1H), 4.88 (t, J = 5.6 Hz, 1H), 4.70 (d, J = 12.0, 1.2 Hz, 1H), 3.99 (dd, J = 12.0, 1.2 Hz, 1H), 3.91 (m, 1H) 2.95 (ddd, J = 16.0, 8.4, 3.2 Hz, 1H), 2.73 (ddd, J = 16.0, 9.6, 3.2 Hz, 1H), 2.55 (ddd, J = 16.0, 8.4, 5.2 Hz, 1H), 2.33-2.17 (m, 4H), 1.90 (m, 1H), 1.79 (s, 1H), 1.66-1.40 (m, 6H), 1.39-1.25 (m, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 174.4, 174.1, 147.6, 141.1, 138.7, 130.1, 129.8, 113.0, 94.6, 83.4, 80.3, 69.7, 61.0, 45.7, 40.1, 33.6, 31.2, 28.1, 27.1, 25.6, 24.4; ¹⁹F NMR (376 MHz, CDCl₃) δ −166.59 (d, J = 4.5 Hz),

5-Fluoro-1-((3aS,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (21)

A solution of compound 20 (2.0 g, 4.58 mmol) in 50 mL of MeOH was treated with 20 mL of 7 N NH₃ in MeOH at 0 °C in a sealed tube. After stirring for 12 h at 80 °C, the solution was concentrated under reduced pressure and the residue was purified on a silica gel pad (CH₂Cl₂:MeOH = 30:1 to 10:1 v/v) to give compound 21 as a beige gum (1.3 g, 4.3 mmol) in 94% yield. MS-ESI⁺m/z 301 (M+H⁺); HRMS-ESI⁺calcd for C₁₃H₁₈FN₂O₅ (M+H⁺) 301.1194, found 301.1193; ¹H NMR (400 MHz, CDCl₃) δ 10.01 (br, 1H), 7.48 (d, J = 5.6 Hz, 1H), 4.77 (dd, J = 7.2, 5.6 Hz, 1H), 4.61 (dd, J = 7.2, 4.8 Hz, 1H), 4.57 (m, 1H), 3.83 (dd, J = 10.4, 4.8 Hz, 1H), 3.74 (dd, J = 10.8, 4.8 Hz, 1H), 2.80 (br, 1H) 2.37-2.27 (m, 2H), 2.12-2.03 (m, 1H), 1.54 (s, 3H), 1.31 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 157.5, 157.2, 149.8, 141.9, 139.6, 127.9, 127.6, 113.2, 83.0, 81.4, 64.2, 63.3, 45.6, 32.0, 27.7, 25.4; ¹⁹F NMR (376 MHz, CDCl₃) δ −169.67 (d, J = 6.0 Hz).

1-((3aS,4R,6R,6aR)-6-((tert-Butyldiphenylsilyloxy)methyl)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-5-fluoropyrimidine-2,4(1H,3H)-dione (22)

To a solution of compound 21 (1.3 g, 4.5 mmol) in 30 mL of anhydrous CH₂Cl₂ was added imidazole (0.74 g, 10.9mmol) and TBDPSCl (1.7 g, 5.4 mmol) at 0 °C under N₂ atmosphere. The reaction mixture was stirred for 12 h at room temperature and treated with 5.0 mL of MeOH. After stirring for 1 h at room temperature, the solution was concentrated under reduced pressure and purified by silica gel column chromatography (hexane:EtOAc = 2:1 to 1:4 v/v) to give compound 22 as an off white foam (2.3 g, 4.3mmol) in 98% yield. MS-ESI⁺m/z 539 (M+H⁺);HRMS-ESI⁺calcd for C₂₉H₃₆FN₂O₅Si (M+H⁺) 539.2372, found 539.2368; ¹H NMR (400 MHz, CDCl₃) δ 9.78 (br, 1H), 7.66-7.62 (m, 4H), 7.40-7.36 (m, 6H), 7.23 (d, J = 5.6 Hz, 1H), 4.71 (m, 1H), 4.63 (dd, J = 6.8, 6.0 Hz, 1H), 4.51 (dd, J = 7.2, 6.8 Hz, 1H), 3.79 (d, J = 5.2 Hz, 2H), 2.33 (m, 1H), 2.25 (m, 1H), 2.00 (q, J = 12.0 Hz, 1H), 1.53 (s, 3H), 1.29 (s, 3H), 1.09 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 157.2, 157.0, 149.7, 142.1, 139.7, 135.83, 135.78, 133.5, 133.4, 130.1, 128.0, 126.6, 126.3, 113.9, 82.8, 80.7, 64.3, 63.8, 45.2, 32.4, 27.8, 27.2, 25.5, 19.6; ¹⁹F NMR (376 MHz, CDCl₃) δ −165.85 (d, J = 4.5 Hz).

4-Amino-1-((3aS,4R,6R,6aR)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-5-fluoropyrimidin-2(1H)-one (23)

To a solution of compound 22 (2.2 g, 4.1 mmol) and DMAP (2.0 g, 16.4 mmol) in 50 mL of anhydrous CH₃CN was added 2,4,6-trisiopropyl benzenesulfonyl chloride (3.1 g, 10.3mmol) and Et₃N (1.7 g, 16.4 mmol) at room temperature under N₂ atmosphere. The reaction mixture was stirred for 48 h at room temperature and treated with 25 mL of 30% NH₄OH solution. After stirring for 4 h at room temperature, the solution was poured into 150 mL of a cold water-CHCl₃ solution (1:2 v/v) and then extracted with CHCl₃ (30 mL × 3). The combined organic layers were washed with a saturated NH₄Cl aqueous solution (100 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH₂Cl₂ to CH₂Cl₂:MeOH = 10:1 v/v) to give compound 23 as a gum (2.0 g, 3.7mmol) in 89% yield. MS-ESI⁺m/z 538 (M+H⁺); HRMS-ESI⁺calcd for C₂₉H₃₇FN₃O₄Si (M+H⁺) 538.2532, found 538.2525; ¹H NMR (400 MHz, CDCl₃) δ 8.83, (br, 1H), 7.66-7.63 (m, 4H), 7.45-7.36 (m, 6H), 7.24 (d, J = 5.6 Hz, 1H), 5.60 (br, 1H), 4.80 (dd, J = 7.2, 5.2 Hz, 1H), 4.52-4.44 (m, 2H), 3.82 (dd, J = 10.0, 4.4 Hz, 1H), 3.74 (dd, J = 10.0, 6.0 Hz, 1H), 2.33-2.25 (m, 2H), 2.16 (m, 1H), 1.50 (s, 3H), 1.26 (s, 3H), 1.07 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 158.1, 154.6, 137.8, 135.8, 135.7, 135.4, 133.7, 133.6, 129.9, 128.4, 128.1, 127.9, 113.3, 83.0, 81.1, 66.1, 64.8, 46.2, 32.8, 27.8, 27.1, 25.5, 19.5; ¹⁹F NMR (376 MHz, CDCl₃) δ −169.48 (d, J = 7.5 Hz).

4-Amino-1-((1R,2S,3R,4R)-2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl)-5-fluoropyrimidin-2(1H)-one (6)

A solution of compound 23 (2.5 g, 4.7mmol) in 100 mL of MeOH was treated with 50 mL of HCl in MeOH at 0 °C. After stirring for 3 h at room temperature, the acidic solution was concentrated under reduced pressure. The residue was dissolved in water (50 mL) and washed with EtOAc (20 mL × 5). The aqueous layer was concentrated under reduced pressure. The residue was dissolved in EtOH (10 mL) and the solution was concentrated under reduced pressure. After this process was repeated 5 times, the final residue was dried under vacuum for 72 h at room temperature to give pure compound 6 as a white amorphous solid (1.4 g, 4.6 mmol) as a HCl salt in 98% yield. MS-ESI⁺m/z 260 (M+H⁺); HRMS-ESI⁺calcd for C₁₀H₁₅FN₃O₄ (M+H⁺) 260.1041, found 260.1039; ¹H NMR (400 MHz, CD₃OD) δ 8.35 (d, J = 7.2 Hz, 1H), 4.70 (q, J = 9.2 Hz, 1H), 4.12 (dd, J = 5.6, 9.2 Hz, 1H), 3.85 (dd, J = 5.6, 3.2 Hz, 1H), 3.53 (d, J = 5.2 Hz, 2H), 2.19 (m, 1H), 2.05 (m, 1H), 1.46 (m, 1H); ¹³C NMR (100 MHz, CD₃OD) δ 154.65, 154.42, 148.52, 139.97, 137.78, 133.74, 133.46, 75.44, 73.49, 64.83, 64.53, 46.50, 28.67; ¹H NMR (400 MHz, DMSO-d₆) δ 9.75 (br, 1H), 9.14 (br, 1H), 8.53 (d, J = 7.2 Hz, 1H), 5.32 (br, 3H), 4.65 (q, J = 9.2 Hz, 1H), 4.01 (dd, J = 9.2, 5.6 Hz, 1H), 3.72 (dd, J = 4.8, 2.4 Hz, 1H), 3.42 (dd, J = 10.4, 6.4 Hz, 1H), 3.35 (dd, J = 10.4, 5.6 Hz, 1H), 2.04 (m, 1H), 1.93 (m, 1H), 1.28 (m, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 153.1, 152.9, 147.8, 136.2, 133.9, 131.8, 131.5, 73.4, 71.2, 62.9, 61.8, 44.81, 27.8; ¹⁹F NMR (376 MHz, CD₃OD) δ −170.99 (d, J = 7.52 Hz).