Five-membered sulfur-containing heterocyclic nucleoside lead compound and application thereof in anti-hand-foot-and-mouth virus active medicine

Abstract

The invention discloses a five-membered sulfur-containing heterocyclic nucleoside lead compound and application thereof in anti-hand-foot-and-mouth virus active drugs, and belongs to the technical field of medicinal chemistry. A series of five-membered sulfur-containing heterocyclic ring nucleoside compounds are synthesized through a Sulfa-Michael / Aldol cascade reaction. A result shows that in an anti-EV-A71 virus test, the five-membered sulfur-containing heterocyclic ring nucleoside lead compounds 33a, 5b and 30b have relatively good anti-EV-A71 virus activity, and a new structural unit is provided for antiviral candidate drug molecules.

Description

Technical Field

[0001] The invention relates to a five-membered sulfur-containing heterocyclic nucleoside lead compound and application thereof in an anti-hand, foot and mouth virus (EV-A71) active drug, belonging to the field of medicinal chemistry. Background Art

[0002] Viruses seriously threaten human life and health and are one of the main causes of human death. The research and development of antiviral drugs has always been one of the important directions in drug development. Artificially synthesized nucleoside compounds are similar in structure to natural nucleoside compounds. After entering the human body, they can inhibit the replication of viruses or tumor cells, achieving the purpose of antiviral or anti-tumor.

[0003] People pay more and more attention to the research and development of nucleoside anti-tumor and antiviral drugs. Five-membered sulfur heterocyclic nucleoside compounds are a unique class of compounds among nucleoside compounds, with unique physical and chemical properties, and have become the focus of nucleoside drug research and development.

[0004] Currently, five-membered sulfur heterocyclic nucleoside compounds are an important class of antiviral drugs on the market, but there are relatively few studies on the antiviral activity of five-membered sulfur heterocyclic nucleoside compounds. Innovative research on five-membered sulfur heterocyclic nucleoside antiviral drugs can not only break the international monopoly on nucleoside drugs, but also has important scientific significance and potential application prospects. Summary of the invention

[0005] In order to overcome the above-mentioned technical defects, the present invention synthesizes a series of five-membered sulfur heterocyclic nucleoside compounds through reactions such as Sulfa-Michael / Aldol tandem reaction, provides nucleoside lead compounds with more functional structures, and discloses a five-membered sulfur heterocyclic nucleoside lead compound with anti-hand, foot and mouth virus (EV-A71) activity.

[0006] The present invention discloses a five-membered sulfur heterocyclic nucleoside lead compound, including its racemate and chiral compound, wherein: the compound ee value is 0-100% (0% corresponds to the racemate), and is a single enantiomerically pure compound or a mixture of two enantiomers in different proportions. Its general structural formula is as follows:

[0007]

[0008] Where: R 1 is selected from hydrogen, halogen, amino, C1-C20 alkyl, C1-C20 alkoxy; R 2 is selected from hydrogen, halogen, C1-C20 alkyl, C1-C20 alkoxy, C1-20 alkylthio, C2-C20 alkynylthio, halogenated C1-C20 alkylthio, benzylthio, phenylthio, substituted phenylthio, di(C1-C8 alkyl)amine, X=CH 2, O, S, NH, N-(C1-C4)alkyl; n = 0-5; R 3 =C1-C8 alkyl, halogen, trifluoromethyl, benzyl, substituted phenylmethyl, morpholinyl; wherein the substituent in the substituted phenyl is halogen or C1-C8 alkyl; R is selected from hydrogen, C1-C20 alkyl.

[0009] Furthermore, under the preferred conditions, R 1 is selected from hydrogen, halogen, amino, C1-C20 alkyl, C1-C20 alkoxy; R 2 is selected from hydrogen, halogen, C1-C20 alkyl, C1-C20 alkoxy, C1-20 alkylthio, C2-C20 alkynylthio, halogenated C1-C20 alkylthio, benzylthio, phenylthio, substituted phenylthio, di(C1-C8 alkyl)amine, X=CH 2 , O, S, NH, N-(C1-C4)alkyl; n = 0-5; R 3 =C1-C8 alkyl, halogen, trifluoromethyl, benzyl, substituted phenylmethyl, morpholinyl; wherein the substituent in the substituted phenyl is halogen or C1-C8 alkyl; R is selected from ethyl.

[0010] Furthermore, under the preferred conditions, R 1 is selected from hydrogen, halogen, amino; R 2 is selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-6 alkylthio, C2-C4 alkynylthio, halogenated C1-C4 alkylthio, benzylthio, phenylthio, substituted phenylthio, di(C1-C2 alkyl)amine, X=CH 2 , O, S, NH, N-(C1-C4)alkyl; n = 1-3; R 3 =C1-C4 alkyl, halogen, trifluoromethyl, benzyl, substituted phenylmethyl, morpholinyl; wherein the substituent in the substituted phenyl is halogen or C1-C4 alkyl; R is selected from ethyl.

[0011] Further, under the most preferred conditions, the structure of the five-membered sulfur heterocyclic nucleoside lead compound is

[0012]

[0013] The present invention also provides a method for synthesizing a five-membered sulfur heterocyclic nucleoside lead compound, comprising the following steps:

[0014]

[0015] Compound 1-42A reacts in the presence of triphenylphosphine, sodium acetate, acetic acid and propiolate to obtain compound 1-42B; then compound 1-42B reacts with 2,5-dihydroxy-1,4-dithiane in the presence of triethylamine to obtain 1a-42a and 1b-42b; then the corresponding chiral compound is obtained after separation by a chiral separation column.

[0016] Furthermore, in the above technical solution, the first step reaction solvent is toluene, and the reaction temperature is reflux.

[0017] Furthermore, in the above technical solution, the second step reaction solvent is dichloromethane, and the reaction temperature is 0-30°C.

[0018] Furthermore, in the above technical scheme, the chiral separation column is separated using a CHI RALPAK IF (IF00CE-RL017, 0.46 cm IDx 25 cm L) chromatographic column: injection volume 10 μL, mobile phase MeOH / DCM-95 / 5 (V / V), flow rate 1.0 mL / min, wavelength UV 254 nm, column temperature 35°C, HPLC: Shimadzu LC-20AD CP-HPLC-08), and the chiral compounds after separation are greater than 98% ee.

[0019] The present invention also provides the use of the five-membered sulfur heterocyclic nucleoside lead compound in the preparation of antiviral drugs.

[0020] Furthermore, in the above technical solution, the antiviral agent is anti-hand, foot and mouth virus (EV-A71).

[0021] The present invention also provides the use of a five-membered sulfur heterocyclic nucleoside lead compound in the preparation of a drug for treating hand, foot and mouth disease.

[0022] The present invention also provides a pharmaceutical composition containing the five-membered sulfur heterocyclic nucleoside lead compound.

[0023] Beneficial effects of the invention:

[0024] 1. The raw materials for the preparation of the five-membered sulfur heterocyclic nucleoside lead compound are simple and easy to obtain, and have broad application prospects.

[0025] 2. The products were tested for antiviral activity, and it was found that compounds 33a, 5b and 30b had good activity against hand, foot and mouth virus (EV-A71). BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the anti-EV-A71 virus activity graph of 33a;

[0027] Figure 2 This is the anti-EV-A71 virus activity graph of 5b;

[0028] Figure 3 This is a graph showing the anti-EV-A71 viral activity of 30b. DETAILED DESCRIPTION

[0029] Example 1

[0030]

[0031] Synthesis of 1a / 1b-42a / 42b.Reagents and conditions:(a)PPh 3 ,NaOAc,HOAc,Ethyl propiolate,toluene,110℃,12h.(b)1,4-dithiane-2,5-diol,Et 3 N(20mol%),CH 2 Cl 2 ,rt,4h.

[0032] Typical operation of the first step: In a round-bottom flask, purine compound A (10 mmol), triphenylphosphine (0.0521 g, 0.2 mL), sodium acetate (0.1641 g, 2 mmol), acetic acid (0.3 mL, 2 mmol) and ethyl propiolate (1.21 mL, 12 mmol) were added to toluene (100 mL). Under nitrogen protection, the mixture was heated and stirred at 110 ° C for 12 h, and dichloromethane was added to extract the reaction mixture 3 times; the organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by silica gel (PE / EA=2 / 1) column chromatography to obtain product B.

[0033] Typical operation of the second step: α-heterocyclic acrylate B (0.05 mmol) and 1,4-disulfane 2,5-diol (0.03 mmol) were added to a round-bottom flask. Then, 20 mmol% triethylamine was added and stirred at room temperature for 4 hours. The reaction product was detected by thin layer chromatography, and the solvent was removed by silica gel (PE / EA=2 / 1) column chromatography to obtain products a and b.

[0034] Structural characterization of representative compounds:

[0035] 1.2.1.(±)-Ethyl-4-hydroxy-3-(9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(1a,1b)

[0036] 1a: white solids, 41.2% yield. 1 H NMR (600 MHz, CDCl 3)δ9.18(s,1H),8.95(s,1H),8.42(s,1H),5.42(d,J=42.6Hz,2H),4.24-4.16(m,2H),3.96(d,J=12.6Hz,1H),3.59(d,J=12Hz,1H),3.19(dd,J=4.8Hz,J=7.2Hz,1H),2.86(dd,J=4.8Hz,J=11.4Hz,1H),1.08(t,J=6.6Hz,3H). 13 C NMR(150MHz,CDCl 3 )δ169.3,152.4,151.5,149.6,144.0,134.3,78.9,74.0,63.3,35.5,35.4,13.9.ESI-HRMS(m / z)calcd C 12 H 15 N 4 O 3 S(M+H) + ,295.0859;found,295.0856.HPLC purity:97.7%.1b:white solids,52.4%yield. 1 H NMR(600MHz,CDCl 3 )δ9.06(s,1H),8.83(s,1H),8.51(s,1H),5.49(t,J=6.0Hz,1H),4.22-4.15(m,2H),3.74(dd,J=12Hz,J=30.0Hz,2H),3.34(q,J=5.4Hz,1H),2.89(q,J=6,1H),1.12(t,J=7.2Hz,3H), 13 CNMR(150MHz,CDCl 3 )δ168.1,152.1,151.7,148.9,145.0,133.7,76.3,73.8,63.1,34.7,34.0,13.8;ESI-HRMS(m / z)calcd C 12 H 14 N 4 O 3 SNa(M+Na) + ,317.0679;found,317.0678.HPLC purity:98.0%.

[0037] 1.2.2.(±)-Ethyl-3-(6-chloro-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(2a,2b)

[0038] 2a:colorless 1iquid,42.4%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.70(s,1H),8.56(s,1H),5.46(s,1H),4.23(t,J=7.2 Hz,2H),3.79(d,J=11.4 Hz,1H),3.64(d,J=11.4Hz,1H),3.32(q,J=6.0 Hz,1H),2.83(dd,J=6.6 Hz,J=11.4 Hz,1H)1.17(t,J=7.2Hz,3H). 13 C NMR(150 MHz,CDCl 3 )δ167.9,152.2,152.0,151.8,144.7,131,7,74.0,63.5,34.9,33.7,14.0;ESI-HRMS(m / z)calcd C 12 H 13 N 4 ClO 3 SNa(M+Na) + ,351.0281;found,351.0289.HPLC purity:98.3%.2b:colorless 1iquid,58.3%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.71(s,1H),8.48(s,1H),5.34(s,1H),5.14(s,1H),4.20(d,J=6.6 Hz,2H),3.97(d,J=12.6 Hz,1H),3.15(dd,J=4.2 Hz,J=12,1H),2.82(dd,J=4.8 Hz,J=11.4 Hz,1H),1.09(t,J=7.2 Hz,3H). 13 C NMR(150 MHz,CDCl 3 )δ169.1,152.1,151.8,151.7,143.8,132.0,78.8,74.2,63.5,53.6,35.5,35.4,14.0;ESI-HRMS(m / z)calcd C 12 H13 N 4 ClO 3 SNa(M+Na) + ,351.0289;found,351.0294.HPLC purity:98.8%.

[0039] 1.2.3.(±)-Ethyl-3-(6-bromo-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(3a,3b)

[0040] 3a:white solid,30.1%yield. 1 H NMR(400 MHz,CDCl 3 )δ8.61(s,1H),8.58(s,1H),5.46(s,1H),4.27-4.17(m,2H),4.15-4.12(m,1H),3.73(dd,J=12.0 Hz,J=28.4 Hz,2H),3.33(q,J=5.6 Hz,1H),2.84(dd,J=6.4 Hz,J=11.2 Hz,1H),1.14(t,J=7.2Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ167.9,151.7,150.9,144.8,143.9,134.1,74.1,63.4,34.7,33.9,13.9.ESI-HRMS(m / z)calcd C 12 H 13 N 4 BrO 3 SNa(M+Na)+,394.9784;found,394.9782.HPLC purity:95.5%.3b:white solid,41.2%yield. 1 H NMR(400 MHz,CDCl 3 )δ8.66(s,1H),8.50(s,1H),5.34(s,1H),5.12(s,1H),4.20(q,J=7.2 Hz,2H),3.96(d,J=12.4 Hz,1H),3.58(d,J=12.8 Hz,1H),3.15(dd,J=4.0 Hz,J=11.6 Hz,1H),2.81(dd,J=4.8 Hz,J=11.6 Hz,1H),1.10(t,J=7.2 Hz,3H). 13CNMR(100 MHz,CDCl 3 )δ169.0,151.7,150.4,144.1,143.7,134.5,78.7,74.3,63.5,35.5,35.3,13.9.ESI-HRMS(m / z)calcd C 12 H 13 N 4 BrO 3 SNa(M+Na) + ,394.9784;found,394.9789.HPLC purity:98.1%.

[0041] 1.2.4.(±)-Ethyl-3-(6-(dimethylamino)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(4a,4b)

[0042] 4a:white solid,38.1%yield. 1 H NMR(400 MHz,CDCl 3 )δ8.49(s,1H),8.19(s,1H),5.84(s,1H),5.32(t,J=5.2 Hz,1H),4.23-4.15(m,8H),3.93(d,J=12.4 Hz,1H),3.57(d,J=12.0 Hz,1H),3.21(q,J=5.2 Hz,1H),2.89(dd,J=5.2 Hz,J=11.6 Hz,1H),1.10(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ69.4,161.6,152.0,151.9,141.0,121.8,79.3,74.1,63.1,54.6,35.6,35.3,14.0.ESI-HRMS(m / z)calcd C 14 H 20 N 5 O 3 S(M+H) + ,338.1281;found,338.1272.HPLC purity:98.9%.4b:white solid,59.2%yield. 1 H NMR(600 MHz,CDCl 3)δ8.22(s,1H),8.00(s,1H),6.28(s,1H),5.40(t,J=6.6,1H),4.25-4.18(m,2H),3.74(d,J=12 Hz,1H),3.54-3.44(m,7H),3.22(dd,J=6.6Hz,J=10.8 Hz,1H),2.81(dd,J=7.2 Hz,J=10.8 Hz,1H),1.18(t,J=7.2 Hz,3H). 13 C NMR(150 MHz,CDCl 3 )δ168.6,155.2,151.8,150.4,137.8,120.1,73.9,63.0,35.3,33.3,14.0.ESI-HRMS(m / z)calcd C 14 H 20 N 5 O 3 S(M+H) + ,338.1281;found,338.1280.HPLC purity:95.2%.

[0043] 1.2.5.(±)-Ethyl-4-hydroxy-3-(6-(piperidin-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(5a,5b)

[0044] 5a:white solid,41.7%yield. 1 H NMR(400 MHz,CDCl 3 )δ8.24(s,1H),7.90(s,1H),5.25(t,J=5.6 Hz,1H),4.28-4.15(m,6H),3.87(d,J=12.0 Hz,1H),3.53(d,J=12.0Hz,1H),3.25(dd,J=6.0,J=11.2 Hz,1H),2.94(dd,J=11.2,J=5.2 Hz,1H),1.76-1.69(m,6H),1.15(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ169.8,154.1,151.9,150.9,136.8,119.8,79.7,74.0,63.0,35.9,35.1,26.3,24.9,14.1.ESI-HRMS(m / z)calcdC 17 H24 N 5 O 3 S(M+H) + ,378.1594;found,378.1597.HPLC purity:98.7%.5b:white solid,51.1%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.19(s,1H),7.99(s,1H),5.39(t,J=6.6,1H),4.24-4.18(m,6H),3.72(d,J=12,1H),3.44(d,J=12,1H),3.21(q,J=6,1H),2.80(q,J=7.2,1H),1.74-1.67(m,6H),1.18(t,J=6.6,3H). 13 C NMR(150 MHz,CDCl 3 )δ168.6,154.1,151.9,150.7,137.5,119.6,73.8,63.0,35.2,33.4,26.3,24.8,14.0.ESI-HRMS(m / z)calcdC 17 H 23 N 5 O 3 SNa(M+Na) + ,400.1414;found,400.1410.HPLC purity:95.8%.

[0045] 1.2.6.(±)-Ethyl-3-(6-ethoxy-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(6a,6b)

[0046] 6a:white solid,39.1%yield. 1 H NMR(600 MHz,CDCl 3)δ8.47(s,1H),8.17(s,1H),5.90(d,J=2.4 Hz,1H),5.31(t,J=5.4 Hz,1H),4.69-4.63(m,2H),4.23-4.15(m,2H),3.92(d,J=12.6 Hz,1H),3.67(d,J=12.0 Hz,1H),3.21(q,J=5.4 Hz,1H),2.89(dd,J=4.8 Hz,J=11.4 Hz,1H),1.51(t,J=7.2 Hz,3H),1.10(t,J=7.2 Hz,3H). 13 C NMR(100MHz,CDCl 3 )δ169.8,161.7,152.4,152.4,141.3,122.1,79.7,74.5,64.0,63.5,36.1,35.7,15.0,14.4.ESI-HRMS(m / z)calcd C 14 H 18 N 4 O 4 SNa(M+Na) + ,361.0941;found,361.0938.HPLCpurity:95.4%.6b:white solid,53.2%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.43(s,1H),8.22(s,1H),5.46(t,J=6.0 Hz,1H),4.64(q,J=7.2 Hz,2H),4.23-4.63(m,2H),4.23-4.16(m,2H),3.74(d,J=12.0 Hz,1H),3.59(d,J=11.4 Hz,1H),3.30(q,J=6.0 Hz,1H),2.87(dd,J=6.0 Hz,J=7.2 Hz,1H),1.51(t,J=7.2 Hz,3H),1.15(t,J=7.2 Hz,3H). 13 C NMR(100MHz,CDCl 3 )δ168.3,161.2,152.0,151.9,141.8,121.4,76.7,74.1,63.6,63.1,35.0,33.8,14.6,13.9.ESI-HRMS(m / z)calcd C 14 H 18 N 4 O 4 SNa(M+Na)+ ,361.0941;found,361.0936.HPLCpurity:96.0%.

[0047] 1.2.7.(±)-Ethyl-4-hydroxy-3-(6-methoxy-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(7a,7b)

[0048] 7a:light yellow solid,32.8%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.49(s,1H),8.19(s,1H),5.31(t,J=7.8 Hz,1H),4.21-4.15(m,5H),3.92(d,J=18.6 Hz,1H),3.57(d,J=18 Hz,1H),3.20(dd,J=7.8 Hz,J=17.4 Hz,1H),2.89(dd,J=7.2 Hz,J=17.4Hz,1H),1.09(t,J=10.2 Hz,3H). 13 C NMR(150 MHz,CDCl 3 )δ169.8,155.2,151.9,150.7,137.1,120.1,79.7,74.0,62.9,53.6,35.9,35.1,14.1.ESI-HRMS(m / z)calcdC 13 H 16 N 4 O 4 SNa(M+Na) + ,347.0784;found,347.0786.HPLC purity:95.1%.7b:light yellow solid,54.1%yield. 1 H NMR(400 MHz,CDCl 3 )δ8.47(s,1H),8.25(s,1H),5.46(t,J=6.4 Hz,1H),4.21(t,J=7.2 Hz,5H),3.76(d,J=11.6 Hz,1H),3.57(d,J=11.2 Hz,1H),3.29(q,J=5.6 Hz,1H),2.86(dd,J=6.4 Hz,J=11.2 Hz,1H),1.16(t,J=7.2 Hz,3H). 13 C NMR(100MHz,CDCl3 )δ168.3,161.6,152.0,151.9,141.9,74.0,63.2,54.6,35.1,33.7,14.0.ESI-HRMS(m / z)calcd C 13 H 16 N 4 O 4 SNa(M+Na) + ,347.0784;found,347.0778.HPLC purity:95.7%.

[0049] 1.2.8.(±)-Ethyl-4-hydroxy-3-(6-(propylthio)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(8a,8b)

[0050] 8a:yellow solid,31.2%yield. 1 H NMR(400 MHz,CDCl 3 )δ8.63(s,1H),8.28(s,1H),5.48(t,J=6.0 Hz,1H),4.22(q,J=7.2 Hz,2H),3.78(d,J=12.0 Hz,1H),3.56(d,J=11.6 Hz,2H),3.37(t,J=7.2 Hz,2H),3.29(dd,J=6.0 Hz,J=11.2,1H),2.86(dd,J=6.8 Hz,J=11.2 Hz,1H),1.81.78(m,2H),1.18(t,J=7.2 Hz,3H),1.09(t,J=7.6,3H). 13 CNMR(100 MHz,CDCl 3 )δ168.3,163.0,151.5,148.4,142.2,131.3,74.0,63.2,35.1,33.6,30.9,22.9,14.0,13.6.ESI-HRMS(m / z)calcd C 15 H 20 N 4 O 3 S 2 Na(M+Na) + ,391.0869;found,391.0870.HPLC purity:98.0%.8b:yellow solid,yield:54.1%. 1H NMR(400 MHz,CDCl 3 )δ8.64(s,1H),8.22(s,1H),5.32(t,J=4.8 Hz,1H),4.25-4.14(m,2H),3.92(d,J=12.4Hz,1H),3.56(d,J=12.4 Hz,1H),3.42-3.31(m,2H),3.19(dd,J=5.2 Hz,J=11.6 Hz,1H),2.87(dd,J=4.8 Hz,J=11.6 Hz,1H),1.86-1.77(m,2H),1.13-1.06(m,6H). 13 C NMR(100 MHz,CDCl 3 )δ169.4,162.9,151.5,148.3,141.2,131.5,79.2,74.0,63.2,35.6,35.3,30.9,22.9,14.0,13.5.ESI-HRMS(m / z)calcd C 15 H 20 N 4 O 3 S 2 Na(M+Na) + ,391.0869;found,391.0865.HPLC purity:96.4%.

[0051] 1.2.9.(±)-Ethyl-4-hydroxy-3-(6-morpholino-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(9a,9b)

[0052] 9a:white solid,24.4%yield. 1 H NMR(400 MHz,CDCl 3 )δ8.27(s,1H),7.94(s,1H),5.26(t,J=5.6 Hz,1H),4.31(s,4H),4.26-4.15(m,2H),3.88(d,J=12.4 Hz,1H),3.83(d,J=4.0 Hz,4H),3.53(d,J=12.0 Hz,1H),3.23(q,J=6.0 Hz,1H),2.92(dd,J=5.2 Hz,J=11.6 Hz,1H),1.15(t,J=6.8 Hz,3H). 13 C NMR(100 MHz,CDCl 3)δ169.7,154.1,151.9,151.1,137.3.120.0,79.6,74.0,67.1,63.0,35.8,35.2,14.1.ESI-HRMS(m / z)calcdC 16 H 22 N 5 O 4 S(M+H) + ,380.1387;found,380.1390.HPLC purity:97.9%.9b:white solid,45.4%yield. 1 H NMR(400 MHz,CDCl 3 )δ8.23(s,1H),8.02(s,1H),5.94(s,1H),5.40(t,J=6.4 Hz,1H),4.29(s,4H),4.21(q,J=7.2 Hz,2H),3.81(t,J=4.8 Hz,4H),3.73(d,J=11.6 Hz,1H),3.46(d,J=11.6 Hz,1H),3.23(dd,J=6.0 Hz,J=10.8 Hz,1H),2.80(dd,J=7.2 Hz,J=10.8 Hz,1H),1.18(t,J=6.8Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ168.5,154.1,151.8,150.9,138.1,119.8,77.3,73.8,67.1,63.0,35.2,33.4,14.0.ESI-HRMS(m / z)calcd C 16 H 21 N 5 O 4 SNa(M+Na) + ,402.1206;found,402.1196.HPLC purity:99.4%.

[0053] 1.2.10.(±)-Ethyl-4-hydroxy-3-(6-thiomorpholino-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(10a,10b)

[0054] 10a:white solid,30.4%yield. 1 H NMR(400 MHz,CDCl 3)δ8.27(s,1H)7.94(s,1H),5.27(t,J=5.6 Hz,1H),4.59(s,4H),4.26-4.18(m,2H),3.88(d,J=12 Hz,1H),3.54(d,J=12 Hz,1H),3.24(q,J=6 Hz,1H),2.93(dd,J=5.2 Hz,J=11.2 Hz,1H),2.76(t,J=5.2 Hz,4H),1.16(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ160.7,153.9,151.9,151.2,137.3,120.0,79.6,74.0,63.0,35.9,35.2,27.5,14.1.ESI-HRMS(m / z)calcdC 16 H 22 ClN 5 O 3 S 2 (M+H) + ,396.1159;found,396.1149.HPLC purity:96.1%.10b:white solid,45.2%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.27(s,1H),7.94(s,1H),6.46(s,1H),5.27(t,J=6Hz,1H)4.59(s,4H),4.25-4.19(m,2H),3.88(d,J=12 Hz,1H),3.54(d,J=12 Hz,1H),3.24(q,J=6 Hz,1H),2.93(q,J=5.4 Hz,1H),2.77(t,J=4.8 Hz,4H),1.16(t,J=7.2 Hz,3H). 13 C NMR(150 MHz,CDCl 3 )δ169.7,153.9,151.9,151.2,120.0,79.6,74.0,63.0,35.9,35.2,27.5,14.1.ESI-HRMS(m / z)calcd C 16 H 21 ClN 5 O 3 S 2 Na(M+Na) + ,418.0978;found,418.0980.HPLC purity:97.4%.

[0055] 1.2.11.(±)-Ethyl-4-hydroxy-3-(6-(4-methylpiperidin-1-yl)-9H-purin-9yl)tetrahydrothiophene-3-carboxylate(11a,11b)

[0056] 11a:white solid,32.7%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.23(s,1H),7.89(s,1H),6.66(s,1H),5.41(s,2H),5.24(t,J=8.4 Hz,1H),4.24-4.16(m,2H),3.86(d,J=18Hz,1H),3.52(d,J=17.4 Hz,1H)3.24(dd,J=6.6 Hz,J=9 Hz,1H),3.08(s,2H),2.93(q,J=7.8 Hz,1H),1.82-1.66(m,2H),1.31-1.19(2H),1.14(t,J=10.2 Hz,3H),0.97(d,J=10.2 Hz,3H). 13 C NMR(150 MHz,CDCl 3 )δ169.8,154.0,151.9,150.9,136.8,119.8,82.3,79.6,73.9,62.9,35.9,35.1,34.4,31.3,22.0,14.0.ESI-HRMS(m / z)calcd C 18 H 25 ClN 5 O 3 SNa(M+Na) + ,392.1751;found,392.1744.HPLCpurity:95.4%.11b:white solid,31.5%yield. 1 H NMR(400 MHz,CDCl 3)δ8.21(s,1H),8.00(s,1H),5.40(t,J=6.8 Hz,4H),4.22(dd,J=7.2 Hz,J=15.6 Hz,2H),3.74(d,J=12 Hz,1H),3.43(d,J=12 Hz,1H),3.22(dd,J=6.0 Hz,J=10.8 Hz,1H),3.09(s,2H),2.81(dd,J=7.2 Hz,J=10.8 Hz,1H),1.83-1.70(m,2H),1.32-1.27(m,2H),1.19(t,J=7.2 Hz,3H),0.98(d,J=6.4 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ168.7,154.1,151.9,150.7,137.5,119.7,73.8,63.0,35.3,34.5,33.3,31.4,22.0,14.0.ESI-HRMS(m / z)calcd C 18 H 25 ClN 5 O 3 SNa(M+Na) + ,392.1751;found,392.1746.HPLC purity:96.7%.

[0057] 1.2.12.(±)-Ethyl-3-(6-(4-fluoropiperidin-1-yl)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(12a,12b)

[0058] 12a:white solid,42.3%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.21(s,1H),7.90(s,1H),6.48(s,1H),5.21(t,J=6 Hz,1H),4.93-4.84(m,1H),4.46(s,2H)4.19-4.13(m,4H),3.83(d,J=12 Hz,1H),3.50(d,J=12.6 Hz,1H),3.18(q,J=6 Hz,1H),2.89(dd,J=4.8Hz,J=11.4 Hz,1H),1.99-1.90(m,4H),1.10(t,J=7.2 Hz,3H). 13C NMR(150 MHz,CDCl 3 )δ169.5,153.8,151.8,150.9,137.0,119.7,88.6,87.5,79.4,73.9,62.8,42.3,35.7,35.0,31.4(Jc-F=19.7),13.9. 19 F NMR(376MHz,CDCl 3 )δ-181.58.ESI-HRMS(m / z)calcdC 17 H 22 FN 5 O 3 SNa(M+Na) + ,396.1500;found,396.1506.HPLC purity:98.9%.12b:whitesolid,53.4%yield. 1 H NMR(400 MHz,CDCl 3 )δ8.24(s,1H),8.03(s,1H),6.10(s,1H),5.41(t.J=6.0 Hz,1H),5.03-4.86(m,1H),4,54(s,2H),4.22(q,J=7.2 Hz,4H),3.75(d,J=12Hz,1H),3.44(d,J=11.6 Hz,1H),3.23(dd,J=10.8 Hz,J=6.0 Hz,1H),2.81(dd,J=10.8Hz,J=7.2 Hz,1H),2.05-1.95(m,4H),1.20(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ168.6,154.1,151.9,150.9,137.9,119.8,87.3,73.8,63.1,35.3,33.3,31.6(d,J C-F =20.2Hz),14.0. 19 F NMR(376MHz,CDCl 3 )δ-181.68.ESI-HRMS(m / z)calcd C 17 H 22 FN 5 O 3 SNa(M+Na) + ,396.1500;found,396.1508.HPLC purity:97.2%.

[0059] 1.2.13.(±)-Ethyl-4-hydroxy-3-(6-(4-(trifluoromethyl)piperidin-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(13a,13b)

[0060] 13a:yellow solid,42.1%yield. 1 H NMR(400 MHz,CDCl 3 )δ8.25(s,1H),7.93(s,1H),6.44(s,1H),5.60(s,2H),5.24(t.J=5.6 Hz,1H),4.23-4.15(m,2H),3.86(d,J=12Hz,1H),3.53(d,J=12 Hz,1H),3.20(q,J=6 Hz,1H),3.08-2.99(m,2H)2.95(q,J=5.6Hz,1H),2.42-2.33(m,1H),2.00(d,J=12.8 Hz,2H),1.69-1.56(m,2H),1.13(t.J=6.8Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ169.6,153.9,151.8,151.0,137.2,128.5,125.8,119.8,79.5,73.9,62.9,44.2,40.7(q,J C-F =27.4 Hz),35.8,35.1,24.7,14.0. 19 F NMR(376MHz,CDCl 3 )δ-73.90.ESI-HRMS(m / z)calcd C 18 H 22 F 3 N 5 O 3 SNa(M+Na) + ,446.1468;found,446.1468.HPLC purity:95.6%.13b:yellow solid,37.5%yield. 1 H NMR(400 MHz,CDCl 3)δ8.24(s,1H)8.03(s,1H),5.97(s,1H),5.63(s,2H),5.41(t.J=6.4 Hz,1H),4.22,(q,J=6.8 Hz,2H),3.75(d,J=12 Hz,1H),3.45(d,J=12 Hz,1H),3.23(dd,J=11.2 Hz,J=6Hz,1H),3.07(t,J=11.6Hz,2H),2.81(dd,J=10.8 Hz,J=7.2 Hz,1H),2.46-2.34(m,1H),2.03(d,J=12.8Hz,2H),1.72-1.61(m,2H),1.25-1.15(m,3H). 13 C NMR(100 MHz,CDCl 3 )δ168.6,154.0,151.9,151.0,138.1,119.8,73.9,63.1,40.8(d,J C-F =27.3),35.3,33.3,24.8,14.0. 19 F NMR(376MHz,CDCl 3 )δ-73.90.ESI-HRMS(m / z)calcd C 18 H 22 F 3 N 5 O 3 SNa(M+Na) + ,446.1468;found,446.1467.HPLC purity:96.7%.

[0061] 1.2.14.(±)-Ethyl-3-(6-(4-chloropiperidin-1-yl)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(14a,14b)

[0062] 14a:light yellow solid,51.7%yield. 1 H NMR(600 MHz,CDCl 3)δ8.23(s,1H),8.02(s,1H),6.03(s,1H),5.40(t,J=6.6 Hz,1H),4.60(s,2H)4.38-4.35(m,1H),4.22(q,J=7.2 Hz,4H),3.74(d,J=12 Hz,1H),3.45(d,J=12 Hz,1H),3.23(dd,J=4.8 Hz,J=10.8 Hz,1H),2.80(dd,J=7.2 Hz,J=10.8 Hz,1H),2.22-2.19(m,2H),2.00-1.96(m,2H),1.19(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ168.5,154.0,151.8,150.9,137.9,119.8,73.8,63.1,57.0,35.4,35.2,33.4,14.0.ESI-HRMS(m / z)calcd C 17 H 23 ClN 5 O 3 S(M+H) + ,412.1205;found,412.1198.HPLC purity:96.1%.14b:light yellow solid,31.0%yield. 1 H NMR(400 MHz,CDCl 3 )δ8.27(s,1H),7.93(s,1H),6.50(s,1H),5.26(t,J=5.6 Hz,1H),4.62(s,2H),4.39-4.34(m,1H),4.27-4.16(m,4H),3.88(d,J=12.0 Hz,1H),3.54(d,J=12.0 Hz,1H),3.24(q,J=6 Hz,1H),2.93(dd,J=5.2 Hz,J=11.6 Hz,1H),2.25-2.17(m,2H),2.02-1.93(m,2H),1.15(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ169.7,154.0,151.9,151.1,137.2,119.9,79.6,74.0,63.0,57.0,35.9,35.4,35.2,14.1.ESI-HRMS(m / z)calcd C 17 H 23 ClN5 O 3 S(M+H) + ,412.1205;found,412.1203.HPLC purity:95.0%.1.2.15.(±)-Ethyl-4-hydroxy-3-(6-(4-morpholinopiperidin-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(15a,15b)

[0063] 15a:white solid,37.7%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.22(s,1H),8.02(s,1H),5.50(s,1H),5.40(t,J=9.6 Hz,1H),4.22(q,J=10.8 Hz,2H),3.76(s,1H),3.72(t,J=6.6 Hz,4H),3.44(d,J=18 Hz,1H),3.22(dd,J=9 Hz,J=16.2 Hz,1H),3.13(s,2H),2.80(dd,J=11.4 Hz,J=16.2 Hz,1H),2.59-2.52(m,5H),2.01(d,J=19.2 Hz,2H),1.62-1.52(m,4H),1.19(t,J=10.8 Hz,3H). 13 C NMR(150 MHz,CDCl 3 )δ169.6,154.0,150.8,137.8,119.8,73.8,67.4,63.1,62.2,49.9,35.3,33.3,28.6,14.0.ESI-HRMS(m / z)calcdC 21 H 31 N 6 O 4 S(M+H) + ,463.2111;found,463.2113.HPLC purity:97.9%.15b:white solid,38.6%yield. 1 H NMR(600 MHz,CDCl 3)δ8.25(s,1H),7.92(s,1H),6.55(s,1H)5.49(s,1H),5.25(t,J=8.4 Hz,1H),4.26-4.17(m,2H),3.88(d,J=18.6 Hz,1H),3.73(t,J=6.6 Hz,4H),3.53(d,J=18 Hz,1H),3.24(dd,J=9 Hz,J=16.8 Hz,1H),3.12(s,2H),2.93(q J=7.8 Hz,1H),2.60-2.53(s,5H),2.02(d,J=18 Hz,2H),1.63-1.52(m,4H),1.15(t,J=10.8Hz,3H). 13 CNMR(100 MHz,CDCl 3 )δ169.8,153.9,151.9,151.0,137.0,119.9,79.6,74.0,67.3,63.0,62.2,49.9,35.9,35.2,29.8,28.6,14.1.ESI-HRMS(m / z)calcd C 21 H 31 N 6 O 4 S(M+H) + ,463.2111;found,463.2120.HPLC purity:95.9%.

[0064] 1.2.16.(±)-Ethyl-3-(6-(4-benzylpiperidin-1-yl)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(16a,16b)

[0065] 16a:white solid,32.4%yield. 1 H NMR(600 MHz,CDCl 3)δ8.18(s,1H),7.97(s,1H),7.28-7.11(m,5H),5.37(t,J=10.0 Hz.3H),4.22-4.16(m,2H),3.71(d,J=17.4Hz,1H),3.41(d,J=17.4 Hz,1H)3.21(dd,J=9.6 Hz,J=16.8 Hz,1H),2.99(s,2H),2.78(dd,J=10.8 Hz,J=16.2 Hz,1H)2.55(d,J=10.8 Hz,2H),1.90-1.78(m,4H),1.35-1.22(m,1H),1.16(t,J=10.8 Hz,3H). 13 C NMR(150 MHz,CDCl 3 )δ168.6,154.0,151.9,150.7,140.2,137.6,129.3,128.4,126.1,119.7,73.8,63.0,43.3,38.5,35.3,33.3,32.4,14.0.ESI-HRMS(m / z)calcd C 24 H 29 N 5 O 3 SNa(M+Na) + ,490.1883;found,490.1886.HPLCpurity:97.9%.16b:white solid,31.8%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.21(s,1H),7.87(s,1H),7.28-7.11(m,5H),5.43(s,2H),5.23(t,J=8.4 Hz,1H),4.23-4.14(m,2H),3.84(d,J=18 Hz,1H),3.50(d,J=18 Hz,1H),3.21(dd,J=9 Hz,J=16.8 Hz,1H),3.00(s,2H),2.91(dd,J=7.8 Hz,J=16.8Hz,1H),2.55(d,J=10.8 Hz,2H),1.90-1.78(m,4H),1.36-1.21(m,1H),1.13(t,J=10.8 Hz,3H). 13 C NMR(150 MHz,CDCl 3)δ169.8,154.0,151.9,150.9,140.2,136.8,129.3,128.4,126.1,119.8,79.7,74.0,62.9,43.3,38.5,35.9,35.1,32.4,14.1.ESI-HRMS(m / z)calcd C 24 H 30 N 5 O 3 S(M+H) + ,468.2064;found,468.2067.HPLC purity:99.7%.

[0066] 1.2.17.(±)-Ethyl-3-(6-(4-(2-fluorobenzyl)piperidin-1-yl)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(17a,17b)

[0067] 17a:white solid,24.6%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.24(s,1H),7.90(s,1H),7.20-7.12(m,2H),7.07-7.00(m,2H),6.63(s,1H),5.45(s,2H),5.25(t,J=6Hz,1H),4.26-4.18(m,1H),3.87(d,J=11.4 Hz,1H),3.53(d,J=12 Hz,1H),3.24(dd,J=6 Hz,J=10.8 Hz,1H),3.03(s,2H),2.94(q,J=4.8 Hz,1H),2.62(d,J=7.2Hz,2H),1.96-1.92(m,1H),1.82(d,J=12.6 Hz,2H),1.38-1.34(m,2H),1.15(t,J=7.2 Hz,3H). 13 C NMR(150MHz,CDCl 3 )δ169.8,162.2,160.6,154.0,151.9,136.8,131.7(Jc-F=5.0 Hz),128.0(Jc- F =7.7 Hz),127.1(Jc- F =11.4 Hz),123.9(Jc- F=3.9Hz),119.8,115.4,(Jc- F =22.1 Hz),79.7,73.9,62.9,37.4,36.1,35.9,35.1,32.4,14.1. 19 F NMR(376MHZ,CDCl 3 )δ-117.84.ESI-HRMS(m / z)calcd C 24 H 29 FN 5 O 3 S(M+H) + ,486.1970;found,486.1980.HPLCpurity:96.4%.17b:white solid,37.8%yield. 1 HNMR(400 MHz,CDCl 3 )δ8.24(s,1H),7.90,(s,1H),7.20-7.00(m,4H),5.47(s,2H),5.26(t,J=5.6 Hz,1H),4.26-4.17(m,2H),3.87(d,J=12.4 Hz,1H),3.53(d,J=12Hz,1H),3.24(dd,J=6.4 Hz,J=11.6 Hz,1H),3.03(s,2H),2.94(dd,J=11.2 Hz,J=5.2 Hz,1H),2.62(d,J=7.2 Hz,2H),1.95(s,2H),1.82(d,J=13.2 Hz,2H),1.64(s,1H),1.15(t,J=17.2 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ169.8,154.0,152.0,150.9,136.8,131.7,128.0(d,J C-F =7.9),127.1(d,J C-F =16.1 Hz),123.9,119.8,115.4(d,J C-F =21.7 Hz),79.7,74.0,63.0,37.4,36.2,35.2,32.3,29.8,14.1. 19 F NMR(376 MHZ,CDCl 3 )δ-117.86.ESI-HRMS(m / z)calcd C 24 H 29 FN 5 O 3S(M+H) + ,486.1970;found,486.1978.HPLC purity:99.6%.

[0068] 1.2.18.(±)-Ethyl-4-hydroxy-3-(6-(4-methylpiperazin-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(18a,18b)

[0069] 18a:white solid,29.5%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.23(s,1H),8.02(s,1H),5.40(t,J=6.6 Hz,1H),4.35(s,2H),4.24-4.20(m,2H),3.74(d,J=11.4 Hz,1H),3.44(d,J=11.4 Hz,1H),3.23(dd,J=6 Hz,J=10.8 Hz,1H),2.80(dd,J=7.2Hz,J=10.8Hz,1H),2.55(t,J=5.4 Hz,4H),2.35(s,3H)1.77(s,2H),1.19(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ168.6,154.1,151.9,150.9,137.9,119.8,73.8,63.1,55.2,46.2,35.3,33.4,14.0.ESI-HRMS(m / z)calcd C 17 H 24 ClN 6 O 3 SNa(M+Na) + ,393.1703;found,393.1701.HPLC purity:98.1%.18b:white solid,41.6%yield. 1 H NMR(600 MHz,CDCl 3)δ8.26(s,1H),7.93(s,1H),5.26(t,J=6 Hz,1H),4.34(s,2H),4.25-4.18(m,2H),3.88(d,J=12 Hz,1H),3.54(d,J=12 Hz,1H),3.24(q,J=5.4 Hz,1H),2.93(q,J=5.4 Hz,1H),2.56(s,4H),2.36(s,3H),1.75(s,2H),1.15(t,J=7.2 Hz,3H). 13 C NMR(150 MHz,CDCl 3 )δ169.8,154.1,151.9,151.0,137.1,119.9,79.6,74.0,63.0,55.2,46.3,35.9,35.2,14.1.ESI-HRMS(m / z)calcdC 17 H 24 ClN 6 O 3 SNa(M+Na) + ,393.1703;found,393.1699.HPLCpurity:95.6%.

[0070] 1.2.19.(±)-Ethyl-3-(6-(azepan-1-yl)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(19a,19b)

[0071] 19a:white solid,31.8%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.25(s,1H),7.88(s,1H),6.84(s,1H)5.30-5.24(m,1H),4.45(s,1H),4.28-4.20(m,3H),3.99(s,1H),3.88(d,J=17.4 Hz,2H),3.55(d,J=18 Hz,1H),3.27(dd,J=9.6 Hz,J=16.8 Hz,1H),2.98(q,J=7.8 Hz,1H),1.89(s,4H),1.62-1.57(m,4H),1.17(t,J=10.8 Hz,3H). 13 C NMR(100 MHz,CDCl 3)δ169.7,154.5,151.8,150.6,136.9,119.6,79.7,73.9,62.8,35.9,35.0,14.0.ESI-HRMS(m / z)calcd C 18 H 26 N 5 O 3 S(M+H) + ,392.1751;found,392.1744.HPLC purity:95.7%.19b:white solid,52.4%yield. 1 H NMR(600MHz,CDCl 3 )δ8.22(s,1H),7.99(s,1H),6.42(s,1H),5.41(t,J=6.6 Hz,1H),4.44(s,1H)4.30-4.20(m,3H),3.97(s,1H),3.85(s,1H),3.75(d,J=11.4 Hz,1H),3.44(d,J=11.4 Hz,1H),3.22(dd,J=6 Hz,J=10.8 Hz,1H),2.83(t,J=7.2 Hz,1H),1.89(s,4H),1.60(s,1H),1.20(t,J=6.6 Hz,3H). 13 C NMR(100MHz,CDCl 3 )δ168.7,154.7,151.9,150.5,137.8,119.6,73.8,63.0,50.0,48.6,35.3,33.3,29.1,27.3,27.0,14.0.ESI-HRMS(m / z)calcd C 18 H 26 N 5 O 3 S(M+H) + ,392.1751;found,392.1758.HPLC purity:95.3%.

[0072] 1.2.20.(±)-Ethyl-4-hydroxy-3-(6-(pyrrolidin-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(20a,20b)

[0073] 20a:white solid,26.5%yield. 1 H NMR(600 MHz,CDCl3 )δ8.27(s,1H),7.87(s,1H),5.26(t,J=6.0 Hz,1H),4.26-4.16(m,4H),3.88(d,J=12.0 Hz,1H),3.76(s,2H),3.55(d,J=12.0 Hz,1H),3.27(dd,J=6.4 Hz,J=11.2 Hz,1H),2.96(dd,J=5.2 Hz,J=11.2 Hz,1H),2.04(d,J=26 Hz,4H),1.16(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ169.7,153.4,152.3,150.3,137.6,120.3,79.9,74.0,62.9,36.0,35.0,14.1.ESI-HRMS(m / z)calcd C 16 H 22 N 5 O 3 S(M+H) + ,364.1438;found,364.1437.HPLC purity:98.4%.20b:whitesolid,58.0%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.22(s,1H),7.96(s,1H),6.43(s,1H),5.41(t,J=6.4 Hz,1H),4.30-4.15(m,4H),3.72(d,J=9.2 Hz,3H),3.47(d,J=11.6 Hz,1H),3.24(dd,J=6 Hz,J=10.8 Hz,1H),2.84(dd,J=6.8 Hz,J=10.8 Hz,1H),2.03(t,J=25.6 Hz,4H),1.18(t,J=6.8Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ168.7,153.3,152.3,150.0,138.3,120.1,74.0,63.0,49.1,47.7,35.2,33.5,26.4,24.4,14.0.ESI-HRMS(m / z)calcd C 16 H 22 N 5 O 3 S(M+H) +,364.1438;found,364.1435.HPLC purity:95.3%.

[0074] 1.2.21.(±)-Ethyl-4-hydroxy-3-(6-(methylthio)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(21a,21b)

[0075] 21a:yellowish solid,33.4%yield. 1 H NMR(400 MHz,CDCl 3 )δ8.63(s,1H),8.22(s,1H),5.74(s,1H),5.30(t,J=5.2 Hz,1H),4.18-4.12(m,2H),3.91(d,J=12.4 Hz,1H),3.56(d,J=12.0 Hz,1H),3.15(dd,J=5.2 Hz,=11.6 Hz,1H),2.86(dd,J=4.8Hz,J=11.6 Hz,1H),2.68(s,3H),1.07(t,J=7.2 Hz,3H). 13 C NMR(150 MHz,CDCl 3 )δ169.1,162.7,151.4,148.2,141.3,131.4,79.0,74.0,63.0,35.5,35.2,13.9,11.8.ESI-HRMS(m / z)calcd C 13 H 16 N 4 O 3 S 2 Na(M+Na) + ,363.0556;found,363.0546.HPLC purity:99.8%.21b:yellowish solid,40.5%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.61(s,1H),8.28(s,1H),5.45(t,J=6.0 Hz,1H),5.04(s,1H),4.23-4.14(m,2H),3.69(dd,J=11.6 Hz,J=36.8 Hz,2H),3.30(q,J=6.0 Hz,1H),2.87(q,J=6.0 Hz,1H),2.67(s,3H),1.12(t,J=7.2 Hz,3H).13 C NMR(100 MHz,CDCl 3 )δ168.2,162.6,151.5,148.3,142.3,131.1,76.5,74.0,63.1,34.8,34.0,13.9,11.9.ESI-HRMS(m / z)calcd C 13 H 16 N 4 O 3 S 2 Na(M+Na) + ,363.0556;found,363.0564.HPLC purity:99.6%.1.2.22.(±)-Ethyl-3-(6-(ethylthio)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(22a,22b)

[0076] 22a:yellow solid,21.5%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.58(s,1H),8.28(s,1H),5.44(q,J=6 Hz,1H),5.12(d,J=5.4 Hz,1H),4.18-4.13(m,2H),3.73(d,J=12 Hz,1H),3.65(d,J=11.4 Hz,1H),3.34-3.28(m,3H),2.86(q,J=5.4 Hz,1H),1.41(t,J=7.8Hz,3H),1.12(t,J=7.2 Hz,3H). 13 C NMR(150 MHz,CDCl 3 )δ168.2,162.3,151.5,148.4,142.3,130.9,76.5,74.0,63.0,34.8,33.9,23.4,14.7,13.9.ESI-HRMS(m / z)calcdC 14 H 18 N 4 O 3 S 2 Na(M+Na) + ,377.0713;found,377.0697.HPLCpurity:99.8%.22b:yellowsolid,22.6%yield. 1 H NMR(600 MHz,CDCl 3)δ8.63(s,1H),8,28(s,1H),5.48(q,J=6 Hz,1H),4.81(d,J=5.4 Hz,1H),4.23-4.19(m,2H),3.77(d,J=12 Hz,1H),3.55(d,J=12 Hz,1H),3.38(q,J=7.2 Hz,2H),3.28(q,J=6 Hz,1H),2.85(dd,J=6.6 Hz,J=11.4 Hz,1H),1.45(t,J=7.2 Hz,3H),1.17(t,J=7.2 Hz,3H). 13 C NMR(150 MHz,CDCl 3 )δ168.2,162.9,151.5,148.5,142.2,131.3,74.0,63.2,35.1,33.6,23.5,14.8,14.0.ESI-HRMS(m / z)calcdC 14 H 18 N 4 O 3 S 2 Na(M+Na) + ,377.0713;found,377.0704.HPLC purity:99.3%.

[0077] 1.2.23.(±)-Ethyl-4-hydroxy-3-(6-(isopropylthio)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(23a,23b)

[0078] 23a:white solid,51.2%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.63(s,1H),8.27(s,1H),5.48(s,1H),4.81(s,1H)4.37-4.33(m,1H),4.24-4.20(m,2H)3.78(d,J=12 Hz,1H),3.53(d,J=12 Hz,1H),3.28(q,J=6 Hz,1H),2.85(dd,J=6.6 Hz,J=10.8 Hz,1H),1.50(d,J=7.2 Hz,6H),1.18(t,J=7.2 Hz,3H). 13 C NMR(150 MHz,CDCl 3)δ168.3,163.2,151.5,148.5,142.1,131.2,74.0,63.3,35.1,34.7,33.6,23.4,23.3,14.0.ESI-HRMS(m / z)calcd C 15 H 20 N 4 O 3 S 2 Na(M+Na) + ,391.0869;found,391.0871.HPLC purity:99.8%.23b:whitesolid,22.4%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.61(s,1H),8.20(s,1H),5.76(s,1H),5.30(s,1H),4.34-4.29(m,1H),4.19-4.15(m,2H)3.91(d,J=12.6 Hz,1H),3.56(d,J=12.6Hz,1H),3.16(dd,J=4.8 Hz,J=11.4 Hz,1H),2.86(dd,J=5.4 Hz,J=12 Hz,1H),1.46(d,J=6.0 Hz,6H),1.09(t,J=6.6 Hz,3H). 13 C NMR(150 MHz,CDCl 3 )δ169.2,162.8,151.4,148.4,141.2,131.2,74.0,63.1,35.5,35.2,34.6,23.3,23.2,13.9.ESI-HRMS(m / z)calcd C 15 H 20 N 4 O 3 S 2 Na(M+Na) + ,391.0869;found,391.0868.HPLC purity:95.1%.

[0079] 1.2.24.(±)-Ethyl-4-hydroxy-3-(6-(prop-2-yn-1-ylthio)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(24a,24b)

[0080] 24a:white solid,41.2%yield. 1H NMR(600 MHz,CDCl 3 )δ8.68(s,1H),8.32(s,1H),5.46(s,1H),4.65(s,1H),4.23-4.18(m,2H),4.16(d,J=2.4 Hz,2H),3.76(d,J=12Hz,1H),3.60(d,J=12 Hz,1H),3.29(q,J=6 Hz,1H),2.85(q,J=6 Hz,1H),2.22(t,J=2.4 Hz,1H),1.16(t,J=7.2 Hz,3H). 13 C NMR(150 MHz,CDCl 3 )δ168.1,160.2,151.6,148.9,142.6,131.0,79.3,74.0,71.3,63.2,35.0,33.7,17.3,14.0.ESI-HRMS(m / z)calcdC 15 H 16 N 4 O 3 S 2 Na(M+Na) + ,387.0556;found,387.0550.HPLCpurity:98.6%.24b:white solid,49.7%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.71(s,1H),8.27(s,1H),5.54(s,1H),5.33(s,1H),4.23-4.19(m,4H),3.93(d,J=12.6 Hz,1H),3.57(d,J=12.6 Hz,1H),3.19(dd,J=4.8Hz,J=12 Hz,1H),2.83(dd,J=4.8Hz,J=11.4 Hz,1H),2.23(t,J=2.4 Hz,1H),1.12(t,J=7.2 Hz,3H). 13 C NMR(150MHz,CDCl 3 )δ169.4,160.3,151.6,148.7,141.7,131.4,79.3,79.1,74.1,71.3,63.3,35.6,35.4,17.3,14.0.ESI-HRMS(m / z)calcd C 15 H 16 N 4 O 3 S 2 Na(M+Na)+ ,387.0556;found,387.0566.HPLC purity:97.2%.

[0081] 1.2.25.(±)-Ethyl-3-(6-(butylthio)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(25a,25b)

[0082] 25a:white solid,45.1%yield. 1 H NMR(400 MHz,DMSO)δ8.72(s,1H),8.68(s,1H),5.93(d,J=5.2 Hz,1H),5.23-5.20(m,1H),4.17-4.07(m,2H),4.00(d,J=11.6Hz,1H),3.62(d,J=11.2 Hz,1H),3.41-3.30(m,3H),2.90(dd,J=3.6 Hz,J=11.2Hz,1H),1.73-1.66(m,2H),1.49-1.39(m,2H),1.04(t,J=7.2 Hz,3H),0.91(t,J=7.2 Hz,3H). 13 CNMR(100 MHz,DMSO)δ167.9,159.8,151.3,148.6,143.8,130.5,73.9,73.8,62.0,35.5,33.7,31.2,27.5,21.4,13.6,13.5.ESI-HRMS(m / z)calcdC 16 H 22 N 4 O 3 S 2 (M+Na) + ,405.1026;found,405.1020.HPLC purity:99.6%.25b:white solid,30.2%yield. 1 H NMR(400 MHz,CDCl 3)δ8.62(s,1H),8.21(s,1H),5.74(s,1H),5.33-5.30(m,1H),4.22-4.15(m,2H),3.91(d,J=12 Hz,1H),3.56(d,J=12.4 Hz,1H),3.40-3.35(m,1H),3.18(q,J=4.8 Hz,1H),2.87(q=3.2,1H),1.80-1.72(m,2H),1.54-1.45(m,2H),1.10(t,J=7.2 Hz,3H),0.94(t,J=7.6 Hz,3H). 13 CNMR(100 MHz,CDCl 3 )δ169.3,162.9,151.5,148.3,141.2,131.4,79.1,74.0,63.0,35.6,35.3,31.5,28.6,22.1,14.0,13.7.ESI-HRMS(m / z)calcd C 16 H 22 N 4 O 3 S 2 (M+Na) + ,405.1026;found,405.1031.HPLC purity:98.6%.

[0083] 1.2.26.(±)-Ethyl-4-hydroxy-3-(6-(pentylthio)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(26a,26b)

[0084] 26a:white solid,43.3%yield. 1 H NMR(400 MHz,CDCl 3)δ8.62(s,1H),8.27(s,1H),5.48(q,J=6.0 Hz,1H),4.80(d,J=6.0 Hz,1H),4.21(q,J=7.2 Hz,2H),3.78(d,J=12 Hz,1H),3.54(d,J=12.0 Hz,1H),3.38(t.J=7.2 Hz,2H),3.28(q,J=6.0 Hz,1H),2.85(dd,J=6.4 Hz,J=11.2 Hz,1H),1.83-1.75(m,2H),1.51-1.33(m,4H),1.17(t,J=6.8 Hz,3H),0.91(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ168.3,163.1,151.5,148.4,142.2,131.3,74.0,63.2,35.1,33.6,31.1,29.2,29.0,22.4,14.1,14.0.ESI-HRMS(m / z)calcd C 17 H 25 N 4 O 3 S 2 (M+H) + ,397.1363;found,397.1354.HPLC purity:99.6%.26b:white solid,25.1%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.62(s,1H),8.21(s,1H),5.75(s,1H),5.32-5.30(m,1H),4.21-4.14(m,2H),3.91(d,J=12.0 Hz,1H),3.56(d,J=12.0 Hz,1H),3.39-3.32(m,2H),3.17(dd,J=4.8 Hz,J=11.4 Hz,1H),2.87(dd,J=4.8 Hz,J=11.4 Hz,1H),1.79-1.74(m,2H),1.47-1.42(m,2H),1.37-1.31(m,2H),1.09(t,J=7.2 Hz,3H),0.88(t,J=7.2 Hz,3H). 13 CNMR(100 MHz,CDCl 3)δ169.1,162.7,151.4,148.2,141.3,131.3,79.0,74.0,63.0,35.5,35.2,31.0,29.0,28.8,22.2,14.0,14.0.ESI-HRMS(m / z)calcd C 17 H 25 N 4 O 3 S 2 (M+H) + ,397.1363;found,397.1361.HPLC purity:95.3%.

[0085] 1.2.27.(±)-Ethy-3-(6-(benzylthio)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(27a,27b)

[0086] 27a:white solid,43.2%yield. 1 H NMR(400 MHz,DMSO)δ8.73(s,1H),8.68(s,1H),7.44(d,J=7.2 Hz,1H),7.31-7.20(m,3H),6.40(d,J=5.6 Hz,1H),5.43(dd,J=5.6Hz,J=9.6 Hz,1H),4.64(dd,J=13.6 Hz,J=16 Hz,2H),4.12-4.00(m,J=13.6 Hz 2H),3.90(d,J=12.4 Hz,1H),3.78(d,J=12.4 Hz,1H),3.26-3.22(m,1H),2.96(dd,J=4 Hz,J=12 Hz,1H),1.00(t,J=7.2 Hz,3H). 3 C NMR(100 MHz,DMSO)δ167.9,159.0,151.2,148.8,144.0,137.8,130.3,129.0,128.5,127.2,73.9,62.0,35.5,33.7,31.6,13.7.ESI-HRMS(m / z)calcd C 19 H 20 N 4 O 3 S 2 (M+Na) +,439.0869;found,439.0872.HPLC purity:99.7%.27b:white solid,42.4%yield. 1 H NMR(400 MHz,DMSO)δ8.75(s,1H),8.74(s,1H),7.47(d,J=7.2 Hz,2H),7.34-7.22(m,3H),5.94(d,J=5.2 Hz,1H),5.24-5.21(m,1H),4.66(dd,J=13.6 Hz,J=18.4Hz,2H),4.15-4.07(m,2H),4.00(d,J=11.6 Hz,1H),3.62(d,J=11.2 Hz,1H),3.40-3.38(m,1H),2.90(dd,J=3.2 Hz,J=11.2 Hz,1H),1.04(t,J=6.8Hz,3H). 13 CNMR(100 MHz,DMSO)δ167.3,159.3,151.3,148.7,143.0,137.7,130.5,129.0,128.5,127.2,76.6,75.4,61.8,35.8,34.6,31.6,13.8.ESI-HRMS(m / z)calcd C 19 H 20 N 4 O 3 S 2 (M+Na) + ,439.0869;found,439.0863.HPLC purity:98.3%.

[0087] 1.2.28.(±)-Ethyl-3-(6-((3-fluorophenyl)thio)-9H-purin-9-yl)-4-Hydroxytetrahydro thiophene-3-carboxylate(28a,28b)

[0088] 28a:white solid,26.4%yield. 1 H NMR(600 MHz,CDCl 3)δ8.21(s,1H),7.90(s,1H),6.48(s,1H),5.21(t,J=6 Hz,1H),4.93-4.84(m,1H),4.46(s,2H)4.19-4.13(m,4H),3.83(d,J=12 Hz,1H),3.50(d,J=12.6 Hz,1H),3.18(q,J=6 Hz,1H),2.89(dd,J=4.8Hz,J=11.4 Hz,1H),1.99-1.90(m,4H),1.10(t,J=7.2 Hz,3H). 13 CNMR(150 MHz,CDCl 3 )δ169.5,153.8,151.8,150.9,137.0,119.7,88.6,87.5,79.4,73.9,62.8,42.3,35.7,35.0,31.4(Jc- F =19.7),13.9. 19 F NMR(376MHz,CDCl 3 )δ-111.38.ESI-HRMS(m / z)calcdC 18 H 17 FN 4 O 3 S 2 (M+Na) + ,443.0618;found,443.0613.HPLC purity:99.0%.28b:white solid,31.3%yield. 1 H NMR(400 MHz,CDCl 3 )δ8.58(s,1H),8.31(s,1H),7.46-7.40(m,3H),7.21-7.16(m,1H),5.33(t,J=4.8 Hz,1H),4.24-4.18(m,2H),3.94(d,J=12.4 Hz,1H),3.58(d,J=12.4 Hz,1H)3.19(dd,J=4.8 Hz,J=11.6 Hz,1H),2.86(dd,J=4.8 Hz,J=11.6 Hz,1H),1.12(t,J=7.2Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ169.4,164.0,161.4(d,J C-F =38Hz),151.9,149.0,142.0,131.3(d,J C-F=3.4 Hz),130.9,130.7(d,J C-F =8.1 Hz),128.8(d,J C-F =8.0 Hz),122.8,122.6,117.2,117.0,79.1,74.1,63.3,35.6,35.4,14.0. 19 F NMR(376MHz,CDCl 3 )δ-111.35.ESI-HRMS(m / z)calcd C 18 H 18 FN 4 O 3 S 2 (M+H) + ,421.0799;found,421.0790.HPLC purity:95.7%.

[0089] 1.2.29.(±)-Ethyl-3-(6-((4-(tert-butyl)phenyl)thio)-9H-purin-9-yl)-4-hydroxytetra hydrothiophene-3-carboxylate(29a,29b)

[0090] 29a:white solid,33.4%yield. 1 H NMR(400 MHz,DMSO)δ8.80(s,1H),8.55(s,1H),7.57-7.51(m,4H),5.95(d,J=5.2 Hz,1H),5.24-5.20(m,1H),4.15-4.08(m,2H),4.01(d,J=11.2 Hz,1H),3.63(d,J=11.2 Hz,1H),3.38(dd,J=5.6 Hz,J=11.2 Hz,1H),2.91(dd,J=3.6 Hz,J=11.2 Hz,1H),1.33(s,9H),1.04(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,DMSO)δ167.9,159.2,152.3,151.5,149.0,144.4,135.3,129.8,126.4,123.1,73.9,62.0,35.5,34.5,33.7,31.0,13.6.ESI-HRMS(m / z)calcdC 22 H 27 N 4 O 3 S2 (M+H) + ,459.1519;found,459.1515.HPLC purity:96.4%.29b:white solid,37.4%yield. 1 H NMR(400 MHz,DMSO)δ8.76(s,1H),8.56(s,1H),7.57-7.51(m,4H),6.42(d,J=5.2 Hz,1H),5.49-5.45(m,1H),4.13-4.03(m,2H),3.92(d,J=12.4 Hz,1H),3.80(d,J=12.0 Hz,1H),3.28(dd,J=5.2Hz,J=12 Hz,1H),2.99(dd,J=3.6 Hz,J=12.0 Hz,1H),1.32(s,10H),1.03(t,J=6.8Hz,3H). 13 CNMR(100 MHz,DMSO)δ167.3,159.5,152.3,151.5,148.9,143.3,135.3,130.0,126.4,123.0,76.6,75.5,61.8,35.8,34.6,34.5,31.0,13.8.ESI-HRMS(m / z)calcdC 22 H 27 N 4 O 3 S 2 (M+H) + ,459.1519;found,459.1514.HPLC purity:96.3%.

[0091] 1.2.30.(±)-Ethyl-3-(6-((3-fluoropropyl)thio)-9H-purin-9-yl)-4-hydroxytetrahydro thiophene-3-carboxylate(30a,30b)

[0092] 30a:white solid,31.4%yield. 1 H NMR(400 MHz,CDCl 3)δ8.63(s,1H),8.30(s,1H),5.47(t,J=6.4 Hz,1H),4.71(s,1H),4.67(t,J=6 Hz,1H),4.55(t,J=6.0 Hz,1H),4.21(q,J=6.8 Hz,2H)3.77(d,J=11.6 Hz,1H),3.56(d,J=11.6 Hz,1H),3.50(t,J=6.8Hz,2H),3.29(q,J=5.6 Hz,1H),2.84(dd,J=6.8 Hz,J=11.2 Hz,1H),2.27-2.14(m,2H),1.17(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ168.2,162.0,151.5.148.6142.4,131.4,83.3,81.6,74.0,63.2,35.1,33.6,30.6(d,J C-F =20Hz),24.8(d,J C-F =5.2 Hz),14.0. 19 F NMR(376MHz,CDCl 3 )δ20.41.ESI-HRMS(m / z)calcd C 15 H 19 FN 4 O 3 S 2 Na(M+Na) + ,409.0775;found,409.0774.HPLC purity:97.1%.30b:white solid,30.2%yield. 1 H NMR(400 MHz,CDCl 3 )δ8.65(s,1H),8.24(s,1H),5.66(s,1H),5.32(t,J=4.8 Hz,1H),4.66(t,J=5.6 Hz,1H),4.54(t,J=6.0 Hz,1H),4.20(dd,J=6.4 Hz,J=13.6 Hz,2H),3.92(d,J=12.4 Hz,1H),3.56(d,J=12.0 Hz,1H),3.50(t,J=6.8 Hz,2H),3.18(dd,J=11.2 Hz,J=4.8 Hz,1H),2.86(dd,J=11.6 Hz,J=5.2 Hz,1H),2.26-2.14(m,2H),1.11(t,J=7.2Hz,3H).13 CNMR(100 MHz,CDCl 3 )δ169.3,161.9,151.5,148.5,141.5,131.5,83.3,81.6,79.1,74.1,63.2,35.42(d,J C-F =25.4 Hz),30.6(d,J C-F =20.1 Hz),24.8(d,J C-F =5.1Hz),14.0. 19 F NMR(376MHz,CDCl 3 )δ-20.45.ESI-HRMS(m / z)calcd C 15 H 19 FN 4 O 3 S 2 Na(M+Na) + ,409.0775;found,409.0768.HPLC purity:97.7%.

[0093] 1.2.31.(±)-Ethyl-3-(6-((3-chloropropyl)thio)-9H-purin-9-yl)-4-hydroxytetrahydro thiophene-3-carboxylate(31a,31b)

[0094] 31a:white solid,42.4%yield. 1 H NMR(400 MHz,CDCl 3 )δ8.59(s,1H),8.31(s,1H),5.44(s,1H),5.01(s,1H),4.20-4.13(m,2H),3.75-3.63(m,4H),3.47(t,J=6.8Hz,2H),3.29(q,J=5.6 Hz,1H),2.85(dd,J=6.4 Hz,J=11.2 Hz,1H),2.26-2.20(m,2H),1.13(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ168.1,161.4,151.5,148.6,142.5,131.0,73.9,63.1,43.5,34.8,33.9,32.2,26.0,13.9.ESI-HRMS(m / z)calcdC 15 H 19 ClN4 O 3 S 2 Na(M+Na) + ,425.0479;found,425.0470.HPLC purity:96.4%.31b:whitesolid,21.7%yield. 1 H NMR(400 MHz,CDCl 3 )δ8.62(s,1H),8.24(s,1H),5.67(s,1H),5.31(s,1H),4.176(q,J=7.2 Hz,2H),3.92(d,J=12 Hz,1H),3.68(t,J=6.4 Hz,2H),3.56(d,J=12.4 Hz,1H),3.50(t,J=6.8 Hz,2H),3.16(dd,J=5.2 Hz,J=11.6 Hz,1H),2.86(dd,J=4.8 Hz,J=11.6 Hz,1H),2.28-2.21(m,2H),1.094(t,J=7.2 Hz,3H). 13 C NMR(100MHz,CDCl 3 )δ169.2,161.7,151.4,148.4,141.5,131.4,79.0,74.1,63.1,43.5,35.5,35.3,32.2,26.0,13.9.ESI-HRMS(m / z)calcd C 15 H 19 ClN 4 O 3 S 2 Na(M+Na) + ,425.0479;found,425.0474.HPLC purity:95.2%.

[0095] 1.2.32.(±)-Ethyl-3-(2-amino-6-(piperidin-1-yl)-9H-purin-9-yl)-4-hydroxytetrahydro thiophene-3-carboxylate(32a,32b)

[0096] 32a:yellow solid,43.0%yield. 1 H NMR(600 MHz,CDCl 3)δ7.60(s,1H),5.18(t,J=5.4 Hz,1H),4.60(s,2H),4.27-4.16(m,4H),3.80(d,J=12 Hz,1H),3.49(d,J=12 Hz,1H),3.24(dd,J=6.6 Hz,J=11.4 Hz,1H),2.97(q,J=5.4 Hz,1H),1.71-1.64(m,6H),1.18(t,J=7.2 Hz,3H). 13 C NMR(150 MHz,CDCl 3 )δ169.9,152.8,134.5,114.7,79.5,73.6,62.7,58.6,36.0,34.9,29.8,26.3,24.9,18.6,14.1.ESI-HRMS(m / z)calcd C 17 H 25 N 6 O 3 S(M+H) + ,393.1703;found,393.1701.HPLC purity:99.0%.32b:yellow solid,32.6%yield. 1 HNMR(600 MHz,CDCl 3 )δ7.72(s,1H),5.34(t,J=6.6 Hz,1H),4.59(s,2H),4.26-4.11(m,4H),3.68(d,J=12 Hz,1H)3.37(d,J=12 Hz,1H),3.17(dd,J=6 Hz,J=10.8 Hz,1H),2.81(dd,J=7.2 Hz,J=10.8Hz,1H),1.72-1.65(m,6H),1.20(t,J=7.2 Hz,3H). 13 C NMR(150 MHz,CDCl 3 )δ168.8,158.5,154.5,152.4,135.1,114.7,73.5,62.8,35.2,33.4,26.3,24.9,14.1.ESI-HRMS(m / z)calcd C 17 H 25 N 6 O 3 S(M+H) + ,393.1703;found,393.1701.HPLCpurity:98.2%.

[0097] 1.2.33.(±)-Ethyl-3-(2-fluoro-6-(piperidin-1-yl)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(33a,33b)

[0098] 33a:yellow solid,36.4%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.01(s,1H),5.44(s,1H),4.35(s,2H),4.23(q,J=7.2,2H),3.94-3.68(m,3H),3.47(d,J=11.6,1H),3.26(dd,J=6,J=11.2,1H),2.84(dd,J=6.4,J=11.2,1H),1.72(s,6H),1.21(t,J=7.6,3H) 13 C NMR(100 MHz,CDCl 3 )δ168.3,159.6,157.5,154.9(d,J C-F =19.6),152.4(d,J C-F =11.5),137.5(d,J C-F =2.9),117.8(d,J C-F =4.3),76.3,73.6,63.1,58.6,35.2,33.9,26.2,24.5,18.5,14.0. 19 F NMR(376MHz,CDCl 3 )δ-50.27.ESI-HRMS(m / z)calcdC 17 H 23 FN 5 O 3 S(M+H) + ,396.1500;found,396.1499.HPLC purity:99.4%.33b:yellow solid,43.6%yield. 1 H NMR(600MHz,CDCl 3)δ7.96(s,1H),5.38(s,1H),5.26-5.24(m,1H),4.60-4.45(m,2H),4.29-4.19(m,2H),3.97(s,1H),3.86(d,J=12,2H),3.49(d,J=12.6,1H),3.13(dd,J=4.8,J=12,1H),3.8(dd,J=3.6,J=11.4,1H),1.16(t,J=7.2,3H). 13 C NMR(150 MHz,CDCl 3 )δ169.7,159.1,157.7,154.9(Jc- F =19.8),152.3(Jc- F =18.6),136.6(J c-F =3),118.0(J c-F =4.4),78.9,73.6,63.1,35.6(J c-F =6.5),24.7,14.0.. 19 F NMR(376MHz,CDCl 3 )δ-50.14.ESI-HRMS(m / z)calcd C 17 H 22 FN 5 O 3 S(M+Na) + ,418.1320;found,418.1322.HPLC purity:96.6%.

[0099] 1.2.34.(±)-Ethyl-3-(2-chloro-6-(piperidin-1-yl)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(34a,34b)

[0100] 34a:white solid,17.6%yield. 1 H NMR(600 MHz,CDCl 3)δ8.01(s,1H),5.41(t,J=6 Hz,1H),4.62(s,2H),4.29-4.20(m,4H),3.69(d,J=12 Hz,1H),3.45(d,J=11.4Hz,1H),3.25(q,J=5.4 Hz,1H),2.82(dd,J=7.2 Hz,J=11.4 Hz,1H),1.74-1.68(m,6H),1.22(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ168.4,154.0,153.7,152.1,137.6,118.4,73.6,63.2,35.3,33.7,29.8,26.3,24.7,14.0.ESI-HRMS(m / z)calcd C 17 H 23 ClN 5 O 3 S(M+H) + ,412.1205;found,412.1210.HPLC purity:96.1%.34b:white solid,21.6%yield 1 H NMR(400 MHz,CDCl 3 )δ7.93(s,1H),5.65(s,1H),5.24(t,J=4.8 Hz,1H),4.31-4.16(m,6H),3.86(d,J=12,6 Hz,1H),3.48(d,J=12Hz,1H),3.16(dd,J=4.8 Hz,J=11.4 Hz,1H),2.86(dd,J=4.8 Hz,J=11.4 Hz,1H),1.73-1.67(m,6H),1.18(t,J=7.2 Hz,3H). 13 CNMR(100 MHz,CDCl 3 )δ169.6,154.0,153.6,152.0,136.8,118.6,79.2,73.7,63.1,35.7,35.4,26.2,24.7,14.0.ESI-HRMS(m / z)calcd C 17 H 23 ClN 5 O 3 S(M+H) + ,412.1205;found,412.1206.HPLCpurity:98.7%.

[0101] 1.2.35.(±)-Ethyl-3-(2-amino-6-(propylthio)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(35a,35b)

[0102] 35a:white solid,41.3%yield. 1 H NMR(400 MHz,CDCl 3 )δ7.90(s,1H),5.42(t,J=5.6 Hz,1H),4.96(s,2H),4.23-4.16(m,2H),3.71(d,J=11.6 Hz,1H),3.44(d,J=11.2Hz,1H),3.27-3.21(m,3H),2.86(dd,J=6.0 Hz,J=10.8 Hz,1H),1.82-1.73(m,2H),1.19(t,J=7.2 Hz,3H),1.05(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ168.5,163.8,158.3,150.0,139.3,125.5,76.7,73.8,63.0,35.1,33.7,30.7,23.0,14.0,13.6.ESI-HRMS(m / z)calcd C 15 H 22 N 5 O 3 S 2 (M+H) + ,384.1159;found,384.1150.HPLC purity:99.7%.35b:white solid,43.5%yield. 1 H NMR(400 MHz,CDCl 3 )δ7.84(s,1H),5.98(s,1H),5.24(t,J=5.2 Hz,1H),4.95(s,2H),4.27-4.14(m,2H),3.82(d,J=12 Hz,1H),3.50(d,J=12.0 Hz,1H),3.28-3.24(m,2H),3.17(q,J=5.6Hz,1H),2.90(dd,J=4.8 Hz,J=11.2 Hz,1H),1.82-1.73(m,2H),1.15(t,J=7.2 Hz,3H),1.05(t,J=7.2 Hz,3H).13 C NMR(100 MHz,CDCl 3 )δ169.7,163.4,158.2,150.3,138.4,125.6,79.0,73.6,63.0,35.6,35.1,30.6,23.0,14.0,13.6.ESI-HRMS(m / z)calcd C 15 H 22 N 5 O 3 S 2 (M+H) + ,384.1159;found,384.1149.HPLC purity:98.4%.1.2.36.(±)-Ethyl-3-(2-fluoro-6-(propylthio)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(36a,36b)

[0103] 36a:white solid,38.5%yield. 1 H NMR(400 MHz,CDCl 3 )δ8.31,(s,1H)5.44,(t,J=6 Hz,1H),4.24(q,J=13.6 Hz,2H),3.72(d,J=11.6 Hz,1H),3.61(d,J=11.6 Hz,1H),3.34-3.29(m,2H),2.85(dd,J=11.2,J=6 Hz,1H),1.86-1.77(m,2H),1.19(t,J=6.8 Hz,3H)1.08(t,J=7.6 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ169.3,166.0(d,J C-F =16.5 Hz),159.7,158.3,152.2(d,J C-F =17.6 Hz),145.2(d,J C-F =3.2 Hz),76.4,75.7,36.0,31.9,23.9,14.1,13.6. 19 F NMR(376MHz,CDCl 3 )δ-49.78.ESI-HRMS(m / z)calcd C 15 H 19 FN 4 O 3 S2 Na(M+Na) + ,409.0775;found,409.0775.HPLCpurity:99.4%.36b:white solid,41.5%yield. 1 HNMR(600 MHz,CDCl 3 )δ8.27(s,1H),5.29(t,J=4.2 Hz,1H),4.25-4.19(m,2H)3.92(d,J=12.6 Hz,1H)3.54(d,J=12.6 Hz,1H),3.36-3.29(m,2H),3.11(dd,J=4.2 Hz,J=12 Hz,1H),3.81(dd,J=4.8 Hz,J=12 Hz,1H),1.84-1.78(m,2H),1.14(t,J=7.2 Hz,3H),1.07(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ169.1,166.1(Jc-F=16.5 Hz),157.6(d,Jc-F=215.0 Hz),150.1(Jc-F=17.3 Hz),141.4(Jc-F=3.2 Hz),129.8(Jc-F=4.2 Hz),78.5,73.8,63.4,35.4(Jc-F=45 Hz),31.3,22.7,14.0,13.5. 19 F NMR(376 MHz,CDCl 3 )δ-49.51.ESI-HRMS(m / z)calcd C 15 H 19 FN 4 O 3 S 2 Na(M+Na) + ,409.0775;found,409.0769.HPLCpurity:99.4%.

[0104] 1.2.37.(±)-Ethyl-3-(2-chloro-6-(propylthio)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(37a,37b)

[0105] 37a:white solid,22.4%yield. 1H NMR(400 MHz,DMSO)δ7.57(s,1H),5.90(d,J=5 Hz,2H),5.17-5.14(m,1H),4.14(q,J=7.2 Hz,2H),3.92(J=11.2 Hz,1H),3.57(d,J=11.2 Hz,1H),3.37(q,J=5.6 Hz,1H),3.33-3.29(m,2H),2.88(dd,J=3.6 Hz,11.6,1H),1.78-1.69(m,2H),1.07(t,J=7.2 Hz,3H),1.00(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,DMSO)δ167.6,162.4,151.8,150.0,144.6,130.0,74.0,73.6,62.1,22.2,13.6,13.1.ESI-HRMS(m / z)calcd C 15 H 19 ClN 4 O 3 S 2 Na(M+Na) + ,425.0479;found,425.0471.HPLC purity:99.8%.37b:white solid,51.6%yield. 1 H NMR(400 MHz,CDCl 3 )δ8.25(s,1H),5.30-5.27(m,1H),5.12-5.11(m,1H),4.28-4.12(m,2H),3.91(d,J=12.4 Hz,1H),3.53(d,J=12.4Hz,1H),3.37-3.27(m,2H),3.12(dd,J=4.4 Hz,J=11.6 Hz,1H),2.85-2.80(m,1H),1.84-1.75(m,1H),1.13(q,J=7.2Hz,3H),1.05(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ168.9,164.7,153.3,149.6,141.5,130.2,78.6,73.9,63.3,35.4(J C-F =13.7 Hz),31.2,22.6,13.9,13.4.ESI-HRMS(m / z)calcd C 15 H 19 ClN 4 O3 S 2 Na(M+Na) + ,425.0479;found,425.0478.HPLC purity:97.9%.

[0106] 1.2.38.(±)-Ethyl-3-(2-amino-6-chloro-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(38a,38b)

[0107] 38a:white solid,71.0%yield. 1 H NMR(600 MHz,MeOD)δ8.31(s,1H),5.35(t,J=5.4 Hz,1H),4.23-4.15(m,2H),4.02(d,J=12.6 Hz,1H),3.73(d,J=12.6 Hz,1H),3.33(s,1H),3.21(q,J=5.4 Hz,1H),3.10(dd,J=4.8 Hz,J=11.4 Hz,1H),1.13(t,J=6.6Hz,3H). 13 C NMR(150 MHz,CDCl 3 )δ69.5,158.5,153.4,152.3,140.4,125.3,78.4,73.5,63.1,35.5,35.3,13.9.ESI-HRMS(m / z)calcd C 12 H 14 ClN 5 O 3 SNa(M+Na) + ,366.0398;found,366.0399.HPLC purity:97.1%.38b:white solid,16.7%yield. 1 HNMR(600 MHz,MeOD)δ8.43(s,1H),5.33(t,J=5.4 Hz,1H),4.23-4.19(m,2H),4.01(d,J=11.4 Hz,1H),3.67(d,J=11.4 Hz,1H),3.43(q,J=6.0 Hz,1H),2.94(dd,J=4.2 Hz,J=10.8 Hz,1H),1.16(t,J=6.6 Hz,3H). 13C NMR(100 MHz,DMSO)δ168.2,159.5,154.4,149.7,142.4,123.2,74.1,73.7,62.1,35.5,33.6,13.8.ESI-HRMS(m / z)calcd C 12 H 14 ClN 5 O 3 SNa(M+Na) + ,366.0398;found,366.0398.HPLCpurity:98.8%.

[0108] 1.2.39.(±)-Ethy-3-(6-chloro-2-fluoro-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(39a,39b)

[0109] 39a:brown solid,21.5%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.61(s,1H),5.40(t,J=6.6 Hz,1H),4.28-4.21(m,2H),3.72(dd,J=12.0 Hz,J=29.4 Hz,2H),3.59(s,1H),3.32(q,J=6.0 Hz,1H),2.82(dd,J=11.4 Hz,J=6.6 Hz,1H),1.18(t,J=7.2 Hz,3H). 13 CNMR(100 MHz,CDCl 3 )δ167.6,145.2,76.6,73.8,63.6,53.6,34.7,33.7(dd,J=100.6),14.0. 19 F NMR(376 MHz,CDCl 3 )δ-48.89.ESI-HRMS(m / z)calcd C 12 H 12 ClFN 4 O 3 SNa(M+Na) + ,369.0195;found,369.0187.HPLC purity:97.5%.39b:brown solid,32.5%yield. 1 H NMR(400 MHz,CDCl 3)δ8.51(s,1H),5.30(s,2H),4.55(s,1H),4.29-4.21(m,2H),3.97(d,J=12.8 Hz,1H),3.56(d,J=12.4 Hz,1H),3.12(dd,J=3.2 Hz,J=11.6 Hz,1H),2.78(dd,J=4.4 Hz,J=11.6 Hz,1H),1.14(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ168.7,158.0,155.8,153.8,144.2(d,J C - F =3.6),130.6,78.3,74.1,63.8,53.6,35.6,35.1,14.0. 19 F NMR(376 MHz,CDCl 3 )δ-48.89.ESI-HRMS(m / z)calcd C 12 H 12 ClFN 4 O 3 SNa(M+Na) + ,369.0195;found,366.0192.HPLC purity:99.0%.

[0110] 1.2.40.(±)-Ethyl-3-(2,6-dichloro-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(40a,40b)

[0111] 40a:white solid,22.6%yield. 1 H NMR(600 MHz,CDCl 3 )δ 1 H NMR(600 MHz,CDCl 3 )δ8.61(s,1H),5.40(q,J=6 Hz,1 H),4.25(q,J=7.2 Hz,2H),3.71(dd,J=12.0Hz,J=31.2 Hz,1H),3.58(d,J=4.8 Hz,1H),3.32(q,J=6.0 Hz,1H),2.81(q,J=6.6 Hz,J=11.4 Hz,1H),1.19(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,CDCl3 )δ167.6,153.6,152.8,152.2,145.4,130.5,76.5,73.9,63.6,34.6,33.7,14.0.ESI-HRMS(m / z)calcdC 12 H 13 N 4 BrO 3 SNa(M+Na) + ,371.9892;found,351.0290.HPLCpurity:99.0%.40b:whitesolid,35.6%yield. 1 H NMR(600 MHz,CDCl 3 )δ8.51(s,1H),5.30(q,J=3.0 Hz,1H),4.65(q,J=1.8 Hz,1H),4.32-4.19(m,2H),3.96(d,J=12.6 Hz,1H),3.55(d,J=12.6 Hz,1H),3.13(dd,J=3.0,J=12.0 Hz,1H),2.78(dd,J=4.2 Hz,J=12.0 Hz,1H),1.15(t,J=7.2Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ168.6,163.1,151.6,148.3,141.2,131.5,79.3,74.1,53.7,35.7,35.2,30.9,22.9,13.6.ESI-HRMS(m / z)calcd C 12 H 13 N 4 BrO 3 SNa(M+Na) + ,371.9892;found,351.0290.HPLC purity:96.0%.

[0112] 1.2.41.(±)-Ethyl-3-(2-chloro-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(41a,41b)

[0113] 41a:white solid,54.6%yield. 1 H NMR(600 MHz,CDCl 3)δ8.96(s,1H),8.56(s,1H),5.44(t,J=6.0 Hz,1H),4.29-4.21(m,2H),3.71(dd,J=12.0 Hz,J=43.8 Hz,2H),3.33(q,J=6.0 Hz,1H),2.83(dd,J=6.6 Hz,J=11.4 Hz,1H),1.18(t,J=7.2Hz,3H). 13 CNMR(150 MHz,CDCl 3 )δ167.9,154.2,153.7,150.6,145.5,132.9,76.4,73.6,63.5,34.8,33.8,14.0;ESI-HRMS(m / z)calcd C 12 H 13 N 4 ClO 3 SNa(M+Na) + ,351.0289;found,351.0295.HPLC purity:99.5%.41b:white solid,27.1%yield. 1 HNMR(600 MHz,CDCl 3 )δ8.99(s,1H),8.48(s,1H),5.34(t,J=4.4 Hz,1H),4.89(s,1H),4.31-4.14(m,2H),3.96(d,J=12.4 Hz,1H),3.56(d,J=12.4 Hz,1H),3.14(dd,J=3.6 Hz,J=11.6 Hz,1H),2.81(dd,J=4.8 Hz,J=11.6 Hz,Hz,1H),1.12(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,CDCl 3 )δ168.9,154.3,153.2,150.8,144.5,133.3,78.4,73.9,63.5,35.6,35.2,13.9;ESI-HRMS(m / z)calcd C 12 H 13 N 4 ClO 3 SNa(M+Na) + ,351.0289;found,351.0290.HPLC purity:99.7%.

[0114] 1.2.42.(±)-Ethy-3-(2-chloro-6-morpholino-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(42a,42b)

[0115] 42a:white solid,18.4%yield. 1 H NMR(400 MHz,CDCl 3 )δ8.06(s,1H),5.42(q,J=6 Hz,1H),4.33(d J=5.2 Hz,3H),4.34-4.19(m,6H),3.82(t,J=5.2 Hz,4H),3.70(d,J=12.0 Hz,1H),3.46(d,J=11.6 Hz,1H)3.26(dd,J=6.0 Hz,J=10.8 Hz,1H),2.81(dd,J=6.8 Hz,J=11.2 Hz,1H),1.23(t,J=6.8 Hz,3H). 13 C NMR(100MHz,CDCl 3 )δ168.3,154.2,153.7,152.4,138.1,118.6,76.6,73.6,67.1,63.3,35.3,33.7,14.0.ESI-HRMS(m / z)calcd C 16 H 20 ClN 5 O 4 SNa(M+Na) + ,436.0817;found,436.0824.HPLC purity:97.6%.42b:white solid,53.2%yield. 1 H NMR(400MHz,CDCl 3 )δ7.98(s,1H),5.49(s,1H),5.26(t,J=4.4Hz,1H),4.32-4.17(m,6H),3.88(d,J=12.4Hz,1H),3.83(t,J=4.8Hz,4H),3.49(d,J=12.4Hz,1H),3.17(dd,J=4.8Hz,J=11.6Hz,1H),2.85(dd,J=4.4Hz,J=11.6Hz,1H)1.18(t,J=7.2Hz,3H). 13 C NMR(100MHz,CDCl 3)δ169.5,154.1,153.6,152.2,137.4,118.8,79.1,73.8,67.1,63.3,35.7,35.5,14.1.ESI-HRMS(m / z)calcd C 16 H 20 C1N 5 O 4 SNa(M+Na) + ,436.0817; found,436.0817.HPLC purity:96.9%.

[0116] The compounds were separated by chiral separation column using CHIRALPAK IF (IF00CE-RL 017, 0.46cm IDx 25cmL) chromatographic column (injection volume 10μL / mobile phase MeOH / DCM-95 / 5 (V / V), flow rate 1.0mL / min, wavelength UV254nm, column temperature 35℃, HPLC: Shimadzu LC-20AD CP-HPLC-08). After separation, the chiral compounds were all greater than 98% ee.

[0117] Example 2

[0118] 2.1 Experimental Materials

[0119] 1. Virus: Enterovirus 71 (EV-A71 Zhenjiang isolate).

[0120] 2. Cells: African green monkey kidney cells (Vero), culture conditions: DMEM + 10% FBS + 1% Pen-Strep; 37°C 5% CO 2 .

[0121] 3. Main reagents

[0122]

[0123] ChamQ SYBR Color qPCR Master Mix Vazyme

[0124]

[0125] 2.2 Research Methods

[0126] 1. Concentration-dependent anti-EV-A71 activity of compounds 33a, 5b and 30b

[0127] (1) Take well-growing, uncontaminated Vero cells and inoculate them in a 12-well plate with complete medium, 4 × 10 cells per well. 5 cells, in 5% CO 2 / Culture in a 37℃ constant temperature incubator for 12-16h until it grows to a single layer.

[0128] (2) The original culture medium was discarded, and compounds 33a, 5b, and 30b were diluted to 5 concentrations in a 2-fold dilution manner with complete culture medium. 1 mL of 5% FBS culture medium was added to the cells and the cells were incubated at 37°C with 5% CO 2 After incubation for 4 hours, the culture medium was discarded, EV-A71 was infected at MOI = 0.001, and 1 mL of compound 33a, 5b and 30b (concentrations as above) diluted in FBS-free culture medium was added; incubation at 37°C for 2 hours, free viruses were washed away, and 1 mL of compound 33a, 5b and 30b (concentrations as above) diluted in 2% FBS culture medium was added; a virus control without compound treatment was set up. 2 / 37℃ constant temperature incubator for 48h.

[0129] (3) RT-qPCR: After 48 h of cell culture, the supernatant was discarded, and the total RNA in the cells was extracted using a nucleic acid extraction kit and then reversed for RT-qPCR to simultaneously detect the intracellular viral load and GAPDH gene expression. The relative expression of the virus in the sample well was calculated based on the relative quantitative amplification region (EV-A715'UTR).

[0130] qPCR Primers:

[0131]

[0132] (4) CCK-8 assay for cytotoxicity of compounds 33a, 5b, and 30b: Vero cells in good growth condition were inoculated in complete medium in 96-well plates, with 1×10 cells per well. 4 In the presence of 5% CO 2 / 37℃ constant temperature incubator for 12-16h; discard the old culture medium, dilute compounds 33a, 5b and 30b with complete culture medium according to the 5-fold dilution method, add 100 μL to each well, and set normal growing cells as cell control and cell-free group as blank zero well; after treatment with compounds 33a, 5b and 30b for 48h, add 10 μL CCK-8 to each well and protect from light for 4h, detect the absorbance at 450nm with an enzyme reader, and calculate the cell survival rate of the compound 33a, 5b and 30b treatment group relative to the cell control group.

[0133] 2.3 Test results:

[0134] Concentration-dependent anti-EV-A71 activity

[0135]

[0136]

[0137] Compound 5 μM rescreening results (compounds with an inhibition rate of EV-A71 greater than 50% at a concentration of 25 μM and compounds with high cytotoxicity at a concentration of 25 μM):

[0138]

[0139]

[0140] The three compounds with the best inhibitory effect at 5 μM concentration

[0141]

[0142] from Figure 1 It can be seen that (±)-33a compound EC 50 =0.9975μM,CC 50 =77.08μM. The selectivity coefficient of the two is SI = CC 50 / EC 50 =77.27.

[0143] from Figure 2 It can be seen that (±)-5b compound EC 50 =0.9000μM,CC 50 =160.6μM. The selectivity coefficient of the two is SI=CC 50 / EC 50 =178.44.

[0144] from Figure 3 It can be seen that (±)-30b compound EC 50 =0.9670μM,CC 50 =65.19μM. The selectivity coefficient of the two is SI=CC 50 / EC 50 =67.14.

[0145] The antiviral activity experiment found that the compounds 33a, 5b and 30b of the present invention have good anti-EV-A71 activity and have good prospects in the preparation of drugs for the treatment of hand, foot and mouth disease and related symptoms.

Claims

1. A five-membered sulfur heterocyclic nucleoside lead compound, including its racemate and chiral compound, characterized in that: The general structure is as follows: Where: R 1 is selected from hydrogen, halogen, amino, C1-C20 alkyl, C1-C20 alkoxy; R 2 is selected from hydrogen, halogen, C1-C20 alkyl, C1-C20 alkoxy, C1-20 alkylthio, C2-C20 alkynylthio, halogenated C1-C20 alkylthio, benzylthio, phenylthio, substituted phenylthio, di(C1-C8 alkyl)amine, X = CH2, O, S, NH, N-(C1-C4)alkyl; n = 0-5; R 3 =C1-C8 alkyl, halogen, trifluoromethyl, benzyl, substituted phenylmethyl, morpholinyl; wherein the substituent in the substituted phenyl is halogen or C1-C8 alkyl; R is selected from hydrogen, C1-C20 alkyl.

2. The five-membered sulfur heterocyclic nucleoside lead compound according to claim 1, characterized in that: R 1 is selected from hydrogen, halogen, amino; R 2 is selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-6 alkylthio, C2-C4 alkynylthio, halogenated C1-C4 alkylthio, benzylthio, phenylthio, substituted phenylthio, di(C1-C2 alkyl)amine, X = CH2, O, S, NH, N-(C1-C4)alkyl; n = 1-3; R 3 =C1-C4 alkyl, halogen, trifluoromethyl, benzyl, substituted phenylmethyl, morpholinyl; wherein the substituent in the substituted phenyl is halogen or C1-C4 alkyl.

3. The five-membered sulfur heterocyclic nucleoside lead compound according to claim 2, characterized in that: R is selected from ethyl.

4. The five-membered sulfur heterocyclic nucleoside lead compound according to claim 3, characterized in that: The structure of the lead five-membered sulfur heterocyclic nucleoside compound is 5. Use of the five-membered sulfur heterocyclic nucleoside lead compound according to any one of claims 1 to 4 in the preparation of antiviral drugs.

6. The use of the five-membered sulfur heterocyclic nucleoside lead compound according to claim 5 in the preparation of antiviral drugs, characterized in that: The antiviral agent is against hand, foot and mouth virus (EV-A71).

7. Use of the five-membered sulfur heterocyclic nucleoside lead compound according to any one of claims 1 to 4 in the preparation of a drug for treating hand, foot and mouth disease.

8. A pharmaceutical composition comprising a compound according to any one of claims 1 to 4.

9. The method for synthesizing the five-membered sulfur heterocyclic nucleoside lead compound as claimed in claim 3, characterized in that: The steps include: Compound 1-42A reacts in the presence of triphenylphosphine, sodium acetate, acetic acid and propiolate to obtain compound 1-42B; then compound 1-42B reacts with 2,5-dihydroxy-1,4-dithiane in the presence of triethylamine to obtain 1a-42a and 1b-42b; finally, the corresponding chiral compound is obtained after separation by a chiral separation column.

10. The method for synthesizing the five-membered sulfur heterocyclic nucleoside lead compound according to claim 9, characterized in that: The first step reaction solvent is toluene, and the reaction temperature is reflux; the second step reaction solvent is dichloromethane, and the reaction temperature is 0-30°C; the third step chiral separation column separation is performed using a CHI RALPAK IF (IF00CE-RL017, 0.46cm IDx 25cm L) chromatographic column.

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