Design, Synthesis, and Anti-RNA Virus Activity of 6′-Fluorinated-Aristeromycin Analogues

Abstract

The 6′-fluorinated aristeromycins were designed as dual-target antiviral compounds aimed at inhibiting both the viral RNA-dependent RNA polymerase (RdRp) and the host cell S-adenosyl-l-homocysteine (SAH) hydrolase, which would indirectly target capping of viral RNA. The introduction of a fluorine at the 6′-position enhanced the inhibition of SAH hydrolase and the activity against RNA viruses. The adenosine and N⁶-methyladenosine analogues 2a–e showed potent inhibition against SAH hydrolase, while only the adenosine derivatives 2a–c exhibited potent antiviral activity against all tested RNA viruses such as Middle East respiratory syndrome-coronavirus (MERS-CoV), severe acute respiratory syndrome-coronavirus, chikungunya virus, and/or Zika virus. 6′,6′-Difluoroaristeromycin (2c) showed the strongest antiviral effect for MERS-CoV, with a ∼2.5 log reduction in infectious progeny titer in viral load reduction assay. The phosphoramidate prodrug 3a also demonstrated potent broad-spectrum antiviral activity, possibly by inhibiting the viral RdRp. This study shows that 6′-fluorinated aristeromycins can serve as starting points for the development of broad-spectrum antiviral agents that target RNA viruses.

Introduction

Over the past 15 years, outbreaks of a number of emerging positive-stranded RNA (+RNA) viruses,¹ such as the severe acute respiratory syndrome coronavirus (SARS-CoV),² Middle East respiratory syndrome coronavirus (MERS-CoV),³ chikungunya virus (CHIKV),⁴ and Zika virus (ZIKV)⁵ have seriously threatened human health and have had a substantial socio-economic impact. SARS-CoV and MERS-CoV cause serious respiratory diseases⁶ that can be fatal in approximately 10 and 35% of cases, respectively. CHIKV is transmitted by mosquitoes and causes a painful arthritis that can persist for months.⁷ ZIKV is also transmitted by mosquitoes,⁸ although sexual transmission⁸ occurs as well. This virus usually causes mild disease, but can cause neurological complications in adults and fetal death or severe complications, including microcephaly in infants when women are infected during pregnancy.⁹ CHIKV and ZIKV have caused massive outbreaks, totaling millions of infections over the past decade. Currently, there are no effective chemotherapeutic agents or vaccines that can prevent or cure infections of any of these four serious pathogens.

The aforementioned viruses belong to the +RNA virus group (Baltimore class IV),¹ which indicates that their genomic RNA has the same polarity as mRNA and can be directly translated by host ribosomes upon release into the cytoplasm of a host cell. After infection, the genomes of these viruses are translated into polyproteins that are subsequently cleaved into individual proteins by viral and/or host proteases. The nonstructural proteins (nsps) of these viruses harbor a variety of enzymatic activities that are required for the replication of the viral RNA and invariably include a RNA-dependent RNA polymerase (RdRp),¹⁰ an enzyme which is not present in uninfected cells. The RdRp transcribes the genomic RNA into a complementary negative-stranded RNA that subsequently serves as the template for the synthesis of new positive-stranded RNA.

Many +RNA viruses (including coronaviruses, CHIKV, and ZIKV) also encode methyltransferases (MTases)¹¹ that are required for methylations of viral mRNA cap structures.¹² Because this capping is crucial for stability and translation of the viral RNA, and evasion of the host innate immune response, the viral MTases are considered promising targets for the development of antiviral therapy.¹² Inhibition of MTases can be indirectly achieved by the inhibition of S-adenosyl-l-homocysteine (SAH) hydrolase.¹³ The SAH hydrolase catalyzes the interconversion of SAH into adenosine and l-homocysteine. Inhibition of this enzyme leads to the accumulation of SAH in the cell, which in turn inhibits S-adenosyl-l-methionine (SAM)-dependent transmethylase reactions by feedback inhibition.^13,14 Most of the viral MTases are dependent on SAM as the only methyl donor. Compounds that target cellular proteins might exhibit a broader spectrum of activity, are less likely to lead to drug-resistance, but have a higher likelihood of toxicity. Compounds that are specifically aimed at viral proteins are expected to be less cytotoxic, but might have a narrower spectrum of antiviral activity and might have a lower barrier antiviral drug-resistance¹⁴ Thus, the approach of targeting cellular proteins such as SAH hydrolase can be considered as a promising strategy for the development of broad-spectrum antiviral agents.¹⁴ A number of compounds have been reported to act as SAH hydrolase inhibitors.¹⁴ Type I inhibitors act through inactivation of the NAD⁺ cofactor, and their inhibitory effect on the catalytic activity of the enzyme can be reversed by the addition of excess NAD⁺.¹⁴ Type II inhibitors are irreversible inhibitors of the SAH hydrolase that form covalent bonds with amino acid residues in the active site of the enzyme. This irreversible inhibition cannot be reversed by the addition of NAD⁺ or adenosine or by dialysis.¹⁴

Because both the viral RdRp and host SAH hydrolase are critical for virus replication, we aimed to design broad-spectrum nucleoside analogue inhibitors that could directly target RdRp activity and/or indirectly inhibit the methylation of viral RNA through their effect on the host SAH hydrolase. Modified nucleosides are usually taken up by the cell via nucleoside transporters and can be successively converted into mono-, di-, and triphosphates by cellular kinases.¹⁵ Then, these modified nucleoside triphosphates (NTPs) can compete with natural NTPs during RNA synthesis or can be incorporated into the nascent viral RNA, leading to chain termination or detrimental mutations.¹⁵

(−)-Aristeromycin (1) is a naturally occurring carbocyclic nucleoside that was originally identified as a metabolite of Streptomyces citricolor in 1967.^16a The first synthesis of 1 as racemate was reported by Clayton and his co-worker,^16b−16d and its asymmetric syntheses have since been reported.^16e−16h It is a type I SAH hydrolase inhibitor and exhibits potent antiviral activity against many viruses.^14a However, it could not be further advanced into clinical development because of its cytotoxicity.¹⁷ Compound 1 was found to be toxic at low concentrations in both adenosine kinase-positive (AK⁺) and AK^– cells. AK⁺ cells were presumably killed by the 5′-phosphorylated form of 1, while the toxicity in AK^– cells was caused by 1 itself.¹⁷ However, this compound is also metabolized into a triphosphate form and has been observed to exert a variety of metabolic effects.¹⁷ We aimed to use 1 as a prototype for the design of dual-target compounds intended at directly inhibiting the viral RdRp and indirectly inhibiting the capping process through targeting of cellular SAH hydrolase.

Since the introduction of a fluorine at the 6′-position of carbocyclic nucleosides has been known to affect biological activities to a significant extent,¹⁸ we aimed to synthesize the 6′-fluorinated-aristeromycin analogues 2 by introducing fluorine at the 6′-position of 1 (Figure 1). Prisbe and his co-workers^18a have reported the synthesis of (±)-6′-α- and (±)-6′-β-fluorinated aristeromycins and their inhibitory activity on SAH hydrolase, but the synthesis and biological activity of (±)-6′,6′-difluoroaristeromycin was not reported, despite the fact that the structure was claimed in the patent.^18b Thus, we set out to synthesize the 6′-fluorinated-aristeromycin analogues 2 in the optically pure d-forms because biological activity can generally be attributed to one enantiomer, the d-isomer. Yin and co-workers^18c reported the elegant synthesis of optically pure (−)-6′-β-fluoro-aristeromycin, but its biological activity was not reported. Their synthetic route involved the 6-β-fluoroazide as the key intermediate, which was synthesized by employing S_N2 fluorination of the 6-α-triflic azide with tris(dimethylamino)sulfur(trimethylsilyl)difluoride, whereas our current approach¹⁹ included the stereoselective electrophilic fluorination of silyl enol ether with Selectfluor as the fluorine source. In addition to the adenosine analogues, aimed at inhibiting SAH hydrolase and/or RdRp, we have also synthesized 6′-fluorinated purine and pyrimidine nucleosides (changes in B of the structures in Figure 1), which could interfere with viral RNA synthesis by targeting the viral RdRp after their phosphorylation by cellular kinases.¹⁵ To bypass the first and rate-limiting 5′-monophosphorylation step, we have also synthesized a phosphoramidate prodrug 3 of nucleoside 2, using the McGuigan ProTides.²⁰ Herein, we report the synthesis of the 6′-fluoro-aristeromycin analogues 2 and 3 and a preliminary characterization of their effect on several +RNA viruses, which provided insight into structure–activity relationships (SARs).

Figure 1.

Rationale for the design of the target nucleosides 2 and 3.

Results and Discussion

Chemistry

For the synthesis of the target nucleosides 2, the key fluorosugars 8a–c were synthesized from d-ribose via electrophilic fluorination, as shown in Scheme 1.

Scheme 1. Synthesis of 6-β-Fluoro-, 6-α-Fluoro-, and 6-Difluorosugar 8a–c.

Reagents and conditions: (a) LiCu(CH₂Ot-Bu)₂; (b) TESCl, LiHMDS, THF, −78 °C, 10 min; (c) Selectfluor, DMF, 0 °C, 12 h; (d) NaBH₄, MeOH, 0 °C, 30 min. (e) LiBH₄, MeOH, 0 °C, 30 min.

d-Ribose was converted to d-cyclopentenone 4 according to our previously published procedure.²¹ The 1,4-conjugated addition of 4 with Gilman reagent yielded the d-cyclopentanone derivative 5.^19,22 Treatment of 5 with lithium hexamethyldisilazide (LiHMDS) followed by trapping with triethylsilyl chloride (TESCl) gave silylenol ether 6, which was treated with (1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate): Selectfluor) in dimethylformamide (DMF) at 0 °C to yield a 5:1 ratio of 6-β-fluorosugar 7a to 6-α-fluorosugar 7b.¹⁹ The stereochemistry of the fluorine in 7a and 7b was confirmed by ¹H NOE experiments. Irradiation of 6-H of 7b gave NOE effects on its 2-H and 5-H, indicating the 6-α-fluoro configuration, but no NOE effects were observed on the same experiment in the case of 7a, confirming the 6-β-fluoro configuration. The configuration of the fluorine in 7b was further confirmed by the X-ray crystal structure obtained after it was converted to the final uracil derivative 2g (Scheme 5). Further electrophilic fluorination of 6-β-fluorosugar 7a or 6-α-fluorosugar 7b under the same conditions yielded the 6,6-difluorosugar 7c, which was equilibrated to form a geminal diol because of the presence of electronegative fluorine atoms. Electrophilic fluorinations with other electrophilic fluorines such as N-fluorobenzenesulfonimide (NFSI) or N-fluoro-O-benzenedisulfonimide (NFOBS) were problematic, resulting in low yields with many side spots. The reduction of 7a–c with sodium borohydride (NaBH₄) or lithium borohydride (LiBH₄) in MeOH resulted in the production of the 1-hydroxyl derivatives 8a–c.

Scheme 5. Synthesis of Fluorinated Pyrimidine Nucleoside Analogues 2f–j.

Reagents and conditions: (a) (E)-3-methoxy-2-propenoyl isocyanate, benzene, 4 Å-MS, DMF, −20 °C to rt, 15 h; (b) 2 M H₂SO₄, dioxane, reflux, 1.5 h; (c) BzCl, pyridine, CH₂Cl₂, rt, 15 h; (d) (i) 1,2,4-triazole, POCl₃, Et₃N, CH₃CN, rt, 15 h. (ii) NH₄OH, dioxane, rt, 15 h. (iii) NH₃/MeOH, rt, 15 h.

As the α-fluoro derivative 8b was obtained as the minor isomer, as shown in Scheme 1, we wanted to improve the stereoselective synthesis of 8b, by using Rubottom²³ oxidation as the key step, as illustrated in Scheme 2. Rubottom oxidation of silylenol ether 6 with osmium tetroxide (OsO₄) and N-methylmorpholine-N-oxide (NMO) followed by trapping with t-butyldimethylsilyl chloride (TBSCl) produced 6-β-alkoxyketone 9 as a single stereoisomer in 53% yield. The reduction of ketone 9 with NaBH₄ gave alcohol 10, which was protected with a benzyl group to give 11. Removal of the t-butyldimethylsilyl (TBS) group in 11 with tetra-n-butylammonium fluoride (TBAF) yielded the 6-β-alcohol 12. To our disappointment, the treatment of 12 with N,N-diethylaminosulfur trifluoride (DAST) gave the desired product, 6-α-fluoride 13a, but also the undesired product 1-β-fluoride 13b at a 1:1 ratio. The formation of 13a (route I) resulted from the direct S_N2 reaction of 12a with fluoride, while 12a was readily converted into the oxonium ion 12b (route II) via its participation of the neighboring benzyl group, which was attacked exclusively by the fluoride at the less sterically hindered 1-position to yield the undesired product 13b (route III). However, the product via route IV was not formed because of the steric effect of the t-butyloxymethyl substituent.

Scheme 2. Synthetic Approach to 6-α-Fluorosugar 8b via Rubottom Oxidation.

Reagents and conditions: (a) (i) OsO₄, NMO·H₂O, THF, rt, 1 h, then NaHCO₃, MeOH, rt, 3 h; (ii) TBSCl, imidazole, DMF, rt, 3 h; (b) NaBH₄, MeOH, rt, 1 h; (c) BnBr, NaH, DMF, 0 °C to rt, 12 h; (d) TBAF, THF, rt, 12 h; (e) DAST, toluene, 0 °C to rt, 2 h.

To avoid the participation of the neighboring group, we considered using a cyclic sulfate substrate with electron-withdrawing property and conformational restraint to be the best choice. Furthermore, cyclic sulfate has the advantage that it can be utilized as a surrogate for epoxide during nucleobase condensation, as shown in Scheme 3. The regioselective cleavage of the 2,3-acetonide in 10 with trimethylaluminum (AlMe₃) followed by treatment of the resulting diol with thionyl chloride (SOCl₂) yielded the 6-β-hydroxyl cyclic sulfite 14 after the removal of the TBS group. The treatment of 14 with DAST yielded the desired 6-α-fluoro cyclic sulfite 15 as a single stereoisomer. The cyclic sulfite 15 was oxidized to form cyclic sulfate 16, which was subsequently condensed with 6-chloropurine anions; however, this resulted in decomposition.¹⁹ Thus, we decided to synthesize the 6-α-fluoro derivative 8b according to Scheme 1.

Scheme 3. Synthetic Approach to 2b via Cyclic Sulfate.

Reagents and conditions: (a) AlMe₃, CH₂Cl₂, −78 °C to rt, 12 h; (b) SOCl₂, Et₃N, CH₂Cl₂, 0 °C, 10 min; (c) TBAF, AcOH, THF, rt, 12 h; (d) DAST, CH₂Cl₂, 0 °C to rt, 4 h; (e) RuCl₃, NaIO₄, CCl₄/CH₃CN/H₂O (1/1/1.5), rt, 20 min; (f) (i) 6-chloropurine, 18-crown-6, NaH, THF, 65 °C, 15 h; (ii) 20% H₂SO₄, rt, 1 h.

Scheme 4 depicts the synthesis of the aristeromycin analogues 2a–e from the 6-β-fluoro-, 6-α-fluoro-, and 6,6-difluorosugars 8a–c.¹⁹ Compounds 8a–c were treated with triflic anhydride (Tf₂O) followed by treatment with sodium azide to give azido derivatives 18a–c. The catalytic hydrogenation of 18a–c yielded the amino derivatives 19a–c, respectively, which are starting compounds for the base-building process. The treatment of 19a–c with 5-amino-4,6-dichloropyrimidine^18a−18c,24 in the presence of N,N-diisopropylethylamine (DIPEA) under microwave radiation conditions yielded 20a–c, which were cyclized with diethoxymethyl acetate^18a−18c,24 in the presence of microwave radiation to produce the 6-chloropurine derivatives 21a–c. The treatment of 21a–c with t-butanolic ammonia followed by the removal of protective groups under acidic conditions yielded the 6′-β-fluoro-, 6′-α-fluoro-, and 6′,6′-difluoroaristeromycins 2a–c, respectively. The structure of compound 2c was confirmed by a single-crystal X-ray analysis (see the Supporting Information).²⁵ The treatment of 21a and 21c with 40% aqueous methylamine followed by aqueous trifluoroacetic acid (TFA) resulted in N⁶-methyl-aristeromycin analogues 2d and 2e, respectively.

Scheme 4. Synthesis of β-Fluoro-, α-Fluoro-, and Difluoro-Aristeromycin Analogues 2a–e.

Reagents and conditions: (a) (i) Tf₂O, pyridine, 0 °C, 30 min; (ii) NaN₃, DMF, 60–100 °C, 4–15 h; (b) Pd/C, H₂, MeOH, rt, 18 h; (c) 5-amino-4,6-dichloropyrimidine, DIPEA, n-BuOH, 170–200 °C, 4–7 h, MW; (d) CH₃C(O)OCH(OEt)₂, 140 °C, 3 h, MW; (e) NH₃/t-BuOH, 120 °C, 15 h; (f) NH₂Me/H₂O, (40 wt %), EtOH, 30 °C, 2 h; (g) 67% aq TFA, 50 °C, 15 h.

The amino derivatives 19a–c were also converted into the pyrimidine nucleoside derivatives 2f–j, as shown in Scheme 5. Treatment of 19a–c with (E)-3-methoxy-2-propenoyl isocyanate, which was prepared by reacting 3-methoxyacryloyl chloride with silver cyanate,²⁶ in benzene produced 22a–c, respectively, which were cyclized with 2 M H₂SO₄ to yield the uridine derivatives 2f–h, respectively. The structures of 2g and 2h were confirmed by the X-ray crystallography (see the Supporting Information) (Scheme 5).²⁷ To synthesize the cytidine derivatives 2i and 2j, compounds 2f and 2h were benzoylated to give 23a and 23b, respectively, which were converted to the cytidine derivatives 2i and 2j using conventional three-step procedures.²⁸

The uracil phosphoramidate analogue Sofosbuvir²⁰ is used in the clinic as a powerful anti-hepatitis C virus agent. Therefore, we have also synthesized the uracil phosphoramidate prodrugs 3b–c and the adenine phosphoramidate prodrug 3a derived from the purine and pyrimidine nucleoside analogues 2a–j by using McGuigan’s ProTide prodrug methodology,²⁰ as shown in Scheme 6. 6′,6′-Difluoro-aristeromycin (2c) was treated with acetone under acidic conditions to give 2,3-acetonide 24. The treatment of 24 with di-tert-butyl dicarbonate (Boc₂O) yielded a mixture of 25a and 25b in a 2:1 ratio, which was converted to the phosphoramidate prodrug 26 by treating with phosphoramiditing reagent (A)²⁹ in the presence of t-butylmagnesium chloride. The treatment of 26 with 50% formic acid produced the final product, prodrug 3a. The monofluoro- and difluoropyrimidine derivatives 2f and 2h were similarly converted to the final prodrugs 3b and 3c.

Scheme 6. Synthesis of Phosphoramidate Prodrugs 3a–c.

Reagents and conditions: (a) cH₂SO₄, acetone, rt, 4 h; (b) (i) TMSOTf, DMAP, HMDS, 75 °C, 2 h; (ii) Boc₂O, THF, rt, 4 h; (iii) MeOH/Et₃N (5:1), 55 °C, 16 h; (c) A, t-BuMgCl, 4 Å-MS, THF, 0 °C to rt, 36 h; (d) 50% HCOOH, rt, 8 h.

Inhibition of SAH Hydrolase

All compounds 1, 2a–j, and 3a–c were assayed for their ability to inhibit recombinant human SAH hydrolase protein, expressed in Escherichia coli JM109, using a 5,5′-dithiobis-2-nitrobenzoate (DTNB) coupled assay as described by Lozada-Ramírez et al.³⁰ As expected, all adenosine derivatives 2a–e potently inhibited SAH hydrolase, but none of the pyrimidine analogues 2f–j showed any inhibitory activity at concentrations up to 100 μM. None of the prodrugs 3a–c exhibited inhibitory activity at concentrations up to 100 μM. This result is not surprising because adenosine is the substrate for SAH hydrolase. Among the adenosine analogues, 6′-β-fluoroaristeromycin (2a) exhibited the most potent inhibitory activity (IC₅₀ = 0.37 μM), which was 3.6-fold more potent than the control 1 (IC₅₀ = 1.32 μM). However, 6′-α-fluoroaristeromycin (2b, IC₅₀ = 9.70 μM) was 26-fold less potent than the corresponding 6′-β-fluoro analogue 2a and 7.4-fold less active than the 6′-unsubstituted compound 1. This indicates that the stereochemistry at the 6′-position is important for inhibitory activity. Interestingly, the introduction of two fluorines at the 6′-position resulted in 2c (IC₅₀ = 1.06 μM), which was slightly more potent than the control 1. The inhibitory activity of the 6′-fluoro-aristeromycin series can be ranked in the following order: 6′-β-F > 6′,6′-F,F > 6′-H > 6′-α-F. The introduction of a methyl group at the N⁶-amino group of 2a, resulting in 2d, decreased the inhibitory activity (IC₅₀ = 4.39 μM) by 11.9-fold, while the addition of a methyl group to the N⁶-amino group of 2c, resulting in 2e, increased the inhibitory activity (IC₅₀ = 0.76 μM) by 1.7-fold. These results demonstrate that the N⁶-methyladenine and the adenine moieties do not lead to a decrease in inhibitory activity.

Antiviral Activity

The novel 6′-fluoro-aristeromycin analogues 2a–j and 3a–c were screened for antiviral activity against a variety of +RNA viruses. The compounds were tested for antiviral activity in cytopathic effect (CPE) reduction assays at 4 concentrations, that is, 150, 50, 16.7, and 5.6 μM by preparing 3-fold serial dilutions. Compounds that demonstrated antiviral activity in this primary screen were further tested more extensively in dose response experiments at 8 different concentrations to determine the EC₅₀. Cytotoxicity (CC₅₀) was determined in parallel in uninfected cells (Table 1).

Table 1. Inhibition of SAH Hydrolase and the Replication of Several +RNA Viruses by All Final Nucleoside Analogues 2a–j and 3a–c^a,b,c,d.

		MERS-CoV			SARS-CoV			ZIKV			CHIKV
compound no.	SAH hydrolase IC50 (μM)	EC50 (μM)	CC50 (μM)	SI	EC50 (μM)	CC50 (μM)	SI	EC50 (μM)	CC50 (μM)	SI	EC50 (μM)	CC50 (μM)	SI
1	1.32	>50	2		>50	>5		0.64	2.4	3.8	0.8	6.3	7.9
2a	0.37	0.20	0.60	3	ND	ND		ND	ND		>100	>100
2b	9.70	ND	ND		ND	ND		2.54	3.97	1.56	0.53	1.32	2.49
2c	1.06	0.2	3.2	16	0.5	5.9	11.8	0.26	>2.5	>9.6	0.13	>1.25	>9.6
2d	4.39	>50	>50		>100	>100		>100	>100		>100	>100
2e	0.76	>50	12.5		>100	>100		>100	>100		>100	>100
2f	>100	>100	>100		>100	>100		>100	>100		>100	>100
2g	>100	>100	>100		>100	>100		>100	>100		>100	>100
2h	>100	>50	>50		>100	>100		>100	>100		>100	>100
2i	>100	>100	>100		>100	>100		>100	>100		>100	>100
2j	>100	>50	>50		>100	>100		>100	>100		>100	>100
3a	>100	9.3	>50		6.8	>25	>3.7	1.75	>25	>14.3	1.95	>12.5	>6.4
3b	>100	>50	>50		>100	>100		>100	>100		>100	>100
3c	>100	>50	>50		>100	>100		>100	>100		>100	>100

^aND: not determined; SI = CC₅₀/EC₅₀.

^bEC₅₀: effective concentration to inhibit the replication of the virus by 50%.

^cCC₅₀: cytotoxic concentration to inhibit the replication of normal cells by 50%.

^dEC₅₀ > 100 indicates that no antiviral activity was observed at the highest concentration tested because either there was no protection or the compound was toxic.

As shown in Table 1, only the adenosine derivatives 2a–c exhibited potent antiviral activities against +RNA viruses, while the other purine N⁶-methyladenine derivatives 2d and 2e and pyrimidine derivatives 2f–j did not show significant antiviral activities, not even at 100 μM. This result suggests that the antiviral activity might be due to an (indirect) effect on viral MTase activity through the inhibition of host SAH hydrolase. Inhibition of the viral RdRp appears not to be important. The mechanism of action of these compounds has been studied in more detail and results will be published elsewhere.

Compound 2a inhibited MERS-CoV replication with an EC₅₀ of 0.20 μM; however, it was also rather cytotoxic, resulting in a selectivity index (SI) of 3. Replacement of the remaining 6′-H in 2a with F resulted in compound 2c, which exhibited a > 5-fold reduction in cytotoxicity, while its antiviral activity remained unchanged, with an EC₅₀ of ∼0.20 μM and an SI of 15 for MERS-CoV. This compound was also active against SARS-CoV with an SI of 12.5, suggesting that it may be a broad-spectrum coronavirus inhibitor. In addition, it also inhibited ZIKV replication with an EC₅₀ of 0.26 μM (SI > 10) and was active against CHIKV with an EC₅₀ of 0.13 μM. Compound 2b showed some inhibitory effects on CHIKV and ZIKV replication, but this was likely due to pleiotropic cytotoxic effects, as the SI was <3. Among the phosphoramidate prodrugs 3a–c, only the adenosine prodrug 3a exhibited significant broad-spectrum antiviral activities, demonstrating that it may inhibit the RdRp of RNA viruses after conversion into the triphosphate form, although it remains to be determined in biochemical assays whether the triphosphate form affects RdRp activity.²⁰ Compound 3a had an EC₅₀ of 9.3 μM for MERS-CoV and 6.8 μM for SARS-CoV, but it also had an SI < 10, and it was therefore not considered a potent inhibitor of coronavirus replication. However, for CHIKV and ZIKV, 3a had EC₅₀ values of 1.95 and 1.75 μM, respectively, with good selectivity indices. Interestingly, the prodrug 3a was less potent, but also much less cytotoxic than the parent compound 2c, which is unusual as regularly the phosphoramidate is more potent than the parent drug.²⁰ The phosphoramidate 3a might be slowly hydrolyzed to the 5′-monophosphate by metabolic enzymes, or to the parent drug 2c by a phosphatase, which could inhibit SAH hydrolase, explaining the observed antiviral effect. Viral load reduction assays were performed with compound 2c by infecting cells with CHIKV, ZIKV, SARS-CoV, and MERS-CoV, followed by treatment with different concentrations of 2c. At 30 hpi (CHIKV) or 48 hpi (ZIKV, SARS- and MERS-CoV), infectious progeny titers in the medium were determined by plaque assay (Figure 2). Treatment with concentrations higher than 1 μM of 2c reduced infectious CHIKV titers by more than 2 log. The effect on ZIKV infectious progeny titers was limited and showed a ∼1 log reduction. For SARS-CoV, the reduction in infectious progeny titer was ∼1.5 log at 2c concentrations above 0.3 μM. The strongest antiviral effect was observed for MERS-CoV, with a ∼2.5 log reduction in infectious progeny titers when infected cells were treated with 2c concentrations above 0.3 μM. Follow-up studies to gain more insights into the mode of action of 2c and 3a and related compounds are currently ongoing, and results will be published elsewhere.

Figure 2.

Effect of 2c on the infectious progeny of CHIKV, ZIKV, SARS-CoV, and MERS-CoV. Cells were infected with the virus indicated on the y-axis of the graph in medium with various concentrations of 2c. Infectious progeny titers were determined by plaque assay (n = 4) and viability of noninfected cells was monitored using the CellTiter 96AQueous Non-Radioactive Cell Proliferation Assay (Promega). Significant differences are indicated by *: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Finally, we measured the log P of the most active compound 2c by the pH-metric method, using a T3 Sirius instrument, because the lipophilicity is a major determinant for compound absorption, distribution in the body, penetration across biological barriers, metabolism, and excretion. The measured log P was 0.02, indicating that it is almost equally partitioned between the lipid and aqueous phases. The relatively low log P of 2c is expected to be overcome by converting it to the phosphoramidate 3a.

Conclusions

We have synthesized the 6′-fluorinated aristeromycin analogues 2a–j, which were designed as dual-target antiviral compounds aimed at inhibiting both the viral RdRp and the host SAH hydrolase. The electrophilic fluorination of silyl enol ether with Selectfluor was the key step in the synthesis. We have also synthesized the phosphoramidate prodrugs 3a–c to determine whether these would inhibit virus replication through an effect on the viral RNA polymerase. Figure 3 depicts the summarized SAR of the synthesized 6′-fluorinated final nucleoside analogues 2a–j and 3a–c concerning the inhibition of human SAH hydrolase and the inhibition of the replication of various +RNA viruses with capped genomes. It was discovered that the introduction of fluorine at the 6′-position increases the inhibitory activity on SAH hydrolase and the replication of selected +RNA viruses. Compared to the 6′-unsubstituted compound 1, the 6′-fluorinated aristeromycin analogues 2a and 2c more potently inhibited SAH hydrolase activity and the replication of MERS-CoV, SARS-CoV, ZIKV, and CHIKV. Among these compounds, 6′-β-fluoroaristeromycin (2a) was the most potent with an IC₅₀ of 0.37 μM for SAH hydrolase activity and an EC₅₀ of 0.20 μM for MERS-CoV replication. There was a correlation between the inhibition of SAH hydrolase and the antiviral activity of the compounds, suggesting that the latter was mainly due to indirect targeting of viral methylation reactions. The SAR studies and a lack of antiviral effects of several purine and pyrimidine analogues suggest that the antiviral effect of 1, 2a, and 2c is unlikely due to targeting of the viral RdRp. Compound 2c appears to be an interesting compound for further development and evaluation as a broad-spectrum antiviral agent, as it inhibited several coronaviruses, CHIKV, and ZIKV. More detailed biological studies on the efficacy of these compounds in virus-infected cells and into their mode of action are currently ongoing and will be published elsewhere.

Figure 3.

Summarized SAR of 6′-fluorinated aristeromycin analogues 2 and 3.

Experimental Section

Chemical Synthesis

General Methods

Proton (¹H) and carbon (¹³C) NMR spectra were obtained on a Bruker AV 400 (400/100 MHz), Bruker AMX 500 (500/125 MHz), JEOL JNM-ECA600 (600/150 MHz), or Bruker AVANCE III 800 (800/200 MHz) spectrometer. Chemical shifts are reported as parts per million (δ) relative to the solvent peak. Coupling constants (J) are reported in hertz. Mass spectra were recorded on a Thermo LCQ XP instrument. Optical rotations were determined on Jasco III in appropriate solvent. UV spectra were recorded on U-3000 made by Hitachi in methanol or water. Infrared spectra were recorded on FT-IR (FTS-135) made by Bio-Rad. Melting points were determined on a Buchan B-540 instrument and are uncorrected. The crude compounds were purified by column chromatography on a silica gel (Kieselgel 60, 70–230 mesh, Merck). Elemental analyses (C, H, and N) were used to determine the purity of all synthesized compounds, and the results were within ±0.4% of the calculated values, confirming ≥95% purity.

(((3aR,6R,6aR)-6-(tert-Butoxymethyl)-2,2-dimethyl-6,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)triethylsilane (6)

To a cooled (−78 °C) solution of 5 (1568.0 mg, 6.470 mmol) in anhydrous tetrahydrofuran (THF; 32.0 mL, 0.2 M) was dropwise added chlorotriethylsilane (5.4 mL, 32.355 mmol), followed by addition of LiHMDS (19.0 mL, 1.0 M solution in THF, 19.0 mmol) under N₂. After being stirred at the same temperature for 10 min, the reaction mixture was quenched with saturated aqueous NH₄Cl (80 mL). The layers were separated, and the aqueous layer was extracted with ethyl acetate (EtOAc; 150 mL). The combined organic layers were washed successively with H₂O and saturated brine, dried over anhydrous MgSO₄, filtered, and evaporated. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 100/1 to 30/1) to give 6 (2267.0 mg, 98%) as colorless oil: [α]_D²⁰ = +36.48 (c 1.23, CHCl₃); ¹H NMR (400 MHz, CDCl₃): δ 4.73 (dd, J = 1.1, 6.0 Hz, 1H), 4.58 (d, J = 2.1 Hz, 1H), 4.36 (d, J = 6.1 Hz, 1H), 3.27 (dd, J = 5.6, 8.6 Hz, 1H), 3.15 (dd, J = 6.6, 8.6 Hz, 1H), 2.72 (dd, J = 5.9, 5.9 Hz, 1H), 1.42 (s, 3H), 1.32 (s, 3H), 1.12 (s, 9H), 0.96 (t, J = 8.0 Hz, 9H), 0.66–0.72 (m, 6H); ¹³C NMR (100 MHz, CDCl₃): δ 154.1, 110.3, 104.4, 82.8, 79.7, 72.5, 63.9, 47.9, 27.4 (3 × CH₃-tert-butyl), 27.3, 25.8, 6.5 (3 × triethylsilyl), 4.6 (3 × triethylsilyl); IR (neat): 2973, 1648, 1363, 1262, 1204, 1056, 851, 748 cm^–1; HRMS (FAB): found, 356.2388 [calcd for C₁₉H₃₆O₄Si⁺ (M + H)⁺, 356.2383].

(3aR,5R,6R,6aR)-6-(tert-Butoxymethyl)-5-fluoro-2,2-dimethyldihydro-3aH-cyclopenta[d][1,3]dioxol-4(5H)-one (7a) and (3aR,5S,6R,6aR)-6-(tert-Butoxymethyl)-5-fluoro-2,2-dimethyldihydro-3aH-cyclopenta[d][1,3]dioxol-4(5H)-one (7b)

To a cooled (0 °C) solution of silyl enol ether 6 (8.75 g, 24.548 mmol) in anhydrous DMF (123.0 mL, 0.20 M) was added 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (13.04 g, 36.824 mmol, Selectfluor) in one portion under N₂. After being stirred at the same temperature for 12 h, the reaction mixture was quenched with saturated aqueous NH₄Cl (130 mL), diluted with EtOAc (130 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2 × 100 mL). The combined organic layers were washed successively with H₂O and saturated brine, dried over anhydrous MgSO₄, filtered, and evaporated. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 40/1 to 20/1) to give 7a and 7b (5.80 g, 91%, total yield, 7a/7b = 5.2:1 by ¹H NMR analysis).

Compound 7a

It was obtained as a white solid; [α]_D²⁵ = −156.69 (c 0.735, CHCl₃); ¹H NMR (400 MHz, CDCl₃): δ 5.29 (dd, J = 8.2, 49.5 Hz, 1H), 4.70 (t, J = 5.7 Hz, 1H), 4.20 (dd, J = 2.4, 6.1 Hz, 1H), 3.61 (dd, J = 1.6, 8.6 Hz, 1H) 3.38–3.41 (m, 1H), 2.75 (d, J = 8.2 Hz, 1H), 1.41 (s, 3H), 1.30 (s, 3H), 1.06 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 203.0 (d, J = 12.9 Hz), 111.4, 88.5 (d, J = 201.5 Hz), 78.2 (d, J = 6.9 Hz), 75.0 (d, J = 3.1 Hz), 74.3, 56.6 (d, J = 6.6 Hz), 40.5 (d, J = 15.5 Hz), 26.8 (3 × CH₃-tert-butyl), 26.2, 23.6; ¹⁹F NMR (376 MHz, CDCl₃): δ −220.60 to –221.14 (m); LRMS (ESI⁺): found, 283.13 [calcd for C₁₃H₂₁FO₄Na⁺ (M + Na)⁺, 283.1322]; Anal. Calcd for C₁₃H₂₁FO₄: C, 59.98; H, 8.13. Found: C, 59.99; H, 8.53.

Compound 7b

It was obtained as a white solid; [α]_D²⁵ = −83.72 (c 0.495, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 5.21–5.36 (ddd, J = 1.3, 4.5, 50.8 Hz, 1H), 4.55 (d, J = 5.9 Hz, 1H), 4.50 (d, J = 5.9 Hz, 1H), 3.63 (d, J = 2.2 Hz, 2H), 2.52–2.58 (m, 1H), 1.41 (s, 3H), 1.33 (s, 3H), 1.13 (s, 9H); ¹³C NMR (150 MHz, CDCl₃): δ 207.8 (d, J = 12.9 Hz), 112.2, 91.9 (d, J = 192.4 Hz), 78.78 (d, J = 3.5 Hz), 78.74, 73.6, 60.5 (d, J = 4.3 Hz), 45.0 (d, J = 17.9 Hz), 27.2 (3 × CH₃-tert-butyl), 26.8, 25.2; ¹⁹F NMR (376 MHz, CDCl₃): δ −196.0 to –196.2 (m); HRMS (FAB): found, 262.1679 [calcd for C₁₃H₂₂FO₄⁺ (M + H)⁺, 261.1505]; Anal. Calcd for C₁₃H₂₁FO₄: C, 59.98; H, 8.13. Found: C, 59.77; H, 8.45.

(3aR,6R,6aR)-6-(tert-Butoxymethyl)-5,5-difluoro-2,2-dimethyldihydro-3aH-cyclopenta[d][1,3]dioxol-4(5H)-one (7c)

It was obtained in 70% yield (mixture of 7c and 7d) as a white solid; [α]_D²⁵ = −4.34 (c 0.21, MeOH); ¹H NMR (7c and 7d mixture, 400 MHz, CDCl₃; 7c and 7d mixture): δ 4.82 (s, 1H), 4.72 (t, J = 6.1 Hz, 1H), 4.52–4.57 (m, 1H), 4.35–4.41 (m, 1H), 4.25 (dd, J = 8.0, 4.0 Hz, 1H), 3.74 (s, 1H), 3.69 (d, J = 8.0 Hz, 1H), 3.67–3.60 (m, 1H), 3.54–3.59 (m, 1H), 3.46 (d, J = 8.3 Hz, 1H), 2.68 (d, J = 17.4 Hz, 1H), 2.53–2.62 (m, 1H), 1.48 (s, 3H), 1.44 (s, 3H), 1.34 (s, 3H), 1.32 (s, 3H), 1.21 (s, 9H), 1.06 (s, 9H).

General Procedure for the Synthesis of 8a–c

To a cooled (0 °C) solution of 7a–c (1 equiv) in MeOH (0.18 M), sodium borohydride or lithium borohydride was added in a single portion in a N₂ atmosphere. After stirring for 30 min at the same temperature, the reaction mixture was neutralized with acetic acid (2 mL) and evaporated. The residue was diluted with saturated aqueous NH₄Cl, and the aqueous layer was extracted with EtOAc (2 × 100 mL). The combined organic layers were dried over anhydrous MgSO₄, filtered, and evaporated. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 20/1) to give 8a–c.

(3aS,4R,5R,6R,6aR)-6-(tert-Butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-ol (8a)

It was obtained in 71% yield as a colorless syrup; [α]_D²⁵ = −47.46 (c 0.395, CHCl₃); ¹H NMR (400 MHz, CDCl₃): δ 4.91 (td, J = 6.6, 52.5 Hz, 1H), 4.51–4.52 (m, 1H), 4.47 (ddd, J = 1.6, 6.3, 7.8 Hz, 1H), 4.26–4.34 (m, 1H), 3.52 (dd, J = 3.3, 8.8 Hz, 1H), 3.36–3.39 (m, 1H), 2.67 (d, J = 7.9 Hz, 1H), 2.46 (br s, 1H), 1.45 (s, 3H), 1.32 (s, 3H), 1.14 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 111.1, 99.5 (d, J = 185.9 Hz), 81.2 (d, J = 4.4 Hz), 76.3 (d, J = 9.0 Hz), 74.0 (d, J = 23.4 Hz), 73.0, 56.8 (d, J = 8.2 Hz), 44.6 (d, J = 18.1 Hz), 27.3 (3 × CH₃-tert-butyl), 26.1, 24.1; ¹⁹F NMR (376 MHz, CDCl₃) −211.0 to −211.21 (m); HRMS (FAB): found, 263.1662 [calcd for C₁₃H₂₄FO₄⁺ (M + H)⁺, 263.1659]; Anal. Calcd for C₁₃H₂₃FO₄: C, 59.52; H, 8.84. Found: C, 59.32; H, 9.15.

(3aS,4R,5S,6R,6aR)-6-(tert-Butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-ol (8b)

It was obtained in 67% yield as a colorless syrup; [α]_D²⁵ = −40.42 (c 0.22, MeOH); ¹H NMR (500 MHz, CDCl₃): δ 4.68 (dd, J = 4.1, 52.4 Hz, 1H), 4.46–4.53 (m, 2H), 4.13–4.24 (m, 1H), 3.33–3.40 (m, 1H), 2.81 (d, J = 11.4 Hz, 1H), 2.50 (dt, J = 2.9, 22.9 Hz, 1H), 1.46 (s, 3H), 1.30 (s, 3H), 1.08 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): δ 111.4, 98.4 (d, J = 181.5 Hz), 82.8, 79.3, 73.8 (d, J = 16.3 Hz), 73.0, 60.6 (d, J = 12.1 Hz), 49.2 (d, J = 18.3 Hz), 27.1 (3 × CH₃-tert-butyl), 26.2, 24.2; HRMS (ESI⁺): found, 285.1480 [calcd for C₁₃H₂₃FNaO₄⁺ (M + Na)⁺, 285.1478]; Anal. Calcd for C₁₃H₂₃FO₄: C, 55.70; H, 7.91. Found: C, 55.40; H, 7.75.

(3aS,4R,6R,6aR)-6-(tert-Butoxymethyl)-5,5-difluoro-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-ol (8c)

It was obtained in 74% yield as a colorless syrup; [α]_D²⁵ = 22.37 (c 0.28, MeOH); ¹H NMR (500 MHz, CDCl₃): δ 4.53 (t, J = 5.7 Hz, 1H), 4.44 (ddd, J = 2.6, 6.4, 8.9 Hz, 1H), 4.20–4.29 (m, 1H), 3.55 (d, J = 8.7 Hz, 1H), 3.39 (d, J = 8.8 Hz, 1H), 2.76 (d, J = 11.5 Hz, 1H), 2.43 (d, J = 17.2 Hz, 1H), 1.46 (s, 3H), 1.31 (s, 3H), 1.12 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): δ 126.9 (dd, J = 252.3, 260.3 Hz), 110.9, 79.6 (d, J = 5.9 Hz), 75.5 (d, J = 11.3 Hz), 73.7 (dd, J = 18.5, 25.8 Hz), 73.4, 57.6 (dd, J = 4.6, 8.5 Hz), 48.7 (t, J = 20.8 Hz), 27.2 (3 × CH₃-tert-butyl), 25.9, 24.2; HRMS (ESI⁺): found, 298.1834 [calcd for C₁₃H₂₆F₂NO₄⁺ (M + NH₄)⁺, 298.1830 ]; Anal. Calcd for C₁₃H₂₂F₂O₄: C, 55.70; H, 7.91. Found: C, 55.45; H, 7.56.

(3aR,5R,6R,6aR)-6-(tert-Butoxymethyl)-5-((tert-butyldimethylsilyl)oxy)-2,2-dimethyldihydro-3aH-cyclopenta[d][1,3]dioxol-4(5H)-one (9)

To a cooled (0 °C) solution of 6 (1275 mg, 3.57 mmol) in anhydrous THF (12 mL, 0.3 M) were added 4-methylmorpholine N-oxide monohydrate (967 mg, 7.15 mmol, 2 equiv) and osmium tetroxide (1000 mg, 3.93 mmol, 1.1 equiv) under a N₂ atmosphere. After stirring for 30 min, to the reaction mixture were added sodium thiosulfate pentahydrate (300 mg), sodium sulfite (300 mg), and acetone (30 mL) and stirred for additional 1 h at the same temperature. The layers were separated, and the aqueous layer was extracted with EtOAc (100 mL). The combined organic layers were washed with H₂O followed by saturated brine, dried over anhydrous MgSO₄, filtered, and evaporated. The residue was used for the next step without further purification. To a solution of above generated intermediate in anhydrous DMF (18 mL, 0.19 M) were added TBSCl (1614 mg, 10.71 mmol) and imidazole (729 mg, 10.71 mmol) under a N₂ atmosphere. After stirring for 3 h at room temperature, the reaction mixture was quenched with saturated aqueous NH₄Cl (50 mL) and diluted with EtOAc (50 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (2 × 50 mL). The combined organic layers were washed successively with H₂O and saturated brine, dried over anhydrous MgSO₄, filtered, and evaporated. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 40/1 to 20/1) to give 9 (705 mg, 53%) as a colorless syrup: [α]_D²⁵ = −103.19 (c 0.30, MeOH); ¹H NMR (400 MHz, CDCl₃): δ 4.65 (d, J = 6.4 Hz, 1H), 4.53 (d, J = 8.0 Hz, 1H), 4.11 (d, J = 6.3 Hz, 1H), 3.61 (dd, J = 1.6, 8.0 Hz, 1H), 3.30 (dd, J = 2.4, 8.1 Hz, 1H), 2.41–2.46 (m, 1H), 1.42 (s, 3H), 1.30 (s, 3H), 1.03 (s, 9H), 0.88 (s, 9H), 0.13 (s, 3H), 0.05 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 207.2, 110.9, 78.1, 75.8, 73.7, 71.3, 56.9, 42.3, 27.0 (3 × CH₃-tert-butyl), 26.4, 25.7 (3 × CH₃-tert-butyl), 23.8, 18.3, −4.4, −5.6; HRMS (FAB⁺) (m/z): found, 373.2398 [calcd for C₁₉H₃₇O₅Si⁺ (M + H)⁺, 373.2410]; Anal. Calcd for C₁₉H₃₆O₅Si: C, 61.25; H, 9.74. Found: C, 61.26; H, 9.75.

(3aS,4R,5R,6R,6aR)-6-(tert-Butoxymethyl)-5-((tert-butyldimethylsilyl)oxy)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-ol (10)

To a cooled (0 °C) solution of 9 (471 mg, 1.26 mmol) in methanol (6.3 mL, 0.2 M) was added sodium borohydride (144 mg, 3.79 mmol, 3 equiv) under a N₂ atmosphere. After being stirred at the same temperature for 1 h, the reaction mixture was diluted with H₂O (20 mL) and EtOAc (20 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (3 × 50 mL). The combined organic layers were washed successively with H₂O and saturated brine, dried over anhydrous MgSO₄, filtered, and evaporated. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 30/1 to 20/1) to give 10 (415 mg, 88%) as a colorless syrup: [α]_D²⁵ = −40.39 (c 0.32, MeOH); ¹H NMR (500 MHz, CDCl₃): δ 4.49 (d, J = 6.1 Hz, 1H), 4.41 (t, J = 6.2 Hz, 1H), 4.07 (t, J = 6.9 Hz, 1H), 3.95 (dd, J = 6.8, 14.7 Hz, 1H), 3.48 (dd, J = 3.9, 8.5 Hz, 1H), 3.32 (dd, J = 4.6, 8.5 Hz, 1H), 2.43 (d, J = 8.4 Hz, 1H), 2.12–2.18 (m, 1H), 1.45 (s, 3H), 1.32 (s, 3H), 1.12 (s, 9H), 0.87 (s, 9H), 0.09 (s, 3H), 0.05 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): δ 110.4, 81.0, 78.8, 77.0, 76.1, 72.6, 57.3, 46.0, 27.4 (3 × CH₃-tert-butyl), 26.2, 25.8 (3 × CH₃-tert-butyl), 24.0, 18.1, −4.5, −5.1; HRMS (FAB⁺) (m/z): found, 375.2584 [calcd for C₁₉H₃₉O₅Si⁺ (M + H)⁺, 375.2567]; Anal. Calcd for C₁₉H₃₈O₅Si: C, 60.92; H, 10.23. Found: C, 60.91; H, 10.25.

(((3aR,4R,5R,6R,6aR)-4-(Benzyloxy)-6-(tert-butoxymethyl)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-yl)oxy)(tert-butyl)dimethylsilane (11)

To a cooled (0 °C) solution of 10 (193 mg, 0.515 mmol) in DMF (5.2 mL, 0.1 M) was added benzyl chloride (0.12 mL, 1.030 mmol, 2.0 equiv) and sodium hydride (41 mg, 1.030 mmol, 2.0 equiv) under a N₂ atmosphere. After being stirred at room temperature for 12 h, the reaction mixture was diluted with H₂O (20 mL) and EtOAc (20 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (3 × 50 mL). The combined organic layers were washed successively with H₂O and saturated brine, dried over anhydrous MgSO₄, filtered, and evaporated. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 50/1) to give 11 (204 mg, 85%) as a colorless syrup: [α]_D²⁵ = −46.64 (c 0.66, MeOH); ¹H NMR (400 MHz, CDCl₃): δ 7.22–7.39 (m, 5H), 4.76 (d, J = 12.4 Hz, 1H), 4.59 (d, J = 12.4 Hz, 1H), 4.45 (d, J = 6.0 Hz, 1H), 4.33–4.37 (m, 2H), 3.83 (dd, J = 5.6, 8.8 Hz, 1H), 3.39 (dd, J = 4.4, 8.8 Hz, 1H), 3.32 (dd, J = 4.0, 8.4 Hz, 1H), 2.05–2.11 (m, 1H), 1.48 (s, 3H), 1.29 (s, 3H), 1.03 (s, 9H), 0.88 (s, 9H), 0.09 (s, 3H), 0.05 (s, 3H); ¹³C NMR (200 MHz, CDCl₃): δ 138.9, 128.4, 128.1, 127.9, 127.7, 127.2, 110.0, 82.1, 80.2, 76.0, 75.6, 72.4, 71.7, 57.5, 45.7, 27.3 (3 × CH₃-tert-butyl), 26.4, 25.8 (3 × CH₃-tert-butyl), 24.2, −4.7, −4.9; HRMS (FAB⁺) (m/z): found, 465.3001 [calcd for C₂₆H₄₅O₅Si⁺ (M + H)⁺, 465.3029]; Anal. Calcd for C₂₆H₄₄O₅Si: C, 67.20; H, 9.54. Found: C, 67.22; H, 9.55.

(3aR,4S,5R,6S,6aR)-4-(Benzyloxy)-6-(tert-butoxymethyl)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-ol (12)

To a cooled (0 °C) solution of 11 (179 mg, 0.385 mmol) in anhydrous THF (3.8 mL, 0.1 M) was added TBAF solution (1.2 mL, 1.0 M solution in THF, 1.2 mmol, 3.0 equiv) under a N₂ atmosphere. After being stirred at room temperature for 12 h, the reaction mixture was diluted with H₂O (30 mL) and EtOAc (30 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (3 × 50 mL). The combined organic layers were washed successively with H₂O and saturated brine, dried over anhydrous MgSO₄, filtered, and evaporated. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 8/1) to give 12 (129 mg, 88%) as a colorless syrup: [α]_D²⁵ = −49.04 (c 0.28, MeOH); ¹H NMR (400 MHz, CDCl₃): δ 7.39 (d, J = 7.2 Hz, 2H), 7.29–7.35 (m, 2H), 7.23–7.28 (m, 1H), 4.85 (d, J = 12.4 Hz, 1H), 4.62 (d, J = 12.4 Hz, 1H), 4.51 (t, J = 6.0 Hz, 1H), 4.40–4.45 (m, 2H), 3.81 (dd, J = 4.8, 7.2 Hz, 1H), 3.58 (dd, J = 3.6, 8.8 Hz, 1H), 3.44 (dd, J = 4.4, 8.8 Hz, 1H), 2.70 (br s, 1H), 2.26–2.32 (m, 1H), 1.48 (s, 3H), 1.31 (s, 3H), 1.08 (s, 9H); ¹³C NMR (200 MHz, CDCl₃): δ 138.5, 128.3 (2 × CH-benzene), 128.0 (2 × CH-benzene), 127.5, 111.1, 82.7, 80.6, 77.2, 76.7, 73.4, 71.9, 59.3, 45.4, 27.2 (3 × CH₃-tert-butyl), 26.5, 24.6; Anal. Calcd for C₂₀H₃₀O₅: C, 68.54; H, 8.63. Found: C, 68.52; H, 8.64.

(3aR,4R,5S,6R,6aR)-4-(Benzyloxy)-6-(tert-butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxole (13a)

To a cooled (0 °C) solution of 12 (20 mg, 0.052 mmol) in anhydrous toluene (2.0 mL, 0.026 M) was dropwise added diethylaminosulfur trifluoride (30 μL, 0.210 mmol, 4.0 equiv) under a N₂ atmosphere. After being stirred at room temperature for 2 h, the reaction mixture was quenched with saturated aqueous NH₄Cl (30 mL) and EtOAc (30 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (3 × 50 mL). The combined organic layers were washed successively with H₂O and saturated brine, dried over anhydrous MgSO₄, filtered, and evaporated. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 30/1) to give 13a (5.6 mg, 30%) and 13b (5.6 mg, 30%) as a colorless syrup.

Compound 13a

[α]_D²⁵ = −26.59 (c 0.22, MeOH); ¹H NMR (500 MHz, CDCl₃): δ 7.25–7.34 (m, 5H), 4.96 (ddd, J = 2.6, 6.8, 52.7 Hz, 1H), 4.72 (dd, J = 0.8, 11.6 Hz, 1H), 4.54 (d, J = 11.6 Hz, 1H), 4.44–4.52 (m, 2H), 4.02–4.09 (m, 1H), 3.41–3.47 (m, 2H), 2.15–2.18 (m, 1H), 1.47 (s, 3H), 1.28 (s, 3H), 1.12 (s, 9H); ¹³C NMR (200 MHz, CDCl₃): δ 137.8, 128.3 (2 × CH-benzyl), 128.1 (2 × CH-benzyl), 127.8, 111.8, 96.0 (d, J = 187.1 Hz), 81.6, 79.3, 78.2 (d, J = 15.7 Hz), 72.6, 71.8, 60.6 (d, J = 11.0 Hz), 50.2 (d, J = 18.7 Hz), 27.0 (3 × CH₃-tert-butyl), 26.6, 24.4; HRMS (FAB⁺) (m/z): found, 353.2121 [calcd for C₂₀H₃₀FO₄⁺ (M + H)⁺, 353.2128]; Anal. Calcd for C₂₀H₂₉FO₄: C, 68.16; H, 8.29. Found: C, 68.13; H, 8.27.

Compound 13b

[α]_D²⁵ = −61.72 (c 0.42, MeOH); ¹H NMR (500 MHz, CDCl₃): δ 7.38 (t, J = 7.3 Hz, 2H), 7.31 (t, J = 7.2 Hz, 2H), 7.25 (d, J = 7.2 Hz, 1H), 5.18 (dt, J = 7.8, 53.7 Hz, 1H), 4.76 (d, J = 12.2 Hz, 1H), 4.66 (d, J = 12.2 Hz, 1H), 4.45–4.49 (m, 1H), 4.41–4.44 (m, 1H), 4.19 (ddd, J = 5.9, 7.7, 16.5 Hz, 1H), 3.45 (dd, J = 3.0, 8.8 Hz, 1H), 3.31–3.34 (m, 1H), 2.37–2.43 (m, 1H), 1.47 (s, 3H), 1.28 (s, 3H), 1.01 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): δ 138.0, 128.3, 127.9 (2 × CH-benzyl), 127.7 (2 × CH-benzyl), 112.2, 103.5, 102.1, 81.5 (d, J = 27.5 Hz), 81.1 (d, J = 20.0 Hz), 72.6, 72.4, 57.6, 48.8 (d, J = 6.2 Hz), 27.4 (3 × CH₃-tert-butyl), 27.1, 25.0; HRMS (FAB⁺) (m/z): found, 353.2131 [calcd for C₂₀H₃₀FO₄⁺ (M + H)⁺, 353.2128]; Anal. Calcd for C₂₀H₂₉FO₄: C, 68.16; H, 8.29. Found: C, 68.13; H, 8.27.

(3aR,4R,5S,6R,6aS)-4-(tert-Butoxy)-5-(tert-butoxymethyl)-6-hydroxytetrahydro-3aH-cyclopenta[d][1,3,2]dioxathiole 2-Oxide (14)

To perform regioselective cleavage, to a cooled (−78 °C) solution of 10 (420 mg, 1.121 mmol) in anhydrous CH₂Cl₂ (5.6 mL, 0.2 M) was dropwise added trimethylaluminum (3.4 mL, 2.0 M solution in hexane, 6.727 mmol, 6.0 equiv) under a N₂ atmosphere. After being stirred at room temperature for 12 h, the reaction mixture was quenched with saturated aqueous NH₄Cl (30 mL) and EtOAc (30 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (3 × 50 mL). The combined organic layers were washed successively with H₂O and saturated brine, dried over anhydrous MgSO₄, filtered, and evaporated. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 10/1) to give diol intermediate (245 mg, 56%) 10a as a colorless syrup. For the introduction of cyclic sulfite, to a cooled (0 °C) solution of diol intermediate 10a (250 mg, 0.639 mmol) in anhydrous CH₂Cl₂ (6.4 mL, 0.1 M) was dropwise added triethylamine (0.3 mL, 2.239 mmol, 3.5 equiv) followed by thionyl chloride (70 μL, 0.959 mmol) under a N₂ atmosphere. After being stirred at room temperature for 30 min, the reaction mixture was quenched with saturated aqueous NH₄Cl (30 mL) and diluted with EtOAc (30 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (3 × 50 mL). The combined organic layers were washed successively with H₂O and saturated brine, dried over anhydrous MgSO₄, filtered, and evaporated. The residue was purified by flash column chromatography (silica gel, hexanes/EtOAc, 10/1) to give cyclic sulfite intermediate 10b (249 mg, 89%) as a colorless syrup. For TBS deprotection, to a cooled (0 °C) solution of 10b (286 mg, 0.654 mmol) in anhydrous THF (6.5 mL, 0.1 M) was added acetic acid (0.13 mL, 0.131 mmol, 0.2 equiv) followed by TBAF solution (2.6 mL, 1.0 M solution in THF, 2.6 mmol, 4.0 equiv) under a N₂ atmosphere. After being stirred at room temperature for 12 h, the reaction mixture was quenched with H₂O (30 mL) and diluted with EtOAc (30 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (3 × 50 mL). The combined organic layers were washed successively with H₂O and saturated brine, dried over anhydrous MgSO₄, filtered, and evaporated. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 6/1) to give 14 (202 mg, 96%, two diastereomers A and B were generated from sulfoxide stereogenic center) as a colorless syrup: for A: ¹H NMR (400 MHz, CDCl₃): δ 5.27 (t, J = 5.4 Hz, 1H), 5.02 (d, J = 5.9 Hz, 1H), 4.79 (s, 1H), 4.44 (dd, J = 4.8, 11.4 Hz, 1H), 4.19 (d, J = 3.9 Hz, 1H), 3.80 (dd, J = 2.6, 9.3 Hz, 1H), 1.90–1.94 (m, 1H), 1.27 (s, 9H), 1.21 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): δ 86.9, 82.6, 74.9, 74.5, 74.1, 69.4, 58.2, 43.6, 28.3 (3 × CH₃-tert-butyl), 27.2 (3 × CH₃-tert-butyl); HRMS (FAB⁺) (m/z): found, 323.1530 [calcd for C₁₄H₂₇O₆S⁺ (M + H)⁺, 323.1528]; for B: ¹H NMR (500 MHz, CDCl₃): δ 4.98–5.07 (m, 2H), 4.79 (d, J = 6.4 Hz, 1H), 4.36 (dd, J = 4.6, 11.5 Hz, 1H), 4.31 (d, J = 4.1 Hz, 1H), 3.84 (d, J = 9.2 Hz, 1H), 3.77 (d, J = 9.3 Hz, 1H), 2.65 (d, J = 10.1 Hz, 1H), 1.25 (s, 9H), 1.21 (s, 9H).

(3aR,4R,5R,6S,6aR)-4-(tert-Butoxy)-5-(tert-butoxymethyl)-6-fluorotetrahydro-3aH-cyclopenta[d][1,3,2]dioxathiole 2-Oxide (15)

To a cooled (0 °C) solution of 14 (33 mg, 0.102 mmol) in anhydrous CH₂Cl₂ (1.5 mL, 0.068 M) was dropwise added diethylaminosulfur trifluoride (60 μL, 0.434 mmol, 4.0 equiv) under a N₂ atmosphere. After being stirred at room temperature for 4 h, the reaction mixture was quenched with saturated aqueous NH₄Cl (30 mL) and diluted with EtOAc (30 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (3 × 50 mL). The combined organic layers were washed successively with H₂O and saturated brine, dried over anhydrous MgSO₄, filtered, and evaporated. The residue was purified by flash column chromatography (silica gel, hexanes/EtOAc, 15/1) to give 15 (12 mg, 37%) as a colorless syrup: ¹H NMR (600 MHz, CDCl₃): δ 5.17 (ddd, J = 4.6, 7.8, 52.7 Hz, 1H), 5.03 (t, J = 8.2 Hz, 1H), 4.92 (ddd, J = 5.0, 8.7, 17.8 Hz, 1H), 4.06 (ddd, J = 7.8, 11.0, 16.5 Hz, 1H), 3.53 (ddd, J = 2.7, 2.7, 6.8 Hz, 1H), 3.44 (dd, J = 2.2, 9.1 Hz, 1H), 2.54–2.58 (m, 1H), 1.17 (s, 18H); ¹³C NMR (125 MHz, CDCl₃): δ 102.1 (d, J = 191.2 Hz), 87.2 (d, J = 28.2 Hz), 81.9 (d, J = 5.8 Hz), 74.5, 72.8, 72.4 (d, J = 19.2 Hz), 55.5, 50.4 (d, J = 6.5 Hz), 28.6 (3 × CH₃-tert-butyl), 27.5 (3 × CH₃-tert-butyl).

(3aR,4R,5R,6S,6aR)-4-(tert-Butoxy)-5-(tert-butoxymethyl)-6-fluorotetrahydro-3aH-cyclopenta[d][1,3,2]dioxathiole 2,2-Dioxide (16)

To a solution of cyclic sulfite 15 (13 mg, 0.040 mmol) in CCl₄/CH₃CN/H₂O (1:1:1.5, total 1.75 mL, 0.14 M) was added in one portion sodium periodate (26 mg, 0.120 mmol), followed by ruthenium(III) chloride trihydrate (2 mg, 0.008 mmol) at room temperature under a N₂ atmosphere. After being stirred at the same temperature for 20 min, the reaction mixture was quenched with H₂O (20 mL) and diluted with CH₂Cl₂ (20 mL). The layers were separated, and the aqueous layer was extracted with CH₂Cl₂ (2 × 50 mL). The combined organic layers were washed successively with H₂O and saturated brine, dried over anhydrous MgSO₄, filtered, and evaporated. The crude product 16 was used for the next step without further purification.

General Procedure for the Synthesis of 18a–c

Triflation

To a cooled (0 °C) solution of 8a–c (1 equiv) in anhydrous pyridine (0.32 M), trifluoromethanesulfonic anhydride (2 equiv) was added dropwise in a N₂ atmosphere. After stirring at the same temperature for 30 min, the reaction mixture was quenched with H₂O (50 mL) and diluted with EtOAc (30 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (2 × 30 mL). The combined organic layers were washed with saturated aqueous CuSO₄ followed by water, dried over anhydrous MgSO₄, filtered, and evaporated. The residue was used for the next step without further purification.

Azidation

To a solution of triflate intermediate (1 equiv) in anhydrous DMF (0.19 M), sodium azide (3 equiv) was added in a single portion at room temperature. After being heated to 60–100 °C and stirred for 4–15 h, the reaction mixture was cooled to room temperature, quenched with H₂O (50 mL), and diluted with EtOAc (50 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with H₂O followed by saturated brine, dried over anhydrous MgSO₄, filtered, and evaporated. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 10/1) to give 18a–c.

(3aS,4S,5R,6R,6aR)-4-Azido-6-(tert-butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxole (18a)

It was obtained in 45% yield as a colorless syrup; [α]_D²⁵ = −24.42 (c 0.016, CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃): δ 5.16 (td, J = 52.4, 3.1 Hz, 1H), 4.66 (t, J = 6.0 Hz, 1H), 4.41 (t, J = 6.5 Hz, 1H), 3.62–3.69 (m, 1H), 3.54 (s, 1H), 3.50 (s, 1H), 2.27–2.36 (m, 1H), 1.47 (s, 3H), 1.29 (s, 3H), 1.16 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): δ 114.1, 96.9 (d, J = 182.6 Hz), 82.0, 80.2, 73.1, 67.9 (d, J = 15.7 Hz), 57.8 (d, J = 7.2 Hz), 49.4 (d, J = 17.6 Hz), 27.3 (3 × CH₃-tert-butyl), 27.1, 24.6; ¹⁹F NMR (376 MHz, CDCl₃) −206.9 to –207.2 (m); IR (neat): 2108 cm^–1; LR-MS (ESI⁺): 310.15 [calcd for C₁₃H₂₂FN₂NaO₃⁺ (M + Na)⁺, 310.1543]; Anal. Calcd for C₁₃H₂₂FN₃O₃: C, 54.34; H, 7.72; N, 14.62. Found: C, 54.35; H, 7.45; N, 14.23.

(3aS,4S,5S,6R,6aR)-4-Azido-6-(tert-butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxole (18b)

It was obtained in 88% yield as a colorless syrup; [α]_D²⁵ = 9.66 (c 0.51, MeOH); ¹H NMR (500 MHz, CDCl₃): δ 4.75 (dt, J = 7.7, 53.0 Hz, 1H), 4.41 (dd, J = 4.5, 6.7 Hz, 1H), 4.22 (t, J = 5.7 Hz, 1H), 4.00 (ddd, J = 5.5, 7.4, 16.6 Hz, 1H), 3.43–3.50 (m, 2H), 2.33–2.44 (m, 1H), 1.50 (s, 3H), 1.27 (s, 3H), 1.15 (s, 9H); ¹³C NMR (150 MHz, CDCl₃): δ 112.7, 95.8 (d, J = 188.9 Hz), 81.0 (d, J = 8.6 Hz), 77.8 (d, J = 7.2 Hz), 73.0, 70.9 (d, J = 20.1 Hz), 57.9, 49.1 (d, J = 18.7 Hz), 27.3 (3 × CH₃-tert-butyl), 27.2, 25.0; IR (neat): 2111 cm^–1; Anal. Calcd for C₁₃H₂₂FN₃O₃: C, 54.34; H, 7.72; N, 14.62. Found: C, 54.12; H, 7.94; N, 14.33.

(3aS,4S,6R,6aR)-4-Azido-6-(tert-butoxymethyl)-5,5-difluoro-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxole (18c)

It was obtained in 75% yield as a colorless syrup; [α]_D²⁵ = −43.39 (c 0.36, MeOH); ¹H NMR (500 MHz, CDCl₃): δ 4.40–4.44 (m, 1H), 4.34–4.39 (m, 1H), 3.87–3.95 (m, 1H), 3.61 (dd, J = 6.5, 9.3 Hz, 1H), 3.48 (t, J = 7.6 Hz, 1H), 2.54–2.66 (m, 1H), 1.49 (s, 3H), 1.28 (s, 3H), 1.17 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): δ 127.1 (dd, J = 255.9, 260.9 Hz), 113.0, 80.0 (d, J = 5.9 Hz), 78.4 (d, J = 5.6 Hz), 73.4, 69.1 (dd, J = 18.8, 25.1 Hz), 57.2 (d, J = 6.4 Hz), 50.8 (t, J = 20.0 Hz), 27.3 (3 × CH₃-tert-butyl), 26.9, 24.7; IR (neat): 2116 cm^–1; Anal. Calcd for C₁₃H₂₁F₂N₃O₃: C, 51.14; H, 6.93; N, 13.76. Found: C, 51.45; H, 7.21; N, 14.10.

General Procedure for the Synthesis of 19a–c

To a suspension of 18a–c (1 equiv) in methanol (0.2 M), 10% palladium on activated carbon (0.03 equiv) was added and stirred overnight at room temperature in a H₂ atmosphere. After filtration, the solvent was removed, and the residue was used for the next step without further purification.

General Procedure for the Synthesis of 20a–c

To a solution of 19a–c (1 equiv) in n-butanol (0.38 M), 5-amino-4,6-dichloro pyrimidine (3–10 equiv) and diisopropylamine (10 equiv) were added. The reaction mixture was placed under microwave irradiation at 170–200 °C for 4–7 h. The solvent was co-evaporated with MeOH, and the residue was purified with column chromatography (silica gel, hexane/EtOAc, 4/1) to give 20a–c, respectively.

N⁴-((3aS,4S,5R,6R,6aR)-6-(tert-Butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)-6-chloropyrimidine-4,5-diamine (20a)

It was obtained in 66% yield from 18a; yellow foam; [α]_D²⁵ = −53.8 (c 0.10, CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃): δ 8.08 (s, 1H), 5.27–5.33 (br s, 1H), 5.24 (td, J = 3.5, 52.9 Hz, 1H), 4.71–4.81 (m, 1H), 4.57 (t, J = 6.1 Hz, 1H), 4.44 (t, J = 6.3 Hz, 1H), 3.58–3.63 (m, 1H), 3.53 (t, J = 9.2 Hz, 1H), 3.39 (br s, 2H), 2.42–2.55 (m, 1H), 1.52 (s, 3H), 1.30 (s, 3H), 1.18 (s, 9H); ¹³C NMR (200 MHz, CDCl₃): δ 154.4, 149.0, 122.4, 113.8, 95.9 (d, J = 178.7 Hz), 84.2, 80.1, 77.1, 73.3, 59.8 (d, J = 15.9 Hz), 58.0 (d, J = 7.0 Hz), 49.4 (d, J = 17.6 Hz), 27.4 (3 × CH₃-tert-butyl), 27.2, 24.8; ¹⁹F NMR (376 MHz, CDCl₃) −212.8 to –213.1 (m); UV (CH₂Cl₂): λ_max 287 nm; LRMS (ESI⁺): found, 388.17 [calcd for C₁₇H₂₇ClFN₄O₃⁺ (M + H)⁺, 389.1756]; Anal. Calcd for C₁₇H₂₆ClFN₄O₃: C, 52.51; H, 6.50; N, 14.45. Found: C, 52.45; H, 6.13; N, 14.15.

N⁴-((3aS,4S,5S,6R,6aR)-6-(tert-Butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)-6-chloropyrimidine-4,5-diamine (20b)

It was obtained in 47% yield from 18b; yellow foam; [α]_D²⁵ = −11.79 (c 0.36, MeOH); ¹H NMR (500 MHz, CDCl₃): δ 8.10 (s, 1H), 5.56 (d, J = 9.2 Hz, 1H), 4.89 (dt, J = 3.1, 51.0 Hz, 1H), 4.77 (dd, J = 9.1, 21.2 Hz, 1H), 4.61 (dd, J = 2.5, 5.0 Hz, 1H), 4.51 (dd, J = 2.4, 6.0 Hz, 1H), 3.60 (dd, J = 2.6, 9.2 Hz, 1H), 3.55 (dd, J = 2.5, 9.3 Hz, 1H), 3.39 (br s, 2H), 2.60 (d, J = 23.5 Hz, 1H), 1.54 (s, 3H), 1.29 (s, 3H), 1.21 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): δ 154.2, 149.6, 143.4, 122.4, 111.7, 101.3 (d, J = 185.1 Hz), 85.5 (d, J = 3.3 Hz), 82.0 (d, J = 2.6 Hz), 74.0, 63.7 (d, J = 26.6 Hz), 60.6 (d, J = 7.1 Hz), 51.3 (d, J = 20.5 Hz), 27.5 (3 × CH₃-tert-butyl), 27.1, 24.9; UV (MeOH): λ_max 297.60, 265.07 nm; HRMS (ESI⁺): found, 389.1762 [calcd for C₁₇H₂₇ClFN₄O₃⁺ (M + H)⁺, 389.1756]; Anal. Calcd for C₁₇H₂₆lFN₄O₃: C, 52.51; H, 6.50; N, 14.45. Found: C, 52.56; H, 6.51; N, 14.43.

N⁴-((3aS,4S,6R,6aR)-6-(tert-Butoxymethyl)-5,5-difluoro-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)-6-chloropyrimidine-4,5-diamine (20c)

It was obtained in 67% yield from 18c; yellow foam; [α]_D²⁵ = −61.76 (c 0.23, MeOH); ¹H NMR (500 MHz, CDCl₃): δ 8.11 (s, 1H), 5.71 (d, J = 10.1 Hz, 1H), 5.03 (t, J = 12.7 Hz, 1H), 4.56 (t, J = 4.6 Hz, 1H), 4.40–4.45 (m, 1H), 3.69 (dd, J = 2.6, 9.5 Hz, 1H), 3.57 (dd, J = 4.4, 9.4 Hz, 1H), 3.38 (br s, 2H), 2.72 (d, J = 14.7 Hz, 1H), 1.53 (s, 3H), 1.44 (s, 3H), 1.25 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): δ 154.5, 149.6, 143.9, 128.0 (dd, J = 257.3, 260.0 Hz), 122.3, 111.7, 84.5, 79.7 (d, J = 4.1 Hz), 74.5, 61.7 (dd, J = 18.1, 31.9 Hz), 58.3 (t, J = 5.8 Hz), 51.6 (t, J = 22.6 Hz), 27.5 (3 × CH₃-tert-butyl), 26.7, 24.6; UV (MeOH): λ_max 297.39, 263.29 nm; HRMS (ESI⁺): found, 407.1658 [calcd for C₁₇H₂₆ClF₂N₄O₃⁺ (M + H)⁺, 407.1661]; Anal. Calcd for C₁₇H₂₅ClF₂N₄O₃: C, 50.19; H, 6.19; N, 13.77. Found: C, 50.11; H, 6.23; N, 13.65.

General Procedure for the Synthesis of 21a–c

A solution of 20a–c in diethoxymethyl acetate (0.15 M) was placed under microwave irradiation at 140 °C for 3 h. The mixture was then co-evaporated with MeOH three times, and the resulting residue was purified with column chromatography (silica gel, hexane/EtOAc, 7/1) to give 21a–c.

9-((3aS,4S,5R,6R,6aR)-6-(tert-Butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)-6-chloro-9H-purine (21a)

It was obtained in 96% yield as yellow foam; [α]_D²⁵ = −29.2 (c 0.17, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃): δ 8.74 (s, 1H), 8.34 (d, J = 2.4 Hz, 1H), 5.28–5.43 (td, J = 2.8, 52.8 Hz, 1H), 5.12–5.23 (m, 2H), 4.61 (t, J = 5.0 Hz, 1H), 3.65–3.69 (m, 1H), 3.61 (t, J = 9.2 Hz, 1H), 2.56–2.71 (m, 1H), 1.56 (s, 3H), 1.32 (s, 3H), 1.17 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 152.3, 151.4, 144.2, 144.1, 131.4, 115.4, 97.7–95.9 (d, J = 181.2 Hz), 82.9, 80.1, 73.5, 63.1 (d, J = 16.1 Hz), 58.0 (d, J = 7.4 Hz), 50.0 (d, J = 17.5 Hz), 27.6 (3 × CH₃-tert-butyl), 27.5, 25.1; ¹⁹F NMR (376 MHz, CDCl₃) −202.6–202.9 (m); UV (CH₂Cl₂): λ_max 271 nm; LRMS (ESI⁺): found, 399.16 [calcd for C₁₈H₂₅ClFN₄O₃⁺ (M + H)⁺, 399.1599]; Anal. Calcd for C₁₈H₂₄ClFN₄O₃: C, 54.20; H, 6.06; N, 14.05. Found: C, 54.12; H, 6.34; N, 14.23.

9-((3aS,4S,5S,6R,6aR)-6-(tert-Butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)-6-chloro-9H-purine (21b)

It was obtained in 76% yield as yellow foam; [α]_D²⁵ = −31.54 (c 0.54, MeOH); ¹H NMR (500 MHz, CDCl₃): δ 8.67 (s, 1H), 8.15 (s, 1H), 5.55 (dt, J = 8.4, 53.6 Hz, 1H), 5.02 (t, J = 6.4 Hz, 1H), 4.84–4.94 (m, 1H), 4.65 (t, J = 5.1 Hz, 1H), 3.53–3.63 (m, 2H), 2.47–2.57 (m, 1H), 1.54 (s, 3H), 1.25 (s, 3H), 1.17 (s, 9H); ¹³C NMR (150 MHz, CDCl₃): δ 151.7, 151.5, 151.3, 144.8, 132.3, 113.1, 93.9 (d, J = 191.0 Hz), 79.1 (d, J = 7.9 Hz), 77.6 (d, J = 7.9 Hz), 73.1, 67.8 (d, J = 20.8 Hz), 58.1, 48.7 (d, J = 18.7 Hz) 27.5 (3 × CH₃-tert-butyl), 27.3, 25.0; UV (MeOH): λ_max 264.36 nm; HRMS (ESI⁺): found, 399.1589 [calcd for C₁₈H₂₅ClFN₄O₃⁺ (M + H)⁺, 399.1599]; Anal. Calcd for C₁₈H₂₄ClFN₄O₃: C, 54.20; H, 6.06; N, 14.05. Found: C, 54.34; H, 6.46; N, 13.99.

9-((3aS,4S,6R,6aR)-6-(tert-Butoxymethyl)-5,5-difluoro-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)-6-chloro-9H-purine (21c)

It was obtained in 92% yield as yellow foam; [α]_D²⁵ = −46.05 (c 0.43, MeOH); ¹H NMR (500 MHz, CDCl₃): δ 8.73 (s, 1H), 8.28 (d, J = 2.1 Hz, 1H), 5.30 (dt, J = 6.9, 20.1 Hz, 1H), 5.10 (t, J = 6.7 Hz, 1H), 4.57–4.62 (m, 1H), 3.63–3.73 (m, 2H), 2.81–2.93 (m, 1H), 1.56 (s, 3H), 1.30 (s, 3H), 1.18 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): δ 152.4, 152.4, 151.3, 143.9 (d, J = 4.0 Hz), 131.2, 125.6 (dd, J = 253.4, 264.6 Hz), 114.0, 79.5 (d, J = 7.7 Hz), 77.9 (d, J = 7.5 Hz), 73.7, 64.6 (dd, J = 19.3, 24.3 Hz), 57.1 (d, J = 7.1 Hz), 50.3 (t, J = 19.8 Hz), 27.3 (3 × CH₃-tert-butyl), 27.2, 25.0; UV (MeOH): λ_max 263.74 nm; HRMS (ESI⁺): found, 417.1500 [calcd for C₁₈H₂₄ClF₂N₄O₃⁺ (M + H)⁺, 417.1505]; Anal. Calcd for C₁₈H₂₃ClF₂N₄O₃: C, 51.86; H, 5.56; N, 13.44. Found: C, 51.56; H, 5.96; N, 13.13.

General Procedure for the Synthesis of 2a–c

To a solution of 21a–c in tert-butanol (2 mL, 0.27 M) contained in a stainless steel bomb reactor, saturated ammonia in tert-butanol (15 mL) was added and the reactor was locked. After being heated to 120 °C with stirring for 15 h, the mixture was cooled to room temperature and co-evaporated with MeOH. Without purification, the residue was added to a TFA/H₂O solution (2:1, v/v, total 15 mL) and heated to 50 °C with stirring for 15 h. After the reaction mixture was evaporated, the residue was purified by column chromatography (silica gel, CH₂Cl₂/MeOH, 9/1) to give 2a–c.

(1R,2S,3S,4R,5R)-3-(6-Amino-9H-purin-9-yl)-4-fluoro-5-(hydroxymethyl)cyclopentane-1,2-diol (2a)

It was obtained in 43% yield as a white solid; mp 172–177 °C; [α]_D²⁵ = −64.49 (c 0.22, MeOH); ¹H NMR (800 MHz, CD₃OD-d₆): δ 8.26 (d, J = 2.0 Hz, 1H), 8.21 (s, 1H), 5.21 (dt, J = 4.0, 54.6, 1H), 4.99 (ddd, J = 3.4, 10.8, 29.5 Hz, 1H), 4.75 (dd, J = 6.7, 9.4 Hz, 1H), 4.02 (dd, J = 4.8, 6.4 Hz, 1H), 3.79–3.85 (m, 2H), 2.42–2.51 (m, 1H); ¹³C NMR (200 MHz, CD₃OD): δ 158.1, 154.6, 152.2, 142.4 (d, J = 3.3 Hz), 120.5, 92.8 (d, J = 180.7 Hz), 74.3, 71.8, 64.0 (d, J = 17.0 Hz), 60.6 (d, J = 10.7 Hz), 54.3 (d, J = 17.9 Hz); ¹⁹F NMR (376 MHz, CD₃OD): δ −204.7 to −205.4 (m); UV (MeOH): λ_max 259.90 nm; HRMS (ESI⁺): found, 284.1161 [calcd for C₁₁H₁₅FN₅O₃⁺ (M + H)⁺, 284.1159]; Anal. Calcd for C₁₁H₁₄FN₅O₃: C, 46.64; H, 4.98; N, 24.72. Found: C, 46.65; H, 5.38; N, 25.10.

(1R,2S,3S,4S,5R)-3-(6-Amino-9H-purin-9-yl)-4-fluoro-5-(hydroxymethyl)cyclopentane-1,2-diol (2b)

It was obtained in 71% yield as a white solid; mp 182–186 °C; [α]_D²⁵ = −11.85 (c 0.26, MeOH); ¹H NMR (500 MHz, CD₃OD): δ 8.19 (s, 1H), 8.18 (s, 1H), 5.40 (ddd, J = 5.2, 7.3, 54.4 Hz, 1H), 5.03 (ddd, J = 7.5, 9.8, 20.7 Hz, 1H), 4.60 (dd, J = 5.1, 9.9 Hz, 1H), 4.05–4.09 (m, 1H), 3.80 (d, J = 5.8 Hz, 2H), 2.28–2.40 (m, 1H); ¹³C NMR (125 MHz, CD₃OD): δ 158.0, 154.3, 151.9, 143.4, 121.6, 95.8 (d, J = 186.4 Hz), 74.2 (d, J = 7.4 Hz), 73.2 (d, J = 3.3 Hz), 68.6 (d, J = 21.1 Hz), 62.6, 54.6 (d, J = 19.0 Hz); ¹⁹F NMR (378 MHz, CD₃OD): δ −185.244 (dt, J = 23.8, 53.7 Hz); UV (MeOH): λ_max 260.88 nm; HRMS (ESI⁺): found, 284.1155 [calcd for C₁₁H₁₅FN₅O₃⁺ (M + H)⁺, 284.1159]; Anal. Calcd for C₁₁H₁₄FN₅O₃: C, 46.64; H, 4.98; N, 24.72. Found: C, 46.38; H, 5.12; N, 24.33.

(1R,2S,3S,5R)-3-(6-Amino-9H-purin-9-yl)-4,4-difluoro-5-(hydroxymethyl)cyclopentane-1,2-diol (2c)

It was obtained in 61% yield as a white solid; mp 180–185 °C; [α]_D²⁵ = −56.51 (c 0.30, MeOH); ¹H NMR (500 MHz, CD₃OD): δ 8.26 (d, J = 19.5 Hz, 1H), 8.20 (s, 1H), 5.33 (dt, J = 10.0, 17.0 Hz, 1H), 4.79 (dd, J = 5.2, 10.6 Hz, 1H, merged with solvent peak), 4.13–4.17 (m, 1H), 3.79–3.91 (m, 2H), 2.60–2.71 (m, 1H); ¹³C NMR (200 MHz, CD₃OD): δ 158.2, 154.8, 152.6, 142.7 (d, J = 2.4 Hz), 125.9 (dd, J = 252.3, 258.4 Hz), 120.6, 73.7 (d, J = 7.3 Hz), 71.8 (d, J = 3.3 Hz), 64.8 (dd, J = 19.4, 23.8 Hz), 59.6 (d, J = 10.8 Hz), 56.4 (t, J = 19.9 Hz); ¹⁹F NMR (378 MHz, CD₃OD): δ −97.5 (d, J = 238.5 Hz), −115.4 (dt, J = 15.9, 238.9 Hz); UV (MeOH): λ_max 259.92 nm; HRMS (ESI⁺): found, 302.1066 [calcd for C₁₁H₁₄F₂N₅O₃⁺ (M + H)⁺, 302.1065]; Anal. Calcd for C₁₁H₁₃F₂N₅O₃: C, 43.86; H, 4.35; N, 23.25. Found: C, 44.17; H, 4.14; N, 23.05.

General Procedure for the Synthesis of 2d and 2e

To a solution of 21a and 21c (0.283 mmol) in EtOH (1.5 mL, 0.19 M) in a sealed glass tube, methylamine (40 wt % in H₂O, 10 mL) was added. After being stirred at room temperature for 2 h, the mixture was concentrated and added to a TFA/H₂O solution (2:1, v/v, total 15 mL) without purification. After being heated to 50 °C with stirring for 15 h, the reaction mixture was evaporated. The residue was purified by column chromatography (silica gel, CH₂Cl₂/MeOH, 9/1) to give 2d and 2e.

(1S,2R,3R,4R,5S)-4-Fluoro-3-(hydroxymethyl)-5-(6-(methylamino)-9H-purin-9-yl)cyclopentane-1,2-diol (2d)

It was obtained in 67% yield as a white solid; mp 197–201 °C; [α]_D²⁵ = −61.46 (c 0.40, MeOH); ¹H NMR (800 MHz, CD₃OD): δ 8.27 (s, 1H), 8.20 (d, J = 18.4 Hz, 1H), 5.21 (dt, J = 4.0, 54.6 Hz, 1H), 4.98 (ddd, J = 3.4, 10.0, 29.6 Hz, 1H), 4.74 (dd, J = 6.7, 9.4 Hz, 1H), 4.01 (dd, J = 4.9, 6.4 Hz, 1H), 3.79–3.85 (m, 2H), 3.11 (br s, 3H), 2.42–2.51 (m, 1H); ¹³C NMR (200 MHz, CD₃OD): δ 157.5, 154.6, 151.1, 141.8 (d, J = 3.7 Hz), 121.1, 92.9 (d, J = 180.8 Hz), 74.3, 71.8, 64.0 (d, J = 17.0 Hz), 60.6 (d, J = 10.5 Hz), 54.3 (d, J = 18.0 Hz), 28.5; ¹⁹F NMR (376 MHz, CD₃OD): δ −206.3 (dt, J = 29.7, 53.4 Hz); UV (MeOH): λ_max 266.89 nm; HRMS (ESI⁺): found, 298.1317 [calcd for C₁₂H₁₇FN₅O₃⁺ (M + H)⁺, 298.1315]; Anal. Calcd for C₁₂H₁₆FN₅O₃: C, 48.48; H, 5.42; N, 23.56. Found: C, 48.50; H, 5.22; N, 23.93.

(1S,2R,3R,5S)-4,4-Difluoro-3-(hydroxymethyl)-5-(6-(methylamino)-9H-purin-9-yl)cyclopentane-1,2-diol (2e)

It was obtained in 76% yield as a white solid; mp 125–129 °C; [α]_D²⁵ = −48.62 (c 0.25, MeOH); ¹H NMR (500 MHz, CD₃OD): δ 8.24 (s, 1H), 8.20 (s, 1H), 5.33 (dt, J = 9.9, 18.4 Hz, 1H), 4.79 (dd, J = 10.3, 10.2 Hz, 1H), 4.17 (s, 1H), 3.81–3.90 (m, 2H), 3.10 (br s, 3H), 2.67 (m, 1H); ¹³C NMR (125 MHz, CD₃OD): δ 157.5, 154.7, 151.5, 142.1, 125.9 (dd, J = 252.4, 258.1 Hz), 121.1, 73.7 (d, J = 7.25 Hz), 71.9 (d, J = 3.1 Hz), 64.7 (dd, J = 20.0, 24.3 Hz), 59.6 (d, J = 10.8 Hz), 56.4 (t, J = 19.9 Hz), 28.6; ¹⁹F NMR (378 MHz, CD₃OD): δ −97.4 (d, J = 238.5 Hz), −115.3 (d, J = 238.9 Hz); UV (MeOH): λ_max 263.72 nm; HRMS (ESI⁺): found, 316.1227 [calcd for C₁₂H₁₆F₂N₅O₃⁺ (M + H)⁺, 316.1221]; Anal. Calcd for C₁₂H₁₅F₂N₅O₃: C, 45.71; H, 4.80; N, 22.21. Found: C, 45.99; H, 4.47; N, 22.02.

General Procedure for the Synthesis of 22a–c

To a cooled (−20 °C) solution of 19a–c (1 equiv) in DMF (0.2 M), 3-methoxyacryloyl isocyanate (2 equiv) in benzene was added dropwise in a N₂ atmosphere. After the reaction mixture was slowly warmed to room temperature for 15 h with stirring, the reaction mixture was filtered with CH₂Cl₂ and co-evaporated with toluene and ethanol. The residue was purified by column chromatography (silica gel, hexane/EtOAc, 1.5/1) to give 22a–c.

(E)-N-(((3aS,4S,5R,6R,6aR)-6-(tert-Butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)carbamoyl)-3-methoxyacrylamide (22a)

It was obtained in 76% yield as a colorless syrup; [α]_D²⁵ = −19.41 (c 0.37, MeOH); ¹H NMR (600 MHz, CDCl₃): δ 10.24 (s, 1H), 9.16 (d, J = 8.2 Hz, 1H), 7.61 (d, J = 12.4 Hz, 1H), 5.35 (d, J = 12.4 Hz, 1H), 5.06 (dt, J = 3.2, 52.7 Hz, 1H), 4.51 (t, J = 6.6 Hz, 1H), 4.29–4.38 (m, 2H), 3.64 (s, 3H), 3.45–3.52 (m, 2H), 2.21–2.31 (m, 1H), 1.41 (s, 3H), 1.21 (s, 3H), 1.10 (s, 9H); ¹³C NMR (150 MHz, CDCl₃): δ 168.0, 163.3, 155.4, 113.7, 97.5, 96.7 (d, J = 178.8 Hz), 84.4, 80.1, 72.9, 58.6 (d, J = 15.8 Hz), 57.8 (d, J = 6.5 Hz), 57.4, 49.8 (d, J = 17.2 Hz), 27.2 (3 × CH₃-tert-butyl), 27.1, 24.6; UV (MeOH): λ_max 243.14 nm; HRMS (ESI⁺): found, 389.2088 [calcd for C₁₈H₃₀FN₂O₆⁺ (M + H)⁺, 389.2088].

(E)-N-(((3aS,4S,5S,6R,6aR)-6-(tert-Butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)carbamoyl)-3-methoxyacrylamide (22b)

It was obtained in 88% yield as a colorless syrup; [α]_D²⁵ = −20.47 (c 0.34, MeOH); ¹H NMR (500 MHz, CDCl₃): δ 10.33 (s, 1H), 8.96 (d, J = 7.4 Hz, 1H), 7.63 (d, J = 12.3 Hz, 1H), 5.39 (d, J = 12.3 Hz, 1H), 4.80 (dt, J = 6.4, 52.5 Hz, 1H), 4.44 (t, J = 5.5 Hz, 1H), 4.33–4.41 (m, 2H), 3.67 (s, 3H), 3.46 (d, J = 32.5 Hz, 2H), 2.33–2.42 (m, 1H), 1.46 (s, 3H), 1.24 (s, 3H), 1.13 (s, 9H); ¹³C NMR (150 MHz, CDCl₃): δ 168.1, 163.2, 155.5, 111.9, 97.9 (d, J = 187.4 Hz), 97.5, 83.3 (d, J = 7.2 Hz), 79.0 (d, J = 6.5 Hz), 73.1, 61.9 (d, J = 23.7 Hz), 58.6 (d, J = 2.1 Hz), 57.4, 49.9 (d, J = 19.4 Hz), 27.3 (3 × CH₃-tert-butyl), 27.2, 25.0; UV (MeOH): λ_max 242.93 nm; HRMS (ESI⁺): found, 389.2098 [calcd for C₁₈H₃₀FN₂O₆⁺ (M + H)⁺, 389.2088].

(E)-N-(((3aS,4S,6R,6aR)-6-(tert-Butoxymethyl)-5,5-difluoro-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)carbamoyl)-3-methoxyacrylamide (22c)

It was obtained in 90% yield as a colorless syrup; [α]_D²⁵ = −40.41 (c 0.52, MeOH); ¹H NMR (500 MHz, CDCl₃): δ 10.26 (s, 1 Η), 9.11 (d, J = 8.7 Hz, 1H), 7.65 (d, J = 12.3 Hz, 1H), 5.37 (d, J = 12.4 Hz, 1H), 4.52–4.62 (m, 1H), 4.39 (s, 2H), 3.67 (s, 3H), 3.53–3.60 (m, 2H), 2.57–2.68 (m, 1H), 1.47 (s, 3H), 1.27 (s, 3H), 1.16 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): δ 167.9, 163.4, 155.7, 126.9 (dd, J = 252.9, 261.3 Hz), 112.4, 97.4, 82.5 (d, J = 6.9 Hz), 78.6 (d, J = 4.9 Hz), 73.5, 60.6 (dd, J = 19.4, 29.2 Hz), 57.4 (d, J = 6.1 Hz), 57.3, 50.8 (t, J = 20.8 Hz), 27.1 (3 × CH₃-tert-butyl), 27.0, 24.9; UV (MeOH): λ_max 242.22 nm; HRMS (ESI⁺): found, 407.1991 [calcd for C₁₈H₂₉F₂N₂O₆⁺ (M + H)⁺, 407.1994].

General Procedure for the Synthesis of 2f–h

To a stirred solution of 22a–c in 1,4-dioxane (3 mL, 2.5 M), 2 M sulfuric acid (0.3 mL) was dropwise added. After refluxing with stirring for 1 h, the reaction mixture was cooled to room temperature and neutralized with DOWEX 66 ion-exchange resin. The mixture was filtered and evaporated. The residue was purified by column chromatography (silica gel, CH₂Cl₂/MeOH, 9/1) to give 2f–h.

1-((1S,2R,3R,4R,5S)-2-Fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopentyl)pyrimidine-2,4(1H,3H)-dione (2f)

It was obtained in 56% yield as a white solid; mp 112–118 °C; [α]_D²⁵ = −77.11 (c 0.20, MeOH); ¹H NMR (500 MHz, CD₃OD): δ 7.70 (dd, J = 1.1, 8.1 Hz, 1H), 5.69 (d, J = 8.0 Hz, 1H), 5.10 (dt, J = 4.1, 55.3 Hz, 1H), 4.91 (dd, J = 3.4, 10.2 Hz, 1H, merged with solvent peak), 4.46 (dd, J = 6.6, 10.1 Hz, 1H), 3.93 (t, J = 4.8 Hz, 1H), 3.70–3.80 (m, 2H), 3.69 (s, 1H), 2.29–2.41 (m, 1H); ¹³C NMR (125 MHz, CD₃OD): δ 166.9, 154.2, 145.5 (d, J = 3.8 Hz), 102.7, 93.0 (d, J = 180.1 Hz), 72.4, 71.7, 64.4 (d, J = 16.6 Hz), 60.6 (d, J = 11.4 Hz), 53.8 (d, J = 17.9 Hz); ¹⁹F NMR (378 MHz, CD₃OD): δ −208.9 (dt, J = 29.9, 59.7 Hz); UV (MeOH): λ_max 264.11 nm; HRMS (ESI⁺): found, 261.0886 [calcd for C₁₀H₁₄FN₂O₅⁺ (M + H)⁺, 261.0887]; Anal. Calcd for C₁₀H₁₃FN₂O₅: C, 46.16; H, 5.04; N, 10.77. Found: C, 45.98; H, 5.44; N, 10.98.

1-((1S,2S,3R,4R,5S)-2-Fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopentyl)pyrimidine-2,4(1H,3H)-dione (2g)

It was obtained in 53% yield as a white solid; mp 195–200 °C; [α]_D²⁵ = −16.89 (c 0.35, MeOH); ¹H NMR (500 MHz, CD₃OD): δ 7.60 (d, J = 7.9 Hz, 1H), 5.69 (d, J = 7.9 Hz, 1H), 5.07–5.21 (ddd, J = 5.10, 6.8, 55.2 Hz, 1H), 4.61–4.69 (ddd, J = 7.3, 8.7, 22.6 Hz, 1H), 4.32 (dd, J = 5.25, 9.0 Hz, 1H), 3.98 (t, J = 3.7 Hz, 1H), 3.70 (m, 2H), 2.24 (m, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 167.1, 153.6, 147.4, 103.5, 94.8 (d, J = 183.9 Hz), 73.4 (d, J = 7.3 Hz), 73.1 (d, J = 22.0 Hz), 72.7 (d, J = 3.5 Hz), 62.3 (d, J = 1.8 Hz), 54.1 (d, J = 18.9 Hz); ¹⁹F NMR (378 MHz, CD₃OD): δ −184.3 (dt, J = 23.8, 53.7 Hz); UV (MeOH): λ_max 265.33 nm; HRMS (ESI⁺): found, 261.0894 [calcd for C₁₀H₁₄FN₂O₅⁺ (M + H)⁺, 261.0887]; Anal. Calcd for C₁₀H₁₃FN₂O₅: C, 46.16; H, 5.04; N, 10.77. Found: C, 46.24; H, 5.23; N, 10.78.

1-((1S,3R,4R,5S)-2,2-Difluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopentyl)pyrimidine-2,4(1H,3H)-dione (2h)

It was obtained in 52% yield as a white solid; mp 164–169 °C; [α]_D²⁵ = −31.06 (c 0.30, MeOH); ¹H NMR (500 MHz, CD₃OD): δ 7.67 (dd, J = 2.3, 8.1 Hz, 1H) 5.71 (d, J = 8.0 Hz, 1H), 5.36 (dt, J = 10.3, 17.7 Hz, 1H), 4.41 (dd, J = 5.15, 10.7 Hz, 1H), 4.07 (m, 1H), 3.73–3.82 (m, 2H), 2.53 (m, 1H); ¹³C NMR (150 MHz, CD₃OD): δ 166.6, 154.1, 145.3 (d, J = 4.3 Hz), 126.8 (dd, J = 252.8, 258.5 Hz), 103.4, 72.5 (d, J = 7.9 Hz), 71.8 (d, J = 2.9 Hz), 64.4 (dd, J = 18.7, 25.1 Hz), 59.5 (d, J = 11.5 Hz), 56.3 (t, J = 20.1 Hz); ¹⁹F NMR (378 MHz, CD₃OD): δ −96.6 (d, J = 238.9 Hz), −116.9 (dt, J = 15.1, 238.5 Hz); UV (MeOH): λ_max 262.41 nm; HRMS (ESI⁺): found, 279.0801 [calcd for C₁₀H₁₃F₂N₂O₅⁺ (M + H)⁺, 279.0793]; Anal. Calcd for C₁₀H₁₂F₂N₂O₅: C, 43.17; H, 4.35; N, 10.07. Found: C, 43.34; H, 4.67; N, 9.94.

General Procedure for the Synthesis of 2i and 2j

Benzoylation

To a cooled (0 °C) solution of 2f or 2h (1 equiv) in CH₂Cl₂ (0.07 M), benzoyl chloride (6 equiv) and pyridine (6.7 equiv) were added in a N₂ atmosphere. After being stirred for 15 h at room temperature, the reaction mixture was quenched with H₂O and extracted with CH₂Cl₂. The organic layers were combined and washed with H₂O followed by brine, dried over MgSO₄, filtered, and evaporated. The residue was purified with column chromatography (silica gel, hexane/EtOAc, 1/1) to give the benzoylated intermediate.

Introduction of Triazole

To a cooled (0 °C) suspension of 1,2,4-triazole (10 equiv) in anhydrous MeCN (0.6 M), phosphoryl chloride (10 equiv) was added dropwise in a N₂ atmosphere. After stirring, the benzoylated intermediate (1 equiv) in MeCN (0.14 M) followed by trimethylamine (10 equiv) were added to the reaction mixture. After additional stirring at room temperature for 15 h, the reaction mixture was evaporated. The reaction mixture was diluted with CH₂Cl₂ and H₂O. The layers were separated, and the organic layers were washed with H₂O, dried over MgSO₄, filtered, and evaporated.

Amination

In the sealed glass tube, the above-generated intermediate in 1,4-dioxane (0.06 M) was added to excess saturated aqueous ammonia at room temperature. After being stirred at the same temperature for 2 h, the reaction mixture was evaporated and purified with flash chromatography (silica gel, CH₂Cl₂/MeOH, 7/1) to give the benzoyl protected cytosine intermediate.

Benzoyl Deprotection

In a sealed glass tube, the above-generated benzoyl-protected cytosine intermediate in MeOH (0.2 M) was added to saturated ammonia in MeOH (0.2 M). After being stirred at the same temperature for 2 d, the reaction mixture was evaporated and diluted with H₂O and CH₂Cl₂. The layers were separated, and the H₂O layers were washed with CH₂Cl₂ 10 times and evaporated to give 2i and 2j, respectively.

4-Amino-1-((1S,2R,3R,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopentyl)pyrimidin-2(1H)-one (2i)

It was obtained in 17% yield as a white solid; mp 230–233 °C; [α]_D²⁵ = −84.26 (c 0.20, MeOH); ¹H NMR (800 MHz, CD₃OD): δ 7.67 (dd, J = 1.3, 7.5 Hz, 1H), 5.88 (d, J = 7.4 Hz, 1H), 5.23 (dt, J = 3.7, 55.4 Hz, 1H), 4.93 (ddd, J = 3.4, 10.3, 30.4 Hz, 1H), 4.44 (dd, J = 6.6, 10.3 Hz, 1H), 3.92 (dd, J = 4.5, 6.3 Hz, 1H), 3.71–3.78 (m, 2H), 2.31–2.40 (m, 1H); ¹³C NMR (200 MHz, CDCl₃): δ 168.3, 160.3, 145.7 (d, J = 3.1 Hz), 96.2, 93.0 (d, J = 179.9 Hz), 72.5, 71.8, 65.3 (d, J = 16.6 Hz), 60.7 (d, J = 11.3 Hz), 53.9 (d, J = 17.9 Hz); ¹⁹F NMR (376 MHz, CD₃OD): δ −209.4 (dt, J = 29.3, 53.4 Hz); UV (MeOH): λ_max 274.67 nm; HRMS (ESI⁺): found, 260.1041 [calcd for C₁₀H₁₅FN₃O₄⁺ (M + H)⁺, 260.1047]; Anal. Calcd for C₁₀H₁₄FN₃O₄: C, 46.33; H, 5.44; N, 16.21. Found: C, 46.71; H, 5.12; N, 15.99.

4-Amino-1-((1S,3R,4R,5S)-2,2-difluoro-4,5-dihydroxy-3 (hydroxymethyl)cyclopentyl)pyrimidin-2(1H)-one (2j)

It was obtained in 20% yield as a white solid; mp 242–245 °C; [α]_D²⁵ = −39.85 (c 0.30, MeOH); ¹H NMR (500 MHz, CD₃OD): δ 7.62 (dd, J = 7.45, 2.35 Hz, 1H), 5.90 (d, J = 7.40 Hz, 1H), 5.51 (dt, J = 18.2, 10.0 Hz, 1H), 4.37 (dd, J = 10.6, 5.25 Hz, 1H), 4.06 (m, 1H), 3.73–3.83 (m, 2H), 2.54 (m, 1H); ¹³C NMR (150 MHz, CD₃OD): δ 168.2, 160.1, 145.7 (d, J = 3.6 Hz), 126.9 (dd, J = 252.1, 259.2 Hz), 96.8, 72.9 (d, J = 8.6 Hz), 71.7 (d, J = 3.6 Hz), 65.1 (dd, J = 18.7, 23.0 Hz), 59.6 (d, J = 10.8 Hz), 56.3 (t, J = 20.1 Hz); ¹⁹F NMR (378 MHz, CD₃OD): δ −97.4 (d, J = 235.9 Hz), −117.4 (dt, J = 14.7, 238.9 Hz); UV (MeOH): λ_max 272.27, 237.93 nm; HRMS (ESI⁺): found, 278.0954 [calcd for C₁₀H₁₄F₂N₃O₄⁺ (M + H)⁺, 278.0952]; Anal. Calcd for C₁₀H₁₃F₂N₃O₄: C, 43.32; H, 4.73; N, 15.16. Found: C, 43.56; H, 4.56; N, 15.44.

General Procedure for the Synthesis of 24, 27a and 27b

To a cooled (0 °C) suspension of 2c, 2f, and 2h (1 equiv) in acetone (0.005 M) were added 1–2 drops of cH₂SO₄ in N₂ (g). After being stirred at room temperature for 4 h, the reaction mixture was neutralized with solid NaHCO₃, filtered, and evaporated under reduced pressure. The residue was further purified by silica gel column chromatography to give 24, 27a, and 27b, respectively.

((3aR,4R,6S,6aS)-6-(6-Amino-9H-purin-9-yl)-5,5-difluoro-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)methanol (24)

It was obtained in 96% yield as a colorless syrup; ¹H NMR (500 MHz, CD₃OD): δ 8.31 (s, 1H), 8.21 (s, 1H), 5.30–5.40 (m, 2H), 4.70 (br s, 1H), 3.94 (dd, J = 6.8, 11.4 Hz, 1H), 3.86 (dd, J = 6.8, 11.4 Hz, 1H), 2.81–2.90 (m, 1H), 1.58 (s, 3H), 1.35 (s, 3H); ¹³C NMR (125 MHz, CD₃OD): δ 163.5 (dd, J = 33.1, 69.2 Hz), 156.5, 152.2, 152.0, 143.4, 128.0 (dd, J = 251.7, 263.6 Hz), 116.0, 80.7 (d, J = 7.3 Hz), 79.6 (d, J = 8.3 Hz), 66.1 (dd, J = 19.2, 22.8 Hz), 59.2 (d, J = 8.0 Hz), 53.8 (t, J = 19.4 Hz), 28.2, 25.9; HRMS (ESI⁺) (m/z): found, 342.1370 [calcd for C₁₄H₁₈F₂N₅O₃⁺ (M + H)⁺, 342.1372]; Anal. Calcd for C₁₄H₁₇F₂N₅O₃: C, 49.27; H, 5.02; N, 20.52. Found: C, 49.28; H, 4.98; N, 20.91.

1-((3aS,4S,5R,6R,6aR)-5-Fluoro-6-(hydroxymethyl)-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (27a)

It was obtained in 98% yield as a colorless syrup; ¹H NMR (500 MHz, CD₃OD): δ 7.75 (dd, J = 1.4, 8.1 Hz, 1H), 5.70 (d, J = 8.1 Hz, 1H), 5.20 (dt, J = 3.1, 54.1 Hz, 1H), 5.01–5.13 (m, 2H), 4.58 (d, J = 6.3 Hz, 1H), 3.73–3.83 (m, 2H), 2.42–2.56 (m, 1H), 1.50 (s, 3H), 1.32 (s, 3H); ¹³C NMR (125 MHz, CD₃OD): δ 166.7, 153.7, 145.3 (d, J = 5.9 Hz), 116.5, 103.2, 99.2 (d, J = 180.2 Hz), 82.2, 82.0, 65.0 (d, J = 15.7 Hz), 60.3 (d, J = 8.7 Hz), 53.2 (d, J = 17.7 Hz), 28.4, 25.9; HRMS (ESI⁺) (m/z): found, 301.1185 [calcd for C₁₃H₁₈FN₂O₅⁺ (M + H)⁺, 301.1194]; Anal. Calcd for C₁₃H₁₇FN₂O₅: C, 52.00; H, 5.71; N, 9.33. Found: C, 52.15; H, 5.47; N, 9.15.

1-((3aS,4S,6R,6aR)-5,5-Difluoro-6-(hydroxymethyl)-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (27b)

It was obtained in 97% yield; ¹H NMR (500 MHz, CD₃OD): δ 7.71 (dd, J = 2.0, 8.1 Hz, 1H), 5.73 (d, J = 8.1 Hz, 1H), 5.33 (dt, J = 6.8, 21.3 Hz, 1H), 4.94 (d, J = 6.8 Hz, 1H), 4.57–4.63 (m, 1H), 3.88 (dd, J = 6.7, 11.4 Hz, 1H), 3.81 (dd, J = 6.7, 11.4 Hz, 1H), 2.68–2.79 (m, 1H), 1.54 (s, 3H), 1.34 (s, 3H); HRMS (ESI⁺) (m/z): found, 319.1104 [calcd for C₁₃H₁₇F₂N₂O₅⁺ (M + H)⁺, 319.1100]; Anal. Calcd for C₁₃H₁₆F₂N₂O₅: C, 49.06; H, 5.07; N, 8.80. Found: C, 49.43; H, 5.47; N, 8.43.

Synthesis of tert-Butyl-(9-((3aS,4S,6R,6aR)-5,5-difluoro-6-(hydroxymethyl)-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)-9H-purin-6-yl)carbamate (25a) and Its N⁶-Di-Boc derivative (25b)

To a suspension of 24 (20 mg, 0.058 mmol) and 4-dimethylaminopyridine (1 mg, 0.0058 mmol) in hexamethyldisilazane (3 mL), trimethylsilyl trifluoromethanesulfonate (TMSOTf; 5 μL) was added dropwise at room temperature in a N₂ atmosphere (g). After being heated to 75 °C with stirring for 2 h, the reaction mixture was evaporated, and anhydrous THF (7 mL) was added. To a cooled (0 °C) reaction mixture, di-t-butyl dicarbonate (63 mg, 0.29 mmol) was added. After stirring for 4 h at room temperature, the reaction mixture was evaporated, and the residue was added to MeOH/trimethylamine (6 mL, 5:1 (v/v)). After heating to 55 °C with stirring for 16 h, the reaction mixture was evaporated, and the residue was purified with column chromatography (silica gel, CH₂Cl₂/MeOH, 50/1) to give 25a (13 mg, 52%) and 25b (8 mg, 25%) as a colorless syrup.

Compound 25a

¹H NMR (500 MHz, CD₃OD): δ 8.59 (s, 1H), 8.49 (s, 1H), 5.36–5.50 (m, 2H), 4.72 (d, J = 5.6 Hz, 1H), 3.95 (dd, J = 6.8, 11.4 Hz, 1H), 3.87 (dd, J = 6.8, 11.4 Hz, 1H), 2.83–2.95 (m, 1H), 1.57 (s, 12H), 1.34 (s, 3H); HRMS (ESI⁺) (m/z): found, 442.1899 [calcd for C₁₉H₂₆F₂N₅O₅⁺ (M + H)⁺, 442.1897].

Compound 25b

¹H NMR (500 MHz, CD₃OD): δ 8.87 (s, 1H), 8.73 (d, J = 1.8 Hz, 1H), 5.46–5.57 (m, 2H), 4.75 (d, J = 5.4 Hz, 1H), 3.95 (dd, J = 6.8, 11.4 Hz, 1H), 3.88 (dd, J = 6.8, 11.4 Hz, 1H), 2.84–2.95 (m, 1H), 1.59 (s, 3H), 1.37 (s, 21H); ¹³C NMR (125 MHz, CD₃OD): δ 156.2, 154.2, 152.2, 152.1 (2 × C(O)-Boc protection group), 147.8 (d, J = 2.4 Hz), 130.6, 128.1 (dd, J = 251.8, 263.3 Hz), 116.0, 86.1, 80.4 (d, J = 7.4 Hz), 79.7 (d, J = 8.2 Hz), 72.7, 66.5 (dd, J = 19.1, 23.1 Hz), 59.2 (d, J = 8.0 Hz), 53.8 (t, J = 19.2 Hz), 28.7 (6 × CH₃-tert-butyl), 28.3, 25.9; HRMS (ESI⁺) (m/z): found, 542.2411 [calcd for C₂₄H₃₄F₂N₅O₇⁺ (M + H)⁺, 542.2421].

Iso-propyl ((S)-(((3aR,4R,6S,6aS)-6-(6-((tert-Butoxycarbonyl)amino)-9H-purin-9-yl)-5,5-difluoro-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)-l-alaninate (26)

To a stirred suspension of 25a (16 mg, 0.036 mmol), 25b (7 mg, 0.012 mmol), and powdered molecular sieves (4 Å, 62 mg) in anhydrous THF (20 mL), tert-butylmagnesium chloride solution (0.26 mL, 1.0 M in THF, 0.26 mmol) was added at 0 °C in a nitrogen atmosphere. After 10 min, a solution of pentafluorophosphoramidate reagent A (47 mg, 0.10 mmol) in THF (12 mL) was slowly added, and the reaction mixture was stirred at room temperature for 36 h. Then, it was quenched by the dropwise addition of methanol (10 mL), filtered, and evaporated. The residue was purified by column chromatography (silica gel, CH₂Cl₂/MeOH, 9/1) to give the phosphoramidate 26 as a colorless liquid (12 mg, 33%): ¹H NMR (500 MHz, CD₃OD): δ 8.59 (s, 1H), 8.45 (s, 1H), 7.37 (d, J = 7.8 Hz, 2H), 7.25 (d, J = 8.1 Hz, 2H), 7.19 (d, J = 7.5 Hz, 1H), 5.50 (dt, J = 5.9, 22.3 Hz, 1H), 5.40–5.45 (m, 1H), 4.92–4.99 (m, 1H), 4.73–4.80 (m, 1H), 4.36–4.50 (m, 2H), 3.86–3.98 (m, 1H), 3.07–3.19 (m, 1H), 1.58 (s, 12H), 1.34 (s, 6H), 1.21 (d, J = 6.2 Hz, 3H), 1.17 (d, J = 6.2 Hz, 3H); HRMS (ESI⁺) (m/z): found, 711.2716 [calcd for C₃₁H₄₂F₂N₆O₉P⁺ (M + H)⁺, 711.2713].

Iso-propyl ((S)-(((1R,3S,4S,5R)-3-(6-Amino-9H-purin-9-yl)-2,2-difluoro-4,5-dihydroxycyclopentyl)methoxy)(phenoxy)phosphoryl)-l-alaninate (3a)

A solution of 26 (15 mg, 0.021 mmol) in 10 mL of formic acid/H₂O (1:1, v/v) was stirred at room temperature for 8 h. After evaporation, the crude product was purified by column chromatography (silica gel, CH₂Cl₂/MeOH, 6/1) to give 3a (9.9 mg, 82%) as a colorless solid: mp 95–100 °C; UV (MeOH): λ_max 259.6 nm; [α]_D²⁵ = −38.06 (c 0.1, MeOH); ¹H NMR (400 MHz, CD₃OD): δ 8.18 (s, 1H), 8.17 (d, J = 1.6 Hz, 1H), 7.35 (d, J = 8.4 Hz, 2H), 7.23 (d, J = 8.6 Hz, 2H), 7.18 (d, J = 8.0 Hz, 1H), 5.26–5.38 (m, 1H), 4.81–4.98 (m, merged with H₂O peak, 1H), 4.74 (dd, J = 4.8, 10.0 Hz, 1H), 4.29–4.43 (m, 2H), 7.17 (br s, 1H), 3.82–3.93 (m, 1H), 2.79–2.94 (m, 1H), 1.32 (d, J = 6.8 Hz, 3H), 1.19 (d, J = 6.2 Hz, 3H), 1.14 (d, J = 6.2 Hz, 3H); ¹³C NMR (150 MHz, CD₃OD): δ 175.2 (d, J = 5.7 Hz), 158.1, 154.7, 153.0, 152.9, 152.6, 142.6, 131.6 (2 × CH-phenyl), 127.0, 124.4 (dd, J = 253.5, 260.6 Hz), 122.2 (d, J = 4.3 Hz), 120.6 (2 × CH-phenyl), 73.3 (d, J = 7.1 Hz), 71.2 (d, J = 5.0 Hz), 70.9, 64.6, 64.1 (dd, J = 5.0, 10.7 Hz), 52.4, 22.6 (d, J = 2.9 Hz, 2 × CH₃), 21.2 (d, J = 6.5 Hz); ¹⁹F NMR (376 MHz, CD₃OD): δ −98.71 (d, J = 238.4 Hz), −115.13 (dt, J = 14.9, 236.4 Hz); HRMS (ESI⁺) (m/z): found, 571.1889 [calcd for C₂₃H₃₀F₂N₆O₇P⁺ (M + H)⁺, 571.1876]; Anal. Calcd for C₂₃H₂₉F₂N₆O₇P: C, 48.42; H, 5.12; N, 14.73. Found: C, 48.74; H, 4.98; N, 14.54.

Iso-propyl ((S)-(((1R,2R,3S,4S,5R)-3-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-fluoro-4,5-dihydroxycyclopentyl)methoxy)(phenoxy)phosphoryl)-l-alaninate (3b)

Introduction of Phosphoramidate

To a cooled (0 °C) suspension of 27a (21 mg, 0.069 mmol) and molecular sieves (4 Å, 35 mg) in anhydrous THF (15 mL, 0.005 M), tert-butylmagnesium chloride solution (0.34 mL, 1.0 M in THF, 0.34 mmol) was added dropwise in a N₂ atmosphere (g). After being stirred for 5 min, a solution of the phosphoramidate reagent A (31 mg, 0.069 mmol) in anhydrous THF (7 mL) was added dropwise, and the reaction mixture was stirred at room temperature for 36 h, quenched with MeOH (5 mL), filtered, and evaporated, and the residue was purified by column chromatograph (silica gel, CH₂Cl₂/MeOH, 24/1) to give phosphoramidate as a colorless liquid (13 mg, 33%): ¹H NMR (500 MHz, CD₃OD): δ 7.73 (dd, J = 1.3, 8.1 Hz, 1H), 7.36 (d, J = 7.8 Hz, 2H), 7.24 (d, J = 7.8 Hz, 2H), 7.19 (d, J = 7.4 Hz, 1H), 5.70 (d, J = 8.1 Hz, 1H), 5.02–5.22 (m, 3H), 4.93–5.01 (m, 1H), 4.66 (d, J = 6.3 Hz, 1H), 4.29 (d, J = 7.6 Hz, 2H), 3.87–3.95 (m, 1H), 2.62–2.73 (m, 1H), 1.51 (s, 3H), 1.34 (d, J = 7.7 Hz, 3H), 1.32 (s, 3H), 1.22 (d, J = 6.2 Hz, 6H); HRMS (ESI⁺) (m/z): found, 570.2003 [calcd for C₂₅H₃₄FN₃O₉P⁺ (M + H)⁺, 570.2011].

Hydrolysis

A solution of phosphoramidate (13 mg, 0.022 mmol) in a formic acid/H₂O solution (1:1, v/v, 7 mL total) was stirred at room temperature for 8 h. The reaction mixture was evaporated and the residue was purified by column chromatography (silica gel, CH₂Cl₂/MeOH, 7/1) to give the phosphoramidate prodrug 3b (10.8 mg, 90%) as a white solid: mp 107–110 °C; UV (MeOH): λ_max 262.8 nm; [α]_D²⁵ = −59.40 (c 0.1, MeOH); ¹H NMR (500 MHz, CD₃OD): δ 7.64 (d, J = 8.1 Hz, 1H), 7.36 (d, J = 7.9 Hz, 2H), 7.23 (d, J = 7.9 Hz, 2H), 7.19 (d, J = 7.4 Hz, 1H), 5.68 (d, J = 8.1 Hz, 1H), 5.04 (dt, J = 4.1, 55.4 Hz, 1H), 4.87–4.98 (m, merged with H₂O peak, 2H), 4.45 (dd, J = 6.6, 9.7 Hz, 1H), 4.26 (d, J = 7.1 Hz, 2H), 3.99 (d, J = 5.4 Hz, 1H), 3.85–3.93 (m, 1H), 2.49–2.60 (m, 1H), 1.33 (d, J = 7.0 Hz, 3H), 1.21 (d, J = 6.1 Hz, 6H); ¹³C NMR (125 MHz, CD₃OD): δ 175.2, 166.9, 154.0, 151.1, 145.4, 131.5 (2 × CH-phenyl), 126.9 (2 × CH-phenyl), 122.2 (d, J = 4.6 Hz), 102.8, 93.3 (d, J = 184.5 Hz), 80.3, 79.9 (d, J = 32.5 Hz), 72.3, 71.2, 70.9, 64.2 (d, J = 16.0 Hz), 52.4, 22.7 (d, J = 9.2 Hz, 2 × CH₃), 21.2 (d, J = 6.8 Hz); ¹⁹F NMR (376 MHz, CD₃OD): δ −208.27 (dt, J = 29.7, 59.4 Hz); HRMS (ESI⁺) (m/z): found, 530.1685 [calcd for C₂₂H₃₀FN₃O₉P⁺ (M + H)⁺, 530.1698]; Anal. Calcd for C₂₂H₂₉FN₃O₉P: C, 49.91; H, 5.52; N, 7.94. Found: C, 50.03; H, 5.32; N, 7.54.

Iso-propyl ((S)-(((1R,3S,4S,5R)-3-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-difluoro-4,5-dihydroxycyclopentyl)methoxy)(phenoxy)phosphoryl)-l-alaninate (3c)

Compound 3c was synthesized according the same procedure used in the preparation of 3b: yield = 30%; white solid; mp 174 °C (decomp); UV (MeOH): λ_max 262.8 nm; [α]_D²⁵ = −19.40 (c 0.1, MeOH); ¹H NMR (500 MHz, CD₃OD): δ 7.53 (dd, J = 2.1, 8.1 Hz, 1H), 7.36 (d, J = 7.8 Hz, 2H), 7.25 (d, J = 7.8 Hz, 2H), 7.20 (d, J = 7.6 Hz, 1H), 5.70 (d, J = 8.1 Hz, 1H), 5.29–5.39 (m, 1H), 4.93–5.02 (m, 1H), 4.30–4.39 (m, 2H), 4.23–4.29 (m, 1H), 4.08 (br s, 1H), 3.84–3.92 (m, 1H), 2.69–2.80 (m, 1H), 1.33 (d, J = 7.1 Hz, 3H), 1.22 (d, J = 6.2 Hz, 6H); ¹⁹F NMR (376 MHz, CD₃OD): δ −98.47 (d, J = 237.2 Hz), −116.91 (dt, J = 17.6, 237.2 Hz); HRMS (ESI⁺) (m/z): found, 548.1619 [calcd for C₂₂H₂₉F₂N₃O₉P⁺ (M + H)⁺, 548.1604]; Anal. Calcd for C₂₂H₂₈F₂N₃O₉P: C, 48.27; H, 5.16; N, 7.68. Found: C, 48.12; H, 4.98; 8.01.

SAH Hydrolase Assay^18e−18g,30

The gene encoding human placental SAH hydrolase was cloned into expression plasmid pPROKcd20. Recombinant SAH hydrolase protein was produced in E. coli JM109 in 50 mM Tris-HCl (pH 7.5) containing 2 mM ethylenediaminetetraacetic acid and was purified by DEAE-cellulose column (2.8 cm × 6 cm), ammonium sulfate fractionation (35–60%), Sephacryl S-300HR (1.0 cm × 105 cm), and DEAE cellulose (2.8 cm × 24 cm). The protein homogeneity was confirmed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein concentration was determined by using the Bradford method. Bovine serum albumin was a standard material for protein assay. Enzyme activity was determined in reaction mixtures (250 μL) that contain 50 mM sodium phosphate (pH 8.0), 2 μM SAH hydrolase (0.5 μM tetrameric form), and varying concentrations of compounds. The reaction mixtures were first preincubated with the compounds for 10 min at 37 °C, after which the reaction was initiated by adding 100 μM SAH. The reaction was allowed to proceed for 20 min, followed by the addition of 5,5′-dithiobis-2-nitrobenzoate (DNTB) to a final concentration of 200 μM. The absorbance of the product 5-thio-2-nitrobenzoic acid (TNB) was measured at 412 nm using a spectrophotometer (Varian, Cary 100). The molar extinction coefficient for TNB (ε₄₁₂ = 13 700 M^–1 cm^–1) was used in calculations to quantify TNB formation.

Cells, Viruses, and Compounds

Vero E6 and Vero CCL81 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Lonza), supplemented with 8% fetal calf serum (FCS; PAA), 2 mM l-glutamine, 100 IU/mL of penicillin and 100 μg/mL of streptomycin, and were grown at 37 °C in a humidified incubator with 5% CO₂. Vero cells were maintained in Eagle’s minimum essential medium (EMEM; Lonza), supplemented with 8% FCS (FCS; PAA), 100 IU/mL of penicillin and 100 μg/mL of streptomycin, and were grown at 37 °C in a humidified incubator with 5% CO₂. Infections were performed in EMEM with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Lonza) supplemented with 2% FCS, l-glutamine, and antibiotics. Infectious clone-derived CHIKV(CHIKV-LS3) was generated as described by Scholte et al.³¹ The ZIKV strain SL0612 was isolated from an infected traveler returning from Suriname as described by van Boheemen et al.³² The Sindbis virus (SINV) strain HR and Semliki forest virus (SFV) strain SFV4 are part of the LUMC virus collection. The MERS-CoV strain EMC/2012 was isolated from patient material in the Dr. Soliman Fakeeh Hospital, Jeddah, Saudi Arabia, and was obtained from Erasmus Medical Center, Rotterdam.³³ The SARS-CoV strain Frankfurt 1 was provided by H. F. Rabenau and H. W. Doerr (Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany).³⁴ The compounds were dissolved in dimethylsulfoxide to obtain 20 mM stock solutions. All work with infectious CHIKV, MERS-CoV, SARS-CoV, and ZIKV was performed inside biosafety cabinets in the BSL-3 facilities of the Leiden University Medical Center.

Antiviral CPE-Reduction Assays

VeroE6 cells were seeded at a density of 5000 cells/well (CHIKV) and 10 000 cells/well (SARS-CoV, SFV and SINV) in a total volume of 100 μL per well in 96-well plates. Vero cells were seeded at a density of 20 000 cells/well when used for MERS-CoV infections, and Vero CCL81 cells were seeded at a density of 5000 cells/well for ZIKA infections under the same conditions as described for Vero E6. The following day, compound dilutions with concentrations of 150, 50, 16.7, and 5.6 μM were prepared in the infection medium by 3-fold serial dilution of the 150 μM solution. After replacing the culture medium with the respective dilutions of the compound, the cells were infected with CHIKV (MOI 0.005), SFV (MOI 0.025), SINV (MOI 0.025), ZIKV (MOI 0.05), MERS-CoV (MOI 0.005), or SARS-CoV (MOI 0.01). Viability assays were conducted in parallel. Each compound was tested at each concentration in quadruplicate (4 biological replicates per concentration). An MTS colorimetric assay was conducted 40 hpi for SFV, 76 hpi for SINV, 72 h hpi for MERS- and SARS-CoV, and 96 hpi for CHIKV and ZIKV by adding 20 μL/well of the CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) reagent (Promega). The assay was stopped after 2–2.5 h by fixing the cells with 37% formaldehyde. The absorbance was measured at 495 nm in a Berthold Mithras LB 940 plate reader, and the values were expressed relative to uninfected (infection) or untreated (viability) samples. The results represent the average of quadruplicate samples expressed as the mean ± SD. Compounds that were found to be protective were further evaluated in CPE reduction assays by testing 8 different concentrations to determine the EC₅₀ as previously described.^31,34 The cytotoxicity (CC₅₀) of the compounds was determined in parallel, and all experiments were performed in quadruplicate. Graph-Pad Prism 8.0.1 was used for EC₅₀ and CC₅₀ determination by nonlinear regression.

Viral Load Reduction Assays

VeroE6 (CHIKV, ZIKV) cells were seeded at a density of 7.5 × 10⁴ cells/well in 0.5 mL DMEM/8%FCS in 24-well cell culture plates and allowed to adhere overnight. For MERS-CoV and SARS-CoV, a cell density of or 6.0 × 10⁴ cells/well of Vero E6 and Vero cells was used, respectively, under the same conditions as described above. The next day, compound dilutions (0–1.5 μM) were prepared in EMEM/2%FCS to which virus was added to yield inocula for infecting the cells with an MOI of 0.1 for CHIKV, MOI of 1 for ZIKV, and MOI of 0.01 for SARS- and MERS-CoV. Cells were incubated at 37 °C with 250 μL/well of the inoculum for 1 h (CHIKV and SARS- and MERS-CoV) or 2 h (ZIKV). After the infection, the cells were washed twice with 1 mL/well warm phosphate-buffered saline and 0.5 mL/well fresh EMEM/2%FCS with different concentrations of compound 2c (0–1.5 μM) was added. The cells were incubated for 30 h (CHIKV) or 48 h (ZIKV, SARS- and MERS-CoV) at 37 °C, after which supernatants were harvested and stored at −80 °C for determination of the infectious virus titer by plaque assay. Viability assays were conducted in parallel as described in the previous paragraph. Plaque assays with CHIKV and SARS-CoV on VeroE6 cells, MERS-CoV on Vero cells, and ZIKV on Vero CCL81 cells were performed as described previously.^31,34a,35 Compound 2c was tested at each concentration in duplicate in two independent experiments (n = 4). Graph-Pad Prism 8.0.1 was used for statistical analysis with a one-way ANOVA multiple comparison test.

Glossary

Abbreviations

RdRp

RNA-dependent RNA polymerase

SAH

S-adenosyl-l-homocysteine

SARS-CoV

severe acute respiratory syndrome coronavirus

CHIKV

chikungunya virus

ZIKV

Zika virus

nsps

nonstructural proteins

MTase

methyltransferase

NTP

nucleoside triphosphate

SAM

S-adenosyl-l-methionine

AK

adenosine kinase

LiHMDS

lithium hexamethyldisilazide

TESCl

triethylsilyl chloride

NFSI

N-fluorobenzenesulfonimide

NFOBS

N-fluoro-O-benzenedisulfonimide

NaBH₄

sodium borohydride

LiBH₄

lithium borohydride

NMO

N-methylmorpholine-N-oxide

TBS

t-butyldimethylsilyl

TBAF

tetra-n-butylammonium fluoride

DAST

N,N-diethylaminosulfur trifluoride

AlMe₃

trimethylaluminum

SOCl₂

thionyl chloride

DIPEA

N,N-diisopropylethylamine

TFA

trifluoroacetic acid

Boc₂O

di-tert-butyl dicarbonate

DNTB

5,5′-dithiobis-2-nitrobenzoate

CPE

cytopathic effect

TMSOTf

trimethylsilyl trifluoromethanesulfonate

DMEM

Dulbecco’s modified Eagle’s medium

FCS

fetal calf serum

NEAA

non-essential amino acid

EMEM

Eagle’s minimum essential medium

SINV

Sindbis virus

SFV

Semliki forest virus

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.9b00781.

• ¹H and ¹³C NMR copies of all final compounds 2a–j and 3a–c (PDF)

• Crystallographic data for 2c, 2g, and 2h (ZIP)

• Molecular formula strings (CSV)

Author Contributions

J.-s.Y. and G.K. contributed equally to this work. All authors have contributed to the manuscript and given approval to the final version of the manuscript.

The authors declare no competing financial interest.