Regio- and Stereoselective Synthesis of 2′-Deoxy-4′-thioguanine Nucleosides: Evaluation of Anti-Hepatitis B Virus Activity and ­Cytotoxicity Leading to Improved Selectivity Index by 4′-C-Cyanation

Abstract

An N ⁹-regio- and β-anomer-selective 4′-thioglycosidation of purine bases has been developed. The reaction between a 2-deoxy-2-iodo-4-thioribofuranosyl glycosyl donor and N-(6-chloro-9H-purin-2-yl)-2-methylpropanamide gave the corresponding 2′-deoxy-4′-thiopurine nucleoside in 87% yield along with its N ⁷-regioisomer in 6% yield, without the formation of the α-anomer. By using a derivative obtained from 17, a practical chemical synthesis of 2′-deoxy-4′-thioguanosine was developed. 4′-α-C-Cyano-2′-deoxy-4′-thioguanosine was synthesized, starting from a 4-(acetoxymethyl)-2-deoxy-2-iodo-4-thioribofuranose derivative as a glycosyl donor. An evaluation of the anti-hepatitis B virus (HBV) activity and the cytotoxicity toward the host cell revealed that 4'-C-cyano-2'-deoxy-4'-thioguanosine exhibited about 100 times more potent anti-HBV activity than 2′-deoxy-4′-thioguanosine with a comparative cytotoxicity, resulting in the identification of a novel molecule having better selectivity index value than that of 2′-deoxy-4′-thioguanosine. This finding might provide a guideline for the development of the next generation of anti-HBV agents.

Keywords

nucleosides - glycals - deoxythioguanosine - medicinal chemistry - cytotoxicity - hepatitis B virus

• References and Notes

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• 14 Purines 17 and 18 N,O-Bis(trimethylsilyl)acetamide (0.22 mL, 0.90 mmol) was added to a suspension of 16 (215.7 mg, 0.90 mmol) in MeCN (5.0 mL) at rt under Ar, and the mixture was stirred for 1 h. A solution of 15 (360.4 mg, 0.60 mmol) in MeCN (7.0 mL)–CH₂Cl₂ (4.0 mL) and TMSOTf (0.22 mL, 1.20 mmol) were added sequentially at –10 °C under Ar. The resulting mixture was stirred at –10 °C for 1 h, 0 °C for 1 h, and r.t. overnight. The mixture was then partitioned between CHCl₃ and sat. aq NaHCO₃, and the organic layer was subjected to column chromatography [silica gel, hexane–EtOAc (7:1)] to give 17 as a colorless foam [yield: 387.6 mg (87%)] and 18 as a colorless syrup [yield: 21.4 mg (6%)]. 17 ¹H NMR (500 MHz, CDCl₃): δ = 0.86–0.90 and 1.07–1.14 (both m, 28 H, Si-i-Pr), 1.32 and 1.33 [both d, J _CH,_CH3 = 3.5 Hz, 6 H, CH(CH₃)₂], 3.41–3.42 [br s, 1 H, CH(CH₃)₂], 3.61 (dd, J _2′,_3′ = 4.6 and J _3′,4′ = 8.6 Hz, 1 H, H-3′), 3.76 (ddd, J _3′,_4′ = 8.6, J _4′,_5′a = 1.7 and J _4′,_5′b = 2.9 Hz, 1 H, H-4′), 4.08 (dd, J _4′,_5′a = 1.7 and J _5′a,_5′b = 12.6 Hz, 1 H, CH_2a-5′), 4.18 (dd, J _4′,_5′b = 2.9 and J _5′a,_5′b = 12.6 Hz, 1 H, CH_2b-5′), 4.73 (d, J _2′,_3′ = 4.6 Hz, 1 H, H-2′), 6.16 (s, 1 H, H-1′), 8.24 (br s, 1 H, NH), 8.66 (s, 1 H, H-8). NOE experiment: H-8/H-2′ and H-1′/H-4′. ¹³C NMR (125 MHz, CDCl₃): δ = 12.6, 13.1, 13.2, 13.3, 16.88, 16.93, 17.0, 17.31, 17.33, 17.5, 19.1, 19.2, 35.2, 37.7, 53.6, 58.2, 64.6, 71.9, 128.5, 143.3, 151.8, 152.0, 152.2, 176.8. HMBC experiment: C-4/H-1′. ESI-MS: m/z = 762 [M + Na]⁺. ESI-HRMS: m/z [M + Na]⁺ calcd for C₂₆H₄₃ClIN₅NaO₄SSi₂: 762.11997; found: 762.11896. UV (MeOH): λ_max 290, 263, 230 nm; λ_min 272 and 248 nm. 18 ¹H NMR (500 MHz, CDCl₃): δ = 0.79–0.85 and 1.07–1.20 (both m, 28 H, Si-i-Pr), 1.29 [d, J _CH,_CH3 = 6.9 Hz, 6 H, CH(CH₃)₂]. 3.12–3.25 [m, 1 H, CH(CH₃)₂], 3.52 (dd, J _2′,_3′ = 4.0 and J _3′,_4′ = 9.2 Hz, 1 H, H-3′), 3.74 (dd, J _3′,_4′ = 9.2 and J _4′,_5′b = 2.3 Hz, 1 H, H-4′), 4.13 (d, J _5′a,_5′b = 13.2 Hz, 1 H, CH_2a-5′), 4.21 (dd, J _4′,_5′b = 2.3 and J _5′a,_5′b = 13.2 Hz, 1 H, CH_2b-5′), 4.53 (d, J _2′,_3′ = 4.0 Hz, 1 H, H-2′), 6.40 (s, 1 H, H-1′), 8.13 (br s, 1 H, NH), 9.25 (s, 1 H, H-8). NOE experiment: H-8/ H-2′ and H-1′/H-4′. ¹³C NMR (125 MHz, CDCl₃): δ = 12.6, 13.1, 13.2, 13.3, 16.8, 16.88, 16.93, 17.27, 17.35, 17.5, 19.19, 19.22, 35.6, 39.4, 53.5, 57.9, 66.5, 71.0, 118.8, 143.2, 149.0, 152.7, 163.9, 176.2. HMBC experiment: C-5/H-1′. ESI-MS: m/z = 762 [M + Na]⁺. ESI-HRMS: m/z [M + Na]⁺ calcd for C₂₆H₄₃ClIN₅NaO₄SSi₂: 762.11997; found: 762.11788. UV (MeOH): λ_max 298 and 236 nm, λ_min 275 nm.
• 15 Purines 21 and 22 N,O-Bis(trimethylsilyl)acetamide (0.24 mL, 0.99 mmol) was added to a suspension of 16 (237.3 mg, 0.99 mmol) in MeCN (6.0 mL) at r.t. under Ar, and the mixture was stirred for 1 h. A solution of 20 (448.3 mg, 0.66 mmol) in MeCN (5.0 mL)–CH₂Cl₂ (3.0 mL) and TMSOTf (0.36 mL, 1.98 mmol) were added sequentially at –10 °C under Ar. The resulting mixture was stirred at –10 °C for 1 h, 0 °C for 1 h, and r.t. overnight. The mixture was partitioned between CHCl₃ and sat. aq NaHCO₃, and the organic layer was subjected to column chromatography [silica gel, hexane–EtOAc (4:1 to 2:1)] to give 21 as a colorless foam [yield: 387.1 mg, (72%)] and 22 as a colorless syrup [yield: 37.2 mg (7%)]. 21 ¹H NMR (500 MHz, CDCl₃): δ = 0.90–0.97 and 1.03–1.12 (both m, 28 H, Si-i-Pr), 1.29 and 1.30 [both d, J _CH,_Me = 3.5 Hz, 6 H, CH(CH₃)₂], 2.16 (s, 3 H, Ac), 3.26–3.27 [br s, 1 H, CH(CH₃)₂], 4.03 (d, J _5′a,_5′b = 12.0 Hz, 1 H, CH_2a-5′), 4.10 (d, J _5′a,_5′b = 12.0 Hz, 1 H, CH_2b-5′), 4.30 (d, J _2′,_3′ = 6.5 Hz, 1 H, H-3′), 4.57 (d, J _CH2a,_CH2b = 9.8 Hz, 1 H, CH_2aOAc), 5.15 (d, J _CH2a,_CH2b = 9.8 Hz, 1 H, CH_2bOAc), 5.34 (dd, J _1′,_2′ = 1.7 and J _2′,_3′ = 6.5 Hz, 1 H, H-2′), 6.24 (d, J _{1′ 2′} = 1.7 Hz, 1 H, H-1′), 8.12 (br s, 1 H, NH), 8.41 (s, 1 H, H-8). NOE experiment: H-8/H-2′, H-8/H-3′, H-8/CH_2a-5′, H-8/CH_2b-5′, H-1′/CH_2b-OAc, H-2′/CH_2a-5′, H-2′/CH_2b-5′. ¹³C NMR (125 MHz, CDCl₃): δ = 12.7, 12.9, 13.0, 13.1, 17.0, 17.1, 17.2, 17.3, 17.4, 19.1, 19.2, 21.0, 35.5, 36.0, 63.18, 65.0, 66.0, 67.0, 76.2, 128.7, 143.0, 151.91, 151.98, 152.2, 170.6, 176.1. ESI-MS: m/z = 834 [M + Na]⁺. FAB-HRMS: m/z [M + Na]⁺ calcd for C₂₉H₄₇ClIN₅NaO₆SSi₂: 834.14110 found: 834.13980. UV(MeOH) λ_max 290, 263 and 230 nm, λ_min 272 and 247 nm. 22 ¹H NMR (500 MHz, CDCl₃): δ = 0.83–0.90 and 1.01–1.13 (both m, 28 H, Si-i-Pr), 1.29 [d, J _CH,_CH3 = 6.9 Hz, 6 H, CH(CH₃)₂], 2.14 (s, 3 H, Ac), 3.09–3.16. [br s, 1 H, CH(CH₃)₂], 4.09 (d, J _5′a,_5′b = 12.1 Hz, 1 H, CH_2a-5′), 4.13 (d, J _5′a,_5′b = 12.1 Hz, 1 H, CH_2b-5′), 4.16 (d, J _2′,_3′ = 6.3 Hz, 1 H, H-3′), 4.53 (d, J _CH2a,_CH2b = 11.5 Hz, 1 H, CH_2aOAc), 4.75 (d, J _2′,_3′ = 6.3 Hz, 1 H, H-2’), 5.28 (dd, J _CH2a,_CH2b = 11.5 Hz, 1 H, CH_2bOAc), 6.47 (s, 1 H, H-1′), 8.14 (br s, 1 H, NH), 9.04 (s, 1 H, H-8). NOE experiment: H-8/H-2′, H-8/H-3′, H-8/CH_2b-5′, H-1′/CH_2b-OAc. ¹³C NMR (125 MHz, CDCl₃): δ = 12.86, 12.90, 13.0, 13.2, 17.0, 17.06, 17.09, 17.2, 17.3, 17.4, 19.2, 20.9, 35.7, 37.2, 62.5, 66.0, 74.9, 118.8, 143.4, 148.1, 152.7, 163.9, 170.4, 175.9. ESI-MS: m/z = 834 [M⁺ + Na]. FAB-HRMS: m/z [M + Na]⁺ calcd for C₂₉H₄₇ClIN₅NaO₆SSi₂: 834.14110; found: 834.13938. UV(MeOH) λ_max 299 and 237 nm, λ_min 275 nm.
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