Structure–Activity Relationship of Truncated 2,8-Disubstituted-Adenosine Derivatives as Dual A_2A/A₃ Adenosine Receptor Antagonists and Their Cancer Immunotherapeutic Activity

Abstract

Based on hA_2AAR structures, a hydrophobic C8-heteroaromatic ring in 5′-truncated adenosine analogues occupies the subpocket tightly, converting hA_2AAR agonists into antagonists while maintaining affinity toward hA₃AR. The final compounds of 2,8-disubstituted-N⁶-substituted 4′-thionucleosides, or 4′-oxo, were synthesized from d-mannose and d-erythrono-1,4-lactone, respectively, using a Pd-catalyst-controlled regioselective cross-coupling reaction. All tested compounds completely antagonized hA_2AAR, including 5d with the highest affinity (K_i,A2A = 7.7 ± 0.5 nM). The hA_2AAR–5d X-ray structure revealed that C8-heteroaromatic rings prevented receptor activation-associated conformational changes. However, the C8-substituted compounds still antagonized hA₃AR. Structural SAR features and docking studies supported different binding modes at A_2AAR and A₃AR, elucidating pharmacophores for receptor activation and selectivity. Favorable pharmacokinetics were demonstrated, in which 5d displayed high oral absorption, moderate half-life, and bioavailability. Also, 5d significantly improved the antitumor effect of anti-PD-L1 in vivo. Overall, this study suggests that the novel dual A_2AAR/A₃AR nucleoside antagonists would be promising drug candidates for immune-oncology.

Graphical Abstract

INTRODUCTION

Although there has been remarkable progress in anticancer therapy, cancer remains the main life-threatening disease.¹ Recently, immuno-oncology agents emerged as the next generation of antitumor drugs for immune checkpoint inhibition.² Among them, monoclonal antibodies (mAbs) have been intensively studied as immune checkpoint inhibitors.³ They block costimulatory molecules on immune cells and inhibit immune-evasion of a tumor, resulting in the reactivation of the immune system. However, mAb immune checkpoint inhibitors have some limitations such as their high cost, prolonged half-life, immune-related adverse effects, and immunogenicity.⁴ To overcome these drawbacks, the development of small molecules as immuno-oncology agents has been highly appealing. A number of target proteins such as toll-like receptors (TLRs), stimulators of interferon genes (STING), adenosine receptors (ARs), and so on have been investigated as the principal targets for the development of small molecule immune-oncology agents.⁵

ARs consist of four subtypes termed A₁, A_2A, A_2B, and A₃. Among these, A_2AAR and A_2BAR couple to G_S protein, which activates adenylate cyclase (AC) to increase the level of cyclic AMP (cAMP), while A₁AR and A₃AR bind to G_i protein, inhibiting the level of cAMP. In a tumor microenvironment (TME), ecto-5′-nucleotidase (CD73) on the cell surface catalyzes the hydrolysis of extracellular adenosine-5′-monophosphate (AMP) to adenosine and phosphate, resulting in high adenosine concentrations. When adenosine is released in the tumor microenvironment, it can activate any of the four AR subtypes to control cAMP levels.⁶ Notably, adenosine activating the A_2AAR, which is overexpressed in effector T cells and then blocks T cell receptor (TCR) signaling, abolishes the immune response.⁷ In addition, A_2AAR also upregulates the expression of negative costimulatory molecules such as programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA4), stimulating immune evasion.⁸ Thus, if A_2AAR is blocked, immune checkpoints by CTLA4, PD-1, or PD-L1 can be inhibited in cancer cells, giving A_2AAR antagonists cancer immunotherapeutic potential. Furthermore, an A_2AAR antagonist is expected to show a synergistic effect in combination with mAb immune checkpoint inhibitors.⁹ In fact, most of the A_2AAR antagonists in clinical trials as cancer immunotherapeutic agents, are coadministered with a mAb immune checkpoint inhibitor as combination therapy.¹⁰

Based on a potent and selective A₃AR agonist, 2-chloro-N⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide (Cl-IB-MECA, 1a),¹³ we have long been interested in discovering new AR ligands with the 4′-thionucleoside skeleton. Among these, 2-chloro-N⁶-(3-iodobenzyl)-4′-thioadenosine-5′-N-methyluronamide (thio-Cl-IB-MECA, 1b)¹⁴ was discovered to be a more potent human (h) A₃AR agonist (K_i = 0.38 ± 0.07 nM) than 1a (K_i = 1.4 ± 0.3 nM) (Chart 1). Truncation of the 5′-uronamide of 1a and 1b converted selective A₃AR agonists 1a and 1b into A₃AR antagonists 2a (K_i = 42.9 ± 8.9 nM) and 2b (K_i = 4.16 ± 0.5 nM).¹⁵ From this study, the amide proton of the 5′-uronamide group of 1a and 1b acted as a hydrogen-bonding donor, which was essential for the induced fit in the receptor required for A₃AR agonism. Compound 2b also showed high binding affinity (K_i = 3.89 ± 1.15 nM) at the rat A₃AR, indicating that it is a species-independent A₃AR antagonist. These truncated nucleosides 2a and 2b were the first examples to show pure A₃AR antagonism with species-independent and high binding affinity with a nucleoside skeleton,¹⁵ potentially for treatment of chronic kidney disease (CKD)¹⁶ and cancer.¹⁷ Further SAR study of 2a and 2b at the C2 and N⁶-positions resulted in the discovery of A_2AAR agonists 3a (K_i = 63.2 ± 15 nM) and 3b (K_i = 7.19 ± 0.6 nM), in which an extended hexynyl group at the C2 position and a free amino group at the N⁶-position were found to act as essential pharmacophores for A_2AAR binding, although they also maintained the antagonistic activity at the hA₃AR.¹⁸

Chart 1.

Rationale for the Design of the Target Nucleosides 5

In order to switch compound 3 to an A_2AAR antagonist, we analyzed agonist- and antagonist-bound co-crystal structures of the A_2AAR. As shown in Figure 1, A_2AAR antagonist (ZM-241385) occupied a subpocket which was expanded by His250^6.52/A2A (Figure 1A, His250^6.52/A2A; Ballesteros–Weinstein residue numbering and name of the receptor are referred to as superscripts.¹⁹). Also, the predicted binding mode of 3a with hA_2AAR indicated that the C8 position is located in a spacious subpocket capable of accommodating a bulky substituent (Figure 1B, yellow). Thus, if we introduce an aromatic ring at the C8 position of 3a, it was hypothesized that it induces tight binding rather than an induced fit required for receptor activation, anticipating the conversion of A_2AAR agonist 3b into A_2AAR antagonist 4. As expected, compound 4 was found to be a full A_2AAR antagonist (K_i = 18.3 ± 4.8 nM). Recently, we reported the X-ray co-crystal structure complexed with A_2AAR antagonist 4,¹¹ confirming our hypothesis that the C8 aromatic ring of 3a tightly binds to the subpocket and serves as the key pharmacophoric feature to discriminate between agonist and antagonist.^18,20 Most known A_2AAR agonists possess a nucleoside structure, while all known A_2AAR antagonists have heterocyclic compounds without a sugar ring.¹⁰ These results indicate that both 2′–OH and 3′–OH groups have a pivotal role for A_2AAR activation but are not essential for an inactive A_2AAR state.²⁰ Namely, despite possessing a nucleoside skeleton containing both 2′–OH and 3′–OH, the compounds of this research exhibited potent A_2AAR antagonism, and it is believed to be the first example.¹¹

Figure 1.

X-ray co-crystal structure of hA^2AAR containing NECA (pink, PDB ID: 2YDV) and ZM-241385 (green, PDB ID: 3EML). (A) Comparison of the binding pocket for the co-crystal structures and its subpocket. (B) Predicted binding mode of 3a with hA_2AAR (yellow, PDB ID: 3EML). (C) Chemical structures of the ligands.

Based on these findings, we carried out a comprehensive SAR study at the C2-, C8-, and N⁶-positions of 4, synthesized the truncated 4′-oxonucleoside derivatives 5a–f and measured their A_2AAR binding affinities.²⁰ Also, we synthesized the corresponding truncated 4′-thionucleosides 5g–v, based on a bioisosteric relationship between oxygen and sulfur, and evaluated them for binding affinities of the four adenosine receptor subtypes.¹²

Herein, we report the design, synthesis, and binding affinity of the final nucleosides 5, modified at the C2, C8, and N⁶-positions. We also report the pharmacokinetic characterization and in vivo antitumor effects of the most potent A_2AAR antagonist in the series for its development as an immuno-oncology agent.

RESULTS AND DISCUSSION

Chemistry.

The 4′-oxonucleoside derivatives 5a–f were synthesized from d-erythrono-1,4-lactone, as shown in Scheme 1. d-Erythrono-1,4-lactone was protected as 2,3-O-acetonide under the standard conditions, which was reduced with diisobutylaluminum hydride (DIBAL-H) to afford lactol 6.¹⁵ Acetylation of lactol 6 afforded the glycosyl donor 7.^15c Condensation of 7 with silylated 6-chloropurine under Vorbrüggen²¹ conditions in the presence of trimethylsilyl trifluoromethansulfonate (TMSOTf) as a Lewis acid yielded the protected β-nucleoside 8 as a single stereoisomer. Treatment of 8 with 1 N HCl followed by the protection of resulting diol with the t-butyldimethylsilyl (TBS) group afforded the di-O-TBS ether, which was subjected to the iodination²² at C2 and C8 positions using a freshly prepared lithium tetramethylpiperidide (LiTMP) to produce 6-chloro-2,8-diiodo-purine derivative 9. Palladium catalyst-controlled regioselective Sonogashira^22–24 coupling of 9 with 1-hexyne in the presence of tetrakis(triphenylphosphine)palladium and cesium carbonate yielded C2-hexynyl derivative 10 as the major regioisomer.²³ Then, another palladium-catalyzed Stille²⁵ coupling of 10 with 2-tributylstannylfuran and–thiophene yielded the C8-furyl derivative 11a and the C8-thienyl derivative 11b, respectively, after desilylation. The regio- and stereochemistry of 11b were confirmed by X-ray crystallography (Scheme 1, please see the Supporting Information for the X-ray crystallographic details of the compound 11b). Compounds 11a and 11b were treated with ammonia, methylamine, and 3-iodobenzylamine to afford 5a–c and 5d–f, respectively.

Scheme 1. Synthesis of Truncated 2-Hexynyl-N⁶,8-Disubstituted-Adenosines 5a–f^a.

^aReagents and conditions: (a) pTSA, 2,2-dimethoxypropane, DMF, 4 h, reflux; (b) DIBAL-H, toluene, −78 °C, 30 min; (c) AC₂O, pyridine, rt, 3 h; (d) 6-chloropurine, HMDS, (NH₄)₂SO₄, TMSOTf, 1,2-dichloroethane, 0 to 80 °C, 15 h; (e) (i) 1 N HCl, THF, rt, 15 h; (ii) TBSCl, imidazole, DMF, rt, 15 h; (iii) tetramethylpiperidine, I₂, n-BuLi, THF, −78 °C, 4 h; (f) Pd(PPh₃)₄, Cs₂CO₃, CuI, 1-hexyne, DMF, rt, 5 h; (g) (i) Pd(PPh₃)₂Cl₂, 2-tributylstannylfuran, THF, 70 °C, 1 h for 11a or Pd(PPh₃)₂Cl₂, 2-tributylstannylthiophene, THF, 60 °C, 1 h for 11b; (ii) Et₃N, Et₃N·3HF, THF, rt, 15 h; (h) NH₃/t-BuOH, 100 °C, 12 h for 5a, 5d and CH₃NH₂·HCl, Et₃N, EtOH, rt, 24 h for 5b, 5e, and 3-iodobenzylamine·HCl, Et₃N, EtOH, rt, 24 h for 5c and 5f.

Next, we synthesized the corresponding 4′-thionucleoside derivatives 5g–v, based on the bioisosteric relationship between oxygen and sulfur. The synthetic strategy to the 4′-thionucleoside derivatives 5g–v was to condense the glycosyl donor 16 with 6-chloro-2,8-diiodopurine 17d or 2,6-dichloro-8-iodopurine 19 under Vorbrüggen²¹ conditions and then apply regioselective C8-Stille²⁵ coupling, C2-Sonogashira^22–24 coupling, and N⁶-amination on the purine moiety. Thus, we first synthesized the glycosyl donor 16 from d-mannose according to our previously published procedure (Scheme 2).²⁶

Scheme 2. Synthesis of the Glycosyl Donor 16^a.

^aReagents and conditions: (a) 2,2-dimethoxypropane, camphorsulfonic acid, acetone, rt, 15 h; (b) NaBH₄, EtOH, rt, 2 h; (c) MsCl, Et₃N, CH₂Cl₂, rt, 1 h; (d) Na₂S, DMF, 80 °C, 15 h; (e) 60% AcOH, rt, 2 h; (f) BzCl, pyridine, CH₂Cl₂, rt, 18 h; (g) 80% AcOH, 70 °C, 12 h; (h) TBSCl, imidazole, DMF, rt, 18 h; (i) NaOMe, MeOH, rt, 2 h; (j) Pb(OAc)₄, EtOAc, rt, 18 h.

d-Mannose was treated with 2,2-dimethoxypropane in the presence of camphorsulfonic acid to give di-O-acetonide, which was reduced with NaBH₄, and the resulting diol was treated with mesyl chloride to give the dimesylate 12 in 82% yield. Treatment of 12 with Na₂S in DMF at 80 °C afforded the 4-thiosugar, which was selectively hydrolyzed with 60% acetic acid to give 5,6-diol 13. As the 2,3-acetonide was problematic for removal under acidic conditions at the final step, the 2,3-O-acetonide was converted to the 2,3-di-O-TBS group as follows. Benzoylation of 13, followed by hydrolysis of 2,3-O-acetonide with 80% acetic acid, yielded 2,3-diol 14. Protection of diol 14 with a TBS group followed by the removal of the benzoyl group afforded 5,6-diol 15. Oxidative cleavage of 15 with Pb(OAc)₄ yielded the glycosyl donor 16.

Then, we synthesized 6-chloro-2,8-diiodopurine 17d and 2,6-dichloro-8-iodopurine 19, which can serve as essential intermediates for selective modification at the C2, C8, and N⁶ positions (Scheme 3). N⁶-Protected 6-chloropurine (17a)²⁷ was treated with LiTMP, prepared by treating tetramethylpiperidine with n-BuLi, followed by treatment with iodine to yield 2,8-diiodo derivative 17c.²² Because the 8-iodo position was sensitive to degradation under acidic conditions, the tetrahydropyran (THP) group of 17c was removed with CuCl₂ to afford 17d. N⁹-Protected 2,6-dichloropurine 17b²⁷ was treated with lithium diisopropylamide (LDA), prepared by treating i-Pr₂NH with n-BuLi, followed by further treatment with iodine to give 8-iodo derivative 18. The removal of the THP group of 18 under mild acidic conditions afforded 19.

Scheme 3. Synthesis of 6-Chloro-2,8-diiodopurine 17d and 2,6-Dichloro-8-iodopurine 19^a.

^aReagents and conditions: (a) tetramethylpiperidine, I₂, n-BuLi, THF, −78 °C, 4 h; (b) CuCl₂, EtOH/H₂O, 85 °C, 5 h; (c) i-Pr₂NH, n-BuLi, I₂, THF, −78 °C, 2 h; (d) PPTS, EtOH, 60 °C, 6 h.

Condensation of 16 with silylated 6-chloro-2,8-diiodopurine in the presence of TMSOTf²¹ as a Lewis acid yielded the protected nucleoside 20 as a single β-anomer without concomitant formation of the N⁷-isomer. The desired C2-selectivity was not observed in substrate 20 when we attempted regioselective Sonogashira coupling, as shown in Scheme 1. Thus, we paid attention to Stille coupling to obtain the desired product. Delightfully, we were able to regioselectively synthesize 8-furyl derivative 21a and the 8-thienyl derivative 21b regioselectively with high yield. The 2-hexynyl derivatives 22a and 22b were then synthesized via another palladium-catalyzed Sonogashira coupling of 21a and 21b with 1-hexyne, respectively, and their structures were confirmed by X-ray crystallography (Scheme 4, please see Supporting Information for the X-ray crystallographic details of the compounds 22a and 22b). N⁶-Substitution of 22a and 22b with ammonia, methylamine, and 3-iodobenzylamine afforded the final nucleosides 5g–i and 5j–l, respectively.

Scheme 4. Synthesis of Truncated 2-Hexynyl-N⁶,C8-Disubstituted-4′-Thioadenosines 5g–l^a.

^aReagents and conditions: (a) 17d, BSA, TMSOTf, CH₃CN, 75 °C, 1.5 h; (b) Pd(dba)₂, 2-tributylstannylfuran, DMF/PhMe, rt, 9 h for 21a or Pd(dba)₂, 2-tributylstannylthiophene, DMF/PhMe, rt, 23 h for 21b; (c) Pd(PPh₃)₂Cl₂, 1-hexyne, CuI, Cs₂CO₃, DMF, rt, 14 h for 22a or 16 h for 22b; (d) n-Bu₄NF, THF, rt, 16 h for 22a or 50 °C, 12 h for 22b; (e) NH₃/t-BuOH, 100 °C, 15 h for 5g, 5j or CH₃NH₂·HCl, Et₃N, EtOH, 50 °C, 23 h for 5h or 28 h for 5k or 3-iodobenzylamine·HCl, Et₃N, EtOH, 50 °C, 24 h for 5i or 75 °C, 20 h for 5l.

Then, we synthesized the N⁶,C8-disubstituted-4′-thioadenosines 5m–v with chloro or p-hydroxyphenethylamine (tyramine) groups at the C2 position because a selective A_2A antagonist, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a]-[1,3,5]triazin-5-ylamino]ethyl) phenol (ZM-241385) possessed a C2-tyramine group²⁹ (Scheme 5). The glycosyl donor 16 was condensed with silylated 2,6-dichloro-8-iodopurine under Vorbrüggen²¹ conditions to afford the condensed nucleoside 23. Compound 23 was subjected to palladium-catalyzed Stille²⁵ coupling with 2-tributylstannylfuran and -thiophene to give 8-furyl- and 8-thienyl derivatives 24a and 24b, respectively. Treatment of 24a and 24b with tetra-n-butylammonium fluoride (TBAF), followed by further treatment of resulting diol derivatives with ammonia, methylamine, and 3-iodobenzylamine afforded the N⁶-amino-, methylamino-, and 3-iodobenzylamino derivatives 5m–o and 5p–r, respectively. Furthermore, 2-chloro-8-furyl-N⁶-amino- and methylamino derivatives 5m and 5n were converted to the corresponding 2-p-hydroxyphenethylamino derivatives 5s and 5t, respectively. Similarly, 8-thienyl derivatives 5p and 5q were converted to 5u and 5v, respectively.

Scheme 5. Synthesis of Truncated 2-Chloro- and 2-p-Hydroxyphenethylamino-N⁶,C8-Disubstituted-4′-Thioadenosines 5m–v^a.

^aReagents and conditions: (a) 19, BSA, TMSOTf, CH₃CN, 75 °C, 1.5 h; (b) Pd(PPh₃)₂Cl₂, 2-tributylstannylfuran, THF, 70 °C, 24 h for 24a or Pd(PPh₃)₂Cl₂, 2-tributylstannylthiophene, THF, 70 °C, 24 h for 24b; (c) TBAF, THF, rt, 1 h; (d) NH₃/t-BuOH, 120 °C, 15 h for 5m, 5p or CH₃NH₂·HCl, Et₃N, EtOH, rt, 24 h for 5n, 5q or 3-iodobenzylamine·HCl, Et₃N, EtOH, rt, 24 h for 5o, 5r; (e) tyramine, DIPEA, 1-butanol, MW, 180 °C, 2 h.

Structure–Activity Relationship Studies and Pharmacological Profiles.

Binding Affinities and cAMP Functional Data to Adenosine Receptor Subtypes.

The binding affinities of all final nucleosides 5a–v at the four subtypes of the hARs were measured using standard radioligands and membrane preparations.²⁹ The hA₁AR and hA₃AR were expressed in Chinese hamster ovary (CHO) cells, and the hA_2AAR and hA_2BAR were expressed in human embryonic kidney (HEK)-293 cells. [₃H](−)-N⁶-2-Phenylisopropyladenosine (25, R-PIA), [₃H]2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamido-adenosine (26, CGS21680), 1,3-[₃H]-dipropyl-8-cyclopentylxanthine (27, DPCPX), and [¹²⁵I]N⁶-(4-amino-3-iodobenzyl)-5′-N-methylcarboxamidoadenosine (28, I-AB-MECA) were used for the hA₁AR, hA_2AAR, hA_2BAR, and hA₃AR, respectively. The percent inhibition of radioligand binding at 10 μM was reported in cases of weak binding. Nonspecific binding was defined using 5′-N-ethylcarboxamidoadenosine (29, NECA).

As shown in Table 1, the addition of a hydrophobic furan or thiophene ring at the C8 position of 3 preserved considerable AR affinity in general and at the A_2AAR, specifically. The furan substitution exhibited comparable hA_2AAR binding affinity with the thiophene substitution, although the orientation of thiophene can be stabilized as a preferred binding pose by the 1,4-N…S noncovalent sulfur interaction between thiophene and N⁷ nitrogen.³⁰ It implies that the interaction between hA_2AAR and the heteroatom of the C8-aromatic ring is not crucial for binding with the receptor. As the C2 position is appended with a rigid and hydrophobic hexyne substituent (5a–5l), the addition of a hydrophobic group at the N⁶-position decreased hA_2AAR binding affinity in the following order: R¹ = H > CH₃ > 3-iodobenzyl. This pattern indicated that a free amino group is essential for appropriate hydrogen bonding in the hA_2AAR, which matches our previous findings.¹⁸ In the case of the binding affinities of C2-hexyne compounds for hA₃AR, the 4′-thionucleoside derivatives (5g–5l) could not tolerate the bulky N⁶-3-iodobenzylamine, as opposed to the 4′-oxonucleoside derivatives (5a–5f) and the series of compounds with C8–H substitution.^18b On the other hand, 2-Cl or 2-tyramine compounds (5m–5v) favored bulkier N⁶-substituents than free amines. These results indicate that the tight binding by both bulky C8-aromatic ring and rigid C2-hexyne can contract the binding pocket near N⁶-position. In the N⁶-NH₂ derivatives, the C2-hexyne compounds maintained substantial binding affinity at the hA_2A and hA₃ARs, but 2-Cl or 2-tyramine substitution decreased the binding affinities at both receptors dramatically. It is surprising in that a C2-tyramine substitution²⁸ was reported to increase the binding affinity at the hA_2AAR. In general, 4′-oxonucleoside derivatives exhibited better binding affinities at the hA_2AAR and hA₃AR than the corresponding 4′-thionucleoside derivatives, among which 5d exhibited the best binding affinity at the hA_2AAR (K_i = 7.7 ± 0.5 nM) along with good binding affinity at the hA₃AR (K_i = 15.6 ± 1.6 nM). Among tested 4′-thionucleoside derivatives, compound 5g exhibited the highest binding affinity (K_i = 13.2 ± 0.2 nM at hA_2AAR) with high selectivity (K_i,5g = 119 ± 39 nM at hA₃AR). These findings provide an essential clue leading to the discovery of selective nucleoside A_2AAR ligands.

Table 1.

Binding Affinities of Compounds 5a–v at Four Subtypes of the hARs

compd no.	X	Y	R1	R2	Ki value (nM ± SEMa or % displacement at 10 μMb)
					hA1AR	hA2AAR	hA2BAR	hA3AR
5a	O	O	H	1-hexyne	183 ± 28	18.0 ± 5.5	327 ± 32	15.5 ± 2.9
5b	O	O	CH3	1-hexyne	3030 ± 50	50.5 ± 12.1	76 ± 1%	2.9 ± 0.7
5c	O	O	3-I-Bn	1-hexyne	13 ± 1%	579 ± 53	4 ± 2%	199 ± 20
5d = 4	O	S	H	1-hexyne	392 ± 99	7.7 ± 0.5	834 ± 11	15.6 ± 1.6
5e	O	S	CH3	1-hexyne	2310 ± 600	126 ± 18	52 ± 1%	2.9 ± 0.5
5f	O	S	3-I-Bn	1-hexyne	2580 ± 460	513 ± 153	2 ± 3%	8.9 ± 4.8
5g	S	O	H	1-hexyne	46 ± 1%	13.2 ± 0.2	1973 ± 251	119 ± 39
5h	S	O	CH3	1-hexyne	17 ± 9%	159 ± 17	41 ± 2%	53.5 ± 16.9
5i	S	O	3-I-Bn	1-hexyne	11 ± 7%	5 ± 5%	3 ± 2%	2 ± 8%
5j	S	S	H	1-hexyne	494 ± 122	61.4 ± 4.1	27 ± 2%	150 ± 6
5k	S	S	CH3	1-hexyne	11 ± 3%	2820 ± 1080	11 ± 2%	206 ± 26
5l	S	S	3-I-Bn	1-hexyne	1 ± 0.2%	2 ± 2%	3 ± 3%	12 ± 5%
5m	S	O	H	Cl	3890 ± 650	140 ± 10	53 ± 3%	855 ± 157
5n	S	O	CH3	Cl	49 ± 1%	752 ± 67	46 ± 1%	94.6 ± 45.2
5o	S	O	3-I-Bn	Cl	4900 ± 1070	336 ± 108	51 ± 1%	89.7 ± 20.4
5p	S	S	H	Cl	1980 ± 140	459 ± 46	62 ± 3%	294 ± 80
5q	S	S	CH3	Cl	26 ± 4%	955 ± 704	17 ± 1%	138 ± 10
5r	S	S	3-I-Bn	Cl	50 ± 2%	720 ± 9	17 ± 2%	137 ± 28
5s	S	O	H	tyramine	46 ± 3%	112 ± 6	227 ± 41	1650 ± 100
5t	S	O	CH3	tyramine	5330 ± 340	468 ± 28	713 ± 88	445 ± 148
5u	S	S	H	tyramine	8050 ± 910	946 ± 94	608 ± 81	3010 ± 1140
5v	S	S	CH3	tyramine	47 ± 2%	917 ± 59	579 ± 135	106 ± 27
3ac	S		H	1-hexyne	39 ± 10%	7.19 ± 0.6	N.D.d	11.8 ± 1.3
3bc	O		H	1-hexyne	740 ± 430	63.2 ± 15	N.D.d	138 ± 44

^aAll binding experiments were performed using adherent CHO cells and HEK293 cells stably transfected with cDNA encoding the appropriate hAR binding was carried out using 1 nM 25, 10 nM 26, 25 nM 27, and 0.2 nM 28 as radioligands for A₁, A_2A, A_2B, and A₃ARs, respectively. Values are expressed as mean ± SEM, n ± 3–4 (outliers eliminated), and are normalized against a nonspecific binder, 29.

^bWhen a percent value is shown, it refers to the percent inhibition of a specific radioligand binding at 10 μM, with nonspecific binding defined using 10 μM of 29.

^cRef 18.

^dNot determined.

Three synthesized compounds were evaluated in functional cAMP assays in hA_2AAR- or hA₃AR-expressing CHO cell lines. As shown in Table 2, no receptor activation at the hA_2AAR at 10 μM concentration for compounds 5a, 5d, and 5g was observed, demonstrating that they act as hA_2AAR full antagonists. Among them, the compound 5d demonstrated the highest potency, and its K_B value was calculated to be 25.8 nM at the hA_2AAR using full agonist NECA (29) (Table S1). Similarly, compounds 5a, 5d, and 5g inhibited hA₃AR activation at 10 μM, and the K_B value of 5d for hA₃AR was calculated to be 107.4 nM, also indicating that it is a full hA₃AR antagonist. It should be noted that the addition of a hydrophobic furan or thiophene at the C8 position could convert A_2AAR agonists into A_2AAR antagonists successfully by inhibiting the interaction for receptor inactivation.

Table 2.

cAMP Functional Assay Data at hA_2AAR and hA³AR Expressed in CHO Cells^a

compound no.	% activation, A2A cAMP assay	% activation, A3 cAMP assay
5a	0.3 ± 0.3	16 ± 4
5d	0.8 ± 0.3	4 ± 4
5g	0.05 ± 0.1	11 ± 5

^aAt a concentration of 10 μM, unless noted.

Molecular Docking Studies.

In order to elucidate the effect of 4′-thioribose, a homology modeling study was performed using the co-crystal structure of A_2AAR containing 5d (Figure 2, PDB ID: 8CU6). Comparison of the binding sites of hA_2AAR and hA₃AR revealed two characteristic differences that can induce variation in binding affinity between the two receptors: the expanded binding pocket by Ser247^6.52/A3 (blue arrow), and the reduced ribose-binding pocket by Leu90^3.32/A3 of hA₃AR (red arrow) (Figure 2A). Molecular docking was then performed for 4′-thionucleoside 5j and 4′-oxonucleoside 5d. Docking with the homology model of hA₃AR showed that the binding pose of 5d was similar to that of hA_2AAR, whereas 5j exhibited a reverse-oriented binding pose due to steric clashes between 4′-thioribose and Leu90^3.32/A3 (Figure 2B). In hA_2AAR, although there were minor steric clashes between Ile274^7.39/A2A/His278^7.43/A2A and 5j, the ligand could still retain its forward-oriented binding pose due to the smaller residue Val84^3.32/A2A compared to Leu90^3.32/A3 (red dashed line, Figure 2C,D).

Figure 2.

Comparison of the crystal structure of hA_2AAR (PDB ID: 8CU6) and the homology model of A₃AR. (A) Binding pocket of the co-crystal structure of hA_2AAR (orange), and the homology model of A₃AR. (green) (B) Predicted binding mode of 5d (cyan) and 5j (magenta) for hA₃AR. (C) Predicted binding mode of 5j (lime) and (D) co-crystal structure of 5d (white) with hA_2AAR. The red dashed line represents steric clashes, and the yellow dashed line represents hydrogen bonding.

Overall, the molecular docking analysis suggests that steric clashes between the sugar moiety and the receptors can be a key factor in the binding of both hA_2AAR and hA₃AR. Although the difference in the bulkiness of ribose can be considered subtle, as observed in the X-ray crystal structure of 11b [C(4′)–O: 1.39 Å in ribose] and 22b [C(4′)–S: 1.82 Å in 4′-thioribose], the results showed it was significant enough to result in several-fold changes in binding affinities and even flip the binding mode in hA₃AR (Figure 2B).

ADME Evaluation of Compound 5d.

We examined the druggability of the most potent compound 5d primarily through in vitro ADME assays to determine its potential as a preclinical candidate. Our results showed that compound 5d did not significantly inhibit five types of CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) and hERG K+ channels (Tables S2 and S3).

We also conducted an in vivo pharmacokinetic study of 5d in mice, which revealed proper distribution (V_ss = 0.69 L/kg) and moderate plasma clearance (CL = 60.5 mL/min/kg). After oral administration, compound 5d was rapidly absorbed (T_max = 0.25 h) with moderate bioavailability (F = 22.81%) and half-life (t_1/2 = 1.99 h) (Table 3).

Table 3.

In Vivo Pharmacokinetic Properties of Compound 5d

PK parameter	route of dosing
	i.v. (n = 3)	p.o. (n = 3)
dose (mg/kg)	2.00	10.00
AUC0–t (h·ng/mL)	550.95	626.55
Cmax (ng/mL)	1984.94	1241.79
Tmax (h)	0.08	0.25
CL ((mL/min)/kg)	60.5
Vss (L/kg)	0.69
t1/2 (h)	0.16	1.99
F (%)		22.81

Synergistic Antitumor Activity of Compound 5d with the Immune Checkpoint Inhibitor in 4T1-Luc Cells Implanted in Mouse Xenograft Models.

We conducted an in vivo test to investigate the antitumor effect of compound 5d and its potential synergistic effect with an immune checkpoint inhibitor. 4T1-Luc tumor-bearing mice were intraperitoneally treated with compound 5d (10 mg/kg), antimouse PD-L1 (10 mg/kg), or a combination of both treatments thrice per week for 21 days. As shown in Figure 3A–D, the single administration of compound 5d resulted in a weak antitumor effect, with a TGI of 15%. However, coadministration of 5d with antimouse PD-L1 demonstrated a substantial increase in TGI up to 77%, while the single administration of antimouse PD-L1 exhibited only 43% of TGI. No adverse effects or body weight loss were observed. In addition, bioluminescence imaging of mice was conducted on the final day of the experiment to precisely quantify the tumor mass. Consistent with the tumor volume decrease, the group of mice treated with combination therapy exhibited a remarkable reduction in signal intensity from the tumor (Figure 3E). Taken together, these results demonstrate that compound 5d effectively improves the antitumor potential of the immune checkpoint inhibitor anti-PD-L1.

Figure 3.

In vivo antitumor activity of the compound 5d and its synergistic effect with immune checkpoint inhibitor. Mice bearing 4T1-Luc tumors were treated thrice per week with vehicle (DMSO/cremophor/normal saline = 5:5:90), 5d (10 mg/kg, i.p.), anti-PD-L1 (10 mg/kg, i.p.), or combination of 5d (10 mg/kg, i.p.), and anti-PD-L1 (10 mg/kg, i.p.). (A) Tumor volume and (B) body weight of the mice bearing 4T1-Luc tumors during administration. (C) Image of excised tumors from the mice and (D) their tumor weight. (E) Luciferase imaging (IVIS) of the mice on the final day of the experiment. Average signal intensity was measured by PerkinElmer Living Image software. *P < 0.05, **P < 0.01, ***P < 0.001 vs the control.

CONCLUSIONS

A series of truncated 4’-thio- and 4’-oxonucleoside derivatives substituted at C2, C8, and N₆-positions were synthesized from d-mannose and d-erythrono-1,4-lactone, respectively. Functional groups such as C2-hexynyl and C8-aryl groups were introduced by palladium catalyst-controlled regioselective cross-coupling.²² From this study, it was discovered that the introduction of hydrophobic rings such as furan and thiophene at the C8 position successfully converted an A_2AAR agonist into an A_2AAR antagonist. The X-ray co-crystal structure of hA_2AAR complexed with antagonist 5d,¹¹ revealed that the subpocket was occupied by heteroaromatic rings, inducing tight binding that can abolish receptor activation. Additionally, the C8 substituent caused the ribose ring to rotate from its canonical orientation, disrupting a crucial interaction for receptor activation. Also, it was elucidated that the C2-hexynyl group formed favorable hydrophobic interactions in a relatively large hydrophobic pocket of the A_2AAR.

4’-Oxonucleoside derivatives, 5a–5f, showed better binding affinities than 4’-thionucleosides. Among them, compound 5d was discovered as the best hA_2AAR antagonist (K_i = 7.7 ± 0.5 nM) along with a high antagonistic binding affinity at the hA₃AR (K_i = 15.6 ± 1.6 nM). Among the 4’-thionucleoside derivatives, 5g–5v, the 4’-thionucleoside 5g exhibited the highest binding affinity at hA_2AAR (K_i = 13.2 ± 0.2 nM), along with a ninefold stronger binding affinity than hA₃AR (K_i = 119 ± 39 nM). According to the molecular docking study, the steric clashes between 4′-thioribose and Leu90^3.32/A3 weaken the binding with A₃AR, flipping the binding pose of the ligand.

The most potent compound 5d also showed good druggability and an acceptable pharmacokinetic profile suitable for a preclinical candidate. Furthermore, 5d exhibited a potent in vivo antitumor effect as a combination therapy with the immune checkpoint inhibitor, anti-PD-L1, without any adverse effects and body weight loss.

A_2AAR antagonists are known to be highly associated with cancer immunotherapy activity. On the other hand, existing research demonstrates that the modulation of A₃AR can exhibit anticancer effects through distinct mechanisms, such as the induction of cell cycle arrest and inhibition of HIF-1α expression.^17,31 Hence, further investigation into the anticancer effects of compound 5d will be conducted to elucidate its potential additive effect achieved through the modulation of A₃AR.

To the best of our knowledge, this is the first SAR study of A_2AAR antagonists with a nucleoside skeleton. The characteristics of the nucleoside moiety can provide beneficial effects such as species independence and pharmacokinetic properties, which are favorable for preclinical and clinical trials, as in the case of the A₃AR antagonist with a nucleoside skeleton.^15a,b Thus, the novel nucleoside dual A_2AAR/A₃AR antagonist 5d can be a promising drug candidate for combination therapy with immune checkpoint inhibitors.

EXPERIMENTAL SECTION

General Procedures.

The study employed various scientific techniques to measure and analyze the physical and chemical properties of synthesized compounds. NMR spectra (¹H/¹³C) were obtained by Jeol JNM-ECZ 400s (400 MHz/100 MHz), Bruker AV500 (500 MHz/125 MHz), or Bruker AV800 (800 MHz/200 MHz) and reported as chemical shifts in parts per million (δ) relative to solvent peaks, with coupling constants (J) expressed in hertz (Hz). Melting points were measured using a Barnstead Electrothermal instrument, while optical rotations were obtained using a Jasco-P2000 instrument. Elemental analyses (C, H, and N) were conducted to assess compound purity, and HPLC (Agilent 1260 infinity series) was also used to determine the purities of the representative compounds using a binary solvent system [0.01 M KH₂PO₄ buffer in H₂O/MeCN] and an Agilent Zorbax Eclipse XDB-C18 column [5.0 μm, 4.6 mm i.d. × 250 mm]. The HPLC purity was more than 95% purity. High-resolution mass spectrometry spectra were obtained using fast atom bombardment (FAB) and electrospray ionization (ESI) methods. Silica gel 60 (230–400 mesh) was used for flash column chromatography, and all solvents were purified and dried before use using standard techniques. Commercially available materials were used without purification unless otherwise stated.

Synthesis and Characterization of the Compounds.

The compound 5d was synthesized in the previous paper through the intermediates 6–10, 11b.¹¹ The glycosylic donor 16²⁶ and the diiodinated purine 17d²³ were prepared by a known procedure.

(2R,3R,4R)-2-(6-Chloro-8-(furan-2-yl)-2-(hex-1-yn-1-yl)-9H-purin-9-yl)tetrahydrofuran-3,4-diol (11a). Stille Coupling.

To a solution of 10 (530 mg, 0.77 mmol) in anhydrous THF (38 mL) were added 2-(tributylstannyl)furan (0.48 mL, 1.53 mmol) and bis-(triphenylphosphine)palladium(II) dichloride (107 mg, 0.15 mmol) at room temperature under N₂. After being refluxed with stirring for 1 h, the reaction mixture was cooled to room temperature and evaporated. The residue was purified by column chromatography (silica gel, hexane/EtOAc, 30/1 to 15/1) to give the intermediate.

TBS Deprotection.

To a cooled (0 °C) solution of the above-generated intermediate in anhydrous THF (30 mL) were dropwise added triethylamine (0.50 mL, 3.83 mmol) and trimethylamine-trihydrofluoride (0.68 mL, 3.83 mmol) at room temperature under N₂. After being stirred at room temperature for 15 h, the reaction mixture was evaporated. The residue was purified by column chromatography (silica gel, hexane/EtOAc, 1/1) to give 11a (130 mg, 42%) as white solid: mp 184 °C; $[\alpha {]}_{\mathrm{D}}^{25}=-34.47$ (c 0.11, MeOH); UV (MeOH) λ_max 225, 239, 331 nm; ¹H NMR (400 MHz, MeOD) δ 7.93 (dd, J = 1.6, 0.8 Hz, 1H), 7.49 (dd, J = 4.0, 1.2 Hz, 1H), 6.77 (dd, J = 3.6, 1.6 Hz, 1H), 6.52 (d, J = 6.4 Hz, 1H), 5.40 (dd, J = 6.4, 4.8 Hz, 1H), 4.68 (dd, J = 9.6, 3.2 Hz, 1H), 4.50–4.54 (m, 1H), 4.03 (dd, J = 9.6, 1.6 Hz, 1H), 2.51 (t, J = 6.8 Hz, 2H), 1.63–1.69 (m, 2H), 1.50–1.59 (m, 2H), 0.99 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, MeOD): δ 154.9, 150.9, 150.2, 148.9, 147.3, 144.9, 132.7, 119.2, 114.4, 93.0, 92.2, 81.4, 77.2, 76.2, 73.7, 32.1, 23.9, 20.3, 14.7; HRMS (FAB): found 403.1165 [calcd for C₁₉H₂₀ClN₄O₄⁺ (M + H)⁺ 403.1173]; Anal. Calcd for C₁₉H₁₉ClN₄O₄: C, 56.65; H, 4.75; N, 13.91. Found: C, 56.91; H, 4.35; N, 13.77.

General Procedure for N⁶-Amination for the Preparation of 5a–5f.

To a solution of 11a or 11b (1.00 equiv) in EtOH (0.20 M) or t-BuOH (0.20 M) were dropwise added appropriate amines (1.50 equiv to excess) at room temperature under N₂. After being stirred at the provided temperature for 12–24 h, the reaction mixture was evaporated. The residue was purified by silica gel column chromatography (CH₂Cl₂/MeOH, 20/1) to give 5a–5f.

(2R,3R,4R)-2-(6-Amino-8-(furan-2-yl)-2-(hex-1-yn-1-yl)-9H-purin-9-yl)tetrahydrofuran-3,4-diol (5a).

11a was dissolved in NH₃/t-BuOH (0.20 M) and stirred for 12 h at 100 °C in a steel bomb. Yield = 78%; white solid; mp 194 °C; $[\alpha {]}_{\mathrm{D}}^{25}=15.64$ (c 2.16, MeOH); UV (MeOH) λ_max 237, 318 nm; ¹H NMR (400 MHz, MeOD): δ 7.82 (dd, J = 1.6, 0.4 Hz, 1H), 7.19 (dd, J = 3.4, 0.4 Hz, 1H), 6.70 (dd, J = 3.5, 1.7 Hz, 1H), 6.31 (d, J = 6.3 Hz, 1H), 5.44 (dd, J = 6.2, 4.8 Hz, 1H), 4.63 (dd, J = 9.6, 3.5 Hz, 1H), 4.47–4.48 (m, 1H), 3.98 (dd, J = 9.6, 1.3 Hz, 1H), 2.44 (t, J = 7.1 Hz, 2H), 1.60–1.63 (m, 2H), 1.49–1.54 (m, 2H), 0.97 (t, J = 7.3 Hz, 3H); ¹³C NMR (100 MHz, MeOD): δ 156.7, 152.0, 147.7, 146.7, 144.8, 144.7, 119.8, 115.5, 113.1, 91.4, 88.1, 81.6, 76.1, 75.0, 72.9, 31.5, 23.1, 19.4, 13.9; HRMS (FAB): found 384.1666 [calcd for C₁₉H₂₂N₅O₄⁺ (M + H)⁺ 384.1672]; Anal. Calcd for C₁₉H₂₁N₅O₄: C, 59.52; H, 5.52; N, 18.27. Found: C, 59.21; H, 5.32; N, 18.56.

(2R,3R,4R)-2-(8-(Furan-2-yl)-2-(hex-1-yn-1-yl)-6-(methylamino)-9H-purin-9-yl)tetrahydrofuran-3,4-diol (5b).

11a and methylamine hydrochloride (1.50 equiv) were dissolved in EtOH (0.20 M) and stirred for 24 h at room temperature. Yield = 80%; white solid; mp 201 °C; $[\alpha {]}_{\mathrm{D}}^{25}=-43.62$ (c 0.09, MeOH); UV (MeOH) λ_max 244, 325 nm; ¹H NMR (800 MHz, DMSO-d₆): δ 7.99 (d, J = 0.8 Hz, 1H), 7.97 (br s, 1H), 7.09 (d, J = 3.4 Hz, 1H), 6.76 (dd, J = 3.0, 1.5 Hz, 1H), 6.11 (d, J = 6.6 Hz, 1H), 5.43 (d, J = 6.5 Hz, 1H), 5.20 (d, J = 3.8 Hz, 1H), 5.17 (q, J = 12.1, 5.5 Hz, 1H), 4.43 (dd, J = 9.2, 3.2 Hz, 1H), 4.30 (d, J = 2.9 Hz, 1H), 3.84 (d, J = 9.2 Hz, 1H), 2.94 (br s, 3H), 2.44 (t, J = 7.0 Hz, 2H), 1.51–1.57 (m, 2H), 1.41–1.47 (m, 2H), 0.92 (t, J = 7.2 Hz, 3H); ¹³C NMR (200 MHz, DMSO-d₆): δ 154.2, 149.7, 145.7, 145.3, 130.8, 130.0, 129.6, 128.2, 119.0, 88.8, 85.6, 81.8, 74.2, 72.5, 70.5, 29.9, 27.0, 21.5, 17.9, 13.4; HRMS (FAB): found 398.1817 [calcd for C₂₀H₂₄N₅O₄⁺ (M + H)⁺ 398.1817]; Anal. Calcd for C₂₀H₂₃N₅O₄: C, 60.44; H, 5.83; N, 17.62. Found: C, 60.84; H, 5.45; N, 17.35.

(2R,3R,4R)-2-(8-(Furan-2-yl)-2-(hex-1-yn-1-yl)-6-((3-iodobenzyl)-amino)-9H-purin-9-yl)tetrahydrofuran-3,4-diol (5c).

11a and 3-iodobenzylamine hydrochloride (1.50 equiv) were dissolved in EtOH (0.20 M) and stirred for 24 h at room temperature. Yield = 68%; pale yellow solid; mp 215 °C; $[\alpha {]}_{\mathrm{D}}^{25}=233.70$ (c 0.055, MeOH); UV (MeOH) λ_max 229, 325 nm; ¹H NMR (400 MHz, MeOD): δ 7.81–7.82 (m, 2H), 7.61 (d, J = 8.0 Hz, 1H), 7.42 (dd, J = 7.6, 0.4 Hz, 1H), 7.19 (dd, J = 3.2, 0.4 Hz, 1H), 7.10 (t, J = 7.6 Hz, 1H), 6.70 (dd, J = 3.6, 1.6 Hz, 1H), 6.33 (d, J = 6.8 Hz, 1H), 5.46 (dd, J = 6.4, 4.8, Hz, 1H), 4.77 (br s, 2H), 4.65 (dd, J = 9.6, 3.6 Hz, 1H), 4.48–4.50 (m, 1H), 3.99 (dd, J = 9.6, 1.2 Hz, 1H), 2.46 (t, J = 7.2 Hz, 2H), 1.60–1.66 (m, 2H), 1.51–1.56 (m, 2H), 0.99 (t, J = 7.2 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆): δ 150.3, 145.59, 145.54, 145.52, 143.3, 142.3, 142.1, 135.9, 135.3, 130.4, 126.71, 126.73, 113.7, 112.1, 94.6, 89.1, 86.0, 74.3, 73.0, 70.5, 40.0, 29.8, 27.8, 21.4, 17.9, 13.4; HRMS (FAB): found 600.1109 [calcd for C₂₆H₂₇IN₅O₄⁺ (M + H)⁺ 600.1108]; Anal. Calcd for C₂₆H₂₆IN₅O₄: C, 52.10; H, 4.37; N, 11.68. Found: C, 52.15; H, 4.32; N, 11.70.

(2R,3R,4R)-2-(6-Amino-2-(hex-1-yn-1-yl)-8-(thiophen-2-yl)-9H-purin-9-yl)tetrahydrofuran-3,4-diol (5d).¹¹

The compound was synthesized by the reported procedure. HPLC purity: 98.73%.

(2R,3R,4R)-2-(2-(Hex-1-yn-1-yl)-6-(methylamino)-8-(thiophen-2-yl)-9H-purin-9-yl)tetrahydrofuran-3,4-diol (5e).

11b and methylamine hydrochloride (1.50 equiv) were dissolved in EtOH (0.20 M) and stirred for 24 h at room temperature. Yield = 76%; white solid; mp 216 °C; $[\alpha {]}_{\mathrm{D}}^{25}=170.00$ (c 0.01, MeOH); UV (MeOH) λ_max 239, 325 nm; ¹H NMR (400 MHz, MeOD): δ 7.71 (dd, J = 5.1, 0.9 Hz, 1H), 7.65 (dd, J = 3.6, 0.9 Hz, 1H), 7.23 (dd, J = 5.0, 3.6 Hz, 1H), 6.11 (d, J = 6.4 Hz, 1H), 5.50 (dd, J = 6.3, 4.8 Hz, 1H), 4.63 (dd, J = 9.6, 3.5 Hz, 1H), 4.46 (t, J = 4.6 Hz, 1H), 3.97 (dd, J = 6.0, 1.2 Hz, 1H), 3.08 (br s, 3H), 2.45 (t, J = 7.2 Hz, 2H), 1.61–1.65 (m, 2H), 1.50–1.55 (m, 2H), 0.98 (t, J = 7.3 Hz, 3H); ¹³C NMR (200 MHz, DMSO-d₆): δ 154.2, 149.7, 145.7, 145.3, 130.8, 130.0, 129.6, 128.2, 119.0, 88.8 (d, J = 7.9 Hz), 85.6, 81.8, 79.1, 74.2 (d, J = 10.5 Hz), 72.6 (d, J = 20.9 Hz), 70.5 (d, J = 21.0 Hz), 29.9, 21.5, 17.9, 13.4; HRMS (FAB): found 414.1605 [calcd for C₂₀H₂₄N₅O₃S⁺ (M + H)⁺ 414.1600]; Anal. Calcd for C₂₀H₂₃N₅O₃S: C, 58.09; H, 5.61; N, 16.94. Found: C, 57.99; H, 5.86; N, 16.67.

(2R,3R,4R)-2-(2-(Hex-1-yn-1-yl)-6-((3-iodobenzyl)amino)-8-(thiophen-2-yl)-9H-purin-9-yl)tetrahydrofuran-3,4-diol (5f).

11b and 3-iodobenzylamine hydrochloride (1.50 equiv) were dissolved in EtOH (0.20 M) and stirred for 24 h at room temperature. Yield = 78%; white solid; mp 225 °C; $[\alpha {]}_{\mathrm{D}}^{25}=285.26$ (c 0.06, MeOH); UV (MeOH) λ_max 231, 326 nm; ¹H NMR (400 MHz, DMSO-d₆): δ 8.55 (br s, 1H), 7.88 (dd, J = 5.1, 0.7 Hz, 1H), 7.74 (s, 1H), 7.58–7.60 (m, 2H), 7.35 (d, J = 6.4 Hz, 1H), 7.29 (dd, J = 4.8, 3.7 Hz, 1H), 7.11 (t, J = 7.7 Hz, 1H), 5.97 (d, J = 6.8 Hz, 1H), 5.51 (d, J = 6.5 Hz, 1H), 5.22–5.24 (m, 2H), 4.41 (dd, J = 9.3, 3.3 Hz, 1H), 3.83 (d, J = 8.9 Hz, 1H), 2.42 (t, J = 7.1 Hz, 2H), 1.52–1.56 (m, 2H), 1.42–1.46 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ 153.6, 150.2, 145.8, 145.5, 142.5, 136.1, 135.4, 130.8, 130.5, 130.2, 129.8, 128.3, 126.8, 118.9, 94.7, 89.0, 81.8, 74.4, 70.6, 42.3, 29.9, 21.5, 18.0, 13.5; HRMS (FAB): found 616.0887 [calcd for C₂₆H₂₇IN₅O₃S⁺ (M + H)⁺ 616.0879]; Anal. Calcd for C₂₆H₂₆IN₅O₃S: C, 50.74; H, 4.26; N, 11.38. Found: C, 50.75; H, 4.30; N, 11.15.

2,6-Dichloro-8-iodo-9-(tetrahydro-2H-pyran-2-yl)-9H-purine (18).

To a cooled (−78 °C) solution of 17b (1.27 g, 4.65 mmol) in anhydrous THF (30 mL) was dropwise added LDA (14.0 mL, 1.0 M in THF/hexanes, 14.0 mmol) under N₂, and the reaction mixture was stirred at the same temperature for 1 h. After adding iodine (5.90 g, 23.25 mmol) in THF (10 mL), the reaction mixture was stirred for 1 h. The reaction mixture was quenched with saturated aqueous Na₂S₂O₃ (50 mL), 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, 2/1) to give 18 (1.50 g, 81%) as a brown solid: ¹H NMR (800 MHz, CDCl₃): δ 5.65 (dd, J = 11.6, 2.4 Hz, 1H), 4.16–4.20 (m, 1H), 3.72 (td, J = 11.6, 2.0 Hz, 1H), 3.03 (ddd, J = 24.0, 12.8, 4.4 Hz, 1H), 2.11–2.19 (m, 1H), 1.57–1.93 (m, 4H); ¹³C NMR (200 MHz, CDCl₃): δ 153.4, 152.6, 150.0, 133.2, 106.4, 86.8, 69.2, 28.7, 24.4, 23.1; HRMS (FAB): found 398.9273 [calcd for C₁₀H₁₀Cl₂IN₄O⁺ (M + H)⁺ 398.9283]; Anal. Calcd for C₁₀H₉Cl₂IN₄O: C, 30.10; H, 2.27; N, 14.04. Found: C, 30.37; H, 1.91; N, 14.01.

2,6-Dichloro-8-iodopurine (19).

To a solution of 18 (3.0 g, 7.52 mmol) in anhydrous EtOH (50.0 mL, 0.15 M) was added PPTS (378 mg, 1.50 mmol) at room temperature under N₂. After being heated at 60 °C for 6 h, the reaction mixture was quenched with triethylamine (1.0 mL) and evaporated. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 1/1) to give 19 (2.25 g, 95%) as a yellow solid: ¹³C NMR (200 MHz, MeOD): δ 163.1, 152.6, 147.0, 134.2, 115.1; HRMS (FAB): found 314.8683 [calcd for C₅H₂Cl₂IN4⁺ (M + H)⁺ 314.8683]; Anal. Calcd for C₅HCl₂IN₄: C, 19.07; H, 0.32; N, 17.79. Found: C, 19.07; H, 0.31; N, 17.71.

9-((2R,3R,4S)-3,4-Bis((tert-butyldimethylsilyl)oxy)-tetrahydrothiophen-2-yl)-6-chloro-2,8-diiodo-9H-purine (20).

To a stirred suspension of 2-chloro-8-iodo-6-chloropurine (17d) (581 mg, 1.84 mmol) in CH₃CN (10.0 mL) was dropwise added BSA (2.40 mL, 9.83 mmol) at room temperature under N₂, and the mixture was heated at 60 °C until obtaining clear brown solution. To the reaction mixture was quickly added 16 (500 mg, 1.23 mmol) in CH₃CN (5.0 mL) and TMSOTf (0.22 mL, 1.23 mmol), and the mixture was stirred at 75 °C for 1.5 h. The reaction mixture was cooled to room temperature, quenched with saturated NaHCO₃ solution (30 mL), and diluted with EtOAc (30 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (3 × 30 mL). The combined organic layers were washed successively with H₂O (50 mL), dried over anhydrous MgSO₄, filtered, and evaporated. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 30/1) to give 20 (569.0 mg, 41%) as brown syrup: ¹H NMR (400 MHz, CDCl₃): δ 6.01 (d, J = 7.2 Hz, 1H), 5.26 (dd, J = 7.2, 2.8 Hz, 1H), 4.55 (dd, J = 5.6, 2.4 Hz, 1H), 3.54 (dd, J = 11.2, 3.2 Hz, 1H), 2.86 (dd, J = 11.2, 2.4 Hz, 1H), 0.98 (s, 9H), 0.74 (s, 9H), 0.17 (s, 3H), 0.16 (s, 3H); ¹³C NMR (100 MHz, MeOD): δ 154.7, 149.9, 136.2, 118.2, 113.3, 95.1, 76.9, 76.7, 75.0, 27.2, 27.1, 19.8, 19.5; HRMS (FAB): found 752.9854 [calcd for C₂₁H₃₆ClI₂N₄O₂SSi₂⁺ (M + H)⁺ 752.9876]; Anal. Calcd for C₂₁H₃₅ClI₂N₄O₂SSi₂: C, 33.49; H, 4.68; N, 7.44. Found: C, 33.87; H, 4.31; N, 7.11.

General Procedure for Stille Coupling for the Preparation of 21a and 21b.

To a stirred solution of 20 (1.00 equiv) in DMF/toluene (v/v = 1/1, 0.040 M) was added bis(dibenzylideneacetone)-palladium(0) (0.10 equiv), followed by 2-(tributylstannyl)furan (1.05 equiv, for 21a), or 2-(tributylstannyl)thiophene (1.05 equiv, for 21b) dropwise at room temperature under N₂. After being stirred for 9 h (for 21a) or 23 h (for 21b) at the same condition, the reaction mixture was quenched with saturated NaHCO₃ solution and diluted with Et₂O. The layers were separated, and the aqueous layer was extracted with Et₂O. 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 to give 21a, or 21b.

9-((2R,3R,4S)-3,4-Bis((tert-butyldimethylsilyl)oxy)-tetrahydrothiophen-2-yl)-6-chloro-8-(furan-2-yl)-2-iodo-9H-purine (21a).

21a was isolated by column chromatography (silica gel, hexanes/EtOAc, 100/3 to 25/2). Yield = 69%; yellow oil; $[\alpha {]}_{\mathrm{D}}^{21}=-73.9$ (c 1.21, MeOH); UV (MeOH) λ_max 326 nm; ¹H NMR (400 MHz, MeOD): δ 7.89 (dd, J = 0.9, 0.7 Hz, 1H), 7.46 (dd, J = 0.9, 2.8 Hz, 1H), 6.79 (dd, J = 3.6, 1.6 Hz, 1H), 6.72 (d, J = 7.8 Hz, 1H), 5.44 (dd, J = 7.8, 2.7 Hz, 1H), 4.61 (q, J = 2.4 Hz, 1H), 3.61 (dd, J = 11.2, 3.0, Hz, 1H), 2.87 (dd, J = 11.2, 2.1 Hz, 1H), 1.00 (s, 9h), 0.63 (s, 9H), 0.20 (s, 3H), 0.17 (s, 3H), −0.07 (s, 3H), −0.54 (s, 3H); ¹³C NMR (200 MHz, MeOD): δ 155.4, 150.8, 149.7, 148.8, 145.0, 134.0, 119.3, 117.2, 114.5, 80.0, 76.2, 66.5, 37.6, 27.1, 26.8, 19.8, 19.4, −3.4, −3.4, −3.5, −4.6. HRMS (ESI): found 693.1027 [calcd for C₂₅H₃₈ClIN₄O₃SSi₂⁺ (M + H)⁺ 692.0936].

9-((2R,3R,4S)-3,4-Bis((tert-butyldimethylsilyl)oxy)-tetrahydrothiophen-2-yl)-6-chloro-2-iodo-8-(thiophen-2-yl)-9H-purine (21b).

21b was isolated by column chromatography (silica gel, hexanes/EtOAc, 40/1). Yield = 44%; yellow oil; $[\alpha {]}_{\mathrm{D}}^{21}=-91.9$ (c 0.18, MeOH); UV (MeOH) λ_max 330 nm; ¹H NMR (400 MHz, MeOD): δ 7.91 (dd, J = 5.1, 0.9 Hz, 1H), 7.81 (dd, J = 3.7, 0.9 Hz, 1H), 7.29 (dd, J = 5.1, 3.7 Hz, 1H), 6.43 (d, J = 8.3 Hz, 1H), 5.53 (dd, J = 8.3, 2.8 Hz, 1H), 4.60 (q, J = 2.5 Hz, 1H), 3.60 (dd, J = 11.5, 2.8 Hz, 1H), 2.87 (dd, J = 11.5, 1.8 Hz, 1H), 0.94 (s, 9H), 0.63 (s, 9H), 0.19 (s, 3H), 0.15 (s, 3H), −0.06 (s, 3H), −0.44 (s, 3H); ¹³C NMR (200 MHz, MeOD): δ 153.66, 151.63, 149.67, 132.37, 131.48, 131.33, 129.33, 128.28, 115.19, 77.73, 73.98, 64.29, 36.03, 25.83, 25.68, 18.17, 17.93, −4.20, −4.36, −4.47, −5.14. HRMS (ESI): found 709.0781 [calcd for C₂₅H₃₈ClIN₄O₂S₂Si₂⁺ (M + H)⁺ 709.0781].

General Procedure for Sonogashira Coupling and Desilylation for the Preparation of 22a and 22b. Sonogashira Coupling.

To a stirred solution of 21a (1.00 equiv, for 22a) or 21b (1.00 equiv, for 22b) in DMF (0.10 M) were added bis(triphenylphosphine)-palladium(II) dichloride (0.10 equiv), copper(I) iodide (0.20 equiv), and cesium carbonate (1.5 equiv), followed by 1-hexyne (1.05 equiv) dropwise at room temperature under N₂. After being stirred for 14 h (for 22a) or 16 h (for 22b) at the same condition, the reaction mixture was quenched with H₂O and diluted with Et₂O. The layers were separated, and the aqueous layer was extracted with Et₂O. 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 to give the coupling products.

TBS Deprotection.

To a solution of coupling product (1.00 equiv) in THF (0.10 M) was added the mixture of tetrabutylammoniumfluoride (1.0 M in THF) and acetic acid (v/v = 10/1, 2.50 equiv) at room temperature under N₂. After being stirred for 16 h (for 22a) or 12 h (for 22b) at 50 °C under N₂, the reaction mixture was quenched by H₂O, and diluted with EtOAc. The layers were separated, and the aqueous layer was extracted with EtOAc. The combined organic layers were dried over anhydrous MgSO₄, filtered, and evaporated. The residue was purified by column chromatography to give 22a or 22b.

(2R,3R,4S)-2-(6-Chloro-8-(furan-2-yl)-2-(hex-1-yn-1-yl)-9H-purin-9-yl)tetrahydro-thiophene-3,4-diol (22a). Sonogashira Coupling.

The coupling product was isolated by column chromatography (silica gel, hexanes/EtOAc, 100/3 to 50/2). Yield = 73%; yellow oil; $[\alpha {]}_{\mathrm{D}}^{21}=-56.2$ (c 0.84, MeOH); UV (MeOH) λ_max 330 nm; ¹H NMR (400 MHz, MeOD): δ 7.89 (d, J = 1.8 Hz, 1H), 7.46 (dd, J = 3.6, 0.8 Hz, 1H), 6.79 (dd, J = 3.4, 1.6 Hz, 1H), 6.72 (d, J = 7.8 Hz, 1H), 5.48 (dd, J = 7.5, 3.0 Hz, 1H), 4.66 (q, J = 2.6 Hz, 1H), 3.63 (dd, J = 11.2, 3.0 Hz, 1H), 2.88 (dd, J = 11.4, 2.3 Hz, 1H), 2.53 (t, J = 6.9 Hz, 2H), 1.64–1.71 (m, 2H), 1.51–1.60 (m, 2H), 1.00 (s, 9H), 1.00 (t, J = 7.2 Hz, 3H), 0.20 (s, 3H), 0.17 (s, 3H), −0.07 (s, 3H), −0.55 (s, 3H); ¹³C NMR (200 MHz, MeOD): δ 154.9, 151.2, 150.4, 148.7, 147.0, 145.2, 133.0, 119.1, 114.5, 92.5, 81.5, 79.9, 76.3, 66.5, 37.5, 32.1, 27.1, 26.8, 23.9, 20.4, 19.8, 19.4, 14.8, −3.4, −3.5, −3.6, −4.7. HRMS (ESI): found 647.2664 [calcd for C₃₁H₄₈ClN₄O₃SSi₂⁺ (M + H)⁺ 646.2596].

TBS Deprotection.

22a was isolated by column chromatography (silica gel, CH₂Cl₂/MeOH, 50/1). Yield = 94%; yellow solid; $[\alpha {]}_{\mathrm{D}}^{21}=-110.8$ (c 0.13, MeOH); UV (MeOH) λ_max 330 nm; ¹H NMR (400 MHz, MeOD): δ 7.95 (d, J = 1.4 Hz, 1H), 7.51 (d, J = 3.7 Hz, 1H), 6.79 (dd, J = 3.7, 1.8 Hz, 1H), 6.61 (d, J = 7.8 Hz, 1H), 5.61 (dd, J = 7.8, 3.7 Hz, 1H), 4.59 (q, J = 2.8 Hz, 1H), 3.74 (dd, J = 11.5, 3.2 Hz, 1H), 2.97 (dd, J = 11.5, 1.8 Hz, 1H), 2.52 (t, J = 6.9 Hz, 2H), 1.63–1.70 (m, 2H), 1.50–1.60 (m, 2H), 1.00 (t, J = 7.1 Hz, 3H); ¹³C NMR (200 MHz, MeOD): δ 154.9, 151.0, 150.4, 148.8, 146.9, 145.0, 133.0, 118.9, 114.5, 92.2, 81.5, 79.2, 75.5, 67.7, 37.8, 32.2, 23.9, 20.3, 14.7. HRMS (FAB): found 419.0944 [calcd for C₁₉H₂₀ClN₄O₃S⁺ (M + H)⁺ 419.0945].

(2R,3R,4S)-2-(6-Chloro-2-(hex-1-yn-1-yl)-8-(thiophen-2-yl)-9H-purin-9-yl)tetrahydro-thiophene-3,4-diol (22b). Sonogashira Coupling.

The coupling product was isolated by column chromatography (silica gel, hexanes/EtOAc, 100/3 to 50/2). Yield = 64%; colorless oil; $[\alpha {]}_{\mathrm{D}}^{21}=-56.2$ (c 0.84, MeOH); ¹H NMR (400 MHz, MeOD): δ 7.91 (dd, J = 5.3, 1.1 Hz, 1H), 7.83 (dd, J = 3.7, 0.9 Hz, 1H), 7.30 (dd, J = 5.3, 3.9 Hz, 1H), 6.44 (d, J = 8.2 Hz, 1H), 5.57 (dd, J = 8.0, 3.0 Hz, 1H), 4.64 (q, J = 2.4 Hz, 1H), 3.64 (dd, J = 11.4, 3.2 Hz, 1H), 2.88 (dd, J = 11.4, 2.3 Hz, 1H), 2.54 (t, J = 7.1 Hz, 2H), 1.64–1.72 (m, 2H), 1.51–1.60 (m, 2H), 1.01 (t, J = 7.3 Hz, 3H), 0.94 (s, 9H), 0.63 (s, 9H), 0.19 (s, 3H), 0.15 (s, 3H), −0.06 (s, 3H), −0.47 (s, 3H); ¹³C NMR (200 MHz, MeOD): δ 155.1, 154.7, 151.2, 147.0, 133.8, 133.3, 132.9, 131.1, 130.1, 92.5, 81.5, 79.5, 75.9, 66.3, 37.2, 32.1, 27.1, 26.8, 23.9, 20.3, 19.7, 19.4, 14.7, −3.4, −3.4, −3.6, −4.5. HRMS (ESI): found 663.2450 [calcd for C₃₁H₄₈ClN₄O₂S₂Si₂⁺ (M + H)⁺ 663.2440].

TBS Deprotection.

22b was isolated by column chromatography (silica gel, CH₂Cl₂/MeOH, 50/1). Yield = 73%; white solid; $[\alpha {]}_{\mathrm{D}}^{21}=-94.8$ (c 0.52, MeOH); UV (MeOH) λ_max 326 nm; ¹H NMR (400 MHz, MeOD): δ 7.90 (dd, J = 3.7, 0.9 Hz, 1H), 7.88 (dd, J = 5.1, 1.4 Hz, 1H), 7.32 (dd, J = 5.1, 3.7 Hz, 1H), 6.33 (d, J = 7.8 Hz, 1H), 5.65 (q, J = 3.8 Hz, 1H), 4.56 (td, J = 3.4, 1.7 Hz, 1H), 3.73 (dd, J = 11.5, 3.7 Hz, 1H), 2.96 (dd, J = 11.5, 1.8 Hz, 1H), 2.52 (t, J = 6.9 Hz, 2H), 1.63–1.70 (m, 2H), 1.50–1.59 (m, 2H), 1.00 (t, J = 7.1 Hz, 3H); ¹³C NMR (200 MHz, MeOD): δ 155.2, 154.6, 150.9, 146.9, 133.7, 133.4, 132.9, 131.2, 130.3, 92.1, 81.5, 78.9, 75.3, 67.5, 37.6, 32.2, 23.9, 20.3, 14.7. HRMS (FAB): found 435.0717 [calcd for C₁₉H₂₀ClN₄O₃S⁺ (M + H)⁺ 435.0711].

General Procedure for N⁶-Amination for the Preparation of 5g–5l.

To a solution of 22a or 22b (1.00 equiv) in EtOH (0.20 M) or t-BuOH (0.20 M) were dropwise added appropriate amines (1.50 equiv to excess) at room temperature under N₂. After being stirred at the provided temperature for 12–28 h, the reaction mixture was evaporated. The residue was purified by silica gel column chromatography (CH₂Cl₂/MeOH, 20/1) to give 5g–5l.

(2R,3R,4S)-2-(6-Amino-8-(furan-2-yl)-2-(hex-1-yn-1-yl)-9H-purin-9-yl)tetrahydro-thiophene-3,4-diol (5g).

22a was dissolved in NH₃/t-BuOH (0.20 M) and stirred for 15 h at 100 °C in a steel bomb. Yield = 44%; white solid; mp 220 °C; $[\alpha {]}_{\mathrm{D}}^{21}=-205.2$ (c 0.05, MeOH); UV (MeOH) λ_max 238, 320 nm; ¹H NMR (400 MHz, MeOD): δ 7.85 (d, J = 1.8 Hz, 1H), 7.23 (d, J = 3.2 Hz, 1H), 6.72 (q, J = 1.7 Hz, 1H), 6.41 (d, J = 7.8 Hz, 1H), 5.65 (dd, J = 3.2, 1.8 Hz, 1H), 4.56 (td, J = 3.4, 1.8 Hz, 1H), 3.73 (dd, J = 11.4, 3.7 Hz, 1H), 2.92 (dd, J = 11.4, 1.8 Hz, 1H), 2.46 (t, J = 6.9 Hz, 2H), 1.60–1.68 (m, 2H), 1.49–1.58 (m, 2H), 0.99 (t, J = 7.3 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ 155.68, 150.44, 145.49, 145.48, 143.53, 142.14, 118.89, 113.36, 112.20, 85.70, 81.64, 75.98, 72.14, 63.88, 35.65, 29.93, 21.51, 17.97, 13.48. HRMS (FAB): found 400.1443 [calcd for C₁₉H₂₂N₅O₃S⁺ (M + H)⁺ 400.1441]; HPLC purity: 99.50%.

(2R,3R,4S)-2-(8-(Furan-2-yl)-2-(hex-1-yn-1-yl)-6-(methylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol (5h).

22a and methylamine hydrochloride (1.50 equiv) were dissolved in EtOH (0.20 M) and stirred for 23 h at 50 °C. Yield = 65%; white solid; mp 210 °C; $[\alpha {]}_{\mathrm{D}}^{21}=-135.8$ (c 0.06, MeOH); UV (MeOH) λ_max 244, 326 nm; ¹H NMR (400 MHz, MeOD): δ 7.82 (s, 1H), 7.19 (d, J = 3.7 Hz, 1H), 6.70 (dd, J = 3.4, 1.6 Hz, 1H), 6.40 (d, J = 7.8 Hz, 1H), 5.65 (dd, J = 7.6, 3.4 Hz, 1H), 4.56 (q, J = 2.8 Hz, 1H), 3.73 (dd, J = 11.5, 3.7 Hz, 1H), 3.10 (s, 3H), 2.92 (dd, J = 11.5, 1.4 Hz, 1H), 2.47 (t, J = 6.9 Hz, 2H), 1.62–1.69 (m, 2H), 1.49–1.59 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H); ¹³C NMR (200 MHz, MeOD): δ 157.08, 148.45, 147.27, 145.83, 144.92, 121.48, 115.62, 113.89, 88.49, 82.80, 79.02, 75.52, 66.78, 37.49, 32.38, 23.94, 20.35, 14.76. HRMS (FAB): found 414.1600 [calcd for C₂₀H₂₄N₅O₃S⁺ (M + H)⁺ 414.1607]; HPLC purity: 99.00%.

(2R,3R,4S)-2-(8-(Furan-2-yl)-2-(hex-1-yn-1-yl)-6-((3-iodobenzyl)-amino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol (5i).

22a and 3-iodobenzylamine hydrochloride (1.50 equiv) were dissolved in EtOH (0.20 M) and stirred for 24 h at 50 °C. Yield = 79%; white solid; mp 238 °C; $[\alpha {]}_{\mathrm{D}}^{21}=79.11$ (c 0.07, MeOH); UV (MeOH) λ_max 246, 325 nm; ¹H NMR (400 MHz, DMSO-d₆): δ 8.62 (s, 1H), 8.03 (d, J = 1.4 Hz, 1H), 7.75 (s, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.14 (d, J = 3.2 Hz, 1H), 7.11 (d, J = 7.8 Hz, 1H), 6.79 (dd, J = 3.2, 1.8 Hz, 1H), 6.20 (d, J = 7.8 Hz, 1H), 5.47 (d, J = 6.4 Hz, 1H), 5.38–5.41 (m, 2H), 4.63 (s, 2H), 4.38 (s, 1H), 3.48 (dd, J = 11.2, 3.4 Hz, 1H), 2.81 (d, J = 10.5 Hz, 1H), 2.44 (t, J = 7.1 Hz, 2H), 1.51–1.59 (m, 2H), 1.40–1.49 (m, 2H), 0.92 (t, J = 7.1 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ 153.75, 149.88, 145.60, 145.33, 143.51, 142.50, 142.26, 136.05, 135.45, 130.51, 126.78, 119.32, 113.51, 112.26, 94.74, 86.10, 81.90, 76.12, 72.15, 63.94, 42.26, 35.69, 29.91, 21.56, 18.06, 13.53. HRMS (FAB): found 616.0879 [calcd for C₂₆H₂₇IN₅O₃S⁺ (M + H)⁺ 616.0876]; HPLC purity: 95.75%.

(2R,3R,4S)-2-(6-Amino-2-(hex-1-yn-1-yl)-8-(thiophen-2-yl)-9H-purin-9-yl)tetrahydro-thiophene-3,4-diol (5j).

22b was dissolved in NH₃/t-BuOH (0.20 M) and stirred for 15 h at 100 °C in a steel bomb. Yield = 83%; white solid; mp 197 °C; $[\alpha {]}_{\mathrm{D}}^{21}=-141.6$ (c 0.15, MeOH); UV (MeOH) λ_max 236, 319 nm; ¹H NMR (400 MHz, DMSO-d₆): δ 7.88 (d, J = 4.1 Hz, 1H), 7.64 (d, J = 3.2 Hz, 1H), 7.45 (s, 2H), 7.31 (dd, J = 5.1, 3.7 Hz, 1H), 6.05 (d, J = 7.8 Hz, 1H), 5.43–5.49 (d, J = 6.8 Hz, 1H), 5.49 (d, J = 6.8 Hz, 1H), 5.46 (qd, J = 7.2, 2.8 Hz, 1H), 5.35 (d, J = 3.7 Hz, 1H), 4.37 (d, J = 1.8 Hz, 1H), 3.47 (dd, J = 11.0, 3.2 Hz, 1H), 2.80 (d, J = 10.6 Hz, 1H), 2.43 (t, J = 7.1 Hz, 2H), 1.50–1.58 (m, 2H), 1.39–1.48 (m, 2H), 0.92 (t, J = 7.4 Hz, 3H); ¹³C NMR (200 MHz, DMSO-d₆): δ 155.46, 150.78, 145.56, 145.29, 130.90, 129.99, 128.97, 128.21, 118.73, 85.60, 81.63, 75.58, 71.92, 63.94, 35.56, 29.89, 21.48, 17.94, 13.44. HRMS (FAB): found 416.1219 [calcd for C₁₉H₂₂N₅O₂S₂⁺ (M + H)⁺ 416.1215]; HPLC purity: 99.56%.

(2R,3R,4S)-2-(2-(Hex-1-yn-1-yl)-6-(methylamino)-8-(thiophen-2-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol (5k).

22b and methylamine hydrochloride (1.50 equiv) were dissolved in EtOH (0.20 M) and stirred for 28 h at 50 °C. Yield = 61%; white solid; mp 238 °C; $[\alpha {]}_{\mathrm{D}}^{21}=237.09$ (c 0.04, DMSO); UV (MeOH) λ_max 242, 326 nm; ¹H NMR (400 MHz, DMSO-d₆): δ 7.89 (s, 1H), 7.87 (dd, J = 5.0, 0.9 Hz, 1H), 7.64 (dd, J = 3.7, 0.9 Hz, 1H), 7.31 (dd, J = 5.0, 3.7 Hz, 1H), 6.06 (d, J = 8.2 Hz, 1H), 5.51 (d, J = 6.4 Hz, 1H), 5.42–5.46 (m, 1H), 5.37 (d, J = 3.2 Hz, 1H), 4.37 (d, J = 1.4 Hz, 1H), 3.48 (dd, J = 11.2, 3.4 Hz, 1H), 2.92 (s, 3H), 2.81 (dd, J = 11.2, 1.1 Hz, 1H), 2.45 (t, J = 7.3 Hz, 2H), 1.53–1.60 (m, 2H), 1.40–1.49 (m, 2H), 0.93 (t, J = 7.1 Hz, 3H); ¹³C NMR (200 MHz, MeOD): δ 154.32, 149.77, 145.37, 145.27, 130.96, 129.96, 128.91, 128.24, 119.31, 85.72, 81.97, 75.70, 71.95, 63.95, 35.59, 29.93, 27.08, 21.59, 18.03, 13.47. HRMS (FAB): found 430.1372 [calcd for C₂₀H₂₄N₅O₂S₂⁺ (M + H)⁺ 430.1371]; HPLC purity: 99.17%.

(2R,3R,4S)-2-(2-(Hex-1-yn-1-yl)-6-((3-iodobenzyl)amino)-8-(thiophen-2-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol (5l).

22b and 3-iodobenzylamine hydrochloride (1.50 equiv) were dissolved in EtOH (0.20 M) and stirred for 20 h at 75 °C. Yield = 78%; white solid; mp 236 °C; $[\alpha {]}_{\mathrm{D}}^{21}=100.99$ (c 0.11, DMSO); UV (MeOH) λ_max 248, 326 nm; ¹H NMR (400 MHz, DMSO-d₆): δ 8.53 (s, 1H), 7.89 (d, J = 5.0 Hz, 1H), 7.76 (s, 1H), 7.65 (d, J = 3.2 Hz, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 5.5 Hz, 1H), 7.31 (t, J = 4.1 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), 6.07 (d, J = 8.2 Hz, 1H), 5.52 (d, J = 5.0 Hz, 1H), 5.44 (t, J = 3.0 Hz, 1H), 5.38 (s, 1H), 4.62 (s, 2H), 4.37 (s, 1H), 3.47 (dd, J = 11.0, 2.7 Hz, 1H), 2.81 (d, J = 11.0 Hz, 1H), 2.44 (t, J = 7.1 Hz, 2H), 1.52–1.59 (m, 2H), 1.40–1.49 (m, 2H), 0.93 (t, J = 7.1 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ 153.59, 150.29, 145.80, 145.23, 142.58, 136.12, 135.49, 130.92, 130.55, 130.21, 129.13, 128.39, 126.87, 119.23, 94.77, 86.12, 81.96, 75.77, 72.01, 64.06, 54.98, 42.32, 35.67, 29.95, 21.62, 18.10, 13.58. HRMS (FAB): found 632.0653 [calcd for C₂₆H₂₇IN₅O₂S₂⁺ (M + H)⁺ 632.0651].

9-((2R,3R,4S)-3,4-Bis((tert-butyldimethylsilyl)oxy)-tetrahydrothiophen-2-yl)-6-chloro-2,8-diiodo-9H-purine (23).

To a stirred solution of 2,6-dichloro-8-iodopurine (19) (581 mg, 1.84 mmol) in CH₃CN (10.0 mL) was dropwise added BSA (2.4 mL, 9.83 mmol) at room temperature under N₂, and the reaction mixture was heated at 60 °C until obtaining a clear solution. To the reaction mixture was dropwise added 16 (500 mg, 1.23 mmol) in CH₃CN (5.0 mL) and TMSOTf (0.22 mL, 1.23 mmol). After being stirred at 75 °C for 1.5 h, the reaction mixture was cooled to room temperature and evaporated. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 10/1) to give 23 (400 mg, 54%) as a colorless syrup: $[\alpha {]}_{\mathrm{D}}^{25}=-1.02$ (c 37.53, MeOH); UV (MeOH) λ_max 229, 286 nm; ¹H NMR (400 MHz, DMSO-d₆): δ 5.98 (d, J = 7.2 Hz, 1H), 5.08 (dd, J = 7.2, 2.8 Hz, 1H), 4.64 (dd, J = 5.2, 2.8 Hz, 1H), 3.43 (dd, J = 11.2, 3.2 Hz, 1H), 2.78 (dd, J = 11.6, 2.8 Hz, 1H), 0.91 (s, 9H), 0.62 (s, 9H), 0.11 (s, 3H), 0.09 (s, 3H), −0.1 (s, 3H), −0.53 (s, 3H); ¹³C NMR (200 MHz, DMSO-d₆): δ 152.9, 150.5, 148.3, 133.5, 128.2, 77.1, 73.3, 66.5, 35.4, 25.6, 25.2, 17.7, 17.3, −4.6, −4.7, −4.7, −5.8; HRMS (FAB): found 661.0459 [calcd for C₂₁H₃₆Cl₂IN₄O₂SSi₂⁺ (M + H)⁺ 661.0451]; Anal. Calcd for C₂₁H₃₅ClI₂N₄O₂SSi₂: C, 33.49; H, 4.68; N, 7.44. Found: C, 33.11; H, 5.08; N, 7.19.

General Procedure for Stille Coupling for the Preparation of 24a and 24b.

To a solution of 23 (2.90 g, 4.38 mmol) in anhydrous THF (0.10 M) was added bis(triphenylphosphine)palladium(II) dichloride (0.10 equiv), 2-(tributylstannyl)furan (1.50 equiv, for 24a), or 2-(tributylstannyl)thiophene (1.50 equiv, for 24b) at room temperature under N₂. After being heated reflux with stirring for 24 h, the reaction mixture was cooled to room temperature and evaporated. The residue was purified by column chromatography to give 24a or 24b.

9-((2R,3R,4S)-3,4-Bis((tert-butyldimethylsilyl)oxy)-tetrahydrothiophen-2-yl)-2,6-dichloro-8-(furan-2-yl)-9H-purine (24a).

24a was isolated by column chromatography (silica gel, hexanes/EtOAc, 20/1). Yield = 71%; colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}=-25.42$ (c 0.045, MeOH); UV (MeOH) λ_max 322 nm; ¹H NMR (400 MHz, CDCl₃): δ 7.66–7.69 (m, 1H), 7.38 (d, J = 3.2 Hz, 1H), 6.65–6.66 (m, 2H), 5.32 (dd, J = 6.8, 2.8 Hz, 1H), 4.56–4.60 (m, 1H), 3.57 (dd, J = 10.8, 3.2 Hz, 1H), 2.86 (dd, J = 11.2, 3.2 Hz, 1H), 0.96 (s, 9H), 0.65 (s, 9H), 0.16 (s, 3H), 0.14 (s, 9H), −0.11 (s, 3H), −0.52 (s, 3H); ¹³C NMR (150 MHz, MeOD): δ 155.3, 152.8, 151.5, 149.7, 148.0, 144.3, 132.6, 118.6, 113.8, 79.3, 75.4, 65.8, 36.8, 26.4, 26.0, 19.0, 18.6, −4.2, −4.2, −4.3, −5.5; HRMS (FAB): found 601.1652 [calcd for C₂₅H₃₉Cl₂N₄O₃SSi₂⁺ (M + H)⁺ 601.1651]; Anal. Calcd for C₂₅H₃₈Cl₂N₄O₃SSi₂: C, 49.90; H, 6.37; N, 9.31. Found: C, 49.94; H, 6.36; N, 9.37.

9-((2R,3R,4S)-3,4-Bis((tert-butyldimethylsilyl)oxy)-tetrahydrothiophen-2-yl)-2,6-dichloro-8-(thiophen-2-yl)-9H-purine (24b).

24b was isolated by column chromatography (silica gel, hexanes/EtOAc, 40/1). Yield = 56%; colorless oil; $[\alpha {]}_{\mathrm{D}}^{25}=66.89$ (c 0.10, DMSO); UV (MeOH) λ_max 253, 315 nm; ¹H NMR (400 MHz, CDCl₃): δ 7.77 (d, J = 4.4 Hz, 1H), 7.65 (d, J = 5.2 Hz, 1H), 7.19 (dd, J = 5.2, 4.0 Hz, 1H), 6.40 (d, J = 7.6 Hz, 1H), 5.39 (dd, J = 7.6, 2.8 Hz, 1H), 4.52–4.56 (m, 1H), 3.58 (dd, J = 10.8, 2.4 Hz, 1H), 2.84 (dd, J = 10.8, 2.4 Hz, 1H), 0.92 (s, 9H), 0.65 (s, 9H), 0.14 (s, 3H), 0.12 (s, 3H), −0.11 (s, 3H), −0.46 (s, 3H); ¹³C NMR (150 MHz, CDCl₃): δ 154.4, 152.4, 151.9, 151.0, 135.8, 131.5, 131.4, 129.5, 128.3, 77.8, 74.0, 64.4, 36.0, 25.9, 25.7, 18.2, 18.0, −4.2, −4.3, −4.4, −5.2; HRMS (FAB): found 617.1452 [calcd for C₂₅H₃₉Cl₂N₄O₂S₂Si₂⁺ (M + H)⁺ 617.1451]; Anal. Calcd for C₂₅H₃₈Cl₂N₄O₂S₂Si₂: C, 48.60; H, 6.20; N, 9.07. Found: C, 48.66; H, 6.24; N, 9.02.

General Procedure for Desilylation and N⁶-Amination for the Synthesis of 5m–5r.

To a solution of 24a or 24b (1.00 equiv) in anhydrous THF (0.10 M) was dropwise added TBAF (2.50 equiv) at room temperature. The reaction mixture was stirred at the same temperature for 1 h and evaporated. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 5/1 to 3/1) to give a diol intermediate. To a solution of the diol intermediate (1.00 equiv) in EtOH (0.20 M) or t-BuOH (0.20 M) were dropwise added appropriate amines (1.50 equiv to excess) at room temperature under N₂. After being stirred at the provided temperature for 15–24 h, the reaction mixture was evaporated. The residue was purified by silica gel column chromatography (CH₂Cl₂/MeOH, 20/1) to give 5m–5r.

(2R,3R,4S)-2-(6-Amino-2-chloro-8-(furan-2-yl)-9H-purin-9-yl)-tetrahydrothiophene-3,4-diol (5m).

Yield = 56%; pale yellow solid; mp 241 °C; $[\alpha {]}_{\mathrm{D}}^{25}=-14.13$ (c 0.045, MeOH); UV (MeOH) λ_max 242, 305 nm; ¹H NMR (400 MHz, MeOD): δ 7.82 (d, J = 1.8 Hz, 1H), 7.20 (d, J = 2.7 Hz, 1H), 6.70 (dd, J = 3.7, 1.8 Hz, 1H), 6.37 (d, J = 7.8 Hz, 1H), 5.54 (dd, J = 7.3, 3.6 Hz, 1H), 4.54 (dd, J = 5.4, 3.6 Hz, 1H), 3.67 (dd, J = 11.4, 3.2 Hz, 1H), 2.91 (dd, J = 11.4, 1.8 Hz, 1H); ¹³C NMR (150 MHz, MeOD): δ 158.6, 155.5, 153.5, 147.4, 145.5, 145.2, 120.4, 115.8, 113.9, 79.1, 75.5, 67.0, 37.5; HRMS (FAB): found 354.0416 [calcd for C₁₃H₁₃ClN₅O₃S⁺ (M + H)⁺ 354.0428]; Anal. Calcd for C₁₃H₁₂ClN₅O₃S: C, 44.13; H, 3.42; N, 19.80. Found: C, 43.91; H, 3.14; N, 19.95.

(2R,3R,4S)-2-(2-Chloro-8-(furan-2-yl)-6-(methylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol (5n).

Yield = 55%; white solid; mp 224 °C; $[\alpha {]}_{\mathrm{D}}^{25}=-8.10$ (c 0.06, MeOH); UV (MeOH) λ_max 245, 310 nm; ¹H NMR (400 MHz, MeOD): δ 7.80 (d, J = 1.9 Hz, 1H), 7.16 (d, J = 3.1 Hz, 1H), 6.68 (dd, J = 3.7, 1.8 Hz, 1H), 6.36 (d, J = 7.3 Hz, 1H), 5.54 (dd, J = 7.8, 3.6 Hz, 1H), 4.54 (dd, J = 5.0, 3.1 Hz, 1H), 3.67 (dd, J = 11.4, 3.6 Hz, 1H), 3.07 (br s, 3H), 2.91 (dd, J = 11.4, 2.2 Hz, 1H); ¹³C NMR (200 MHz, MeOD): δ 157.8, 155.8, 152.3, 147.2, 145.7, 144.6, 121.2, 115.6, 113.8, 79.1, 75.5, 66.9, 37.5, 28.4; HRMS (FAB): found 368.0587 [calcd for C₁₄H₁₅ClN₅O₃S⁺ (M + H)⁺ 368.0584]; Anal. Calcd for C₁₄H₁₄ClN₅O₃S: C, 45.72; H, 3.84; N, 19.04. Found: C, 45.75; H, 4.06; N, 19.39.

(2R,3R,4S)-2-(2-Chloro-8-(furan-2-yl)-6-((3-iodobenzyl)amino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol (5o).

Yield = 64%; white solid; mp 219 °C; $[\alpha {]}_{\mathrm{D}}^{25}=-52.49$ (c 0.13, MeOH); UV (MeOH) λ_max 248, 311 nm; ¹H NMR (800 MHz, DMSO-d₆): δ 9.06 (t, J = 6.0 Hz, 1H), 8.03 (s, 1H), 7.75 (s, 1H), 7.60 (d, J = 7.7 Hz, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.12–7.14 (m, 2H), 6.79 (s, 1H), 6.17 (d, J = 7.8 Hz, 1H), 5.48 (d, J = 6.2 Hz, 1H), 5.35 (br s, 1H), 5.30 (br s, 1H), 4.57–4.63 (m, 2H), 4.38 (s, 1H), 3.46 (dd, J = 11.3, 3.4 Hz, 1H), 2.80 (d, J = 11.2 Hz, 1H); ¹³C NMR (200 MHz, DMSO-d₆): δ 154.5, 152.5, 150.5, 145.6, 143.1, 142.1, 141.7, 136.0, 135.5, 130.5, 126.8, 118.9, 113.5, 112.2, 94.7, 76.2, 72.0, 64.1, 42.5, 35.6; HRMS (FAB): found 569.9866 [calcd for C₂₀H₁₈ClIN₅O₃S⁺ (M + H)⁺ 569.9864]; Anal. Calcd for C₂₀H₁₇ClIN₅O₃S: C, 42.16; H, 3.01; N, 12.29. Found: C, 42.55; H, 2.97; N, 12.61.

(2R,3R,4S)-2-(6-Amino-2-chloro-8-(thiophen-2-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol (5p).

Yield = 60%; white solid; mp 235 °C; $[\alpha {]}_{\mathrm{D}}^{25}=-51.90$ (c 0.205, MeOH); UV (MeOH) λ_max 246, 303 nm; ¹H NMR (600 MHz, MeOD): δ 7.73 (dd, J = 5.0, 0.9 Hz, 1H), 7.71 (dd, J = 3.6, 0.9 Hz, 1H), 7.25 (dd, J = 4.9, 3.6 Hz, 1H), 6.20 (d, J = 7.3 Hz, 1H), 5.56 (dd, J = 7.8, 3.6 Hz, 1H), 4.52–4.53 (m, 1H), 3.67 (dd, J = 11.4, 3.1 Hz, 1H), 2.91 (dd, J = 11.4, 1.8 Hz, 1H); ¹³C NMR (200 MHz, MeOD): δ 158.5, 155.3, 153.9, 148.7, 132.2, 131.77, 131.74, 130.0, 120.4, 78.8, 75.4, 66.9, 37.5; HRMS (FAB): found 370.0194 [calcd for C₁₃H₁₃ClN₅O₂S₂⁺ (M + H)⁺ 370.0199]; Anal. Calcd for C₁₃H₁₂ClN₅O₂S₂: C, 42.23; H, 3.29; N, 18.95. Found: C, 42.61; H, 3.01; N, 18.91.

(2R,3R,4S)-2-(2-Chloro-6-(methylamino)-8-(thiophen-2-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol (5q).

Yield = 58%; white solid; mp 236 °C; $[\alpha {]}_{\mathrm{D}}^{25}=-81.42$ (c 0.10, MeOH); UV (MeOH) λ_max 249, 311 nm; ¹H NMR (400 MHz, MeOD): δ 7.71 (dd, J = 5.0, 1.3 Hz, 1H), 7.68 (dd, J = 3.9, 0.9 Hz, 1H), 7.24 (dd, J = 5.0, 3.6 Hz, 1H), 6.19 (d, J = 7.3 Hz, 1H), 5.56 (dd, J = 7.3, 3.2 Hz, 1H), 4.52–4.53 (m, 1H), 3.67 (dd, J = 11.4, 3.6 Hz, 1H), 3.06 (br s, 3H), 2.90 (dd, J = 11.4, 1.8 Hz, 1H); ¹³C NMR (200 MHz, MeOD): δ 160.3, 157.8, 155.8, 152.8, 148.1, 132.5, 131.5, 129.9, 121.0, 78.8, 75.4, 66.9, 37.4, 28.4; HRMS (FAB): found 384.0349 [calcd for C₁₄H₁₅ClN₅O₂S₂⁺ (M + H)⁺ 384.0356]; Anal. Calcd for C₁₄H₁₄ClN₅O₂S₂: C, 43.80; H, 3.68; N, 18.24. Found: C, 43.81; H, 3.60; N, 17.99.

(2R,3R,4S)-2-(2-Chloro-6-((3-iodobenzyl)amino)-8-(thiophen-2-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol (5r).

Yield = 55%; white solid; mp 207 °C; $[\alpha {]}_{\mathrm{D}}^{25}=-77.58$ (c 0.12, MeOH); UV (MeOH) λ_max 252, 310 nm; ¹H NMR (6 00 MHz, MeOD): δ 7.79 (br s, 1H), 7.71 (d, J = 5.0 Hz, 1H), 7.69 (d, J = 3.6 Hz, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.40 (d, J = 7.8 Hz, 1H), 7.24 (dd, J = 5.0, 3.7 Hz, 1H), 7.09 (t, J = 7.8 Hz, 1H), 6.20 (d, J = 7.8 Hz, 1H), 5.57 (dd, J = 7.8, 3.6 Hz, 1H), 4.66–4.74 (m, 2H), 4.51–4.53 (m, 1H), 3.67 (dd, J = 11.4, 3.6 Hz, 1H), 2.90 (dd, J = 11.4, 1.8 Hz, 1H); ¹³C NMR (200 MHz, DMSO-d₆): δ 154.4, 152.3, 150.9, 145.6, 141.8, 136.1, 135.5, 130.5, 130.4, 130.2, 129.2, 128.2, 126.8, 118.8, 94.7, 75.9, 71.8, 64.1, 42.5, 35.6; HRMS (FAB): found 585.9620 [calcd for C₂₀H₁₈ClIN₅O₂S₂⁺ (M + H)⁺ 585.9635]; Anal. Calcd for C₂₀H₁₇ClIN₅O₂S₂: C, 41.00; H, 2.92; N, 11.95. Found: C, 41.40; H, 2.50; N, 11.55.

General Procedure for N²-Amination for the Synthesis of 5s-5v.

To a solution of 5m, 5n, 5p, and 5q in anhydrous 1-butanol (0.10 M) was dropwise added N,N-diisopropylethylaminetriethylamine (10.00 equiv) followed by tyramine (5.00 equiv) at room temperature. After being heated with microwave at 180 °C for 2 h, the reaction mixture was cooled to room temperature and evaporated. The residue was purified by silica gel column chromatography (CH₂Cl₂/MeOH, 25/1) to give 5s–5v.

(2R,3R,4S)-2-(6-Amino-8-(furan-2-yl)-2-((4-hydroxyphenethyl)-amino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol (5s).

Yield = 63%; yellow solid; mp 232 °C; $[\alpha {]}_{\mathrm{D}}^{25}=16.16$ (c 0.10, MeOH); UV (MeOH) λ_max 228, 324 nm; ¹H NMR (800 MHz, MeOD): δ 7.75 (d, J = 1.2 Hz, 1H), 7.05 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 3.2 Hz, 1H), 6.69–6.71 (m, 2H), 6.66 (dd, J = 3.4, 1.8 Hz, 1H), 6.27 (d, J = 7.2 Hz, 1H), 5.72–5.75 (m, 1H), 4.53 (dd, J = 5.6, 3.3 Hz, 1H), 3.59 (dd, J = 11.5, 3.6 Hz, 1H), 3.56 (t, J = 7.2 Hz, 2H), 2.87 (dd, J = 11.5, 2.2 Hz, 1H), 2.83 (t, J = 7.2 Hz, 2H); ¹³C NMR (200 MHz, DMSO-d₆): δ 158.7, 156.0, 155.4, 154.8, 144.3, 144.2, 138.0, 132.1, 130.3, 130.0, 129.3, 115.0, 114.9, 111.7, 111.3, 79.1, 78.9, 75.7, 72.6, 35.7, 34.5; HRMS (FAB): found 455.1439 [calcd for C₂₁H₂₃N₆O₄S⁺ (M + H)⁺ 455.1435]; Anal. Calcd for C₂₁H₂₂N₆O₄S: C, 55.49; H, 4.88; N, 18.49. Found: C, 55.51; H, 4.85; N, 18.51.

(2R,3R,4S)-2-(8-(Furan-2-yl)-2-((4-hydroxyphenethyl)amino)-6-(methylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol (5t).

Yield = 65%; pale yellow solid; mp 262 °C; $[\alpha {]}_{\mathrm{D}}^{25}=-3.12$ (c 8.67, MeOH); UV (MeOH) λ_max 219, 287, 327 nm; ¹H NMR (800 MHz, MeOD): δ 7.72 (d, J = 1.2 Hz, 1H), 7.05 (d, J = 8.4 Hz, 2H), 6.99 (d, J = 3.1 Hz, 1H), 6.70–6.71 (m, 2H), 6.63 (dd, J = 3.4, 1.8 Hz, 1H), 6.26 (d, J = 7.2 Hz, 1H), 5.73 (br s, 1H), 4.53 (dd, J = 3.2, 5.6 Hz, 1H), 3.60 (dd, J = 11.4, 3.5 Hz, 1 H), 3.58 (s, 1H), 3.57 (s, 1H), 3.02 (br s, 3H), 2.87 (dd, J = 11.5, 2.1 Hz, 1H), 2.83 (t, J = 7.4 Hz, 2H); ¹³C NMR (200 MHz, DMSO-d₆): δ 158.7, 155.4, 155.3, 155.2, 151.4, 144.4, 144.2, 137.6, 130.0, 129.3, 115.0, 114.3, 111.7, 111.1, 75.8, 72.6, 63.9, 62.7, 43.1, 35.7, 34.6, 26.8; HRMS (FAB): found 469.1670 [calcd for C₂₂H₂₅N₆O₄S⁺ (M + H)⁺ 469.1658]; Anal. Calcd for C₂₂H₂₄N₆O⁴S: C, 56.40; H, 5.16; N, 17.94. Found: C, 56.12; H, 5.00; N, 17.86.

(2R,3R,4S)-2-(6-Amino-2-((4-hydroxyphenethyl)amino)-8-(thiophen-2-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol (5u).

Yield = 66%; pale yellow solid; mp 165 °C; $[\alpha {]}_{\mathrm{D}}^{25}=-91.28$ (c 0.185, MeOH); UV (MeOH) λ_max 231, 324 nm; ¹H NMR (800 MHz, DMSO-d₆): δ 9.15 (s, 1H), 7.74 (dd, J = 5.0, 0.9 Hz, 1H), 7.48 (dd, J = 3.4, 0.7 Hz, 1H), 7.24 (dd, J = 5.1, 3.6 Hz, 1H), 7.02 (d, J = 7.9 Hz, 2H), 6.79 (br s, 2H), 6.68–6.69 (m, 2H), 6.39 (t, J = 5.7 Hz, 1H), 5.98 (d, J = 7.6 Hz, 1H), 5.62 (br s, 1H), 5.46 (br s, 1H), 5.26 (br s, 1H), 4.36 (s, 1H), 3.44 (d, J = 10.9 Hz, 1H), 3.41 (dd, J = 14.8, 5.4 Hz, 2H), 2.78 (dd, J = 11.2, 1.7 Hz, 1H), 2.73–2.75 (m, 2H); ¹³C NMR (200 MHz, DMSO-d₆): δ 158.6, 155.9, 155.4, 152.7, 141.1, 132.0, 130.1, 129.38, 129.35, 128.3, 127.9, 127.6, 115.08, 115.05, 113.8, 72.5, 63.9, 62.7, 43.1, 35.7, 34.6; HRMS (FAB): found 471.1268 [calcd for C₂₁H₂₃N₆O₃S₂⁺ (M + H)⁺ 471.1273]; Anal. Calcd for C₂₁H₂₂N₆O₃S₂: C, 53.60; H, 4.71; N, 17.86. Found: C, 53.41; H, 4.68; N, 17.94.

(2R,3R,4S)-2-(2-((4-Hydroxyphenethyl)amino)-6-(methylamino)-8-(thiophen-2-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol (5v).

Yield = 60%; pale yellow solid; mp 195 °C; $[\alpha {]}_{\mathrm{D}}^{25}=-0.93$ (c 3.38, MeOH); UV (MeOH) λ_max 219, 331 nm; ¹H NMR (800 MHz, DMSO-d₆): δ 9.15 (s, 1H), 7.73 (dd, J = 5.1, 0.9 Hz, 1H), 7.47 (d, J = 2.9 Hz, 1H), 7.33 (br s, 1H), 7.24 (dd, J = 5.1, 3.6 Hz, 1H), 7.02 (d, J = 8.2 Hz, 2H), 6.68 (d, J = 8.4 Hz, 2H), 6.54 (br s, 1H), 5.98 (d, J = 7.6 Hz, 1H), 5.62 (br s, 1H), 5.44 (br s, 1H), 5.25 (s, 1H), 4.36 (s, 1H), 3.41–3.45 (m, 3H), 2.89 (br s, 2H), 2.75–2.79 (m, 3H); ¹³C NMR (200 MHz, DMSO-d₆): δ 158.6, 155.4, 155.0, 151.9, 140.7, 132.1, 130.1, 130.1, 129.3, 128.2, 127.9, 127.4, 115.0, 115.0, 114.1, 75.6, 72.5, 63.9, 43.1, 35.7, 34.6, 26.8; HRMS (FAB): found 485.1432 [calcd for C₂₂H₂₅N₆O₃S₂⁺ (M + H)⁺ 485.1430]; Anal. Calcd for C₂₂H₂₄N₆O₃S₂: C, 54.53; H, 4.99; N, 17.34. Found: C, 54.31; H, 4.67; N, 17.36.

Molecular Docking Study.

The crystal structures of A_2AAR containing the A_2AAR agonist (NECA) or antagonists (LJ-4517, ZM-241385) were used for the molecular docking study. (PDB ID: 2YDV, 3EML, 8CU6). The preparation of ligands and proteins and homology modeling of A₃AR with the crystal structure of A_2AAR (PDB ID: 8CU6) were performed by Maestro 13.0 (Schrödinger, LLC, New York, NY, 2021). The grid generation and ligand docking were progressed in a Glide docking program in Maestro 13.0, and the predicted binding mode was calculated as XP-precision.

Pharmacological Assays.

Cell Culture and Membrane Preparation.

HEK293 cells expressing recombinant hA₁AR, hA_2AAR, or A₃AR were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 μmol/mL glutamine. Cells were collected by scraping into ice-cold PBS buffer. After homogenization and suspension, cells were centrifuged at 1000g or 10 min, and the pellet was discarded. The suspension was then recentrifuged at 20,000g for 60 min at 4 °C. The resultant pellets were resuspended in buffer containing 3 units/mL adenosine deaminase (from bovine spleen, Worthington Biochemical Corp., Lakewood, NJ) and incubated at 37 °C for 30 min. The aliquots of membrane preparation were kept at −80 °C until further use.

Binding Assays at hA₁AR and hA_2AAR.

For binding to hA₁AR, 50 μL of increasing concentrations of a test ligand and 50 μL of 25 (1.0 nM, PerkinElmer, Boston, MA) were incubated with membrane preparations (40 μg/tube) at 25 °C for 60 min in 50 mM Tris·HCl buffer (pH 7.4) containing 10 mM MgCl₂ in a total assay volume of 200 μL. Nonspecific binding was determined using 10 μM of 29. For hA_2AAR binding, membrane preparations (20 μg/tube) were incubated at 25 °C for 60 min with a final concentration of 26 (5 nM, American Radiolabeled Chemicals, Inc., St. Louis, MO) in a mixture containing 50 μL of increasing concentrations of a test ligand and 200 μL of 50 mM Tris·HCl, pH 7.4, containing 10 mM MgCl₂. 29 (10 μM) was used to define nonspecific binding. The reaction was terminated by filtration with GF/B filters. Filters were placed in scintillation vials containing 5 mL of Hydrofluor scintillation liquid and counted using a PerkinElmer Tricarb 2810TR liquid scintillation counter.

Binding Assays at hA_2BAR.

Adenosin A_2B receptor competition binding experiments were carried out in a multiscreen GF/C 96-well plate. In each well was incubated 25 μg of membranes from the Euroscreen HEK-A2B cell line and prepared in our laboratory (lot: A009/14–02–2020, protein concentration = 5254.8 μg/mL), 25 nM of 27 (140 Ci/mmol, 1 mCi/mL, PerkinElmer NET974001MC) and compounds studied and standard. Nonspecific binding was determined in the presence of 1000 μM of 29 (Sigma E2397). The reaction mixture (Vt: 250 μL/well) was incubated at 25 °C for 30 min, 200 μL was transferred to GF/C 96-well plate (Millipore, Madrid, Spain) pretreated with binding buffer (Tris-HCl 50 mM, EDTA 1 mM, MgCl₂ 5 mM, bacitracin 100 μg/μL, adenosine deaminase 2 U/mL, pH = 6.5), which was then filtered and washed 4 times with 250 μL wash buffer (Tris-HCl 50 mM, EDTA 1 mM, MgCl₂ 5 mM, pH = 6.5) before measuring in a microplate β scintillation counter (MicroBeta Trilux, PerkinElmer, Madrid, Spain).

Binding Assay at hA₃AR.

Each tube in the binding assay contained 100 μL of membrane suspension (10 μg protein), 50 μL of 28 (0.2 nM, PerkinElmer, Boston, MA), and 50 μL of increasing concentrations of the test ligands in Tris·HCl buffer (50 mM, pH 8.0) containing 10 mM MgCl₂. Nonspecific binding was determined using 10 μM of 29. The mixtures were incubated at 25 °C for 60 min. Binding reaction was terminated by filtration through Whatman GF/B filters using a MT-24 cell harvester (Brandel, Gaithersburg, MD, USA). Filters were washed 3 times with 9 mL of ice-cold buffer. Filters were counted using a PerkinElmer Cobra II γ-counter.

Cyclic AMP Accumulation Assay.

Human adenosine receptor functional experiments were carried out in the CHO-A_2A cell line and the CHO-A₃ cell line for hA_2AAR and hA₃AR, respectively. The day before the assay, the cells are seeded on the 96 well culture plate (Falcon 353072). The cells are washed with wash buffer [Dulbecco’s modified Eagle’s medium nutrient mixture F-12 ham (Sigma D8062), 25 mM Hepes; pH = 7.4]. Wash buffer is replaced by incubation buffer [Dulbecco’s modified Eagle’s medium nutrient mixture F-12 ham (Sigma D8062), 25 mM Hepes, 30 μM rolipram (Sigma R6520); pH = 7.4]. Test compounds are added and incubated at 37 °C for 15 min. Afterward, 1 μM of 29 (Sigma E2387) is added and incubated at 37 °C for 15 min. Also, for hA₃AR, FSK (Sigma F3917) is added and incubated at 37 °C for 5 min. After incubation, the amount of cAMP is determined using the cAMP Biotrak Enzymeimmunoassay (EIA) System Kit (GE Healthcare RPN225).

Statistical Analysis.

Binding and functional parameters were calculated using Prism 5.0 software (GraphPAD, San Diego, CA, USA). IC₅₀ values obtained from competition curves were converted to K_i values using the Cheng–Prusoff equation.³² Data were expressed as mean ± standard error of the mean.

Pharmacokinetic Assay.

For pharmacokinetic studies, CrljOr-i:CD1 male mice were obtained as weanlings of 6 weeks of age upon arrival (provided by DBL Co., Ltd.). This study was reviewed and approved by the Institutional Animal Care and Use Committees (IACUC) of Chaon Co., Ltd. (IACUC number: CE21077). Mice weighed about 31.4 g; animals were 7 weeks old at the start of dosing. For the single pharmacokinetics (i.v.: 2 mg/5 mL/kg; oral: 10 mg/10 mL/kg) of compound 5d in mice, the dosing solution was 5% DMSO/40% PEG 400/55% DW (v/v/v). This study was conducted using a cross-over (n = 3) design. Blood samples (0.1 mL) were collected from the retro-orbital plexus at 5, 15, and 30 min and 1, 2, 4, 8, 12, and 24 h postdose for the i.v. group and 15, 30 min, and 1, 2, 4, 8, 12, and 24 h postdose for the oral group. The collected blood samples were placed in heparinized tubes (5 IU/mL). Plasma was obtained by centrifugation at 5000 rpm for 5 min at 4 °C. Aliquots of plasma (30–40 μL) were transferred to polypropylene tubes and stored at −15 °C until analysis. LC–MS/MS was used to determine the concentration of the compound 5d at plasma. To measure the concentration of compound 5d at plasma, selectivity, linearity, and stability in plasma (at 10 °C for 4 h) were confirmed. In vivo PK data were analyzed using Phoenix WinNonlin software (Phoenix WinNonlin 8.3). A noncompartment model analysis was performed to obtain the following parameters: AUC_0–t, C₀ or C_max, T_max, clearance (CL), volume of distribution (V_ss), half-life (t_1/2), and bioavailability (F).

In Vivo Tumor Xenograft Model.

All animal experiments were conducted according to the guidelines approved by the Seoul National University Institutional Animal Care and Use Committee (IACUC; permission number: SNU-190408–5). Female mice (BALB/c, aged 4–5 weeks, weighing 18 g) were purchased from the Central Laboratory Animal, Inc. (Seoul, Korea), and they were housed under pathogen-free conditions with a 12 h light–dark schedule. 4T1-Luc cells were then injected subcutaneously into the flanks of mice (1 × 10⁶ cells in 200 μL of 50:50 PBS/Matrigel), and tumors were allowed to grow for 7 days until their volume reached approximately 50 mm³. The mice were randomized into vehicle control and treatment groups (n = 5). Vehicle control (DMSO/cremophor/normal saline = 5:5:90), 5d (10 mg/kg body weight), antimouse PD-L1 (10 mg/kg body weight), or a combination of 5d (10 mg/kg body weight) and antimouse PD-L1 (10 mg/kg body weight) was administered intraperitoneally (i.p.) 3 times per week for 21 days; mice were then euthanized 1 week later. Tumors were excised, weighed, and frozen for further biochemical analysis. Tumor volume was measured using an electronic caliper according to the following formula: tumor volume (mm³) = 3.14 × length × width × height/6. Toxicity was evaluated based on body weight loss. Bioluminescence images of mice were acquired on the final day of the experiment. Mice were anesthetized, positioned in the IVIS (PerkinElmer, Waltham, MA, USA), and imaged 15 min after injection of d-luciferin (150 mg/kg, Gold Biotechnology) resuspended in PBS.

ABBREVIATIONS

AUC

area under the curve

AR

adenosine receptor

cAMP

cyclic adenosine-5′-monophosphate

BSA

bis(trimethylsilyl)-acetamide

CGS21680

2-[p-(2-carboxyethyl)-phenylethylamino]-5′-N-ethylcar-boxamidoadenosine

CHO

Chinese hamster ovary

CKD

chronic kidney disease

Cl-IB-MECA

2-chloro-N⁶-(3-iodobenzyl)-5′-N-methylcarbamoyladenosine

CYP

cytochrome P450

DIBAL-H

diisobutylaluminum hydride

HEK

human embryonic kidney

hERG

human ether-a-go-go related gene

I-AB-MECA

N⁶-(4-amino-3-iodobenzyl)-5′-N-methylcarboxamidoadenosine

IVIS

in vivo imaging system

LDA

lithium diisopropylamide

LiTMP

lithium tetramethylpiperidide

mAb

monoclonal antibody

NECA

5′-N-ethylcarboxamidoadenosine

PBS

phosphate buffered saline

PD-L1

programmed death-ligand 1

PKC

protein kinase C

PLC

phospholipase C

PPTS

pyridinium p-toluenesulfonate

pTSA

para-toluene sulfonic acid

R-PIA

(−)-N⁶-2-phenylisopropyl adenosine

SAR

structure—activity relationship

TBAF

tetra-n-butylammonium fluoride

TBS

t-butyldimethylsilyl

thio-Cl-IB-MECA

2-chloro-N⁶-(3-iodobenzyl)-5′-N-methylcarbamoyl–4′-thioadenosine

TMSOTf

trimethylsilyl trifluoromethanesulfonate

Tris-HCl

tris(hydroxymethyl)aminomethane hydrochloride