Thiocyclic nucleoside derivatives and their synthesis and anti-colon cancer activity applications

Abstract

The present invention discloses thioheterocyclic nucleoside derivatives and their synthesis and anti-colon cancer activity applications, belonging to the technical field of pharmaceutical chemistry. A series of thioheterocyclic nucleoside derivatives were synthesized by an asymmetric sulfonamide-Michael / aldol cascade reaction, including racemate, 3S,4S-configuration, 3R,4R-configuration, 3S,4R-configuration and 3R,4S-configuration, with the general structure being (* represents a chiral center). The results showed that in the in vitro colon cancer cell line (HCT116) test, the anti-tumor activity of the racemate and the enantiomeric configuration was not much different. Among them, racemates 33a and 36b showed strong inhibitory ability to inhibit proliferation in the in vitro colon cancer cell line, IC 50 reached 0.27μM and 0.49μM, which were significantly better than the control drugs 5-fluorouracil and cisplatin (IC 50 9.84 μM and 5.29 μM, respectively).

Description

Technical Field

[0001] The present invention relates to a five-membered sulfur heterocyclic nucleoside lead compound, its synthesis and application in anti-colon cancer activity, and belongs to the technical field of medicinal chemistry. Background Art

[0002] Cancer cells undergo uncontrolled and abnormal growth. Normal cells have regulatory mechanisms that ensure they grow, divide, and die in a controlled manner. However, cancer cells often evade these normal regulatory signals, leading to uncontrolled proliferation. Heterocyclic nucleoside analogs, particularly purine nucleoside analogs such as nerabine, azacytidine, folosidine, clofarabine, fludarabine, and clabine, have been widely used in clinical applications. Substitution of these purine nucleosides at the 2- and 6-positions with groups such as aryl, halogen, and alkyl groups is key to their potent antitumor activity.

[0003] Compared to oxygen, sulfur atoms in compound structures exhibit significant differences in chemical reactivity, protein recognition, and metabolic stability. Therefore, modifying nucleoside structures into sulfur-containing heterocycles may yield effective anti-tumor drugs. Summary of the Invention

[0004] To provide nucleoside lead compounds with a wider range of functional structures, this invention discloses five-membered sulfur heterocyclic nucleoside lead compounds with anti-colon cancer activity. A series of sulfur heterocyclic nucleoside derivatives, including racemic, 3S,4S-, 3R,4R-, 3S,4R-, and 3R,4S-configurations, were synthesized via an asymmetric sulfonamide-Michael / aldol cascade reaction. Based on this, 84 new compounds were designed and synthesized, and their antitumor activities were evaluated in vitro and in vivo.

[0005] The five-membered sulfur heterocyclic nucleoside lead compound of the present invention has the following general structural formula: Wherein, * represents a chiral center, and each general structure includes racemate, 3S,4S-configuration, 3R,4R-configuration, 3S,4R-configuration and 3R,4S-configuration. 1 、R 2 Each is independently selected from hydrogen, halogen, amino, alkylamino, C1-C4 alkoxy, para-substituted piperidine, cycloheximide, pyrrole, propylthio; R is selected from C1-C4 alkyl.

[0006] More preferably, R 1 is hydrogen, fluorine, or chlorine; R 2 It is hydrogen, chlorine, methoxy, amino, methylamino, dimethylamino, piperidine, propylthio, pyrrole, methylpiperazine, and cycloheximide.

[0007] More preferably, R 1 is hydrogen; R 2The sulfhydryl group is p-methylpiperidine, p-trifluoromethylpiperidine, p-fluoromethylpiperidine, p-chloropiperidine, p-morpholinopperidine, p-difluorobenzylpiperidine, methyl sulfide, ethyl sulfide, propyl sulfide, propynyl sulfide, butyl sulfide, pentyl sulfide, or benzyl sulfide.

[0008] More preferably, R 1 is fluorine; R 2 is piperidine, propylthio; R is ethyl.

[0009] More preferably, the 3S,4S-configuration, 3R,4R-configuration, 3S,4R-configuration and 3R,4S-configuration generally have antitumor activities comparable to those of the racemate.

[0010] The present invention also provides the use of the five-membered sulfur heterocyclic nucleoside lead compound in the preparation of anti-colon cancer active drugs.

[0011] Furthermore, in the above technical solution, the anti-colon cancer activity is selected from anti-HCT116 activity.

[0012] Furthermore, in the above technical solution, under the preferred conditions, the five-membered sulfur heterocyclic nucleoside lead compound is: R 1 is fluorine; R 2 For piperidine, propylthio.

[0013] The present invention also provides an anti-colon cancer active drug, the active ingredient of which includes the above-mentioned five-membered sulfur heterocyclic nucleoside lead compound.

[0014] Furthermore, in the above technical solution, the anti-colon cancer activity is selected from anti-HCT116 activity.

[0015] Furthermore, in the above technical solution, under the preferred conditions, the five-membered sulfur heterocyclic nucleoside lead compound in the active ingredient is: R 1 is fluorine; R 2 is piperidine, propylthio; R is ethyl.

[0016] The present invention also provides a method for synthesizing the five-membered sulfur heterocyclic nucleoside compound of the above-mentioned general structural formula, comprising the following steps:

[0017]

[0018] Step 1: Compound 1-42A reacts with propionate in the presence of PPh3 and NaOAc in acetic acid and toluene solvent at 100-110°C to obtain intermediate 1B-42B;

[0019] Step 2: Intermediate 1B-42B reacts with 1,4-disulfane-2,5-diol in the presence of triethylamine in dichloromethane solvent to obtain the racemates of intermediates 1a-42a and 1b-42b;

[0020] Step 3: The 1a-42a and 1b-42b racemates were resolved by chiral HPLC to yield 3S,4S-, 3R,4R-, 3S,4R-, and 3R,4S-configured products. Chiral HPLC analysis was performed using a Thermo Scientific Dionex Ultimate 3000 and an Agilent Technologies 1260 Infinity.

[0021] In order to investigate the antitumor activity of five-membered sulfur heterocyclic nucleosides, a series of five-membered sulfur heterocyclic nucleosides with different substituents at the 2 / 6 position of purine and the 1' position of the sugar ring were synthesized via asymmetric sulfonamide-Michael / aldol cascade reactions.

[0022] The experimental results showed that: in the racemate, compounds 33a and 36b showed strong inhibitory ability against colon cancer cell line (HCT116) in vitro, IC 50 The results reached 0.27μM and 0.49μM, which were significantly better than the control drugs 5-fluorouracil and cisplatin (IC 50 9.84μM and 5.29μM, respectively. Among chiral structures, 3S,4S-, 3R,4R-, 3S,4R-, and 3R,4S-configured pentacyclic sulfur nucleosides exhibited comparable antitumor activity to racemates 33a and 36b. These results provide a foundational building block for the design of pentacyclic sulfur nucleoside lead compounds with anti-colon cancer activity, enabling further structural refinement and engineering.

[0023] Through the synthesis of five-membered sulfur heterocyclic nucleosides and the evaluation of their anti-tumor activity in vitro and in vivo, we obtained anti-tumor lead compounds or preferred drug molecules with better activity, provided reasonable synthesis strategies and methods for five-membered sulfur heterocyclic nucleoside drugs, and laid a good foundation for the development of anti-tumor five-membered sulfur heterocyclic nucleoside drugs with independent intellectual property rights. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the effect of compound 33a on HCT116 cell cycle in Example 4;

[0025] Figure 2 This is a graph showing the effect of compound 36b on HCT116 cell cycle in Example 4;

[0026] Figure 3 Figure 33a and 36b show the effects of 33a and 36b on cell apoptosis;

[0027] Figure 4 is the apoptosis protein analysis diagram;

[0028] Figure 5 is the mitochondrial membrane potential (MMP) measurement diagram;

[0029] Figure 6 This is a diagram of the cell colony formation experiment;

[0030] Figure 7 is the effect diagram of cell invasion;

[0031] Figure 8 This is a diagram showing the effect of 33a on transplanted tumors;

[0032] Figure 9 HE staining picture. DETAILED DESCRIPTION

[0033] Example 1:

[0034] Ethyl 2-(9H-purin-9-yl)acrylate (43.6 mg, 0.2 mmol), 1,4-disulfane-2,5-diol (18.2 mg, 0.12 mmol), triethylamine (4.0 mg, 20 mol%), and dichloromethane (4 mL) were added to a reaction tube. TLC confirmed completion after 12 hours. The mixture was spin-dried and the product was isolated by flash chromatography using a PE / EA solvent system (PE / EA = 2 / 1).

[0035] Characterization data of representative compounds are as follows:

[0036] (±)-4-Hydroxy-3-(9-hydrogen-purin-9-yl)tetrahydrothiophene-3-carboxylic acid ethyl ester (1a, 1b)

[0037] 1a: white solids, 41.2% yield. 1 H 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=12.6 Hz, 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, 2 H),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%.

[0038] (±)-3-(6-Chloro-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (2a, 2b)

[0039] 2a: colorless 1iquid, 42.4% yield. 1 H NMR (600MHz, CDCl3) δ8.70(s,1H),8.56(s,1H),5.46(s,1H),4.23(t,J=7.2Hz,2H),3.79(d,J=11.4Hz,1H ), 3.64 (d, J = 11.4Hz, 1H), 3.32 (q, J = 6.0Hz, 1H), 2.83 (dd, J = 6.6Hz, J = 11.4Hz, 1H) 1.17 (t, J = 7.2Hz, 3H). 13 CNMR(150MHz, 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.HPLCpurity:98.3%.2b:colorless 1iquid,58.3%yield. 1 H NMR (600MHz, CDCl3) δ8.71(s,1H),8.48(s,1H),5.34(s,1H),5.14(s,1H),4.20(d,J=6.6Hz,2H),3.97(d ,J=12.6Hz,1H),3.15(dd,J=4.2Hz,J=12,1H),2.82(dd,J=4.8Hz,J=11.4Hz,1H),1.09(t,J=7.2Hz,3H). 13 C NMR(150MHz, 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%.

[0040] (±)-3-(6-Bromo-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (3a,3b)

[0041] 3a: white solid, 30.1% yield. 1 H NMR (400MHz, 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=12.0Hz,J=28.4Hz,2H),3.33(q,J=5.6Hz,1H),2.84(dd,J=6.4Hz,J=11.2Hz,1H),1.14(t,J=7.2Hz,3H). 13 C NMR(100MHz, 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 13N4BrO3SNa(M+Na)+, 394.9784; found, 394.9782. HPLC purity: 95.5%. 3b: white solid, 41.2% yield. 1 H NMR (400MHz, CDCl3) δ8.66(s,1H),8.50(s,1H),5.34(s,1H),5.12(s,1H),4.20(q,J=7.2Hz,2H),3.96(d,J=12.4Hz,1H ), 3.58 (d, J = 12.8Hz, 1H), 3.15 (dd, J = 4.0Hz, J = 11.6Hz, 1H), 2.81 (dd, J = 4.8Hz, J = 11.6Hz, 1H), 1.10 (t, J = 7.2Hz, 3H). 13 CNMR(100MHz, 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%.

[0042] (±)-3-(6-Dimethylamino-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (4a,4b)

[0043] 4a: white solid, 38.1% yield. 1 H NMR (400MHz, CDCl3) δ8.49(s,1H),8.19(s,1H),5.84(s,1H),5.32(t,J=5.2Hz,1H),4.23-4.15(m,8H),3.93(d,J=1 2.4Hz, 1H), 3.57 (d, J = 12.0Hz, 1H), 3.21 (q, J = 5.2Hz, 1H), 2.89 (dd, J = 5.2Hz, J = 11.6Hz, 1H), 1.10 (t, J = 7.2Hz, 3H). 13 C NMR(100MHz, 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 20N5O3S(M+H) + , 338.1281; found, 338.1272. HPLC purity: 98.9%. 4b: white solid, 59.2% yield. 1 H NMR (600MHz, 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=12Hz ,1H),3.54-3.44(m,7H),3.22(dd,J=6.6Hz,J=10.8Hz,1H),2.81(dd,J=7.2Hz,J=10.8Hz,1H),1.18(t,J=7.2Hz,3H). 13 C NMR(150MHz, 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%.

[0044] (±)-3-(6-(piperidin-1-yl)-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (5a,5b)

[0045] 5a: white solid, 41.7% yield. 1 H NMR (400MHz, CDCl3) δ8.24(s,1H),7.90(s,1H),5.25(t,J=5.6Hz,1H),4.28-4.15(m,6H),3.87(d,J=12.0Hz,1H),3.53(d, J=12.0Hz,1H),3.25(dd,J=6.0,J=11.2Hz,1H),2.94(dd,J=11.2,J=5.2Hz,1H),1.76-1.69(m,6H),1.15(t,J=7.2Hz,3H). 13 C NMR(100MHz, 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)calcd C 17 H24 N5O3S(M+H) + , 378.1594; found, 378.1597. HPLC purity: 98.7%. 5b: white solid, 51.1% yield. 1 HNMR (600MHz, 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(150MHz, 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)calcd C 17 H 23 N5O3SNa(M+Na) + ,400.1414; found,400.1410.HPLC purity:95.8%.

[0046] (±)-3-(6-Ethoxy-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (6a,6b)

[0047] 6a: white solid, 39.1% yield. 1 H NMR (600MHz, CDCl3) δ8.47(s,1H),8.17(s,1H),5.90(d,J=2.4Hz,1H),5.31(t,J=5.4Hz,1H),4.69-4.63(m,2H),4.23-4.15(m,2H),3.92(d,J =12.6Hz,1H),3.67(d,J=12.0Hz,1H),3.21(q,J=5.4Hz,1H),2.89(dd,J=4.8Hz,J=11.4Hz,1H),1.51(t,J=7.2Hz,3H),1.10(t,J=7.2Hz,3H). 13C 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 (600MHz, CDCl3) δ8.43(s,1H),8.22(s,1H),5.46(t,J=6.0Hz,1H),4.64(q,J=7.2Hz,2H),4.23-4.63(m,2H),4.23-4.16(m,2H),3.74(d, J=12.0Hz,1H),3.59(d,J=11.4Hz,1H),3.30(q,J=6.0Hz,1H),2.87(dd,J=6.0Hz,J=7.2Hz,1H),1.51(t,J=7.2Hz,3H),1.15(t,J=7.2Hz,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%.

[0048] (±)-3-(6-Methoxy-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (7a,7b)

[0049] 7a: light yellow solid, 32.8% yield. 1H NMR (600MHz, CDCl3) δ8.49(s,1H),8.19(s,1H),5.31(t,J=7.8Hz,1H),4.21-4.15(m,5H),3.92(d,J=18.6Hz,1H), 3.57(d,J=18Hz,1H), 3.20(dd,J=7.8Hz,J=17.4Hz,1H), 2.89(dd,J=7.2Hz,J=17.4Hz,1H), 1.09(t,J=10.2Hz,3H). 13 C NMR (150MHz, CDCl3) δ169.8,155.2,151.9,150.7,137.1,120.1,79.7,74.0,62.9,53.6,35.9,35.1,1 4.1.ESI-HRMS(m / z)calcd C 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 (400MHz, CDCl3) δ8.47(s,1H),8.25(s,1H),5.46(t,J=6.4Hz,1H),4.21(t,J=7.2Hz,5H),3.76(d,J=11.6H z,1H),3.57(d,J=11.2Hz,1H),3.29(q,J=5.6Hz,1H),2.86(dd,J=6.4Hz,J=11.2Hz,1H),1.16(t,J=7.2Hz,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%.

[0050] (±)-3-(6-propylthio-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (8a,8b)

[0051] 8a: yellow solid, 31.2% yield. 1H NMR(400MHz,CDCl3)δ8.63(s,1H),8.28(s,1H),5.48(t,J=6.0Hz,1H),4.22(q,J=7.2Hz,2H),3.78(d,J=12.0Hz,1H),3.56(d,J=11.6Hz,2H),3.37(t,J=7.2Hz,2H),3.29(dd,J=6.0Hz,J=11.2,1H),2.86(dd,J=6.8Hz,J=11.2Hz,1H),1.81.78(m,2H),1.18(t,J=7.2Hz,3H),1.09(t,J=7.6,3H). 13 C NMR(100MHz,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(400MHz,CDCl3)δ8.64(s,1H),8.22(s,1H),5.32(t,J=4.8Hz,1H),4.25-4.14(m,2H),3.92(d,J=12.4Hz,1H),3.56(d,J=12.4Hz,1H),3.42-3.31(m,2H),3.19(dd,J=5.2Hz,J=11.6Hz,1H),2.87(dd,J=4.8Hz,J=11.6Hz,1H),1.86-1.77(m,2H),1.13-1.06(m,6H). 13 C NMR(100MHz,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%.

[0052] (±)-3-(6-Morpholin-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (9a,9b)

[0053] 9a:white solid,24.4%yield. 1 H NMR (400MHz, CDCl3) δ8.27(s,1H),7.94(s,1H),5.26(t,J=5.6Hz,1H),4.31(s,4H),4.26-4.15(m,2H),3.88(d,J=12.4Hz,1H), 3.83(d,J=4.0Hz,4H), 3.53(d,J=12.0Hz,1H), 3.23(q,J=6.0Hz,1H), 2.92(dd,J=5.2Hz,J=11.6Hz,1H), 1.15(t,J=6.8Hz,3H). 13 C NMR(100MHz, 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)calcd C 16 H 22 N5O4S(M+H) + , 380.1387; found, 380.1390. HPLC purity: 97.9%. 9b: white solid, 45.4% yield. 1 HNMR (400MHz, CDCl3) δ8.23(s,1H),8.02(s,1H),5.94(s,1H),5.40(t,J=6.4Hz,1H),4.29(s,4H),4.21(q,J=7.2Hz,2H),3.81(t,J=4.8Hz,4H ), 3.73 (d, J = 11.6Hz, 1H), 3.46 (d, J = 11.6Hz, 1H), 3.23 (dd, J = 6.0Hz, J = 10.8Hz, 1H), 2.80 (dd, J = 7.2Hz, J = 10.8Hz, 1H), 1.18 (t, J = 6.8Hz, 3H). 13 C NMR(100MHz, 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%.

[0054] (±)-3-(6-Thiomorpholin-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (10a, 10b)

[0055] 10a: white solid, 30.4% yield. 1 H NMR (400MHz, CDCl3) δ8.27(s,1H)7.94(s,1H),5.27(t,J=5.6Hz,1H),4.59(s,4H),4.26-4.18(m,2H),3.88(d,J=12Hz,1H) ,3.54(d,J=12Hz,1H),3.24(q,J=6Hz,1H),2.93(dd,J=5.2Hz,J=11.2Hz,1H),2.76(t,J=5.2Hz,4H),1.16(t,J=7.2Hz,3H). 13 C NMR(100MHz, 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)calcd C 16 H 22 ClN5O3S2(M+H) + , 396.1159; found, 396.1149. HPLC purity: 96.1%. 10b: white solid, 45.2% yield. 1 H NMR (600MHz, 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 =12Hz,1H),3.54(d,J=12Hz,1H),3.24(q,J=6Hz,1H),2.93(q,J=5.4Hz,1H),2.77(t,J=4.8Hz,4H),1.16(t,J=7.2Hz,3H). 13 C NMR(150MHz, 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 21ClN5O3S2Na(M+Na) + ,418.0978; found,418.0980.HPLC purity:97.4%.

[0056] (±)-3-(6-(4-methylpiperidin-1-yl)-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (11a, 11b)

[0057] 11a: white solid, 32.7% yield. 1 H NMR(600MHz, CDCl3)δ8.23(s,1H),7.89(s,1H),6.66(s,1H),5.41(s,2H),5.2 4(t,J=8.4Hz,1H),4.24-4.16(m,2H),3.86(d,J=18Hz,1H),3.52(d,J=17.4Hz ,1H)3.24(dd,J=6.6Hz,J=9Hz,1H),3.08(s,2H),2.93(q,J=7.8Hz,1H),1.82- 1.66(m,2H),1.31-1.19(2H),1.14(t,J=10.2Hz,3H),0.97(d,J=10.2Hz,3H). 13 C NMR (150MHz, 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. HPLC purity: 95.4%. 11b: white solid, 31.5% yield. 1H NMR (400MHz, CDCl3) δ8.21 (s, 1H), 8.00 (s, 1H), 5.40 (t, J = 6.8Hz, 4H), 4.22 (d d,J=7.2Hz,J=15.6Hz,2H),3.74(d,J=12Hz,1H),3.43(d,J=12Hz,1H),3.22(dd ,J=6.0Hz,J=10.8Hz,1H),3.09(s,2H),2.81(dd,J=7.2Hz,J=10.8Hz,1H),1.83 -1.70(m,2H),1.32-1.27(m,2H),1.19(t,J=7.2Hz,3H),0.98(d,J=6.4Hz,3H). 13 C NMR(100MHz, 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.HPLCpurity:96.7%.

[0058] (±)-3-(6-(4-Fluoropiperidin-1-yl)-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (12a, 12b)

[0059] Compounds 12a,12b were prepared as described for the preparation of1a,1b except ethyl 2-(6-(4-fluoropiperidin-1-yl)-9H-purin-9-yl)acrylate was used in place of ethyl2-(9H-purin-9-yl)acrylate.12a: white solid, 42.3% yield. 1H NMR(600MHz,CDCl3)δ8.21(s,1H),7.90(s,1H),6.48(s,1H),5.21(t,J=6Hz,1H),4.93-4.84(m,1H),4.46(s,2H)4.19-4.13(m,4H),3.83(d,J=12Hz,1H),3.50(d,J=12.6Hz,1H),3.18(q,J=6Hz,1H),2.89(dd,J=4.8Hz,J=11.4Hz,1H),1.99-1.90(m,4H),1.10(t,J=7.2Hz,3H). 13 C NMR(150MHz,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)calcd C 17 H 22 FN5O3SNa(M+Na) + ,396.1500;found,396.1506.HPLC purity:98.9%.12b:white solid,53.4%yield. 1 HNMR(400MHz,CDCl3)δ8.24(s,1H),8.03(s,1H),6.10(s,1H),5.41(t.J=6.0Hz,1H),5.03-4.86(m,1H),4,54(s,2H),4.22(q,J=7.2Hz,4H),3.75(d,J=12Hz,1H),3.44(d,J=11.6Hz,1H),3.23(dd,J=10.8Hz,J=6.0Hz,1H),2.81(dd,J=10.8Hz,J=7.2Hz,1H),2.05-1.95(m,4H),1.20(t,J=7.2Hz,3H). 13 C NMR(100MHz,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. 19 F NMR(376MHz,CDCl3)δ-181.68.ESI-HRMS(m / z)calcd C17 H 22 FN5O3SNa(M+Na) + ,396.1500; found,396.1508.HPLC purity:97.2%.

[0060] (±)-3-(6-(4-trifluoromethylpiperidin-1-yl)-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (13a, 13b)

[0061] 13a: yellow solid, 42.1% yield. 1 H NMR(400MHz, CDCl3)δ8.25(s,1H),7.93(s,1H),6.44(s,1H),5.60(s,2H),5 .24(tJ=5.6Hz,1H),4.23-4.15(m,2H),3.86(d,J=12Hz,1H),3.53(d,J=12H z,1H),3.20(q,J=6Hz,1H),3.08-2.99(m,2H)2.95(q,J=5.6Hz,1H),2.42-2 .33(m,1H),2.00(d,J=12.8Hz,2H),1.69-1.56(m,2H),1.13(tJ=6.8Hz,3H). 13 CNMR(100MHz, 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 =27.4Hz),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.HPLCpurity:95.6%.13b:yellow solid,37.5%yield. 1H NMR(400MHz, CDCl3)δ8.24(s,1H)8.03(s,1H),5.97(s,1H),5.63(s,2H),5.41(tJ= 6.4Hz,1H),4.22,(q,J=6.8Hz,2H),3.75(d,J=12Hz,1H),3.45(d,J=12Hz,1H),3.23 (dd,J=11.2Hz,J=6Hz,1H),3.07(t,J=11.6Hz,2H),2.81(dd,J=10.8Hz,J=7.2Hz,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 (100MHz, 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%.

[0062] (±)-3-(6-(4-chloropiperidin-1-yl)-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (14a, 14b)

[0063] White solid; 14a: light yellow solid, 51.7% yield. 1 H NMR (600MHz, CDCl3) δ8.23 (s, 1H), 8.02 (s, 1H), 6.03 (s, 1H), 5.40 (t, J = 6.6Hz ,1H),4.60(s,2H)4.38-4.35(m,1H),4.22(q,J=7.2Hz,4H),3.74(d,J=12Hz,1 H),3.45(d,J=12Hz,1H),3.23(dd,J=4.8Hz,J=10.8Hz,1H),2.80(dd,J=7.2Hz ,J=10.8Hz,1H),2.22-2.19(m,2H),2.00-1.96(m,2H),1.19(t,J=7.2Hz,3H).13 C NMR (100MHz, 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 (400MHz, CDCl3) δ8.27(s,1H),7.93(s,1H),6.50(s,1H),5.26(t,J=5. 6Hz,1H),4.62(s,2H),4.39-4.34(m,1H),4.27-4.16(m,4H),3.88(d,J=12. 0Hz,1H),3.54(d,J=12.0Hz,1H),3.24(q,J=6Hz,1H),2.93(dd,J=5.2Hz,J =11.6Hz,1H),2.25-2.17(m,2H),2.02-1.93(m,2H),1.15(t,J=7.2Hz,3H). 13 C NMR(100MHz, 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%.

[0064] (±)-3-(6-(4-morpholinopiperidin-1-yl)-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (15a, 15b)

[0065] 15a:white solid,37.7%yield. 1H NMR(600MHz,CDCl3)δ8.22(s,1H),8.02(s,1H),5.50(s,1H),5.40(t,J=9.6Hz,1H),4.22(q,J=10.8Hz,2H),3.76(s,1H),3.72(t,J=6.6Hz,4H),3.44(d,J=18Hz,1H),3.22(dd,J=9Hz,J=16.2Hz,1H),3.13(s,2H),2.80(dd,J=11.4Hz,J=16.2Hz,1H),2.59-2.52(m,5H),2.01(d,J=19.2Hz,2H),1.62-1.52(m,4H),1.19(t,J=10.8Hz,3H). 13 C NMR(150MHz,CDCl3)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)calcd C 21 H 31 N6O4S(M+H) + ,463.2111;found,463.2113.HPLC purity:97.9%.15b:white solid,38.6%yield. 1 H NMR(600MHz,CDCl3)δ8.25(s,1H),7.92(s,1H),6.55(s,1H)5.49(s,1H),5.25(t,J=8.4Hz,1H),4.26-4.17(m,2H),3.88(d,J=18.6Hz,1H),3.73(t,J=6.6Hz,4H),3.53(d,J=18Hz,1H),3.24(dd,J=9Hz,J=16.8Hz,1H),3.12(s,2H),2.93(q J=7.8Hz,1H),2.60-2.53(s,5H),2.02(d,J=18Hz,2H),1.63-1.52(m,4H),1.15(t,J=10.8Hz,3H). 13 CNMR(100MHz,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 31N6O4S(M+H) + ,463.2111; found,463.2120.HPLC purity:95.9%.

[0066] (±)-3-(6-(4-Benzylpiperidin-1-yl)-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (16a, 16b)

[0067] 16a:white solid,32.4%yield. 1 H NMR (600MHz, CDCl3) δ8.18 (s, 1H), 7.97 (s, 1H), 7.28-7.11 (m, 5H), 5.37 (t, J = 10. 0Hz.3H),4.22-4.16(m,2H),3.71(d,J=17.4Hz,1H),3.41(d,J=17.4Hz,1H)3.21( dd,J=9.6Hz,J=16.8Hz,1H),2.99(s,2H),2.78(dd,J=10.8Hz,J=16.2Hz,1H)2.55 (d,J=10.8Hz,2H),1.90-1.78(m,4H),1.35-1.22(m,1H),1.16(t,J=10.8Hz,3H). 13 C NMR (150MHz, CDCl3) δ168.6,154.0,151.9,150.7,140.2,137.6,129.3,128.4,12 6.1,119.7,73.8,63.0,43.3,38.5,35.3,33.3,32.4,14.0.ESI-HRMS(m / z)calcd C 24 H 29 N5O3SNa(M+Na) + ,490.1883; found,490.1886.HPLC purity:97.9%.16b:whitesolid,31.8%yield. 1HNMR(600MHz, CDCl3)δ8.21(s,1H),7.87(s,1H),7.28-7.11(m,5H),5.43(s,2H),5 .23(t,J=8.4Hz,1H),4.23-4.14(m,2H),3.84(d,J=18Hz,1H),3.50(d,J=18Hz,1H), 3.21(dd,J=9Hz,J=16.8Hz,1H),3.00(s,2H),2.91(dd,J=7.8Hz,J=16.8Hz,1H),2. 55(d,J=10.8Hz,2H),1.90-1.78(m,4H),1.36-1.21(m,1H),1.13(t,J=10.8Hz,3H). 13 C NMR (150MHz, 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)calcdC 24 H 30 N5O3S(M+H) + ,468.2064; found,468.2067.HPLC purity:99.7%.

[0068] (±)-3-(6-(4-p-Oxybenzylpiperidin-1-yl)-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (17a, 17b)

[0069] 17a:white solid,24.6%yield. 1 H NMR (600MHz, 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=11.4Hz,1H),3.53(d,J= 12Hz,1H),3.24(dd,J=6Hz,J=10.8Hz,1H),3.03(s,2H),2.94(q,J=4.8Hz,1H),2.62(d,J=7.2 Hz,2H),1.96-1.92(m,1H),1.82(d,J=12.6Hz,2H),1.38-1.34(m,2H),1.15(t,J=7.2Hz,3H).13 C NMR(150MHz,CDCl3)δ169.8,162.2,160.6,154.0,151.9,136.8,131.7(Jc-F=5.0Hz),128.0(Jc- F =7.7Hz),127.1(Jc- F =11.4Hz),123.9(Jc- F =3.9Hz),119.8,115.4,(Jc- F =22.1Hz),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 FN5 O3S(M+H) + ,486.1970;found,486.1980.HPLC purity:96.4%.17b:white solid,37.8%yield. 1 H NMR(400MHz,CDCl3)δ8.24(s,1H),7.90,(s,1H),7.20-7.00(m,4H),5.47(s,2H),5.26(t,J=5.6Hz,1H),4.26-4.17(m,2H),3.87(d,J=12.4Hz,1H),3.53(d,J=12Hz,1H),3.24(dd,J=6.4Hz,J=11.6Hz,1H),3.03(s,2H),2.94(dd,J=11.2Hz,J=5.2Hz,1H),2.62(d,J=7.2Hz,2H),1.95(s,2H),1.82(d,J=13.2Hz,2H),1.64(s,1H),1.15(t,J=17.2Hz,3H). 13 C NMR(100MHz,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 =16.1Hz),123.9,119.8,115.4(d,J C-F =21.7Hz),79.7,74.0,63.0,37.4,36.2,35.2,32.3,29.8,14.1. 19FNMR(376MHZ,CDCl3)δ-117.86.ESI-HRMS(m / z)calcd C 24 H 29 FN5O3S(M+H) + ,486.1970; found,486.1978.HPLCpurity:99.6%.

[0070] (±)-3-(6-(4-methylpiperazin-1-yl)-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (18a, 18b)

[0071] 18a:white solid,29.5%yield. 1 H NMR (600MHz, CDCl3) δ8.23(s,1H),8.02(s,1H),5.40(t,J=6.6Hz,1H),4.35(s,2H),4.24-4.20(m,2H),3.74(d,J=11.4Hz,1H),3.44(d,J=11.4H z,1H),3.23(dd,J=6Hz,J=10.8Hz,1H),2.80(dd,J=7.2Hz,J=10.8Hz,1H),2.55(t,J=5.4Hz,4H),2.35(s,3H)1.77(s,2H),1.19(t,J=7.2Hz,3H). 13 C NMR(100MHz, 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.HPLCpurity:98.1%.18b:white solid,41.6%yield. 1H NMR (600MHz, CDCl3) δ8.26 (s, 1H), 7.93 (s, 1H), 5.26 (t, J = 6Hz, 1H), 4.34 (s, 2H), 4.25-4.18 (m, 2H), 3.88 (d, J = 12Hz, 1H), 3.5 4(d,J=12Hz,1H),3.24(q,J=5.4Hz,1H),2.93(q,J=5.4Hz,1H),2.56(s,4H),2.36(s,3H),1.75(s,2H),1.15(t,J=7.2Hz,3H). 13 C NMR (150MHz, 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.HPLC purity:95.6%.

[0072] (±)-3-(6-Azepanyl-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (19a, 19b)

[0073] 19a:white solid,31.8%yield. 1 H NMR(600MHz, 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=17.4H z,2H),3.55(d,J=18Hz,1H),3.27(dd,J=9.6Hz,J=16.8Hz,1H),2.98(q,J=7.8Hz,1H),1.89(s,4H),1.62-1.57(m,4H),1.17(t,J=10.8Hz,3H). 13 C NMR(100MHz, 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. 1 H NMR(600MHz, CDCl3)δ8.22(s,1H),7.99(s,1H),6.42(s,1H),5.41(t,J=6.6Hz,1H),4.44(s,1H)4.30-4.20(m,3H),3.97(s,1H),3.85(s,1H),3.75 (d,J=11.4Hz,1H),3.44(d,J=11.4Hz,1H),3.22(dd,J=6Hz,J=10.8Hz,1H),2.83(t,J=7.2Hz,1H),1.89(s,4H),1.60(s,1H),1.20(t,J=6.6Hz,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%.

[0074] (±)-3-(6-Pyrrolyl-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (20a, 20b)

[0075] 20a:white solid,26.5%yield. 1 H NMR (600MHz, CDCl3) δ8.27(s,1H),7.87(s,1H),5.26(t,J=6.0Hz,1H),4.26-4.16(m,4H),3.88(d,J=12.0Hz,1H),3.76(s,2H),3.55 (d,J=12.0Hz,1H),3.27(dd,J=6.4Hz,J=11.2Hz,1H),2.96(dd,J=5.2Hz,J=11.2Hz,1H),2.04(d,J=26Hz,4H),1.16(t,J=7.2Hz,3H). 13C NMR(100MHz, CDCl3)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)calcdC 16 H 22 N5O3S(M+H) + , 364.1438; found, 364.1437. HPLC purity: 98.4%. 20b: white solid, 58.0% yield. 1 H NMR (600MHz, CDCl3) δ8.22(s,1H),7.96(s,1H),6.43(s,1H),5.41(t,J=6.4Hz,1H),4.30-4.15(m,4H),3.72(d,J=9.2Hz,3H),3.47( d, J=11.6Hz, 1H), 3.24 (dd, J=6Hz, J=10.8Hz, 1H), 2.84 (dd, J=6.8Hz, J=10.8Hz, 1H), 2.03 (t, J=25.6Hz, 4H), 1.18 (t, J=6.8Hz, 3H). 13 C NMR (100MHz, CDCl3) 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 N5O3S(M+H) + ,364.1438; found,364.1435.HPLC purity:95.3%.

[0076] (±)-3-(6-Methylthio-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (21a, 21b)

[0077] 21a: yellowish solid, 33.4% yield. 1H NMR (400MHz, CDCl3) δ8.63(s,1H),8.22(s,1H),5.74(s,1H),5.30(t,J=5.2Hz,1H),4.18-4.12(m,2H),3.91(d,J=12.4Hz,1H), 3.56(d,J=12.0Hz,1H),3.15(dd,J=5.2Hz,=11.6Hz,1H),2.86(dd,J=4.8Hz,J=11.6Hz,1H),2.68(s,3H),1.07(t,J=7.2Hz,3H). 13 C NMR (150MHz, CDCl3) 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)calcdC 13 H 16 N4O3S2Na(M+Na) + , 363.0556; found, 363.0546. HPLC purity: 99.8%. 21b: yellowish solid, 40.5% yield. 1 H NMR (600MHz, CDCl3) δ8.61(s,1H),8.28(s,1H),5.45(t,J=6.0Hz,1H),5.04(s,1H),4.23-4.14(m,2H),3.69(d d, J=11.6Hz, J=36.8Hz, 2H), 3.30 (q, J=6.0Hz, 1H), 2.87 (q, J=6.0Hz, 1H), 2.67 (s, 3H), 1.12 (t, J=7.2Hz, 3H). 13 C NMR(100MHz, CDCl3)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 N4O3S2Na(M+Na) + ,363.0556; found,363.0564.HPLCpurity:99.6%.

[0078] (±)-3-(6-Ethylthio-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (22a, 22b)

[0079] 22a:yellow solid,21.5%yield. 1 H NMR(600MHz,CDCl3)δ8.58(s,1H),8.28(s,1H),5.44(q,J=6Hz,1H),5.12(d,J=5.4Hz,1H),4.18-4.13(m,2H),3.73(d,J=12Hz,1H),3.65(d,J=11.4Hz,1H),3.34-3.28(m,3H),2.86(q,J=5.4Hz,1H),1.41(t,J=7.8Hz,3H),1.12(t,J=7.2Hz,3H). 13 C NMR(150MHz,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)calcd C 14 H 18 N4O3S2Na(M+Na) + ,377.0713;found,377.0697.HPLC purity:99.8%.22b:yellow solid,22.6%yield. 1 HNMR(600MHz,CDCl3)δ8.63(s,1H),8,28(s,1H),5.48(q,J=6Hz,1H),4.81(d,J=5.4Hz,1H),4.23-4.19(m,2H),3.77(d,J=12Hz,1H),3.55(d,J=12Hz,1H),3.38(q,J=7.2Hz,2H),3.28(q,J=6Hz,1H),2.85(dd,J=6.6Hz,J=11.4Hz,1H),1.45(t,J=7.2Hz,3H),1.17(t,J=7.2Hz,3H). 13 C NMR(150MHz,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)calcd C 14 H 18 N4O3S2Na(M+Na) + ,377.0713;found,377.0704.HPLC purity:99.3%.

[0080] (±)-3-(6-Isopropylthio-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (23a, 23b)

[0081] 23a:white solid,51.2%yield. 1 H NMR(600MHz, 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=12Hz, 1H), 3.53 (d, J = 12Hz, 1H), 3.28 (q, J = 6Hz, 1H), 2.85 (dd, J = 6.6Hz, J = 10.8Hz, 1H), 1.50 (d, J = 7.2Hz, 6H), 1.18 (t, J = 7.2Hz, 3H). 13 C NMR (150MHz, 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)calcdC 15 H 20 N4O3S2Na(M+Na) + ,391.0869; found,391.0871.HPLC purity:99.8%.23b:whitesolid,22.4%yield. 1 H NMR(600MHz, 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=12.6Hz,1H), 3.56(d,J=12.6Hz,1H), 3.16(dd,J=4.8Hz,J=11.4Hz,1H), 2.86(dd,J=5.4Hz,J=12Hz,1H), 1.46(d,J=6.0Hz,6H), 1.09(t,J=6.6Hz,3H). 13 C NMR (150MHz, 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)calcdC 15 H 20 N4O3S2Na(M+Na)+ ,391.0869; found,391.0868.HPLC purity:95.1%.

[0082] (±)-3-(6-allylthio-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (24a, 24b)

[0083] 24a:white solid,41.2%yield. 1 H NMR (600MHz, 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=2.4Hz,2H),3.76( d,J=12Hz,1H),3.60(d,J=12Hz,1H),3.29(q,J=6Hz,1H),2.85(q,J=6Hz,1H),2.22(t,J=2.4Hz,1H),1.16(t,J=7.2Hz,3H). 13 C NMR(150MHz, 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)calcd C 15 H 16 N4O3S2Na(M+Na) + , 387.0556; found, 387.0550. HPLC purity: 98.6%. 24b: white solid, 49.7% yield. 1 H NMR (600MHz, 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=12.6Hz,1H),3.57(d,J =12.6Hz,1H),3.19(dd,J=4.8Hz,J=12Hz,1H),2.83(dd,J=4.8Hz,J=11.4Hz,1H),2.23(t,J=2.4Hz,1H),1.12(t,J=7.2Hz,3H). 13C 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%.

[0084] (±)-3-(6-Butylthio-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (25a, 25b)

[0085] 25a:white solid,45.1%yield. 1 H NMR(400MHz,DMSO-d6)δ8.72(s,1H),8.68(s,1H),5.93(d,J=5.2Hz,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 .2Hz,1H),3.41-3.30(m,3H),2.90(dd,J=3.6Hz,J=11.2Hz,1H),1.73-1.6 6(m,2H),1.49-1.39(m,2H),1.04(t,J=7.2Hz,3H),0.91(t,J=7.2Hz,3H). 13 C NMR(100MHz,DMSO-d6)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)calcd C 16 H 22 N4O3S2(M+Na) + , 405.1026; found, 405.1020. HPLC purity: 99.6%. 25b: white solid, 30.2% yield. 1H NMR (400MHz, CDCl3) 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=12Hz,1H),3.56(d,J=12.4Hz,1H) ,3.40-3.35(m,1H),3.18(q,J=4.8Hz,1H),2.87(q=3.2,1H),1.80-1.72 (m,2H),1.54-1.45(m,2H),1.10(t,J=7.2Hz,3H),0.94(t,J=7.6Hz,3H). 13 CNMR(100MHz, CDCl3)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 N4O3S2(M+Na) + ,405.1026; found,405.1031.HPLC purity:98.6%.

[0086] (±)-3-(6-pentylthio-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (26a, 26b)

[0087] 26a:white solid,43.3%yield. 1 H NMR (400MHz, CDCl3) δ8.62(s,1H),8.27(s,1H),5.48(q,J=6.0Hz,1H),4.80(d,J =6.0Hz,1H),4.21(q,J=7.2Hz,2H),3.78(d,J=12Hz,1H),3.54(d,J=12.0Hz,1H), 3.38(tJ=7.2Hz,2H),3.28(q,J=6.0Hz,1H),2.85(dd,J=6.4Hz,J=11.2Hz,1H),1. 83-1.75(m,2H),1.51-1.33(m,4H),1.17(t,J=6.8Hz,3H),0.91(t,J=7.2Hz,3H). 13C NMR (100MHz, 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)calcdC 17 H 25 N4O3S2(M+H) + , 397.1363; found, 397.1354. HPLC purity: 99.6%. 26b: white solid, 25.1% yield. 1 H NMR(600MHz, CDCl3)δ8.62(s,1H),8.21(s,1H),5.75(s,1H),5.32-5.30(m,1H),4.2 1-4.14(m,2H),3.91(d,J=12.0Hz,1H),3.56(d,J=12.0Hz,1H),3.39-3.32(m,2H),3. 17(dd,J=4.8Hz,J=11.4Hz,1H),2.87(dd,J=4.8Hz,J=11.4Hz,1H),1.79-1.74(m,2H) ,1.47-1.42(m,2H),1.37-1.31(m,2H),1.09(t,J=7.2Hz,3H),0.88(t,J=7.2Hz,3H). 13 CNMR(100MHz, 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%.

[0088] (±)-3-(6-Benzylthio-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (27a, 27b)

[0089] 27a:white solid,43.2%yield. 1H NMR(400MHz,DMSO-d6)δ8.73(s,1H),8.68(s,1H),7.44(d,J=7.2Hz,1H),7.31-7.20(m,3H),6.40(d,J=5.6Hz,1H),5.43(dd,J=5.6Hz,J=9.6Hz,1H),4.64(dd,J=13.6Hz,J=16Hz,2H),4.12-4.00(m,J=13.6Hz 2H),3.90(d,J=12.4Hz,1H),3.78(d,J=12.4Hz,1H),3.26-3.22(m,1H),2.96(dd,J=4Hz,J=12Hz,1H),1.00(t,J=7.2Hz,3H). 3 C NMR(100MHz,DMSO-d6)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 N4O3S2(M+Na) + ,439.0869;found,439.0872.HPLC purity:99.7%.27b:whitesolid,42.4%yield. 1 HNMR(400MHz,DMSO-d6)δ8.75(s,1H),8.74(s,1H),7.47(d,J=7.2Hz,2H),7.34-7.22(m,3H),5.94(d,J=5.2Hz,1H),5.24-5.21(m,1H),4.66(dd,J=13.6Hz,J=18.4Hz,2H),4.15-4.07(m,2H),4.00(d,J=11.6Hz,1H),3.62(d,J=11.2Hz,1H),3.40-3.38(m,1H),2.90(dd,J=3.2Hz,J=11.2Hz,1H),1.04(t,J=6.8Hz,3H). 13 C NMR(100MHz,DMSO-d6-d6)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 20N4 O3S2(M+Na) + ,439.0869; found,439.0863.HPLC purity:98.3%.

[0090] (±)-3-(6-m-Fluorophenylthio-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (28a, 28b)

[0091] 28a:white solid,26.4%yield. 1 H NMR (600MHz, CDCl3) δ8.21 (s, 1H), 7.90 (s, 1H), 6.48 (s, 1H), 5.21 (t, J = 6Hz, 1H), 4.93-4.84 (m, 1H), 4.46 (s, 2H) 4.19-4.13 (m, 4H), 3.83 (d,J=12Hz,1H),3.50(d,J=12.6Hz,1H),3.18(q,J=6Hz,1H),2.89(dd,J=4.8Hz,J=11.4Hz,1H),1.99-1.90(m,4H),1.10(t,J=7.2Hz,3H). 13 CNMR (150MHz, 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-111.38.ESI-HRMS(m / z)calcd C 18 H 17 FN4O3S2(M+Na) + , 443.0618; found, 443.0613. HPLC purity: 99.0%. 28b: white solid, 31.3% yield. 1H NMR (400MHz, 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=4.8Hz,1H),4.24-4.18(m,2H),3.94 (d,J=12.4Hz,1H),3.58(d,J=12.4Hz,1H)3.19(dd,J=4.8Hz,J=11.6Hz,1H),2.86(dd,J=4.8Hz,J=11.6Hz,1H),1.12(t,J=7.2Hz,3H). 13 C NMR(100MHz,CDCl3)δ169.4,164.0,161.4(d,J C-F =38Hz),151.9,149.0,142.0,131.3(d,J C-F =3.4Hz),130.9,130.7(d,J C-F =8.1Hz),128.8(d,J C-F =8.0Hz),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%.

[0092] (±)-3-(6-(4-tert-Butylphenylthio)-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (29a, 29b) 29a: white solid, 33.4% yield. 1 H NMR (400MHz, DMSO-d6) δ8.80(s,1H),8.55(s,1H),7.57-7.51(m,4H),5.95(d,J=5.2Hz,1H),5.24-5.20(m,1H),4.15-4.08(m,2H),4.01(d,J= 11.2Hz, 1H), 3.63 (d, J = 11.2Hz, 1H), 3.38 (dd, J = 5.6Hz, J = 11.2Hz, 1H), 2.91 (dd, J = 3.6Hz, J = 11.2Hz, 1H), 1.33 (s, 9H), 1.04 (t, J = 7.2Hz, 3H). 13C NMR (100MHz, DMSO-d6) 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)calcd C 22 H 27 N4O3S2(M+H) + , 459.1519; found, 459.1515. HPLC purity: 96.4%. 29b: white solid, 37.4% yield. 1 H NMR (400MHz, DMSO-d6) δ8.76(s,1H),8.56(s,1H),7.57-7.51(m,4H),6.42(d,J=5.2Hz,1H),5.49-5.45(m,1H),4.13-4.03(m,2H),3.92(d,J= 12.4Hz, 1H), 3.80 (d, J = 12.0Hz, 1H), 3.28 (dd, J = 5.2Hz, J = 12Hz, 1H), 2.99 (dd, J = 3.6Hz, J = 12.0Hz, 1H), 1.32 (s, 10H), 1.03 (t, J = 6.8Hz, 3H). 13 CNMR(100MHz,DMSO-d6)δ167.3,159.5,152.3,151.5,148.9,143.3,135.3,130.0,1 26.4,123.0,76.6,75.5,61.8,35.8,34.6,34.5,31.0,13.8.ESI-HRMS(m / z)calcdC 22 H 27 N4O3S2(M+H) + ,459.1519; found,459.1514.HPLC purity:96.3%.

[0093] (±)-3-(6-(3-Fluoropropylthio)-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (30a, 30b)

[0094] 30a:white solid,31.4%yield. 1H NMR(400MHz,CDCl3)δ8.63(s,1H),8.30(s,1H),5.47(t,J=6.4Hz,1H),4.71(s,1H),4.67(t,J=6Hz,1H),4.55(t,J=6.0Hz,1H),4.21(q,J=6.8Hz,2H)3.77(d,J=11.6Hz,1H),3.56(d,J=11.6Hz,1H),3.50(t,J=6.8Hz,2H),3.29(q,J=5.6Hz,1H),2.84(dd,J=6.8Hz,J=11.2Hz,1H),2.27-2.14(m,2H),1.17(t,J=7.2Hz,3H). 13 C NMR(100MHz,CDCl3)δ168.2,162.0,151.5.148.6 142.4,131.4,83.3,81.6,74.0,63.2,35.1,33.6,30.6(d,J C-F =20Hz),24.8(d,J C-F =5.2Hz),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. 1 H NMR(400MHz,CDCl3)δ8.65(s,1H),8.24(s,1H),5.66(s,1H),5.32(t,J=4.8Hz,1H),4.66(t,J=5.6Hz,1H),4.54(t,J=6.0Hz,1H),4.20(dd,J=6.4Hz,J=13.6Hz,2H),3.92(d,J=12.4Hz,1H),3.56(d,J=12.0Hz,1H),3.50(t,J=6.8Hz,2H),3.18(dd,J=11.2Hz,J=4.8Hz,1H),2.86(dd,J=11.6Hz,J=5.2Hz,1H),2.26-2.14(m,2H),1.11(t,J=7.2Hz,3H). 13CNMR (100MHz, 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 =25.4Hz),30.6(d,J C-F =20.1Hz),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.HPLCpurity:97.7%.

[0095] (±)-3-(6-(3-chloropropylthio)-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (31a, 31b)

[0096] 31a:white solid,42.4%yield. 1 H NMR (400MHz, 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=5.6Hz,1H),2.85(dd,J=6.4Hz,J=11.2Hz,1H),2.26-2.20(m,2H),1.13(t,J=7.2Hz,3H). 13 C NMR(100MHz, 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)calcd C 15 H 19 ClN4O3S2Na(M+Na) + , 425.0479; found, 425.0470. HPLC purity: 96.4%. 31b: white solid, 21.7% yield. 1HNMR(400MHz, CDCl3)δ8.62(s,1H),8.24(s,1H),5.67(s,1H),5.31(s,1H), 4.176(q,J=7.2Hz,2H),3.92(d,J=12Hz,1H),3.68(t,J=6.4Hz,2H),3.56(d ,J=12.4Hz,1H),3.50(t,J=6.8Hz,2H),3.16(dd,J=5.2Hz,J=11.6Hz,1H),2 .86(dd,J=4.8Hz,J=11.6Hz,1H),2.28-2.21(m,2H),1.094(t,J=7.2Hz,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%.

[0097] (±)-3-(2-Amino-6-piperidinyl-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (32a, 32b) 32a: yellow solid, 43.0% yield. 1 H NMR (600MHz, CDCl3) δ7.60 (s, 1H), 5.18 (t, J = 5.4Hz, 1H), 4.60 (s, 2H), 4.27-4.16 (m, 4H), 3.80 (d, J = 12Hz, 1H), 3.49 (d,J=12Hz,1H),3.24(dd,J=6.6Hz,J=11.4Hz,1H),2.97(q,J=5.4Hz,1H),1.71-1.64(m,6H),1.18(t,J=7.2Hz,3H). 13 C NMR (150MHz, 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(600MHz, CDCl3)δ7.72(s,1H),5.34(t,J=6.6Hz,1H),4.59(s,2H),4.26-4.11(m,4H),3.68(d,J=12Hz,1H)3.37(d,J =12Hz,1H),3.17(dd,J=6Hz,J=10.8Hz,1H),2.81(dd,J=7.2Hz,J=10.8Hz,1H),1.72-1.65(m,6H),1.20(t,J=7.2Hz,3H). 13 C NMR(150MHz, 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 H 25 N6O3S(M+H) + ,393.1703; found,393.1701.HPLC purity:98.2%.

[0098] (±)-3-(2-Fluoro-6-piperidinyl-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (33a, 33b)

[0099] 33a: yellow solid, 36.4% yield. 1 H NMR (600MHz, 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 (100MHz, 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)calcd C 17 H 23 FN5O3S(M+H) + , 396.1500; found, 396.1499. HPLC purity: 99.4%. 33b: yellow solid, 43.6% yield. 1 HNMR(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), 3.83(dd,J=3.6,J=11.4,1H), 1.16(t,J=7.2,3H). 13 C NMR (150MHz, 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),78.9,7 3.6,63.1,35.6(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.HPLCpurity:96.6%.

[0100] (±)-3-(2-chloro-6-piperidinyl-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (34a, 34b)

[0101] 34a:white solid,17.6%yield. 1H NMR (600MHz, CDCl3) δ8.01 (s, 1H), 5.41 (t, J = 6Hz, 1H), 4.62 (s, 2H), 4.29-4.20 (m, 4H), 3.69 (d, J = 12Hz, 1H), 3.45 (d ,J=11.4Hz,1H),3.25(q,J=5.4Hz,1H),2.82(dd,J=7.2Hz,J=11.4Hz,1H),1.74-1.68(m,6H),1.22(t,J=7.2Hz,3H). 13 C NMR(100MHz, 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 (400MHz, CDCl3) δ7.93(s,1H),5.65(s,1H),5.24(t,J=4.8Hz,1H),4.31-4.16(m,6H),3.86(d,J=12,6Hz,1H),3.48(d,J =12Hz,1H),3.16(dd,J=4.8Hz,J=11.4Hz,1H),2.86(dd,J=4.8Hz,J=11.4Hz,1H),1.73-1.67(m,6H),1.18(t,J=7.2Hz,3H). 13 C NMR (100MHz, 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%.

[0102] (±)-3-(2-Amino-6-propylthio-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (35a,35b)

[0103] 35a:white solid,41.3%yield. 1 H NMR(400MHz,CDCl3)δ7.90(s,1H),5.42(t,J=5.6Hz,1H),4.96(s,2H),4.23-4.16(m,2H),3.71(d,J=11.6Hz,1H),3.44(d,J=11.2Hz,1H),3.27-3.21(m,3H),2.86(dd,J=6.0Hz,J=10.8Hz,1H),1.82-1.73(m,2H),1.19(t,J=7.2Hz,3H),1.05(t,J=7.2Hz,3H). 13 C NMR(100MHz,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)calcdC 15 H 22 N5O3S2(M+H) + ,384.1159;found,384.1150.HPLC purity:99.7%.35b:white solid,43.5%yield. 1 H NMR(400MHz,CDCl3)δ7.84(s,1H),5.98(s,1H),5.24(t,J=5.2Hz,1H),4.95(s,2H),4.27-4.14(m,2H),3.82(d,J=12Hz,1H),3.50(d,J=12.0Hz,1H),3.28-3.24(m,2H),3.17(q,J=5.6Hz,1H),2.90(dd,J=4.8Hz,J=11.2Hz,1H),1.82-1.73(m,2H),1.15(t,J=7.2Hz,3H),1.05(t,J=7.2Hz,3H). 13 C NMR(100MHz,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%.

[0104] (±)-3-(2-Fluoro-6-propylthio-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (36a, 36b)

[0105] 36a:white solid,38.5%yield. 1 H NMR (400MHz, CDCl3) δ8.31, (s, 1H) 5.44, (t, J = 6Hz, 1H), 4.24 (q, J = 13.6Hz, 2H), 3.72 (d, J = 11.6Hz, 1H), 3.61 (d, J = 11.6 Hz,1H),3.34-3.29(m,2H),2.85(dd,J=11.2,J=6Hz,1H),1.86-1.77(m,2H),1.19(t,J=6.8Hz,3H)1.08(t,J=7.6Hz,3H). 13 C NMR(100MHz,CDCl3)δ169.3,166.0(d,J C-F =16.5Hz),159.7,158.3,152.2(d,J C-F =17.6Hz),145.2(d,J C-F =3.2Hz),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. HPLC purity: 99.4%. 36b: white solid, 41.5% yield. 1 H NMR (600MHz, CDCl3) δ8.27(s,1H),5.29(t,J=4.2Hz,1H),4.25-4.19(m,2H)3.92(d,J=12.6Hz,1H)3.54(d,J=12.6Hz,1H),3.36-3.29(m ,2H),3.11(dd,J=4.2Hz,J=12Hz,1H),3.81(dd,J=4.8Hz,J=12Hz,1H),1.84-1.78(m,2H),1.14(t,J=7.2Hz,3H),1.07(t,J=7.2Hz,3H). 13C NMR (100MHz, CDCl3) δ169.1,166.1(Jc-F=16.5Hz),157.6(d,Jc-F=215.0Hz),150.1(Jc-F=17.3Hz),1 41.4(Jc-F=3.2Hz),129.8(Jc-F=4.2Hz),78.5,73.8,63.4,35.4(Jc-F=45Hz),31.3,22.7,14.0,13.5. 19 F NMR(376MHz,CDCl3)δ-49.51.ESI-HRMS(m / z)calcd C 15 H 19 FN4O3S2Na(M+Na) + ,409.0775; found,409.0769.HPLC purity:99.4%.

[0106] (±)-3-(2-chloro-6-propylthio-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (37a, 37b37a: white solid, 22.4% yield. 1 H NMR (400MHz, DMSO-d6) δ7.57(s,1H),5.90(d,J=5Hz,2H),5.17-5.14(m,1H),4.14(q,J=7.2Hz,2H),3.92(J=11.2Hz,1H),3.57(d,J=11.2Hz, 1H),3.37(q,J=5.6Hz,1H),3.33-3.29(m,2H),2.88(dd,J=3.6Hz,11.6,1H),1.78-1.69(m,2H),1.07(t,J=7.2Hz,3H),1.00(t,J=7.2Hz,3H). 13 C NMR(100MHz,DMSO-d6)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. 1HNMR(400MHz, 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=12.4Hz,1H),3.53(d,J=12.4Hz,1H ),3.37-3.27(m,2H),3.12(dd,J=4.4Hz,J=11.6Hz,1H),2.85-2.80(m,1H),1.84-1.75(m,1H),1.13(q,J=7.2Hz,3H),1.05(t,J=7.2Hz,3H). 13 C NMR (100MHz, CDCl3) δ168.9,164.7,153.3,149.6,141.5,130.2,78.6,73.9,63.3,35.4(J C-F =13.7Hz),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%.

[0107] (±)-3-(2-Amino-6-chloro-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (38a,38b)

[0108] 38a:white solid,71.0%yield. 1 H NMR(600MHz,MeOD)δ8.31(s,1H),5.35(t,J=5.4Hz,1H),4.23-4.15(m,2H),4.02(d,J=12.6Hz,1H),3.73(d, J=12.6Hz,1H),3.33(s,1H),3.21(q,J=5.4Hz,1H),3.10(dd,J=4.8Hz,J=11.4Hz,1H),1.13(t,J=6.6Hz,3H). 13 CNMR(150MHz, CDCl3)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 ClN5O3SNa(M+Na) +, 366.0398; found, 366.0399. HPLC purity: 97.1%. 38b: white solid, 16.7% yield. 1 HNMR(600MHz,MeOD)δ8.43(s,1H),5.33(t,J=5.4Hz,1H),4.23-4.19(m,2H),4.01(d,J=11.4Hz,1H),3 .67(d,J=11.4Hz,1H),3.43(q,J=6.0Hz,1H),2.94(dd,J=4.2Hz,J=10.8Hz,1H),1.16(t,J=6.6Hz,3H). 13 C NMR(100MHz,DMSO-d6)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 ClN5O3SNa(M+Na) + ,366.0398; found,366.0398.HPLC purity:98.8%.

[0109] (±)-3-(2-Fluoro-6-chloro-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (39a,39b)

[0110] 39a:brown solid,21.5%yield. 1 H NMR (600MHz, CDCl3) δ8.61 (s, 1H), 5.40 (t, J = 6.6Hz, 1H), 4.28-4.21 (m, 2H), 3.72 (dd, J = 12.0Hz, J = 29. 4Hz, 2H), 3.59 (s, 1H), 3.32 (q, J = 6.0Hz, 1H), 2.82 (dd, J = 11.4Hz, J = 6.6Hz, 1H), 1.18 (t, J = 7.2Hz, 3H). 13 C NMR (100MHz, CDCl3) δ 167.6, 145.2, 76.6, 73.8, 63.6, 5 3.6, 34.7, 33.7 (dd, J = 100.6), 14.0. 19 F NMR(376MHz,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 (400MHz, CDCl3) δ8.51(s,1H),5.30(s,2H),4.55(s,1H),4.29-4.21(m,2H),3.97(d,J=12.8Hz,1H),3.56( d, J=12.4Hz, 1H), 3.12 (dd, J=3.2Hz, J=11.6Hz, 1H), 2.78 (dd, J=4.4Hz, J=11.6Hz, 1H), 1.14 (t, J=7.2Hz, 3H). 13 C NMR (100MHz, 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(376MHz,CDCl3)δ-48.89.ESI-HRMS(m / z)calcd C 12 H 12 ClFN4O3SNa(M+Na) + ,369.0195; found,366.0192.HPLCpurity:99.0%.

[0111] (±)-3-(2-chloro-6-chloro-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (40a, 40b)

[0112] 40a:white solid,22.6%yield. 1 H NMR (600 MHz, CDCl3) δ 1 H NMR (600MHz, CDCl3) δ8.61 (s, 1H), 5.40 (q, J = 6Hz, 1H), 4.25 (q, J = 7.2Hz, 2H), 3.71 (dd, J = 12.0Hz, J = 31.2Hz ,1H),3.58(d,J=4.8Hz,1H),3.32(q,J=6.0Hz,1H),2.81(q,J=6.6Hz,J=11.4Hz,1H),1.19(t,J=7.2Hz,3H). 13C NMR(100MHz, 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)calcd C 12 H 13 N4BrO3SNa(M+Na) + , 371.9892; found, 351.0290. HPLC purity: 99.0%. 40b: white solid, 35.6% yield. 1 H NMR (600MHz, CDCl3) δ8.51 (s, 1H), 5.30 (q, J = 3.0Hz, 1H), 4.65 (q, J = 1.8Hz, 1H), 4.32-4.19 (m, 2H), 3.9 6(d,J=12.6Hz,1H), 3.55(d,J=12.6Hz,1H), 3.13(dd,J=3.0,J=12.0Hz,1H), 2.78(dd,J=4.2Hz,J=12.0Hz,1H), 1.15(t,J=7.2Hz,3H). 13 C NMR (100MHz, 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.HPLCpurity:96.0%.

[0113] (±)-3-(2-Chloro-9-hydrogen-purin-9-yl)-4-hydroxy-tetrahydrothiophene-3-carboxylic acid ethyl ester (41a, 41b)

[0114] 41a:white solid,54.6%yield. 1 H NMR (600MHz, CDCl3) δ8.96(s,1H),8.56(s,1H),5.44(t,J=6.0Hz,1H),4.29-4.21(m,2H),3.71(dd,J=1 2.0Hz, J=43.8Hz, 2H), 3.33 (q, J=6.0Hz, 1H), 2.83 (dd, J=6.6Hz, J=11.4Hz, 1H), 1.18 (t, J=7.2Hz, 3H). 13C NMR(150MHz, 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.HPLCpurity:99.5%.41b:white solid,27.1%yield. 1 HNMR (600MHz, CDCl3) δ8.99 (s, 1H), 8.48 (s, 1H), 5.34 (t, J = 4.4Hz, 1H), 4.89 (s, 1H), 4.31-4.14 (m, 2H), 3.96 (d, J = 12.4Hz, 1H), 3.56 (d, J = 12.4Hz, 1H), 3.14 (dd, J = 3.6Hz, J = 11.6Hz, 1H), 2.81 (dd, J = 4.8Hz, J = 11.6Hz, Hz, 1H), 1.12 (t, J = 7.2Hz, 3H). 13 C NMR(100MHz, 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)calcdC 12 H 13 N4ClO3SNa(M+Na) + ,351.0289; found,351.0290.HPLC purity:99.7%.

[0115] (±)-3-(2-chloro-6-morpholin-9-hydrogen-purin-9-yl)-4-tetrahydrothiophene-3-carboxylic acid ethyl ester (42a, 42b)

[0116] 42a:white solid,18.4%yield. 11H NMR (400 MHz, CDCl3) δ 8.06 (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 13C NMR (100 MHz, CDCl3) δ 168.3, 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 for C 16 11 20 11ClN5O4SNa (M + Na) + , 436.0817; found, 436.0824. HPLC purity: 97.6%. 42b: white solid, 53.2% yield. 1 1H NMR (400 MHz, CDCl3) δ 7.98 (s, 1H), 5.49 (s, 1H), 5.26 (t, J = 4.4 Hz, 1H), 4.32 - 4.17 (m, 6H), 3.88 (d, J = 12.4 Hz, 1H), 3.83 (t, J = 4.8 Hz, 4H), 3.49 (d, J = 12.4 Hz, 1H), 3.17 (dd, J = 4.8 Hz, J = 11.6 Hz, 1H), 2.85 (dd, J = 4.4 Hz, J = 11.6 Hz, 1H), 1.1A8 (t, J = 7.2 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 169.5, 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 for C 16 11 20 11ClN5O4SNa (M + Na) + , 436.0817; found, 436.0817. HPLC purity: 96.9%.[[ID=,19]]

[0117] Example 2:

[0118] Chiral HPLC separation was used for 3S, 4S-, 3R, 4R-, 3S, 4R- and 3R, 4S-. HPLC columns (Daicel Chiral Technology (China) Co., Ltd.) were used for chiral HPLC analysis using a Thermo Scientific Dionex Ultimate 3000 and an Agilent Technologies 1260 Infinity device. The chiral HPLC analysis was performed using a Thermo Scientific UltiMate 3000 high performance liquid chromatography with a UV / visible detector and an Agilent C 18 The target compounds were analyzed using a 5 μm column (4.6 mm×150 mm).

[0119] Example 3:

[0120] The present invention uses the above method to synthesize a series of racemic, 3S, 4S-configuration, 3R, 4R-configuration, 3S, 4R-configuration and 3R, 4S-pentacyclic sulfur nucleoside compounds, and the cell activity of these compounds against tumor cells is tested using the CCK-8 method. MCF-7, A375 and HeLa cells are cultured in DMEM medium, HCT116 cells are cultured in McCoy's 5A medium, DU145 and CT26 cells are cultured in RPMI 1640 medium, and RPMI 1640 medium is supplemented with 10% FBS and 1% penicillin-streptomycin solution (10000U / mL penicillin and 10000μg / mL streptomycin) at 37°C and 5% CO2. The cells are seeded in 96-well plates (4×10 3 Cells / well) were cultured overnight and then treated with different concentrations (1-50 μM) of compounds. Cell viability was determined 48 h later using the CCK-8 assay. The absorbance of each well was measured at a wavelength of 450 nm using a microplate reader (Multiskan FC, Thermo). The experiment was performed in triplicate. The compound concentration that inhibited cell growth by 50% (IC) was calculated based on the dose-response curve of each cell line. 50 ). Data are the mean ± SD values ​​of three independent experiments.

[0121] The anti-tumor activity results of the five-membered sulfur heterocyclic nucleoside compounds are as follows (Tables 1-4):

[0122]

[0123] Table 1 Inhibitory effects of compounds 1a-8a and 1b-8b on cancer cell proliferation

[0124]

[0125]

[0126]

[0127] Table 2 Inhibitory effects of compounds 9a-20a and 9b-20b on cancer cell proliferation

[0128]

[0129]

[0130]

[0131]

[0132] Table 3 Inhibitory effects of compounds 21a-31a and 21b-31b on cancer cell proliferation

[0133]

[0134]

[0135]

[0136]

[0137] Table 4 Inhibitory effects of compounds 32a-37a and 32b-37b on cancer cell proliferation

[0138]

[0139]

[0140] Compounds 33a and 36b, two with excellent antitumor activity, were selected. Four chiral configurations of 33a and 36b, including 3S,4S, 3R,4R, 3S,4R, and 3R,4S, were synthesized and their antitumor activity was investigated using CCK-8. The results showed that the chiral 3S,4S, 3R,4R, 3S,4R, and 3R,4S five-membered sulfur heterocyclic nucleosides exhibited comparable antitumor activity to the racemates 33a and 36b (Table 5).

[0141] Table 5 Inhibitory effects of compounds 33a / 33aa / 33ab and 36b / 36ba / 36bb on cancer cell proliferation

[0142]

[0143] Example 4

[0144] Cell cycle assay: Cells were seeded in 6-well plates (5×105 cells / well). The next day, compounds 33a or 36b at the specified concentrations were added to the wells and incubated at 37°C, 5% CO2 for 48 hours. Cells were harvested and frozen overnight in 70% alcohol. Cells were then stained with propidium iodide (PI) dye in the dark for 15 minutes, and cellular DNA content was measured by flow cytometry (BD Bioscience). The experimental results showed that compounds 33a and 36b could induce a slight cell cycle arrest at the G2 / M phase ( Figure 1 、 2 Therefore, compounds 33a and 36b have a certain inhibitory effect on cell division and can arrest cancer cells in the G2 / M phase.

[0145] Apoptosis test: HCT116 cells were seeded in 6-well plates (8×10 5 cells / well). The next day, compound 33a or 36b at the specified concentration was added to the wells and incubated at 37°C, 5% CO2 for 48 hours. The cells were harvested and stained with Annexin V-FITC and PI for 15 minutes at room temperature. Apoptotic cells were analyzed by flow cytometry (BD Bioscience). The results showed that the total number of cells induced to apoptosis increased with increasing concentrations of 33a or 36b, indicating that compound 33a or 36b can induce apoptosis in a concentration-dependent manner ( Figure 3 ).

[0146] Western Blot: Whole proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blotted with primary antibodies against caspase-3, Bax, Bcl-2, β-actin, and β-tubulin. Protein bands were analyzed using ImageJ software, and protein expression levels were expressed as a percentage difference compared to the control group. Figure 4 As shown, the protein levels of Bax and cleaved caspase-3 were increased, and the expression of Bcl-2 was significantly decreased.

[0147] Mitochondrial membrane potential (MMP) assay: Mitochondrial damage was imaged using Mito-Tracker Green (Beyotime, Jiangsu, China) or cell-permeable JC-1 dye according to the manufacturer's instructions and observed using flow cytometry (BD Biosciences). Figure 5 ). This experiment found that after treating cells with different concentrations of 33a for 48 hours, red fluorescence was downregulated, indicating a decrease in mitochondrial membrane potential. In summary, compound 33a can reduce mitochondrial membrane potential and induce apoptosis in HeLa cells.

[0148] Cell colony formation assay: HCT116 cells were seeded in 6-well plates (1.5×10 3 Different concentrations of compound 33a or 36b were added to the wells and incubated for 48 hours. The culture medium was changed every 72 hours. After 14 days, the cells were fixed with methanol for 30 minutes, stained with 0.1% crystal violet solution at room temperature for 30 minutes, washed three times with PBS, and dried. Then, the cells were photographed. Figure 6 It can be seen that the number of cell colony formation is inhibited as the concentration of 33a or 36b increases, which indicates that compound 33a or 36b can inhibit the formation of cancer cell colonies in a dose-dependent manner and has good anti-tumor activity.

[0149] Cell invasion: Transwell chambers (24 wells) (Corning, USA) were coated with Matrigel (BD Biosciences, USA), with 1 × 10 cells per well. 5 Cells were seeded in the upper chamber in serum-free medium and treated with the specified dose of compound 33a or 36b. Complete medium was placed in the lower chamber. After 24 hours, cells that had penetrated the wells were stained with 0.1% crystal violet solution and observed using a Leica dil fluorescence microscope (Leica, Germany). Under normal circumstances, cells would migrate through the membrane formed by the Matrigel to the lower chamber, but after the interference of 33a or 36b, the number of HCT116 cells that penetrated the Matrigel was reduced ( Figure 7 This indicates that 33a or 36b inhibits the invasion of HCT116 cells in a concentration gradient manner.

[0150] All animal experiments and experimental procedures were approved by the Animal Ethics Committee of the School of Chemistry and Chemical Engineering, Henan Normal University, China. CT26 cells (ATCC, Rockville, MD, USA) were used for tumor allograft experiments. SPF-grade BALB / c mice (male, 22 ± 2 g, Kawen Laboratory Animal Co., Ltd., Changzhou, China) were randomly divided into four experimental groups (n = 6): a control group, a positive control 5-FU (55 mg / kg), and 33a (20 mg / kg and 55 mg / kg). Mice were subcutaneously injected with CT26 cells (1 × 107 per mouse). The diameter of the transplanted tumors in the mice was measured with a vernier caliper. After tumors grew to approximately 120 mm3, 33a (55 mg / kg or 20 mg / kg), 5-FU (55 mg / kg), or control solvent (0.2 mL) were injected intraperitoneally daily for 14 consecutive days. The antitumor activity of the compounds was evaluated by measuring tumor diameter. Tumor diameter and body weight were measured every 2 days. After 15 days, the tumor tissue was surgically removed, weighed and photographed. The formula for calculating tumor volume (TV) is V = 1 / 2 × a × b 2, where a is length and b is width. The samples were used for histological examination. The formula for calculating the tumor growth inhibition rate TGI (%) is TGI = (1-Tw / Cw) × 100%, where Tw and Cw are the average tumor weights of the treatment group and the model group on the 15th day, respectively. Compound 33a had a significant dose-dependent inhibitory effect on both tumor volume and tumor weight, with TGI values ​​of 75.79% and 54.74%, respectively. Compound 33a had no significant toxic effect on body weight, indicating the safety of 33a ( Figure 8 、 9 ).

[0151] Example 5

[0152] The synthesized five-membered sulfur heterocyclic nucleoside compounds were evaluated for their antitumor activity in vitro and in vivo, and the optimal compounds were selected to determine their overall structure-activity relationship. To assess their spectrum of antiproliferative activity, selected compounds (5a / 5b, 8a / 8b, 33a / 33b, and 36a / 36b) were further tested in a CCK-8 assay against MCF-7 (human breast cancer), A375 (human malignant melanoma), DU145 (human prostate cancer), and CT26 (mouse colon cancer) cell lines. The selected compounds exhibited moderate to potent antitumor activity against all four cell lines, demonstrating their broad-spectrum antitumor activity (Table 6).

[0153] Table 6 Inhibitory effects of compounds 5a / 5b, 8a / 8b, 33a / 33b and 36a / 36b on cancer cell proliferation

[0154]

[0155]

[0156] The above embodiments illustrate the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions merely illustrate the principles of the present invention. Various variations and improvements are possible without departing from the scope of the present invention. Any structural changes to the five-membered sulfur heterocyclic nucleoside are considered to fall within the scope of protection of the present invention.

Claims

1. Use of a five-membered sulfur heterocyclic nucleoside lead compound in the preparation of an anti-colon cancer drug, wherein the specific 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 an anti-colon cancer active drug, characterized in that: The anti-colon cancer activity is selected from the anti-HCT116 cell activity.

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