Five-membered sulfur-containing heterocyclic nucleoside lead compounds and their applications in anti-hand, foot and mouth disease virus active drugs

Abstract

The application discloses five-membered sulfur-containing heterocyclic nucleoside leading compounds and application thereof in anti-hand-foot-mouth-disease virus active drugs, and belongs to the technical field of medicinal chemistry. A series of five-membered sulfur-containing heterocyclic nucleoside compounds are synthesized through Sulfa-Michael / Aldol tandem reaction. Results show that in anti-EV-A71 virus testing, the five-membered sulfur-containing heterocyclic nucleoside leading compounds 33a, 5b and 30b have relatively good anti-EV-A71 virus activity, and new structural units are provided for candidate drug molecules for anti-virus.

Description

Technical Field

[0001] The present 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 pose a serious threat to human life and health and are one of the leading causes of death. The development of antiviral drugs has always been a key focus in pharmaceutical research. Synthetic nucleoside compounds, structurally similar to natural nucleoside compounds, can inhibit viral or tumor cell replication upon entry into the human body, achieving antiviral or antitumor effects.

[0003] The research and development of nucleoside anti-tumor and antiviral drugs is gaining increasing attention. Five-membered sulfur heterocyclic nucleosides, a unique class of nucleoside compounds with distinctive physical and chemical properties, have become a key focus of nucleoside drug research and development.

[0004] Currently, pentacyclic sulfur nucleoside compounds are an important class of marketed antiviral drugs, but research on their antiviral activity is relatively limited. Innovative research on pentacyclic sulfur nucleoside antiviral drugs could not only break the international monopoly on nucleoside drugs, but also have 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 has an ee value of 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 R is selected from hydrogen, halogen, amino, C1-C20 alkyl, C1-C20 alkoxy; 2 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)amino, 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.

[0009] Furthermore, under the preferred conditions, R 1 R is selected from hydrogen, halogen, amino, C1-C20 alkyl, C1-C20 alkoxy; 2 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)amino, 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 ethyl.

[0010] Furthermore, under the preferred conditions, R 1 is selected from hydrogen, halogen, amino; R 2 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)amino, 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; R is selected from ethyl.

[0011] Furthermore, 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; compound 1-42B then reacts with 2,5-dihydroxy-1,4-dithiane in the presence of triethylamine to obtain 1a-42a and 1b-42b; and the corresponding chiral compounds are then separated 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 solvent for the second step reaction is dichloromethane, and the reaction temperature is 0-30°C.

[0018] Furthermore, in the above technical solution, 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 all greater than 98% ee.

[0019] The present invention also provides the use of a 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 easily available, 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 a graph showing the anti-EV-A71 viral activity of 33a;

[0027] Figure 2 This is a graph showing the anti-EV-A71 viral activity 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) PPh3, NaOAc, HOAc, Ethyl propiolate, toluene, 110℃, 12h. (b) 1,4-dithiane-2,5-diol, Et3N (20mol%), CH2Cl2, 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. Dichloromethane was added to extract the reaction mixture three 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 Step 2 procedure: 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 analyzed 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. 1H NMR(600MHz,CDCl3)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,CDCl3)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 N4O3S(M+H) + ,295.0859;found,295.0856.HPLC purity:97.7%.1b:white solids,52.4%yield. 1 H NMR(600MHz,CDCl3)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,CDCl3)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 N4O3SNa(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,CDCl3)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,CDCl3)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 N4ClO3SNa(M+Na) + ,351.0281;found,351.0289.HPLC purity:98.3%.2b:colorless 1iquid,58.3%yield. 1 H NMR(600 MHz,CDCl3)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,CDCl3)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 H 13 N4ClO3SNa(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,CDCl3)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,CDCl3)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 N4BrO3SNa(M+Na)+,394.9784;found,394.9782.HPLC purity:95.5%.3b:white solid,41.2%yield. 1 H NMR(400 MHz,CDCl3)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). 13 CNMR(100 MHz,CDCl3)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 N4BrO3SNa(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,CDCl3)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,CDCl3)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 N5O3S(M+H) + ,338.1281;found,338.1272.HPLC purity:98.9%.4b:white solid,59.2%yield. 1 H NMR(600 MHz,CDCl3)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). 13C NMR(150 MHz,CDCl3)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 N5O3S(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,CDCl3)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,CDCl3)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 H 24 N5O3S(M+H) + ,378.1594;found,378.1597.HPLC purity:98.7%.5b:white solid,51.1%yield. 1H NMR(600 MHz,CDCl3)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,CDCl3)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 N5O3SNa(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,CDCl3)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,CDCl3)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 N4O4SNa(M+Na)+ ,361.0941;found,361.0938.HPLCpurity:95.4%.6b:white solid,53.2%yield. 1 H NMR(600 MHz,CDCl3)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,CDCl3)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 N4O4SNa(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,CDCl3)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). 13C NMR(150 MHz,CDCl3)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 N4O4SNa(M+Na) + ,347.0784;found,347.0786.HPLC purity:95.1%.7b:light yellow solid,54.1%yield. 1 H NMR(400 MHz,CDCl3)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 N4O4SNa(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. 1H NMR(400 MHz,CDCl3)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,CDCl3)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 N4O3S2Na(M+Na) + ,391.0869;found,391.0870.HPLC purity:98.0%.8b:yellow solid,yield:54.1%. 1 H NMR(400 MHz,CDCl3)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,CDCl3)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 N4O3S2Na(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,CDCl3)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,CDCl3)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 N5O4S(M+H) + ,380.1387;found,380.1390.HPLC purity:97.9%.9b:white solid,45.4%yield. 1 H NMR(400 MHz,CDCl3)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). 13C NMR(100 MHz,CDCl3)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 N5O4SNa(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,CDCl3)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,CDCl3)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 ClN5O3S2(M+H) + ,396.1159;found,396.1149.HPLC purity:96.1%.10b:white solid,45.2%yield. 1H NMR(600 MHz,CDCl3)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,CDCl3)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 ClN5O3S2Na(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,CDCl3)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). 13C NMR(150 MHz,CDCl3)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 ClN5O3SNa(M+Na) + ,392.1751;found,392.1744.HPLCpurity:95.4%.11b:white solid,31.5%yield. 1 H NMR(400 MHz,CDCl3)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,CDCl3)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 ClN5O3SNa(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. 1H NMR(600 MHz,CDCl3)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 C NMR(150 MHz,CDCl3)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,CDCl3)δ-181.58.ESI-HRMS(m / z)calcdC 17 H 22 FN5O3SNa(M+Na) + ,396.1500;found,396.1506.HPLC purity:98.9%.12b:whitesolid,53.4%yield. 1 H NMR(400 MHz,CDCl3)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,CDCl3)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. 19F NMR(376MHz,CDCl3)δ-181.68.ESI-HRMS(m / z)calcd C 17 H 22 FN5O3SNa(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,CDCl3)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,CDCl3)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,CDCl3)δ-73.90.ESI-HRMS(m / z)calcd C 18 H 22 F3N5O3SNa(M+Na) + ,446.1468;found,446.1468.HPLC purity:95.6%.13b:yellow solid,37.5%yield. 1H NMR(400 MHz,CDCl3)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,CDCl3)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,CDCl3)δ-73.90.ESI-HRMS(m / z)calcd C 18 H 22 F3N5O3SNa(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. 1H NMR(600 MHz,CDCl3)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,CDCl3)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 ClN5O3S(M+H) + ,412.1205;found,412.1198.HPLC purity:96.1%.14b:light yellow solid,31.0%yield. 1 H NMR(400 MHz,CDCl3)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,CDCl3)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 ClN5O3S(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, CDC13) δ 8.22 (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, CDC13) δ 169.6, 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) calcd for C 21 H 31 N6O4S (M+H) + ,463.2111; found, 463.2113. HPLC purity: 97.9%. 15b: white solid, 38.6% yield. 1H NMR(600 MHz,CDCl3)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,CDCl3)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 N6O4S(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. 1H NMR (600 MHz, CDC13) δ 8.18 (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 = 17.4 Hz, 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, CDC13) δ 168.6, 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 for C 24 H 29 N5O3SNa (M + Na) + , 490.1883; found, 490.1886. HPLC purity: 97.9%. 16b: white solid, 31.8% yield. 1 H NMR (600 MHz, CDC13) δ 8.18 (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 = 17.4 Hz, 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). 13C NMR(150 MHz,CDCl3)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 N5O3S(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,CDCl3)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,CDCl3)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,CDCl3)δ-117.84.ESI-HRMS(m / z)calcd C 24 H 29 FN5O3S(M+H) + ,486.1970;found,486.1980.HPLCpurity:96.4%.17b:white solid,37.8%yield. 1 HNMR(400 MHz,CDCl3)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,CDCl3)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,CDCl3)δ-117.86.ESI-HRMS(m / z)calcd C 24 H 29 FN5O3S(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,CDCl3)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,CDCl3)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 ClN6O3SNa(M+Na) + ,393.1703;found,393.1701.HPLC purity:98.1%.18b:white solid,41.6%yield. 1 H NMR(600 MHz,CDCl3)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). 13C NMR(150 MHz,CDCl3)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 ClN6O3SNa(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,CDCl3)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,CDCl3)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 N5O3S(M+H) + ,392.1751;found,392.1744.HPLC purity:95.7%.19b:white solid,52.4%yield. 1H NMR(600MHz,CDCl3)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,CDCl3)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 N5O3S(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). 13C NMR (100 MHz, CDC13) δ 169.7, 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 for C2iH25N4O5S (M+H) 449.1516; found, 449.1516. HPLC purity: 98.4%. 16 H 22 N5O3S (M+H) + , 364.1438; found, 364.1437. HPLC purity: 98.4%. 20b: white solid, 58.0% yield. 1 H NMR (600 MHz, CDC13) δ 8.22 (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 = 6.8 Hz, 3H). 13 C NMR (100 MHz, CDC13) δ 168.7, 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 for C2iH25N4O5S (M+H) 449.1516; found, 449.1516. HPLC purity: 98.4%. 16 H 22 N5O3S (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. 1H NMR (400 MHz, CDC13) δ 8.63 (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 = 4.8 Hz, J = 11.6 Hz, 1H), 2.68 (s, 3H), 1.07 (t, J = 7.2 Hz, 3H). 13 C NMR (150 MHz, CDC13) δ 169.1, 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 for C 13 H 16 N4O3S2Na (M + Na) + , 363.0556; found, 363.0546. HPLC purity: 99.8%. 21b: yellowish solid, 40.5% yield. 1 H NMR (600 MHz, CDC13) δ 8.61 (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, CDC13) δ 168.2, 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 for C 13 H 16 N4O3S2Na (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,CDCl3)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,CDCl3)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 N4O3S2Na(M+Na) + ,377.0713;found,377.0697.HPLCpurity:99.8%.22b:yellowsolid,22.6%yield. 1 H NMR(600 MHz,CDCl3)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). 13C NMR(150 MHz,CDCl3)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 N4O3S2Na(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,CDCl3)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,CDCl3)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 N4O3S2Na(M+Na) + ,391.0869;found,391.0871.HPLC purity:99.8%.23b:whitesolid,22.4%yield. 1H NMR(600 MHz,CDCl3)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,CDCl3)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 N4O3S2Na(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. 1 H NMR(600 MHz,CDCl3)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). 13C NMR(150 MHz,CDCl3)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 N4O3S2Na(M+Na) + ,387.0556;found,387.0550.HPLCpurity:98.6%.24b:white solid,49.7%yield. 1 H NMR(600 MHz,CDCl3)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,CDCl3)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 N4O3S2Na(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. 1H NMR (400 MHz, DMSO) δ 8.72 (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 = 11.6 Hz, 1H), 3.62 (d, J = 11.2 Hz, 1H), 3.41-3.30 (m, 3H), 2.90 (dd, J = 3.6 Hz, J = 11.2 Hz, 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) δ 167.9, 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) calcd for C 16 H 22 N4O3S2(M+Na) + , 405.1026; found, 405.1020. HPLC purity: 99.6 %. 25b: white solid, 30.2 % yield. 1 H NMR (400 MHz, CDCl3) δ 8.62 (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, CDCl3) δ 169.3, 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 for C 16 H 22 N4O3S2(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,CDCl3)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,CDCl3)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 N4O3S2(M+H) + ,397.1363;found,397.1354.HPLC purity:99.6%.26b:white solid,25.1%yield. 1H NMR(600 MHz,CDCl3)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,CDCl3)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 N4O3S2(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. 1H NMR (400 MHz, DMSO) δ 8.73 (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 = 5.6 Hz, 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) δ 167.9, 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 for C 19 H 20 N4O3S2 (M + Na) + , 439.0869; found, 439.0872. HPLC purity: 99.7%. 27b: white solid, 42.4% yield. 1 H NMR (400 MHz, DMSO) δ 8.75 (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 C NMR (100 MHz, DMSO) δ 167.3, 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 for C 19 H20 N4O3S2(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, CDC13) δ 8.21 (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 = 4.8 Hz, J = 11.4 Hz, 1H), 1.99-1.90 (m, 4H), 1.10 (t, J = 7.2 Hz, 3H). 13 CNMR (150 MHz, CDC13) δ 169.5, 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 (376 MHz, CDC13) δ -111.38. ESI-HRMS (m / z) calcd for C 18 H 17 FN4O3S2(M+Na) + ,443.0618; found, 443.0613. HPLC purity: 99.0%. 28b: white solid, 31.3% yield. 1H NMR(400 MHz,CDCl3)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,CDCl3)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,CDCl3)δ-111.35.ESI-HRMS(m / z)calcd C 18 H 18 FN4O3S2(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. 1H 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 N4O3S2(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 N4O3S2(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,CDCl3)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,CDCl3)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,CDCl3)δ20.41.ESI-HRMS(m / z)calcd C 15 H 19 FN4O3S2Na(M+Na) + ,409.0775;found,409.0774.HPLC purity:97.1%.30b:white solid,30.2%yield. 1H NMR(400 MHz,CDCl3)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,CDCl3)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,CDCl3)δ-20.45.ESI-HRMS(m / z)calcd C 15 H 19 FN4O3S2Na(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. 1H NMR(400 MHz,CDCl3)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,CDCl3)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 ClN4O3S2Na(M+Na) + ,425.0479;found,425.0470.HPLC purity:96.4%.31b:whitesolid,21.7%yield. 1 H NMR(400 MHz,CDCl3)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,CDCl3)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 ClN4O3S2Na(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,CDCl3)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,CDCl3)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 N6O3S(M+H) + ,393.1703;found,393.1701.HPLC purity:99.0%.32b:yellow solid,32.6%yield. 1 HNMR(600 MHz,CDCl3)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,CDCl3)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 H25 N6O3S(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,CDCl3)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,CDCl3)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,CDCl3)δ-50.27.ESI-HRMS(m / z)calcdC 17 H 23 FN5O3S(M+H) + ,396.1500;found,396.1499.HPLC purity:99.4%.33b:yellow solid,43.6%yield. 1H NMR(600MHz,CDCl3)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,CDCl3)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,CDCl3)δ-50.14.ESI-HRMS(m / z)calcd C 17 H 22 FN5O3S(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,CDCl3)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). 13C NMR(100 MHz,CDCl3)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 ClN5O3S(M+H) + ,412.1205;found,412.1210.HPLC purity:96.1%.34b:white solid,21.6%yield 1 H NMR(400 MHz,CDCl3)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,CDCl3)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 ClN5O3S(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. 1H NMR(400 MHz,CDCl3)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,CDCl3)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 N5O3S2(M+H) + ,384.1159;found,384.1150.HPLC purity:99.7%.35b:white solid,43.5%yield. 1 H NMR(400 MHz,CDCl3)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,CDCl3)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 N5O3S2(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,CDCl3)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,CDCl3)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,CDCl3)δ-49.78.ESI-HRMS(m / z)calcd C 15 H 19 FN4O3S2Na(M+Na) + ,409.0775;found,409.0775.HPLCpurity:99.4%.36b:white solid,41.5%yield. 1HNMR(600 MHz,CDCl3)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,CDCl3)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,CDCl3)δ-49.51.ESI-HRMS(m / z)calcd C 15 H 19 FN4O3S2Na(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 ClN4O3S2Na(M+Na) + ,425.0479;found,425.0471.HPLC purity:99.8%.37b:white solid,51.6%yield. 1 H NMR(400 MHz,CDCl3)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,CDCl3)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 ClN4O3S2Na(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) δ 8.31 (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 = 6.6 Hz, 3H). 13 C NMR (150 MHz, CDC13) δ 69.5, 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 for C 12 H 14 ClN5O3SNa (M+Na) + ,366.0398; found, 366.0399. HPLC purity: 97.1%. 38b: white solid, 16.7% yield. 1 H NMR (600 MHz, MeOD) δ 8.43 (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). 13 C NMR (100 MHz, DMSO) δ 168.2, 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 for C 12 H 14 ClN5O3SNa (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,CDCl3)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,CDCl3)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,CDCl3)δ-48.89.ESI-HRMS(m / z)calcd C 12 H 12 ClFN4O3SNa(M+Na) + ,369.0195;found,369.0187.HPLC purity:97.5%.39b:brown solid,32.5%yield. 1 H NMR(400 MHz,CDCl3)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,CDCl3)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,CDCl3)δ-48.89.ESI-HRMS(m / z)calcd C 12 H 12 ClFN4O3SNa(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,CDCl3)δ 1 H NMR(600 MHz,CDCl3)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 N4BrO3SNa(M+Na) + ,371.9892;found,351.0290.HPLCpurity:99.0%.40b:whitesolid,35.6%yield. 1H NMR(600 MHz,CDCl3)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,CDCl3)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 N4BrO3SNa(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,CDCl3)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,CDCl3)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 N4ClO3SNa(M+Na) +,351.0289;found,351.0295.HPLC purity:99.5%.41b:white solid,27.1%yield. 1 HNMR(600 MHz,CDCl3)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,CDCl3)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 N4ClO3SNa(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,CDCl3)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). 13C NMR(100MHz, CDCl3)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 ClN5O4SNa(M+Na) + , 436.0817; found, 436.0824. HPLC purity: 97.6%. 42b: white solid, 53.2% yield. 1 H NMR (400MHz, CDCl3) 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, CDCl3) 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 ClN5O4SNa(M+Na) + ,436.0817; found,436.0817.HPLC purity:96.9%.

[0116] The compounds were separated by chiral separation column using a CHIRALPAK IF (IF00CE-RL 017, 0.46 cm IDx 25 cmL) 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). 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% CO2.

[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 the Vero cells with good growth status and no contamination and inoculate them into 12-well plates with complete culture medium, 4×10 cells per well. 5 The cells were cultured in a 5% CO2 / 37°C constant temperature incubator for 12-16 hours until they grew to a monolayer.

[0128] (2) Discard the original culture medium and dilute compounds 33a, 5b, and 30b to five concentrations using complete culture medium in a two-fold dilution pattern. 1 mL of compound 33a, 5b, and 30b diluted in 5% FBS medium was added to the cells and incubated at 37°C with 5% CO2 for 4 h. The culture medium was discarded and the cells were infected with EV-A71 at an MOI of 0.001. 1 mL of compound 33a, 5b, and 30b diluted in FBS-free medium (same concentrations as above) was added. The cells were incubated at 37°C for 2 h, free viruses were washed away, and 1 mL of compound 33a, 5b, and 30b diluted in 2% FBS medium (same concentrations as above) was added. A virus control without compound treatment was also set up. The cells were cultured in a 5% CO2 / 37°C constant temperature incubator for 48 h.

[0129] (3) RT-qPCR: After 48 h of cell culture, the supernatant was discarded and total RNA was extracted from the cells using a nucleic acid extraction kit. RT-qPCR was then performed to simultaneously detect the intracellular viral load and GAPDH gene expression. The relative viral expression in the sample wells was calculated based on the relative quantitative amplification region (EV-A71 5'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 seeded in 96-well plates with complete culture medium, and 1×10 cells were added to each well. 4 The cells were cultured in a 5% CO2 / 37°C constant temperature incubator for 12-16 hours; the old culture medium was discarded, and compounds 33a, 5b, and 30b were diluted 5-fold with complete culture medium, and 100 μL was added to each well. At the same time, normal growing cells were set as cell controls and a cell-free group was set as blank zero wells; after treatment with compounds 33a, 5b, and 30b for 48 hours, 10 μL of CCK-8 was added to each well and protected from light for 4 hours. The absorbance at 450 nm was detected using a microplate reader, and the cell viability of the compound 33a, 5b, and 30b-treated groups relative to the cell control group was calculated.

[0133] 2.3 Test results:

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

[0135]

[0136]

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

[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 The compound EC of (±)-30b can be seen 50 = 0.9670 μM, CC 50 = 65.19 μM. The selectivity coefficient is SI = CC 50 / EC 50 = 67.14.

[0145] The compounds 33a, 5b and 30b of the present application are found to have good anti-EV-A71 activity by the anti-viral activity experiment, and have good prospects in the preparation of drugs for treating hand-foot-mouth disease and related symptoms.

Claims

1. Use of a five-membered sulfur heterocyclic nucleoside lead compound in the preparation of an anti-hand, foot and mouth disease virus drug, wherein the structure of the five-membered sulfur heterocyclic nucleoside lead compound is as follows:

2. The use of the five-membered sulfur heterocyclic nucleoside lead compound according to claim 1 in the preparation of anti-hand, foot and mouth disease virus drugs, wherein the structure of the five-membered sulfur heterocyclic nucleoside lead compound is 33a, 5b or 30b.

3. Use of the five-membered sulfur heterocyclic nucleoside lead compound according to claim 1 or 2 in the preparation of anti-hand, foot and mouth disease virus drugs, wherein the hand, foot and mouth disease virus is EV-A71.

Images