Synthesis and anti-HCV activity of β-d-2′-deoxy-2′-α-chloro-2′-β-fluoro and β-d-2′-deoxy-2′-α-bromo-2′-β-fluoro nucleosides and their phosphoramidate prodrugs

Abstract

We report herein the synthesis and evaluation of a series of β-d-2′-deoxy-2′-α-chloro-2′-β-fluoro and β-d-2′-deoxy-2′-α-bromo-2′-β-fluoro nucleosides along with their corresponding phosphoramidate prodrugs. Key intermediates, lactols 11 and 12, were obtained by a diastereoselective fluorination of protected 2-deoxy-2-chloro/bromo-ribonolactones 7 and 8. All synthesized nucleosides and prodrugs were evaluated with a hepatitis C virus (HCV) subgenomic replicon system.

1. Introduction

According to WHO, an estimated 71 million people are chronically infected worldwide with Hepatitis C Vims (HCV) and among them a significant number will develop cirrhosis or liver cancer.¹ However, since 2011 and the first approved direct acting antivirals (DAAs), boceprevir and telaprevir, a revolution occurred in the treatment of HCV infection. Today, several oral regimens combining different pangenotypic DAAs with different modes of action are available and a sustained virologic response (SVR) of almost 100% can be reached after 8 to 12 weeks of treatment.² To date, sofosbuvir (SOF) is the only nucleoside analog approved for the treatment of HCV infection and it is the pillar of the most prescribed treatments (Vozevi and Epcluza). Because nucleoside analogs targeting the HCV NS5B polymerase generally display broader activity against HCV genotypes (GT) along with a favorable safety profile and a high genetic barrier to resistance, they remain the gold standard for the treatment of HCV.³ This idea was reinforced by the recent discovery of treatment naïve patients from Equatorial Guinea, with a new HCV GT-1 subtype naturally bearing resistance-associated mutations to NS5A inhibitors, another class of DAA.⁴

Based on the success of 2’-halogeno compounds such as SOF⁵ but also the anticancer drug gemcitabine 2⁶, our group, and others, focused on the development of new 2’-dihalogeno nucleosides as potential anti-HCV agents. In 2017, Pinho et al. first reported the discovery of the β-d-2’-deoxy-2’-dichlorouridine nucleotide prodmg 3 as a potent inhibitor of HCV replication.⁷ Our group also reported the discovery of β-d-2’-deoxy-2’-β-chloro,2’-α-fluoro-, β-d-2’-deoxy-2’-dibromo-and β-d-2’-deoxy-2’-β-bromo, 2’-α-fluoro-uridine prodrugs 4a⁸, 5⁹ and 4b,¹⁰ as three potent anti-HCV agents (Figure 1). As part of our ongoing HCV drug discovery program, we wish to report herein, the synthesis and antiviral evaluation of new series of 2’-α-chloro,2’-β-fluoro and 2’-α-bromo,2’-β-fluoro nucleosides and their corresponding phosphoramidate prodrugs.

Fig. 1.

Structures of sofosbuvir, gemcitabine, known 2’-deoxy-2’-dihalogeno nucleoside prodrugs, 3-5 and targeted 2’-β-chloro-2’-α-fluoro and 2’-β-bromo,2’-α-fluoro nucleotides analogs.

2. Results and Discussion

2.1. Chemistry

Diastereoselective synthesis of key 2-chloro-2-fluoro and 2-bromo-2-fluoro lactols 11 and 12 was performed by adapting the chemistry developed by Cen et al. for the stereoselective synthesis of the 2-Br, 2-F lactone 10¹¹ (Scheme 1). Thus, protected lactone 6 was first converted to its corresponding trimethylsilyl enol ether followed by direct chlorination or bromination in the presence of either N-chlorosuccimimide (NCS) or N-bromosuccimimide (NBS) to give the chloro and bromo lactones 7 and 8, as diastereomeric mixtures. Subsequent fluorination in presence of lithium hexamethyldisilazide (LiHMDS) and N-fluorobenzenesulfonimide (NFSI) afforded selectively the dihalogeno lactones 9 and 10. Reduction with lithium tri-tert-butoxyaluminium hydride in tetrahydrofuran (THF) gave the corresponding lactols 11 and 12 as a mixture of α and β- anomers (ratio ~2:1). With these two key lactols in hand, the corresponding pyrimidine and purine nucleoside analogs along with their phosphoramidate prodrugs were prepared following the chemistry depicted in Schemes 2–7.

Scheme 1.

Synthesis of ribolactols 11 and 12. (a) i) TEA, TMSOTf, DCM, 0 °C, 30 min; ii) NCS or NBS, DCM, 0 °C, 1 h, 77% (7), 82% (8) over two steps; (b) NFSI, LiHMDS, THF, −78 °C, 1 h, 55% (9), 64% (10); (c) LiAl(OtBu)₃H, THF, 0 °C to rt, 1 h, 97% (11), 98% (12).

Scheme 2.

Synthesis of uridine analog phosphoramidates 19 and 20. (a) BzCl, TEA, DCM, 0 °C to rt, 16 h, 96% (13) and 83% (14); (b) uracil, BSA, CH₃CN, 60 °C, 15 min, then TMSOTf, MW, 150 °C, 6 min (15), 10 min (16), 30% (15a), 12% (15b), 22% (16a), 12% (16b); (c) TBAF, THF, rt, 1 h, 70% (17), 90% (18); (d) 21, NMI, THF, rt, 1 h, 34% (19), 46% (20).

Scheme 7.

Synthesis of guanosine analog phosphoramidates 50 and 51. (a) 2-bis-N-Boc-6-Cl purine, PPh₃, DIAD, 70 °C, 1 h, 54% (44a), 12% (44b), 20% (45a), 6% (45b); (b) TBAF, THF, rt, 1 h, 89% (46), 60% (48); (c) HCOOH/H₂O (4/1), 60 °C, 16 h, 54% (48), 50% (49); (d) i) 21 or 52, tBuMgCl, THF, 0 °C to rt, 1 h; ii) HCOOH/H₂O (4/1), 60 °C, 4 h, 24% (50), 22% (51) over two steps.

Thus, benzoylated derivatives 13 and 14, formed by reaction of benzoyl chloride with lactols 11 and 12, were reacted under classical Vorbrüggen type conditions in presence of persilylated uracil and trimethylsilyl trifluoromethanesulfonate (TMSOTf) under microwave irradiation to give α and β anomers 15a-b and 16a-b. At this stage, α and β isomers were separated by column chromatography and the isolated β isomers 16a-b were deprotected by treatment with tetra-N-butylammonium fluoride (TBAF) to give nucleosides 17 and 18 in 70% and 90% yield respectively. In order to express their therapeutic effect, nucleoside analogs need to be sequentially phosphorylated onto their corresponding 5’-triphosphate forms. However, the first phosphorylation step has often been identified as the limiting step and that is why nucleoside monophosphate prodrugs, which are able to deliver intracellularly a nucleoside monophosphate, have been developed.¹² Among all the prodrugs that have emerged, McGuigan’s Protides remains the most popular and have been clinically validated with the FDA-approval of drugs such as sofosbuvir 1 (HCV) or tenofovir disoproxyl fumarate (TDF) (HIV and HBV). Hence, phosphoramidate prodrugs 19 and 20 (R_P:S_P ~ 1:1) were prepared from nucleosides 17 and 18 by reaction with the phenyl L-isopropylalaninyl phosphorochloridate reagent 21 in presence of N-methylimidazole (Scheme 2).

In a similar manner, glycosylation of benzoylated derivativatives 13 and 14 with persilylated N-Bz-cytosine in the presence of TMSOTf at 150 °C for 12 min under microwave irradiation gave compounds 22 and 23 as a mixture of α/β anomers, easily separable by flash column chromatography on silica gel. In this case, in-situ 5′-TBDMS cleavage was observed probably due to the longer reaction time. Removal of the remaining TBDMS group with TBAF followed by debenzoylation in a saturated solution of ammonia in methanol afforded nucleosides 26 and 27 in 71% and 96% yield, respectively (Scheme 3).

Scheme 3.

Synthesis of cytidine nucleoside analogs 26 and 27. (a) N-Bz-cytosine, BSA, CH₃CN, 60 °C, 15 min, then TMSOTf, 150 °C, 12 min, MW, 37% (22a), 12% (22b), 21% (23a), 14% (23b); (b) TBAF, THF, rt, 1 h, 73% (24), 90% (25); (c) NH₃/MeOH, rt, 16 h, 71% (26), 96% (27).

Due to the low solubility of nucleosides 26 and 27 in solvents, and combination of solvents, commonly used for the formation of phosphoramidates (THF, THF/ACN or THF/DMF), alternative approaches were investigated to synthesize the corresponding cytidine phosphoramidate prodrugs 32 and 35. We first introduced the prodrug moiety onto the more soluble N-benzoylated intermediates 24 and 25 but were finally unable to find suitable conditions to debenzoylate the protected prodrug without substantial degradation. The preparation of prodrug 32 was eventually achieved by introduction of a more labile benzyloxycarbonyl group (Cbz).¹³ Thus, nucleoside 27 was first reacted with TBDMSCl in presence of imidazole followed by treatment with CbzCl and DMAP to give the fully protected nucleoside 29. TBDMS groups were then removed in the presence of Et₃N.3HF to give the desired 4-N-Cbz protected nucleoside 30. Finally, reaction with phenyl L-isopropylalaninyl phosphorochloridate reagent 21 in the presence of N-methyl imidazole and hydrogenolysis of the Cbz-protected cytidine ProTide intermediate 31 gave the corresponding phosphoramidate prodrug 32 (Scheme 4).

Scheme 4.

Synthesis of cytidine analog phosphoramidate 32. (a) TBDMSCl, imidazole, DCM, rt, 16 h, 83%; (b) CbzCl, DMAP, DCM, rt, 1.5 h, 74%; (c) Et₃N.3HF, THF, rt, 36 h 67%; (d) 21, NMI, THF, rt, 4 h, 20%; (e) Pd/C, EtOH, 1,4-cyclohexadiene, rt, 2 h, 55%.

For the synthesis of 2’-β-chloro-2’-α-fluoro cytidine phosphoramidate prodrug 35, we investigated the protection of the 3’-hydroxyl with a Boc group in order to favor the 5’-regioselectivity of the phosphorylation and increase the solubility of the nucleoside.¹⁴ Thus, nucleoside 26 was selectively protected in the presence of di-tert-butyl dicarbonate in dioxane/water (4:1) to give compound 33 in 30% yield. Compound 33 was then reacted with phenyl L-isopropylalaninyl phosphorochloridate reagent 21 in the presence of tert-butyl magnesium chloride to give the phosphoramidate prodrug 34, in 89% yield. Final treatment of compound 34 with a 4 M solution of HCl in dioxane to remove the Boc group gave the desired phosphoramidate prodrug 35 (Scheme 5).

Scheme 5.

Synthesis of cytidine analog phosphoramidate 35. (a) Boc₂O, Na₂CO₃, dioxane/water 4:1, rt, 48 h, 30%; (b) 21, tBuMgCl, THF, 0 °C to rt, 1 h, 89%; (c) HCl/dioxane 4M, 2 h, rt, 50%.

Purine nucleosides 36, 37, 44 and 45 were prepared according to the chemistry described in Scheme 6 and 7. Thus, coupling of lactols 11 and 12 with bis-N-Boc protected adenine in the presence of DIAD and PPh₃ gave 36 and 37 as a mixture of α/β anomers (ratio 4:1) separable by flash chromatography on silica gel. Deprotection of the silyl groups with TBAF in THF lead to the bis-N-Boc protected adenine derivatives 38 and 39, which were treated with a 4 M solution of HCl in dioxane to give the desired adenosine nucleosides 40 and 41. The corresponding monophosphate prodrugs 42 and 43 were obtained by reaction of protected intermediates 38 and 40 with phenyl L-isopropylalaninyl phosphorochloridate 21 in presence of tert-butyl magnesium chloride followed by treatment with a 4 M solution of HCl in dioxane (Scheme 6).

Scheme 6.

Synthesis of adenosine analog phosphoramidates 42 and 43. (a) bis-N-Boc A, PPh₃, DIAD, 70 °C, 1 h, 27% (36a), 7% (36b), 44% (37a), 12% (37b); (b) TBAF, THF, rt, 1 h, 99% (38), 70% (39); (c) 4 M HCl/dioxane, rt, 1 h, 61% (40), 60% (41); (d) i) 21, tBuMgCl, THF, rt, 1 h; ii) 4 M HCl/dioxane, rt, 1 h, 29% (42), 20% (43) over two steps.

Similarly, reaction of the 2-bis-N-Boc-amino-6-chloro-9H-purine with lactols 11 and 12 under Mitsunobu conditions gave compounds 44 and 45 as a mixture of α/β anomers (ratio 4:1) separable by flash chromatography. Treatment of 44a and 44b with TBAF afforded bis-N-Boc protected nucleosides 46 and 47 which were treated with 75% aqueous formic acid to give targeted nucleosides 48 and 49.¹⁵ Compound 46 was reacted with L-isopropylalaninyl phosphorochloridate 21 in presence of tert-butyl magnesium chloride and then treated with 75% aqueous formic acid to give the targeted phophoramidate prodrug 50 as a S_p/R_p mixture, in 24% yield over two steps.

Interestingly, our first attempts to prepare monophosphate prodrugs 51 from protected intermediates 47 using phenyl L-isopropylalaninyl phosphorochloridate 21 were unsuccessful and led to the formation of 3’,5’-di-phosphorylated derivatives. In order to avoid this problem, we turned our attention to the pentafluoro phenyl reagent 52. Indeed, this diastereomerically pure reagent (S_p isomer), used to synthesize sofosbuvir on a large scale, is known to be selective to the 5’-position when used at low temperature.¹⁶ Thus, reaction of compound 47 with pentafluoro phenyl phosphoramidate 52 in the presence of tert-butyl magnesium chloride followed by treatment with an aqueous solution of formic acid gave the desired phosphoramidate prodrug 51 (S_p isomer) in 22% yield over two steps (Scheme 7).

All α and β anomers were identified using 1H 2D-NOESY experiments. Fig. 2 shows an example of the NOE effect observed for each isomers. In the case of the β-anomer clear NOE interaction were observed between H2 of the adenine nucleobase and H3’ and H5’ of the sugar as well as between H1’ and H4’. On the other hand, NOE interactions between the H2 and H4’, H1’ and H5’ were observed for the α-anomer (Fig. 2).

Fig. 2.

Anomer assignment for compound 37a and 37b via NOE experiments.

Despite what was reported by Cen et al.,¹¹ the stereochemistry at the 2’-position could not be clearly established through common NMR experiments. Therefore, suitable crystals were grown from compounds 15a and 22a which allowed us to confirm the R-configuration of C-2’ of our nucleosides through X-ray diffraction analysis (Fig. 3).

Fig. 3.

Single crystal X-ray structure of compound 15a (top) and 22a (bottom).

2.2. Antiviral activity and cytotoxicity

Nucleoside analogs 17, 18, 26, 27, 40, 41, 48, 49 and their corresponding phosphoramidate prodrugs 19, 20, 32, 35, 42, 43, 50, 51 were evaluated for inhibition of HCV genotype 1b RNA replication in Huh-7 cells using a subgenomic HCV replicon system.¹⁷ Cytotoxicity in Huh-7 cells was determined simultaneously by extraction and amplification of both HCV RNA and cellular ribosomal RNA (rRNA).¹⁸ In addition, cytotoxicity was determined in primary human peripheral blood mononuclear (PBM) cells, human lymphoblastoid cells (CEM), and African Green monkey Vero cells (Table 1).^19,20 Except for 2’-Br,2’-F cytidine derivative 27, which displayed an EC₅₀ of 5.1 μM, none of the nucleosides showed anti-HCV activity at concentration up to 10 μM. On the other hand, monophosphate prodrugs 19, 20, 32, 35 and 42 had EC_50’s in the low to submicromolar range (EC₅₀ between 0.4 and 2.7 μM). This difference of potency between the nucleosides and their prodrug counterparts indicates that, once more, the monophosphorylation of modified nucleosides can be problematic and that making prodrug is an unavoidable step in the development of nucleoside analogs. Unfortunately, the increase in potency also came with an increase of cytotoxicity, especially towards Huh-7 cells. Indeed, compounds 19, 20, 27, 32, 35 and 42 displayed CC₅₀ in the low micromolar range in this specific cell line. Interestingly, neither the guanosine analogs 48 and 49 nor their corresponding monophosphate prodrugs (50 and 51) showed anti-HCV activity at concentration up to 10 μM. The unusual anti-HCV activity profile observed in these two series of 2’-dihalo nucleoside analogs versus what we have reported previously⁸,Error! Bookmark not defined. For related 2’-halo series of nucleoside analogs is a strong indicator that the observed anti-HCV activity presented here is largely, if not completely, due to the cytotoxicity toward the Huh-7 replicon cell line.

Table 1.

HCV genotype 1b replicon activity and cytotoxicity of nucleosides 17, 18, 26, 27, 40, 41, 48, 49 and their phosphoramidate prodrugs 19, 20, 32, 35, 42, 43, 50, 51.

Compound	Anti-HCV activity (μM)		Cytotoxicity, CC50 (μM)
	EC50	EC90	Huh-7	PBM	CEM	Vero
17	> 10	> 10	> 100	> 100	> 100	> 100
18	> 10	> 10	> 100	> 100	> 100	> 100
19	0.9 ± 0.3	3.7 ± 1.1	1.3	> 100	90	> 100
20	0.4 ± 0.3	2.0 ± 0.9	8.0	> 100	> 100	> 100
26	> 10	> 10	12	9.3	11	63
27	5.1 ± 0.9	> 10	2.9	> 100	3.2	2.9
32	2.4 ± 0.1	≥ 10	6.8	> 100	> 100	> 100
35	0.7 ± 0.2	3.4 ± 1.7	23	> 100	> 100	> 100
40	> 10	> 10	> 100	> 100	> 100	> 100
41	> 10	> 10	> 100	> 100	> 100	> 100
42	2.7 ± 1.3	≥ 10	1.6	26	15	> 100
43	> 10	> 10	6.9	19	29	71
48	> 10	> 10	> 100	> 100	> 100	> 100
49	> 10	> 10	> 100	> 100	> 100	> 100
50	> 10	> 10	> 100	> 100	84	> 100
51	> 10	> 10	> 100	> 100	> 100	> 100
SOF	0.05 ± 0.02	0.37 ± 0.25	> 100	> 100	> 100	> 100

3. Conclusion

Sixteen 2’-α-chloro,2’-β-fluoro and 2’-α-bromo,2’-β-fluoro nucleosides analogs along with their corresponding phosphoramidate prodrugs were prepared from key dihalogeno lactols 11 and 12. Among them, compounds 19, 20, 27, 32, 35 and 42 displayed anti-HCV activity in the low to sub-micromolar range. Unfortunately cytotoxicity was observed for most of these compounds in Huh-7 cells, precluding them for further development for HCV. Based on this study as well as previously reported studies,²¹ having a 2’-F group in the beta position seems to significantly increase the probability of cytotoxicity.

4. Experimental section

4.1. General

Anhydrous solvents were purchased from Aldrich Chemical Company, Inc. (Milwaukee). Reagents were purchased from commercial sources. Unless noted otherwise, the materials used in the examples were obtained from readily available commercial suppliers or synthesized by standard methods known to one skilled in the art of chemical synthesis. Microwave reactions were performed with a CEM discover explorer 12 hybrid. ¹H, ¹³C and ³¹P NMR spectra were taken on a Bruker Ascend^™ 400 spectrometer at rt, and reported in ppm downfield from internal tetramethylsilane (for ¹H-NMR). NMR processing was performed with Mnova (Mestrelab Research). Deuterium exchange and decoupling experiments were utilized to confirm proton assignments. Signal multiplicities are represented by s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quadruplet), br (broad), bs (broad singlet), m (multiplet). All J-values are in Hz and calculated by Mnova programs. Mass spectra were determined on a Micromass Platform LC spectrometer using electrospray ionization. Analytic TLC was performed on Analtech GHLF silica gel plates, and preparative TLC on Analtech GF silica gel plates. Column chromatography was performed on Combiflash R_f200.

4.2. Synthetic procedures

4.2.1. (3R/S,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-chlorodihydrofuran-2(3H)-one (7)

To a solution of compound 6 (5 g, 13.9 mmol) in DCM (180 mL) at 0 °C was added triethylamine (11.6 mL, 83.1 mmol) followed by TMSOTf (7.54 mL, 41.6 mmol). The reaction mixture was stirred at 0 °C for 30 minute and a solution of N-chlorosuccinimide (2.8 g, 21.0 mmol) in DCM (36 mL) was added. After stirring at 0 °C for another 1 h, the reaction mixture was poured into a saturated solution of NaHCO₃ (200 mL) and extracted with DCM (2 × 150 mL). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexane/ethyl acetate 40:1) to afford compound 7 as a 1:1.3 mixture of diastereoisomers (4.22 g, 77%). ¹H NMR (400 MHz, CDCl₃): 4.59-4.62 (m, 1.5H), 4.48 (m, 0.5H), 4.38-4.42 (m, 1.5H), 4.16-4.19 (m, 1H), 3.90-3.95 (m, 1.5H), 3.76-3.81 (m, 1.5H), 0.8 and 0.9 (each s, 27H), 0.14-0.08 (m, 18H). ¹³C NMR (100 MHz, CDCl₃): δ 171.4, 169.7, 86.0, 83.8, 75.1, 70.6, 61.5, 59.9, 59.2, 55.9, 25.9, 25.7, 18.4, 18.0, −4.9, −5.3, −5.4, −5.5. MS (HR-ESI) for C₁₇H₃₆ClO₄Si₂ [(M+H)⁺]. Calcd: m/z 395.1841, Found: m/z 395.1834.

4.2.2. (3R/S,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-bromodihydrofuran-2(3H)-one (8)

To a solution of compound 6 (7.0 g, 19.4 mmol) in DCM (250 mL) at 0 °C was added triethylamine (16.3 mL, 117 mmol) followed by TMSOTf (10.6 mL, 58.3 mmol). The solution was stirred at 0 °C for 30 minutes then, at 0 °C, a solution of N- bromosuccinimide (5.2 g, 29.1 mmol) in DCM (75 mL) was added. After stirring at 0 °C for another 1 h, the reaction mixture was poured into a saturated solution of NaHCO₃ (300 mL) and extracted with DCM (2 × 200 mL). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexane/ethyl acetate 40:1) to afford compound 8 as a 1:1.5 mixture of diastereoisomers (7.0 g, 82%). ¹H NMR (400 MHz, CDCl₃): δ 4.71 – 4.67 (m, 1H), 4.49 (d, J = 5.6 Hz, 1H), 4.42 – 4.37 (m, 3H), 4.33 (dt, J = 4.7, 2.2 Hz, 1H), 4.25 – 4.20 (m, 1H), 3.98 – 3.92 (m, 1H), 3.83 – 3.74 (m, 3H), 0.93 – 0.86 (m, 48H), 0.20 – 0.04 (m, 33H).¹³C NMR (100 MHz, CDCl₃): δ 170.6 (d, J = 80.9 Hz), 85.2 (d, J = 28.2 Hz), 75.6, 69.6, 68.8, 60.3, 46.13 (d, J = 3.6 Hz), 39.0, 25.8, 25.6, 21.1, 18.2 18.0, 14.21, −4.9, −5.3, −5.4, −5.5. MS (HR-ESI) for C₁₇H₃₆BrO₄Si₂ [(M+H)⁺]. Calcd: m/z 439.1335. Found: m/z 439.1330.

4.2.3. (3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-chloro-3-fluorodihydrofuran-2(3H)-one (9)

Compound 7 (4 g, 10.1 mmol) and NFSI (4.78 g, 15.2 mmol) were dissolved in anhydrous THF (80 mL). The solution was cooled to −78 °C, and a 1 M solution of LiHMDS in THF (13.2 mL, 13.2 mmol) was added dropwise. The reaction mixture was stirred at −78 °C for 1 h, and quenched with a saturated solution of NH₄Cl (100 mL). The mixture was allowed to warm to rt, and the aqueous layer was extracted with ethyl acetate (3 × 100 mL). The organic layers were combined, washed with a saturated solution of NaHCO₃, water and brine, dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (hexane/ethyl acetate 40:1) to afford compound 9 (2.25 g, 55%). ¹H NMR (400 MHz CDCl₃): 4.79 (dd, J = 14.5 Hz, J = 8.4 Hz, 1H), 4.09 (dt, J = 1.9 Hz, 1H), 4.01 (dt, J = 2.3 Hz, 1H), 3.80 (two d, J = 1.9 Hz, 1H), 0.9 (ds, 18H), 0.09 (four s, 12H). ¹⁹F NMR (376 MHz, CDCl₃): −127.5 (d, J = 14.3 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 165.5, 104.4 (d, J = 264 Hz), 80.9, 71.9, 25.9, 25.6, 18.4, 18.2, −4.6, −5.1, −5.3, −5.4. MS (HR-ESI) for C₁₇H₃₅ClFO₄Si₂ [(M+H)⁺]. Calcd: m/z 413.1740. Found: m/z 413.1746.

4.2.4. (3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethysilyl)oxy)methyl)-3-bromo-3-fluorodihydrofuran-2(3H)-one (10)

Compound 8 (7.0 g, 15.9 mmol) and NFSI (7.5 g, 23.8 mmol) were dissolved in anhydrous THF (140 mL). The solution was cooled to −78 °C and a 1 M solution of LiHMDS in THF (20.7 mL, 20.7 mmol) was added dropwise. The reaction mixture was stirred at −78 °C for another 1 h, and was quenched with a saturated solution of NH₄Cl (200 mL). The mixture was allowed to warm to rt, and the aqueous layer was extracted with ethyl acetate (3 × 150 mL). The organic layers were combined, washed with a saturated solution of NaHCO₃, water and brine, dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexane/ethyl acetate 40:1) to afford compound 10 (4.6 g, 64%). ¹H NMR (400 MHz CDCl₃): δ 4.54 (dd, J = 15.3, 8.0 Hz, 1H), 4.04 – 4.00 (m, 1H), 3.98 (dd, J = 2.6, 1.8 Hz, 1H), 3.81 – 3.75 (m, 1H), 0.94 (s, 9H), 0.87 (s, 9H), 0.18 (s, 3H), 0.14 (s, 3H), 0.07 (d, J = 2.6 Hz, 6H).¹⁹F NMR (376 MHz, CDCl₃): δ −126.98 (dd, J = 15.5 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 165.7, 100.7, 97.9, 80.6, 72.1, 58.1, 25.7, 25.6, 18.2, 18.0, −4.6, −5.1, −5.4, −5.5. MS (HR-ESI) for C₁₇H₃₅BrO₄Si₂ [(M+H)⁺]. Calcd: m/z 457.1241. Found: m/z 457.1236.

4.2.5. (2R/S,3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethysilyl)oxy)methyl)-3-chloro-3-fluorotetrahydrofuran-2-ol (11)

To a solution of compound 9 (5.37 g, 13 mmol) in anhydrous THF (90 mL) was added dropwise LiAlH[OC(CH₃)₃]₃ (32.5 mL, 32.48 mmol, 1 M in THF) at 0 °C. The reaction mixture was stirred at rt for 1 h. The reaction was quenched with a saturated solution of NH₄Cl (50 mL) and extracted with ethyl acetate (100 mL). The organic layer was washed with water, brine, dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The crude residue was purified by flash column chromatography on silica gel (hexane/ethyl acetate 20:1) to afford compound 11 (5.23 g, 97%). ¹H NMR (400 MHz, CDCl₃): 5.25 (ddd, J = 12.5 Hz, J = 6.3 Hz, J = 0.9 Hz, 0.5H), 5.16 (d, J = 9.6 Hz, 1H), 4.66 (dd, J = 12.5 Hz, J = 6.7 Hz, 1H), 4.39 (ddd, J = 11.7 Hz, J = 4.0 Hz, J = 0.9 Hz, 0.5H), 4.07-4.11 (m, 0.5H), 3.92 (dt, J = 1.9 Hz, 1H), 3.82 (d, J = 9.3 Hz, 1H), 3.78 (dt, J = 2.5 Hz, 1H), 3.72 and 3.69 (each dd, J = 4.0 Hz, J = 1.8 Hz, 0.5H), 3.61-3.66 (m, 1.5H), 3.50 (d, J = 12.2 Hz, 0.5H), 0.93, 0.92, 0.90 (each s, 27 H), 0.19, 0.16, 0.15, 0.1, 0.07, 0.00 (each s, 18 H). ¹⁹F NMR (376 MHz, CDCl₃): −122.59 (dd, J = 12.2 Hz, J = 6.4 Hz), −130.55 (d, J = 12.5 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 116.1 (d, J = 249 Hz), 115.1 (d, J = 263 Hz), 99.5, 99.2, 98.9, 84.5, 82.7, 82.6, 74.7, 74.5, 72.2, 72.0, 62.0, 61.2, 26.0, 25.7, 18.49, 18.47, 18.2, 18.1, −4.4, −4.7, −4.8, −5.3, −5.3, −5.4 MS (HR-ESI) for C₁₇H₃₆ClFNaO₄Si₂ [(M+Na)⁺]. Calcd: m/z 437.1722, Found: m/z 437.1717.

4.2.6. (2R/S,3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-bromo-3-fluorotetrahydrofuran-2-ol (12)

To a solution of compound 10 (3.5 g, 7.65 mmol) in anhydrous THF (70 mL) was added dropwise LiAlH[OC(CH₃)₃]₃ (11.5 mL, 11.5 mmol, 1 M in THF) at 0 °C. The reaction mixture was stirred at rt for 1 h. The reaction was quenched with a saturated solution of NH₄Cl (50 mL) and extracted with ethyl acetate (100 mL). The organic layer was washed with water, brine, dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The crude residue was purified by flash column chromatography on silica gel (hexane/ethyl acetate 20:1) to afford compound 12 (3.43 g, 98%). ¹H NMR (400 MHz, CDCl₃): δ 5.32 (dd, J = 9.0, 1.3 Hz, 1H), 5.18 (ddd, J = 12.4, 6.3, 0.9 Hz, 0.5H), 4.49 (dd, J = 13.5, 6.7 Hz, 1H), 4.39 (ddd, J = 12.1, 4.6, 1.0 Hz, 0.5H), 4.06 – 3.99 (m, 0.5H), 3.92 (dt, J = 6.7, 2.0 Hz, 1H), 3.87 (d, J = 9.2 Hz, 1H), 3.79 (t, J = 2.4 Hz, 0.5H), 3.76 (d, J = 2.5 Hz, 1H), 3.67 (m, 1H), 3.63 (d, J = 1.7 Hz, 1H), 3.61 (d, J = 1.7 Hz, 0.5H), 3.49 (d, J = 12.4 Hz, 0.5H), 0.96 – 0.87 (m, 27H), 0.22 – 0.04 (m, 18H).¹⁹F NMR (376 MHz, CDCl₃): −120.06 (dd, J = 12.2 Hz, J = 6.4 Hz), −127.23 (d, J = 13.4 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 113.5, 110.8, 99.9 (d, J = 21.4 Hz), 99.0 (d, J = 31.9 Hz), 83.6, 82.3 (d, J = 8.7 Hz), 74.5 (d, J = 24.9 Hz), 72.1 (d, J = 22.3 Hz), 61.7, 60.9, 28.4 – 24.1 (m), 18.2 (d, J = 29.8 Hz).

4.2.7. (2R/S,3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethysilyl)oxy)methyl)-3-chloro-3-fluorotetrahydrofuran-2-yl benzoate (13)

To a solution of compound 11 (4.72 g, 11.4 mmol) in DCM (100 mL) was added triethylamine (3.96 mL, 28.4 mmol). The reaction was cooled to 0 °C and benzoyl chloride (1.97 mL, 17.0 mmol) was introduced dropwise. After being stirred for 30 min at 0 °C, the mixture was warmed to rt and stirred for 16 h. The reaction was quenched with methanol (1 mL) and then was washed with water and brine. The organic layer was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel (hexane/ethyl acetate 20:1) to afford compound 13 (5.65 g, 96%). ¹H NMR (400 MHz, CDCl₃): 8.10 and 8.05 (m, 2.2H), 7.57-7.62 (m, 1.2H), 7.42-7.47 (m, 2.4H), 6.54 and 6.46 (m, 1.25H), 4.80 (dd, J = 12.5 Hz, J = 7.9 Hz, 1H), 4.37 (dd, J = 18.1 Hz, J = 4.7 Hz, 0.25 H), 4.21 (m, 0.25 H), 3.85-3.91 (m, 2H), 3.76-3.84 (m, 0.5H), 3.68-3.72 (m, 1H), 0.95 and 0.78 (each s, 22H), 0.07, 0.08, 0.16, 0.20 and 0.22 (each s, 15 H). ¹⁹F NMR (376 MHz, CDCl₃): −115.15 (dd, J = 17.8 Hz, J = 7.7 Hz) and 128.53 (d, J = 12.1 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 164.9, 133.7, 130.3, 130.2, 129.4, 128.6, 114.0 (d, J = 273 Hz), 97.7, 87.4, 82.5, 72.0, 62.0, 60.8, 25.7, 25.8, 25.9, 26.0, 18.5, 18.2, −4.5, −4.7, −4.8, −5.2, −5.3, −5.4. MS (HR-ESI) for C₂₄H₄₀ClFNaO₅Si₂ [(M+Na)⁺]. Calcd: m/z 541.1985, Found: m/z 541.1978.

4.2.8. (2R/S,3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-bromo-3-fluorotetrahydrofuran-2-yl benzoate (14)

To a solution of compound 12 (744 mg, 1.62 mmol) in DCM (40 mL) was added triethylamine (0.34 mL, 2.43 mmol). The reaction was cooled to 0 °C and benzoyl chloride (0.23 mL, 1.94 mmol) was introduced dropwise. After being stirred for 30 min at 0 °C, the mixture was warmed to rt and stirred for 16 h. The reaction was quenched with methanol (0.20 mL) and then washed with water and brine. The organic layer was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel (hexane/ethyl acetate 20:1) to afford compound 14 (760 mg, 83%). ¹H NMR (400 MHz, CDCl₃): 8.13-8.11 (m, 1H), 8.06-8.03 (m, 2H), 7.62-7.58 (m, 2H), 7.47-7.43 (m, 3H), 6.64 (m, 1H), 6.56 (m, 0.5H), 4.59 (dd, J = 12.5 Hz, J = 7.9 Hz, 1H), 4.37 (dd, J = 18.1 Hz, J = 4.7 Hz, 1H), 4.20 (m, 1H), 3.90-3.86 (m, 2H), 3.81-3.77 (m, 1H), 3.72-3.68 (m, 1H), 0.95 and 0.78 (each s, 22H), 0.25, 0.20, 0.18, 0.09, 0.08, 0.07 (each s, 15 H). ¹⁹F NMR (376 MHz, CDCl₃): −115.18 (dd, J = 17.8 Hz, J = 7.7 Hz) and 127.00 (d, J = 12.1 Hz). MS (HR-ESI) for C₂₄H₄₀BrFO₅Si₂ [(M+H)⁺]. Calcd: m/z 563.1582, Found: m/z 563.1588.

4.2.9. 1-((2S,3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-chloro-3-fluorotetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (15a) and 1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-chloro-3-fluorotetrahydrofuran-2-yl)pyrimidine-2,4(1H, 3H)-dione (15b)

A solution of compound 13 (1.14 g, 2.19 mmol), uracil (0.5 g, 4.46 mmol) and BSA (2.65 mL, 10.8 mmol) in acetonitrile (10 mL) was stirred at 60 °C for 15 minutes before addition of TMSOTf (2.22 mL, 12.3 mmol). The reaction vessel was then placed into the cavity of microwave reactor and irradiated for 6.5 min at 150 °C. The reaction was quenched by addition of a 5% aqueous solution of NaHCO₃ at 0 °C. The aqueous layer was extracted with ethyl acetate (20 mL), and the combined organic layers were washed with a saturated solution of NaHCO₃, water, and brine. The solution was dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/ethyl acetate 2:1) to afford 15a (350 mg, 32%) and 15b (140 mg, 13%). 15a: ¹H NMR (400 MHz, CDCl₃): δ 8.58 (s, 1H), 7.49 (d, J = 8.04 Hz, 1H), 6.45 (d, J = 10.5 Hz, 1H), 5.72 (dd, J = 8.3 Hz, J = 2.3 Hz, 1H), 4.61 (dd, J = 12.3 Hz, J = 4.8 Hz, 1H) 4.20 (m, 1H), 3.75 (m, 2H), 0.93 (two s, 18H), 0.08 (four s, 12H). ¹⁹F NMR (376 MHz, CDCl₃): −114.05 (t, J = 11.3 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 162.7, 150.2, 140.4, 117.9 (d, J = 252 Hz), 102.0, 88.2, 86.1, 74.8, 61.3, 26.0, 25.7, 18.4, 18.1, −4.7, −4.9, −5.3, −5.32. MS (HR-ESI) for C₂₁H₃₈ClFN₂NaO₅Si₂ [(M+Na)⁺]. Calcd: m/z 531.1890, Found: m/z 531.1882. 15b: ¹H NMR (400 MHz, CDCl₃): δ 8.64 (s, 1H), 7.73 (dd, J = 8.3 Hz, J = 1.5 Hz, 1H), 6.37 (d, J = 5.6 Hz, 1H), 5.72 (dd, J = 8.3 Hz, J = 2.4 Hz, 1H), 4.42 (dd, J = 16.3 Hz, J = 7.3 Hz, 1H, 3.96 (dt, J = 2.7 Hz 1H), 3.86 (m, 1H), 3.79 (m, 1H), 0.93 (two s, 18H), 0.08 (four s, 12H). ¹⁹F NMR (376 MHz, CDCl₃): −122.39 (dd, J = 16.04 Hz, J = 4.7 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 162.6, 150.1, 139.6, 114.6 (d, J = 259 Hz), 102.7, 87.8, 82.0, 73.2, 60.3, 26.0, 5.7, 18.5, 18.2, −4.39, −4.91, −5.29, −5.39. MS (HR-ESI) for C₂₁H₃₈ClFN₂NaO₅Si₂ [(M+Na)⁺]. Calcd: m/z 531.1890. Found: m/z 531.1884.

4.2.10. 1-((2S,3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-bromo-3-fluorotetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (16a) and 1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-bromo-3-fluorotetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (16b)

A solution of compound 14 (1.00 g, 1.77 mmol), uracil (0.22 g, 1.95 mmol) and BSA ((1.29 mL, 5.31 mmol) in acetonitrile (10 mL) was stirred at 60 °C for 15 min before addition of TMSOTf (0.96 mL, 5.31 mmol). The reaction vessel was then placed into the cavity of microwave reactor and irradiated for 6.5 min at 150 °C. The reaction was quenched by addition of a 5% aqueous solution of NaHCO₃ at 0 °C. The aqueous layer was extracted with ethyl acetate (20 mL), and the combined organic layers were washed with a saturated solution of NaHCO₃, water, and brine. The solution was dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/ethyl acetate 2:1) to afford 16a (220 mg, 22%) and 16b (120 mg, 12%). 16a: ¹H NMR (400 MHz, CDCl₃): δ 7.50 (d, J = 8.24 Hz, 1H), 6.42 (d, J = 10.6 Hz, 1H), 5.73 (d, J = 8.3 Hz, 1H), 4.53 (dd, J = 12.3 Hz, J = 4.8 Hz, 1H) 4.18 (m, 1H), 3.73 (m, 2H), 0.95 (two s, 18H), 0.15 (threes, 12H). ¹⁹F NMR (376 MHz, CDCl₃): −111.15 (t, J = 11.3 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 163.3, 150.3, 140.3, 116.7 (d, J = 252 Hz), 114.1 101.8, 88.4 (d, J = 150 Hz), 85.3, 74.8 (d, J = 103 Hz), 61.1, 25.9, 25.7, 18.5, 18.0, −4.5, −4.7, −4.9, −5.4, −5.5. 16b: ¹H NMR (400 MHz, CDCl₃): δ 9.05 (s, 1H), 7.76 (dd, J = 8.3 Hz, J = 1.5 Hz, 1H), 6.56 (d, J = 5.6 Hz, 1H), 5.71 (d, J = 8.0 Hz, 1H), 4.25 (dd, J = 16.3 Hz, J = 7.3 Hz, 1H, 3.97 (dt, J = 2.7 Hz 1H), 3.85 (m, 1H), 3.77 (m, 1H), 0.93 (two s, 18H), 0.12 (three s, 12H). ¹⁹F NMR (376 MHz, CDCl₃): −120.56 (dd, J = 16.04 Hz, J = 4.7 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 162.8, 150.1, 139.5, 111.8, 109.1, 102.5, 88.7, 81.6, 73.2, 60.0, 25.7, 18.4, 18.0, −4.4, −4.9, −5.4, −5.5 MS (HR-ESI) for C₂₁H₃₈BrFN₂O₅Si₂ [(M+H)⁺]. Calcd: m/z 553.1565, Found: m/z 553.1567.

4.2.11. 1-((2R,3R,4R,5R)-3-Chloro-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione (17)

To a solution of compound 15b (21 mg, 0.041 mmol) in anhydrous THF (1 mL) was added TBAF (91 μL, 0.091 mmol, 1 M in THF). The reaction mixture was stirred for 1 h at rt and then quenched with a saturated solution of NH₄Cl. The aqueous layer was extracted with ethyl acetate (3 × 5 mL) and the combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 10:1) to afford compound 17 (8 mg, 70%). ¹H NMR (400 MHz, MeOD): δ 7.95 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H), 6.33 (d, J = 8.4 Hz, 1H), 5.72 (d, J = 8.8 Hz, 1H), 4.36 (dd, J = 17.0 Hz, J = 6.9 Hz, 1H), 3.9 (m, 2H), 3.78 (m, 1H). ¹⁹F NMR (376 MHz, MeOD): −124.82 (dd, J = 17.4 Hz, J = 8.5 Hz). ¹³C NMR (100 MHz, MeOD): δ 165.7, 152.1, 142.2, 116.3 (d, J = 258 Hz), 102.8, 88.8, 83.6, 74.1, 60.6. MS (HR-ESI) for C₉H₁₁ClFN₂O₅ [(M+H)⁺]. Calcd: m/z 281.0341, Found: m/z 281.0333.

4.2.12. 1-((2R,3R,4R,5R)-3-Bromo-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (18)

To a solution of compound 16b (60 mg, 0.14 mmol) in anhydrous THF (4 mL) was added TBAF (310 μL, 0.31 mmol, 1 M in THF). The reaction mixture was stirred for 1 h at rt and then quenched with a saturated solution of NH₄Cl. The aqueous layer was extracted with ethyl acetate (3 × 10 mL) and the combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 10:1) to afford compound 18 (41 mg, 90%). ¹H NMR (400 MHz, MeOD): δ 7.97 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H), 6.49 (d, J = 8.4 Hz, 1H), 5.72 (d, J = 8.8 Hz, 1H), 4.22 (dd, J = 17.0 Hz, J = 6.9 Hz, 1H), 3.90 (m, 2H), 3.78 (m, 1H). ¹⁹F NMR (376 MHz, MeOD): −124.12 (dd, J = 17.4 Hz, J = 8.5 Hz). ¹³C NMR (100 MHz, MeOD): δ 164.3, 153.5, 140.8, 128.6, 122.2, 120.1, 112.5, 101.3, 88.3, 81.9, 72.7, 68.1, 59.1, 50.5, 20.4. MS (HR-ESI) for C₉H₃₁₀BrFN₂O₅ [(M+H)⁺]. Calcd: m/z 324.9835, Found: m/z 324.9833.

4.2.13. Isopropyl ((((2R,3R,4R,5R)-4-chloro-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate, diastereomeric mixture (19)

To a solution of compound 17 (30 mg, 0.11 mmol) in THF (2 mL) was added N-methylimidazole (68 μL, 0.85 mmol) then (2S)-isopropyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (0.21 g, 0.69 mmol) in THF (0.69 mL). The mixture was stirred at rt for 1 h, quenched with water and extracted with ethyl acetate (5 mL). The organic layer was washed with water twice, dried over Na₂SO₄, filtrated and concentrated under reduced pressure. The crude product was purified by flash chromatography (CH₂Cl₂/MeOH 20:1) to afford compound 19 as a R_P:S_P, diastereomeric mixture (~1:1) (20 mg, 34%). ¹H NMR (400 MHz, MeOD): δ 7.66 and 7.58 (each dd, J = 8.3 Hz, J = 2.4 Hz, 1H), 7.35-7.38 (m, 2H), 7.20-7.27 (m, 3H), 6.33-6.37 (m, 1H), 5.69 and 5.64 (each d, J = 8.3 Hz, 1H), 4.93-4.99 (m, 1H), 4.29-4.41 (m, 3H), 4.10 (m, 1H), 3.9 (m, 1H), 1.29-1.36 (m, 3H), 1.22-1.24 (m 6H). ¹⁹F NMR (376 MHz, MeOD): −124.88 (m). ³¹P NMR (162 MHz, MeOD): 3.65, 3.58. ¹³C NMR (100 MHz, MeOD): δ 173.2, 162.8, 150.5, 150.3, 140.1, 130.1, 125.6, 120.1, 113.7 (d, J = 257 Hz), 103.0, 87.3, 80.4, 73.4, 69.8, 64.2, 50.6, 21.8, 20.9. MS (HR-ESI) for C₂₁H₂₇ClFN₃O₉P [(M+H)₊]. Calcd: m/z 550.1157, Found: m/z 550.1149.

4.2.14. Isopropyl ((((2R,3R,4R,5R)-4-bromo-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate, diastereomeric mixture (20)

To a solution of compound 18 (22 mg, 0.077 mmol) in THF (2 mL) was added N-methylimidazole (31 μL, 0.39 mmol) then (2S)-isopropyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate 21 (0.071 g, 0.23 mmol) in THF (0.23 mL). The mixture was stirred at rt for 1 h, quenched with water and extracted with ethyl acetate (5 mL). The organic layer was washed with water twice, dried over Na₂SO₄, filtrated and concentrated under reduced pressure. The crude product was purified by flash chromatography (CH₂Cl₂/MeOH 20:1) to afford compound 20 as a diastereomeric mixture of R_P:S_P (~1:1) (18 mg, 46%). ¹H NMR (400 MHz, MeOD): δ 7.66 and 7.60 (each dd, J = 8.3 Hz, J = 2.4 Hz, 1H), 7.38-7.34 (m, 2H), 7.27-7.20 (m, 3H), 6.52-6.47 (m, 1H), 5.69 and 5.64 (each d, J = 8.3 Hz, 1H), 4.99 (m, 1H), 4.40-4.30 (m, 1H), 4.29-4.20 (m, 1H), 4.15-4.05 (m, 1H), 3.95-3.85 (m, 1H) 1.35-1.31 (m, 3H), 1.25-1.21 (m 6H). ¹⁹F NMR (376 MHz, MeOD): −124.53 (m). ³¹P NMR (162 MHz, MeOD): 3.64, 3.57. ¹³C NMR (100 MHz, MeOD): δ 173.2, 164.1, 150.5, 140.1, 129.4, 124.9, 120.0, 101.7, 88.0, 80.4, 68.8, 64.8, 50.3, 20.5, 18.9.: MS (HR-ESI) for C₂₁H₂₇BrFN₃O₉P [(M+H)⁺]. Calcd: m/z 594.0652, Found: m/z 594.0652

4.2.15. N-(1-((2S,3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-3-chloro-3-fluoro-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (22a) and N-(1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-3-chloro-3-fluoro-5-(hydroxymethyl)tetrahydrofurcm-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (22b)

A solution of compound 13 (1.0 g, 1.92 mmol), 4-N-benzoylcytosine (660 mg, 3.06 mmol) and BSA (1.9 mL, 7.66 mmol) in acetonitrile (10 mL) was stirred at rt for 15 min before addition of TMSOTf (3.11 mL, 17.23 mmol). The reaction vessel was then placed into the cavity of a microwave reactor and irradiated for 12 min at 150 °C. The reaction was quenched by addition of 5% aqueous solution of NaHCO₃ (20 mL) at 0 °C. The aqueous layer was extracted with ethyl acetate (3 × 20 mL), and the combined organic layers were washed with a saturated solution of NaHCO₃, water, and brine. The solution was dried over Na₂SO₄, and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (hexanes/ethyl acetate 2:1) to afford 22a (352 mg, 37%) and 22b (120 mg, 13%). 22a: ¹H NMR (400 MHz, CDCl₃): δ 8.95 (br s, 1H), 7.91 (d, J = 7.6 Hz, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.61 (m, 2H), 7.51 (m, 2H), 6.77 (d, J = 8.1 Hz, 1H), 4.73 (dd, J = 12.3 Hz, J = 7.1 Hz, 1H), 4.20 (m, 1H), 3.92 (m, 1H), 3.75 (m, 1H), 2.76 (br s, 1H), 0.92 (s, 9H), 0.17 (s, 6H). ¹⁹F NMR (376 MHz, CDCl₃): −116.55 (s). ¹³C NMR (100 MHz, CDCl₃): δ 166.6, 162.9, 155.2, 145.1, 133.5, 133.0, 129.2, 127.8, 117.7 (d, J = 258 Hz), 96.6, 88.2, 84.8, 74.5, 60.6, 25.7, 18.3, −4.6, −4.9. MS (HR-ESI) for C₂₂H₃₀ClFN₃O₅Si [(M+H)⁺]. Calcd: m/z 498.1627, Found: m/z 498.1624. 22b: ¹H NMR (400 MHz, CDCl₃): δ 8.81 (br s, 1H), 8.17 (d, J = 7.3 Hz, 1H), 7.86 (d, J = 7.3 Hz, 1H), 7.60 (m, 2H), 7.49 (m, 2H), 6.55 (d, J = 6.8 Hz, 1H), 4.51 (dd, J = 16.6 Hz, J = 7.4 Hz, 1H), 4.09 (m, 1H), 3.95 (m, 1H), 3.84 (m, 1H), 2.7 (br s, 1H), 0.93 (s, 9H), 0.15 (s, 6H). ¹⁹F NMR (376 MHz, CDCl₃): −122.31 (s). ¹³C NMR (100 MHz, CDCl₃): δ 170.8, 162.9, 155.1, 145.3, 133.4, 133.0, 129.1, 127.8, 114.5 (d, J = 260 Hz), 97.4, 89.2, 82.2, 73.4, 59.7, 25.8, 18.1, −4.6, −4.9. MS (HR-ESI) for C₂₂H₃₀ClFN₃O₅Si [(M+H)⁺]. Calcd: m/z 498.1627, Found: m/z 498.1620.

4.2.16. N-(1-((2S,3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-3-bromo-3-fluoro-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (23a) and N-(1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-3-bromo-3-fluoro-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (23b)

A solution of compound 14 (1.5 g, 2,67 mmol), 4-N-benzoylcytosine (634 mg, 2.94 mmol) and BSA (1.94 mL, 8.01 mmol) in acetonitrile (10 mL) was stirred at rt for 15 min before addition of TMSOTf (1.45 mL, 8.01 mmol). The reaction vessel was then placed into the cavity of microwave reactor and irradiated for 12 min at 150 °C. The reaction was quenched by addition of a 5% aqueous solution of NaHCO₃ (20 mL) at 0 °C. The aqueous layer was extracted with ethyl acetate (3 × 20 mL), and the combined organic layers were washed with a saturated solution of NaHCO₃, water, and brine. The solution was dried over Na₂SO₄, and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (hexanes/ethyl acetate 2:1) to afford 23a (300 mg, 21%) and 23b (200 mg, 14%). 23a: ¹H NMR (400 MHz, CDCl₃) δ 7.89-7.84 (m, 3H), 7.59 (t, J = 14.8 Hz, 2H), 7.50 (t, J = 15.2 Hz, 2H), 6.68 (d, J = 8.0 Hz, 1H), 4.60 (dd, J = 13.5, 7.2 Hz, 1H), 4.18-4.15 (m, 1H), 3.92 – 3.70 (m, 2H), 2.52 (BR S, 1H), 0.91 (s, 9H), 0.17 (s, 3H), 0.15 (s, 3H). ¹⁹F NMR (376 MHz, CDCl₃) δ −113.25 (s). ¹³C NMR (100 MHz, CDCl₃): δ 170.8, 162.9, 155.1, 144.9, 133.3, 129.1, 127.6, 117.1, 114.4, 96.2, 88.2 (d, J = 137.6 Hz), 83.9, 74.5 (d, J = 94.9 Hz), 60.3, 25.6, 18.0, −4.5, −4.9. MS (HR-ESI) for C₂₂H₂₉BrFN₃O₅Si [(M+H)⁺]. Calcd: m/z 542.1122. Found: m/z 542.1117. 23b: ¹H NMR (400 MHz, CDCl₃) δ 8.28 (d, J = 7.5 Hz, 1H), 7.91 (d, J = 7.5 Hz, 2H), 7.63-7.58 (m, 2H), 7.52-7.48 (m, 2H), 6.71 (d, J = 5.6 Hz, 1H), 4.36 (dd, J = 17.1, 7.6 Hz, 1H), 4.14 – 4.08 (m, 1H), 3.95 (dt, J = 4.9, 2.2 Hz, 1H), 3.84 (dd, J = 12.4, 2.8 Hz, 1H), 0.94 (s, 9H), 0.17 (s, 3H), 0.15 (s, 3H). ¹⁹F NMR (CDCl₃, 376 MHz) δ −121.28 (dd, J = 12.0, 7.8 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 170.9, 162.6, 155.2, 145.4, 133.5, 132.7, 129.0, 127.9, 111.5, 97.2, 81.6, 73.2, 59.4, 29.7, 25.6, 18.0, −4.5, −4.9. MS (HR-ESI) for C₂₂H₂₉BrFN₃O₅Si [(M+H)⁺]. Calcd: m/z 542.1122. Found: m/z 542.1117.

4.2.17. N-(1-((2R,3R,4R,5R)-3-Chloro-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (24)

To a solution of compound 22b (70 mg, 0.14 mmol) in anhydrous THF (2 mL) was added TBAF (180 μL, 0.18 mmol, 1 M in THF). The reaction mixture was stirred for 1 h at rt and then quenched with a saturated solution of NH₄Cl (4 mL). The aqueous layer was extracted with ethyl acetate (3 × 4 mL) and the combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 10:1) to afford compound 24 (39 mg, 73%). ¹H NMR (400 MHz, MeOD): δ 8.51 (d, J = 7.6 Hz, 1H), 7.97 (m, 2H), 7.64 (m, 2H), 7.52 (m, 2H), 6.51 (d, J = 6.1 Hz, 1H), 4.44 (dd, J = 16.1 Hz, J = 7.3 Hz, 1H), 3.93-4.01 (m, 2H), 3.83 (m, 1H). ¹⁹F NMR (376 MHz, MeOD): −124.97 (dd, J = 16.6 Hz, J = 5.3 Hz). ¹³C NMR (100 MHz, MeOD): δ 169.1, 165.4, 157.7, 146.4, 134.59, 134.17, 129.8, 129.2, 116.2 (d, J = 257 Hz), 98.5, 90.0, 83.4, 73.6, 60.3. MS (HR-ESI) for C₁₆H₁₆ClFN₃O₅ [(M+H)⁺]. Calcd: m/z 384.0763, Found: m/z 384.0753.

4.2.18. N-(1-((2R,3R,4R,5R)-3-bromo-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (25)

To a solution of compound 23b (40 mg, 0.074 mmol) in anhydrous THF (2 mL) was added TBAF (96 μL, 0.096 mmol, 1 M in THF). The reaction mixture was stirred for 1 h at rt and then quenched with a saturated solution of NH₄Cl (4 mL). The aqueous layer was extracted with ethyl acetate (3 × 4 mL) and the combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 10:1) to afford compound 25 (28 mg, 90%). ¹H NMR (400 MHz, MeOD) δ (ppm) 8.56 (d, J = 7.6 Hz, 1H), 7.98 (d, J = 6.8 Hz, 2H), 7.67 – 7.53 (m, 4H), 6.68 (d, J = 5.6 Hz, 1H), 4.28 (dd, J = 16.8, 7.6 Hz, 1H), 4.01 – 3.93 (m, 2H), 3.83 (dd, J = 12,0,2.4 Hz, 1H). ¹⁹F NMR (376 MHz, MeOD) δ −123.50 (d, J = 18.4 Hz). ¹³C NMR (100 MHz, MeOD): δ 169.1, 164., 157.7, 144.9, 133.2, 132.7, 128.4, 127.8, 112.3, 97.1, 89.5, 81.7, 72.5, 58.7, 58.1. MS (HR-ESI) for C₁₆H₁₅BrFN₃O₅ [(M+H)⁺]. Calcd: m/z 428.0257. Found: m/z 428.0249.

4.2.19. 4-Amino-1-((2R,3R,4R,5R)-3-chloro-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (26)

A solution of compound 24 (39 mg, 0.1 mmol) in a saturated solution of ammonia in MeOH (2 mL) was stirred at rt for 16 h The solution was evaporated to dryness under reduced pressure and co-evaporated several times with methanol. The residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 4:1) to afford compound 26 (20 mg, 71%). ¹H NMR (400 MHz, MeOD): δ 7.95 (dd, J = 7.6 Hz, J = 1.9 Hz, 1H), 6.45 (d, J = 8.2 Hz, 1H), 5.93 (d, J = 7.3 Hz, 1H), 4.36 (dd, J = 17.2 Hz, J = 7.2 Hz, 1H), 3.94 (m, 1H), 3.89 (m, 1H), 3.8 (m, 1H). ¹⁹F NMR (376 MHz, MeOD): −124.72 (dd, J = 16.7 Hz, J = 7.6 Hz). ¹³C NMR (100 MHz, MeOD): δ 167.6, 158.0, 143.7, 116.4 (d, J = 258 Hz), 96.2, 89.3, 83.1, 74.1, 60.7. MS (HR-ESI) for C₉H₁₂ClFN₃O₄ [(M+H)⁺]. Calcd: m/z 280.0500, Found: m/z 280.0492.

4.2.20. 4-Amino-1-((2R,3R,4R, 5R)-3-bromo-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(lH)-one (27)

A solution of compound 25 (32 mg, 0.073 mmol) in a saturated solution of ammonia in MeOH (2 mL) was stirred at rt for 16 h. The solution was evaporated to dryness under reduced pressure and co-evaporated several times with methanol. The residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 4:1) to afford compound 27 (23 mg, 96%). ¹H NMR (400 MHz, MeOD) δ 7.96 (dd, J = 7.6, 1.8 Hz, 1H), 6.60 (d, J = 8.2 Hz, 1H), 5.90 (d, J = 7.6 Hz, 1H), 4.20 (dd, J = 17.6, 7.2 Hz, 1H), 3.96 – 3.89 (m, 1H), 3.89 – 3.84 (m, 1H), 3.78 (dd, J = 12.4, 3.3 Hz, 1H). ¹⁹F NMR (376 MHz, MeOD) δ −123.93 (m). ¹³C NMR (100 MHz, MeOD): δ 165.6, 155.8, 144.7, 141.6, 132.2, 130.9, 128.5, 112.5, 109.8, 94.7, 93.3, 88.9, 88.8, 81.6, 729, 72.6, 59.1, 29.3 MS (HR-ESI) for C₉H₁₂BrFN₃O₄ [(M+H)⁺]. Calcd: m/z 323.9995, Found: m/z 323.9993.

4.2.21. 4-Amino-1-((2R,3R,4R,5R)-3-bromo-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-one (28)

Imidazole (18 mg, 0.26 mmol) was added to a mixture of compound 27 (26 mg, 0.086 mmol) and TBDMSCl (26 mg, 0.17 mmol) in DCM (4 mL). The mixture was stirred for 16h at rt, quenched with water and the crude mixture was extracted with ethyl acetate (5 mL). The organic layer was washed with a saturated aqueous solution of NH₄Cl, dried over Na₂SO₄, filtrated and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel using CH₂Cl₂/MeOH (10:1) to afford compound 28 (27 mg, 83%). ¹H NMR (CDCl₃, 400 MHz) δ (ppm) 7.75 (dd, J = 7.5, 1.0 Hz, 1H), 6.69 (d, J = 5.7 Hz, 1H), 5.72 (d, J = 7.5 Hz, 1H), 4.23 (dd, J = 16.8, 7.5 Hz, 1H), 3.97 (dt, J = 11.6, 2.6 Hz, 1H), 3.83 (dd, J = 7.5, 2.3 Hz, 1H), 3.78 (dd, J = 11.7, 2.2 Hz, 1H), 0.93 (s, 9H), 0.92 (s, 9H), 0.16 (s, 3H), 0.11 (s, 9H). ¹⁹F NMR (CDCl₃, 376 MHz) δ −121.70 (dd, J = 16.9, 4.6 Hz). ¹³C NMR (CDCl₃, 101 MHz) δ 165.98 (s), 155.80 (s), 141.02 (s), 112.36 (s), 109.65 (s), 95.12 (s), 89.34 (d, J = 19.0 Hz), 81.23 (s), 73.50 (d, J = 25.3 Hz), 60.34 (s), 26.14 (s), 25.84 (s), 18.60 (s), 18.25 (s), −4.12 (s), −4.71 (s), −5.19 (s), −5.26 (s). MS (HR-ESI) for C₂₁H₃₉BrFN₃O₄Si₂ [(M+H)⁺]. Calcd: m/z 552.1647. Found: m/z 552.1642.

4.2.22. Benzyl (1-((2R,3R,4R,5R)-3-bromo-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (29)

To a solution of compound 28 (27 mg, 0.049 mmol) and benzyl chloroformate (21 μL, 0.15 mmol) in DCM (5 mL) was added DMAP (36 mg, 0.29 mmol). The reaction was stirred at rt for 1.5 h, quenched with water (5 mL) and extracted with ethyl acetate (5 mL). The organic layer was washed with HCl IN, NaHCO₃, brine, dried over Na₂SO₄, filtrated and concentrated under reduced pressure. The crude product was purified by flash chromatography (hexane/ethyl acetate 1:1) to afford compound 29 (25 mg, 74%). ¹H NMR (CDCl₃, 400 MHz) δ (ppm) 8.18 (d, J = 7.6 Hz, 1H), 7.75 (s, 1H), 7.37 (brs, 5H), 7.22 (d, J = 7.6 Hz, 1H), 6.72 (d, J = 4.5 Hz, 1H), 5.22 (brs, 2H), 4.26 (dd, J = 16.6, 7.8 Hz, 1H), 4.02 (d, J = 11.8 Hz, 1H), 3.90 (d, J =7.7 Hz, 1H), 3.81 (dd, J = 11.8, 1.9 Hz, 1H), 0.96 (s, 9H), 0.93 (s, 9H), 0.16 (s, 3H), 0.14 (s, 3H), 0.13 (s, 3H), 0.11 (s, 3H). ¹⁹F NMR (CDCl₃, 376 MHz) δ −122.22 (d, J = 16.6 Hz). MS (HR-ESI) for C₂₉H₄₆BrFN₃O₆Si₂ [(M+H)⁺]. Calcd: m/z 686.2014. Found: m/z 686.2009.

4.2.23. Benzyl (1-((2R,3R,4R,5R)-3-bromo-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (30)

To a solution of compound 29 (25 mg, 0.036 mmol) in THF (4 mL) was added Et₃N.3HF (0.06 mL, 0.36 mmol). The reaction mixture was stirred for 36 h at rt then Et₃N (0.06 mL) was added to quench the reaction. After removal of the volatiles under reduced pressure, the crude product was purified using silica gel chromatography (CH₂Cl₂/MeOH 10:1) to afford compound 30 (11 mg, 67%).¹H NMR (MeOD, 400 MHz) δ (ppm) 8.44 (dd, J = 1.1 Hz, 1H), 7.50 – 7.26 (m, 6H), 6.64 (d, J = 6.2 Hz, 1H), (s, 2H), 4.25 (dd, J = 17.2, 7.6 Hz, 1H), 3.98 – 3.91 (m, 2H), (dd, J = 12.5, 2.9 Hz, 1H). ¹⁹F NMR (MeOD, 376 MHz) δ −124.35 (dd, J = 17.0, 4.8 Hz). ¹³C NMR (100 MHz, MeOD): δ 163.9, 156.1, 153.1, 144.5, 135.7, 128.2, 127.9, 112.2, 109.5, 95.5, 89.6, 89.4, 81.7 (d, J= 23.56 Hz), 72.4, 72.2, 67.3, 58.8. MS (HR-ESI) for C₁₇H₁₇BrFN₃O₆ [(M+H)⁺]. Calcd: m/z 458.0363. Found: m/z 458.0355.

4.2.24. Isopropyl ((((2R,3R,4R,5R)-5-(4-(((benzyloxy)carbonyl)amino)-2-oxopyrimidin-1(2H)-yl)-4-bromo-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate, diastereomeric mixture (31)

To a solution of compound 30 (35 mg, 0.076 mmol) in THF (3 mL) was added NMI (30 μL, 0.38 mmol) then (2S)-isopropyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate 21 (70 mg, 0.23 mmol) in THF (0.23 mL). The mixture was stirred at rt for 4 h, quenched with water (1 mL) and extracted with ethyl acetate (2 mL). The organic layer was washed with water twice, dried over Na₂SO₄, filtrated and concentrated under reduced pressure. The crude product was purified by flash chromatography (CH₂Cl₂/MeOH 20:1) to afford compound 31 (11 mg, 20%) as a diastereomeric mixture of R_P/S_P (~1:1).¹H NMR (MeOD, 400 MHz) δ (ppm) 8.00 (ddd, J = 43.8, 7.7, 1.8 Hz, 1H), 7.47 – 7.15 (m, 11H), 6.65 (dd, J = 9.0 Hz, 1H), 5.24 (s, 2H), 5.04–4.94 (m, 1H), 4.59 – 4.35 (m, 2H), 4.26 – 4.19 (m, 1H), 4.16 – 4.12 (m, 1H), 3.97 – 3.86 (m, 1H), 1.38 – 1.31 (m, 3H), 1.22 (dd, J = 6.2, Hz, 6H). ¹⁹F NMR (MeOD, 376 MHz) δ −124.22 – −124.67 (m). ³¹P NMR (MeOD, 162 MHz) δ 3.66 (d, J = 13.5 Hz). LR-MS: calculated for C₂₉H₃₄BrFN₄O₁₀P 727.12, found 726.11, 728.20,.

4.2.25. Isopropyl ((((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4-bromo-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate, diastereomeric mixture (32)

To a round bottom flask charged with compound 31 (23 mg, 0.032 mmol) in ethanol (2 mL) was added 1,4-cyclohexadiene (0.1 mL, 0.70 mmol) and Pd/C (10 mg, 0.01 mmol). After 2 h at 25 °C, the mixture was filtrated through a pad of celite and the filtrate was then concentrated under reduced pressure. Purification by flash column chromatography on silica gel (CH₂Cl₂/MeOH 10:1) gave nucleotide 32 in 55% yield (13 mg) as a diastereomeric mixture of R_p/S_p (~1:1). ¹H NMR (MeOD, 400 MHz) δ (ppm) 7.61 (ddd, J = 28.8, 7.6, 2.3 Hz, 1H), 7.40 – 7.34 (m, 2H), 7.27 – 7.20 (m, 3H), 6.61 (dd, J = 11.2, 8.4 Hz, 1H), (dd, J = 12.0, 7.6 Hz, 1H), 5.04 – 4.95 (m, 1H), 4.54 – 4.31 (m, 2H), 4.19 (ddd, J = 17.5, 6.5, 2.0 Hz, 1H), 4.10 (brs, 1H), 3.93 – 3.88 (m, 1H), 1.37 – 1.31 (m, 3H), 1.29 (brs, 2H), 1.25 – 1.20 (m, 4H). ¹⁹F NMR (MeOD, 376 MHz) δ −124.46 (m). ³¹P NMR (MeOD, 162 MHz) δ 3.60, 3.53. ¹³C NMR (100 MHz, MeOD): δ 171.5, 165.5, 155.1, 150.2, 143.8, 130.1, 121.3, 106.1, 98.1, 94.1, 78.4, 76.7, 69.5, 63.0, 46.9, 21.6, 19.1. MS (HR-ESI) for C₂₁H₂₈BrFN₄O₈P [(M+H)⁺]. Calcd: m/z 593.0812, Found: m/z 593.0813.

4.2.26. (2R,3R,4R,5R)-5-(4-Amino-2-oxopyrimidin-1(2H)-yl)-4-chloro-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl tert-butyl carbonate (33)

To a solution of compound 26 (109 mg, 0.39 mmol) and di-tert-butyl dicarbonate (85 mg, 0.39 mmol) in dioxane (8 mL) was added a solution of Na₂CO₃ (207 mg, 1.9 mmol) in water (2 mL). The reaction mixture was stirred at rt for 48 h and diluted with water (4 mL). The product was extracted with ethyl acetate (5 mL), washed with water, brine, dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 10:1) to afford compound 33 (44 mg, 30%). ¹H NMR (400 MHz, MeOD): δ 7.80 (dd, J = 7.6 Hz, J = 2.8 Hz, 1H), 6.45 (d, J = 6.4 Hz, 1H), 5.93 (d, J = 7.9 Hz, 1H), 5.31 (dd, J = 16.9 Hz, J = 6.2 Hz, 1H), 4.10 (m, 1H), 3.92 (m, 1H), 3.78 (m, 1H), 1.50 (s, 9H). ¹⁹F NMR (376 MHz, MeOD): −124.07 (t, J = 14.7 Hz). ¹³C NMR (100 MHz, MeOD): δ 167.6, 157.8, 153.2, 143.0, 113.8 (d, J = 257 Hz), 96.5, 89.1, 85.0, 81.3, 76.9, 60.8, MS (HR-ESI) for C₁₄H₂₀ClFN₃O₆ [(M+H)⁺]. Calcd: m/z 380.1025, Found: m/z 380.1017.

4.2.27. Isopropyl ((((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-((tert-butoxycarbonyl)oxy)-4-chloro-4-fluorotetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate diastereomeric mixture (34)

To a solution of compound 33 (44 mg, 0.11 mmol) in THF (3 mL) was added tert-butylmagnesium chloride (180 μL, 0.18 mmol, 1 M in THF) at 0 °C. The reaction mixture was stirred at 0 °C for 20 min and a solution of L-isopropylalaninyl phosphorochloridate 21 (105 mg, 0.34 mmol) in THF (0.34 mL) was added. The reaction mixture was stirred for 1 h at rt. The solvent was evaporated and the residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 10:1) to afford compound 34 as a diastereomeric mixture of R_P/S_P (~1:1) (67 mg, 89%). ¹H NMR (400 MHz, CDCl₃): 7.28-7.35 (m, 3H), 7.13-7.23 (m, 3H), 6.51-6.57 (m, 1H), 5.76 and 5.68 (each d, J = Hz, 1H), 5.17-5.22 (m, 1H), 4.97-5.00 (m, 1H), 4.29-4.45 (m, 2H), 4.21 (m, 1H), 3.91-3.99 (m, 2H), 1.51 (s, 9H), 1.36-1.41 (m, 3H), 1.16-1.23 (m, 6H). ¹⁹F NMR (376 MHz, CDCl₃): −125.8 (s), 126.1 (s). ³²P NMR (162 MHz, CDCl₃): 2.51, 2.38. ¹³C NMR (100 MHz, CDCl₃): δ 173.0, 165.7, 155.5, 151.7, 150.7, 142.2, 129.93, 129.88, 125.3, 120.3, 111.5 (d, J = 254 Hz), 95.5, 87.1, 84.7, 69.5, 64.5, 50.5, 27.7, 21.8, 21.0. MS (HR-ESI) for C₂₆H₃₆ClFN₄O₁₀P [(M+H)⁺]. Calcd: m/z 649.1842, Found: m/z 649.1837.

4.2.28. Isopropyl ((((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4-chloro-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy) (phenoxy)phosphoryl)-L-alaninate, diastereomeric mixture (35)

Compound 34 (67 mg, 0.1 mmol) was treated with a 4 M solution of HCl in dioxane for 2 h at rt. Solvents were removed under vacuum and the residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 10:1) to afford compound 35 as a diastereomeric mixture of R_p/S_p (~1:1) (28 mg, 50%). ¹H NMR (400 MHz, MeOD): δ 7.64 & 7.56 (each dd, J = 7.6 Hz, J = 2.3 Hz, 1H), 7.34-7.40 (m, 2H), 7.20-7.27 (m, 3H), 6.44-6.49 (m, 1H), 5.85 & 5.89 (each d, J = 7.6 Hz, 1H), 4.96-5.00 (m, 1H), 4.35-4.43 (m, 2H), 4.30 (m, 1H), 4.10 (m, 1H), 3.91 (m, 1H), 1.34 (m, 3H), 1.22 (m, 6H). ¹⁹F NMR (376 MHz, MeOD): −124.83 (s). ³¹P NMR (162 MHz, MeOD): 3.60, 3.54. ¹³C NMR (100 MHz, MeOD): δ 174.5, 167.6, 157.9, 152.1, 130.8, 126.3, 121.4, 115.8 (d, J = 257 Hz), 96.5, 89.1, 81.4, 74.9, 70.2, 65.9, 51.8, 21.9, 20.5. MS (HR-ESI) for C₂₁H₂₈ClFN₄O₈P [(M+H)⁺]. Calcd: m/z 549.1317, Found: m/z 549.1313.

4.2.29. 6-bis(tert-Butoxycarbonyl)amino-9-(3,5-di-O-tert-butyldimethylsilyl-2’-deoxy-2’-chloro-2’-fluoro-D-ribofuranosyl)purine (36)

To a solution of compound 11 (0.5 g, 1.2 mmol), 6-bis(tert-butoxycarbonyl)amino-9H-purine (0.2 g, 1.29 mmol), triphenylphosphine (0.47 g, 1.79 mmol) in THF (10 mL) was added DIAD (0.36 mL, 1.79 mmol) dropwise at 0 °C. The reaction mixture was heated at 70 °C for 1 h, and then diluted with ethyl acetate. The organic layer was washed with water and brine, dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/ethyl acetate 9:1) to afford 36a (236 mg, 27%) and 36b (59 mg, 7%). 36a: ¹H NMR (400 MHz, CDCl₃): δ 8.77 (s, 1H), 8.45 (s, 1H), 6.67 (d, J = 9.0 Hz, 1H), 4.77 (dd, J = 13.0 Hz, 4.8 Hz, 1H), 4.36 (m, 1H), 3.83 (m, 2H), 1.44 (s, 18H), 0.92 (two s, 18H), 0.10 (four s, 12H). ¹⁹F NMR (MHz, CDCl₃): −116.34 (t, J = 11.2 Hz, 1F). ¹³C NMR (100 MHz, CDCl₃): δ 152.6, 151.8, 151.34, 144.1, 131.5, 116.9 (d, J = 251 Hz), 87.6, 86.5, 74.9, 61.5, 26.0, 25.7, 18.5, −4.6, −4.8, −5.3, −5.4. MS (HR-ESI) for C₃₂H₅₆ClFN₅O₇Si₂ [(M+H)⁺]. Calcd: m/z 732.3391, Found: m/z 732.3394. 36b: ¹H NMR (400 MHz, CDCl₃): δ 8.89(s, 1H), 8.44 (s, 1H), 6.56 (d, J = 10.6 Hz, 1H), 4.63 (dd, J = 15.8 Hz, 5.5 Hz, 1H), 4.03 (m, 1H), (m, 1H), 3.87 (m, 1H), 1.44 (s, 18H), 0.95 (two s, 18H), 0.14 (four s, 12H). ¹⁹F NMR (MHz, CDCl₃): −123.24 (t, J = 12.5 Hz, 1F). ¹³C NMR (100 MHz, CDCl₃): δ 153.4, 152.7, 150.7, 143.0, 128.7, 114.1 (d, J = 262 Hz), 87.3, 83.9, 83,7, 74.1, 61.1, 27.9, 26.1, 25.4, 18.6, 18.2, −3.4, −4.5, −4.8, −5.2. MS (HR-ESI) for C₃₂H₅₆ClFN₅O₇Si₂ [(M+H)⁺]. Calcd: m/z 732.3391, Found: m/z 732.3390.

4.2.30. 6-bis(tert-Butoxycarbonyl)amino-9-(3,5-di-O-tert-butyldimethylsilyl-2’-deoxy-2’-bromo-2’-fluoro-D-ribofuranosyl)purine (37a and 37b)

To a solution of compound 12 (1.0 g, 2.17 mmol), 6-bis(tert-butoxycarbonyl)amino-9H-purine (0.80 g, 2.39 mmol) and triphenylphosphine (0.85 g, 0.33 mmol) in THF (25 mL) was added DIAD (0.64 mL, 0.33 mmol) dropwise at 0 °C. The reaction mixture was heated at 70 °C for 1 h, and then diluted with ethyl acetate, washed with water and brine, dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/ethyl acetate 9:1) to afford 37a (756 mg, 44%) and 37b (190 mg, 12%). 37a: ¹H NMR (400 MHz, CDCl₃): δ 8.87 (s, 1H), 8.41 (s, 1H), 6.65 (d, J = 9.6 Hz, 1H), 4.70 (dd, J = 13.5, 5.2 Hz, 1H), 4.46 – 4.26 (m, 1H), 3.91 – 3.73 (m, 2H), 1.41 (s, 18H), 0.93 (two s, 18H), 0.31 – 0.02 (m, 12H). ¹⁹F NMR (MHz, CDCl₃): −113.87 (t, J = 11.2 Hz, 1F). ¹³C NMR (101 MHz, CDCl₃): δ 153.1, 152.5, 150.6, 150.2, 143.2, 88.1, 87.7, 85.7, 83.8, 75.2, 75.0, 61.4, 31.6, 27.8, 25.9, 22.7, 18.4, 18.1. −4.5, −4.8, −5.3, −5.4. MS (HR-ESI) for C₃₂H₅₅BrFN₅O₇Si₂ [(M+H)⁺]. Calcd: m/z 776.2885, Found: m/z 776.2883. 37b: ¹H NMR (400 MHz, CDCl₃): δ 8.91 (s, 1H), (s, 1H), 6.74 (d, J = 9.2 Hz, 1H), 4.55 (dd, J = 13.5, 5.2 Hz, 1H), 4.07 – 4.04 (m, 1H), 4.02 – 3.88 (m, 2H), 1.45 (s, 18H), 0.97 (s, 18H), 0.20 – 0.15 (m, 12H). ¹⁹F NMR (MHz, CDCl₃): −122.96 (m, 1F). ¹³C NMR (101 MHz, CDCl₃) : δ 153.2, 152.5, 150.2, 143.9, 142.8, 128.5, 111.5, 108.6, 88.3, 88.1, 83.8, 83,2, 74.1, 73.8, 60.9, 27.7, 26.0, 25.6, 18.5, 18.0, −4.4, −4.8, −5.3, −5.4. MS (HR-ESI) for C₃₂H₅₅BrFN₅O₇Si₂ [(M+H)⁺]. Calcd: m/z 776.2885, Found: m/z 776.2883.

4.2.31. 6-bis(tert-Butoxycarbonyl)amino-9-(2’-deoxy-2’-chloro-2’-fluoro-β-D-ribofuranosyl)purine (38)

To a solution of compound 36 (103 mg, 0.14 mmol) in anhydrous THF (5 mL) was added TBAF (350 μL, 0.35 mmol, 1 M in THF). The reaction mixture was stirred for 1 h at rt and quenched with a saturated solution of NH₄Cl (5 mL). The aqueous layer was extracted with ethyl acetate and the combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 10:1) to afford compound 38 (70 mg, 99%). ¹H NMR (400 MHz, CDCl₃): δ 8.87 (s, 1H), 8.62 (s, 1H), 6.54 (d, J = 8.7 Hz, 1H), 5.19 (br s, 1H), 4.73 (dd, J = 15.4 Hz, 6.0 Hz, 1H), 4.37 (br s, 1H), 4.13 (m, 1H), 4.03 (m, 1H), 3.93 (m, 1H), 1.38 (s, 18H). ¹⁹F NMR (MHz, CDCl₃): −123.52 (dd, J = 14.7 Hz, J = 8.8 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 153.2, 152.7, 150.4, 144.0, 128.8, 114.3 (d, J = 253.6 Hz), 88.3, 84.7, 83.4, 73.0, 72.7, 60.4, 27.8. MS (HR-ESI) for C₂₀H₂₈ClFN₅O₇ [(M+H)⁺]. Calcd: m/z 504.1661, Found: m/z 504.1658.

4.2.32. 6-bis(tert-Butoxycarbonyl)amino-9-(2’-deoxy-2’-bromo-2’-fluoro-β-D-ribofuranosyl)purine (39)

To a solution of compound 37 (190 mg, 0.14 mmol) in anhydrous THF (10 mL) was added TBAF (610 μL, 0.61 mmol, 1 M in THF). The reaction mixture was stirred for 1 h at rt and quenched with a saturated solution of NH₄Cl (10 mL). The aqueous layer was extracted with ethyl acetate (3 × 10 mL) and the combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 10:1) to afford compound 39 (93 mg, 70%). ¹H NMR (400 MHz, CDCl₃): δ 8.89 (s, 1H), 8.49 (d, J = 1.6 Hz, 1H), 6.69 (d, J = 9.5 Hz, 1H), 4.71 (dd, J = 15.7, 5.9 Hz, 1H), 4.19 (dt, J = 6.0, 3.0 Hz, 1H), 4.09 (d, J = 12.7 Hz, 1H), 3.98 (d, J = 12.4 Hz, 2H), 1.43 (s, 18H). ¹⁹F NMR (MHz, CDCl₃): −121.02 (dd, J = 14.7 Hz, J = 8.8 Hz). ¹³C NMR (101 MHz, CDCl₃) δ 153.1, 152.5, 150.7, 150.3, 128.9, 111.8, 89.7, 89.5, 84.2, 83.1, 83.1, 73.5, 73.3, 60.6, MS (HR-ESI) for C₂₀H₂₈BrFN₅O₇ [(M+H)⁺]. Calcd: m/z 548.1156, Found: m/z 548.1132.

4.2.33. (2R, 3R, 4R, 5R)-5-(6-Amino-9H-purin-9-yl)-4-chloro-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol (40)

Compound 38 (0.06 g, 0.12 mmol) was dissolved in a 4 M solution of HCl in dioxane (2 mL). The reaction mixture was stirred for 1 h at rt. Volatiles were removed under vacuum and the residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 4:1) to afford compound 40 (21 mg, 61%). ¹H NMR (400 MHz, MeOD): δ 8.41 (s, 1H), 8.23 (s, 1H), (d, J = 11.2 Hz, 1H), 4.57 (dd, J = 16.7 Hz, J = 6.1 Hz, 1H), (m, 1H), 3.98 (m, 1H), 3.88 (m, 1H). ¹⁹F NMR (MHz, MeOD): −123.99 (t, J = 14.2 Hz). ¹³C NMR (100 MHz, MeOD): δ 157.0, 154.2, 150.3, 141.2, 119.32 115.2 (d, J = 258 Hz), 88.5, 74.3, 61.2. MS (HR-ESI) for C₁₀H₁₂ClFN₅O₃ [(M+H)⁺]. Calcd: m/z 304.0613, Found: m/z 304.0605.

4.2.34. (2R, 3R, 4R, 5R)-5-(6-Amino-9H-purin-9-yl)-4-bromo-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol (41)

Compound 39 (0.014 g, 0.026 mmol) was dissolved in a 4 M solution of HCl in dioxane (1 mL). The reaction mixture was stirred for 1 h at rt. Volatiles were removed under vacuum and the residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 4:1) to afford compound 41 (5.2 mg, 60%). ¹H NMR (400 MHz, MeOD) δ 8.34 (s, 1H), 8.13 (s, 1H), 6.51 (d, J = 10.5 Hz, 1H), 4.40 (dd, J = 16.7, 6.1 Hz, 1H), 3.94 (m, 1H), 3.86-3.73 (m, 1H), 3.63-3.54 (d, J = 4.8 Hz, 1H). ¹⁹F NMR (MHz, MeOD): −122.50 (dd, J = 12.12 Hz, J = 12.0 Hz). ¹³C NMR (101 MHz, MeOD) δ 156.0, 152.8, 149.4, 139.7, 139.7, 112.0, 88.3, 88.1, 83.1, 83.1, 73.4, 73.1, 59.9. MS (HR-ESI) for C₁₀H₁₂BrFN₅O₃ [(M+H)⁺]. Calcd: m/z 348.0107, Found: m/z 348.0107.

4.2.35. Isopropyl ((((2R,3R,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-chloro-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy) (phenoxy)phosphoryl)-L-alaninate, diastereomeric mixture (42)

To a solution of compound 38 (50 mg, 0.099 mmol) in THF (2 mL) was added tert-butylmagnesium chloride (248 μL, 0.248 mmol, 1 M in THF) The reaction mixture was stirred for 20 minutes at 0 °C before addition of L-isopropylalaninyl phosphorochloridate 21 (90 mg, 0.29 mmol) in THF (0.29 mL). The reaction mixture was stirred 1 h at rt. The volatiles were evaporated and the residue was directly treated, without further purification, with a 4 M solution of HCl in dioxane (2 mL). The reaction mixture was stirred 1 h at rt. Solvents were removed under vacuum and the residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 4:1) to afford compound 42 as a diastereomeric mixture of R_p/S_p (~1:1) (16 mg, 29% over two steps). ¹H NMR (400 MHz, CDCl₃): δ 8.37 (s, 1H), 8.07 & 7.98 (each s, 1H), 7.28-7.34 (m, 2H), 7.1-7.23 (m, 3H), 6.47 (dd, J = 12.4 Hz, J = 2.8 Hz, 1H), 6.04 (d, J = 11.6 Hz, 2H), 5.59 (br s, 1H), 4.94-5.01 (m, 1H), 4.55-4.68 (m, 1H), 4.38-4.48 (m, 2H), 4.09-4.28 (m, 2H), 3.92-4.04 (m, 1H), 1.33-1.38 (two s, 3H), 1.16-1.22 (m, 6H). ¹⁹F NMR (MHz, CDCl₃): −123.44 (dd, J = 14.7 Hz), −124.17 (t, J = 14.7 Hz). ³¹P NMR (MHz, CDCl₃): 3.01, 2.88. ¹³C NMR (100 MHz, CDCl₃): δ 173.2, 153,7, 150.6, 150.3, 139.4, 129.9, 125.4, 120.3, 119.1, 113.5 (d, J = 256 Hz), 86.8, 81.1, 74.1, 69.7, 64.9, 60.6, 50.6, 21.7, 20.9. MS (HR-ESI) for C₂₂H₂₈ClFN₆O₇P [(M+H)⁺]. Calcd: m/z 573.1430, Found: m/z 573.1424.

4.2.36. Isopropyl ((((2R,3R,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-bromo-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate, diastereomeric mixture (43)

To a solution of compound 39 (70 mg, 0.128 mmol) in THF (2 mL) tert-butylmagnesium chloride (319 μL, 0.319 mmol, 1 M in THF) was added and the reaction mixture was stirred for 20 minutes at 0 °C. A solution of the phosphorochloridate 21 (46 mg, 0.153 mmol) in THF (0.153 mL) was then added and the reaction mixture was stirred for 1 h at rt. The volatiles were removed under vacuum and the residue was directly treated, without further purification, with a 4 M solution of HCl in dioxane (2 mL). The reaction mixture was stirred 1 h at rt. Solvents were removed under vacuum and the residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 4:1) to afford compound 43 as a diastereomeric mixture of R_p/S_p (~1:1) (16 mg, 20% over two steps). ¹H NMR (400 MHz, CDCl₃) δ 8.38 (d, J = 5.1 Hz, 1H), 8.05 (d, J = 2.8 Hz, 1H), 7.99 – 7.91 (m, 1H), 7.38 – 7.28 (m, 2H), 7.25 – 7.14 (m, 3H), 6.67 – 6.59 (m, 1H), 5.85 (s, 2H), 5.00 (q, J = 7.2, 6.7 Hz, 2H), 4.57 (d, J = 4.7 Hz, 1H), 4.48 – 4.39 (m, 3H), 4.25 (d, J = 5.0 Hz, 1H), 4.09 – 3.93 (m, 3H), 3.79 – 3.71 (m, 1H), 1.37 (dd, J =6.9, 2.6 Hz, 4H), 1.30 – 1.15 (m, 14H). ¹⁹F NMR (MHz, CDCl₃): −121.51 (t, J = 14.7 Hz), −122.53 (t, J = 14.7 Hz). ³¹P NMR (MHz, CDCl₃): 3.42, 3.07. ¹³C NMR (101 MHz, CDCl₃) δ 173.0, 155.4, 153.5, 150.5, 150.2, 139.4, 129.8, 129.7, 125.4, 125.3, 120.1, 120.1, 87.6, 69.6, 50.5, 29.7, 21.7, 21.6, 20.9, 20.8. MS (HR-ESI) for C₂₂H₂₈BrFN₆O₇P [(M+H)⁺]. Calcd: m/z 617.0924, Found: m/z 617.0925.

4.2.37. 2-bis(tert-Butoxycarbonyl)amino-6-chloro-9-(3,5-di-O-tert-butyldimethylsilyl-2’-deoxy-2’-chloro-2’-fluoro-D-ribofuranosyl)purine (44a and 44b)

To a solution of compound 11 (3.0 g, 7.22 mmol), 2-bis(tert-butoxycarbonyl)amino-6-chloro-9H-purine (2.94 g, 7.95 mmol) and triphenylphosphine (2.84 g, 10.83 mmol) in THF (50 mL) was added DIAD (2.13 mL, 10.83 mmol) dropwise at 0 °C. The reaction mixture was heated at 70 °C for 1 h, and then diluted with ethyl acetate (50 mL). The organic layer was washed with water, and brine, dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/ethyl acetate 9:1) to afford 44a (3.0 g, 54%) and 44b (0.67 g, 12%). 44a: ¹H NMR (400 MHz, CDCl₃): δ 8.45 (s, 1H), 6.60 (d, J = 9.0 Hz, 1H), 4.74(dd, J = 12.9 Hz, J = 4.4 Hz, 1H), 4.37 (m, 2H), 3.82 (m, 1H), 1.41 (s, 18H), 0.93 (two s, 18H), 0.15 (four s, 12H). ¹⁹F NMR (MHz, CDCl₃): −115.58 (t, J = 10.9 Hz, 1F). ¹³C NMR (100 MHz, CDCl₃): δ 152.6, 151.4, 150.4, 144.8, 129.8, 117.1 (d, J = 251 Hz), 87.8, 85.6, 83.8, 75.0, 61.5, 28.0, 26.0, 25.7, 18.5, 18.2, −4.5, −4.9, −5.28, −5.31. MS (HR-ESI) for C₃₂H₅₅Cl₂FN₅O₇Si₂ [(M+H)⁺]. Calcd: m/z 766.3001, Found: m/z 766.2998. 44b: ¹H NMR (400 MHz, CDCl₃): δ 8.52 (s, 1H), 6.49 (d, J = 9.0 Hz, 1H), 4.60 (dd, J = 13.0 Hz, 4.8 Hz, 1H), 4.00 (m, 2H), 3.88 (m, 1H), 1.41 (s, 18H), 0.99 (two s, 18H), 0.15 (s, 12H). ¹⁹F NMR (MHz, CDCl₃): −123.58 (t, J = 11.8 Hz, 1F). ¹³C NMR (100 MHz, CDCl₃): δ 152.7, 151.5, 150.4, 144.3, 129.9, 113.9 (d, J = 258 Hz), 87.4, 83.8, 73.6, 61.0, 27.9, 26.1, 25.7, 18.8, 18.1, −4.5, −4.9, −5.28, −5.31. MS (HR-ESI) for C₃₂H₅₅Cl₂FN₅O₇Si₂ [(M+H)⁺]. Calcd: m/z 766.3001, Found: m/z 766.3001.

4.2.38. 2-bis(tert-Butoxycarbonyl)amino-6-chloro-9-(3,5-di-O-tert-butyldimethylsilyl-2’-deoxy-2’-bromo-2’-fluoro-D-ribofuranosyl)purine (45a and 45b)

To a solution of compound 12 (2.60 g, 5.66 mmol), 2-bis(tert-butoxycarbonyl)amino-6-chloro-9H-purine (2.29 g, 6.23 mmol), triphenylphosphine (2.19 g, 8.49 mmol) in THF (50 mL), DIAD (1.67 mL, 8.49 mmol) was added dropwise at 0 °C. The reaction mixture was heated at 70 °C for 1 h, and then diluted with ethyl acetate (50 mL). The organic layer was washed with water, and brine, dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexanes/ethyl acetate 9:1) to afford 45a (0.95 g, 20%) and 45b (0.26 g, 6%). 45a: ¹H NMR (400 MHz, CDCl₃) δ 8.48 (s, 1H), 6.58 (d, J = 9.7 Hz, 1H), 4.69 (dd, J = 13.5, 5.1 Hz, 1H), 4.41 – 4.31 (m, 1H), 3.83 (m, 2H), 1.43 (s, 18H), 0.94 (d, J = 11.1 Hz, 18H), 0.21-0.10 (m, 12H). ¹⁹F NMR (MHz, CDCl₃): −113.37 (t, J = 10.9 Hz, 1F). ¹³C NMR (101 MHz, CDCl₃) δ 150.25, 144.56, 129.80, 88.34, 87.97, 85.84, 83.66, 61.28, 60.40, 28.22, 27.83, 25.99, 25.86, 25.65, 25.60, 21.05, 18.34, 18.08, 14.20, −4.60, −4.87, −5.40, −5.44. [(M+H)⁺] for C₃₂H₅₄BrClFN₅0₇Si₂. Calcd: m/z 810.2496, Found: m/z 810.2495. 45b: ¹H NMR (400 MHz, CDCl₃): δ 8.55 (s, 1H), 6.65 (d, J = 8.4 Hz, 1H), 4.49 (dd, J = 15.3, 5.7 Hz, 1H), 4.06-3.89 (m, 1H), 3.86 (m, 1H), 1.41 (s, 18H), 0.95 (d, J = 8.6 Hz, 18H), 0.15 (m, 12H). ¹⁹F NMR (MHz, CDCl₃): −121.88 (t, J = 11.8 Hz, 1F). ¹³C NMR (101 MHz, CDCl₃) δ 152.6, 150.2, 144.9, 88.4, 83.7, 83.3, 73.8, 73.5, 60.8, 30.9, 27.8, 26.0, 25.6, 21.0, 18.5, 18.0, 14.2, −4.4, −4.7, −5.2, −5.3. [(M+H)⁺] for C₃₂H₅₄BrClFN₅0₇Si₂. Calcd: m/z 810.2496, Found: m/z 810.2495.

4.2.39. 2-bis(tert-Butoxycarbonyl)amino-6-chloro-9-(2’-deoxy-2’-chloro-2’-fluoro-β-D-ribofuranosyl)purine (46)

To a solution of compound 44 (0.3 g, 0.39 mmol) in anhydrous THF (10 mL) was added TBAF (0.97 mL, 0.98 mmol, 1 M in THF). The reaction mixture was stirred for 1 h at rt and quenched with a saturated solution of NH₄Cl (10 mL). The aqueous layer was extracted with ethyl acetate (10 mL) and the combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 10:1) to afford compound 46 (188 mg, 89%). ¹H NMR (400 MHz, CDCl₃): δ 8.74 (s, 1H), 6.49 (d, J = 7.2 Hz, 1H), 4.80 (dd, J = 14.8 Hz, J = 6.03 Hz, 1H), 4.68 (br s, 1H), 4.14 (m, 1H), 4.07 (m, 1H), 3.97 (m, 1H), 3.86 (br s, 1H), 1.39 (s, 18H). ¹⁹F NMR (MHz, CDCl₃): −123.64 (dd, J = 13.88 Hz, J = 6.4 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 152.5, 152.3, 151.7, 150.5, 145.1, 130.0, 114.2 (d, J = 260 Hz), 88.5, 84.4, 83.2, 72.5, 60.4, 27.9. MS (HR-ESI) for C₂₀H₂₆Cl₂FN₅NaO₇ [(M+Na)⁺]. Calcd: m/z 560.1091, Found: m/z 560.1084.

4.2.40. 2-bis(tert-Butoxycarbonyl)amino-6-chloro-9-(2’-deoxy-2’-bromo-2’-fluoro-β-D-ribofuranosyl)purine (47)

To a solution of compound 45 (0.24 g, 0.296 mmol) in anhydrous THF (10 mL) was added TBAF (0.74 mL, 0.741 mmol, 1 M in THF). The reaction mixture was stirred for 1 h at rt and quenched with a saturated solution of NH₄Cl (10 mL). The aqueous layer was extracted with ethyl acetate (3×10 mL) and the combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 10:1) to afford compound 47 (100 mg, 60%). ¹H NMR (400 MHz, CDCl₃) δ 8.74 (s, 1H), 6.66 (d, J = 7.5 Hz, 1H), 4.70 (dd, J = 15.7, 6.6 Hz, 1H), 4.54 (br s, 1H), 4.14 (m, 1H), 4.09 (m, 1H), 3.99 (m, 1H), 1.40 (s, 18H). ¹⁹F NMR (MHz, CDCl₃): −121.55 (dd, J = 13.88 Hz, J = 6.4 Hz). ¹³C NMR (101 MHz, CDCl₃) δ 152.4, 152.1, 151.6, 150.3, 144.9, 129.9, 111.5, 108.8, 89.6, 89.4, 84.3, 82.8, 82.8, 72.8, 72.5, 60.2, 53.0, 27.8. MS (HR-ESI) for C₂₀H₂₆BrClFN₅O₇ [(M+H)⁺]. Calcd: m/z 582.0766. Found: m/z 582.0761.

4.2.41. 2-Amino-9-((2R,3R,4R,5R)-3-chloro-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one (48)

Compound 46 (40 mg, 0.074 mmol) was dissolved in a mixture of formic acid/H₂O (4:1) and stirred at 60 °C for 16 h. Volatiles were removed under reduced pressure and the residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 8:2) to afford compound 48 (12.8 mg, 54%). ¹H NMR (400 MHz, CDCl₃): δ 8.04 (s, 1H), 6.27 (d, J = 10.8 Hz, 1H), 4.53 (dd, J = 16.5 Hz, J = 6.04 Hz, 1H), 3.96 (m, 1H), 3.91 (m, 1H), 3.81 (m, 1H). ¹⁹F NMR (MHz, CDCl₃): −124.85 (t, J = 12.9 Hz, 1F). ¹³C NMR (100 MHz, CDCl₃): δ 159.3, 155.8, 153.4, 137.8, 117.04, 115.82 (d, J = 258 Hz), 88.1, 84.3, 74.3, 61.3. MS (HR-ESI) for C₁₀H₁₂ClFN₅O₄ [(M+H)⁺]. Calcd: m/z 320.0562, Found: m/z 320.0554.

4.2.42. 2-amino-9-((2R,3R,4R,5R)-3-bromo-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one (49)

Compound 47 (50 mg, 0.086 mmol) was dissolved in a mixture of formic acid/H₂O (4:1) (2 mL) and stirred at 60 °C for 16 h. Volatiles were removed under reduced pressure and the residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 8:2) to afford compound 49 (15.6 mg, 50%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.73 (br s, 1H), 7.94 (s, 1H), 6.72 (d, J = 5.6 Hz, 1H), 6.62 (br s, 1H), 6.27 (d, J = 9.2 Hz, 1H), 5.26 (t, J = 5.5 Hz, 1H), 4.34 (dd, J = 16.2, 5.6 Hz, 1H), 3.83 (m, 1H), 3.73 – 3.58 (m, 2H). ¹⁹F NMR (MHz, DMSO-d₆): −121.02 (t, J = 12.9 Hz, 1F). ¹³C NMR (101 MHz, DMSO-d₆) δ 157.0, 154.6, 151.8, 135.6, 116.4, 87.2, 87.0, 59.9. MS (HR-ESI) for C₁₀H₁₂BrFN₅O₄ [(M+H)⁺]. Calcd: m/z 364.0056, Found: m/z 364.0054.

4.2.43. Isopropyl ((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-chloro-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate, diastereomeric mixture (50)

To a solution of compound 46 (50 mg, 0.093 mmol) in THF (2 mL) tert-butylmagnesium chloride (232 μL, 0.232 mmol, 1 M in THF) was added and the reaction mixture was stirred for 20 minutes at 0 °C. Then L-isopropylalaninyl phosphorochloridate 21 (42 mg, 0.139 mmol) 1M in THF (0.139 mL) was added and the reaction mixture was stirred 1 h at rt. The solvent was evaporated and the residue was directly treated, without further purification, with a mixture of formic acid/H₂O (4:1) (2 mL). The reaction mixture was stirred 4 h at 60 °C. Solvents were removed under vacuum and the residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 4:1) to afford compound 50 as a diastereomeric mixture R_p/S_p (~1:1) (12.9 mg, 24%). ¹H NMR (400 MHz, CDCl₃): δ 7.84 & 7.80 (each s, 1H), 7.35-7.31 (m, 2H), 7.25-7.17 (m, 3H), 6.29 & 6.26 (m, 1H), 4.98-4.93 (m, 2H), 4.60-4.50 (m, 1H), 4.48-4.39 (m, 2H), 4.20-4.13 (m, 1H), 3.93-3.86 (m, 1H), 1.32 (m, 3H), 1.22- 1.18 (m, 6H). ¹⁹F NMR (MHz, CDCl₃): −124.55 (t, J = 12.9 Hz, 1F). ³¹P NMR (MHz, CDCl₃): 3.66, 3.59. ¹³C NMR (100 MHz, CDCl₃): δ 174.5, 159.31, 155.8, 153.4, 152.1, 137.8, 130.8, 126.2, 121.4, 117.1, 115.3 (d, J = 255 Hz), 88.0, 82.4, 74.9, 70.2, 66.3, 51.7, 21.9, 20.4. MS (HR-ESI) for C₂₂H₂₈ClFN₆O₈P [(M+H)⁺]. Calcd: m/z 589.1379, Found: m/z 589.1372.

4.2.44. Isopropyl ((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-bromo-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate, diastereomeric mixture (51)

To a solution of compound 47 (50 mg, 0.086 mmol) in THF (2 mL) tert-butylmagnesium chloride (215 μL, 0.215 mmol, 1 M in THF) was added and the reaction mixture was stirred for 20 minutes at 0 °C. Then (S)-2-[(S)-(2,3,4,5,6-pentafluorophenoxy)-phenoxyphosphorylamino] propionic acid isopropyl ester, 52 (58 mg, 0.120 mmol) 1M in THF (0.120 mL) was added and the reaction mixture was stirred 2 h at rt. The solvent was evaporated and the residue was directly treated, without further purification, with a mixture of formic acid/H₂O (4:1) (2 mL). The reaction mixture was stirred 4 h at 60 °C. Solvents were removed under vacuum and the residue was purified by flash column chromatography on silica gel (CH₂Cl₂/MeOH 4:1) to afford compound 51 as a S_p isomer (11.9 mg, 22%). ¹H NMR (400 MHz, MeOD δ 7.72 (dd, J = 12.3, 2.9 Hz, 1H), 7.35-7.27 (m, 2H), 7.19 – 7.03 (m, 3H), 6.32 (m 1H), 4.73 – 4.25 (m, 4H), 4.08 – 4.03 (m, 1H), 3.82 – 3.77 (m, 1H), 3.70 – 3.50 (m, 1H), 1.35 – 1.03 (m, 10H). ¹⁹F NMR (MHz, MeOD): −122.80 (d, J = 14.6 Hz, 1F). ³¹P NMR (MHz, MeOD): 3.84. ¹³C NMR (100 MHz, MeOD): δ 173.2, 157.8, 154.3, 152.0, 150.8, 136.4, 129.4, 124.8, 120.0, 115.7, 111.3, 87.6, 80.9, 74.0, 68.7, 65.4, 510.3, 20.5, 19.0. MS (HR-ESI) for C₂₂H₂₈BrFN₆O₈P [(M+H)⁺]. Calcd: m/z 633.0873, Found: m/z 633.0874.