Sulfur heterocyclic nucleoside derivative and synthesis and colon cancer resisting activity application thereof

Abstract

The invention discloses a sulfur heterocyclic nucleoside derivative as well as synthesis and anti-colon cancer activity application thereof, and belongs to the technical field of medicinal chemistry. A series of sulfur heterocyclic nucleoside derivatives are synthesized through an asymmetric sulfanilamide-Michael / aldol cascade reaction and comprise a racemate, a 3S, 4S-configuration, a 3R, 4R-configuration, a 3S, 4R-configuration and a 3R, 4S-configuration, and the general formula structure is # imgabs0 # (* is a chiral center). Results show that in an in-vitro colon cancer cell line cell (HCT116) test, the racemate and enantiomer configuration have little difference in anti-tumor activity. Wherein the racemate 33a and the racemate 36b show relatively strong proliferation inhibition capacity in an in-vitro colon cancer cell line, IC50 reaches 0.27 [mu] M and 0.49 [mu] M and is obviously superior to that of contrast drugs 5-fluorouracil and cis-platinum (IC50 is 9.84 [mu] M and 5.29 [mu] M respectively).

Description

Technical Field

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

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

[0003] Compared with oxygen, sulfur atoms in compound structures have significant differences in chemical reactivity, protein molecular recognition, and metabolic stability. Therefore, modifying the nucleoside structure to contain sulfur heterocycles may produce effective anti-tumor drugs. Summary of the invention

[0004] In order to provide nucleoside lead compounds with more functional structures, the present invention discloses a five-membered sulfur heterocyclic nucleoside lead compound with anti-colon cancer activity. In terms of synthesis method: a series of sulfur heterocyclic nucleoside derivatives, including racemate, 3S, 4S-configuration, 3R, 4R-configuration, 3S, 4R-configuration and 3R, 4S-configuration, were synthesized by asymmetric sulfonamide-Michael / aldol cascade reaction. On this basis, 84 new compounds were designed and synthesized, and their antitumor activities in vitro and in vivo were evaluated.

[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, p-substituted piperidine, cycloheximide, pyrrole, propylthio; R is selected from C1-C4 alkyl.

[0006] More preferably, R 1 is hydrogen, fluorine, 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 2It is p-methylpiperidine, p-trifluoromethylpiperidine, p-fluoromethylpiperidine, p-chloropiperidine, p-morpholinepiperidine, p-difluorobenzylpiperidine, methyl sulfide, ethyl sulfide, propylthio, propynyl sulfide, butyl sulfide, pentyl sulfide, 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 the same antitumor activity as the racemate.

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

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

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

[0013] The present invention also provides an anti-colon cancer active drug, the active component 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] Further, 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 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 a temperature of 100-110°C to obtain intermediate 1B-42B;

[0019] Step 2: The 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 separated by chiral HPLC to obtain 3S, 4S-, 3R, 4R-, 3S, 4R- and 3R, 4S-configuration products. The chiral HPLC analysis was performed using Thermo Scientific Dionex Ultimate 3000 and Agilent Technologies 1260 Infinity.

[0021] In order to study 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 by asymmetric sulfonamide-Michael / aldol cascade reactions.

[0022] The experimental results showed that among the racemates, compounds 33a and 36b showed strong inhibitory ability against colon cancer cell line (HCT116) in vitro, with IC 50 The results showed that the concentrations of 5-fluorouracil and cisplatin in the control group were 0.27μM and 0.49μM, which were significantly better than those of the control drugs 5-fluorouracil and cisplatin (IC 50 The chiral structures of the five-membered sulfur heterocyclic nucleoside compounds with 3S, 4S-, 3R, 4R-, 3S, 4R- and 3R, 4S-configurations were comparable to the antitumor activities of the racemates 33a and 36b. The above results provide the basic structural units of the five-membered sulfur heterocyclic nucleoside lead compounds for the design of drugs with anti-colon cancer activity, on which further structural subtle modifications and transformations can be made.

[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 the effect of compound 33a in Example 4 on the cell cycle of HCT116;

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

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

[0027] Figure 4 This is a diagram of apoptotic protein analysis;

[0028] Figure 5 This is the diagram for determination of mitochondrial membrane potential (MMP);

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

[0030] Figure 7 The impact diagram for cell invasion;

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

[0032] Fig. 9 HE staining picture. DETAILED DESCRIPTION

[0033] Embodiment 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 the reaction tube. After 12 hours, TLC monitoring confirmed completion. The system was spin-dried and the mixture was purified by flash chromatography using a PE / EA solvent system (PE / EA=2 / 1) to isolate the product.

[0035] The characterization data of representative compounds are as follows:

[0036] (±)-Ethyl-4-hydroxy-3-(9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(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,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%.

[0038] (±)-Ethyl-3-(6-chloro-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(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] (±)-Ethyl-3-(6-bromo-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(3a,3b)

[0041] 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 13N4BrO3SNa(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%.

[0042] (±)-Ethyl-3-(6-(dimethylamino)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(4a,4b)

[0043] 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). 13C 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). 13 C 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%.

[0044] (±)-Ethyl-4-hydroxy-3-(6-(piperidin-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(5a,5b)

[0045] 5a:white solid,41.7%yield. 1H 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. 1 H 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%.

[0046] (±)-Ethyl-3-(6-ethoxy-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(6a,6b)

[0047] 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=7.2Hz,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%.

[0048] (±)-Ethyl-4-hydroxy-3-(6-methoxy-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(7a,7b)

[0049] 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). 13 C 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). 13C 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] (±)-Ethyl-4-hydroxy-3-(6-(propylthio)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(8a,8b)

[0051] 8a:yellow solid,31.2%yield. 1 H 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%. 1H 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.HPLCpurity:96.4%.

[0052] (±)-Ethyl-4-hydroxy-3-(6-morpholino-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(9a,9b)

[0053] 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 16H 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). 13 C 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%.

[0054] (±)-Ethyl-4-hydroxy-3-(6-thiomorpholino-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(10a,10b)

[0055] 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. 1 H 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%.

[0056] (±)-Ethyl-4-hydroxy-3-(6-(4-methylpiperidin-1-yl)-9H-purin-9yl)tetrahydrothiophene-3-carboxylate(11a,11b)

[0057] 11a:white solid,32.7%yield. 1H 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). 13 C 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%.

[0058] (±)-Ethyl-3-(6-(4-fluoropiperidin-1-yl)-9H-purin-9-yl)-4-hydroxytetrahydrothio phene-3-carboxylate(12a,12b)

[0059] Compounds 12a,12b were prepared as described for the preparationof1a,1b exceptethyl 2-(6-(4-fluoropiperidin-1-yl)-9H-purin-9-yl)acrylate wasused in place of ethyl2-(9H-purin-9-yl)acrylate.12a:white solid,42.3%yield. 1 H 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=4.8 Hz,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)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(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=12 Hz,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 =20.2 Hz),14.0. 19 F NMR(376MHz,CDCl3)δ-181.68.ESI-HRMS(m / z)calcdC 17 H 22 FN5O3SNa(M+Na) + ,396.1500;found,396.1508.HPLCpurity:97.2%.

[0060] (±)-Ethyl-4-hydroxy-3-(6-(4-(trifluoromethyl)piperidin-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(13a,13b)

[0061] 13a:yellow solid,42.1%yield. 1H 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. 1 H 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,JC-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] (±)-Ethyl-3-(6-(4-chloropiperidin-1-yl)-9H-purin-9-yl)-4-hydroxytetrahydrothioph ene-3-carboxylate(14a,14b)

[0063] White solid;14a:light yellow solid,51.7%yield. 1 H 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)calcdC 17 H 23 ClN5O3S(M+H) + ,412.1205;found,412.1198.HPLCpurity:96.1%.14b:light yellowsolid,31.0%yield. 1H 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=12.0Hz,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.HPLCpurity:95.0%.

[0064] (±)-Ethyl-4-hydroxy-3-(6-(4-morpholinopiperidin-1-yl)-9H-purin-9-yl)tetrahydro thiophene-3-carboxylate(15a,15b)

[0065] 15a:white solid,37.7%yield. 1 H NMR(600 MHz,CDCl3)δ8.22(s,1H),8.02(s,1H),5.50(s,1H),5.40(t,J=9.6 Hz,1H),4.22(q,J=10.8 Hz,2H),3.76(s,1H),3.72(t,J=6.6 Hz,4H),3.44(d,J=18 Hz,1H),3.22(dd,J=9 Hz,J=16.2 Hz,1H),3.13(s,2H),2.80(dd,J=11.4 Hz,J=16.2 Hz,1H),2.59-2.52(m,5H),2.01(d,J=19.2 Hz,2H),1.62-1.52(m,4H),1.19(t,J=10.8 Hz,3H). 13C NMR(150 MHz,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)calcdC 21 H 31 N6O4S(M+H) + ,463.2111;found,463.2113.HPLC purity:97.9%.15b:white solid,38.6%yield. 1 H 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%.

[0066] (±)-Ethyl-3-(6-(4-benzylpiperidin-1-yl)-9H-purin-9-yl)-4-hydroxytetrahydro thiophene-3-carboxylate(16a,16b)

[0067] 16a:white solid,32.4%yield. 1H NMR(600 MHz,CDCl3)δ8.18(s,1H),7.97(s,1H),7.28-7.11(m,5H),5.37(t,J=10.0 Hz.3H),4.22-4.16(m,2H),3.71(d,J=17.4Hz,1H),3.41(d,J=17.4 Hz,1H)3.21(dd,J=9.6 Hz,J=16.8 Hz,1H),2.99(s,2H),2.78(dd,J=10.8 Hz,J=16.2 Hz,1H)2.55(d,J=10.8 Hz,2H),1.90-1.78(m,4H),1.35-1.22(m,1H),1.16(t,J=10.8 Hz,3H). 13 C NMR(150 MHz,CDCl3)δ168.6,154.0,151.9,150.7,140.2,137.6,129.3,128.4,126.1,119.7,73.8,63.0,43.3,38.5,35.3,33.3,32.4,14.0.ESI-HRMS(m / z)calcd C 24 H 29 N5O3SNa(M+Na) + ,490.1883;found,490.1886.HPLCpurity:97.9%.16b:white solid,31.8%yield. 1 HNMR(600 MHz,CDCl3)δ8.21(s,1H),7.87(s,1H),7.28-7.11(m,5H),5.43(s,2H),5.23(t,J=8.4 Hz,1H),4.23-4.14(m,2H),3.84(d,J=18 Hz,1H),3.50(d,J=18Hz,1H),3.21(dd,J=9 Hz,J=16.8 Hz,1H),3.00(s,2H),2.91(dd,J=7.8 Hz,J=16.8 Hz,1H),2.55(d,J=10.8 Hz,2H),1.90-1.78(m,4H),1.36-1.21(m,1H),1.13(t,J=10.8 Hz,3H). 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.HPLCpurity:99.7%.

[0068] (±)-Ethyl-3-(6-(4-(2-fluorobenzyl)piperidin-1-yl)-9H-purin-9-yl)-4-hydroxytetra hydrothiophene-3-carboxylate(17a,17b)

[0069] 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 FN5 O3S(M+H) + ,486.1970;found,486.1980.HPLCpurity:96.4%.17b:white solid,37.8%yield. 1 H NMR(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=12 Hz,1H),3.24(dd,J=6.4 Hz,J=11.6 Hz,1H),3.03(s,2H),2.94(dd,J=11.2Hz,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 FNMR(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%.

[0070] (±)-Ethyl-4-hydroxy-3-(6-(4-methylpiperazin-1-yl)-9H-purin-9-yl)tetrahydrothio phene-3-carboxylate(18a,18b)

[0071] 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%.

[0072] (±)-Ethyl-3-(6-(azepan-1-yl)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(19a,19b)

[0073] 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%.

[0074] (±)-Ethyl-4-hydroxy-3-(6-(pyrrolidin-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(20a,20b)

[0075] 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,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)calcd C 16 H 22 N5O3S(M+H) + ,364.1438;found,364.1437.HPLC purity:98.4%.20b:whitesolid,58.0%yield. 1 H NMR(600 MHz,CDCl3)δ8.22(s,1H),7.96(s,1H),6.43(s,1H),5.41(t,J=6.4 Hz,1H),4.30-4.15(m,4H),3.72(d,J=9.2 Hz,3H),3.47(d,J=11.6 Hz,1H),3.24(dd,J=6 Hz,J=10.8 Hz,1H),2.84(dd,J=6.8 Hz,J=10.8 Hz,1H),2.03(t,J=25.6 Hz,4H),1.18(t,J=6.8Hz,3H). 13 C NMR(100 MHz,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] (±)-Ethyl-4-hydroxy-3-(6-(methylthio)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(21a,21b)

[0077] 21a:yellowish solid,33.4%yield. 1H NMR(400 MHz,CDCl3)δ8.63(s,1H),8.22(s,1H),5.74(s,1H),5.30(t,J=5.2 Hz,1H),4.18-4.12(m,2H),3.91(d,J=12.4 Hz,1H),3.56(d,J=12.0 Hz,1H),3.15(dd,J=5.2 Hz,=11.6 Hz,1H),2.86(dd,J=4.8Hz,J=11.6 Hz,1H),2.68(s,3H),1.07(t,J=7.2 Hz,3H). 13 C NMR(150 MHz,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)calcd 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,CDCl3)δ8.61(s,1H),8.28(s,1H),5.45(t,J=6.0 Hz,1H),5.04(s,1H),4.23-4.14(m,2H),3.69(dd,J=11.6 Hz,J=36.8 Hz,2H),3.30(q,J=6.0 Hz,1H),2.87(q,J=6.0 Hz,1H),2.67(s,3H),1.12(t,J=7.2 Hz,3H). 13 C NMR(100 MHz,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.HPLC purity:99.6%.(±)-Ethyl-3-(6-(ethylthio)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(22a,22b)

[0078] 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.HPLC purity: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=7.2Hz,3H). 13 C 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%.

[0079] (±)-Ethyl-4-hydroxy-3-(6-(isopropylthio)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(23a,23b)

[0080] 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. 1 H 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). 13C 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)calcdC 15 H 20 N4O3S2Na(M+Na) + ,391.0869;found,391.0868.HPLC purity:95.1%.

[0081] (±)-Ethyl-4-hydroxy-3-(6-(prop-2-yn-1-ylthio)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(24a,24b)

[0082] 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). 13 C 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. 1H 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%.

[0083] (±)-Ethyl-3-(6-(butylthio)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(25a,25b)

[0084] 25a:white solid,45.1%yield. 1 H NMR(400 MHz,DMSO-d6)δ8.72(s,1H),8.68(s,1H),5.93(d,J=5.2 Hz,1H),5.23-5.20(m,1H),4.17-4.07(m,2H),4.00(d,J=11.6Hz,1H),3.62(d,J=11.2 Hz,1H),3.41-3.30(m,3H),2.90(dd,J=3.6 Hz,J=11.2Hz,1H),1.73-1.66(m,2H),1.49-1.39(m,2H),1.04(t,J=7.2 Hz,3H),0.91(t,J=7.2 Hz,3H). 13CNMR(100 MHz,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)calcdC 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)δ8.62(s,1H),8.21(s,1H),5.74(s,1H),5.33-5.30(m,1H),4.22-4.15(m,2H),3.91(d,J=12 Hz,1H),3.56(d,J=12.4 Hz,1H),3.40-3.35(m,1H),3.18(q,J=4.8 Hz,1H),2.87(q=3.2,1H),1.80-1.72(m,2H),1.54-1.45(m,2H),1.10(t,J=7.2 Hz,3H),0.94(t,J=7.6 Hz,3H). 13 CNMR(100 MHz,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%.

[0085] (±)-Ethyl-4-hydroxy-3-(6-(pentylthio)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(26a,26b)

[0086] 26a:white solid,43.3%yield. 1H 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. 1 H 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 C17 H 25 N4O3S2(M+H) + ,397.1363;found,397.1361.HPLC purity:95.3%.

[0087] (±)-Ethy-3-(6-(benzylthio)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(27a,27b)

[0088] 27a:white solid,43.2%yield. 1 H NMR(400 MHz,DMSO-d6)δ8.73(s,1H),8.68(s,1H),7.44(d,J=7.2 Hz,1H),7.31-7.20(m,3H),6.40(d,J=5.6 Hz,1H),5.43(dd,J=5.6 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 Hz2H),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=4Hz,J=12 Hz,1H),1.00(t,J=7.2 Hz,3H). 3 C NMR(100 MHz,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:white solid,42.4%yield. 1HNMR(400 MHz,DMSO-d6)δ8.75(s,1H),8.74(s,1H),7.47(d,J=7.2 Hz,2H),7.34-7.22(m,3H),5.94(d,J=5.2 Hz,1H),5.24-5.21(m,1H),4.66(dd,J=13.6 Hz,J=18.4 Hz,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=6.8 Hz,3H). 13 C NMR(100 MHz,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 20 N4O3S2(M+Na) + ,439.0869;found,439.0863.HPLC purity:98.3%.

[0089] (±)-Ethyl-3-(6-((3-fluorophenyl)thio)-9H-purin-9-yl)-4-Hydroxytetrahydrothiophene-3-carboxylate(28a,28b)

[0090] 28a:white solid,26.4%yield. 1 H 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). 13CNMR(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-111.38.ESI-HRMS(m / z)calcdC 18 H 17 FN4O3S2(M+Na) + ,443.0618;found,443.0613.HPLC purity:99.0%.28b:white solid,31.3%yield. 1 H 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=7.2 Hz,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%.

[0091] (±)-Ethyl-3-(6-((4-(tert-butyl)phenyl)thio)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(29a,29b)

[0092] 29a:white solid,33.4%yield. 1 H NMR(400 MHz,DMSO-d6)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(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(400 MHz,DMSO-d6)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=5.2 Hz,J=12 Hz,1H),2.99(dd,J=3.6 Hz,J=12.0 Hz,1H),1.32(s,10H),1.03(t,J=6.8 Hz,3H). 13CNMR(100 MHz,DMSO-d6)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%.

[0093] (±)-Ethyl-3-(6-((3-fluoropropyl)thio)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(30a,30b)

[0094] 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. 1 H 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%.

[0095] (±)-Ethyl-3-(6-((3-chloropropyl)thio)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(31a,31b)

[0096] 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%.(±)-Ethyl-3-(2-amino-6-(piperidin-1-yl)-9H-purin-9-yl)-4-hydroxytetrahydrothio phene-3-carboxylate(32a,32b)

[0097] 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). 13C 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 H 25 N6O3S(M+H) + ,393.1703;found,393.1701.HPLCpurity:98.2%.

[0098] (±)-Ethyl-3-(2-fluoro-6-(piperidin-1-yl)-9H-purin-9-yl)-4-hydroxytetrahydrothio phene-3-carboxylate(33a,33b)

[0099] 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),3.83(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%.

[0100] (±)-Ethyl-3-(2-chloro-6-(piperidin-1-yl)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(34a,34b)

[0101] 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=12 Hz,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%.

[0102] (±)-Ethyl-3-(2-amino-6-(propylthio)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(35a,35b)

[0103] 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%.(±)-Ethyl-3-(2-fluoro-6-(propylthio)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(36a,36b)

[0104] 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%.

[0105] (±)-Ethyl-3-(2-chloro-6-(propylthio)-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(37a,37b

[0106] 37a:white solid,22.4%yield. 1H NMR(400 MHz,DMSO-d6)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(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.HPLCpurity:99.8%.37b:white solid,51.6%yield. 1 HNMR(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=12.4 Hz,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=7.2 Hz,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%.

[0107] (±)-Ethyl-3-(2-amino-6-chloro-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(38a,38b)

[0108] 38a:white solid,71.0%yield. 1 H NMR(600 MHz,MeOD)δ8.31(s,1H),5.35(t,J=5.4 Hz,1H),4.23-4.15(m,2H),4.02(d,J=12.6 Hz,1H),3.73(d,J=12.6 Hz,1H),3.33(s,1H),3.21(q,J=5.4 Hz,1H),3.10(dd,J=4.8 Hz,J=11.4 Hz,1H),1.13(t,J=6.6Hz,3H). 13 C NMR(150 MHz,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(600 MHz,MeOD)δ8.43(s,1H),5.33(t,J=5.4 Hz,1H),4.23-4.19(m,2H),4.01(d,J=11.4 Hz,1H),3.67(d,J=11.4 Hz,1H),3.43(q,J=6.0 Hz,1H),2.94(dd,J=4.2 Hz,J=10.8 Hz,1H),1.16(t,J=6.6 Hz,3H). 13 C NMR(100 MHz,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] (±)-Ethy-3-(6-chloro-2-fluoro-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(39a,39b)

[0110] 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%.

[0111] (±)-Ethyl-3-(2,6-dichloro-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(40a,40b)

[0112] 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%.

[0113] (±)-Ethyl-3-(2-chloro-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(41a,41b)

[0114] 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=6.0Hz,1H),2.83(dd,J=6.6Hz,J=11.4Hz,1H),1.18(t,J=7.2Hz,3H). 13 C 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] (±)-Ethy-3-(2-chloro-6-morpholino-9H-purin-9-yl)-4-hydroxytetrahydrothiophene-3-carboxylate(42a,42b)

[0116] 42a:white solid,18.4%yield. 1 H NMR(400MHz,CDCl3)δ8.06(s,1H),5.42(q,J=6Hz,1H),4.33(d J=5.2Hz,3H),4.34-4.19(m,6H),3.82(t,J=5.2Hz,4H),3.70(d,J=12.0Hz,1H),3.46(d,J=11.6Hz,1H)3.26(dd,J=6.0Hz,J=10.8Hz,1H),2.81(dd,J=6.8Hz,J=11.2Hz,1H),1.23(t,J=6.8Hz,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)calcdC 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%.

[0117] Embodiment 2:

[0118] For 3S, 4S-configuration, 3R, 4R-configuration, 3S, 4R-configuration and 3R, 4S-, chiral HPLC separation was adopted. HPLC column (Daicel Chiral Technology (China) Co., Ltd.), chiral HPLC analysis was performed using Thermo Scientific Dionex Ultimate3000 and Agilent Technologies 1260Infinity device. Thermo Scientific UltiMate 3000 high performance liquid chromatography was used with UV / visible detector and Agilent C 18 The target compounds were analyzed using a 5 μm column (4.6 mm×150 mm).

[0119] Embodiment 3:

[0120] The present invention adopts 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 on tumor cells is tested by 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, RPMI 1640 medium is supplemented with 10% FBS, 1% penicillin-streptomycin solution (10000U / mL penicillin and 10000μg / mL streptomycin), and cultured at 37°C and 5% CO2. The cells are seeded in 96-well plates (4×10 3 cells / well) overnight and then treated with different concentrations (1-50 μM) of compounds. Cell viability was determined 48 h later using the CCK-8 method. 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 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:

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135] Two compounds with good antitumor activity, 33a and 36b, were selected; four configurations of 33a and 36b, 3S, 4S-, 3R, 4R-, 3S, 4R- and 3R, 4S-, were synthesized, and their antitumor activities were studied by CCK-8. The experimental results showed that in the chiral structure, the antitumor activities of 3S, 4S-, 3R, 4R-, 3S, 4R- and 3R, 4S-pentacyclic sulfur nucleoside compounds were equivalent to the racemates 33a and 36b.

[0136]

[0137] Example 4

[0138] Cell cycle test: Cells were seeded in 6-well plates (5×105Cells / well). The next day, the specified concentration of compound 33a or 36b was added to the wells and placed at 37°C, 5% CO2 for 48 hours. The cells were collected and frozen overnight in 70% alcohol. Then, the cells were stained with propidium iodide (PI) dye in the dark for 15 minutes, and the cell DNA content was detected by flow cytometry (BD Bioscience). The experimental results showed that compounds 33a and 36b could induce a slight arrest of the cell cycle to the G2 / M phase ( Figure 1 , 2 ). Therefore, compounds 33a and 36b have a certain inhibitory effect on cell division and can block cancer cells in the G2 / M phase.

[0139] Cell apoptosis test: HCT116 cells were seeded in 6-well plates (8×10 5 cells / well). On the second day, the specified concentration of compound 33a or 36b was added to the wells and placed at 37°C, 5% CO2 for 48 hours. The cells were collected and stained with Annexin V-FITC and PI for 15 minutes at room temperature. The apoptotic cells were analyzed by flow cytometry (BD Bioscience). The results showed that as the concentration of 33a or 36b increased, the total number of cells induced to apoptosis also increased, indicating that compound 33a or 36b can induce cell apoptosis in a concentration-dependent manner ( Figure 3 ).

[0140] Western Blot experiment: The whole protein was separated by polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membrane was blotted with primary antibodies for caspase-3, Bax, Bcl-2, β-actin, and β-tubulin. Protein band analysis was performed using ImageJ software, and the protein expression level was expressed as a percentage difference compared with 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.

[0141] Mitochondrial membrane potential (MMP) assay: Mitochondrial damage was imaged using Mito-Tracker Green (Beyo time, Jiangsu, China) or cell-permeable JC-1 dye as described and observed using flow cytometry (BD Biosciences). Figure 5 ), this experiment found that after treating cells with different concentrations of 33a for 48h, the red fluorescence was downregulated, that is, the mitochondrial membrane potential decreased. In summary, compound 33a can reduce mitochondrial membrane potential and induce apoptosis of HeLa cells.

[0142] Cell colony formation assay: HCT116 cells were seeded in 6-well plates (1.5×10 3 cells / well). Compound 33a or 36b of different concentrations was added to the wells and incubated for 48 h. The culture medium was replaced every 72 h. After 14 days, the cells were fixed with methanol for 30 min, stained with 0.1% crystal violet staining solution at room temperature for 30 min, washed with PBS three times, 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.

[0143] Cell invasion: Transwell chambers (24-well) (Corning, USA) were coated with Matrigel (BD Biosciences, USA), and 1×10 5 Cells were seeded in the upper chamber in serum-free medium and treated with the specified dose of compound 33a or 36b. The complete medium was placed in the lower chamber. After 24 h, the cells that penetrated the wells were stained with 0.1% crystal violet solution and observed with a Leica dil fluorescence microscope (Leica, Germany). Normally, cells will pass through the membrane formed by the matrigel and transfer to the lower chamber, but after the interference of 33a or 36b, the number of HCT116 cells that pass through the matrigel is reduced ( Figure 7 ). This indicates that 33a or 36b inhibits the invasion of HCT116 cells in a concentration gradient manner.

[0144] Transplantation tumor experiment: 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±2g, Kawen Experimental Animal Co., Ltd., Changzhou, China) were selected and randomly divided into 4 experimental groups (n=6): control group, positive control 5-FU (55mg / kg) and 33a (20mg / kg and 55mg / kg). Mice were subcutaneously injected with CT26 cells (1×107 per mouse). The diameter of the transplanted tumors in mice was measured with a vernier caliper. After the tumors grew to about 120mm3, 33a (55mg / kg or 20mg / kg), 5-FU (55mg / kg) or control solvent (0.2mL) were injected intraperitoneally every day for 14 consecutive days. The antitumor activity of the compounds was evaluated by measuring the 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 calculation formula for 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 has a significant dose-dependent inhibitory effect on tumor volume and tumor weight, with TGI values ​​of 75.79% and 54.74%, respectively. Compound 33a has no obvious toxic effect on body weight, indicating the safety of 33a ( Figure 8 , 9 ).

[0145] Example 5

[0146] The synthetic five-membered sulfur heterocyclic nucleoside compounds were evaluated for antitumor activity in vitro and in vivo, and the optimal compounds were selected, with an overall structure-activity relationship. In order to evaluate their antiproliferative activity spectrum, CCK-8 assays were further performed on MCF-7 (human breast cancer), A375 (human malignant melanoma), DU145 (human prostate cancer) and CT26 (mouse colon cancer) cell lines using the selected compounds (5a / 5b, 8a / 8b, 33a / 33b and 36a / 36b). The selected compounds had moderate to potent antitumor activity against the four cell lines, indicating that they have a broad spectrum of antitumor activity (Table 5).

[0147] Table 5 Inhibitory effects of compounds 5a / 5b, 8a / 8b, 33a / 33b and 36a / 36b on cancer cell proliferation

[0148]

[0149]

[0150] The above embodiments describe 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 by the above embodiments, and the above embodiments and the description only illustrate the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and any structural changes of the five-membered sulfur heterocyclic nucleoside, these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A five-membered sulfur heterocyclic nucleoside lead compound, the general structure of which is as follows: * represents chiral center, a and b both include racemate, 3S,4S-configuration, 3R,4R-configuration, 3S,4R-configuration and 3R,4S-configuration; among which: R 1 , R 2 Each independently selected from R 1 , R 2 Each is independently selected from hydrogen, halogen, amino, alkylamino, C1-C4 alkoxy, p-substituted piperidine, cycloheximide, pyrrole, propylthio; R is selected from C1-C4 alkyl.

2. The five-membered sulfur heterocyclic nucleoside lead compound according to claim 1, characterized in that: R 1 is selected from hydrogen or halogen; R 2 is selected from hydrogen, chlorine, bromine, piperidine or propylthio; and R is ethyl.

3. The five-membered sulfur heterocyclic nucleoside lead compound according to claim 2, characterized in that: R 1 is fluorine, R 2 Selected from piperidine or propylthio.

4. The five-membered sulfur heterocyclic nucleoside lead compound according to claim 3, characterized in that: The configuration is selected from 3S,4S-configuration, 3R,4R-configuration, 3S,4R-configuration, and 3R,4S-configuration.

5. Use of the five-membered sulfur heterocyclic nucleoside lead compound according to any one of claims 1 to 4 in the preparation of an anti-colon cancer active drug.

6. The use of the five-membered sulfur heterocyclic nucleoside lead compound according to claim 5 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.

7. An anti-colon cancer active drug, characterized in that: The active ingredient thereof comprises the compound described in any one of claims 1 to 4.

8. The method for synthesizing the five-membered sulfur heterocyclic nucleoside lead compound according to claim 1, characterized in that: The steps include: Step 1: Compound 1-42A reacts with propionate in the presence of PPh3 and NaOAc in acetic acid and toluene solvent at a temperature of 100-110°C to obtain intermediate 1B-42B; Step 2: The 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; Step 3: The racemic forms of 1a-42a and 1b-42b were separated by HPLC to obtain 3S,4S-, 3R,4R-, 3S,4R- and 3R,4S-configuration products.

9. The method for synthesizing the five-membered sulfur heterocyclic nucleoside lead compound according to claim 8, characterized in that: Chiral HPLC analysis was performed using Thermo Scientific Dionex Ultimate 3000 and Agilent Technologies 1260 Infinity.

Images