Synthesis and Modification of Cordycepin-Phosphoramidate ProTide Derivatives for Antiviral Activity and Metabolic Stability

Abstract

In our study, Prodrug nucleoTide (ProTide) technology was applied to cordycepin to enhance its antiviral activities and metabolic stability. Using cordycepin as starting material, we developed a synthetic method to access a series of stereospecific-phosphoramidate derivatives with various ester groups. We also successfully synthesized halogenated cordycepin derivatives via stannylation. Our 17 ProTide-cordycepin derivatives were pharmacologically evaluated for their antiviral activities. Phosphorus diastereomers 22S and 22R showed moderate inhibitory activity against corona and influenza viruses, while these compounds and derivatives (25S, 27S, and 27R) demonstrated promising antiviral efficacy against dengue virus. Pharmacological screening indicated that Sp-isomers generally exhibited slightly greater inhibitory activity than their Rp-isomer counterparts against the dengue virus. The selected ProTides were assessed for their metabolic mechanism and stability via carboxypeptidase and microsomes. The hydrolysis rate of the Rp-isomers was observed to be slightly higher than that of the Sp-isomers, and the addition of a fluorine group also modestly increased this rate and fluorinated 39S extended its half-life compared to nonfluorinated counterparts. These findings suggested not only structure–activity relationships of cordycepin ProTide but also the comprehensive synthetic route to access cordycepin derivatives for further antiviral development.

Introduction

Cordycepin (3′-deoxyadenosine, 1) is a drug-like natural product produced from fungi in the family Cordycipitaceae.^1,2 Cordycepin 1 exhibits a wide variety of pharmacological activities, for example, anti-inflammatory, anticancer, antimicrobial, antibacterial, antimalarial, antiprotozoal, antioxidant, immunomodulatory/immunoregulatory, etc.³⁻⁶ Furthermore, cordycepin has demonstrated promising antiviral properties, especially virulent RNA viruses such as influenza viruses, dengue virus, hepatitis C virus, human immunodeficiency virus, and Zika virus.⁷⁻¹⁰ Recently, cordycepin was discovered to inhibit SARS-CoV-2 replication in vitro (VOC-202012/01 strain), demonstrating superior efficacy compared to the drug remdesivir.¹¹ Several targets of cordycepin were proposed in silico, including RNA-dependent RNA polymerase.¹²

Given its promising antiviral activities and possible drug target, enhancing cordycepin’s drug-like properties could significantly amplify its therapeutic potential. ProTide technology, already successful in developing antiviral drugs like remdesivir (2) and sofosbuvir (3), can achieve this by increasing the lipophilicity of nucleoside compounds. This enhancement improves cellular uptake and leads to the formation of triphosphate active metabolites that inhibit targeted polymerase (Figure 1). For example, in the current anticancer drug discovery program (ClinicalTrials.gov ID NCT03829254), cordycepin (1) underwent structural modifications into NUC-7738, a racemic 5′-aryloxy phosphoramidate ProTide (4).¹⁴ This ProTide compound was discovered to be tolerant to adenosine deaminase (ADA) metabolic deactivation, resulting in increased levels of intracellular nucleoside triphosphate analogue as the active metabolite, whereas the parent cordycepin was subject to ADA simultaneously (Figure 1B).¹⁴⁻¹⁷ In contrast, another trypanocidal discovery project found that 2-fluorocordycepin (5), which lacks a phosphoramidate part, was also tolerant to ADA.¹⁸ Taken together, the racemic phosphoramidate ProTide-cordycepin and the presence of a fluorine group at the C2 position of cordycepin derivatives may contribute to enhancing their biological activities and stability.

Figure 1.

(A) Chemical structures of cordycepin (1), remdesivir (2), sofosbuvir (3), NUC-7738 (4), and 2-fluorocordycepin (5). (B) Intracellular conversion of nucleoside and ProTides forms: Upon entering the cell, ProTide derivatives are metabolized into the aminoacyl phosphoramidate form through esterase and H₂O-mediated hydrolysis of the amino acid ester. Phosphoramidase-type enzymes then hydrolyze the P–N bond, releasing the nucleoside monophosphate. This compound undergoes further phosphorylation to produce an active nucleoside triphosphate analogue. The nucleoside form has poor cell permeability and generates nucleoside monophosphate at a slower rate compared with the ProTide form.

To explore the relationship between the antiviral and stereospecific activities of ProTide-cordycepin, we developed the synthesis of distinct Sp- and Rp-phosphoramidate ProTide-cordycepin derivatives with various ester functionalities. Different substituent groups in the ester moieties of phosphoramidates might influence the hydrolysis rate by esterase or carboxypeptidase-like enzymes upon entering the cell, potentially affecting the production rate of the active nucleoside triphosphate analogue. Additionally, we established a novel synthetic pathway for C-2 halogenated cordycepin ProTides. Our study also involved the preparation of 2′-deoxyadeonsine-phosphoramidate and 2′, 3′-dideoxyadenosine-phosphoramidate to compare their antiviral properties to the cordycepin (1)-phosphoramidate counterpart. These compounds have been evaluated against a spectrum of viruses, including coronavirus (SARS-CoV-2 delta and omicron strain), dengue virus (DENV1), and influenza (H1N1) virus. Moreover, we also investigated their metabolic stability using NMR analysis and fluorescence measurement of hydrolysis reactions catalyzed by mimicking carboxypeptidase Y as well as in vitro microsomal stability assays.

Results and Discussion

In order to prepare our series of phosphoramidate ProTide analogues, the retrosynthetic analysis toward cordycepin 1 as the starting material was illustrated in Scheme 1. Sp- and Rp-Phosphoramidate ProTide-cordycepin 2 with diverse ester functionalities (R₁ including methyl, ethyl, isopropyl, tert-butyl, 2-ethylbutyl, and benzyl esters) would be coupled from known racemic phosphoramidate reagent 3¹⁹ and protected nucleoside 4. On the other hand, the coupling between commercially available Sp-phosphoramidate reagent 6 and protected halogenated nucleoside 7 (R₂ including fluorine, bromine, and iodine groups) was carried out to provide Sp-halogenated cordycepin-phosphoramidate ProTides. The halogenated nucleoside at the C2-position (7) could be synthesized from 2-stannylated cordycepin (8), which was prepared from cordycepin (1) via orthogonal protection and stannylation using the modified method by Tanaka and several research groups.²⁰⁻²⁴

Scheme 1. Retrosynthetic Analysis of Cordycepin-Phosphoramidate ProTide Derivatives Using Cordycepin (1) as the Starting Material.

In our experiment, the syntheses of Sp- and Rp-phosphoramidate ProTide-cordycepin 22–27 were prepared from cordycepin (1) (Scheme 2). Initially, the aim was to synthesize the primary alcohol nucleoside 4 as a key intermediate from 1. The TBS group was chosen as a protecting group for the 2′-hydroxy group as it could presumably be removed under mild acidic conditions in the final step. Selective protection of the secondary alcohol moiety of diol 1 was achieved in two steps: protection of both the 2′ and 5′ hydroxyl groups with TBSCl and removal of the 5′ TBS protecting group of 9 using a solution of trifluoroacetic acid (TFA) in water and THF in a ratio of (1:1:4) at 0 °C to give the corresponding primary alcohol nucleoside 4 in 72% over two steps.²⁵ Intermediate 4 was subsequently coupled with corresponding racemic phosphonate reagent 3(19) to provide racemic intermediate 10–15 in the presence of MgCl₂ and base in a good yield.²⁶ Subsequently, the racemic 10–15 were desilylated in TFA, water, and THF in a ratio of 1:1:1 at 0 °C to room temperature for 24 h, providing racemic phosphoramidate ProTide 16–21. Lastly, all racemic mixtures were isolated to obtain single isomers of Sp and Rp-22–27 in 29–42% yields using preparative chiral column chromatography with a Lux 5 μm Cellulose-2 LC Column. Chirality at the phosphorus atom was confirmed by ³¹P NMR spectroscopy, with the Sp-isomer resonating upfield and the Rp-isomer downfield, consistent with trends in similar nucleoside analog ProTides.²⁷⁻³⁰ Moreover, the diastereomeric purity of these compounds was found to exceed 95% de by chiral HPLC analysis.

Scheme 2. Synthesis and Isolation of Single Diastereomer Cordycepin-Phosphoramidate Derivatives (Compounds 22–27).

Next, to investigate the effect of C-2 substitution, the syntheses of Sp-halogenated cordycepin-phosphoramidate derivatives were performed using 2-stannylated cordycepin intermediate 8 as the key intermediate. Slightly modified from previous reports,^20,24 the preparation of intermediate 8 was achieved in three steps from silyl ether 9 (Scheme 3). First, a chlorodediazoniation reaction was performed using intermediate 9 in the presence of tert-butyl nitrite and TMSCl in order to introduce an electronegative chlorine atom at the C6 position to afford 6-chloropurine derivative 28. This step enhances the electron deficiency in the purine ring, facilitating functionalization at the C2 position of 6-chloropurine in 28. To achieve this, the lithiation-mediated stannyl transfer of 6-chloropurine nucleoside 28, as reported by Tanaka and co-workers, was employed.²¹ Upon treatment of 28 with freshly prepared lithium 2,2,6,6-tetramethylpiperidide (LiTMP) derived from n-BuLi and 2,2,6,6-tetramethylpiperidine (TMP), the C8-lithiated species was initially formed due to the higher acidity of the C8-hydrogen compared to the C2-hydrogen. Subsequent reaction of this C8-lithiated intermediate with tributyltin chloride (SnBu₃Cl) smoothly produced 6-chloro-2-(tributylstannyl)purine (29) as the sole product with a 78% yield. This result arose from the anionic transfer of the stannyl group from the C8 to the C2 position, which is stable under lithiation conditions, leading to its accumulation.^21,24 Finally, stannyl compound 29 was subjected to nucleophilic substitution in the presence of methanolic ammonia in a sealed tube at 100 °C, yielding the desired intermediate 8 in a moderate yield (55%).²¹ The yield of intermediate 8 was compromised due to the instability of the tributylstannyl group under these conditions, resulting in a reversion to silyl ethers 9 and 28 with yields of 25% and 10%, respectively. Nevertheless, these compounds could be reclaimed for further use.

Scheme 3. Synthesis of 2-Stannylated Cordycepin Intermediate 8.

Upon obtaining the 2-stannylated cordycepin intermediate (8), the subsequent step was to introduce a halogen group at the C2 position. The synthesis of fluoro-, iodo-, and bromocordycepins is shown in Scheme 4. Modified from a previous report,²¹ electrophilic fluorination, iodination, and bromination of compound 8 were executed utilizing XeF₂/AgTf, iodine (I₂), and N-bromosuccinimide (NBS) to yield compounds 30, 31, and 32 in 65%, 70%, and 76%, respectively. In contrast, the installation of a chlorine group at the C2 position using N-chlorosuccinimide (NCS) was not successful due to decomposition. Then, these obtained halogenated compounds 30–32 were treated with TFA/H₂O/THF (1:1:4) at 0–25 °C, providing compounds 33–35 in 77%, 82%, and 70% yield, respectively. The syntheses of C2-substituted cordycepin-phosphoramidate ProTides (39S–41S) were accomplished in two steps from nucleoside 33–35, employing a synthetic procedure similar to that described for Sp- and Rp-Phosphoramidate ProTide-cordycepin, as illustrated in Scheme 2. Compounds 33–35 were treated with commercially available phosphate reagent 6 followed by removal of the remaining TBS group to obtain the desired phosphoramidated and halogenated ProTide-cordycepin with yields of 65%, 62%, and 65%, respectively. The diastereomeric purity of final compounds 39S–41S was also found to exceed 95% de by chiral HPLC analysis.

Scheme 4. Synthesis of C2-Halogenated Cordycepin-Phosphoramidate (39S–41S).

In addition, the preparation of 2′-deoxyadeonsine-phosphoramidate (44S) and 2′,3′-dideoxyadenosine-phosphoramidate (45S) was performed in order to investigate SAR of hydroxy group at 2′ and 3′ (Scheme 5). The syntheses of both compounds were achieved by coupling commercially available 2′-deoxyadenosine (42) and 2′,3′-dideoxyadenosine (43) with phosphate reagent 6, providing the desired compound with yields of 31% and 55%, respectively.

Scheme 5. Synthesis of 2′-Deoxyadeonsine-Phosphoramidate (44S) and 2′,3′-Dideoxyadenosine-Phosphoramidate (45S).

Our synthetic phosphoramidate ProTides were evaluated for their in vitro antiviral activity against three RNA viruses, including SARS-CoV-2 virus (Delta and Omicron-XBB.1.16 variants), dengue virus serotype 1 (DENV-1), and influenza virus (H1N1). All compounds were screened for preliminary antiviral activity at 25 μM, revealing that most of the compounds showed no antiviral effects. However, only 22S, 22R, 25S, 27S, 27R, and 39S exhibited promising inhibition against one of these three viruses. Hence, these active and parent compounds were selected for further IC₅₀ measurement and cytotoxicity testing against the African green monkey kidney fibroblast (Vero) cells (Table 1). Initial findings demonstrated that the presence of a hydroxy group at the 2′ position, as observed in these active compounds, is crucial for antiviral inhibition. Notably, 44S and 45S did not exhibit any inhibitory effects against the tested viruses, whereas 22S showed inhibitory activities against all three viruses. Therefore, the absence of this hydroxy group at 2′ or its presence at 3′ or neither of them significantly reduces antiviral efficacy. Furthermore, we also found that most of these active compounds were noncytotoxic to Vero cells at 50 μg/mL; however, 22R and 39S showed sign of toxicity, with IC₅₀ values of 65.38 and 15.35 μM, respectively. These results suggest that differences between Sp- and Rp-isomers as well as the presence of a fluorine group in ProTide-cordycepin derivatives might influence their toxicity to Vero cells.

Table 1. Selected Antiviral and Cytotoxic Activities of Synthetic ProTides against Three Viruses (SARS-CoV-2 Virus (Delta and Omicron-XBB.1.16 variants), Dengue Virus Serotype 1 (DENV-1), and Influenza Virus (H1N1)^e.

	antiviral activities, IC50 (μM)				cytotoxicity, IC50 (μM)
compounds	SARS-CoV-2 (Delta)a	SARS-CoV-2 (Omicron-XBB.1.16)b	dengue serotype 1 (DENV-1)c	influenza (H1N1)d	Vero cells
cordycepin (1)	>200	N/A	87.50 ± 29.60	5.73 ± 0.36	N/A
22S	69.8 ± 9.91	N/A	1.76 ± 0.77	43.7 ± 3.67	N/A
22R	49.4 ± 8.62	N/A	5.53 ± 1.41	23.4 ± 1.80	65.38 ± 8.85
25S		N/A	13.50 ± 5.91	N/A	N/A
25R		N/A	N/A	N/A	N/A
27S		N/A	2.80 ± 0.90	N/A	N/A
27R		N/A	3.07 ± 0.14	N/A	N/A
39S		N/A	71.20 ± 32.30	N/A	15.35 ± 6.99
remdesivir (2)		4.99 ± 0.07	0.06 ± 0.01	N/A	N/A
molnupiravir (46)	2.32 ± 0.45	8.60 ± 1.92	N/A	20.2 ± 1.49	N/A

^aThe virus and the test substances are added to CaLu-3 cells.

^bThe virus and the test substances are added to Vero-E6 cells.

^cThe virus and the test substances are added to K562-CD209 cells.

^dThe virus and the test substances are added to Madin-Darby Canine Kidney (MDCK) cells.

^eAntiviral activity N/A: the dose–response experiment was not performed due to no inhibitory properties against corresponding virus at 25 μM in our preliminary screening (see Supporting Information); Cytotoxicity N/A: no toxicity was observed at 50 μg/mL or approximately 90 μM.

In our assay evaluating the anti-SARS-CoV-2 activity against the Delta and Omicron strains, we observed that cordycepin (1) exhibited no inhibitory effects on these variants, despite demonstrating potent inhibitory activity against the VOC-202012/01 strain in a previous reported.¹¹ Nevertheless, compared to compound 1 as a parent compound, compounds 22S and 22R exhibited marginal inhibitory properties against the Delta variant of the SARS-CoV-2 virus, with IC₅₀ values of 69.8 and 49.4 μM, respectively, albeit with much lower efficacy in inhibiting this virus variant compared to the standard drug molnupiravir (46) (IC₅₀ = 2.32 μM). For the Delta variant, a limited number of compounds were screened, placing greater focus on the new Omicron variant. In the case of the SARS-CoV-2 Omicron-XBB.1.16 variant, none of our synthetic compounds displayed inhibitory activity at 25 μM, resulting in no further testing for the dose–response assay. Similarly, only the standard drugs remdesivir (2) and molnupiravir (46) demonstrated strong anti-SARS-CoV-2 activity against this variant, with IC₅₀ values of 4.99 and 8.60 μM, respectively. It should be noted that our synthetic cordycepin-ProTides did not exhibit enhanced antiviral efficacy against the SARS-CoV-2 virus of both variants. We anticipated that the difference in inhibition activity of compounds targeting the RdRp between Delta and Omicron variants of SARS-CoV-2 despite no mutations at the target binding site, might be due to the influence of other factors, including replication rate and virulence. The Omicron variant favors an endocytic entry path into host cells, which is different from the Delta variant. This suggests that the mutations in the Omicron spike protein affect its mechanism of cell entry, potentially influencing RdRp function once the virus is inside the host cell.³¹ Another finding was that the ProTide technology promoted the inhibitory activities, so the polymerase could be a target of cordycepin.

On the other hand, preliminary screening results indicated that 22S, 22R, 25S, 27S, 27R, and 39S possessed promising inhibitory activity against dengue virus serotype 1 (DENV-1) without inducing cytotoxicity in their host cells (K562). Although parent cordycepin (1) showed low activities against dengue virus with an IC₅₀ value of 87.5 μM, the ProTides 22S, 22R, 27S, and 27R, bearing a large ester group (2-ethylbutyl and benzyl moiety, respectively), demonstrated the high level of inhibition with IC₅₀ values of 1.76, 5.53, 2.80, and 3.07 μM, respectively. For the small ester group, compound 25S, an ethyl-substituted phosphoramidate derivative, also showed relatively strong anti-DENV-1 activity with an IC₅₀ value of 13.5 μM, whereas the 25R isomer exhibited no anti-DENV-1 activity. Moreover, other halogenated compounds showed no activity at 25 μM and fluorinated compound 39S exhibited sign of cytotoxicity against Vero cells as aforementioned. Notably, these results indicated that S-forms of 22, 25, and 27 showed significantly greater inhibitory activities compared to R-forms isomers. It might be attributed to differences in biotransformation into the active form, similar to those observed with S- and R-sofosbuvir.²⁹ However, all compounds were less potent in inhibiting dengue virus serotype 1 compared with remdesivir (2), which demonstrated impressive efficacy with an IC₅₀ value of 0.058 μM.

Likewise, all compounds were primarily screened at 25 μM against influenza (H1N1) in MDCK cells. None of the cordycepin ProTides were active, whereas cordycepin (1), as a nucleoside, showed good inhibitory activity with an IC₅₀ value of 5.73 μM, stronger than that of molnupiravir (46) (IC₅₀ = 20.2 μM). However, we selected present ProTides 22S and 22R for the dose–response assay (IC₅₀ = 43.7 and 23.4 μM, respectively). These results indicated that ProTide compounds 22S and 22R exhibited inferior efficacy compared to that of nucleoside 1. Evaluating the anti-influenza activity of these synthetic ProTides necessitates further investigation as these results were obtained from canine kidney cells, which might exhibit differences in cellular uptake and biotransformation compared to human cells. Alternatively, the target of cordycepin against influenza might not be polymerase.

To investigate further on the enzymatic activation process, the efficiency of ProTides depends on their active nucleotide forms mediated by host esterases, including carboxypeptidases. The proposed mechanism for metabolic activation pathway of cordycepin ProTides is shown in Scheme 6.^13,32 These enzymes hydrolyze the ester bond of the amino acid moiety, leading to the generation of intermediate A. Subsequently, five-membered cyclic intermediate B is formed followed by another round of spontaneous hydrolysis reactions. Consequently, the P–N bond undergoes cleavage, converting the compound to the corresponding aminoacyl phosphoramidate (intermediate C). This compound is critically important as it leads to the formation of the bioactive compound (5′-nucleoside triphosphate).

Scheme 6. Proposed Mechanism for the Metabolic Activation Pathway of Cordycepin ProTides.

To explore the mechanism of bioactivation and assess the metabolic stability of our ProTides, enzymatic hydrolysis studies of ester bonds were performed using the ³¹P NMR technique and fluorescence measurement at the characteristic wavelength of phenol in the presence of yeast carboxypeptidase Y (CPY) as a representative model of human carboxypeptidase. This approach specifically highlighted the catalytic role of Cathepsin A in ProTide metabolism in the human liver. Only the active compounds Sp-22, Rp-22, and Sp-39 were chosen for these studies because their interesting antiviral activities as well as core structures include both isomers and a halogen group at the C2 position.

First, the kinetic study of 22S, 22R, and 39S hydrolysis with yeast CPY, determined by ³¹P NMR spectroscopy, was conducted to examine intermediates A and C. In the ³¹P NMR experiments of 22S and 22R compounds, chemical shifts at 3.95 and 3.56 ppm, respectively, were observed (Figure 2A,B). When carboxypeptidase Y enzyme was added, it was observed that the signal intensity corresponding to 22S gradually diminished and completely disappeared within 5 h. For 22R, it vanished within 3 h. The emergence of new signal intensities of these compounds was found at approximately 4.70 and 7.12 ppm, corresponding to intermediate A and intermediate C, respectively, according to the previous reported pattern.³² Therefore, 22S undergoes hydrolysis by the carboxypeptidase Y enzyme more slowly than does its stereoisomer 22R. In the case of 2-fluorocordycepin phosphoramidate ProTide (39S), as illustrated in Figure 2C, the experiment revealed a chemical shift of the starting material at 3.74 ppm. Upon the addition of carboxypeptidase Y enzyme, 39S underwent hydrolysis to form intermediate C at 7.13 ppm within 3 h, but detection of intermediate A of 39S was not clear because of noise background under the similar condition. Although compound 39S contains the same Sp form as 22S, the presence of a fluorine group might lead to an increased rate of hydrolysis by CPY. Notably, these NMR results were obtained using a representative enzyme at high concentration, and the hydrolysis rates of these compounds showed minimal variation.

Figure 2.

³¹P NMR spectra showing the time course of metabolic transformation for (A) compound 22S, (B) compound 22R, and (C) compound 39S, catalyzed by carboxypeptidase Y (CYP).

In order to monitor compound hydrolysis in a time-resolved and kinetic manner, we developed a new fluorescence-based assay. Unlike the NMR assay, this assay exploits the release of the phenol group upon spontaneous cyclization of intermediate A into intermediate B (Scheme 6). Test compounds were diluted in PBS buffer at pH 7.4, and CPY was added to initiate the reaction, leading to a rapid increase of fluorescence at 305 nm (Figure 3A). We noted that a lag phase is observed in the first 2 min, which corresponds to the accumulation of intermediate A. Following this, Michaelis–Menten curves were recorded for compounds 22S, 22R, and 39S (Figure 3A–C), and V_max and K_M values were extracted. These values encompassed two reactions: ester hydrolysis by CPY and the spontaneous cyclization of intermediate A (which is intramolecular and independent of substrate concentration).

Figure 3.

Activation of ProTides compounds by CPY monitored by fluorescence. (A) Representative curves obtained for 22S, 22R, and 39S at 1 mM, (B) Michaelis–Menten curve for 22S, (C) Michaelis–Menten curve for 22R, and (D) Michaelis–Menten curve for 22S.

From this experiment, 22R appears to be hydrolyzed slightly faster than 22S (V_max of 0.019 mM min^–1 vs 0.0122 mM min^–1, p = 0.017) but shows weaker binding (K_M of 0.46 mM vs 0.17 mM, p = 0.0055). If one considers k_cat/K_M parameters, 22S is a more efficient substrate for CPY than 22R. If comparing 22S with 39S, which have the same stereochemistry but differs by one fluorine atom, compounds do not show any significant difference in reaction rate (V_max of 0.0122 mM min^–1 vs 0.0135 mM min^–1, p = 0.46). Yet, the presence of the fluorine atom results in a significantly weaker enzyme affinity (K_M of 0.40 mM vs 0.17 mM, p = 0.041), making 22S an overall more efficient substrate.

Lastly, to further explore metabolic mimicry, human microsomal stability assays for cordycepin and its ProTide were carried out for the same derivatives, including 22S, 22R, and 39S, as shown in Table 2. Comparing to standard drugs, including carbamazepine and propranolol, our compounds were rapidly metabolized in the presence of human liver microsomes (HLM) supplemented with and without NADPH (see Supporting Information Figure 4). Therefore, the fast metabolic labile could stem from phase I metabolism (simple ester hydrolysis) and metabolic stability. Notably, 2-fluorocordycepin phosphoramidate (39S) has a significantly extended half-life, approximately 2-fold that of 22S, indicating greater stability. These findings align with the research reported by Vodnala and co-workers¹⁸ as well as the effect on fluorination of cordycepin.

Table 2. Metabolic Stability of Cordycepin and Its Derivatives by a Microsomal Stability Assay.

	human liver microsomes NADPH (n= 3)
test compounds	Clint(μL/min/mg protein)	t1/2(min)	Clintclass
carbamazepine	<3	>180	low
propranolol	32.12 ± 4.89	54.86 ± 9.00	moderate
cordycepin (1)	141.78 ± 10.20	12.27 ± 0.88	high
22S	2117.29 ± 89.73	0.82 ± 0.04	high
22R	2613.26 ± 245.39	0.67 ± 0.06	high
39S	1228.96 ± 101.93	1.42 ± 0.12	high

Conclusions

In conclusion, we have successfully developed synthetic methods from cordycepin and obtained both Sp- and Rp-phosphoramidate ProTide 22–27 with diverse ester functionalities. In addition, the C2 halogenated Sp-39, 40, and 41 were efficiently accessed via the stannylated intermediate. These novel synthetic ProTides were evaluated for their antiviral activity against three RNA viruses. In contrast to previous research, cordycepin and our ProTides did not show inhibitory activities against SARS-CoV-2, but cordycepin showed greater activity against influenza in comparison to its ProTides. Notably, 22S, 27S, and 27R displayed intriguing activity against dengue virus without cytotoxicity to Vero cells, and the Sp-isomer generally demonstrated enhanced efficacy. These ProTides are promising candidates for further dengue drug development. To understand the effect of the newly synthesized ProTide compound on biotransformation, in vitro metabolic studies were performed. As a result, the addition of Sp-cordycepin ProTide and fluorine could delay the intrinsic metabolic effect. These findings underscore the importance of structural modifications and chirality of ProTides in biological activity and metabolic stability, informing the design of next-generation antiviral agents.

Experimental Section

General Information

All reactions were conducted under an argon or nitrogen atmosphere in oven- or flamed-dried glassware. Solvents were used as received from the suppliers. All other reagents were obtained from commercial sources and used without further purification. Racemic phosphoramidate reagent 3 was stock chemical and made in our research group.¹⁹ Column chromatography was performed on Silica gel 60 (0.063–0.200 mm, Merck) and Ultra-Performance Flash Purification Chromatography (UPFP), Interchim Generation 5-PF-5.125. Preparative chiral column chromatography using Lux 5 μm Cellulose-2, Column size 250 mm × 21.2 mm was used to separate all racemic mixture. Thin-layer chromatography (TLC) was performed on Silica gel 60 F₂₅₄ (Merck). ¹H, ¹³C, ³¹P and ¹⁹F NMR Spectroscopic data were reported in ppm on the δ scale and recorded on 400 and 500 MHz Bruker Mercury-400 plus or Bruker/Advance 400 NMR spectrometer. The data are presented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, br = broad, and app = apparent), coupling constant (s) in hertz (Hz). High-resolution mass spectra (HRMS) were obtained on a micrOTOF mass spectrometer (Bruker Daltonics) using the ESI technique. Diastereomeric purity was determined by ultrahigh-performance liquid chromatography (UHPLC) on Vanquish with Chromeleon 7 software using CHIRALPAK IG-U 1.6 μm 100 × 3.0 mm column and a gradient mobile phase consisting of methanol (phase A) and acetonitrile (phase B). The gradient was as follows: isocratic for 3 min at 100% A, from 3 to 12 min decreased to 5% A, from 12 to 13 min raised to 95% A, and finally column equilibrated at 100% A until 15 min. Flow rate was 0.4 mL/min, and temperature was 40 °C.

General Procedure for Silyl Deprotection at the 5′-Position

To a solution of silyl ether derivatives (1.0 equiv) in anhydrous THF (0.16 M) at 0 °C under an argon atmosphere was added a solution of TFA:H₂O (1:1, 0.33 M). The reaction was allowed to react at 25 °C for 4–8 h, monitored by TLC. Then, the reaction was neutralized with saturated aqueous NaHCO₃ and extracted with EtOAc (×3). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude residue was purified by column chromatography to give the primary alcohol derivative.

General Procedure for Phosphorylation

To a solution of primary alcohol derivative (1 equiv) in anhydrous THF (0.065 M) were added phosphate reagent (1.5 equiv) and MgCl₂ (1.5 equiv) and stirred at 50 °C. After stirring at 50 °C for 10 min, N,N-diisopropylethylamine (DIEA) (2.5 equiv) was added and stirred at this temperature for 2–6 h monitored by TLC. The reaction mixture was then cooled to room temperature and diluted with H₂O. The organic phase was extracted with EtOAc (×4), washed with saturated aqueous NaHCO₃ and brine, dried over with anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude residue was purified by column chromatography to afford the phosphoramidate ProTide-cordycepin derivative.

General Procedure for Silyl Deprotection at the 2′-Position

To a solution of phosphoramidate ProTide-cordycepin derivative (1 equiv) in anhydrous THF (0.22 M) at 0 °C under an argon atmosphere was added a solution of TFA:H₂O (1:1, 0.1 M). The reaction was stirred at 0 °C to room temperature for 18–24 h, monitored by TLC, and then concentrated under reduced pressure. Purification of the crude residue by column chromatography yielded the desired product.

9-((2R,3R,5S)-3-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-9H-purin-6-amine (9)

To a solution of cordycepin (1) (5.00 g, 19.9 mmol) in anhydrous DMF (46 mL, 0.43 M) at room temperature under an argon atmosphere were added imidazole (8.94 g, 131.34 mmol) and tert-butyldimethylsilyl chloride, TBSCl) (9.90 g, 65.67 mmol). The reaction was stirred at room temperature for 20 h, then quenched with saturated aqueous NH₄Cl (150 mL), and extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated in vacuo. The crude residue was purified by column chromatography (10–30% EtOAc/hexanes) to afford 4 (8.63 g, 90%) as a white solid; ¹H NMR (500 MHz, DMSO-d₆): δ 8.30 (s, 1H), 8.13 (s, 1H), 7.29 (br s, 2H), 5.88 (d, J = 2.3 Hz, 1H), 4.73–4.67 (m, 1H), 4.43–4.35 (m, 1H), 3.94 (dd, J = 11.5, 3.0 Hz, 1H), 3.76 (dd, J = 11.5, 3.6 Hz, 1H), 2.33–2.25 (m, 1H), 1.96–1.89 (m, 1H), 0.88 (s, 9H), 0.82 (s, 9H), 0.06 (s, 6H), 0.00 (s, 3H), −0.00 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆): δ 155.9, 152.6, 148.9, 138.2, 118.9, 90.3, 80.0, 76.5, 63.9, 33.9, 25.8 (3C), 25.5 (3C), 18.0, 17.6, −5.0, −5.1, −5.4, −5.4; HRMS (ESI) m/z calcd for C₂₂H₄₂N₅O₃Si₂ [M + H]⁺ 480.2821, found 480.2825.

((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)methanol (4)

Alcohol 4 was prepared from 9 (6.52 g, 13.6 mmol) using the general procedure for silyl deprotection at the 5′-position. The crude residue was purified by column chromatography (40% EtOAc/hexanes–100% EtOAc) to give 4 (3.98 g, 80%) as a white solid; ¹H NMR (500 MHz, DMSO-d₆): δ 8.38 (s, 1H), 8.14 (s, 1H), 7.32 (br s, 2H), 5.84 (d, J = 3.0 Hz, 1H), 5.30–5.23 (m, 1H), 4.79–4.73 (m, 1H), 4.39–4.31 (m, 1H), 3.72 (d, J = 12.0 Hz, 1H), 3.53 (d, J = 12.0 Hz, 1H), 2.32–2.24 (m, 1H), 1.97–1.90 (m, 1H), 0.81 (s, 9H), −0.03 (s, 3H), −0.05 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆): δ 156.0, 152.4, 148.8, 139.0, 119.1, 90.6, 80.3, 75.8, 62.5, 34.3, 25.5 (3C), 17.6, −5.1, −5.1; HRMS (ESI) m/z calcd for C₁₆H₂7N₅NaO₃Si [M + Na]⁺ 388.1775, found 388.1771.

2-Ethylbutyl ((((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy) tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (10)

Compound 10 was prepared from alcohol 4 (1.10 g, 3 mmol) and phosphate reagent 3a (2.02 g, 4.5 mmol) using the general procedure for phosphorylation. The crude residue was purified by column chromatography (70% EtOAc/hexanes–100% EtOAc) to afford 10 as a white solid (1.70 g, 84%): ¹H NMR (500 MHz, DMSO-d₆): δ 8.27–8.25 (m, 1H), 8.14 (s, 1H), 7.37–7.26 (m, 4H), 7.22–7.13 (m, 3H), 6.09–5.98 (m, 1H), 5.89 (dd, J = 7.5, 2.3 Hz, 1H), 4.85–4.77 (m, 1H), 4.58–4.45 (m, 1H), 4.33–4.10 (m, 2H), 3.97–3.76 (m, 3H), 2.35–2.26 (m, 1H), 2.04–1.94 (m, 1H), 1.46–1.36 (m, 1H), 1.30–1.15 (m, 7H), 0.86–0.72 (m, 15H), 0.02 to −0.07 (m, 6H); The ratio of two diastereomers Sp:Rp was 1.6:1 based on ³¹P NMR (202 MHz, DMSO-d₆, δ 3.91 (Rp), 3.62 (Sp)); HRMS (ESI) m/z calcd for C₃₁H₄₉N₆NaO7PSi [M + Na]⁺ 699.3062, found 699.3064.

Isopropyl ((((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy) tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (11)

Compound 11 was prepared from alcohol 4 (0.3565 g, 1 mmol) and phosphate reagent 3b (0.612 g, 1.5 mmol) using the general procedure for phosphorylation. The crude residue was purified by column chromatography (70% EtOAc/hexanes–100% EtOAc) to provide 11 as a white solid (0.471 g, 74%): ¹H NMR (500 MHz, DMSO-d₆): δ 8.26 (s, 1H), 8.14 (s, 1H), 7.38–7.25 (m, 4H), 7.22–7.12 (m, 3H), 6.04–5.94 (m, 1H), 5.92–5.87 (m, 1H), 4.87–4.77 (m, 2H), 4.59–4.46 (m, 1H), 4.32–4.10 (m, 2H), 3.82–3.67 (m, 1H), 2.36–2.28 (m, 1H), 2.04–1.95 (m, 1H), 1.22–1.15 (m, 3H), 1.14–1.06 (m, 6H), 0.82 (s, 9H), – 0.01– – 0.05 (m, 6H); the ratio of two diastereomers Sp:Rp was 1.4:1 based on ³¹P NMR (202 MHz, DMSO-d₆, δ 3.90 (Rp), 3.66 (Sp)); HRMS (ESI) m/z calcd for C₂₈H₄₃N₆NaO7PSi [M + Na]⁺ 657.2592, found 657.2595.

Methyl ((((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (12)

Compound 12 was prepared from alcohol 4 (0.73 g, 2 mmol) and phosphate reagent 3c (1.14 g, 3 mmol) using the general procedure for phosphorylation. The crude residue was purified by column chromatography (70% EtOAc/hexanes–100% EtOAc) to give 12 as a white solid (1.05 g, 87%): ¹H NMR (500 MHz, CD₃OD) δ 8.31–8.27 (m, 1H), 8.21–8.18 (m, 1H), 7.37–7.29 (m, 2H), 7.25–7.14 (m, 3H), 6.00–5.96 (m, 1H), 4.92–4.87 (m, 1H), 4.71–4.61 (m, 1H), 4.49–4.37 (m, 1H), 4.33–4.25 (m, 1H), 3.97–3.84 (m, 1H), 3.67–3.60 (m, 3H), 2.41–2.32 (m, 1H), 2.11–2.04 (m, 1H), 1.34–1.24 (m, 3H), 0.91–0.85 (m, 9H), 0.08–0.01 (m, 6H); the ratio of two diastereomers Sp:Rp was 1.4:1 based on ³¹P NMR (202 MHz, DMSO-d₆, δ 4.03 (Rp), 3.79 (Sp)); HRMS (ESI) m/z calcd for C₂₆H₃₉N₆NaO₇PSi [M + Na]⁺ 629.2279, found 629.2276.

Ethyl ((((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (13)

Compound 13 was prepared from alcohol 4 (0.73 g, 2 mmol) and phosphate reagent 3d (1.18 g, 3 mmol) using a general procedure for phosphorylation. The crude residue was purified by column chromatography (70% EtOAc/hexanes–100% EtOAc) to give 13 as a white solid (1.01 g, 81%): ¹H NMR (500 MHz, CD₃OD): δ (ppm) 8.32–8.27 (m, 1H), 8.22–8.19 (m, 1H), 7.37–7.30 (m, 2H), 7.25–7.14 (m, 3H), 5.98–5.97 (m, 1H), 4.93–4.87 (m, 1H), 4.70–4.61 (m, 1H), 4.49–4.38(m, 1H), 4.33–4.26 (m, 1H), 4.14–4.06 (m, 2H), 3.96–3.84 (m, 1H), 2.41–2.32 (m, 1H), 2.14–2.05 (m, 1H), 1.32–1.17 (m, 6H), 0.89–0.87 (m, 9H), 0.09–0.00 (m, 6H); the ratio of two diastereomers Sp:Rp was 1.3:1 based on ³¹P NMR (202 MHz, DMSO-d₆, δ 4.06 (Rp), 3.81 (Sp)); HRMS (ESI) m/z calcd for C₂₇H₄₁N₆NaO₇PSi [M + Na]⁺ 643.2436, found 643.2421.

tert-Butyl ((((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy) tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (14)

Compound 14 was prepared from alcohol 4 (0.73 g, 2 mmol) and phosphate reagent 3e (1.27 g, 3 mmol) using a general procedure for phosphorylation. The crude residue was purified by column chromatography (70% EtOAc/hexanes–100% EtOAc) to give 14 as a white solid (1.00 g, 77%): ¹H NMR (500 MHz, CD₃OD): δ (ppm) 8.31–8.26 (m, 1H), 8.22–8.18 (m, 1H), 7.37–7.29 (m, 2H), 7.26–7.13 (m, 3H), 5.99–5.95 (m, 1H), 4.91–4.87 (m, 1H), 4.71–4.61 (m, 1H), 4.48–4.38 (m, 1H), 4.34–4.26 (m, 1H), 3.84–3.74 (m, 1H), 2.40–2.31 (m, 1H), 2.11–2.05 (m, 1H), 1.43–1.39 (m, 9H), 1.30–1.22 (m, 3H), 0.89–0.86 (m, 9H), 0.08–0.01 (m, 6H); the ratio of two diastereomers Sp:Rp was 1.4:1 based on ³¹P NMR (202 MHz, DMSO-d₆, δ 4.10 (Rp), 3.90 (Sp)); HRMS (ESI) m/z calcd for C₂₉H₄₅N₆NaO₇PSi [M + Na]⁺ 671.2749, found 671.2750.

Benzyl ((((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy) tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (15)

Compound 15 was prepared from alcohol 4 (0.73 g, 2 mmol) and phosphate reagent 3e (1.37 g, 3 mmol) using a general procedure for phosphorylation. The crude residue was purified by column chromatography (70% EtOAc/hexanes–100% EtOAc) to give 15 as a white solid (1.17 g, 85%): ¹H NMR (500 MHz, CD₃OD) δ 8.32–8.24 (m, 1H), 8.19 (s, 1H), 7.36–7.24 (m, 7H), 7.23–7.13 (m, 3H), 5.97–5.93 (m, 1H), 5.15–5.04 (m, 2H), 4.88–4.80 (m, 1H), 4.64–4.55 (m, 1H), 4.43–4.34 (m, 1H), 4.27–4.18 (m, 1H), 4.02–3.90 (m, 1H), 2.33–2.23 (m, 1H), 2.05–1.96 (m, 1H), 1.33–1.25 (m, 3H), 0.88 (s, 9H), 0.08–0.00 (m, 6H); the ratio of two diastereomers Sp:Rp was 1.5:1 based on ³¹P NMR (202 MHz, DMSO-d₆, δ 4.11 (Rp), 3.71 (Sp)); HRMS (ESI) m/z calcd for C₃₂H₄₃N₆NaO₇PSi [M + Na]⁺ 705.2592, found 705.2596.

2-Ethylbutyl ((S)-(((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (22S) and 2-Ethylbutyl ((R)-(((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (22R)

Compounds 22S and 22R were synthesized from 10 (1.51 g, 2.23 mmol) using the general procedure for silyl deprotection at the 2′-position to give racemic product 16 (0.845 g, 67%), which was purified by preparative chiral column chromatography (5–7% MeOH/ACN; flow rate 6 mL/min) to afford two separable diastereomers: 22R (Rp-isomer, white solid, 140 mg, 33%): ¹H NMR (500 MHz, CD₃OD): δ 8.28 (s, 1H), 8.21 (s, 1H), 7.34–7.28 (m, 2H), 7.22–7.13 (m, 3H), 6.02 (d, J = 1.8 Hz, 1H), 4.74–4.66 (m, 2H), 4.51–4.43 (m, 1H), 4.34–4.27 (m, 1H), 4.07–3.95 (m, 2H), 3.94–3.84 (m, 1H), 2.40–2.29 (m, 1H), 2.14–2.04 (m, 1H), 1.53–1.42 (m, 1H), 1.37–1.25 (m, 7H), 0.86 (t, J = 7.5 Hz, 6H); ¹³C NMR (100 MHz, CD₃OD): δ 175.1 (d, ³J_C–P = 4.4 Hz), 157.2, 153.7, 152.1 (d, ²J_C–P = 6.7 Hz), 150.1, 140.5, 130.7 (2C), 126.1, 121.4 (2C, d, ³J_C–P = 4.7 Hz), 120.4, 93.1, 80.4 (d, ³J_C–P = 8.7 Hz), 76.6, 68.3 (d, ²J_C–P = 5.3 Hz), 68.0, 51.7, 41.7, 34.6, 24.2, 24.2, 20.4 (d, ³J_C–P = 7.4 Hz), 11.3, 11.2; ³¹P NMR (202 MHz, CD₃OD): δ 3.98; HRMS (ESI) m/z calcd for C₂₅H₃₅N₆NaO7P [M + Na]⁺ 585.2197, found 585.2195. The diastereopurity of 22R was determined to be 99% de: retention time = 3.723 min; 22S (Sp-isomer, white solid, 170 mg, 40%): ¹H NMR (500 MHz, CD₃OD): δ 8.27 (s, 1H), 8.21 (s, 1H), 7.36–7.29 (m, 2H), 7.22 (d, J = 8.4 Hz, 2H), 7.17 (t, J = 7.4 Hz, 1H), 6.01 (d, J = 1.6 Hz, 1H), 4.75–4.70 (m, 1H), 4.69–4.62 (m, 1H), 4.43–4.36 (m, 1H), 4.31–4.24 (m, 1H), 4.00 (dd, J = 10.9, 5.8 Hz, 1H), 3.96–3.86 (m, 2H), 2.41–2.32 (m, 1H), 2.13–2.05 (m, 1H), 1.49–1.39 (m, 1H), 1.36–1.25 (m, 7H), 0.93–0.76 (m, 6H); ¹³C NMR(126 MHz, CD₃OD): δ 174.9 (d, ³J_C–P = 5.2 Hz), 157.3, 153.8, 152.1 (d, ²J_C–P = 7.2 Hz), 150.1, 140.5, 130.7 (2C), 126.1, 121.3 (2C, d, ³J_C–P = 4.8 Hz), 120.5, 93.2, 80.4 (d, ³J_C–P = 8.5 Hz), 76.6, 68.6 (d, ²J_C–P = 5.3 Hz), 68.0, 51.5, 41.7, 34.9, 24.2, 24.2, 20.6 (d, ³J_C–P = 6.6 Hz), 11.3, 11.2; ³¹P NMR (202 MHz, CD₃OD): δ 3.78; HRMS (ESI) m/z calcd for C₂₅H₃₅N₆NaO7P [M + Na]⁺ 585.2197, found 585.2191. The diastereopurity of 22S was determined to be 98% de: retention time = 6.203 min.

Isopropyl ((S)-(((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (23S) and Isopropyl ((R)-(((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (23R)

Compounds 23S and 23R were synthesized from 11 (0.980 g, 1.45 mmol) using the general procedure for silyl deprotection at the 2′-position to give racemic product 17 (0.542 g, 72%), which was purified by preparative chiral column chromatography (3% MeOH/ACN; flow rate 6 mL/min) to afford two separable diastereomers: 23R (Rp-isomer, white solid, 81 mg, 30%): ¹H NMR (500 MHz, CD₃OD): δ (ppm) 8.28 (s, 1H), 8.21 (s, 1H), 7.33–7.28 (m, 2H), 7.21–7.13 (m, 3H), 6.02 (d, J = 1.9 Hz, 1H), 4.98–4.90 (m, 1H), 4.75–4.67 (m, 2H), 4.49–4.44 (m, 1H), 4.34–4.27 (m, 1H), 3.86–3.78 (m, 1H), 2.40–2.32 (m, 1H), 2.13–2.06 (m, 1H), 1.25 (dd, J = 7.2, 1.1 Hz, 3H), 1.21–1.18 (m, 6H); ¹³C NMR (126 MHz, CD₃OD): δ (ppm) 174.5 (d, ³J_C–P = 4.7 Hz), 157.2, 153.6, 152.1 (d, ²J_C–P = 6.6 Hz), 150.2, 140.5, 130.7 (2C), 126.1, 121.4 (2C, d, ³J_C–P = 4.7 Hz), 120.4, 93.1, 80.4 (d, ³J_C–P = 8.7 Hz), 76.6, 70.1, 68.3 (d, ²J_C–P = 5.2 Hz), 51.7, 34.7, 21.9, 21.8, 20.3 (d, ³J_C–P = 7.2 Hz); ³¹P NMR (202 MHz, CD₃OD): δ 4.02; HRMS (ESI) m/z calcd for C₂₂H₂₉N₆NaO7P [M + Na]⁺ 543.1728, found 543.1727. The diastereopurity of 23R was determined to be 100% de: retention time = 2.449 min; 23S (Sp-isomer, white solid, 95 mg, 35%): ¹H NMR (500 MHz, CD₃OD): δ (ppm) 8.32 (s, 1H), 8.25 (s, 1H), 7.35–7.30 (m, 2H), 7.21 (d, J = 8.5 Hz, 2H), 7.17 (t, J = 7.3 Hz, 1H), 6.03 (d, J = 1.6 Hz, 1H), 4.95–4.88 (m, 1H), 4.76–4.73 (m, 1H), 4.71–4.65 (m, 1H), 4.44–4.37 (m, 1H), 4.31–4.24 (m, 1H), 3.91–3.82 (m, 1H), 2.43–2.34 (m, 1H), 2.14–2.06 (m, 1H), 1.29 (d, J = 7.1 Hz, 3H), 1.21–1.14 (m, 6H); ¹³C NMR (126 MHz, CD₃OD): δ 174.3 (d, ³J_C–P = 5.4 Hz), 157.3, 153.8, 152.1 (d, ²J_C–P = 6.8 Hz), 150.2, 140.5, 130.7 (2C), 126.1, 121.3 (2C, d, ³J_C–P = 4.8 Hz), 120.5, 93.2, 80.3 (d, ³J_C–P = 8.5 Hz), 76.5, 70.0, 68.6 (d, ²J_C–P = 5.3 Hz), 51.6, 34.9, 21.9, 21.8, 20.5 (d, ³J_C–P = 6.5 Hz); ³¹P NMR (202 MHz, CD₃OD): δ (ppm) 3.84; HRMS (ESI) m/z calcd for C₂₂H₂₉N₆NaO7P [M + Na]⁺ 543.1728, found 543.1727. The diastereopurity of 23S was determined to be 97% de: retention time = 7.810 min.

Methyl ((S)-(((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (24S) and methyl ((R)-(((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (24R)

Compounds 24S and 24R were synthesized from 12 (0.910 g, 1.5 mmol) using the general procedure for silyl deprotection at the 2′-position to give racemic product 18 (0.474 g, 64%), which was purified by preparative chiral column chromatography (3% MeOH/ACN; flow rate 6 mL/min) to afford two separable diastereomers: 24R (Rp-isomer, white solid, 90 mg, 37%): ¹H NMR (500 MHz, CD₃OD): δ 8.28 (s, 1H), 8.21 (s, 1H), 7.34–7.29 (m, 2H), 7.21–7.14 (m, 3H), 6.02 (d, J = 1.8 Hz, 1H), 4.76–4.67 (m, 2H), 4.46 (ddd, J = 11.4, 5.6, 2.7 Hz, 1H), 4.29 (ddd, J = 11.4, 5.9, 3.7 Hz, 1H), 3.87–3.78 (m, 1H), 3.64 (s, 3H), 2.38 (ddd, J = 13.5, 9.4, 5.7 Hz, 1H), 2.09 (ddd, J = 13.4, 6.1, 2.5 Hz, 1H), 1.25 (dd, J = 7.2, 1.1 Hz, 1H); ¹³C NMR (126 MHz, CD₃OD): 175.5 (d, ³J_C–P = 4.0 Hz), 157.3, 153.8, 152.1 (d, ²J_C–P = 6.6 Hz), 150.2, 140.4, 130.7 (2C), 126.1, 121.4 (2C, d, ³J_C–P = 4.8 Hz), 120.4, 93.1, 80.4 (d, ³J_C–P = 8.7 Hz), 76.6, 68.1 (d, ²J_C–P = 5.2 Hz), 52.7, 51.4, 34.6, 20.2 (d, ³J_C–P = 7.4 Hz); ³¹P NMR (202 MHz, CD₃OD): δ 3.98; HRMS (ESI) m/z calcd for C₂₀H₂₅N₆NaO₇P [M + Na]⁺ 515.1415, found 515.1416. The diastereopurity of 24R was determined to be 100% de: retention time = 2.782 min; 24S (Sp-isomer, white solid, 101 mg, 42%): ¹H NMR (500 MHz, CD₃OD): δ 8.26 (s, 1H), 8.21 (s, 1H), 7.35–7.30 (m, 2H), 7.24–7.14 (m, 3H), 6.01 (d, J = 1.6 Hz, 1H), 4.75–4.72 (m, 1H), 4.70–4.64 (m, 1H), 4.42–4.36 (m, 1H), 4.31–4.24 (m, 1H), 3.96–3.88 (m, 1H), 3.61 (s, 3H), 2.42–2.34 (m, 1H), 2.13–2.07 (m, 1H), 1.29 (d, J = 7.1 Hz, 3H); ¹³C NMR (126 MHz, CD₃OD): 175.2 (d, ³J_C–P = 5.2 Hz), 157.3, 153.8, 152.1 (d, ²J_C–P = 6.7 Hz), 150.2, 140.5, 130.7 (2C), 126.1, 121.3 (2C, d, ³J_C–P = 4.8 Hz), 120.5, 93.2, 80.3 (d, ³J_C–P = 8.5 Hz), 76.6, 68.6 (d, ²J_C–P = 5.3 Hz), 52.7, 51.4, 34.8, 20.4 (d, ³J_C–P = 6.5 Hz); ³¹P NMR (202 MHz, CD₃OD): δ 3.81; HRMS (ESI) m/z calcd for C₂₀H₂₅N₆NaO₇P [M + Na]⁺ 515.1415, found 515.1414. The diastereopurity of 24S was determined to be 98% de: retention time = 5.692 min.

Ethyl ((S)-(((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (25S) and ethyl ((R)-(((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (25R)

Compounds 25S and 25R were synthesized from 13 (0.931 g, 1.5 mmol) using the general procedure for silyl deprotection at the 2′-position to give racemic product 19 (0.513 g, 68%), which was purified by preparative chiral column chromatography (3–5% MeOH/ACN; flow rate 6 mL/min) to afford two separable diastereomers: 25R (Rp-isomer, white solid, 89 mg, 35%): ¹H NMR (500 MHz, CD₃OD): δ (ppm) 8.28 (s, 1H), 8.21 (s, 1H), 7.34–7.28 (m, 2H), 7.21–7.13 (m, 3H), 6.02 (d, J = 1.5 Hz, 1H), 4.76–4.67 (m, 2H), 4.49–4.43 (m, 1H), 4.32–4.26 (m, 1H), 4.14–4.06 (m, 2H), 3.87–3.79 (m, 1H), 2.41–2.33 (m, 1H), 2.12–2.06 (m, 1H), 1.26 (d, J = 7.2 Hz, 3H), 1.20 (t, J = 7.1 Hz, 3H); ¹³C NMR (126 MHz, CD₃OD) δ 175.0 (d, ³J_C–P = 4.4 Hz), 157.3, 153.8, 152.1 (d, ²J_C–P = 6.7 Hz), 150.2, 140.4, 130.7 (2C), 126.1, 121.4 (2C, d, ³J_C–P = 4.8 Hz), 120.4, 93.1, 80.4 (d, ³J_C–P = 8.6 Hz), 76.6, 68.2 (d, ²J_C–P = 5.1 Hz), 62.3, 51.6, 34.7, 20.3 (d, ³J_C–P = 7.3 Hz), 14.4; ³¹P NMR (202 MHz, CD₃OD): δ 4.01; HRMS (ESI) m/z calcd for C₂₁H₂₇N₆NaO₇P [M + Na]⁺ 529.1571, found 529.1570. The diastereopurity of 25R was determined to be 99% de: retention time = 2.708 min; 25S (Sp-isomer, white solid, 98 mg, 38%): ¹H NMR (500 MHz, CD₃OD): δ (ppm) 8.26 (s, 1H), 8.21 (s, 1H), 7.35–7.29 (m, 2H), 7.24–7.13 (m, 3H), 6.01 (d, J = 1.6 Hz, 1H), 4.76–4.71 (m, 1H), 4.71–4.64 (m, 1H), 4.43–4.37 (m, 1H), 4.31–4.24 (m, 1H), 4.13–4.01 (m, 2H), 3.94–3.86 (m, 1H), 2.42–2.35 (m, 1H), 2.12–2.06 (m, 1H), 1.30 (d, J = 7.1 Hz, 3H), 1.18 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 174.8 (d, ³J_C–P = 5.4 Hz), 157.3, 153.8, 152.1 (d, ²J_C–P = 6.9 Hz), 150.1, 140.5, 130.7 (2C), 126.1, 121.3 (2C, d, ³J_C–P = 4.8 Hz), 120.4, 93.2, 80.3 (d, ³J_C–P = 8.5 Hz), 76.6, 68.6 (d, ²J_C–P = 5.5 Hz), 62.3, 51.4, 34.9, 20.4 (d, ³J_C–P = 6.5 Hz), 14.4; ³¹P NMR (202 MHz, CD₃OD): δ 3.83; HRMS (ESI) m/z calcd for C₂₁H₂₇N₆NaO₇P [M + Na]⁺ 529.1571, found 529.1577. The diastereopurity of 25S was determined to be 99% de: retention time = 7.271 min.

tert-Butyl ((S)-(((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (26S) and tert-Butyl ((R)-(((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (26R)

Compounds 26S and 26R were synthesized from 14 (0.973 g, 1.5 mmol) using the general procedure for silyl deprotection at the 2′-position to give racemic product 20 (0.510 g, 64%), which was purified by preparative chiral column chromatography (2% MeOH/ACN; flow rate 10 mL/min) to give two separable diastereomers: 26R (Rp-isomer, white solid, 74 mg, 29%): ¹H NMR (500 MHz, CD₃OD): δ 8.27 (s, 1H), 8.21 (s, J = 4.0 Hz, 1H), 7.35–7.27 (m, 2H), 7.23–7.13 (m, 3H), 6.01 (d, J = 1.8 Hz, 1H), 4.75–4.67 (m, 2H), 4.49–4.44 (m, 1H), 4.35–4.28 (m, 1H), 3.80–3.69 (m, 1H), 2.40–2.32 (m, 1H), 2.12–2.05 (m, 1H), 1.41 (s, 9H), 1.24 (d, J = 7.1 Hz, 3H); ¹³C NMR (126 MHz, CD₃OD) δ 174.3 (d, ³J_C–P = 4.8 Hz), 157.3, 153.8, 152.1 (d, ²J_C–P = 6.8 Hz), 150.2, 140.4, 130.7 (2C), 126.1, 121.4 (2C, d, ³J_C–P = 4.8 Hz), 120.4, 93.1, 82.5, 80.4 (d, ³J_C–P = 8.6 Hz), 76.6, 68.3 (d, ²J_C–P = 5.2 Hz), 52.2, 34.7, 28.1 (3C), 20.4 (d, ³J_C–P = 7.2 Hz); ³¹P NMR (202 MHz, CD₃OD): δ 4.06; HRMS (ESI) m/z calcd for C₂₃H₃₁N₆NaO₇P [M + Na]⁺ 557.1884, found 557.1874. The diastereopurity of 26R was determined to be 100% de: retention time = 2.229 min; 26S (Sp-isomer, white solid, 92 mg, 36%): ¹H NMR (500 MHz, CD₃OD): δ 8.27 (s, 1H), 8.21 (s, 1H), 7.35–7.30 (m, 2H), 7.24–7.15 (m, 3H), 6.01 (d, J = 1.8 Hz, 1H), 4.75–4.71 (m, 1H), 4.70–4.64 (m, 1H), 4.43–4.38 (m, 1H), 4.30–4.25 (m, 1H), 3.84–3.75 (m, 1H), 2.44–2.37 (m, 1H), 2.14–2.06 (m, 1H), 1.40 (s, J = 8.0 Hz, 9H), 1.27 (d, J = 7.0 Hz, 3H); ¹³C NMR (126 MHz, CD₃OD) δ 174.0 (d, ³J_C–P = 5.6 Hz), 157.3, 153.8, 152.1 (d, ²J_C–P = 7.0 Hz), 150.2, 140.5, 130.7 (2C), 126.1, 121.3 (2C, d, ³J_C–P = 4.8 Hz), 120.5, 93.2, 82.6, 80.3 (d, ³J_C–P = 8.4 Hz), 76.5, 68.6 (d, ²J_C–P = 5.4 Hz), 52.1, 34.9, 28.1 (3C), 20.7 (d, ³J_C–P = 6.4 Hz); ³¹P NMR (202 MHz, CD₃OD): δ 3.95; HRMS (ESI) m/z calcd for C₂₃H₃₁N₆NaO₇P [M + Na]⁺ 557.1884, found 557.1882. The diastereopurity of 26S was determined to be 95% de: retention time = 5.608 min.

Benzyl ((S)-(((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (27S) and Benzyl ((R)-(((2S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (27R)

Compounds 27S and 27R were synthesized from 15 (1.02 g, 1.5 mmol) using the general procedure for silyl deprotection at the 2′-position to give racemic product 21 (0.614 g, 72%), which was purified by preparative chiral column chromatography (3% MeOH/ACN; flow rate 15 mL/min) to give two separable diastereomers: 27R (Rp-isomer, white solid, 95 mg, 31%): ¹H NMR (500 MHz, CD₃OD): δ (ppm) 8.23 (s, 1H), 8.20 (s, 1H), 7.35–7.24 (m, 7H), 7.21–7.12 (m, 3H), 5.98 (d, J = 1.6 Hz, 1H), 5.10–5.03 (m, 2H), 4.69–4.57 (m, 2H), 4.40–4.32 (m, 1H), 4.25–4.18 (m, 1H), 4.01–3.92 (m, 1H), 2.34–2.26 (m, 1H), 2.06–2.00 (m, 1H), 1.30 (d, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 174.8 (d,³J_C–P = 4.1 Hz), 157.3, 153.8, 152.1, 150.1, 140.4, 137.2, 130.7 (2C), 129.5 (2C), 129.3 (3C), 126.1, 121.4 (2C, d, ³J_C–P = 4.7 Hz), 120.4, 93.1, 80.4 (d, ³J_C–P = 8.6 Hz), 76.6, 68.2 (d, ²J_C–P = 5.1 Hz), 67.9, 51.7, 34.5, 20.2 (d, ³J_C–P = 7.4 Hz); ³¹P NMR (202 MHz, CD₃OD): δ 4.03; HRMS (ESI) m/z calcd for C₂₆H₂₉N₆NaO₇P [M + Na]⁺ 591.1728, found 591.1731. The diastereopurity of 27R was determined to be 99% de: retention time = 4.358 min; 27S (Sp-isomer, white solid, 108 mg, 35%): ¹H NMR (500 MHz, CD₃OD): δ (ppm) 8.25 (s, 1H), 8.20 (s, 1H), 7.34–7.25 (m, 7H), 7.19–7.12 (m, 3H), 5.99 (d, J = 1.3 Hz, 1H), 5.14–5.07 (m, 2H), 4.72–4.68 (m, 1H), 4.66–4.60 (m, 1H), 4.42–4.36 (m, 1H), 4.25–4.20 (m, 1H), 3.96–3.88 (m, 1H), 2.34–2.26 (m, 1H), 2.08–2.01 (m, 1H), 1.27 (d, J = 7.1 Hz, 3H); ¹³C NMR (126 MHz, CD₃OD) δ 174.6 (d, ³J_C–P = 5.0 Hz), 157.3, 153.8, 152.0 (d, ²J_C–P = 6.7 Hz), 150.1, 140.4, 137.2, 130.7 (2C), 129.5 (2C), 129.3, 129.2 (2C), 126.1, 121.3 (2C, d, ³J_C–P = 4.8 Hz), 120.5, 93.2, 80.3 (d, ³J_C–P = 8.5 Hz), 76.6, 68.5 (d, ²J_C–P = 5.3 Hz), 67.8, 51.5, 34.8, 20.3 (d, ³J_C–P = 6.6 Hz); ³¹P NMR (202 MHz, CD₃OD): δ 3.71; HRMS (ESI) m/z calcd for C₂₆H₂₉N₆NaO₇P [M + Na]⁺ 591.1728, found 591.1725. The diastereopurity of 27S was determined to be 96% de: retention time = 6.214 min.

9-((2R,3R,5S)-3-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-6-chloro-9H-purine (28)

To a solution of silyl ether 9 (1.0 g, 2.08 mmol) in anhydrous CH₂Cl₂ (20 mL, 0.1 M) were added tert-butyl nitrite (1.0 mL, 8.33 mmol, 4 equiv) and chlorotrimethylsilane (530 μL, 4.17 mmol, 2 equiv) at 0 °C to room temperature under an argon atmosphere for 5 h. The reaction mixture was quenched with aqueous NaHCO₃ (25 mL) and extracted with CH₂Cl₂ (3 × 20 mL). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. Purification of the crude residue by column chromatography (10–20% EtOAc/hexanes) yielded 28 as a white solid (728 mg, 70%); ¹H NMR (400 MHz, DMSO-d₆) δ 8.82 (s, 1H), 8.78 (s, 1H), 6.02 (d, J = 1.4 Hz, 1H), 4.79–4.77 (m, 1H), 4.48–4.44 (m, 1H), 4.00 (dd, J = 11.7, 2.9 Hz, 1H), 3.79 (dd, J = 11.7, 2.9 Hz, 1H), 2.27 (ddd, J = 13.2, 9.7, 5.0 Hz, 1H), 1.90 (ddd, J = 13.2, 5.7, 2.2 Hz, 1H), 0.85 (s, 9H), 0.84 (s, 9H), 0.05 (s, 3H), 0.04 (s, 9H); ¹³C NMR (100 MHz, DMSO-d₆) δ 151.6, 151.0, 149.1, 144.6, 131.4, 91.3, 81.1, 76.6, 63.5, 33.5, 25.8 (3C), 25.5 (3C), 18.1, 17.6, – 4.9, – 5.0, – 5.4 (2C); HRMS (ESI) m/z calcd for C₂₂H₃₉ClN₄NaO₃Si₂ [M + Na]⁺ 521.2147, found 521.2145.

9-((2R,3R,5S)-3-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-6-chloro-2-(tributylstannyl)-9H-purine (29)

To a solution of 2,2,6,6-tetramethylpiperidide (TMP) (1.3 mL, 7.97 mmol, 5 equiv) in anhydrous THF (8 mL, 0.1 M) was slowly added n-BuLi (1.6 M in hexanes) (5 mL, 7.95 mmol, 5 equiv) at −78 °C under an argon atmosphere. The reaction mixture was stirred at this temperature for 1 h before the addition of a solution of compound 28 (800 mg, 1.59 mmol) in anhydrous THF (5.5 mL, 0.3 M). After stirring for 1 h, to the reaction mixture was added Bu₃SnCl (2.2 mL, 7.97 mmol, 5 equiv), and the mixture was stirred at −78 °C for 30 min. The reaction was warmed to room temperature, quenched with aqueous NH₄Cl (20 mL), and extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated in vacuo. The crude residue was purified by column chromatography (5–10% EtOAc/hexanes) yielded 6-chloro-2-(tributylstannyl) purine (29) as a light yellow oil (980 mg, 78%); ¹H NMR (400 MHz, CDCl₃) δ 8.60 (s, 1H), 6.13 (d, J = 1.2 Hz, 1H), 4.63–4.57 (m, 2H), 4.16 (dd, J = 11.7, 2.4 Hz, 1H), 3.80 (dd, J = 11.7, 2.4 Hz, 1H), 2.32 (ddd, J = 13.0, 10.0, 4.6 Hz, 1H), 1.88 (ddd, J = 13.0, 5.4, 1.9 Hz, 1H), 1.69–1.56 (m, 6H), 1.38–1.19 (m, 12H), 0.95 (s, 9H), 0.91 (s, 9H), 0.91–0.87 (m, 9H), 0.15–0.14 (m, 9H), 0.09 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 181.4, 150.1, 148.9, 142.3, 130.7, 91.7, 81.4, 77.4, 63.5, 33.4, 28.8 (3C), 27.1 (3C), 25.9 (3C), 25.5 (3C), 18.5, 17.8, 13.6 (3C), 10.5 (3C), −4.6, −5.0, −5.3, −5.4; HRMS (ESI) m/z calcd for C₃₄H₆₅ClN₄NaO₃Si₂Sn [M + Na]⁺ 811.3203, found 811.3200.

9-((2R,3R,5S)-3-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-2-(tributylstannyl)-9H-purin-6-amine (8)

To a solution of 29 (300 mg, 0.38 mmol) in THF (2 mL, 0.1 M) was added ammonia 7 N in MeOH (35 mL, 1.9 mmol, 5 equiv) at room temperature under an argon atmosphere. The reaction mixture was heated at 100 °C for 9 h. The reaction was warmed to room temperature and concentrated under reduced pressure, and purification of the crude residue by column chromatography (20–40% EtOAc/hexanes) yielded 8 as a light yellow oil (165 mg, 55%); ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (s, 1H), 7.03 (brs, 2H), 5.89 (d, J = 2.2 Hz, 1H), 4.85–4.83 (m, 1H), 4.37–4.34 (m, 1H), 3.89 (dd, J = 11.4, 3.4 Hz, 1H), 3.74 (dd, J = 11.4, 4.1 Hz, 1H), 2.38–2.32 (m, 1H), 1.94–1.88 (m, 1H), 1.61–1.40 (m, 6H), 1.32–1.23 (m, 6H), 1.16–0.95 (m, 6H), 0.85 (s, 9H), 0.82–0.68 (m, 18H), 0.01 (s, 6H), −0.02 (s, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ178.5, 154.3, 148.1, 138.1, 118.4, 90.7, 80.0, 76.1, 64.2, 34.4, 28.6 (3C), 26.7 (3C), 25.8 (3C), 25.5 (3C), 18.0, 17.6, 13.5 (3C), 9.8 (3C), −5.0, −5.1, −5.4 (2C); HRMS (ESI) m/z calcd for C₃₄H₆₇N₅NaO₃Si₂Sn [M + Na]⁺ 792.3702, found 792.3701.

9-((2R,3R,5S)-3-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-2-fluoro-9H-purin-6-amine (30)

To a solution of 2-stannylated cordycepin intermediate 8 (150 mg, 0.195 mmol) in anhydrous CH₂Cl₂ (9 mL, 0.022 M) were added 2,6-di-tert-butyl-4-methylpyridine (40.2 mg, 0.195 mmol, 1.0 equiv), XeF₂ (82.7 mg, 0.49 mmol, 2.5 equiv), and silver triflate (125.5 mg, 0.49 mmol, 2.5 equiv), and the mixture was stirred at room temperature for 30 min under an argon atmosphere. The reaction mixture was quenched with NaHCO₃ (15 mL) and extracted with CH₂Cl₂ (3 × 10 mL). The combined organic phases were washed with brine, dried over anhydrous Na₂SO₄ and concentrated in vacuo. Purification of the crude residue by column chromatography (5–10% EtOAc/hexanes) afforded 30 as a white solid (64 mg, 65%); ¹H NMR (400 MHz, CDCl₃) δ 8.28 (s, 1H), 6.56 (br s, 2H), 5.90 (d, J = 1.5 Hz, 1H), 4.60–4.53 (m, 1H), 4.12 (dd, J = 11.6, 2.6 Hz, 1H), 3.76 (dd, J = 11.6, 2.6 Hz, 1H), 2.27–2.20 (m, 1H), 1.84 (ddd, J = 13.0, 5.6, 2.2 Hz, 1H), 0.93 (s, 9H), 0.89 (s, 9H), 0.14 (s, 3H), 0.12 (s, 3H), 0.11 (s, 3H), 0.09 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 159.1 (d, ¹J_C–F = 210.2 Hz), 157.2 (d, ²J_C–F = 20.1 Hz), 150.7 (d, ²J_C–F = 19.8 Hz), 139.3 (d, ⁴J_C–F = 3.0 Hz), 118.2 (d, ³J_C–F = 4.0 Hz), 92.0, 81.5, 77.4, 63.7, 33.6, 26.1 (3C), 25.8 (3C), 18.6, 18.0, −4.6, −4.9, −5.1, −5.3. ¹⁹F NMR (470 MHz, CDCl₃) δ – 51.57; HRMS (ESI) m/z calcd for C₂₂H₄₀FN₅NaO₃Si₂ [M + Na]⁺ 520.2551, found 520.2550.

9-((2R,3R,5S)-3-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-2-iodo-9H-purin-6-amine (31)

To a solution of 2-stannylated cordycepin intermediate 8 (100 mg, 0.13 mmol) in anhydrous THF (4.5 mL, 0.030 M) was added iodine (50 mg, 0.195 mmol, 1.5 equiv) at room temperature under an argon atmosphere, and the mixture was stirred for 30 min. The reaction mixture was quenched with 10% Na₂S₂O₃ (10 mL) and extracted with CH₂Cl₂ (3 × 5 mL). The combined organic phases were washed with brine, dried over anhydrous Na₂SO₄, and concentrated in vacuo. Purification of the crude residue by column chromatography (5–10% EtOAc/hexanes) yielded 31 as a light yellow oil (56 mg, 70%); ¹H NMR (400 MHz, CDCl₃) δ 8.26 (s, 1H), 6.33 (brs, 2H), 5.91 (d, J = 1.0 Hz, 1H), 4.59–4.54 (m, 2H), 4.15 (dd, J = 11.6, 2.5 Hz, 1H), 3.77 (dd, J = 11.6, 2.5 Hz, 1H), 2.16 (ddd, J = 13.0, 10.2, 4.6 Hz, 1H), 1.79 (ddd, J = 13.0, 5.3, 1.8 Hz, 1H), 0.93 (s, 9H), 0.92 (s, 9H), 0.21 (s, 3H), 0.14 (s, 3H), 0.13 (s, 3H), 0.11 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 155.2, 149.5, 138.9, 120.0, 119.4, 92.3, 81.8, 77.6, 63.4, 33.2, 26.1 (3C), 25.8 (3C), 18.7, 18.1, −4.4, −4.9, −5.1, −5.3; HRMS (ESI) m/z calcd for C₂₂H₄₀IN₅NaO₃Si₂ [M + Na]⁺ 628.1612, found 628.1607.

2-Bromo-9-((2R,3R,5S)-3-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-9H-purin-6-amine (32)

To a solution of 2-stannylated cordycepin intermediate 8 (290 mg, 0.375 mmol) in anhydrous THF (17 mL, 0.022 M) was added NBS (167 mg, 0.9375 mmol, 2.5 equiv) at room temperature under an argon atmosphere, and the mixture was stirred for 1 h. The reaction mixture was quenched with aqueous NaHCO₃ (20 mL) and extracted with CH₂Cl₂ (3 × 10 mL). The combined organic phases were washed with brine, dried over anhydrous Na₂SO₄, and concentrated in vacuo. Purification of the crude residue by column chromatography (10–20% EtOAc/hexanes) yielded 32 as a light yellow oil (160 mg, 76%); ¹H NMR (400 MHz, CDCl₃) δ 8.32 (s, 1H), 6.45 (brs, 2H), 5.94 (d, J = 1.1 Hz, 1H), 4.62–4.56 (m, 2H), 4.16 (dd, J = 11.7, 2.5 Hz, 1H), 3.78 (dd, J = 11.7, 2.5 Hz, 1H), 2.20 (ddd, J = 13.0, 10.0, 4.6 Hz, 1H), 1.82 (ddd, J = 13.0, 5.4, 1.9 Hz, 1H), 0.94 (s, 9H), 0.93 (s, 9H), 0.21 (s, 3H), 0.15 (s, 3H), 0.14 (s, 3H), 0.13 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 156.0, 150.0, 144.6, 139.2, 119.3, 92.3, 81.8, 77.6, 63.5, 33.2, 26.1 (3C), 25.8 (3C), 18.6, 18.1, −4.5, −4.9, −5.1, −5.3; HRMS (ESI) m/z calcd for C₂₂H₄₀BrN₅NaO₃Si₂ [M + Na]⁺ 580.1751, found 580.1748.

((2S,4R,5R)-5-(6-Amino-2-fluoro-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)methanol (33)

Alcohol 33 was prepared from 30 (750 mg, 1.50 mmol) using the general procedure for silyl deprotection at the 5′-position. The crude residue was purified by column chromatography (40% EtOAc/hexanes–100% EtOAc) to give 33 (440 mg, 77%) as a white solid; ¹H NMR (400 MHz, CD₃OD) δ 8.36 (s, 1H), 5.83 (d, J = 3.0 Hz, 1H), 4.81–4.77 (m, 1H), 4.52–4.47 (m, 1H), 3.93 (dd, J = 12.4, 2.7 Hz, 1H), 3.68 (dd, J = 12.4, 3.4 Hz, 1H), 2.35–2.28 (m, 1H), 2.06–2.00 (m, 1H), 0.87 (s, 9H), 0.05 (s, 3H), 0.02 (s, 3H). ¹³C NMR (100 MHz, CD₃OD) δ 160.4 (d, ¹J_C–F = 209.4 Hz), 159.0 (d, ²J_C–F = 20.4 Hz), 151.5 (d, ²J_C–F = 19.7 Hz), 141.1 (d, ⁴J_C–F = 3.1 Hz), 118.8 (d, ³J_C–F = 4.2 Hz), 93.3, 82.3, 77.5, 64.2, 35.3, 26.1 (3C), 18.7, −4.8, −4.9. ¹⁹F NMR (470 MHz, CD₃OD) δ −53.45; HRMS (ESI) m/z calcd for C₁₆H₂₆FN₅NaO₃Si [M + Na]⁺ 406.1687, found 406.1681.

((2S,4R,5R)-5-(6-Amino-2-iodo-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)methanol (34)

Alcohol 34 was prepared from 31 (75 mg, 0.124 mmol) using the general procedure for silyl deprotection at the 5′-position. The crude residue was purified by column chromatography (40% EtOAc/hexanes–100% EtOAc) to give 34 (49 mg, 82%) as a white solid; ¹H NMR (400 MHz, CD₃OD) δ 8.32 (s, 1H), 5.84 (d, J = 2.7 Hz, 1H), 4.80–4.77 (m, 1H), 4.53–4.48 (m, 1H), 3.94 (dd, J = 12.4, 2.7 Hz, 1H), 3.67 (dd, J = 12.4, 3.4 Hz, 1H), 2.25 (ddd, J = 13.5, 8.1, 5.6 Hz, 1H), 2.02–1.96 (m, 1H), 0.89 (s, 9H), 0.07 (s, 3H), 0.06 (s, 3H); ¹³C NMR (100 MHz, CD₃OD) δ 157.0, 150.3, 140.6, 120.4, 120.4, 120.4, 93.5, 82.5, 77.6, 64.0, 35.0, 26.2 (3C), 18.8, – 4.7, – 4.8; HRMS (ESI) m/z calcd for C₁₆H₂₆IN₅NaO₃Si [M + Na]⁺ 514.0747, found 514.0742.

((2S,4R,5R)-5-(6-Amino-2-bromo-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)methanol (35)

Alcohol 35 was prepared from 32 (160 mg, 0.283 mmol) using the general procedure for silyl deprotection at the 5′-position. The crude residue was purified by column chromatography (40% EtOAc/hexanes–100% EtOAc) to give 35 (86 mg, 70%) as a white solid; ¹H NMR (400 MHz, CD₃OD) δ 8.36 (s, 1H), 5.85 (d, J = 2.9 Hz, 1H), 4.81–4.78 (m, 1H), 4.53–4.48 (m, 1H), 3.93 (dd, J = 12.4, 2.7 Hz, 1H), 3.67 (dd, J = 12.4, 3.4 Hz, 1H), 2.27 (ddd, J = 13.4, 7.9, 5.7 Hz, 1H), 2.01 (ddd, J = 12.9, 6.6, 4.0 Hz, 1H), 0.89 (s, 9H), 0.06 (s, 3H), 0.05 (s, 3H); ¹³C NMR (100 MHz, CD₃OD) δ 157.8, 151.0, 145.8, 141.1, 119.9, 93.5, 82.5, 77.6, 64.1, 35.1, 26.1 (3C), 18.8, −4.7, −4.9; HRMS (ESI) m/z calcd for C₁₆H₂₆BrN₅NaO₃Si [M + Na]⁺ 466.0886, found 466.0880.

(S)-2-Ethylbutyl 2-(((S)-(((2S,4R,5R)-5-(6-Amino-2-fluoro-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (36S)

ProTide product 36S was prepared from alcohol 33 (250 mg, 0.65 mmol) and phosphate reagent ((S)-2-ethylbutyl 2-(((S)-(4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (6)) (468 mg, 1.06 mmol) using a general procedure for phosphorylation. The crude residue was purified by column chromatography (70% EtOAc/hexanes–100% EtOAc) to afford 36S (270 mg, 60%) as a white solid; ¹H NMR (400 MHz, CD₃OD): δ 8.21 (s, 1H), 7.36–7.32 (m, 2H), 7.26–7.23 (m, 2H), 7.19–7.16 (m, 1H), 5.86 (d, J = 2.2 Hz, 1H), 4.83–4.80 (m, 1H), 4.66–4.61 (m, 1H), 4.44–4.39 (m, 1H), 4.32–4.26 (m, 1H), 4.03–3.99 (m, 1H), 3.96–3.90 (m, 2H), 2.32–2.25 (m, 1H), 2.05–1.99 (m, 1H), 1.47–1.39 (m, 1H), 1.35–1.27 (m, 7H), 0.89 (s, 9H), 0.87–0.83 (m, 6H), 0.09 (s, 3H), 0.08 (s, 3H). ¹³C NMR (100 MHz, CD₃OD) δ 174.9 (d, ³J_C–P = 5.2 Hz), 160.5 (d, ¹J_C–F = 208.9 Hz), 159.0 (d, ²J_C–F = 20.5 Hz), 152.1 (d, ²J_C–P = 6.6 Hz), 151.7 (d, ²J_C–F = 19.9 Hz), 140.5 (d, ⁴J_C–F = 2.8 Hz), 130.7 (2C), 126.15, 121.3 (2C, d, ³J_C–P = 4.9 Hz), 118.7, 93.1, 80.2 (d, ³J_C–P = 8.5 Hz), 77.7, 68.5 (d, ²J_C–P = 5.3 Hz), 68.0, 51.5, 41.6, 35.5, 26.1 (3C), 24.2, 24.1, 20.6 (d, ³J_C–P = 6.5 Hz), 18.7, 11.3, 11.2, – 4.7, – 4.8; ³¹P NMR (162 MHz, CD₃OD): δ 3.77. ¹⁹F NMR (470 MHz, CD₃OD) δ – 53.06; HRMS (ESI) m/z calcd for C₃₁H₄₈FN₆NaO₇PSi [M + Na]⁺ 717.2973, found 717.2968.

2-Ethylbutyl ((S)-(((2S,4R,5R)-5-(6-Amino-2-iodo-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-l-alaninate (37S)

ProTide product 37S was prepared from alcohol 34 (60 mg, 0.127 mmol) and phosphate reagent ((S)-2-ethylbutyl 2-(((S)-(4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (6)) (90 mg, 0.20 mmol) using the general procedure for phosphorylation. The crude residue was purified by column chromatography (70% EtOAc/hexanes–100% EtOAc) to afford 37S (63 mg, 65%) as a colorless solid; ¹H NMR (400 MHz, CD₃OD): δ 8.16 (s, 1H), 7.34 (dd, J = 8.7, 7.0 Hz, 2H), 7.27–7.24 (m, 2H), 7.20–7.16 (m, 1H), 5.88 (d, J = 1.6 Hz, 1H), 4.83–4.81 (m, 1H), 4.70–4.63 (m, 1H), 4.44 (ddd, J = 11.4, 5.9, 2.8 Hz, 1H), 4.31 (ddd, J = 11.4, 6.2, 4.4 Hz, 1H), 4.00 (dd, J = 10.9, 5.8 Hz, 1H), 3.96–3.88 (m, 2H), 3.32–3.30 (m, 1H), 2.20 (ddd, J = 13.2, 9.7, 5.2 Hz, 1H), 1.99 (ddd, J = 13.2, 5.9, 2.4 Hz, 1H), 1.46–1.40 (m, 1H), 1.34–1.28 (m, 7H), 0.92 (s, 9H), 0.87–0.83 (m, 6H), 0.15 (s, 3H), 0.13 (s, 3H);¹³C NMR (100 MHz, CD₃OD) δ 174.9 (d, ³J_C–P = 5.1 Hz), 157.0, 152.1 (d, ²J_C–P = 6.6 Hz), 150.4, 139.9, 130.7 (2C), 126.1, 121.3 (2C, d, ³J_C–P = 5.1 Hz), 120.6, 120.3, 93.6, 80.6 (d, ³J_C–P = 8.4 Hz), 77.9, 68.4 (d, ²J_C–P = 5.3 Hz), 68.1, 51.5, 41.6, 35.3, 26.2 (3C), 24.2, 24.1, 20.6 (d, ³J_C–P = 6.6 Hz), 18.8, 11.3, 11.2, – 4.5, – 4.7; ³¹P NMR (162 MHz, CD₃OD): δ 3.77; HRMS (ESI) m/z calcd for C₃₁H₄₈IN₆NaO₇PSi [M + Na]⁺ 825.2034, found 825.2028.

2-Ethylbutyl ((S)-(((2S,4R,5R)-5-(6-Amino-2-bromo-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-l-alaninate (38S)

ProTide product 38S was prepared from alcohol 35 (70 mg, 0.160 mmol) and phosphate reagent ((S)-2-ethylbutyl 2-(((S)-(4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (6)) (109 mg, 0.24 mmol) using a general procedure for phosphorylation. The crude residue was purified by column chromatography (70% EtOAc/hexanes–100% EtOAc) to afford 38S (80 mg, 68%) as a colorless solid; ¹H NMR (400 MHz, CD₃OD): δ 8.22 (s, 1H), 7.36–7.32 (m, 2H), 7.25–7.23 (m, 2H), 7.20–7.16 (m, 1H), 5.88 (d, J = 2.0 Hz, 1H), 4.82–4.81 (m, 1H), 4.46–4.41 (m, 1H), 4.33–4.27 (m, 1H), 3.99 (dd, J = 10.9, 5.8 Hz, 1H), 3.95–3.88 (m, 2H), 2.26–2.19 (m, 1H), 2.01 (ddd, J = 13.2, 6.0, 2.7 Hz, 1H), 1.45–1.39 (m, 1H), 1.33–1.25 (m, 7H), 0.89 (s, 9H), 0.86–0.82 (m, 6H), 0.12 (s, 6H); ¹³C NMR (101 MHz, CD₃OD) δ 175.1 (d, ³J_C–P = 5.2 Hz), 157.7, 152.0 (d, ²J_C–P = 6.8 Hz), 151.0, 145.9, 140.4, 130.8 (2C), 126.2, 121.2 (2C, d, ³J_C–P = 4.8 Hz), 119.7, 93.4, 80.4 (d, ³J_C–P = 8.5 Hz), 77.8, 68.5 (d, ²J_C–P = 5.3 Hz), 68.1, 51.5, 41.5, 35.3, 26.2 (3C), 24.1, 24.1, 20.6 (d, ³J_C–P = 6.5 Hz), 18.8, 11.3, 11.2, – 4.6, – 4.7; ³¹P NMR (162 MHz, CD₃OD): δ 3.74; HRMS (ESI) m/z calcd for C₃₁H₄₈BrN₆NaO₇PSi [M + Na]⁺ 777.2172, found 777.2170.

(S)-2-Ethylbutyl 2-(((S)-(((2S,4R,5R)-5-(6-Amino-2-fluoro-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (39S)

Compound 39S was prepared from compound 36S (200 mg, 0.29 mmol) using the general procedure for silyl deprotection at the 2′-position. The crude residue was purified by column chromatography (5% MeOH/CH₂Cl₂) to give 39S (Sp-isomer, white solid, 110 mg, 65%); ¹H NMR (400 MHz, CD₃OD): δ 8.20 (s, 1H), 7.34–7.29 (m, 2H), 7.21–7.19 (m, 2H), 7.17–7.13 (m, 1H), 5.90 (s, 1H), 4.72–4.69 (m, 1H), 4.67–4.63 (m, 1H), 4.42–4.37 (m, 1H), 4.30–4.24 (m, 1H), 4.02–3.97 (m, 1H), 3.95–3.88 (m, 2H), 2.39–2.31 (m, 1H), 2.09–2.04 (m, 1H), 1.46–1.40 (m, 1H), 1.32–1.27 (m, 7H), 0.86–0.82 (m, 6H); ¹³C NMR (101 MHz, CD₃OD) δ 174.9 (d, ³J_C–P = 5.3 Hz), 160.5 (d, ¹J_C–F = 208.9 Hz), 159.0 (d, ²J_C–F = 20.4 Hz), 152.1 (d, ²J_C–P = 6.7 Hz), 151.6 (d, ²J_C–F = 19.7 Hz), 140.6 (d, ⁴J_C–F = 3.0 Hz), 130.7 (2C), 126.1, 121.3 (2C, d, ³J_C–P = 4.8 Hz), 118.7, 93.3, 80.4 (d, ³J_C–P = 8.5 Hz), 76.5, 68.6 (d, ²J_C–P = 5.3 Hz), 68.0, 51.5, 41.7, 34.9, 24.2, 24.1, 20.6 (d, ³J_C–P = 6.6 Hz), 11.3, 11.2; ³¹P NMR (162 MHz, CD₃OD): δ 3.76; ¹⁹F NMR (470 MHz, CD₃OD): δ – 53.18; HRMS (ESI) m/z calcd for C₂₅H₃₄FN₆NaO₇P [M + Na]⁺ 603.2108, found 603.2108. The diastereopurity of 39S was determined to be 98% de: retention time = 2.657 min.

2-Ethylbutyl ((S)-(((2S,4R,5R)-5-(6-Amino-2-iodo-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (40S)

Compound 40S was prepared from compound 37S (56 mg, 0.07 mmol) using the general procedure for silyl deprotection at the 2′-position. The crude residue was purified by column chromatography (5% MeOH/CH₂Cl₂) to give 40S (Sp-isomer, colorless solid, 28 mg, 62%); ¹H NMR (400 MHz, CD₃OD): δ 8.14 (s, 1H), 7.34–7.30 (m, 2H), 7.23–7.20 (m, 2H) 7.18–7.14 (m, 1H), 5.94 (d, J = 1.5 Hz, 1H), 4.68–4.65 (m, 2H), 4.40 (ddd, J = 11.3, 5.9, 2.8 Hz, 1H), 4.29 (ddd, J = 11.3, 6.2, 4.7 Hz, 1H), 4.00 (dd, J = 10.9, 5.8 Hz, 1H), 3.96–3.88 (m, 2H), 2.35 (ddd, J = 13.4, 9.8, 5.6 Hz, 1H), 2.06 (ddd, J = 13.4, 6.0, 2.2 Hz, 1H), 1.47–1.40 (m, 1H), 1.34–1.27 (m, 7H), 0.87–0.83 (m, 6H); ¹³C NMR (100 MHz, CD₃OD) δ 174.9 (d, ³J_C–P = 5.0 Hz), 157.0, 152.0 (d, ²J_C–P = 7.0 Hz), 150.5, 140.2, 130.7 (2C), 126.1, 121.3 (2C, d, ³J_C–P = 4.9 Hz), 120.6, 120.3, 93.4, 80.6 (d, ³J_C–P = 8.5 Hz), 76.7, 68.5 (d, ²J_C–P = 5.5 Hz), 68.0, 51.5, 41.6, 34.8, 24.2, 24.1, 20.6 (d, ³J_C–P = 6.6 Hz), 11.3, 11.3; ³¹P NMR (162 MHz, CD₃OD): δ 3.71; HRMS (ESI) m/z calcd for C₂₅H₃₄IN₆NaO₇P [M + Na]⁺ 711.1169, found 711.1162. The diastereopurity of 40S was determined to be 97% de: retention time = 3.936 min.

2-Ethylbutyl ((S)-(((2S,4R,5R)-5-(6-Amino-2-bromo-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (41S)

Compound 41S was prepared from compound 38S (74 mg, 0.098 mmol) using the general procedure for silyl deprotection at the 2′-position. The crude residue was purified by column chromatography (5% MeOH/CH₂Cl₂) to give 41S (Sp-isomer, colorless solid, 37 mg, 65%); ¹H NMR (400 MHz, CD₃OD): δ 8.19 (s, 1H), 7.34–7.30 (m, 2H), 7.23–7.20 (m, 2H), 7.18–7.14 (m, 1H), 5.94 (d, J = 1.6 Hz, 1H), 4.69–4.64 (m, 2H), 4.41 (ddd, J = 11.3, 5.9, 2.8 Hz, 1H), 4.33–4.25 (m, 1H), 4.00 (dd, J = 10.9, 5.8 Hz, 1H), 3.96–3.89 (m, 2H), 2.38–2.31 (m, 1H), 2.06 (ddd, J = 13.4, 5.9, 2.2 Hz, 1H), 1.47–1.41 (m, 1H), 1.34–1.27 (m, 7H), 0.87–0.83 (m, 6H); ¹³C NMR (100 MHz, CD₃OD) δ 174.9 (d, ³J_C–P = 5.2 Hz), 157.8, 152.0 (d, ²J_C–P = 6.7 Hz), 151.0, 145.9, 140.5, 130.7 (2C), 126.1, 121.3 (2C, d, ³J_C–P = 4.8 Hz), 119.8, 93.4, 80.6 (d, ³J_C–P = 8.5 Hz), 76.6, 68.5 (d, ²J_C–P = 5.3 Hz), 68.0, 51.5, 41.6, 34.7, 24.2, 24.1, 20.6 (d, ³J_C–P = 6.5 Hz), 11.3, 11.2; ³¹P NMR (162 MHz, CD₃OD): δ 3.73; HRMS (ESI) m/z calcd for C₂₅H₃₄BrN₆NaO₇P [M + Na]⁺ 663.1308, found 663.1308. The diastereopurity of 41S was determined to be 95% de: retention time = 3.728 min.

2-Ethylbutyl ((S)-(((2R,3S,5R)-5-(6-Amino-9H-purin-9-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy) (phenoxy)phosphoryl)-l-alaninate (44S)

The mixture of 2′-deoxyadeonsine (42) (0.200 g, 0.796 mmol), 2-(((S)-(4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (6)) (0.538 g, 1.19 mmol), and MgCl₂ (0.114 g, 1.19 mmol) in THF (8.0 mL) and DMF (4.0 mL) were stirred at 50 °C for 10 min. Then, N,N-diisopropylethylamine or DIEA (0.35 mL, 1.99 mmol) was added to the reaction mixture and stirred for 5 h. After removal of THF, the residue was extracted with EtOAc and water and washed with brine solution and sat. NaHCO₃. The combined organic layers were evaporated and purified by column chromatography (hexane:EtOAc:MeOH (50:50:0 to 0:100:0 to 0:97:3)) to give 44S (Sp-isomer, white solid, 0.140 g, 31%); ¹H NMR (500 MHz, DMSO) δ 8.28 (s, 1H), 8.13 (s, 1H), 7.37–7.30 (m, 2H), 7.27 (s, 2H), 7.22–7.12 (m, 3H), 6.36 (t, J = 6.9 Hz, 1H), 6.01 (dd, J = 13.0, 10.1 Hz, 1H), 5.47 (d, J = 4.3 Hz, 1H), 4.49–4.41 (m, 1H), 4.26–4.19 (m, 1H), 4.09–3.99 (m, 2H), 3.93 (dd, J = 10.9, 5.8 Hz, 1H), 3.89–3.77 (m, 2H), 2.80–2.72 (m, 1H), 2.30 (ddd, J = 13.3, 6.3, 3.4 Hz, 1H), 1.47–1.36 (m, 1H), 1.30–1.17 (m, 7H), 0.83–0.75 (m, 6H); ¹³C NMR (126 MHz, CD₃OD) δ 173.5 (d, ³J_C–P = 5.2 Hz), 155.9, 152.4, 150.7 (d, ²J_C–P = 6.9 Hz), 148.9, 139.4, 129.3, 124.7, 119.9 (d, ³J_C–P = 4.8 Hz), 119.1, 85.4 (d, ³J_C–P = 8.2 Hz), 84.3, 70.9, 66.7, 66.3 (d, ²J_C–P = 5.5 Hz), 50.1, 40.2, 39.5, 22.8, 22.8, 19.2 (d, ³J_C–P = 6.5 Hz), 9.9, 9.9; ³¹P NMR (202 MHz, DMSO) δ 3.68; HRMS (ESI) m/z calcd for C₂₅H₃₅N₆NaO₇P [M + Na]⁺ 585.2203, found 585.2208. The diastereopurity of 44S was determined to be 90% de: retention time = 5.554 min.

2-Ethylbutyl ((S)-(((2S,5R)-5-(6-Amino-9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)(phenoxy) phosphoryl)-l-alaninate (45S)

The mixture of 2′,3′-dideoxyadenosine (43) (50 mg, 0.213 mmol), 2-(((S)-(4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (6)) (144 mg, 0.320 mmol), and MgCl₂ (30 mg, 0.320 mmol) in THF (2.0 mL) and DMF (1.0 mL) were stirred at 50 °C for 10 min. Then, DIEA (93 μL, 0.533 mmol) was added to the reaction mixture, and the mixture was stirred for 5 h. After removal of THF, the residue was extracted with EtOAc and water and washed with brine solution and sat. NaHCO₃. The combined organic layers were evaporated and purified by column chromatography (hexane:EtOAc:MeOH (50:50:0 to 0:100:0 to 0:97:3)) to provide 45S (Sp-isomer, white solid, 0.140 g, 31%); ¹H NMR (500 MHz, CD₃OD) δ 8.29 (s, 1H), 8.20 (s, 1H), 7.35–7.28 (m, 2H), 7.22–7.12 (m, 3H), 6.30 (dd, J = 6.8, 3.7 Hz, 1H), 4.44–4.37 (m, 1H), 4.34 (ddd, J = 11.1, 6.1, 3.2 Hz, 1H), 4.24 (ddd, J = 11.1, 6.2, 5.0 Hz, 1H), 4.00 (dd, J = 10.9, 5.8 Hz, 1H), 3.97–3.83 (m, 2H), 2.64–2.46 (m, 2H), 2.25–2.14 (m, 2H), 1.48–1.40 (m, 1H), 1.36–1.25 (m, 7H), 0.90–0.82 (m, 6H); ¹³C NMR (126 MHz, CD₃OD) δ 174.9 (d, ³J_C–P = 5.3 Hz), 157.2, 153.7, 152.1 (d, ²J_C–P = 6.9 Hz), 150.0, 140.6, 130.7 (2C), 126.0, 121.3 (2C, d, ³J_C–P = 4.8 Hz), 120.4, 86.8, 81.1 (d, ³J_C–P = 8.4 Hz), 68.9 (d, ²J_C–P = 5.5 Hz), 68.0, 51.5, 41.7, 33.0, 26.8, 24.2, 24.2, 20.5 (d, ³J_C–P = 6.5 Hz), 11.3, 11.2; ³¹P NMR (202 MHz, CD₃OD) δ 3.73; HRMS (ESI) m/z calcd for C₂₅H₃₅N₆NaO₆P [M + Na]⁺ 569.2253, found 569.2243. The diastereopurity of 45S was determined to be 86% de: retention time = 10.072 min.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsbiomedchemau.4c00071.

• ¹H, ¹³C, ³¹P and ¹⁹F NMR spectroscopic data for all compounds; preliminary screening of antiviral activities at a concentration of 25 μM; detailed cytotoxicity, antiviral activity, kinetic study of an enzymatic hydrolysis and phenol fluorescence assays; material and methods of in vitro pharmacokinetic study; and the remaining percentage of the test compounds from microsomal stability assay (PDF)

Author Contributions

CRediT: Aticha Thiraporn data curation, formal analysis, investigation, methodology, validation, visualization, writing - original draft; Thanat Tiyasakulchai data curation, formal analysis, investigation, methodology, writing - original draft; Thitiphong Khamkhenshorngphanuch data curation, investigation, methodology; Marie Hoarau data curation, formal analysis, investigation, writing - original draft; Ratthiya Thiabma data curation, formal analysis, investigation, writing - original draft; Suppachoke Onnome data curation, formal analysis, investigation; Amporn Suphatrakul data curation, formal analysis, investigation; Jaraspim Narkpuk data curation, formal analysis, investigation; Chanya Srisaowakarn data curation, formal analysis, investigation; Suwimon Manopwisedjaroen data curation, formal analysis, investigation; Kitlada Srichomthong data curation, formal analysis, investigation; Suradej Hongeng conceptualization, resources, supervision; Arunee Thitithanyanont conceptualization, investigation, supervision; Peera Jaru-ampornpan conceptualization, formal analysis, investigation, supervision, writing - review & editing; Sewan Theeramunkong conceptualization, investigation, writing - review & editing; Bunpote Siridechadilok conceptualization, formal analysis, investigation, supervision, writing - review & editing; Nitipol Srimongkolpithak conceptualization, funding acquisition, investigation, methodology, project administration, resources, supervision, writing - review & editing.

The authors declare no competing financial interest.