Azolo substitution into the purine scaffold in nucleoside cyclic 3',5'-phosphorothioates

Abstract

Azolation in the 8-position in the purine scaffold of cAMP (adenosine 3′,5′-cyclic monophosphate) and cAMPS (adenosine 3′,5′-cyclic monophosphorothioate) provided derivatives with an azole ring directly attached to the purine via an annular azole nitrogen. Electrophilic bromination in the 8-position was followed by nucleophilic substitution with metalated azoles to afford 8-imidazo and 8-triazolo derivatives. The substrates were appropriately protected (S_p)-3′,5′-cyclic N-benzylphosphoramidate. A subsequent carbon disulfide promoted thiation reaction afforded corresponding (R_p)-8-azolo-3′.5′-cAMPS products. The reactions were stereoselective. The products as tri-n-butylammonium salts were soluble in organic solvents and were purified by chromatography. The ammonium salts were converted to sodium salts.

Graphical abstract

Introduction

The heterocyclic purine framework is widely incorporated into essential biomolecular systems and constitutes an important part of medicinal chemistry. Adenine and guanine are common purine nucleobases in such frameworks as exemplified by adenosine in Fig. 1. In this report the emphasis is on development of methodology for introduction or exchange of substituents in the purine 8-position [1]. The 8-position in the purine scaffold is active in both electrophilic and nucleophilic substitution reactions [2]. 8-Bromides or 8-chlorides are useful substrates for nucleophilic substitutions affording 8-aza, 8-oxa, or 8-thia derivatives [3,4,5]. More recently, 8-nitro analogues have become available by nucleophilic displacements [6]. 8-Carbylation is achieved by Pd-promoted organometallic cross-coupling reactions with metalated carbocyclic and heterocyclic arenes [7]. Even heteroatom substituents can be introduced by cross-coupling procedures [8]. In this manner a number of 8-substituted purine skeletons have become available.

Fig. 1

Cyclic nucleotide targets

In (R_P)-adenosine-3′,5′-cyclic phosphorothioic acid (cAMPS), one of the oxygen atoms pendant from the phosphorus atom in (R_P)-adenosine-3′,5′-cyclic phosphoric acid (cAMP) has been replaced by a sulfur atom in a stereoselective manner. The resulting (R_p)- and (S_p)-isomers of cAMPS are stereochemically stable and useful for various bioscreening programs associated with cyclic adenosine and cyclic guanosine monophosphates (cGMP). Both cAMP and cGMP are important secondary messenger in regulating a wide range of cell functions in response to specific hormones [9, 10]. We have reported a method for stereocontrolled preparation of 8-substituted (R_P)-adenosine-3′,5′-cyclic phosphorothioic acids that possess protein kinase antagonistic activity and has a stimulating effect on the immune system [2]. In this report, we describe sp²-hybridized amino-nitrogen hinged to the 8-position in the purine skeleton by substitution reactions from the corresponding bromides or chlorides. Iodides will react in a similar manner but are less readily available intermediates. The products were cAMPS derivatives with sp²-hybridized azole nitrogen in the form of imidazole and triazoles attached to the 8-position in the nucleotide. The nucleophiles were metalated azoles. The triazolo heterocycles are π-electron deficient, and both the 1,2,3-triazoles and 1,2,4-triazoles possess low basicity. In contrast imidazole behaves as a base and nucleophile. Acyclic 8-azido derivatives were included as a less polarized species.

Results and discussion

The generally low solubility of nucleosides and nucleotides in organic solvents may be partly overcome by initial conversions to amidates [2]. A solution of the (S_p)-8-bromo amidate 1 and the sodium salts of the azoles in DMF afforded the 8-azolo products. We have previously described methodology for stereoselective preparation of amidates [2]. Substitution of intermediate amidates with imidazole as sodium salt in DMF proceeded readily at elevated temperature to afford the imidazo derivative 2 (Scheme 1). 1,2,4-Triazole was reacted similarly, but the heating time was increased to 20 h because of lower reaction rate. Exclusive formation of the N-1 product 3 was observed. The proton NMR spectra were used for assignment of structures to the regioisomers. Substitution at the annular 4-nitrogen atom will yield a symmetrical triazole derivative with the same chemical shifts for the two annular triazolo protons. Different chemical shifts for the two hydrogen atoms in the azole ring in the product were observed. The unsymmetrical structure 3 was assigned to the product.

Two regioisomers were formed with 1,2,3-triazole as a reactant, viz. the symmetrical structure 4 and its isomer 5 in the ratio 3:2 (Scheme 2). The isomers were separated by flash chromatography. The symmetrical structure has the same chemical shift for both triazolo protons in the ¹H NMR spectra whereas the second isomer showed two different chemical shifts for the annular triazolo protons. 1,2,3-Triazoles carrying a methyl (6) or a chloro substituent (7) in the 4-position (Scheme 2) were reacted similarly with sodium hydride as base in DMF. The 4-methyltriazole afforded the regioisomers 8 and 10 in the ratio 6:1. The 4-chlorotriazole afforded a 3:1 ratio of the regioisomers 9 and 11 that were separated by flash chromatography. The ¹H shift in the methyl triazole isomers differed significantly, and isomer structures could be assigned. The major isomer was fluorescent in UV light. The chemical shifts for the triazolo proton in the two chloro isomers were almost the same. The major isomer was fluorescent and was tentatively assigned structure 8. The 5-methyl and 5-chloro triazole reactants 6 and 7 were available by literature procedures from 1-SEM-protected 1,2,3-triazol [11]. Finally, the acyclic 8-azido derivative was prepared by heating the bromide 1 with sodium azide in DMF to afford the 8-azido derivative 12. The latter is also a potential intermediate for 8-amino derived products.

Scheme 3 illustrates adaption of the Stec thiylation reaction for generation of thiophosphoric acids [12]. Initial proton abstraction from the benzylic amino group in the phosphoramidates 2–5, 8, 9 by LDA in THF at − 70 to − 40 °C afforded a lithium ylide that reacted with carbon disulfide. A sulfur atom in the intermediate becomes a nucleophile and a cyclization reaction occurs. Cleavage of the P–N bond occurs with retention of the true configuration at the phosphorus atom. The hydrophobic nature of the bulky TBDMS-protecting group attached to the 2′-hydroxy group leads to precipitation of the thioic acid from the aqueous mixture. The product was desilylated by ammonium fluoride in methanol at 45 °C to afford the target compounds (Scheme 3). The 8-azido derivative 12 was thiated in the same manner.

The deprotected products were isolated as tributylammonium salts after addition of tributylamine to the acid. The tributylammonium salts 15, 16, 20–22, 23, 24 of the products were soluble in polar organic solvents and could be purified by recrystallization or by flash chromatography on silica gel using CH₂Cl₂:MeOH:NBu₃. The azido amidate 12 reacted in the same manner to furnish the phosphorothioic acid 24. The tributylammonium salts were converted into sodium salts 25–31 by dissolution of the ammonium salts in methanolic sodium hydroxide. Precipitation of the salts was by addition of diethyl ether (Scheme 4).

The products were subjected to T cell proliferation assays by established methodologies [2, 9, 13]. Comparison with previously reported systems indicate low specific effect by the nature of the annular heteroatom(s) in the five-membered hetarene in the purine 8-position.

Conclusion

Methods for preparation of novel 8-imidazolo and 8-triazolo derivatives of cAMP and cAMPS have been developed. Azolation in the 8-position in the purine scaffold of cAMP and cAMPS provides derivatives with annular sp²-hybridized azole amino-nitrogen attached directly to the purine ring. In the process, an appropriately silyl protected (S_p)-8-bromoadenosine 3′,5′-cyclic N-benzylphosphoramidate is aminated with a metalated azole as nucleophile, followed by stereoselective thiation at the phosphorus atom to deliver adenosine 3′,5′-cyclic phosphorothioates with retention of the configuration at the phosphorus atom. The (R_p).8-azolo-cAMPS products are analogues of cAMP that regulates a broad range of essential cellular functions.

Experimental

¹H NMR spectra were recorded in CDCl₃ or MeOH-d₄ at 200 and 300 MHz. The ¹³C NMR spectra were recorded at 75 and 100 MHz. Chemical shifts are reported in ppm using CHCl₃ (7.24 ppm) and CDCl₃ (77 ppm) as references, and in MeOH-d₄, 3.30 ppm in ¹H NMR and 49.0 ppm in ¹³C NMR. The ³¹P spectra were recorded in CDCl₃, MeOH-d₄ at 81 MHz or 121 MHz with a Bruker DPX 200 or 300 instrument with 85% H₃PO₄ as an external reference. Mass spectra were recorded at 70 eV with a Fisons VG Prospectrometer. The spectra are presented as m/z (% relative intensity). Electrospray spectra were obtained with a Micromass QTOF 2 W spectrometer with electrospray ionisation quadrupole time of flight. Merck silica gel 60 (230–400) was used for flash chromatography.

(S _p)-2′O-(tert-Butyldimethylsilyl)-8-(imidazol-1-yl)adenosine-3′,5′-cyclic N-benzylphosphoramidate (2, C₂₆H₃₅N₈O₅PSi)

A solution of 143 mg imidazole (2.1 mmol) in 5 cm³ dry DMF was added slowly via a syringe to an oven-dried flask containing 84 mg powdered NaH (60% dispersion in mineral oil, 2.1 mmol) and 8 cm³ anhydrous DMF under argon. The mixture was stirred at room temperature for 30 min before a solution of 1.22 g (S_p)-8-bromoadenosine-2-O′-(tert-butyldimethylsilyl)-3′,5′-cyclic N-benzylphosphoramidate (1, 2.0 mmol) in 8 cm³ DMF was added. The mixture was stirred at room temperature for 30 min and finally at 80 °C for 4 h. The solvent was removed at reduced pressure, and the residual material subjected to flash chromatography on silica gel using 10% methanol in CH₂Cl₂. Yield: 586 mg (49%) of a white solid material; ¹H NMR (CDCl₃, 300 MHz): δ = − 0.08 (3H, s, Si-Me), − 0.03 (3H, s, Si-Me), 0.71 (9H, s, Si-tBu), 3.84–3.88 (1H, m, NH), 4.11–4.18 (3H, m), 4.42–4.54 (3H, m), 5.19 (1H, d, J = 5.1 Hz), 5.43 (1H, s), 5.57–5.62 (1H, m), 6.05 (2H, br s, NH₂), 7.26 (1H, s, H-imidazole), 7.27–7.31 (5H, m), 7.36 (1H, s, H-imidazole), 7.96 (1H, s, H-imidazole), 8.27 (1H, s, H-2) ppm; ¹³C NMR (CDCl₃, 75 MHz): δ = − 5.2, − 4.7, 18.0, 25.5, 45.3, 68.2, 71.3, 73.4, 76.3 (partly overlapped with CDCl₃ peak), 93.0, 117.3, 119.65, 127.1, 127.6, 128.6, 131.1, 137.7, 138.6, 140.2, 149.5, 154.1, 155.45 ppm; ³¹P NMR (CDCl₃, 121 MHz): δ = 7.46 ppm; HRMS (electrospray, TOF, ES⁺): m/z = 599.2313, calc. for C₂₆H₃₅N₈O₅PSi + H⁺ 599.2315.

(S _p)-2′O-(tert-Butyldimethylsilyl)-8-(1,2,4-triazol-1-yl)adenosine-3′,5′-cyclic N-benzylphosphoramidate (3, C₂₅H₃₄N₉O₅PSi)

A solution of 145 mg 1,2,4-triazole (2.1 mmol) in 5 cm³ dry DMF was added slowly via a syringe to an oven-dried flask containing 84 mg powdered NaH (60% dispersion in mineral oil, 2.1 mmol) and 8 cm³ anhydrous DMF under argon. The mixture was stirred at room temperature for 30 min before a solution of 1.22 g (S_p)-8-bromoadenosine-2′O-(tert-butyldimethylsilyl)-3′,5′-cyclic N-benzylphosphoramidate (1, 2.0 mmol) in 8 cm³ DMF was added. The mixture was stirred at room temperature for 30 min and finally at 85 °C overnight (20 h). The solvent was distilled off at reduced pressure, and the residual material subjected to flash chromatography on silica gel using 8% methanol in CH₂Cl₂ to afford 659 mg (55%) of the triazole product 3 as a white solid. ¹H NMR (CDCl₃, 300 MHz): δ = 0.02 (6H, s, 2 × Si-Me), 0.81 (9H, s, Si-tBu), 3.78–3.82 (1H, m, NH), 4.13–4.18 (3H, m), 4.40–4.59 (3H, m), 5.07 (1H, d, J = 5.1 Hz), 5.76–5.79 (1H, m), 6.06 (2H, br s, NH₂), 6.56 (1H, s), 7.24–7.30 (5H, m), 8.17 (1H, s, H-triazole), 8.27 (1H, s, H-2), 8.88 (1H, s, H-triazole) ppm; ¹³C NMR (CDCl₃, 75 MHz): δ = -5.2, -4.6, 18.1, 25.5, 45.4, 68.3, 71.4, 73.4, 76.3, 93.45, 117.3, 127.1, 127.55, 128.6, 138.65, 139.2, 145.4, 150.1, 153.6, 154.1, 155.4 ppm; ³¹P NMR (CDCl₃, 121 MHz): δ = 7.53 ppm; HRMS (electrospray, TOF, ES⁺): m/z = 600.2263, calc. for C₂₅H₃₄N₉O₅PSi + H⁺ 600.2268.

(S _p)-2′O-(tert-Butyldimethylsilyl)-8-(1,2,3-triazol-2-yl)adenosine-3′,5′-cyclic N-benzylphosphoramidate (4, C₂₅H₃₄N₉O₅PSi) and (S _p)-2′O-(tert-butyldimethylsilyl)-8-(1,2,3-triazol-1-yl)adenosine-3′,5′-cyclic N-benzylphosphoramidate (5, C₂₅H₃₄N₉O₅PSi)

A solution of 211 mg 1,2,3-triazole (3.06 mmol) in 5 cm³ dry DMF was added slowly via a syringe to an oven-dried flask containing 122 mg powdered NaH (60% dispersion in mineral oil, 3.06 mmol) and 10 cm³ anhydrous DMF under argon. The mixture was stirred at room temperature for 30 min before a solution of 1.70 g (S_p)-8-bromoadenosine-2'O-(tert-butyldimethylsilyl)-3′,5′-cyclic N-benzylphosphoramidate (1, 2.78 mmol) in 10 cm³ DMF was added. The mixture was stirred at room temperature for 30 min and finally at 85 °C overnight (20 h). The solvent was removed at reduced pressure, and the residual material subjected to flash chromatography on silica gel using 8% MeOH in CH₂Cl₂ to furnish the white product as a mixture (4 + 5), yield 880 mg (53%) in ratio 3:2 by ¹H NMR spectroscopy analysis. The isomers were subjected twice to separation by flash chromatography using 8% methanol in CH₂Cl₂. Yields: 365 mg of isomer 4 and 210 mg of isomer 5.

4: ¹H NMR (CDCl₃, 300 MHz): δ = 0.00 (3H, s, Si-Me), 0.02 (3H, s, Si-Me), 0.80 (9H, s, Si-tBu), 3.52–3.56 (1H, m, NH), 4.14–4.21 (3H, m), 4.39–4.63 (3H, m), 5.04 (1H, d, J = 5.2 Hz), 5.80–5.85 (1H, m), 5.97 (2H, br s, NH₂), 6.51 (1H, s), 7.24–7.32 (5H, m), 7.99 (2H, s, H-triazole), 8.35 (1H, s, H-2) ppm; ¹³C NMR (CDCl₃, 75 MHz): δ = − 5.25, − 4.6, 18.1, 25.5, 45.4, 68.4, 71.6, 73.6, 76.3, 93.8, 117.3, 127.1, 127.6, 128.6, 137.7, 138.8, 141.0, 149.9, 154.3, 155.6 ppm; ³¹P NMR (CDCl₃, 121 MHz): δ = 7.47 ppm; HRMS (electrospray, TOF, ES⁺): m/z = 600.2263, calc. for C₂₅H₃₄N₉O₅PSi + H⁺ 600.2268.

5: ¹H NMR (CDCl₃, 200 MHz): δ = 0.025 (3H, s, Si-CH₃), 0.04 (3H, s, Si-Me), 0.81 (9H, s, Si-tBu), 3.86–3.92 (1H, m, NH), 4.11–4.22 (3H, m), 4.35–4.57 (3H, m), 5.12 (1H, d, J = 5.1 Hz), 5.71–5.79 (1H, m), 6.11 (2H, br s, NH₂), 6.48 (1H, s), 7.24–7.30 (5H, m), 7.86 (1H, d, J = 1 Hz, H-triazole), 8.26 (1H, s, H-2), 8.30 (1H, d, J = 1 Hz, H-triazole) ppm; ¹³C NMR (CDCl₃, 75 MHz): δ = -5.15, -4.7, 18.1, 25.5, 45.4, 68.3, 71.4, 73.3, 76.3, 93.8, 117.5, 125.5, 127.1, 127.5, 128.6, 133.9, 138.7, 139.3, 150.1, 154.2, 155.65 ppm; ³¹P NMR (CDCl₃, 121 MHz): δ = 7.58 ppm; HRMS (electrospray, TOF, ES⁺): m/z = 600.2285, calc. for C₂₅H₃₄N₉O₅PSi + H⁺ 600.2268.

(S _P)-2′O-(tert-Butyldimethylsilyl)-8-(4-methyl-1,2,3-triazol-2-yl)adenosine-3′,5′-cyclic N-benzylphosphoramidate (8, C₂₆H₃₆N₉O₅PSi)

A solution of 146 mg 4-methyl-1,2,3-triazole (1.75 mmol) in 5 cm³ dry DMF was added slowly via a syringe to an oven-dried flask containing 70 mg powdered NaH (60% dispersion in mineral oil, 1.75 mmol) and 5 cm³ anhydrous DMF under argon. The mixture was stirred for 30 min at room temperature before a solution of 915 mg (S_P)-8-bromoadenosine-2'O-(tert-butyldimethylsilyl)-3′,5′-cyclic N-benzylphosphoramidate (1, 1.5 mmol) in 7 cm³ DMF was added. The mixture was stirred at room temperature for 30 min and then at 90 °C overnight (20 h). The solvent was removed at reduced pressure, and the residual material subjected to flash chromatography on silica gel using 8% methanol in CH₂Cl₂ to afford 380 mg (41%) of (8 + 10) as a mixture in the ratio 6:1 (¹H NMR) as a white solid material. The products were separated and purified by flash chromatography twice. Yield of 8: 265 mg; ¹H NMR (CDCl₃, 200 MHz): δ = -0.02 (3H, s, Si-Me), 0.03 (3H, s, Si-Me), 0.82 (9H, s, Si-tBu), 2.46 (3H, s, Me), 3.36–3.46 (1H, m, NH), 4.13–4.21 (3H, m), 4.40–4.59 (3H, m), 5.05 (1H, d, J = 5.3 Hz), 5.75–5.85 (3H, m), 6.69 (1H, s), 7.24–7.32 (5H, m), 7.74 (1H, s, H-triazole), 8.35 (1H, s, H-2) ppm; ¹³C NMR (CDCl₃, 75 MHz): δ = − 5.25, -4.6, 10.8, 18.1, 25.5, 45.4, 68.5, 71.4, 73.6, 76.6 (partly overlapped with CDCl₃ peak), 93.8, 117.3, 127.1, 127.6, 128.6, 137.6, 138.7, 141.3, 148.1, 150.0, 153.9, 155.3 ppm; ³¹P NMR (CDCl₃, 121 MHz): δ = 7.88 ppm; HRMS (electrospray, TOF, ES⁺): m/z = 614.2418, calc. for C₂₆H₃₆N₉O₅PSi + H⁺ 614.2424.

(S _P)-2′O-(tert-Butyldimethylsilyl)-8-(4-chloro-1,2,3-triazol-2-yl)adenosine-3′,5′-cyclic N-benzylphosphoramidate (11, C₂₅H₃₃ClN₉O₅PSi)

A solution of 124 mg 4-chloro-1,2,3-triazole (1.2 mmol) in 5 cm³ dry DMF was added slowly via a syringe to an oven-dried flask containing 48 mg powdered NaH (60% dispersion in mineral oil, 1.2 mmol) and 3 cm³ anhydrous DMF under argon. The mixture was stirred at room temperature for 30 min before a solution of 611 mg (S_p)-8-bromoadenosine-2'O-(tert-butyldimethylsilyl)-3′,5′-cyclic N-benzylphosphoramidate (1, 1.0 mmol) in 5 cm³ DMF was added. The mixture was stirred at room temperature for 30 min and finally at 85 °C overnight (20 h). The solvent was removed at reduced pressure, and the residual material subjected to flash chromatography on silica gel using 5% MeOH in CH₂Cl₂ to afford 350 mg (55%) of (9 + 11) as a white solid mixture in the ratio 3:1 (¹H NMR). Pure 9 was isolated after flash chromatography twice. Yield: 215 mg; ¹H NMR (CDCl₃, 300 MHz): δ = 0.03 (6H, s, 2 × Si-Me), 0.82 (9H, s, Si-tBu), 3.65–3.68 (1H, m, NH), 4.14–4.19 (3H, m), 4.38–4.57 (3H, m), 5.06 (1H, d, J = 5.1 Hz), 5.77–5.83 (1H, m), 6.05 (2H, br s, NH₂), 6.45 (1H, s), 7.25–7.32 (5H, m), 7.87 (1H, s, H-triazole), 8.31 (1H, s, H-2) ppm; ¹³C NMR (CDCl₃, 75 MHz): δ = -5.2, -4.6, 18.1, 25.5, 45.4, 68.3, 71.5, 73.5, 76.2, 93.7, 117.2, 127.1, 127.6, 128.6, 136.3, 138.7, 140.3, 141.1, 149.9, 154.4, 155.6 ppm; ³¹P NMR (CDCl₃, 121 MHz): δ = 7.97 ppm; HRMS (electrospray, TOF, ES⁺): m/z = 634.1887, calc. for C₂₅H₃₃ClN₉O₅PSi + H⁺ 634.1878.

(S _p)-8-Azidoadenosine-2′O-(tert-butyldimethylsilyl)-3′,5′-cyclic N-benzylphosphoramidate (12, C₂₃H₃₂N₉O₅PSi)

A solution of 1.22 g (S_p)-8-bromoadenosine-2′O-(tert-butyldimethylsilyl)-3′,5′-cyclic N-benzylphosphoramidate (1, 2.0 mmol) in 20 cm³ DMF was stirred together with 390 mg NaN₃ (6.0 mmol) at 60 °C for 20 h. The solvent was distilled off at reduced pressure, and the residual material subjected to flash chromatography on silica gel using 10% methanol. ¹H NMR (CDCl₃, 200 MHz): δ = 0.03 (3H, s, Si-Me), 0.04 (3H, s, Si-Me), 0.85 (9H, s, Si-tBu), 3.45–3.55 (1H, m, NH), 4.09–4.21 (3H, m), 4.40–4.56 (3H, m), 4.85 (1H, d, J = 5.1 Hz), 5.44–5.49 (1H, m), 5.50 (2H, br s, NH₂), 5.79 (1H, s), 7.27–7.33 (5H, m), 8.20 (1H, s, H-2) ppm; ¹³C NMR (CDCl₃, 75 MHz): δ = -5.0, − 4.7, 18.1, 25.6, 45.4, 68.4, 71.1, 73.1, 76.6 (partly overlapped with CDCl₃ peak), 91.8, 117.8, 127.1, 127.6, 128.6, 138.7, 144.8, 150.0, 152.6, 153.5 ppm; ³¹P NMR (CDCl₃, 121 MHz): δ = 7.43 ppm; HRMS (electrospray, TOF, ES⁺): m/z = 574.2101, calc. for C₂₃H₃₂N₉O₅PSi + H⁺ 574.2111.

(R _p)-8-(Imidazol-1-yl)adenosine-3′,5′-cyclic thiophosphoric acid tri-n-butylammonium salt (15, C₁₃H₁₃N₇O₅PS)

A 1.8 M solution of LDA in THF/heptane/ethylbenzene (0.37 cm³, 0.66 mmol) was added to a solution of 358 mg (S_P)-2′O-(tert-butyldimethylsilyl)-8-(imidazol-1-yl)adenosine-3′,5′-cyclic N-benzylphosphoramidate (2, 0.6 mmol) in 10 cm³ dry THF at − 78 °C. The mixture was stirred under argon at this temperature for 10 min before 0.10 cm³ CS₂ (1.8 mmol) was added. The cooling bath was removed after 10 min. The reaction mixture was stirred at room temperature for 3 h before the solvent was partially removed at reduced pressure. Addition of hexane to the residual solution precipitated (R_P)-2′O-(tert-butyldimethylsilyl)-8-(imidazol-1-yl)adenosine-3′,5′-cyclic thiophosphoric acid lithium salt (13) as a solid. The product was dried and dissolved in 10 cm³ MeOH and 310 mg NH₄F (8.4 mmol) added to the solution. The mixture was stirred at 45 °C for 4 h, 166 mg nBu₃N (0.9 mmol) added and the solvent was removed at reduced pressure. The residue was subjected to flash chromatography on silica gel using CH₂Cl₂:CH₃OH:nBu₃N (90:10:1). A second flash chromatography operation using CH₂Cl₂:CH₃OH:nBu₃N (95:5:1) gave the ammonium salt 15, that contained some nBu₃N. The crude product was purified by dissolution in CH₂Cl₂ and reprecipitation by addition of hexane. Yield: 160 mg (45% from 2); ¹H NMR (CDCl₃, 300 MHz): δ = 0.93 (9H, t, J = 7.3 Hz, 3 × Me), 1.34–1.42 (6H, m, 3 × CH₂), 1.65–1.72 (6H, m, 3 × CH₂), 2.99–3.04 (6H, m, 3 × CH₂), 4.25–4.39 (3H, m), 5.19 (1H, d, J = 5.0 Hz), 5.35–5.47 (1H, m), 5.61 (1H, s), 6.04 (2H, br s, NH₂), 7.19 (1H, s, H-imidazole), 7.44 (1H, s, H-imidazole), 8.10 (1H, s, H-imidazole), 8.19 (1H, s, H-2) ppm; ¹³C NMR (CDCl₃, 75 MHz): δ = 13.6, 20.1, 25.2, 51.9, 67.0, 71.2, 72.0, 76.2, 91.3, 117.4, 119.8, 130.7, 138.0, 140.8, 149.7, 153.25, 155.2 ppm; ³¹P NMR (CDCl₃, 121 MHz): δ = 56.39 ppm; HRMS (electrospray, TOF, ES⁻): m/z = 410.0422, calc. for C₁₃H₁₃N₇O₅PS 410.0436.

(R _p)-8-(1,2,4-Triazol-1-yl)adenosine-3′,5′-cyclic thiophosphoric acid tri-n-butylammonium salt (16, C₁₂H₁₂N₈O₅PS⁻)

A 1.8 M solution of LDA in THF/heptane/ethylbenzene (0.57 cm³, 1.02 mmol) was added to a solution of 560 mg (S_P)-2′O-(tert-butyldimethylsilyl)-8-(1,2,4-triazol-1-yl)adenosine-3',5'-cyclic N-benzylphosphoramidate (3, 0.93 mmol) in 15 cm³ dry THF at − 40 °C. The mixture was stirred under argon at this temperature for 10 min before 0.17 cm³ CS₂ (2.79 mmol) was added. The cooling bath was removed after 10 min and the reaction mixture stirred at room temperature for 3 h before the solvent was partially removed at reduced pressure. Addition of hexane to the residual solution precipitated the solid (R_P)-2′O-(tert-butyldimethylsilyl)-8-(1,2,4-triazol-1-yl)adenosine-3',5'-cyclic thiophosphoric acid lithium salt (14). The dried salt was dissolved in 15 cm³ methanol, 482 mg NH₄F (13.0 mmol) added, and the mixture stirred at 45 °C for 4 h. nBu₃N (258 mg, 1.4 mmol) was added to the solution and the solvent was removed at reduced pressure. The residue was subjected to flash chromatography on silica gel using CH₂Cl₂:CH₃OH:nBu₃N (90:10:1). Repeated flash chromatography using CH₂Cl₂:CH₃OH:n-Bu₃N (93:7:1) gave the ammonium salt 16, that contained some nBu₃N. The product was purified by dissolution in CH₂Cl₂ and reprecipitation by hexane addition. Yield: 255 mg (46% from 3); ¹H NMR (CDCl₃, 300 MHz): δ = 0.94 (9H, t, J = 7.3 Hz, 3 × Me), 1.34–1.42 (6H, m, 3 × CH₂), 1.69–1.74 (6H, m, 3 × CH₂), 2.99–3.04 (6H, m, 3 × CH₂), 4.29–4.43 (3H, m), 5.06 (1H, d, J = 5.2 Hz), 5.52–5.57 (1H, m), 5.87 (2H, br s, NH₂), 6.43 (1H, s), 8.21 (1H, s, H-triazole), 8.26 (1H, s, H-2), 8.89 (1H, s, H-triazole) ppm; ¹³C NMR (CDCl₃, 75 MHz): δ = 13.6, 20.1, 25.2, 51.9, 67.0, 71.9, 72.0, 76.2, 91.7, 117.3, 139.6, 145.5, 150.2, 153.5, 153.8, 155.2 ppm; ³¹P NMR (CDCl₃, 121 MHz): δ = 56.69 ppm; HRMS (electrospray, TOF, ES⁻): m/z = 411.0391, calc. for C₁₂H₁₂N₈O₅PS⁻ 411.0389.

(R _p)-8-(1,2,3-Triazol-2-yl)adenosine-3′,5′-cyclic thiophosphoric acid tri-n-butylammonium salt (20, C₁₂H₁₂N₈O₅PS⁻)

A 1.8 M solution of LDA in THF/heptane/ethylbenzene (0.3 cm³, 0.55 mmol) was added to a solution of 300 mg (S_P)-2’O-(tert-butyldimethylsilyl)-8-(1,2,3-triazol-2-yl)adenosine-3',5'-cyclic N-benzylphosphoramidate (4, 0.5 mmol) in 10 cm³ dry THF at − 50 °C. The mixture was stirred under argon at this temperature for 10 min before 0.09 cm³ CS₂ (1.5 mmol) was added. The cooling bath was removed after 10 min and the reaction mixture stirred at room temperature for 3 h before the solvent was partially removed at reduced pressure. Addition of hexane to the residual solution precipitated a solid that was filtered off. The product was (R_P)-2′O-(tert-butyldimethylsilyl)-8-(1,2,3-triazol-2-yl)adenosine-3′,5′-cyclic thiophosphoric acid lithium salt (17). The solid product was dried, dissolved in 5 cm³ methanol and 259 mg NH₄F (7 mmol) was added. The mixture was stirred at 45 °C for 4 h before 140 mg nBu₃N (0.75 mmol) was added. The solvent was removed at reduced pressure and the residual material subjected to flash chromatography on silica gel using CH₂Cl₂:CH₃OH:nBu₃N (90:10:1). The product was the ammonium salt 20 that contained some nBu₃N. The product was purified by dissolution in CH₂Cl₂ and reprecipitation by addition of hexane. Yield: 125 mg (42% from 4); ¹H NMR (CDCl₃, 300 MHz): δ = 0.94 (9H, t, J = 7.3 Hz, 3 × Me), 1.35–1.42 (6H, m, 3 × CH₂), 1.69–1.73 (6H, m, 3 × CH₂), 2.99–3.04 (6H, m, 3 × CH₂), 4.30–4.45 (3H, m), 5.06 (1H, d, J = 5.2 Hz), 5.59–5.65 (1H, m), 5.99 (2H, br s, NH₂), 6.47 (1H, s), 8.00 (2H, s, H-triazole), 8.27 (1H, s, H-2) ppm; ¹³C NMR (CDCl₃, 75 MHz): δ = 13.6, 20.1, 25.2, 51.9, 67.1, 72.1, 72.3, 76.1, 92.0, 117.3, 137.9, 141.4, 149.9, 153.5, 155.4 ppm; ³¹P NMR (CDCl₃, 121 MHz): δ = 56.08 ppm; HRMS (electrospray, TOF, ES⁻): m/z = 411.0379, calc. for C₁₂H₁₂N₈O₅PS⁻ 411.0389.

(R _p)-8-(4-Methyl-1,2,3-triazol-2-yl)adenosine-3′,5′-cyclic phosphoric acid tri-n-butylammonium salt (21, C₁₃H₁₄N₈O₅PS⁻)

A 1.8 M solution of LDA in THF/heptane/ethylbenzene (0.21 cm³, 0.38 mmol) was added to a solution of 215 mg (S_P)-2'O-(tert-butyldimethylsilyl)-8-(4-methyl-1,2,3-triazol-2-yl)adenosine-3′,5′-cyclic N-benzylphosphoramidate (8, 0.35 mmol) in 7 cm³ dry THF at − 50 °C. The mixture was stirred under argon at this temperature for 10 min before 0.07 cm³ CS₂ (1.05 mmol) was added. The cooling bath was removed after 10 min, the mixture stirred at room temperature for 3 h before the solvent was partially removed at reduced pressure. Addition of hexane to the residual solution precipitated the solid (R_P)-2′O-(tert-butyldimethylsilyl)-8-(4-methyl-1,2,3-triazol-2-yl)adenosine-3′,5′-cyclic thiophosphoric acid lithium salt (18). The product was dried, dissolved in 4 cm³ MeOH and 181 mg NH₄F (4.9 mmol) added. The mixture was stirred at 45 °C for 4 h, 97 mg nBu₃N (0.52 mmol) added and the solvent was removed at reduced pressure. The residue was subjected to flash chromatography on silica gel using CH₂Cl₂:CH₃OH:nBu₃N (90:10:1). A second flash chromatography operation using CH₂Cl₂:CH₃OH:nBu₃N (93:7:1) gave the ammonium salt 23, that contained some nBu₃N. The product was purified by dissolution in CH₂Cl₂ and reprecipitation after hexane addition. Yield: 84 mg (39% from 8); ¹H NMR (CDCl₃, 200 MHz): δ = 0.92 (9H, t, J = 7.3 Hz, 3 × CH₃), 1.32–1.40 (6H, m, 3 × CH₂), 1.61–1.65 (6H, m, 3 × CH₂), 2.44 (3H, s, Me), 2.82–2.90 (6H, m, 3 × CH₂), 4.34–4.41 (3H, m), 5.03 (1H, d, J = 5.2 Hz), 5.59–5.65 (1H, m), 5.86 (2H, br s, NH₂), 6.54 (1H, s), 7.75 (1H, s, H-triazole), 8.24 (1H, s, H-2) ppm; ¹³C NMR (CDCl₃, 75 MHz): δ = 10.9, 13.6, 20.1, 25.2, 51.9, 67.1, 72.3, 72.4, 76.2, 92.0, 117.3, 137.9, 141.7, 148.1, 149.9, 153.5, 155.4 ppm; ³¹P NMR (CDCl₃, 121 MHz): δ = 56.61 ppm; HRMS (electrospray, TOF, ES⁻): m/z = 4,250,539, calc. for C₁₃H₁₄N₈O₅PS⁻ 425.0545.

(R _p)-8-(4-Chloro-1,2,3-triazol)-2-yl)adenosine-3',5'-cyclic thiophosphoric acid tri-n-butylammonium salt (22, C₁₂H₁₁ClN₈O₅PS⁻)

A 1.8 M solution of LDA in THF/heptane/ethylbenzene (0.21 cm³, 0.38 mmol) was added to a solution of 220 mg (S_P)-2'O-(tert-butyldimethylsilyl)-8-(4-chloro-1,2,3-triazol-2-yl)adenosine-3',5'-cyclic N-benzylphosphoramidate (9, 0.35 mmol) in 7 cm³ dry THF at − 50 °C. The mixture was stirred under argon at this temperature for 10 min before 0.07 cm³ CS₂ (1.05 mmol) was added. The reaction mixture was stirred at room temperature for 3 h before the solvent was partially removed at reduced pressure. Addition of hexane to the residual solution precipitated the solid (R_P)-2′O-(tert-butyldimethylsilyl)-8-(4-chloro-1,2,3-triazol-2-yl)adenosine-3',5'-cyclic thiophosphoric acid lithium salt (19). The product was dried, dissolved in 4 cm³ methanol and 181 mg NH₄F (4.9 mmol) added. The mixture was stirred at 45 °C for 4 h, 97 mg nBu₃N (0.52 mmol) added and the solvent was removed at reduced pressure. The residue was subjected to flash chromatography on silica gel using CH₂Cl₂:CH₃OH:n-Bu₃N (90:10:1). A second flash chromatography operation using CH₂Cl₂:CH₃OH:n-Bu₃N (93:7:1) gave the ammonium salt 22, which contained some n-Bu₃N. The product was purified by dissolution in CH₂Cl₂ and reprecipitation after hexane addition. Yield: 92 mg (41% from 9); ¹H NMR (CDCl₃, 300 MHz): δ = 0.94 (9H, t, J = 7.3 Hz, 3 × Me), 1.34–1.42 (6H, m, 3 × CH₂), 1.65–1.72 (6H, m, 3 × CH₂), 2.94–3.03 (6H, m, 3 × CH₂), 4.30–4.40 (3H, m), 5.05 (1H, d, J = 5.2 Hz), 5.54–5.59 (1H, m), 5.94 (2H, br s, NH₂), 6.38 (1H, s), 7.89 (1H, s, H-triazole), 8.27 (1H, s, H-2) ppm; ¹³C NMR (MeOH-d₄, 75 MHz): δ = 13.6, 20.1, 25.2, 51.9, 67.2, 72.1, 72.2, 76.2, 92.0, 117.2, 136.5, 140.7, 141.1, 149.9, 153.7, 155.4 ppm; ³¹P NMR (CDCl₃, 121 MHz): δ = 56.83 ppm; HRMS (electrospray, TOF, ES⁻): m/z = 444.9990, calc. for C₁₂H₁₁ClN₈O₅PS⁻ 444.9999.

(R _p)-8-(1,2,3-Triazol-1-yl)adenosine-3′,5′-cyclic thiophosphoric acid tri-n-butylammonium salt (23, C₁₂H₁₂N₈O₅PS⁻)

A 1.8 M solution of LDA in THF/heptane/ethylbenzene (0.2 cm³, 0.34 mmol) was added to a solution of 180 mg (S_P)-2′O-(tert-butyldimethylsilyl)-8-(1,2,3-triazol-1-yl)adenosine-3′',5′-cyclic N-benzylphosphoramidate (5, 0.3 mmol) in 7 cm³ dry THF at − 50 °C. The mixture was stirred under argon at this temperature for 10 min before 0.06 cm³ CS₂ (0.9 mmol) was added. The reaction mixture was stirred at room temperature for 3 h before the solvent was partially removed at reduced pressure. Addition of hexane to the residual solution precipitated the solid (R_P)-2′O-(tert-butyldimethylsilyl)-8-(1,2,3-triazol-1-yl)adenosine-3′,5′-cyclic thiophosphoric acid lithium salt. The latter was dried, dissolved in 4 cm³ MeOH and 155 mg NH₄F (4.2 mmol) was added. The mixture was stirred at 45 °C for 4 h, 85 mg n-Bu₃N (0.45 mmol) was added, the solvent removed at reduced pressure and the residue subjected to flash chromatography on silica gel using CH₂Cl₂:CH₃OH:nBu₃N (90:10:1). The flash chromatography was repeated using CH₂Cl₂:CH₃OH:nBu₃N (93:7:1). The ammonium salt 23 thus obtained contained some n-Bu₃N and was further purified by dissolution in CH₂Cl₂ and reprecipitation by hexane addition. Yield: 70 mg (40% from 5); ¹H NMR (CDCl₃, 300 MHz): δ = 0.94 (9H, t, J = 7.3 Hz, 3 × Me), 1.34–1.42 (6H, m, 3 × CH₂), 1.69–1.74 (6H, m, 3 × CH₂), 2.99–3.04 (6H, m, 3 × CH₂), 4.30–4.41 (3H, m), 5.11 (1H, d, J = 5.2 Hz), 5.46–5.50 (1H, m), 5.87 (2H, br s, NH₂), 6.29 (1H, s), 7.88 (1H, d, J = 1 Hz, H-triazole), 8.28 (1H, s, H-2), 8.30 (1H, d, J = 1 Hz, H-triazole) ppm; ¹³C NMR (CDCl₃, 75 MHz): δ = 13.6, 20.1, 25.2, 51.9, 67.0, 71.9, 72.1, 76.2, 92.0, 117.5, 125.8, 133.9 137.9, 149.1, 154.3, 155.6 ppm; ³¹P NMR(CDCl₃, 121 MHz): δ = 56.00 ppm; HRMS (electrospray, TOF, ES⁻): m/z = 411.0382, calc. for C₁₂H₁₂N₈O₅PS⁻ 411.0389.

(R _p)-8-Azidoadenosine-3′,5′-cyclic thiophosphoric acid tri-n-butylammonium salt (24, C₁₀H₁₀N₈O₅PS⁻)

A 1.8 M Solution of LDA in THF/heptane/ethylbenzene (0.61 cm³, 1.1 mmol) was added to a solution of 575 mg (S_P)-8-azidoadenosine-2′O-(tert-butyldimethylsilyl)-3′,5′-cyclic N-benzylphosphoramidate (12, 1.0 mmol) in 15 cm³ dry THF at − 78 °C. The mixture was stirred under argon at this temperature for 10 min before 0.18 cm³ CS₂ (3.0 mmol) was added. The reaction mixture was stirred at room temperature for 3 h before the solvent was partially removed at reduced pressure. Addition of hexane to the residual solution precipitated a solid that was filtered off. The resulting (R_P)-8-azidoadenosine-2′O-(tert-butyldimethylsilyl)-3′,5′-cyclic thiophosphoric acid lithium salt was dried and dissolved in 10 cm³ methanol and 476 mg NH₄F (14.0 mmol) added. The mixture was stirred at 45 °C for 4 h, 277 mg nBu₃N (1.5 mmol) added and the solvent removed at reduced pressure. The residue was subjected to flash chromatography on silica gel using CH₂Cl₂:CH₃OH:nBu₃N (90:10:1). Repetitive flash chromatography using CH₂Cl₂:CH₃OH:n-Bu₃N (93:7:1) gave the ammonium salt 24, that contained some nBu₃N. The product was purified by dissolution in CH₂Cl₂ and reprecipitation by addition of hexane. Yield: 273 mg (48% from 12); ¹H NMR (CDCl₃, 200 MHz): δ = 0.94 (9H, t, J = 7.3 Hz, 3 × CH₃), 1.33–1.44 (6H, m, 3 × CH₂), 1.63–1.79 (6H, m, 3 × CH₂), 2.99–3.04 (6H, m, 3 × CH₂), 4.30–4.41 (3H, m), 4.89 (1H, d, J = 5.0 Hz), 5.27–5.33 (1H, m), 5.58 (2H, br s, NH₂), 5.84 (1H, s), 8.14 (1H, s, H-2) ppm; ¹³C NMR (CDCl₃, 75 MHz): δ = 13.6, 20.1, 25.2, 51.9, 67.1, 71.6, 71.7, 76.2, 90.0, 117.8, 145.2, 150.0, 152.0, 153.4 ppm; ³¹P NMR (CDCl₃, 121 MHz): δ = 56.72 ppm; HRMS (electrospray, TOF, ES⁻): m/z = 385.0245, calc. for C₁₀H₁₀N₈O₅PS⁻ 385.0232.

(R _p)-8-(Imidazol-1-yl)adenosine-3′,5′-cyclic thiophosphoric acid sodium salt (25, C₁₃H₁₃N₇O₅PS⁻)

(R_p)-8-(Imidazol-1-yl)adenosine-3′,5′-cyclic thiophosphoric acid tri-n-butylammonium salt (15, 130 mg, 0.21 mmol) was dissolved in 0.1 M NaOH in MeOH (2.1 cm³). The sodium salt was precipitated by addition of diethyl ether. The solvent was decanted and the precipitate washed with diethyl ether. The ether was removed and the white solid sodium salt dried under vacuum. Yield: 81 mg (88%); ¹H NMR (MeOH-d₄, 300 MHz): δ = 4.22–4.35 (3H, m), 5.14 (1H, d, J = 5.2 Hz), 5.35–5.42 (1H, m), 5.51 (1H, s), 7.26 (1H, s, H-imidazole), 7.66 (1H, s, H-imidazole), 8.20 (1H, s, H-imidazole), 8.25 (1H, s, H-2) ppm; ¹³C NMR (MeOH-d₄, 75 MHz): δ = 68.3, 72.5, 73.4, 77.5, 93.1, 118.3, 121.9, 130.7, 139.65, 141.7, 150.6, 154.75, 157.3 ppm; ³¹P NMR (MeOH-d₄, 121 MHz): δ = 57.92 ppm; HRMS (electrospray, TOF, ES⁻): m/z = 410.0425, calc. for C₁₃H₁₃N₇O₅PS⁻ 410.0436.

(R _p)-8-(1,2,4-Triazol-1-yl)adenosine-3′,5′-cyclic thiophosphoric acid sodium salt (26, C₁₂H₁₂N₈O₅PS⁻)

Product 26 was prepared as above from (R_P)-8-(1,2,4-triazol-1-yl)adenosine-3′,5′-cyclic thiophosphoric acid tri-n-butylammonium salt (16) in 0.1 M NaOH in MeOH. Yield: 92%; ¹H NMR (MeOH-d₄, 200 MHz): δ = 4.12–4.35 (3H, m), 5.03 (1H, d, J = 5.3 Hz), 5.45–5.52 (1H, m), 6.27 (1H, s), 8.26 (1H, s, H-triazole), 8.34 (1H, s, H-2), 9.16 (1H, s, H-triazole) ppm; ¹³C NMR (MeOH-d₄, 75 MHz): δ = 68.3, 73.1, 73.3, 77.4, 93.55, 118.3, 140.7, 147.6, 151.3, 154.4, 154.9, 157.5 ppm; ³¹P NMR (MeOH-d₄, 121 MHz): δ = 58.06 ppm; HRMS (electrospray, TOF, ES⁻): m/z = 411.0390, calc. for C₁₂H₁₂N₈O₅PS⁻ 411.0389.

(R _p)-8-(1,2,3-Triazol-2-yl)adenosine-3′,5′-cyclic thiophosphoric acid sodium salt (27, C₁₂H₁₂N₈O₅PS⁻)

Product 27 was prepared as above from (R_P)-8-(1,2,3-triazol-2-yl)adenosine-3′,5′-cyclic thiophosphoric acid tri-n-butylammonium salt (20) in 0.1 M NaOH in MeOH. Yield: 90%; ¹H NMR (MeOH-d₄, 300 MHz): δ = 4.16–4.40 (3H, m), 5.03 (1H, d, J = 5.3 Hz), 5.41–5.49 (1H, m), 6.30 (1H, s), 8.18 (2H, s, H-triazole), 8.28 (1H, s, H-2) ppm; ¹³C NMR (MeOH-d₄, 75 MHz): δ = 68.3, 73.2, 73.3, 77.3, 93.9, 118.0, 139.3, 142.3, 151.1, 155.1, 157.5 ppm; ³¹P NMR (MeOH-d₄, 121 MHz): δ = 57.47 ppm; HRMS (electrospray, TOF, ES⁻): m/z = 411.0386, calc. for C₁₂H₁₂N₈O₅PS⁻ 411.0389.

(R _p)-8-(4-Methyl-1,2,3-triazol-2-yl)adenosine-3′,5′-cyclic phosphoric acid sodium salt (28, C₁₃H₁₄N₈O₅PS⁻)

Product 28 was prepared as above from (R_P)-8-(4-methyl-1,2,3-triazol-2-yl)adenosine-3′,5′-cyclic thiophosphoric acid tri-n-butylammonium salt (21) in 0.1 M NaOH in MeOH. Yield: 91%; ¹H NMR (MeOH-d₄, 200 MHz): δ = 2.46 (3H, s, CH₃), 4.14–4.34 (3H, m), 5.02 (1H, d, J = 5.4 Hz), 5.41–5.50 (1H, m), 6.39 (1H, s), 7.96 (1H, s, H-triazole), 8.26 (1H, s, H-2) ppm; ¹³C NMR (MeOH-d₄, 75 MHz): δ = 10.6, 68.3, 73.2, 73.3, 77.3, 93.9, 117.95, 139.1, 142.6, 149.7, 151.0, 154.8, 157.3 ppm; ³¹P NMR (MeOH-d₄, 121 MHz): δ = 57.95 ppm; HRMS (electrospray, TOF, ES⁻): m/z = 425.0533, calc. for C₁₃H₁₄N₈O₅PS⁻ 425.0545.

(R _p)-8-(4-Chloro-1,2,3-triazol-2-yl)adenosine-3′,5′-cyclic thiophosphoric acid sodium salt (29, C₁₂H₁₁ClN₈O₅PS⁻)

Product 29 was prepared as above from (R_P)-8-(4-chloro-1,2,3-triazol-2-yl)adenosine-3′,5′-cyclic thiophosphoric acid tri-n-butylammonium salt (22) in 0.1 M NaOH in MeOH. Yield: 87%; ¹H NMR (MeOH-d₄, 300 MHz): δ = 4.16–4.35 (3H, m), 5.04 (1H, d, J = 5.3 Hz), 5.43–5.48 (1H, m), 6.20 (1H, s), 8.22 (1H, s, H-triazole), 8.28 (1H, s, H-2) ppm; ¹³C NMR (MeOH-d₄, 75 MHz): δ = 68.3, 73.2, 73.4, 77.3, 93.8, 118.0, 137.8, 141.7, 142.1, 151.0, 155.25, 157.5 ppm; ³¹P NMR (MeOH-d₄, 121 MHz): δ = 57.31 ppm; HRMS (electrospray, TOF, ES⁻): m/z = 444.9991, calc. for C₁₂H₁₁ClN₈O₅PS⁻ 444.9999.

(R _p)-8-(1,2,3-Triazol-1-yl)adenosine-3′,5′-cyclic thiophosphoric acid sodium salt (30, C₁₂H₁₂N₈O₅PS⁻)

Product 30 was prepared as above from (R_P)-8-(1,2,3-triazol-1-yl)adenosine-3′,5′-cyclic thiophosphoric acid tri-n-butylammonium salt (23) in 0.1 M NaOH in MeOH. Yield: 92%; ³¹P NMR (MeOH-d₄, 121 MHz): δ = 57.3 ppm; HRMS (electrospray, TOF, ES⁻): m/z = 411.0380, calc. for C₁₂H₁₂N₈O₅PS⁻ 411.0389.

(R _p)-8-Azidoadenosine-3′,5′-cyclic thiophosphoric acid sodium salt (31, C₁₂H₁₂N₈O₅PS⁻)

Product 31 was prepared as above from (R_P)-8-azidoadenosine-3′,5′-cyclic thiophosphoric acid tri-n-butylammonium salt (24) in 0.1 M NaOH in MeOH. Yield: 87%; ¹H NMR (MeOH-d₄, 300 MHz): δ = 4.15–4.33 (3H, m), 4.78 (1H, d, J = 5.3 Hz), 5.22–5.28 (1H, m), 5.84 (1H, s), 8.12 (1H, s, H-2) ppm; ¹³C NMR (MeOH-d₄, 75 MHz): δ = 68.4, 73.1, 73.2, 77.5, 91.7, 118.4, 146.4, 151.1, 153.2, 155.6 ppm; ³¹P NMR (MeOH-d₄, 121 MHz): δ = 58.26 ppm; HRMS (electrospray, TOF, ES⁻): m/z = 385.0224, calc. for C₁₀H₁₀N₈O₅PS⁻ 385.0232.