Nucleoside analogue and application thereof

Abstract

The invention discloses a nucleoside analogue and application thereof. The invention discloses a nucleoside compound with a relatively good inhibiting effect on RNA (Ribonucleic Acid) viruses. The structure of the nucleoside compound is as shown in a formula I which is described in the specification. The compound shows good inhibitory activity on coronavirus, and has great potential and application prospects in the aspect of being developed into a coronavirus inhibitor. # imgabs0 #

Description

[0001] This application claims the priority of Chinese Patent Application CN2023116828124 with a filing date of December 8, 2023. This application incorporates the entire text of the above-mentioned Chinese patent application by reference. Technical Field

[0002] The present invention relates to the technical field of medicinal chemistry, and particularly to a nucleoside analog and its application. Background Art

[0003] Coronaviruses (CoVs) are a large group of enveloped positive-sense single-stranded RNA viruses that are widely present in nature and can infect multiple classes of vertebrates including humans. According to the phylogenetic tree, CoVs can be divided into four genera: α, β, γ, and δ. Among the currently known seven CoVs that can infect humans, the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which can cause severe diseases, all belong to the β genus of coronaviruses.

[0004] Since human infections with CoVs (HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1) usually only cause general respiratory symptoms similar to influenza, CoVs did not attract much attention in the early stage (before 2001), and there were no targeted drugs clinically. Therefore, the development of drugs against CoVs is not only a crucial topic for human public health and health but also a significant topic for the development of the national economy. Summary of the Invention

[0005] The purpose of the present invention is to provide a class of nucleoside compounds that have good inhibitory effects on RNA viruses. The compounds exhibit good inhibitory activity against coronaviruses and have great potential and application prospects in developing into coronavirus inhibitors.

[0006] Another purpose of the present invention is to provide the use of the above-mentioned nucleoside compounds.

[0007] The above purposes of the present invention are achieved through the following solutions:

[0008] A nucleoside analog having the structure shown in Formula I, its pharmaceutically acceptable salts, stereoisomers, solvates or prodrugs,

[0009]

[0010] wherein X is H, -CH 2 OR 0 , F, N 3 , C 2-4 alkynyl or -CH 2 F;

[0011] R 0 is H or C 1-4 alkyl, C 1-4 haloalkyl;

[0012] R 1 is H, -COR 11 or -P(O)(OR 5 )NHR 4 ;

[0013] R 11 is C 1-4 alkyl, C 1-4 haloalkyl or amino-substituted C 1-4 alkyl;

[0014] R 2 is C 1-4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, phenyl, benzyl, C 3-7 heteroaryl, R 21 substituted C 1-4 alkyl, R 21 substituted C 2-4 alkenyl, R 21 substituted C 2-4 alkynyl, R 21 substituted phenyl, R 21 substituted benzyl, R 21 substituted C 3-7 heteroaryl or C 6-12 fused heterocycle;

[0015] R 21 is halogen, phenyl, halophenyl, C 1-4 alkyl-substituted phenyl or halo- and C 1-4 alkyl-disubstituted phenyl;

[0016] R 3 is C 1-4 alkyl, C 1-4 alkoxy, halo C 1-4 alkyl or halo C 1-4 alkoxy;

[0017] R 4 is H, C 1-4 alkyl, -R 41 C(O)OR 42 or -R 41 OC(O)R 42 ; wherein R 41 and R 42 are each independently C 1-4 alkylene or C 1-4 alkyl;

[0018] R 5 is phenyl, halogenated phenyl, benzyl or halogenated benzyl.

[0019] In one embodiment, R 41 is C 1-4 alkylene.

[0020] In one embodiment, R 41 is C 1-3 alkylene.

[0021] In one embodiment, R 41 is methylene, methylene-methyl, ethylene, propylene or isopropylidene.

[0022] In one embodiment, R 41 is methylene, methylene-methyl or ethylene.

[0023] In one embodiment, R 4 is methylene C(O)OR 42 methylene(methyl)C(O)OR 42 ethylene C(O)OR 42 n-propylene C(O)OR 42 isopropylidene C(O)OR 42 methylene OC(O)R 42 methylene(methyl)OC(O)R 42 ethylene OC(O)R 42 n-propylene OC(O)R 42 or isopropylidene OC(O)R 42 ;

[0024] R 42 is C 1-4 alkyl.

[0025] In one embodiment, R 42 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl.

[0026] In one embodiment, R 42 is methyl, ethyl, propyl or isopropyl.

[0027] In one embodiment, R 42 is ethyl, propyl or isopropyl.

[0028] In one embodiment, R 5 is phenyl or halogenated phenyl.

[0029] In one embodiment, R 5is benzyl or halogenated benzyl.

[0030] In one embodiment, R 5 is phenyl, benzyl or halogenated benzyl.

[0031] In one embodiment, R 5 is phenyl, halogenated phenyl or benzyl.

[0032] The aforementioned halogenated phenyl or halogenated benzyl means that one or more H on the phenyl or benzyl are replaced by a halogen element, and the halogen element is F, Cl or Br.

[0033] In one embodiment, R 5 is phenyl, fluorophenyl, difluorophenyl, trifluorophenyl, benzyl, fluorobenzyl, difluorobenzyl or trifluorobenzyl.

[0034] In one embodiment, R 5 is phenyl or fluorophenyl.

[0035] In one embodiment, R 5 is phenyl, fluorophenyl, benzyl or fluorobenzyl.

[0036] In one embodiment, R 11 is C 1-4 alkyl, C 1-4 haloalkyl or -C(NH 2 )C 1-3 alkyl.

[0037] In one embodiment, R 11 is C 1-4 alkyl or -C(NH 2 )C 1-3 alkyl.

[0038] In one embodiment, R 11 is methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, -C(NH 2 )methyl, -C(NH 2 )ethyl, -C(NH 2 )propyl or -C(NH 2 )isopropyl.

[0039] In one embodiment, R 11 is propyl, isopropyl, butyl, tert-butyl, -C(NH 2 )propyl or -C(NH 2 )isopropyl.

[0040] In one embodiment, R 1is H, -CO-methyl, -CO-ethyl, -CO-propyl, -CO-isopropyl, -CO-butyl, -CO-tert-butyl, -CO-C(NH 2 )-methyl, -CO-C(NH 2 )-ethyl, -CO-C(NH 2 )-propyl, -CO-C(NH 2 )-isopropyl, -P(O)(OR 5 )NH-R 41 C(O)OR 42 or -P(O)(OR 5 )NH--R 41 OC(O)R 42 ;

[0041] R 5 is phenyl or halogenated phenyl; R 41 and R 42 are each independently C 1-4 alkylene or C 1-4 alkyl.

[0042] In one embodiment, R 1 is H, -CO-propyl, -CO-isopropyl, -CO-C(NH 2 )-propyl, -CO-C(NH 2 )-isopropyl, -P(O)(OR 5 )NHCH 2 (CH 3 )C(O)O isopropyl.

[0043] In one embodiment, R 21 is halogen, phenyl, 4-halogenated phenyl, 2,4-dihalogenated phenyl, 3,4-dihalogenated phenyl, 2,3-dihalogenated phenyl or 3,5-dihalogenated phenyl.

[0044] In one embodiment, R 21 is halogen, phenyl, 4-halogenated phenyl, 2,4-dihalogenated phenyl, 3,4-dihalogenated phenyl, 4-C 1-3 alkyl-substituted phenyl, 2,4-di-C 1-3 alkyl-substituted phenyl, 4-halo-3-C 1-3 alkyl-substituted phenyl, 3-halo-4-C 1-3 alkyl-substituted phenyl, 4-halo-2-C 1-3 alkyl-substituted phenyl or 2-halo-4-C 1-3 alkyl-substituted phenyl.

[0045] In one embodiment, R 21is halogen, phenyl, 4-halophenyl, 2,4-dihalophenyl, or 3,4-dihalophenyl.

[0046] In one embodiment, R 21 is halogen.

[0047] In one embodiment, R 21 is phenyl.

[0048] In one embodiment, R 21 is 4-halophenyl.

[0049] In one embodiment, R 21 is 2,4-dihalophenyl.

[0050] In one embodiment, R 21 is 3,4-dihalophenyl.

[0051] In one embodiment, R 2 is C 1-4 alkyl, C 2-4 alkynyl, phenyl, benzyl, C 3-7 heteroaryl, R 21 substituted C 2-4 alkynyl, R 21 substituted phenyl, R 21 substituted benzyl, R 21 substituted C 3-7 heteroaryl, or C 6-12 fused heterocycle.

[0052] In one embodiment, R 2 is phenyl, C 3-7 heteroaryl, R 21 substituted C 2-4 alkynyl, R 21 substituted phenyl, R 21 substituted C 3-7 heteroaryl, or C 6-12 fused heterocycle.

[0053] In one embodiment, R 2 is C 3-7 heteroaryl, R 21 substituted C 2-4 alkynyl, R 21 substituted C 3-7 heteroaryl, or C 6-12 fused heterocycle.

[0054] In one embodiment, the C 2 in R 3-7 heteroaryl is furan, pyrrole, thiophene, pyrazole, imidazole, thiazole, pyridine, or pyrimidine.

[0055] In one embodiment, R 2 the C in 6-12 fused heterocycle is benzofuran, indole, isoindole or indazole.

[0056] In one embodiment, R 2 is phenyl, benzyl, furan, thiophene, pyrazole, imidazole, thiazole, pyrimidine, benzofuran, indole, halo C 2-4 alkynyl, phenyl-substituted C 2-4 alkynyl, 4-halophenyl-substituted C 2-4 alkynyl, 2,4-dihalophenyl-substituted C 2-4 alkynyl or 3,4-dihalophenyl-substituted C 2-4 alkynyl.

[0057] In one embodiment, R 2 is phenyl, furan, thiophene, pyrazole, imidazole, thiazole, pyrimidine, benzofuran, phenyl-substituted C 2-4 alkynyl, 4-halophenyl-substituted C 2-4 alkynyl or 2,4-dihalophenyl-substituted C 2-4 alkynyl.

[0058] In one embodiment, R 2 is furan, thiophene, pyrazole, thiazole, benzofuran, 4-halophenyl-substituted C 2-4 alkynyl or 2,4-dihalophenyl-substituted C 2-4 alkynyl.

[0059] In one embodiment, R 2 is furan.

[0060] In one embodiment, R 2 is thiophene.

[0061] In one embodiment, R 2 is pyrazole.

[0062] In one embodiment, R 2 is thiazole.

[0063] In one embodiment, R 2 is benzofuran.

[0064] In one embodiment, R 2 is 4-halophenyl-substituted C 2-4 alkynyl.

[0065] In one embodiment, R 2 is 2,4-dihalophenyl-substituted C2-4 Alkynyl group.

[0066] In one embodiment, R 3 is C 1-4 alkyl, C 1-4 alkoxy, or halo C 1-4 haloalkyl.

[0067] In one embodiment, R 3 is C 1-4 alkyl or C 1-4 alkoxy.

[0068] In one embodiment, R 3 is C 1-2 alkyl or C 1-2 alkoxy.

[0069] In one embodiment, R 0 is H or C 1-2 alkyl or C 1-2 haloalkyl.

[0070] In one embodiment, R 0 is H or methyl.

[0071] In one embodiment, X is H, -CH 2 OH, -CH 2 OCH 3 3, F, N 3 3, ethynyl, or -CH 2 2F.

[0072] In one embodiment, X is H.

[0073] In one embodiment, X is -CH 2 2OH or -CH 2 2OCH 3 3.

[0074] In one embodiment, X is F.

[0075] In one embodiment, X is N 3 3.

[0076] In one embodiment, X is ethynyl.

[0077] In one embodiment, X is -CH 2 2F.

[0078] In one embodiment, the compound has any of the following structures:

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104] The present invention also protects a pharmaceutical composition, namely a composition comprising any of the above nucleoside analogs, or pharmaceutically acceptable salts thereof, or stereoisomers, or solvates or prodrugs, and pharmaceutically acceptable excipients or carriers.

[0105] The present invention also protects the use of the above nucleoside analogs, or pharmaceutically acceptable salts thereof, or stereoisomers, or solvates, or prodrugs, or the aforementioned pharmaceutical composition in the preparation of a drug for preventing and treating RNA virus infection diseases.

[0106] In one embodiment, the RNA virus is from the Coronaviridae family or Flavivirus.

[0107] In one embodiment, the coronaviruses include: SARS-CoV, MERS-CoV, and SARS-CoV-2.

[0108] Compared with the prior art, the present invention has the following beneficial effects:

[0109] The compounds of the present invention have good inhibitory effects on RNA viruses and also show good inhibitory effects on various coronaviruses, and have great potential and application prospects in the development of coronavirus inhibitors. Detailed Embodiments

[0110] The following further elaborates the present invention in conjunction with specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0111] Example 1 Compound 1 (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(5-iodo-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol

[0112]

[0113] 1) (2R,3S,4R)-2-(hydroxymethyl)-5-methoxytetrahydrofuran-3,4-diol

[0114]

[0115] Under 0 °C and an argon atmosphere, sulfuric acid (1.5 mL, 0.027 mol) was added dropwise to a methanol (300 mL) solution of D-ribose (25.0 g, 0.17 mol). The resulting mixture was stirred at room temperature for 24 hours. Sodium bicarbonate solid (4.54 g, 0.054 mol) was added to the reaction solution and stirred for neutralization. After filtration and rotary evaporation, the title compound was obtained as a pale yellow oil (27.5 g, 100%).

[0116] 2) (2R,3R,4R)-3,4-Bis(benzyloxy)-2-((benzyloxy)methyl)-5-methoxytetrahydrofuran

[0117]

[0118] Under 0 °C and an argon atmosphere, 60% sodium hydride (30.2 g, 0.755 mol) was added portionwise to an anhydrous DMF (500 mL) solution of (2R,3S,4R)-2-(hydroxymethyl)-5-methoxytetrahydrofuran-3,4-diol (27.5 g, 0.167 mol). After addition, the mixture was stirred at room temperature for 30 minutes. Then, benzyl bromide (100.5 g, 0.587 mol) was added dropwise at 0 °C. After addition, the reaction was carried out at room temperature for 16 hours. The reaction solution was slowly poured into a saturated ammonium chloride solution, and then extracted with ethyl acetate. The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and rotary evaporated. The resulting residue was purified by column chromatography (petroleum ether:ethyl acetate = 6:1) to obtain the title compound as a pale yellow oil (72 g, 99%). LCMS [M+NH 4 + : 452.2.

[0119] 3) (2S,3R,4S,5R)-4-(Benzyloxy)-5-((benzyloxy)methyl)-2-methoxytetrahydrofuran-3-ol

[0120]

[0121] Under 0 °C and an argon atmosphere, tin(IV) chloride (4.3 mL, 36.82 mmol) was added dropwise to a dichloromethane (160 mL) solution of (2R,3R,4R)-3,4-bis(benzyloxy)-2-((benzyloxy)methyl)-5-methoxytetrahydrofuran (16.0 g, 36.82 mmol). After addition, the reaction continued for 24 hours. The reaction solution was poured into a saturated sodium bicarbonate solution and stirred, filtered through diatomaceous earth, separated, the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, rotary evaporated, and the resulting residue was purified by column chromatography (petroleum ether:ethyl acetate = 3:1) to obtain the title compound as a pale yellow oil (9.5 g, 73%). LCMS [M+NH 4 + : 362.2. 13C-NMR (500 MHz, CD 3 Cl) δ 137.9, 137.8, 128.5, 128.4, 127.9, 127.8, 127.7, 127.6, 103.0, 82.0, 76.4, 73.5, 73.0, 71.8, 70.0, 55.6.

[0122] 4) (2S,4R,5R)-4-(Benzyloxy)-5-((benzyloxy)methyl)-2-methoxytetrahydrofuran-3(2H)-one

[0123]

[0124] At 0 °C, Dess-Martin periodinane (20.0 g mL, 46.89 mmol) was added to a solution of (2S,3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-methoxytetrahydrofuran-3-ol (9.5 g, 27.58 mmol) in dichloromethane (100 mL), and then the mixture was allowed to warm to room temperature and react for 24 h. Methyl tert-butyl ether (100 mL) was added to the reaction mixture and stirred. The mixture was filtered through diatomaceous earth and rinsed with methyl tert-butyl ether. The filtrate was washed successively with sodium thiosulfate solution, saturated sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give the title compound as a pale yellow oil (9.0 g, 95%). LCMS [M+NH 4 + : 360.2.

[0125] 5) (2S,3R,4R,5R)-3-Allyl-4-(benzyloxy)-5-((benzyloxy)methyl)-2-methoxytetrahydrofuran-3-ol

[0126]

[0127] At -78 °C under an argon atmosphere, allylmagnesium bromide (266 mL, 266 mmol, 1 M in THF) was added dropwise to a solution of (2S,3R,4R,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-methoxydihydrofuran-3(2H)-one (30.4 g, 88.8 mmol) in tetrahydrofuran (240 mL). After stirring at -78 °C for 4 h, the mixture was warmed to room temperature and reacted for 2 h. The reaction was quenched by dropwise addition of saturated ammonium chloride solution, water (300 mL) was added, and the mixture was extracted with ethyl acetate (300 mL x 2). The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated in vacuo, and the resulting residue was purified by column chromatography (petroleum ether:ethyl acetate = 4:1) to give the title compound as a colorless oil (22.2 g, 65%). LCMS [M+NH 4 + ​: 402.2.

[0128] 6) (3R,4R,5R)-3-Allyl-4-(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2,3-diol

[0129]

[0130] Dissolve (2S,3R,4R,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-methoxydihydrofuran-3(2H)-one (22.13 g, 57.5 mmol) in formic acid (326 mL) and water (81 mL), heat to 60 °C and react overnight. After the reaction solution is cooled to room temperature, most of the solvent is removed by concentration under reduced pressure. Add water (100 mL), extract with dichloromethane (200 mL x 2), combine the organic layers, wash with saturated brine and saturated sodium bicarbonate solution, then dry over anhydrous sodium sulfate, evaporate to dryness. The obtained residue is purified by column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain the title compound as a pale yellow oil (18.6 g, 87%). LCMS [M+H] + : 371.2.

[0131] 7) (2S,3R,4R,5R)-3-Allyl-4-(benzyloxy)-5-((benzyloxy)methyl)-2-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3-ol

[0132]

[0133] Under an argon atmosphere at 0 °C, add tributylphosphine (25 mL, 121.1 mmol) and 1,1'-azobis(N,N'-dimethylformamide) (28 g, 111.1 mmol) to a solution of (3R,4R,5R)-3-allyl-4-(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2,3-diol (28 g, 75.7 mmol) in acetonitrile (1.26 L). After addition, warm to room temperature and stir. Meanwhile, add 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (13.9 g, 90.8 mmol) and DMF (80 mL) to another round-bottom flask, cool to 0 °C, add sodium hydride (3.6 g, 90.8 mmol) portionwise, continue stirring for 30 minutes, and add this mixture to the above reaction. The resulting mixture is stirred at 45 °C overnight. Cool to 0 °C, quench the reaction with 1N HCl, add water (1 L), extract with ethyl acetate (1 L x 2), combine the organic layers, wash with saturated brine, dry over anhydrous sodium sulfate, evaporate to dryness. The obtained residue is purified by column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain the title compound as a pale yellow oil (26.4 g, 69%). LCMS [M+H] + : 506.2.1 1H NMR (400 MHz, CDCl 3 ) δ 8.61 (s, 1H), 7.68 (d, J = 2.1 Hz, 1H), 7.49–7.19 (m, 11H), 6.51 (d, J = 2.0 Hz, 1H), 6.44 (s, 1H), 5.60 (m, 1H), 4.87 (d, J = 10.1 Hz, 1H), 4.77–4.64 (m, 2H), 4.56 (m, 2H), 4.49 (d, J = 11.6 Hz, 1H), 4.28–4.20 (m, 2H), 3.84 (d, J = 11.0 Hz, 1H), 3.58 (d, J = 11.0 Hz, 1H), 3.37 (s, 1H), 2.02–1.84 (m, 2H).

[0134] 8) (2S,3R,4R,5R)-4-(Benzyloxy)-5-((benzyloxy)methyl)-2-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3-(2-hydroxyethyl)tetrahydrofuran-3-ol

[0135]

[0136] To a mixture of (2S,3R,4R,5R)-3-allyl-4-(benzyloxy)-5-((benzyloxy)methyl)-2-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3-ol (2.67 g, 5.3 mmol) in tetrahydrofuran (30 mL), tert-butanol (30 mL) and water (5 mL) was added potassium osmate dihydrate (98 mg, 0.27 mmol) and NMO (2.48 g, 10.6 mmol). The resulting mixture was reacted at room temperature for 16 h. The solvent was evaporated, and the residue was co-evaporated twice with toluene. The resulting residue was dissolved in tetrahydrofuran (30 mL) and water (5 mL), and sodium periodate (1.72 g, 7.95 mmol) was added. The mixture was stirred at room temperature for 3 h. The reaction solution was poured into water (30 mL), and extracted with ethyl acetate (40 mL x 2). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, evaporated, and the resulting residue was dissolved in ethanol (30 mL). The solution was cooled to 0 °C, and sodium borohydride (302 mg, 7.95 mmol) was added. The reaction was carried out at 0 °C for 1 h. The solvent was evaporated, water (30 mL) was added, and the mixture was extracted with ethyl acetate (30 mL x 3). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, evaporated, and the resulting residue was purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to give the title compound as a colorless oil (1.75 g, 65%). LCMS [M+H] + : 510.2.

[0137] (4R,5R,7R,8R)-8-(Benzyloxy)-7-((benzyloxy)methyl)-1,6-dioxaspiro[3.4]octan-5-yl)-4-chloro-7H-pyrrolo[2,3-d]pyrimidine

[0138]

[0139] To a solution of (2S,3R,4R,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3-(2-hydroxyethyl)tetrahydrofuran-3-ol (1.75, 3.44 mmol) in dichloromethane (30 mL) was added methanesulfonyl chloride (787 mg, 6.88 mmol), and the resulting mixture was stirred at room temperature for 3 hours. The solvent was evaporated, and the resulting residue was purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to give 1.72 g of the mesylate intermediate. The mesylate intermediate was dissolved in tetrahydrofuran (30 mL), protected by argon, cooled to 0 °C, and sodium hydride (351 mg, 8.78 mmol) was added portionwise. The resulting mixture was reacted at room temperature for 1 hour.

[0140] The reaction solution was poured into water (30 mL), extracted with ethyl acetate (30 mL x 2), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, the solvent was evaporated, and the resulting residue was purified by column chromatography (petroleum ether:ethyl acetate = 2:1) to give the title compound as a pale yellow oil (1.16 g, 69%). LCMS [M+H] + : 492.2.

[0141] (4R,5R,7R,8R)-8-(Benzyloxy)-7-((benzyloxy)methyl)-1,6-dioxaspiro[3.4]octan-5-yl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidine

[0142]

[0143] Under an argon atmosphere, tetrakis(triphenylphosphine)palladium (1.2 g, 0.69 mmol) and trimethylaluminum (10 mL, 10 mmol, 1 M in hexanes) were added to a solution of (4R,5R,7R,8R)-8-(benzyloxy)-7-((benzyloxy)methyl)-1,6-dioxaspiro[3.4]octan-5-yl)-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (3.5 g, 6.9 mmol) in tetrahydrofuran (70 mL). The resulting mixture was heated to reflux for 3 hours. After cooling in an ice bath, the reaction was quenched by dropwise addition of 1 N HCl, and the mixture was extracted with ethyl acetate. The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The resulting residue was purified by column chromatography (ethyl acetate) to give the title compound as a pale yellow oil (2.99 g, 89%). LCMS [M+H] + : 472.2.

[0144] 11) (4R,5R,7R,8R)-7-(Hydroxymethyl)-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol

[0145]

[0146] 10% palladium on carbon (300 mg, containing about 50% water) was added to an ethanol solution (15 mL) of (4R,5R,7R,8R)-8-(benzyloxy)-7-((benzyloxy)methyl)-1,6-dioxaspiro[3.4]octan-5-yl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidine (450 mg, 0.95 mmol). The reaction system was purged with hydrogen three times and heated to 70 °C for reaction overnight. After the reaction solution was cooled, it was concentrated under reduced pressure. The resulting residue was purified by column chromatography (DCM:MeOH = 10:1) to give the title compound as a white solid (248 mg, 89%). LCMS [M+H] + : 292.1. 1 H NMR (500 MHz, CD 3 OD) δ 8.69 (s, 1H), 7.70 (d, J = 3.6 Hz, 1H), 6.76 (d, J = 3.7 Hz, 1H), 6.66 (s, 1H), 4.51 (t, J = 7.6 Hz, 2H), 4.36 (d, J = 9.4 Hz, 1H), 3.98 (d, J = 11.1 Hz, 1H), 3.820–3.78 (m, 2H), 2.73 (s, 3H), 2.54–2.49 (m, 1H), 1.99–1.94 (m, 1H).

[0147] (4R,5R,7R,8R)-7-(Hydroxymethyl)-5-(5-iodo-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol

[0148] Under an argon atmosphere, NIS (1.16 g, 5.15 mmol) was added to a solution of (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol (1 g, 3.43 mmol) in DMF (20 mL). The reaction solution was heated to 50 °C and stirred overnight. After the reaction solution was cooled, a saturated solution of sodium thiosulfate was added to quench the reaction. Water (20 mL) was added, and the mixture was extracted with ethyl acetate (30 mL x 3). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (DCM:MeOH = 10:1) to give the title compound as a white solid (1.3 g, 91%). LCMS [M+H] + : 418.1. 1 H NMR (500 MHz, CD 3 OD) δ 8.68 (s, 1H), 7.96 (s, 1H), 6.64 (s, 1H), 5.50 (d, J = 15.0 Hz, 1H), 4.52 (dd, J = 11.7, 7.6 Hz, 2H), 4.33 (d, J = 8.8 Hz, 1H), 3.98 (d, J = 10.3 Hz, 1H), 3.80 (d, J = 9.6 Hz, 2H), 2.96 (s, 3H), 2.52 (dt, J = 11.8, 7.8 Hz, 1H), 2.15–1.85 (m, 1H).

[0149] Example 2 Compound 2 (4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol

[0150]

[0151] To a solution of (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(5-iodo-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol (267 mg, 0.64 mmol) in anhydrous tetrahydrofuran (5 mL) was added Pd(PPh 3 ) 2 Cl 2(45 mg, 0.064 mmol), CuI (24 mg, 0.128 mmol), 1-bromo-4-ethynyl-2-fluorobenzene (166 mg, 0.83 mmol) and triethylamine (194 mg, 1.92 mmol). After the reaction system was purged with argon, it was heated to 50 °C and reacted overnight. The reaction solution was poured into water (10 mL), extracted with ethyl acetate (15 mL x 2), the organic layers were combined, dried over anhydrous sodium sulfate, concentrated in vacuo, and the resulting residue was purified by column chromatography (dichloromethane:methanol = 50:1 to 20:1) to give the title compound as a white solid (253 mg, 81%). LCMS [M+H] + : 488.1. 1 H NMR (500 MHz, CD 3 OD) δ 8.75 (s, 1H), 8.12 (s, 1H), 7.65 (t, J = 7.8 Hz, 1H), 7.37 (dd, J = 9.3, 1.6 Hz, 1H), 7.26 (d, J = 8.3 Hz, 1H), 6.69 (s, 1H), 4.61 (s, 2H), 4.54 (m, 2H), 4.37 (d, J = 9.2 Hz, 1H), 4.01 (d, J = 10.8 Hz, 1H), 3.82 (m, 2H), 2.99 (s, 3H), 2.58 - 2.53 (m, 1H), 2.09–2.04 (m, 1H).

[0152] Example 3 Compound 3 (4R,5R,7R,8R)-5-(5-((4-bromo-2-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol

[0153]

[0154] The preparation method of Compound 3 was referred to Example 2, except that 1-bromo-4-ethynyl-3-fluorobenzene was used instead of 1-bromo-4-ethynyl-2-fluorobenzene, and the product was obtained as a white solid (210 mg, 78%). LCMS [M+H] + : 488.1. 11H NMR (500 MHz, CDOD) δ 8.79 (s, 1H), 7.64 (s, 1H), 7.37 (t, J = 7.8 Hz, 1H), 7.32 (t, J = 7.8 Hz, 2H), 7.26 (s, 1H), 6.39 (s, 1H), 4.68–4.44 (m, 2H), 4.08 (d, J = 11.9 Hz, 1H), 4.00–3.84 (m, 2H), 3.49 (s, 1H), 3.02 (s, 3H), 2.74–2.62 (m, 1H), 2.14–1.98 (m, 1H).

[0155] Example 4 Compound 4 (4R,5R,7R,8R)-5-(5-((4-Bromo-3-fluorophenyl)ethynyl)-4-methoxy-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol

[0156]

[0157] 1) (4R,5R,7R,8R)-8-(Benzyloxy)-7-((benzyloxy)methyl)-1,6-dioxaspiro[3.4]octan-5-yl)-4-methoxy-7H-pyrrolo[2,3-d]pyrimidine

[0158] To a solution of (4R,5R,7R,8R)-8-(benzyloxy)-7-((benzyloxy)methyl)-1,6-dioxaspiro[3.4]octan-5-yl)-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (491 mg, 1.0 mmol) in methanol (6 mL) was added sodium methoxide (270 mg, 5.0 mmol). The resulting mixture was stirred at room temperature for 20 h. The reaction was neutralized with acetic acid and concentrated in vacuo. The residue obtained was purified by column chromatography (ethyl acetate) to give the title compound as a pale yellow oil (428 mg, 88%). LCMS [M+H] + : 488.2.

[0159] 2) (4R,5R,7R,8R)-7-(Hydroxymethyl)-5-(5-iodo-4-methoxy-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol (4R,5R,7R,8R)-7-(Hydroxymethyl)-5-(5-iodo-4-methoxy-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol was prepared with reference to Example 1, except that (4R,5R,7R,8R)-8-(benzyloxy)-7-((benzyloxy)methyl)-1,6-dioxaspiro[3.4]octan-5-yl)-4-methoxy-7H-pyrrolo[2,3-d]pyrimidine was used instead of (4R,5R,7R,8R)-8-(benzyloxy)-7-((benzyloxy)methyl)-1,6-dioxaspiro[3.4]octan-5-yl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidine, and the title compound was obtained as a white solid (309 mg, 81%). LCMS [M+H] + : 434.0.

[0160] 3) (4R,5R,7R,8R)-5-(5-((4-Bromo-2-fluorophenyl)ethynyl)-4-methoxy-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol

[0161] The preparation method of Compound 4 was referred to Example 2, except that (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(5-iodo-4-methoxy-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol was used instead of (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(5-iodo-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol, and the title compound was obtained as a white solid (55 mg, 87%). LCMS [M+H] + : 504.0. 1 H NMR (500 MHz, CDCl 3)δ8.49(s,1H),7.54(t,J=7.6Hz,1H),7.49(s,1H),7.28(d,J=8.9Hz,1H),7.20(d,J=8.1Hz,1H),6.31(s,1H),4.57(s,1H),4.50(dd,J=16.0,8.3Hz,2H),4.19(s,3H),4.07(d,J=12.3Hz,1H),3.96(d,J=6.2Hz,1H),3.89(d,J=12.1Hz,1H),3.06–2.93(m,1H),2.72–2.60(m,1H),2.13–1.99(m,1H),1.68(s,2H).

[0162] Example 5 Compound 5 (4R,5R,7R,8R)-5-(5-((4-Bromo-2-fluorophenyl)ethynyl)-4-methoxy-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol

[0163]

[0164] The preparation method of Compound 5 refers to Example 4, except that 1-bromo-4-ethynyl-3-fluorobenzene is used instead of 1-bromo-4-ethynyl-2-fluorobenzene, and the product is a white solid (51 mg, 78%). LCMS [M+H] + : 504.0. 1 H NMR(500MHz,CDCl 3 )δ8.46(s,1H),7.49(s,1H),7.37(t,J=7.8Hz,1H),7.29(m,2H),6.30(s,1H),4.55(d,J=6.8Hz,1H),4.53–4.42(m,2H),4.16(s,3H),4.06(dd,J=12.5,2.0Hz,1H),3.94(d,J=6.9Hz,1H),3.87(dd,J=12.5,2.0Hz,1H),3.02(s,1H),2.77–2.53(m,1H),2.03(m,1H).

[0165] Example 6 Compound 6 (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methyl-5-(pyrimidin-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol

[0166]

[0167] To a solution of (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(5-iodo-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol (120 mg, 0.29 mmol) in DMF (3 mL) was added Pd(PPh 3 ) 2 Cl 2 (20 mg, 0.029 mmol) and 2-(tributylstannyl)pyrimidine (321 mg, 0.87 mmol). After the reaction system was purged with argon, it was heated to 100 °C and reacted overnight. After the reaction solution was cooled to room temperature and concentrated in vacuo, the resulting residue was purified by column chromatography (dichloromethane:methanol = 50:1 to 20:1) to give Compound 6 as a white solid (45 mg, 42%). LCMS [M+H] + : 370.1. 1 H NMR (500 MHz, MeOD) δ 8.84 (d, J = 3.6 Hz, 2H), 8.76 (s, 1H), 8.44 (s, 1H), 7.35 (t, J = 4.1 Hz, 1H), 6.79 (s, 1H), 4.55 (dd, J = 14.7, 7.0 Hz, 2H), 4.42 (d, J = 9.1 Hz, 1H), 4.01 (d, J = 12.2 Hz, 1H), 3.90–3.74 (m, 2H), 3.03 (s, 3H), 2.65–2.51 (m, 1H), 2.08 (dd, J = 19.4, 7.9 Hz, 1H).

[0168] Example 7 Compound 7 (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methyl-5-(thiazol-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol

[0169]

[0170] The preparation method of Compound 7 was referred to Example 6, except that 2-(tributylstannyl)thiazole was used instead of 2-(tributylstannyl)pyrimidine, and the product was obtained as a white solid (70 mg, 64%). LCMS [M+H] + : 375.1. 1 H NMR (500 MHz, CDCl 3)δ8.79(s,1H),7.87(d,J=3.3Hz,1H),7.79(s,1H),7.32(d,J=3.3Hz,1H),6.50(s,1H),4.66–4.42(m,3H),4.06(d,J=11.2Hz,2H),3.98–3.85(m,2H),3.27(s,1H),2.88(s,3H),2.64(dt,J=12.2,8.2Hz,1H),2.17–1.88(m,1H).

[0171] Example 8 Compound 8 (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methoxy-5-(pyrimidin-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol

[0172]

[0173] The preparation method of Compound 8 refers to Example 6, except that (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(5-iodo-4-methoxy-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol is used instead of (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(5-iodo-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol, and the product is a white solid (61 mg, 69%). LCMS [M+H] + : 386.1. 1 H NMR(500MHz,CDCl 3 )δ8.70(d,J=4.6Hz,1H),8.50(s,1H),8.01(s,1H),7.07(t,J=4.5Hz,1H),6.50(s,1H),4.70(s,1H),4.51(t,J=7.3Hz,2H),4.12(s,3H),4.06(d,J=12.5Hz,1H),3.89(dd,J=23.6,9.9Hz,2H),3.25(d,J=7.6Hz,1H),2.71–2.62(m,1H),2.15–1.95(m,2H).

[0174] Example 9 Compound 9 (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methoxy-5-(thiazol-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol

[0175]

[0176] The preparation method of Compound 9 was referred to Example 7, except that (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(5-iodo-4-methoxy-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol was used instead of (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(5-iodo-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol, and the product obtained was a white solid (64 mg, 71%). LCMS [M+H] + : 391.1. 1 H NMR (500 MHz, CDCl 3 ) δ 8.48 (s, 1H), 8.02 (s, 1H), 7.60 (d, J = 3.2 Hz, 1H), 7.13 (d, J = 3.1 Hz, 1H), 6.54 (s, 1H), 4.73 (s, 1H), 4.53 (t, J = 7.6 Hz, 2H), 4.17 (s, 3H), 4.09 (d, J = 11.0 Hz, 1H), 3.90 (t, J = 18.3, 5.6 Hz, 2H), 3.47 (s, 1H), 2.83–2.61 (m, 1H), 2.21–2.04 (m, 1H).

[0177] Example 10 Compound 10 (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methyl-5-(thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol

[0178]

[0179] To a solution of (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(5-iodo-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol (100 mg, 0.24 mmol) in water (2 mL) and acetonitrile (1 mL) were added thiophene-2-boronic acid (46 mg, 0.36 mmol), palladium(II) acetate (5.4 mg, 0.024 mmol), trisodium salt of triphenylphosphine trisulfonate (25 mg, 0.048 mmol) and sodium carbonate (76 mg, 0.72 mmol). After the reaction system was purged with argon, it was heated to 100 °C and reacted for 3 hours. After the reaction solution was cooled to room temperature, it was neutralized with 1N HCl, concentrated in vacuo, and the resulting residue was dissolved in dichloromethane and methanol. The insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography (dichloromethane:methanol = 50:1 to 20:1) to give compound 10 as a white solid (62 mg, 69%). LCMS [M+H] + : 374.1. 1 H NMR (500 MHz, CD 3 OD) δ 8.69 (s, 1H), 8.03 (s, 1H), 7.47 (d, J = 5.2 Hz, 1H), 7.18 (d, J = 2.6 Hz, 1H), 7.14 (dd, J = 5.1, 3.6 Hz, 1H), 6.49 (s, 1H), 4.32 (d, J = 9.0 Hz, 1H), 4.10–3.94 (m, 2H), 3.83 (dd, J = 12.6, 2.8 Hz, 1H), 3.67 (td, J = 11.2, 5.6 Hz, 1H), 3.56–3.44 (m, 1H), 2.60 (s, 3H), 1.85–1.71 (m, 1H), 1.59–1.50 (m, 1H).

[0180] Example 11 Compound 11 (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methyl-5-(thiophen-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol

[0181]

[0182] The preparation method of compound 11 was referred to Example 10, except that thiophene-3-boronic acid was used instead of thiophene-2-boronic acid, and the product was obtained as a white solid (66 mg, 73%). LCMS [M+H] + : 374.1. 1 H NMR (500 MHz, CDCl 3) δ 8.77 (s, 1H), 7.39 (s, 2H), 7.11 (d, J = 10.0 Hz, 2H), 6.40 (s, 1H), 4.61 (s, 1H), 4.57 (s, 2H), 4.06 (d, J = 11.2 Hz, 1H), 4.00–3.80 (m, 2H), 2.70 (s, 1H), 2.63 (s, 3H), 2.15 (s, 1H), 1.83 (s, 2H).

[0183] Example 12 Compound 12 (4R,5R,7R,8R)-5-(5-(Furan-3-yl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol

[0184]

[0185] The preparation method of Compound 12 refers to Example 10, except that furan-3-boronic acid is used instead of thiophene-2-boronic acid, and the product is a white solid (59 mg, 69%). LCMS [M+H] + : 358.1. 1 H NMR (500 MHz, CDCl 3 ) δ 8.72 (s, 1H), 7.51 (s, 2H), 7.30 (s, 1H), 6.51 (s, 1H), 6.43 (s, 1H), 4.64–4.43 (m, 3H), 4.02 (d, J = 12.3 Hz, 1H), 3.87 (dd, J = 24.8, 10.0 Hz, 2H), 2.70–2.59 (m, 4H), 2.15–2.06 (m, 1H), 1.80 (s, 2H).

[0186] Example 13 Compound 13 (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methyl-5-(1-methyl-1H-pyrazol-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol

[0187]

[0188] The preparation method of Compound 13 refers to Example 10, except that (1-methyl-1H-pyrazol-3-yl)boronic acid is used instead of thiophene-2-boronic acid, and the product is a white solid (56 mg, 63%). LCMS [M+H] + : 372.2. 1 H NMR (500 MHz, CDCl 3)δ8.74(s,1H),7.48(s,1H),7.40(d,J=1.2Hz,1H),6.42(s,1H),6.34(d,J=1.5Hz,1H),4.63(d,J=6.9Hz,1H),4.48(t,J=7.6Hz,2H),4.21(s,1H),4.04(d,J=12.4Hz,1H),4.00–3.82(m,5H),3.19(s,1H),2.75(s,3H),2.64(dt,J=12.2,7.7Hz,1H),2.09(dt,J=12.2,7.9Hz,1H).

[0189] Example 14 Compound 14 (4R,5R,7R,8R)-7-(Hydroxymethyl)-5-(4-methyl-5-(1-methyl-1H-pyrazol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol

[0190]

[0191] The preparation method of Compound 14 refers to Example 10, except that (1-methyl-1H-pyrazol-5-yl)boronic acid is used instead of thiophene-2-boronic acid, and the product is a white solid (61 mg, 69%). LCMS [M+H] + : 372.2. 1 H NMR(500MHz,CDCl 3 )δ8.75(d,J=2.6Hz,1H),7.59(d,J=3.4Hz,1H),7.52(s,1H),6.58(d,J=3.2Hz,1H),6.30(s,1H),4.52(dd,J=18.0,5.7Hz,2H),4.41(d,J=4.1Hz,1H),3.99(d,J=11.6Hz,1H),3.91–3.75(m,2H),3.66(d,J=3.4Hz,3H),2.64–2.54(m,1H),2.37(d,J=3.3Hz,3H),2.12–2.00(m,1H).

[0192] Example 15 Compound 15 (4R,5R,7R,8R)-7-(Hydroxymethyl)-5-(4-methyl-5-(1H-pyrazol-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol

[0193]

[0194] The preparation method of Compound 15 refers to Example 10, except that 1H-pyrazole-3-boronic acid is used instead of thiophene-2-boronic acid, and the product is a white solid (42 mg, 49%). LCMS [M+H] + : 358.1. 1 H NMR (500 MHz, CDCl 3 ) δ 8.75 (s, 1H), 7.63 (d, J = 1.7 Hz, 1H), 7.58 (s, 1H), 6.69 (s, 1H), 6.45 (d, J = 1.7 Hz, 1H), 4.60 (dd, J = 14.8, 6.5 Hz, 1H), 4.56–4.47 (m, 2H), 4.07 (d, J = 11.8 Hz, 1H), 3.94–3.82 (m, 2H), 3.44 (s, 1H), 2.83–2.40 (m, 6H), 2.14–2.05 (m, 1H).

[0195] Example 16 Compound 16 (4R,5R,7R,8R)-5-(5-(benzofuran-2-yl)-4-methyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol

[0196]

[0197] The preparation method of Compound 16 refers to Example 10, except that benzofuran-2-boronic acid is used instead of thiophene-2-boronic acid, and the product is a white solid (69 mg, 71%). LCMS [M+H] + : 408.2. 1 H NMR (500 MHz, CDCl 3 ) δ 8.81 (s, 1H), 7.70 (s, 1H), 7.61 (d, J = 7.4 Hz, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.39–7.25 (m, 2H), 6.88 (s, 1H), 6.46 (s, 1H), 4.62 (t, J = 7.1 Hz, 1H), 4.53 (t, J = 7.5 Hz, 2H), 4.09 (d, J = 12.1 Hz, 1H), 4.02–3.85 (m, 2H), 3.77 (s, 1H), 3.04 (s, 1H), 2.85 (s, 3H), 2.78–2.60 (m, 1H), 2.14 (dt, J = 11.1, 7.9 Hz, 1H).

[0198] Example 17 Compound 17 (4R,5R,7R,8R)-7-(Hydroxymethyl)-5-(4-methoxy-5-(thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol

[0199]

[0200] The preparation method of Compound 17 refers to Example 10, except that (4R,5R,7R,8R)-7-(Hydroxymethyl)-5-(5-iodo-4-methoxy-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol is used instead of (4R,5R,7R,8R)-7-(Hydroxymethyl)-5-(5-iodo-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol, and the product is a white solid (75 g, 83%). LCMS [M+H] + : 390.1. 1 H NMR (400 MHz, CDCl 3 ) δ 8.46 (s, 1H), 7.46 (d, J = 2.9 Hz, 1H), 7.37 (s, 1H), 7.33–7.19 (m, 1H), 7.08 (t, J = 4.2 Hz, 1H), 6.30 (s, 1H), 4.62 (d, J = 6.2 Hz, 1H), 4.59–4.36 (m, 2H), 4.14 (s, 3H), 4.07 (d, J = 12.3 Hz, 1H), 3.95 (d, J = 6.5 Hz, 1H), 3.88 (d, J = 12.4 Hz, 1H), 3.02 (s, 1H), 2.67 (dt, J = 12.1, 7.7 Hz, 1H), 2.12 (dt, J = 11.8, 8.1 Hz, 1H).

[0201] Example 18 Compound 18 (4R,5R,7R,8R)-5-(5-(Furan-2-yl)-4-methoxy)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol

[0202]

[0203] The preparation method of Compound 18 refers to Example 17, except that furan-2-boronic acid is used instead of thiophene-2-boronic acid, and the product is a white solid (72 mg, 83%). LCMS [M+H] + : 374.1. 1 H NMR (500 MHz, CDCl 3)δ8.81(s,1H),7.70(s,1H),7.61(d,J=7.4Hz,1H),7.52(d,J=7.9Hz,1H),7.39–7.25(m,2H),6.88(s,1H),6.46(s,1H),4.62(t,J=7.1Hz,1H),4.53(t,J=7.5Hz,2H),4.09(d,J=12.1Hz,1H),4.02–3.85(m,2H),3.77(s,1H),3.04(s,1H),2.85(s,3H),2.78–2.60(m,1H),2.14(dt,J=11.1,7.9Hz,1H).

[0204] Example 19 Compound 19 (4R,5R,7R,8R)-5-(5-(Furan-3-yl)-4-methoxy)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol

[0205]

[0206] The preparation method of Compound 19 refers to Example 17, except that furan-3-boronic acid is used instead of thiophene-2-boronic acid, and the product is a white solid (70 mg, 81%). LCMS [M+H] + : 374.1. 1 H NMR(500MHz,CDCl 3 )δ8.44(s,1H),7.96(s,1H),7.45(d,J=1.4Hz,1H),7.28(s,1H),6.67(d,J=0.9Hz,1H),6.31(s,1H),4.59(t,J=7.2Hz,1H),4.54–4.42(m,2H),4.29(s,1H),4.14(s,3H),4.05(dd,J=12.4,1.4Hz,1H),3.97–3.90(m,1H),3.87(d,J=10.8Hz,1H),3.10(d,J=8.1Hz,1H),2.73–2.58(m,1H),2.13–1.99(m,1H).

[0207] Example 20 Compound 20 (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methoxy-5-(thiophen-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol

[0208]

[0209] The preparation method of Compound 20 refers to Example 17, except that thiophene-3-boronic acid is used instead of thiophene-2-boronic acid, and the product is a white solid (57 mg, 63%). LCMS [M+H] + : 374.1. 1 H NMR (400 MHz, CDCl 3 ) δ 8.46 (s, 1H), 7.69 (s, 1H), 7.45–7.30 (m, 3H), 6.32 (s, 1H), 4.63 (d, J = 5.2 Hz, 1H), 4.49 (dd, J = 16.7, 8.3 Hz, 2H), 4.15 (s, 3H), 4.07 (d, J = 12.3 Hz, 1H), 3.96 (d, J = 6.3 Hz, 1H), 3.88 (d, J = 12.4 Hz, 1H), 2.99 (s, 1H), 2.79–2.56 (m, 1H), 2.11 (m, 1H).

[0210] Example 21 Compound 21 (4R,5R,7R,8R)-5-(5-(Benzofuran-2-yl)-4-methoxy)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol

[0211]

[0212] The preparation method of Compound 21 refers to Example 17, except that benzofuran-2-boronic acid is used instead of thiophene-2-boronic acid, and the product is a white solid (71 mg, 73%). LCMS [M+H] + : 424.1. 1 H NMR (400 MHz, CDCl 3 ) δ 8.49 (s, 1H), 7.72 (s, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.36 (s, 1H), 7.32–7.16 (m, 2H), 6.38 (s, 1H), 4.63 (d, J = 7.0 Hz, 1H), 4.57–4.43 (m, 2H), 4.22 (s, 3H), 4.09 (dd, J = 12.3, 1.6 Hz, 1H), 4.02–3.85 (m, 2H), 2.68 (dt, J = 12.3, 8.1 Hz, 1H), 2.09 (dt, J = 12.2, 7.9 Hz, 1H).

[0213] Example 22 Compound 22 (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methoxy-5-(1-methyl-1H-pyrazol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol

[0214]

[0215] The preparation method of Compound 22 refers to Example 17, except that (1-methyl-1H-pyrazol-5-yl)boronic acid is used instead of thiophene-2-boronic acid, and the product is a white solid (66 mg, 74%). LCMS [M+H] + : 388.2. 1 H NMR (500 MHz, CDCl 3 ) δ 8.50 (s, 1H), 7.53 (d, J = 1.7 Hz, 1H), 7.32 (s, 1H), 6.47–6.25 (m, 2H), 4.61 (s, 1H), 4.52 (dd, J = 15.3, 6.9 Hz, 2H), 4.13–4.03 (m, 4H), 3.97 (d, J = 6.8 Hz, 1H), 3.89 (d, J = 11.6 Hz, 1H), 3.80 (d, J = 4.7 Hz, 3H), 3.03 (s, 1H), 2.76–2.58 (m, 1H), 2.22–2.08 (m, 1H).

[0216] Example 23 Compound 23 (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methoxy-5-(1-methyl-1H-pyrazol-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol

[0217]

[0218] The preparation method of Compound 23 refers to Example 17, except that (1-methyl-1H-pyrazol-3-yl)boronic acid is used instead of thiophene-2-boronic acid, and the product is a white solid (59 mg, 66%). LCMS [M+H] + : 388.2. 1 H NMR (400 MHz, CDCl 3)δ8.50(s,1H),7.51(s,1H),7.34(s,1H),6.39(s,1H),6.34(s,1H),4.60(d,J=6.9Hz,1H),4.52(t,J=7.6Hz,2H),4.14–4.02(m,4H),3.96(d,J=6.9Hz,1H),3.89(d,J=12.4Hz,1H),3.79(s,3H),3.12(s,1H),2.68(dt,J=12.3,7.6Hz,1H),2.13(dt,J=12.1,7.8Hz,1H),1.85(s,1H).

[0219] Example 24 Compound 24 (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methoxy-5-(1H-pyrazol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol

[0220]

[0221] The preparation method of Compound 24 refers to Example 17, except that 1H-pyrazole-5-boronic acid is used instead of thiophene-2-boronic acid, and the product is a white solid (39 mg, 45%). LCMS [M+H] + : 374.1. 1 H NMR (500 MHz, CDCl 3 , CD 3 OD) δ8.46(s,1H),7.82(s,1H),7.59(s,1H),6.70(d,J=24.8Hz,2H),4.63–4.48(m,2H),4.35(d,J=8.9Hz,1H),4.17(s,3H),4.01(d,J=10.7Hz,1H),3.90–3.78(m,2H),2.56(dt,J=11.8,8.2Hz,1H),2.12–1.93(m,1H).

[0222] Example 25 Compound 25 (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methoxy-5-(1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol

[0223]

[0224] The preparation method of Compound 25 was referred to Example 17, except that 1H-pyrazole-4-boronic acid was used instead of thiophene-2-boronic acid, and the product was obtained as a white solid (44 mg, 51%). LCMS [M+H] + : 374.1. 1 H NMR (500 MHz, CDCl 3 , CD 3 OD) δ 8.41 (s, 1H), 7.92 (s, 2H), 7.62 (s, 1H), 6.62 (s, 1H), 4.61–4.39 (m, 2H), 4.37 (d, J = 8.7 Hz, 1H), 4.14 (s, 3H), 4.02 (dd, J = 13.3, 3.0 Hz, 1H), 3.83 (d, J = 9.0 Hz, 2H), 2.53 (m, 1H), 2.16–1.88 (m, 1H).

[0225] Example 26 Compound 26 ((S)-(((4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-8-hydroxy-1,6-dioxaspiro[3.4]octan-7-yl)methoxy(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0226]

[0227] Under -20 °C and argon atmosphere, to a solution of (4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol (60 mg, 0.12 mmol) in anhydrous tetrahydrofuran (2 mL) was added tert-butylmagnesium chloride (0.36 mL, 0.36 mmol, 1.0 M in THF), and the mixture was stirred at room temperature for 20 minutes. Then a solution of N-((S)-(pentafluorophenoxy)(phenoxy)phosphoryl)-L-alanine isopropyl ester (72 mg, 0.16 mmol) in tetrahydrofuran (0.5 mL) was added dropwise, and the reaction was continued at room temperature overnight. The reaction was quenched by adding methanol (1 mL), the reaction solution was poured into water (10 mL), and extracted with ethyl acetate (10 mL x 2). The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by thick preparative plate (dichloromethane:methanol = 20:1) to obtain the title compound as a white solid (47 mg, 51%). LCMS [M+H] + : 757.3. 1 H NMR (500 MHz, CDCl 3)δ8.83(s,1H),7.68(m,1H),7.51(m,1H),7.42–7.22(m,6H),7.15(t,J=7.0Hz,1H),6.72(s,1H),5.02(m,1H),4.62(m,3H),4.41(m,2H),4.08–3.95(m,2H),3.91(t,J=10.4Hz,1H),3.61(s,1H),3.02(s,3H),2.74–2.52(m,1H),2.15–1.95(m,1H),1.36(d,J=6.9Hz,3H),1.18(d,J=6.1Hz,6H).

[0228] Example 27 Compound 27 ((S)-(((4R,5R,7R,8R)-5-(5-((4-bromo-2-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-8-hydroxy-1,6-dioxaspiro[3.4]octan-7-yl)methoxy(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0229]

[0230] The preparation method of Compound 27 refers to Example 26, except that (4R,5R,7R,8R)-5-(5-((4-bromo-2-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol is used instead of (4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol, and the product is a white solid (46 mg, 50%). LCMS [M+H] + : 757.3. 1 H NMR (500 MHz, CDCl 3 )δ8.82(s,1H),7.63(s,1H),7.40–7.19(m,7H),7.11(t,J=7.0Hz,1H),6.69(s,1H),4.96(dt,J=12.4,6.2Hz,1H),4.58(m,3H),4.38(m,2H),4.05–3.93(m,2H),3.88(t,J=10.4Hz,1H),3.60(s,1H),3.00(s,3H),2.69–2.56(m,1H),2.13–1.93(m,1H),1.35(d,J=6.9Hz,3H),1.17(d,J=6.1Hz,6H).

[0231] Example 28 Compound 28 ((S)-(((4R,5R,7R,8R)-8-hydroxy-5-(4-methyl-5-(pyrimidin-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-7-yl)methoxy(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0232]

[0233] The preparation method of Compound 28 refers to Example 26, except that (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methyl-5-(pyrimidin-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol is used instead of (4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol, and the product is a white solid (54 mg, 52%). LCMS [M+H] + : 639.3. 1 H NMR (500 MHz, CDCl 3 ) δ 8.84 (s, 1H), 8.76 (d, J = 4.7 Hz, 2H), 8.32 (s, 1H), 7.22 (t, J = 7.6 Hz, 2H), 7.16–7.11 (m, 3H), 7.08 (t, J = 7.2 Hz, 1H), 6.82 (s, 1H), 4.94 (dt, J = 12.4, 6.0 Hz, 1H), 4.76–4.67 (m, 2H), 4.57–4.45 (m, 4H), 4.14 (dd, J = 16.3, 8.7 Hz, 1H), 4.00 (d, J = 8.1 Hz, 1H), 3.45 (s, 1H), 3.10 (s, 3H), 2.65 (dd, J = 19.5, 8.1 Hz, 1H), 2.26–2.17 (m, 1H), 1.32 (d, J = 6.9 Hz, 3H), 1.17 (d, J = 6.1 Hz, 6H).

[0234] Example 29 Compound 29 ((S)-(((4R,5R,7R,8R)-8-hydroxy-5-(4-methyl-5-(thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-7-yl)methoxy(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0235]

[0236] The preparation method of Compound 29 was referred to Example 26, except that (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methoxy-5-(thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol was used instead of (4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol, and the product was obtained as a white solid (47 mg, 55%). LCMS [M+H] + : 659.3. 1 H NMR (500 MHz, CDCl 3 ) δ 8.80 (s, 1H), 7.35 (d, J = 3.0 Hz, 1H), 7.25 (dd, J = 17.0, 8.4 Hz, 4H), 7.13 (dd, J = 12.3, 6.6 Hz, 4H), 6.73 (s, 1H), 4.92 (dt, J = 12.4, 6.1 Hz, 1H), 4.62 (dd, J = 14.3, 6.8 Hz, 1H), 4.59–4.49 (m, 2H), 4.45–4.31 (m, 2H), 4.00–3.87 (m, 2H), 3.78 (t, J = 10.4 Hz, 1H), 2.73–2.63 (m, 1H), 2.57 (s, 3H), 2.11 (dt, J = 12.1, 7.7 Hz, 1H), 1.24 (d, J = 6.9 Hz, 3H),

[0237] 1.16 (dd, J = 14.1, 6.2 Hz, 6H).

[0238] Example 30 Compound 30 ((S)-(((4R,5R,7R,8R)-5-(5-(furan-3-yl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-8-hydroxy-1,6-dioxaspiro[3.4]octan-7-yl)methoxy(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0239]

[0240] The preparation method of Compound 30 refers to Example 26, except that (4R,5R,7R,8R)-5-(5-(furan-3-yl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol is used instead of (4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol, and the product is a white solid (41 mg, 47%). LCMS [M+H] + : 627.2. 1 H NMR (500 MHz, CDCl 3 ) δ 8.79 (s, 1H), 7.48 (d, J = 16.4 Hz, 2H), 7.34–7.19 (m, 3H), 7.20–7.08 (m, 3H), 6.70 (s, 1H), 6.51 (s, 1H), 4.92 (dt, J = 12.5, 6.2 Hz, 1H), 4.61 (dd, J = 14.9, 6.7 Hz, 1H), 4.57–4.49 (m, 2H), 4.43–4.28 (m, 2H), 3.91 (q, J = 8.2 Hz, 2H), 3.69 (t, 1H), 3.36 (s, 1H), 2.71–2.59 (m, 4H), 2.13–2.04 (m, 1H), 1.83 (s, 1H), 1.25 (d, J = 7.0 Hz, 3H), 1.16 (dd, J = 12.9, 6.2 Hz, 6H).

[0241] Example 31 Compound 31 ((S)-(((4R,5R,7R,8R)-8-hydroxy-5-(4-methoxy-5-(1-methyl-1H-pyrazol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-7-yl)methoxy(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0242]

[0243] The preparation method of Compound 31 was referred to Example 26, except that (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methyl-5-(1-methyl-1H-pyrazol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol was used instead of (4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol, and the product was a white solid (53 mg, 61%). LCMS [M+H] + : 641.3. 1 H NMR (500 MHz, CDCl 3 ) δ 8.83 (s, 1H), 7.52 (d, J = 1.7 Hz, 1H), 7.35 (s, 1H), 7.24 (t, 3H), 7.15–7.01 (m, 3H), 6.70 (s, 1H), 6.27 (d, J = 1.7 Hz, 1H), 4.91 (dt, J = 12.5, 6.3 Hz, 1H), 4.62 (dt, J = 8.6, 6.5 Hz, 1H), 4.58–4.49 (m, 2H), 4.43–4.27 (m, 2H), 3.97–3.92 (m, 1H), 3.88 (dd, J = 16.1, 8.8 Hz, 1H), 3.79–3.71 (m, 1H), 3.66 (s, 3H), 3.57 (d, J = 9.8 Hz, 1H), 2.65 (dt, J = 11.9, 8.5 Hz, 1H), 2.36 (s, 3H), 2.13–2.02 (m, 1H), 1.24 (d, J = 7.0 Hz, 3H), 1.17 (dd, J = 9.6, 6.3 Hz, 6H).

[0244] Example 32 Compound 32 ((S)-(((4R,5R,7R,8R)-8-hydroxy-5-(4-methyl-5-(1-methyl-1H-pyrazol-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-7-yl)methoxy(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0245]

[0246] The preparation method of Compound 32 was referred to Example 26, except that (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methyl-5-(1-methyl-1H-pyrazol-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol was used instead of (4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol, and the product was obtained as a white solid (55 mg, 64%). LCMS [M+H] + : 641.3. 1 H NMR (500 MHz, CDCl 3 ) δ 8.78 (s, 1H), 7.47 (s, 1H), 7.35 (d, J = 2.0 Hz, 1H), 7.24 (d, J = 7.8 Hz, 2H), 7.19–7.04 (m, 3H), 6.70 (s, 1H), 6.37 (d, J = 2.1 Hz, 1H), 4.92 (dt, J = 12.5, 6.3 Hz, 1H), 4.61 (dd, J = 14.9, 6.6 Hz, 1H), 4.52 (m, 2H), 4.47–4.30 (m, 2H), 4.04–3.75 (m, 5H), 2.77 (s, 3H), 2.62 (dt, J = 12.0, 8.5 Hz, 1H), 2.09 (dt, J = 12.0, 8.5 Hz, 1H), 1.91 (s, 3H), 1.26 (d, J = 6.9 Hz, 3H), 1.15 (dd, J = 8.7, 6.3 Hz, 6H).

[0247] Example 33 Compound 33 ((S)-(((4R,5R,7R,8R)-8-hydroxy-5-(4-methoxy-5-(thiazol-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-7-yl)methoxy(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0248]

[0249] The preparation method of Compound 33 was referred to Example 26, except that (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methyl-5-(thiazol-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol was used instead of (4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol, and the product was obtained as a white solid (52 mg, 60%). LCMS [M+H] + : 644.2. 1 H NMR (500 MHz, CDCl 3 ) δ 8.83 (s, 1H), 7.87 (d, J = 3.2 Hz, 1H), 7.73 (s, 1H), 7.32–7.18 (m, 4H), 7.19–7.04 (m, 3H), 6.72 (s, 1H), 4.92 (dt, J = 12.5, 6.2 Hz, 1H), 4.64 (dd, J = 14.7, 6.7 Hz, 1H), 4.60–4.47 (m, 2H), 4.44–4.31 (m, 2H), 4.09–3.87 (m, 3H), 3.48 (d, J = 9.8 Hz, 1H), 2.89 (s, 3H), 2.63 (dt, J = 11.9, 8.2 Hz, 1H), 2.17–2.06 (m, 1H), 1.28 (d, J = 6.3 Hz, 3H), 1.22–1.05 (m, 6H).

[0250] Example 34 Compound 34 ((S)-(((4R,5R,7R,8R)-5-(5-(benzofuran-2-yl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-8-hydroxy-1,6-dioxaspiro[3.4]octan-7-yl)methoxy(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0251]

[0252] The preparation method of Compound 34 was referred to Example 26, except that (4R,5R,7R,8R)-5-(5-(benzofuran-2-yl)-4-methyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol was used instead of (4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol, and the product was obtained as a white solid (42 mg, 50%). LCMS [M+H] + : 677.4. 1 H NMR (500 MHz, CDCl 3 ) δ 8.86 (s, 1H), 7.67 (s, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.24 (m, 4H), 7.14 (d, J = 7.9 Hz, 2H), 7.08 (t, J = 7.2 Hz, 1H), 6.85 (s, 1H), 6.76 (s, 1H), 4.90 (dt, J = 12.4, 6.2 Hz, 1H), 4.64 (dd, J = 14.6, 6.7 Hz, 1H), 4.56 (m, 2H), 4.48–4.34 (m, 2H), 3.94 (m, 2H), 3.76 (t, J = 10.4 Hz, 1H), 3.42 (s, 1H), 2.84 (s, 3H), 2.67 (dt, J = 11.9, 8.2 Hz, 1H), 2.10 (dt, J = 12.1, 8.5 Hz, 1H), 1.26 (d, J = 6.8 Hz, 3H), 1.14 (t, J = 6.5 Hz, 6H).

[0253] Example 35 Compound 35 ((S)-(((4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methoxy-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-8-hydroxy-1,6-dioxaspiro[3.4]octan-7-yl)methoxy(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0254]

[0255] The preparation method of Compound 35 was referred to Example 26, except that (4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methoxy-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol was used instead of (4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol, and the product was obtained as a white solid (51 mg, 66%). LCMS [M+H] + : 773.3. 1 H NMR (500 MHz, CDCl 3 ) δ 8.52 (s, 1H), 7.48 (t, J = 27.4 Hz, 2H), 7.35–7.20 (m, 4H), 7.20–7.03 (m, 3H), 6.65 (s, 1H), 4.97 (dt, J = 12.5, 6.2 Hz, 1H), 4.70–4.48 (m, 3H), 4.46–4.28 (m, 2H), 4.17 (s, 3H), 4.04–3.92 (m, 2H), 3.88 (t, J = 10.4 Hz, 1H), 3.51 (d, J = 9.1 Hz, 1H), 2.61 (dt, J = 11.8, 8.3 Hz, 1H), 2.09–1.93 (m, 2H), 1.34 (d, J = 7.0 Hz, 3H), 1.18 (dd, J = 5.9, 4.1 Hz, 6H).

[0256] Example 36 Compound 36 ((S)-(((4R,5R,7R,8R)-5-(5-((4-bromo-2-fluorophenyl)ethynyl)-4-methoxy-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-8-hydroxy-1,6-dioxaspiro[3.4]octan-7-yl)methoxy(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0257]

[0258] The preparation method of Compound 36 was referred to Example 26, except that (4R,5R,7R,8R)-5-(5-((4-bromo-2-fluorophenyl)ethynyl)-4-methoxy-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol was used instead of (4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol, and the product was obtained as a white solid (53 mg, 69%). LCMS [M+H] + : 773.3. 1 H NMR (500 MHz, CDCl 3 ) δ 8.52 (s, 1H), 7.47 (s, 1H), 7.32–7.21 (m, 8H), 7.12 (t, J = 7.1 Hz, 1H), 6.64 (s, 1H), 4.96 (dt, J = 12.5, 6.2 Hz, 1H), 4.61 (dd, J = 14.9, 6.5 Hz, 1H), 4.58–4.50 (m, 2H), 4.42–4.30 (m, 2H), 4.17 (s, 3H), 3.99 (dd, J = 16.0, 8.7 Hz, 1H), 3.93 (d, J = 9.0 Hz, 1H), 3.81–3.71 (m, 1H), 3.41 (s, 1H), 2.61 (dt, J = 11.8, 8.5 Hz, 1H), 2.00 (m, 1H), 1.35 (d, J = 7.0 Hz, 3H), 1.18 (dd, J = 6.2, 3.4 Hz, 6H).

[0259] Example 37 Compound 37 ((S)-(((4R,5R,7R,8R)-5-(5-(furan-3-yl)-4-methoxy-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-8-hydroxy-1,6-dioxaspiro[3.4]octan-7-yl)methoxy(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0260]

[0261] The preparation method of Compound 37 was referred to Example 26, except that (4R,5R,7R,8R)-5-(5-(furan-3-yl)-4-methoxy)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol was used instead of (4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol, and the product was obtained as a white solid (63 mg, 74%). LCMS [M+H] + : 643.2. 1 H NMR (400 MHz, CDCl 3 ) δ 8.49 (s, 1H), 7.97 (s, 1H), 7.37 (s, 1H), 7.35–7.22 (m, 3H), 7.19 (d, J = 7.7 Hz, 2H), 7.13 (t, J = 7.0 Hz, 1H), 6.75 (s, 1H), 6.67 (s, 1H), 4.93 (dt, J = 11.9, 6.0 Hz, 1H), 4.68–4.46 (m, 3H), 4.39 (d, J = 7.8 Hz, 2H), 4.15 (s, 3H), 4.02–3.85 (m, 2H), 3.74 (t, J = 10.2 Hz, 1H), 3.32 (d, J = 9.9 Hz, 1H), 2.63 (dd, J = 19.3, 8.2 Hz, 1H), 2.04 (dd, J = 18.9, 7.8 Hz, 1H), 1.28 (d, J = 7.1 Hz, 3H), 1.16 (t, J = 7.3 Hz, 6H).

[0262] Example 38 Compound 38 ((S)-(((4R,5R,7R,8R)-8-hydroxy-5-(4-methoxy-5-(thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-7-yl)methoxy(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0263]

[0264] The preparation method of Compound 38 was referred to Example 26, except that (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methoxy-5-(thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol was used instead of (4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol, and the product was obtained as a white solid (52 mg, 61%). LCMS [M+H] + : 659.2. 1 H NMR (500 MHz, CDCl 3 ) δ 8.50 (s, 1H), 7.43 (d, J = 2.7 Hz, 1H), 7.31 (s, 1H), 7.29–7.22 (m, 3H), 7.21–7.14 (m, 3H), 7.11 (t, J = 7.3 Hz, 1H), 7.02 (dd, J = 5.1, 3.6 Hz, 1H), 6.63 (s, 1H), 4.93 (dt, J = 12.5, 6.3 Hz, 1H), 4.65–4.48 (m, 3H), 4.44–4.34 (m, 2H), 4.14 (s, 3H), 4.01–3.90 (m, 2H), 3.72 (t, J = 2.7 Hz, 1H), 3.33 (d, J = 9.7 Hz, 1H), 2.65 (dt, J = 12.0, 8.6 Hz, 1H), 2.08 (dt, J = 12.1, 8.7, 1H), 1.28 (d, J = 7.1 Hz, 3H), 1.16 (dd, J = 9.0, 6.3 Hz, 6H).

[0265] Example 39 Compound 39 ((S)-(((4R,5R,7R,8R)-8-hydroxy-5-(4-methoxy-5-(pyrimidin-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-7-yl)methoxy(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0266]

[0267] The preparation method of Compound 39 refers to Example 26, except that (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methoxy-5-(pyrimidin-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol is used instead of (4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol, and the product is a white solid (50 mg, 59%). LCMS [M+H] + : 655.2. 1 H NMR (400 MHz, CDCl 3 ) δ 8.77 (d, J = 4.8 Hz, 1H), 8.56 (s, 1H), 8.14 (s, 1H), 7.26–7.03 (m, 6H), 6.78 (s, 1H), 4.93 (dt, J = 12.5, 6.2 Hz, 1H), 4.75–4.63 (m, 2H), 4.53 (m 2H), 4.44 (d, J = 8.8 Hz, 1H), 4.21–4.11 (m, 1H), 3.99 (d, J = 8.6 Hz, 1H), 2.63 (dt, J = 11.8, 8.4 Hz, 1H), 2.25–2.16 (m, 1H), 1.30 (d, J = 7.1 Hz, 3H), 1.17 (d, J = 6.3 Hz, 6H).

[0268] Example 40 Compound 40 ((S)-(((4R,5R,7R,8R)-8-hydroxy-5-(4-methoxy-5-(thiophen-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-7-yl)methoxy(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0269]

[0270] The preparation method of Compound 40 refers to Example 26, except that (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methoxy-5-(thiophen-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol is used instead of (4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol, and the product is a white solid (43 mg, 51%). LCMS [M+H] + : 659.2.1 H NMR (400 MHz, CDCl 3 ) δ 8.50 (s, 1H), 7.68 (s, 1H), 7.42 (d, J = 4.7 Hz, 1H), 7.34 (s, 1H), 7.27 (d, J = 8.3 Hz, 3H), 7.18 (d, J = 7.7 Hz, 2H), 7.12 (t, J = 7.2 Hz, 1H), 6.70 (s, 1H), 4.92 (dt, J = 12.1, 6.0 Hz, 1H), 4.64–4.48 (m, 3H), 4.38 (s, 2H), 4.14 (s, 3H), 3.93 (d, J = 7.5 Hz, 2H), 3.82 (t, J = 10.4 Hz, 1H), 3.54 (s, 1H), 2.62 (m, 1H), 2.14–2.03 (m, 1H), 1.27 (d, J = 6.8 Hz, 3H), 1.18–1.11 (m, 6H).

[0271] Example 41 Compound 41 ((S)-(((4R,5R,7R,8R)-5-(5-(Benzofuran-2-yl)-4-methoxy-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-8-hydroxy-1,6-dioxaspiro[3.4]octan-7-yl)methoxy(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0272]

[0273] The preparation method of Compound 41 refers to Example 26, except that (4R,5R,7R,8R)-5-(5-(Benzofuran-2-yl)-4-methoxy)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol is used instead of (4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol, and the product is a white solid (43 mg, 53%). LCMS [M+H] + : 693.2. 1 H NMR (400 MHz, CDCl 3)δ8.53(s,1H),7.67(s,1H),7.61–7.54(m,1H),7.47–7.35(m,2H),7.27–7.15(m,6H),7.07(t,J=7.0Hz,1H),6.67(s,1H),4.90(dt,J=12.5,6.2Hz,1H),4.63–4.51(m,3H),4.47–4.39(m,2H),4.25(s,3H),4.09–3.93(m,2H),3.80(t,J=10.2Hz,1H),2.68(dt,J=12.1,8.4Hz,1H),2.15–2.05(m,1H),1.31(d,J=7.0Hz,3H),1.12(t,J=5.7Hz,6H).

[0274] Example 42 Compound 42 ((S)-(((4R,5R,7R,8R)-8-Hydroxy-5-(4-methoxy-5-(1-methyl-1H-pyrazol-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-7-yl)methoxy(phenoxy)phosphoryl)-L-alanine isopropyl ester

[0275]

[0276] The preparation method of Compound 42 was referred to Example 26, except that (4R,5R,7R,8R)-7-(hydroxymethyl)-5-(4-methoxy-5-(1-methyl-1H-pyrazol-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,6-dioxaspiro[3.4]octan-8-ol was used instead of (4R,5R,7R,8R)-5-(5-((4-bromo-3-fluorophenyl)ethynyl)-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-8-ol, and the product was obtained as a white solid (48 mg, 57%). LCMS [M+H] + : 657.3. 1 H NMR (500 MHz, CDCl 3)δ8.49(s,1H),7.77(s,1H),7.35(d,J=2.1Hz,1H),7.31–7.21(m,2H),7.18(d,J=8.5Hz,2H),7.10(t,J=7.3Hz,1H),6.82(d,J=2.2Hz,1H),6.70(s,1H),4.91(dt,J=12.5,6.3Hz,1H),4.63(dd,J=10.5,4.3Hz,1H),4.60–4.44(m,5H),4.15(s,3H),4.09–4.00(m,1H),3.99–3.93(m,1H),3.85(s,3H),2.63(dt,J=12.0,7.9Hz,1H),2.20–2.10(m,3H),1.26(d,J=7.1Hz,3H),1.15(dd,J=6.0,4.8Hz,6H).

[0277] 1. Activity test

[0278] Test for the activity of the compound to inhibit coronavirus replication

[0279] The following activity screening test was commissioned to the P3 Laboratory of Guangzhou National Laboratory and carried out according to the conventional virus activity test conditions

[0280] 1. Technical content: Verify the antiviral (SARS-CoV-2) activity of the above synthetic compound in the Vero E6 cell model

[0281] 2. Technical methods and routes: Vero E6 cells were plated one day before infection, 1.5×10 4 / well; on the day of infection, prepare the test compound solution (multiple concentration gradients), add it to the cell plate, incubate in a 37°C incubator for 2 h, then add the SARS-CoV-2 virus solution (infection titer 0.02 MOI), incubate in a 37°C incubator for 24 h, add 200 μL of 4% PFA to each well to fix for 1 h, discard the liquid, add 300 μL of 4% PFA to each well, and take it out for staining after thorough disinfection. Discard the fixing solution, wash the plate 3 times with PBS, incubate with 1% BSA in 0.2% Triton for blocking and permeabilization for 30 minutes, primary antibody: SARS-CoV / SARS-CoV-2 Nucleocapsid Rabbit PAb; secondary antibody: Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG(H+L), incubate with DAPI in the dark for 15 min; wash the plate with PBS, retain the last liquid, and analyze by scanning the plate with a Nexcelom Celigo Imaging Cytometer to calculate the inhibition rate. At the same time, set up DMSO solvent control and positive drug control

[0282] Referring to the above test procedure, the SARS-CoV-2 virus can be replaced with other coronaviruses for testing, such as MERS-CoV, etc.

[0283] 3. Experimental results.

[0284] The test results of some compounds in the test compounds are shown in Table 1.

[0285] Table 1 Antiviral test results of compounds

[0286]

[0287]

[0288] A: Indicates that the inhibition rate is ≥90%;

[0289] B: Indicates that 80% ≤ inhibition rate < 90% (including the lower limit, not including the upper limit);

[0290] C: Indicates that 70% ≤ inhibition rate < 80%;

[0291] D: Indicates that 60% ≤ inhibition rate < 70%;

[0292] E: Indicates that the inhibition rate < 60%.

[0293] The compounds described in the present invention have good inhibitory effects on coronaviruses. At a concentration of 10 μM, the inhibitory effect of the compounds on coronaviruses is greater than 80%, and more preferably, the inhibitory effect is greater than 90%; the inhibitory IC 50 is less than 2 μM; preferably, less than 1 μM; more preferably, less than 0.5 μM; and can be prepared into related drugs for preventing / treating coronavirus infections.

[0294] II. Enzyme activity inhibition test

[0295] In vitro enzymology experiment in the metastable complex system of SARS-CoV-2

[0296] A1. Preparation of SARS-CoV-2 nsp12, nsp8, and nsp7 proteins

[0297] 1. Using the expression plasmids of pET22b_nsp12, pET28a_nsp8, and pET28a_nsp7, and adopting the traditional "IPTG induction - T7 RNA polymerase transcription" system, overexpression is carried out respectively in Escherichia coli BL21(DE3).

[0298] 2. The collected expressed bacterial liquid was subjected to high-pressure crushing and then centrifuged at high speed to remove the precipitate. The supernatant was filtered and then separated and purified by nickel column affinity chromatography, ion chromatography, and gel filtration chromatography. The purified protein samples were measured for concentration, and nsp12, nsp8, and nsp7 were diluted to 240 μM, 1000 μM, and 1000 μM respectively, and then aliquoted and stored at -80 °C in a refrigerator for later use (Wu et al., 2021).

[0299] A2. Preparation of RNA used in in vitro enzymatic experiments

[0300] 1. The RNA template T33-1 was prepared and purified by the "T7 RNA polymerase - glmS" ribozyme method (Kieft and Batey. RNA 2004; Batey and Kieft. RNA 2007);

[0301] 2. The RNA template T33-1 with a final concentration of 40 μM was annealed with primer P10 at a molar ratio of 1:1.1 at 45 °C for 3 min and then left at room temperature for 10 min.

[0302] A3. In vitro enzymatic experiments of nucleoside drug 13b in the SARS-CoV-2 metastable complex system

[0303] 1. HEPES 7.0, MgCl 2 , DTT, nsp12 / nsp8 / nsp7, T33-1 / P10, NTP, and 13b were melted on ice;

[0304] 2. Prepare an RNA-free 0.6 mL centrifuge tube and sequentially add HEPES 7.0 with a final concentration of 50 mM, MgCl 2 with a final concentration of 5 mM, and DTT with a final concentration of 4 mM, and mix well on ice;

[0305] 3. Prepare an RNA-free 0.6 mL centrifuge tube, mix nsp12, nsp8, and nsp7 at a molar ratio of 1:2:1 until the final concentration of nsp12 is 120 μM, and mix well on ice;

[0306] 4. Add T33-1 / P10 to the solution in step 2 until the final concentration is 4 μM, and mix well on ice;

[0307] 5. Add the protein complex in step 3 to the solution in step 4 until the final concentration of nsp12 is 6 μM, and mix well on ice;

[0308] 6. Add CTP or CTP and ATP or CTP and 13b to the solution in step 5 until the final concentration of NTP is 300 μM, and mix well on ice;

[0309] 7. Add DEPC H 2 O to make up the volume of the reaction system in Step 6 to 20 μL;

[0310] 8. Incubate the reaction solution in Step 7 at 25 °C for 5 / 20 / 60 min;

[0311] 9. Terminate the reaction in Step 8 with 20 μL of 2× stop solution and mix well on ice.

[0312] A4. Denaturing polyacrylamide gel electrophoresis

[0313] 1. Boil the sample at 100 °C for 45 s to unwind the template-product double strand, and then immediately place it on ice;

[0314] 2. Electrophoretically separate the sample in a 5 mL urea (7 M)-20% polyacrylamide gel, and use a Tanon vertical electrophoresis tank to electrophorese at a constant voltage of 200 V for about 2 hours until the bromophenol blue dye migrates to the bottom of the gel;

[0315] 3. Stain with Stains-All staining solution for 45 min;

[0316] 4. Decolorize in water and then scan with an Epson scanner;

[0317] 5. Perform semi-quantitative analysis on the scanned results using Image J software.

[0318] B In vitro enzymology experiments in the stable complex system of the novel coronavirus

[0319] B1. Preparation of the novel coronavirus nsp12, nsp8, and nsp7 proteins

[0320] The procedure is the same as A1.

[0321] B2. Preparation of the RNA used in the in vitro enzymology experiments

[0322] 1. The RNA template T56 is prepared and purified by the "T7 RNA polymerase-glmS" ribozyme method (Kieft and Batey. RNA 2004; Batey and Kieft. RNA 2007);

[0323] 2. Anneal the initial amount of 15 nmol of the RNA template T56 with the primer P10 at a molar ratio of 1:3.1 at 45 °C for 3 min and then leave it at room temperature for 10 min.

[0324] B3. Preparation of the stable complex of the novel coronavirus

[0325] 1. Mix HEPES 7.0, MgCl 2, DTT, nsp12 / nsp8 / nsp7, T56 / P10, NTP, and 13b were thawed on ice;

[0326] 2. Prepare an RNA-free 1.5 mL centrifuge tube and sequentially add HEPES 7.0 at a final concentration of 50 mM, MgCl 2 at 5 mM, and DTT at 4 mM, and mix well on ice;

[0327] 3. Prepare an RNA-free 1.5 mL centrifuge tube, mix nsp12, nsp8, and nsp7 in a molar ratio of 1:2:1 until the final concentration of nsp12 is 120 μM, and mix well on ice;

[0328] 4. Add T56 / P10 to the solution in step 2 to a final concentration of 10 μM, and mix well on ice;

[0329] 5. Add the protein complex from step 3 to the solution in step 4 to make the final concentration of nsp12 12 μM, and mix well on ice;

[0330] 6. Add CTP and ATP separately to the solution in step 5 to make the final concentration of NTP 300 μM, and mix well on ice;

[0331] 7. Add DEPC H 2 O to make the volume of the reaction system in step 6 up to 1500 μL;

[0332] 8. Incubate the reaction solution in step 7 at 25 °C for 120 min;

[0333] 9. Separate and purify the reaction solution in step 8 through a high-resolution anion column (Mono Q);

[0334] 10. Exchange the complex obtained from the separation and purification in step 9 into a solution containing 50 mM HEPES 7.0, 100 mM NaCl, 4 mM MgCl 2 and 4 mM DTT;

[0335] 11. Concentrate the nsp12-nsp8-nsp7-RNA complex obtained in step 10, measure its concentration, and dilute it to 4 μM.

[0336] B4. In vitro enzymatic experiment of nucleoside drug 13b in the stable complex system of novel coronavirus

[0337] 1. Add 20 μL of GTP and ATP or GTP and 13b respectively to the above 4 μM complex, so that the final concentration of NTP

[0338] is 100 μM, and incubate at 25 °C for different times;

[0339] 2. Terminate the reaction in Step 1 with an equal volume of 2× stop solution and mix well on ice.

[0340] B5. Denaturing polyacrylamide gel electrophoresis

[0341] 1. Add the DNA complementary to T56 to the sample at a molar ratio of 1:3.

[0342] 2. Boil at 100 °C for 45 s, and then slowly cool it to room temperature.

[0343] 3. Electrophoretically separate the sample in a 5 mL urea (7M)-20% polyacrylamide gel using a Tanon vertical electrophoresis tank at a constant voltage of 200 V for about 2 hours until the bromophenol blue dye migrates to the bottom of the gel.

[0344] 4. Stain with Stains-All staining solution for 45 min.

[0345] 5. Decolorize in water and then scan with an Epson scanner.

[0346] 6. Perform semi-quantitative analysis on the scanned results using Image J software.

[0347] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description and ideas. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A nucleoside analog having a structure as shown in Formula I, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, in, X is H, -CH2OR0, F, N3, C 2-4 Alkynyl or -CH2F; R0 is H or C 1-4 Alkyl, C 1-4 Haloalkyl; R1 is H, -COR 11 or -P(O)(OR5)NHR4; R 11 C 1-4 Alkyl, C 1-4 Haloalkyl or amino substituted C 1-4 alkyl; R2 is C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, phenyl, benzyl, C 3-7 Heteroaryl, R 21 Substituted C 1-4 Alkyl, R 21 Substituted C 2-4 Alkenyl, R 21 Substituted C 2-4 Alkynyl, R 21 Substituted phenyl, R 21 Substituted benzyl, R 21 Substituted C 3-7 Heteroaryl or C 6-12 Condensed and heterocyclic rings; R 21 is halogen, phenyl, halogenated phenyl, C 1-4 Alkyl substituted phenyl or halogenated and C 1-4 Alkyl disubstituted phenyl; R3 is C 1-4 Alkyl, C 1-4 Alkoxy, halogenated C 1-4 Alkyl or halogenated C 1-4 Alkoxy; R4 is H, C 1-4 Alkyl, -R 41 C(O)OR 42 or -R 41 OC(O)R 42 ; where R 41 and R 42 Each independently is C 1-4 Alkylene or C 1-4 alkyl; R5 is phenyl, halogenated phenyl, benzyl or halogenated benzyl.

2. The nucleoside analog according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that: The R 41 C 1-4 Alkylene; Or, the R 41 C 1-3 Alkylene; Or, the R 41 It is methylene, methylene-methyl, ethylene, propylene or isopropylene.

3. The nucleoside analog according to claim 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that: The R4 is a methylene C(O)OR 42 、Methylene(methyl)C(O)OR 42 、ethylene C(O)OR 42 、propylidene C(O)OR 42 、Isopropylidene C(O)OR 42 、Methylene OC(O)R 42 、Methylene(methyl)OC(O)R 42 、ethylene OC(O)R 42 、PropyleneOC(O)R 42 or isopropylidene OC(O)R 42 ; said R 42 C 1-4 alkyl.

4. The nucleoside analogue according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that: The R 42 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl; Or, the R 42 is methyl, ethyl, propyl or isopropyl; Or, the R 42 It is ethyl, propyl or isopropyl.

5. The nucleoside analogue according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that: The R5 is phenyl or halogenated phenyl; Or, R5 is benzyl or halogenated benzyl; Or, R5 is phenyl, benzyl or halogenated benzyl; Or, R5 is phenyl, halogenated phenyl or benzyl; The halogenated phenyl group or halogenated benzyl group means that one or more H on the phenyl group or benzyl group is replaced by a halogen element, wherein the halogen element is F, Cl or Br.

6. The nucleoside analogue according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that: The R5 is phenyl, fluorophenyl, difluorophenyl, trifluorophenyl, benzyl, fluorobenzyl, difluorobenzyl or trifluorobenzyl.

7. The nucleoside analogue according to any one of claims 1 to 6, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that: The R 11 C 1-4 Alkyl, C 1-4 Haloalkyl or -C(NH2)C 1-3 alkyl; Or, the R 11 C 1-4 Alkyl or -C(NH2)C 1-3 alkyl; Or, the R 11 is methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, -C(NH2)methyl, -C(NH2)ethyl, -C(NH2)propyl or -C(NH2)isopropyl; Or, the R 11 It is propyl, isopropyl, butyl, tert-butyl, -C(NH2)propyl or -C(NH2)isopropyl.

8. The nucleoside analogue according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that: The R1 is H, -CO-methyl, -CO-ethyl, -CO-propyl, -CO-isopropyl, -CO-butyl, -CO-tert-butyl, -CO-C(NH2)methyl, -CO-C(NH2)ethyl, -CO-C(NH2)propyl, -CO-C(NH2)isopropyl, -P(O)(OR5)NH-R 41 C(O)OR 42 or-P(O)(OR5)NH--R 41 OC(O)R 42 ; Said R5 is phenyl or halogenated phenyl; R 41 and R 42 Each independently is C 1-4 Alkylene or C 1-4 alkyl; Or, R1 is H, -CO-propyl, -CO-isopropyl, -CO-C(NH2)propyl, -CO-C(NH2)isopropyl, -P(O)(OR5)NHCH2(CH3)C(O)Oisopropyl.

9. The nucleoside analogue according to any one of claims 1 to 8, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that: The R 21 is halogen, phenyl, 4-halophenyl, 2,4-dihalophenyl, 3,4-dihalophenyl, 2,3-dihalophenyl or 3,5-dihalophenyl; Or, the R 21 is halogen, phenyl, 4-halophenyl, 2,4-dihalophenyl, 3,4-dihalophenyl, 4-C 1-3 Alkyl substituted phenyl, 2,4-diC 1-3 Alkyl substituted phenyl, 4-halo-3-C 1-3 Alkyl substituted phenyl, 3-halo-4-C 1-3 Alkyl substituted phenyl, 4-halo-2-C 1-3 Alkyl substituted phenyl or 2-halo-4-C 1-3 Alkyl substituted phenyl; or, said R 21 It is halogen, phenyl, 4-halophenyl, 2,4-dihalophenyl, 3,4-dihalophenyl.

10. The nucleoside analogue according to any one of claims 1 to 9, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that: R2 is C 1-4 Alkyl, C 2-4 Alkynyl, phenyl, benzyl, C 3-7 Heteroaryl, R 21 Substituted C 2-4 Alkynyl, R 21 Substituted phenyl, R 21 Substituted benzyl, R 21 Substituted C 3-7 Heteroaryl or C 6-12 Condensed and heterocyclic rings; Preferably, R2 is phenyl, C 3-7 Heteroaryl, R 21 Substituted C 2-4 Alkynyl, R 21 Substituted phenyl, R 21 Substituted C 3-7 Heteroaryl or C 6-12 Condensed and heterocyclic rings; Preferably, R2 is C 3-7 Heteroaryl, R 21 Substituted C 2-4 Alkynyl, R 21 Substituted C 3-7 Heteroaryl or C 6-12 Condensed heterocyclic ring.

11. The nucleoside analogue according to any one of claims 1 to 10, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that: The C of R2 3-7 Heteroaryl is furan, pyrrole, thiophene, pyrazole, imidazole, thiazole, pyridine or pyrimidine.

12. The nucleoside analogue according to any one of claims 1 to 11, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that: The C of R2 6-12 The fused heterocyclic ring is benzofuran, indole, isoindole or indazole.

13. The nucleoside analogue according to any one of claims 1 to 12, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that: R2 is phenyl, benzyl, furan, thiophene, pyrazole, imidazole, thiazole, pyridine pyrimidine, benzofuran, indole, halogenated C 2-4 Alkynyl, phenyl substituted C 2-4 Alkynyl, 4-halophenyl substituted C 2-4 Alkynyl, 2,4-dihalophenyl substituted C 2-4 Alkynyl or 3,4-dihalophenyl substituted C 2-4 Alkynyl; Or, R2 is phenyl, furan, thiophene, pyrazole, imidazole, thiazole, pyridine pyrimidine, benzofuran, phenyl substituted C 2-4 Alkynyl, 4-halophenyl substituted C 2-4 Alkynyl or 2,4-dihalophenyl substituted C 2-4 Alkynyl; Or, R2 is furan, thiophene, pyrazole, thiazole, benzofuran, 4-halophenyl substituted C 2-4 Alkynyl or 2,4-dihalophenyl substituted C 2-4 Alkynyl.

14. The nucleoside analogue according to any one of claims 1 to 13, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that: R3 is C 1-4 Alkyl, C 1-4 Alkoxy or halogenated C 1-4 Haloalkyl; Or, said R3 is C 1-4 Alkyl or C 1-4 Alkoxy; Or, said R3 is C 1-2 Alkyl or C 1-2 Alkoxy.

15. The nucleoside analogue according to any one of claims 1 to 13, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that: The R0 is H or C 1-2 Alkyl or C 1-2 Haloalkyl; Or, the R0 is H or methyl.

16. The nucleoside analogue according to any one of claims 1 to 15, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that: Said X is H, -CH2OH, -CH2OCH3, F, N3, ethynyl or -CH2F.

17. The nucleoside analogue according to any one of claims 1 to 16, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, characterized in that: The compound has any of the following structures:

18. A pharmaceutical composition, characterized in that The invention comprises the nucleoside analogue according to any one of claims 1 to 17, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and a pharmaceutically acceptable excipient or carrier.

19. Use of the nucleoside analogue according to any one of claims 1 to 17, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof, or the pharmaceutical composition according to claim 16 in the preparation of a medicament for preventing and treating RNA virus infection diseases.

20. The use according to claim 19, characterized in that The RNA virus is a Coronaviridae or Flavivirus.

21. The use according to claim 19 or 20, characterized in that The coronaviruses include: SARS-CoV, MERS-CoV and SARS-CoV-2.

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