Five-membered sulfur-containing heterocyclic ring nucleoside compound applied to anti-hand-foot-and-mouth virus active medicine

Abstract

The invention discloses a five-membered sulfur-containing heterocyclic ring nucleoside compound applied to anti-hand-foot-and-mouth virus active drugs, and belongs to the technical field of medicinal chemistry. A series of five-membered sulfur-containing heterocyclic ring nucleoside compounds are synthesized through a two-step Michael addition reaction: # imgabs0 # imgabs1 # (* is a chiral center). A result shows that in a hand and foot EV-A71 virus resisting test, the five-membered sulfur-containing heterocyclic ring nucleoside lead compound 22d has relatively high EV-A71 virus resisting activity, and a structural reference is provided for antiviral candidate drug molecules.

Description

Technical Field

[0001] The invention relates to a five-membered sulfur-containing heterocyclic nucleoside compound used in an anti-hand, foot and mouth virus active drug, belonging to the field of pharmaceutical chemistry. Background Art

[0002] Nucleoside drugs are currently commonly used in antiviral and anti-tumor fields due to their unique molecular structure. Antiviral nucleoside drugs include acyclovir, ribavirin, penciclovir, deciclovir, entecavir, sofosbuvir, zidovudine and lamivudine, and anti-tumor nucleoside drugs include cytarabine, doxifluridine, decitabine, etc.

[0003] Hand, foot and mouth disease is an enterovirus, an infectious disease caused by enterovirus, mostly occurring in children under 5 years old, and can cause herpes on the hands, feet, mouth and other parts of the body. In a few children, complications such as myocarditis, pulmonary edema, and aseptic meningoencephalitis may occur. EV71 enterovirus is the most common, and it can also be combined with other respiratory viruses, such as adenovirus, syncytial virus, etc. The disease is common worldwide, and there are cases in my country every year, and the case fatality rate is high to a certain extent.

[0004] Currently, five-membered sulfur heterocyclic nucleoside compounds are an important class of antiviral drugs on the market. In the past, five-membered sulfur heterocyclic nucleosides were mainly used to fight HIV and HBV, but there are relatively few studies on the activity of five-membered sulfur heterocyclic nucleoside compounds against hand, foot and mouth virus (EV-A71). Innovative research on five-membered sulfur heterocyclic nucleoside anti-hand, foot and mouth virus (EV-A71) drugs can not only break the international monopoly on nucleoside drugs, but also has important scientific significance and potential application prospects. Summary of the invention

[0005] In order to overcome the above technical defects, the present invention has developed more functional nucleoside compounds based on the previous research of the research group, and disclosed a five-membered sulfur heterocyclic nucleoside lead compound with anti-hand, foot and mouth virus (EV-A71) activity. The nucleoside compounds of the present invention synthesize a series of five-membered sulfur heterocyclic nucleoside compounds through reactions such as two-step Michael addition reaction, and this type of structure also includes three structural types: racemate, 3S, 4R-configuration and 3R, 4S-configuration.

[0006] The chiral five-membered sulfur heterocyclic nucleoside lead compound of the present invention has the following general structural formula:

[0007] Among them, * represents the chiral center, and each general structure includes three structures: racemate, 3S, 4R-configuration and 3R, 4S-configuration.

[0008] Furthermore, in the above technical solution, R 1 , R 2Each is independently selected from hydrogen, halogen, C1-C4 alkoxy, benzyl, amino, C1-C4 alkylamino, di-C1-C4 alkylamino; R 3 Selected from alkyl acetate.

[0009] Furthermore, in the above technical solution, R 1 is hydrogen, halogen, C1-C4 alkoxy, benzyl, amino, C1-C4 alkylamino, or di-C1-C4 alkylamino; R 2 is hydrogen, chlorine; R 3 It is ethyl acetate, methyl acetate, and isopropyl acetate.

[0010] Furthermore, in the above technical solution, R 1 is dimethylamino, R 2 is chlorine, R 3 It is ethyl acetate.

[0011] Furthermore, in the above technical solution, the compound is selected from 3S, 4R-configuration or 3R, 4S-configuration; for 3S, 4R-configuration and 3R, 4S-configuration, chiral HPLC is used to separate the racemic compound.

[0012] Further, under the most preferred conditions, the structure of the five-membered sulfur heterocyclic nucleoside compound is

[0013]

[0014] The present invention also provides the use of the five-membered sulfur heterocyclic nucleoside compound in the preparation of antiviral drugs.

[0015] Furthermore, in the above technical solution, the antiviral agent is anti-hand, foot and mouth virus (EV-A71).

[0016] The present invention also provides the use of the five-membered sulfur heterocyclic nucleoside compound in the preparation of a medicine for treating hand, foot and mouth disease.

[0017] The present invention also provides a pharmaceutical composition containing the five-membered sulfur heterocyclic nucleoside compound, wherein the active ingredient of the pharmaceutical composition comprises the chiral five-membered sulfur heterocyclic nucleoside compound and a pharmaceutically acceptable salt thereof.

[0018] Furthermore, in the above technical solution, the pharmaceutically acceptable salt includes a salt formed by an acyclic nucleoside compound and an organic acid or an inorganic acid. The organic acid is selected from one or more of malic acid, lactic acid, camphorsulfonic acid, citric acid, fumaric acid or oxalic acid, and the inorganic acid is selected from one or more of phosphoric acid, hydrohalic acid, sulfuric acid or nitric acid.

[0019] Furthermore, in the above technical solution, the antiviral activity is anti-hand, foot and mouth virus (EV-A71) activity.

[0020] In order to study the antiviral 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 two-step Michael addition reaction.

[0021] The experimental results showed that the chiral five-membered sulfur-containing heterocyclic nucleoside compound 22d had an in vitro IC of EV-A71 cells. 50 The above results provide a basic structural unit of chiral five-membered sulfur heterocyclic nucleoside compounds for the design of drugs with anti-hand, foot and mouth virus (EV-A71) activity, on which further structural subtle modifications and transformations can be made.

[0022] Through the synthesis of chiral five-membered sulfur heterocyclic nucleosides and the evaluation of their anti-tumor activity in vitro and in vivo, we obtained lead compounds or preferred drug molecules with good activity against hand, foot and mouth virus (EV-A71), provided reasonable synthesis strategies and methods for five-membered sulfur heterocyclic nucleoside drugs, and laid a good foundation for the development of antiviral five-membered sulfur heterocyclic nucleoside drugs with independent intellectual property rights. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the 22d anti-EV-A71 virus activity. DETAILED DESCRIPTION

[0024] Embodiment 1 Embodiment 1:

[0025]

[0026] (a)Ethyl(triphenylphosphoranylidene)acetate,DCM,40℃,4h.(b)Methylpropiolate(R 3 =CO2Me), Ethyl propiolate(R 3 =CO2Et),Propiolic acid isopropylester(R 3 =CO2i-Pr),PPh3,HOAC,NaOAc,Toluene,110℃,Refluex,N2.(c)19,NaH,DCM,RT,N2.

[0027] Synthesis of compounds 22s, 22t, 23s and 23t:

[0028]

[0029] Reagents andConditions:(a)Pd / C,H2,MeOH,Rt.

[0030] Synthesis of compounds 22u and 22v:

[0031]

[0032] ReagentsandConditions:(a)m-CPBA(1equiv.),DCM,Rt.(b)m-CPBA(2equiv.).DCM,Rt.

[0033] Representative experimental operation: In a reaction bottle, 1,4-dithio-2,5-diol 18 (20 mmol, 3.0448 g), (ethoxycarbonylmethylene)triphenylphosphine (24 mmol, 8.3609 g) and dichloromethane (100 mL) were added in sequence, and the reaction was stirred at room temperature for 4 h. The reaction was completed by TLC detection, and the mixture was concentrated and purified to obtain compound 19 (colorless oil).

[0034] When there are different substituents at the 6-position of purine, the synthesis method is similar. Take 6-chloropurine as an example: under nitrogen protection, 6-chloropurine (20 mmol, 3.0814 g), triphenylphosphine (0.4 mmol, 0.0142 g), sodium acetate (4 mmol, 0.3281 g) and toluene (150 mL) were added in a round-bottom flask in sequence. After stirring evenly, glacial acetic acid (4 mmol, 0.6 mL) and ethyl propiolate (24 mmol, 2.42 mL) were added. The reaction was stirred at room temperature for 12 h. The reaction was completed by TLC detection. The mixture was extracted, dried, concentrated, and separated and purified to obtain compound 21 (white solid).

[0035] Synthesis of five-membered sulfur-containing heterocyclic nucleoside compounds 22 and 23 series: When the purine 6-position carries different substituents, the method for synthesizing five-membered sulfur-containing heterocyclic nucleoside compounds at the 9-position is similar. Take the purine 6-position as Cl and the 9-position as ethyl propiolate as an example: under nitrogen protection, compound 19 (0.2mmol, 50.4mg), sodium hydride (0.6mmol, 60.0mg) and dichloromethane (2mL) were added to the reaction bottle in sequence. After stirring evenly, compound 21 (0.3mmol, 46.9mg) was added. The reaction was stirred at room temperature for 2h. The reaction was completed by TLC detection, and the reaction was quenched with ammonium chloride aqueous solution. The mixture was extracted, dried and concentrated. After separation and purification, compounds 22 and 23 were obtained, both of which were white solids. In addition, compounds 22 and 23 are racemic. Characterization data of representative compounds are as follows:

[0036] (Rac)ethyl-3-(6-chloro-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)tetrahydrothiophene-3-carboxylate(22a,23a).white solid.73.4mg,92%yield.mp:87.8-89.2℃.22a: 1 HNMR(600MHz,CDCl3)δ8.71(s,1H),8.36(s,1H),4.20-4.14(m,4H),3.93-3.85(m,3H),3.15-3.11(m,1H),2.94-2.90(m,1H),2.81(dd,J=16.8,3.6Hz,1H),2.58-2.52(m,1H),1.27(t,J=7.2Hz,3H),1.10(t,J=7.2Hz,3H); 13 C NMR(150MHz,CDCl3)δ171.0,167.5,151.9,151.8,151.6,142.9,132.1,75.0,63.2,61.4,45.6,37.0,34.7,34.6,14.2,13.9;ESI-HRMS(m / z)calcd C 16 H 19 ClN4NaO4S(M+Na + ),421.0708;found,421.0703.23a: 1 HNMR(600MHz,CDCl3)δ8.78(s,1H),8.69(s,1H),4.32-4.25(m,1H),4.24-4.17(m,1H),4.14-4.04(m,2H),3.73-3.68(m,1H),3.66-3.60(m,1H),3.50(d,J=12.6Hz,1H),3.44(dd,J=10.8,6.6Hz,1H),3.13(dd,J=16.2,2.4Hz,1H),2.47(t,J=10.8Hz,1H),1.60-1.54(m,1H),1.19(t,J=7.2Hz,3H),1.12(t,J=7.2Hz,3H); 13 C NMR(150MHz,CDCl3)δ171.1,167.5,152.1,151.8,144.0,131.4,73.4,63.2,61.1,46.9,40.2,34.6,33.5,29.8,14.2,13.9;ESI-HRMS(m / z)calcdC 16 H19 ClN4NaO4S(M+Na + ),421.0708;found,421.0707.

[0037] (Rac)ethyl-3-(6-bromo-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)tetrahydrothiophene-3-carboxylate(22b,23b).Prepared analogously to compounds22a and 23a.Colorless oil.67.6 mg,78%yield.22b: 1 H NMR(600 MHz,CDCl3)δ8.66(s,1H),8.36(s,1H),4.20-4.14(m,4H),3.92-3.85(m,3H),3.12(dd,J=11.4,6.6 Hz,1H),2.94-2.90(m,1H),2.81(dd,J=16.8,3.6 Hz,1H),2.56(dd,J=16.8,10.2 Hz,1H),1.29-1.25(m,3H),1.11(t,J=7.2 Hz,3H); 13 C NMR(150 MHz,CDCl3)δ171.0,167.5,151.7,150.7,143.7,142.7,134.7,75.1,63.2,61.4,45.6,37.0,34.7,34.5,14.2,13.9;ESI-HRMS(m / z)calcd C 16 H 19 BrN4NaO4S(M+Na + ),465.0203;found,465.0200.23b: 1H NMR(600 MHz,CDCl3)δ8.80(s,1H),8.65(s,1H),4.33-4.26(m,1H),4.24-4.18(m,1H),4.12-4.04(m,2H),3.70(d,J=12.6 Hz,1H),3.66-3.60(m,1H),3.50(d,J=13.2 Hz,1H),3.44(dd,J=11.4,6.6 Hz,1H),3.13(dd,J=16.2,3.0Hz,1H),2.47(t,J=10.8 Hz,1H),1.62-1.53(m,1H),1.20(t,J=7.2 Hz,3H),1.13(t,J=7.2 Hz,3H); 13 C NMR(150 MHz,CDCl3)δ171.0,167.4,151.7,150.8,143.9,143.8,133.9,73.4,63.2,61.1,46.8,40.1,34.5,33.4,14.2,13.9;ESI-HRMS(m / z)calcd C 16 H 20 BrN4O4S(M+H + ),443.0383;found,443.0390.

[0038] (Rac)ethyl-4-(2-ethoxy-2-oxoethyl)-3-(6-(propylthio)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate(22c,23c).Prepared analogously to compounds22a and 23a.Colorless oil.77.2 mg,88%yield.3a: 1 H NMR(600 MHz,CDCl3)δ8.66(s,1H),8.16(s,1H),4.21-4.14(m,4H),3.92-3.84(m,3H),3.37(t,J=7.2 Hz,2H),3.15(dd,J=11.4,6.0 Hz,1H),2.92(dd,J=11.4,5.4 Hz,1H),2.85(dd,J=16.8,3.0 Hz,1H),2.56(dd,J=16.8,10.2 Hz,1H),1.86-1.78(m,2H),1.28(t,J=7.2 Hz,3H),1.13-1.06(m,6H); 13C NMR(150 MHz,CDCl3)δ171.2,167.9,162.2,151.8,148.6,140.0,131.8,74.5,62.9,61.3,45.6,37.1,34.7,34.4,30.7,22.9,14.3,14.0,13.5;ESI-HRMS(m / z)calcdC 19 H 26 N4NaO4S2(M+Na + ),461.1288;found,461.1282.3b: 1 H NMR(600 MHz,CDCl3)δ8.62(s,1H),8.58(s,1H),4.31-4.25(m,1H),4.23-4.17(m,1H),4.11-4.04(m,2H),3.68(d,J=13.2Hz,1H),3.70-3.58(m,1H),3.49(d,J=12.6 Hz,1H),3.43-3.32(m,3H),3.17-3.13(m,1H),2.48(t,J=10.8 Hz,1H),1.87-1.80(m,2H),1.61-1.55(m,1H),1.20(t,J=7.2 Hz,3H),1.14-1.07(m,6H); 13 C NMR(150MHz,CDCl3)δ171.3,167.9,162.3,151.7,148.7,141.1,130.9,72.8,62.9,60.9,46.8,40.1,34.5,33.5,30.8,22.9,14.2,13.9,13.5;ESI-HRMS(m / z)calcd C 19 H 26 N4NaO4S2(M+Na + ),461.1288;found,461.1285.

[0039] (Rac)ethyl-3-(6-(dimethylamino)-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)tetrahydr-othiophene-3-carboxylate(22d,23d).white solid.64.4 mg,79%yield.mp:112.2-113.7℃.Prepared analogously to compounds 22a and 23a.22d: 1HNMR(600 MHz,CDCl3)δ8.27(s,1H),7.93(s,1H),4.21-4.11(m,5H),3.88(d,J=12.6 Hz,1H),3.80-3.73(m,2H),3.51(s,5H),3.17(dd,J=11.4,6.0 Hz,1H),2.91(dd,J=11.4,6.0Hz,1H),2.82(dd,J=16.8,3.6 Hz,1H),2.51(dd,J=16.8,6.6 Hz,1H),1.26(t,J=7.2Hz,3H),1.09(t,J=7.2 Hz,3H); 13 C NMR(150 MHz,CDCl3)δ171.4,168.4,155.0,152.3,150.9,135.3,120.6,73.9,62.7,61.3,45.5,37.4,34.8,34.4,14.3,14.0;ESI-HRMS(m / z)calcd C 18 H 25 N5NaO4S(M+Na + ),430.1519;found,430.1516.23d: 1 H NMR(400 MHz,CDCl3)δ8.36(s,1H),8.24(s,1H),4.30-4.14(m,2H),4.11-4.01(m,2H),3.65-3.44(m,9H),3.41-3.36(m,1H),3.23-3.17(m,1H),2.49(t,J=10.8Hz,1H),1.64-1.55(m,1H),1.19(t,J=7.2Hz,3H),1.11(t,J=7.2 Hz,3H); 13 CNMR(150 MHz,CDCl3)δ171.6,168.5,155.0,152.1,151.0,136.6,119.7,72.3,62.6,60.8,46.7,40.1,34.6,33.7,14.2,13.9;ESI-HRMS(m / z)calcd C 18 H 26 N5O4S(M+H + ),408.1700;found,408.1700.

[0040] (Rac)ethyl-3-(6-(diethylamino)-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)tetrahydro-othiophene-3-carboxylate(22e,23e).Prepared analogously tocompounds 22a and23a.Colorless oil.78.4 mg,90%yield.22e: 1 H NMR(600 MHz,CDCl3)δ8.25(s,1H),7.93(s,1H),4.24-4.11(m,5H),4.06-3.84(m,4H),3.80-3.72(m,2H),3.18(dd,J=11.4,6.6 Hz,1H),2.91-2.87(m,1H),2.84-2.80(m,1H),2.54-2.48(m,1H),1.30-1.23(m,9H),1.13(t,J=7.2 Hz,3H); 13 C NMR(150 MHz,CDCl3)δ171.4,168.4,153.9,152.5,150.9,135.3,120.0,73.8,62.7,61.2,45.6,37.4,34.8,34.4,14.3,14.0;ESI-HRMS(m / z)calcd C 20 H 30 N5O4S(M+H + ),436.2013;found,436.2006.23e: 1 HNMR(600 MHz,CDCl3)δ8.36(s,1H),8.22(s,1H),4.33-4.25(m,1H),4.22-4.16(m,1H),4.12-3.70(m,6H),3.64-3.54(m,2H),3.47(d,J=12.6 Hz,1H),3.38(dd,J=10.8,6.6 Hz,1H),3.22(dd,J=16.8,2.4 Hz,1H),2.51(t,J=10.8 Hz,1H),1.63(dd,J=16.8,11.4 Hz,1H),1.30(t,J=7.2Hz,6H),1.21-1.18(m,3H),1.14(t,J=7.2 Hz,3H); 13C NMR(150 MHz,CDCl3)δ171.8,168.6,153.9,152.4,151.1,136.7,119.2,72.3,62.7,60.8,46.8,40.2,34.7,33.8,14.3,14.0;ESI-HRMS(m / z)calcdC 20 H 30 N5O4S(M+H + ),436.2013;found,436.2019.

[0041] (Rac)ethyl-4-(2-ethoxy-2-oxoethyl)-3-(6-methoxy-9H-purin-9-yl)tetrahydrothioph-ene-3-carboxylate(22f,23f).Prepared analogously to compounds22a and 23a.Colorless oil.66.3 mg,84%yield.22f: 1 H NMR(600 MHz,CDCl3)δ8.48(s,1H),8.13(s,1H),4.20-4.11(m,7H),3.88-3.82(m,3H),3.14(dd,J=11.4,6.0 Hz,1H),2.89(dd,J=11.4,5.4 Hz,1H),2.80(dd,J=16.8,3.6 Hz,1H),2.54(dd,J=16.8,10.2Hz,1H),1.25(t,J=7.2 Hz,3H),1.07(t,J=7.2 Hz,3H); 13 C NMR(150 MHz,CDCl3)δ171.2,168.0,161.3,152.2,152.0,139.7,122.1,74.5,62.9,61.3,54.3,45.6,37.2,34.7,34.5,14.2,13.9;ESI-HRMS(m / z)calcd C 17 H 22 N4NaO5S(M+Na + ),417.1203;found,417.1202.23f: 1HNMR(600 MHz,CDCl3)δ8.55(s,1H),8.47(s,1H),4.32-4.16(m,5H),4.11-4.03(m,2H),3.69-3.58(m,2H),3.50(d,J=12.6 Hz,1H),3.40(dd,J=11.4,6.6 Hz,1H),3.15(dd,J=16.8,3.0 Hz,1H),2.48(t,J=10.8Hz,1H),1.57(dd,J=16.2,11.4 Hz,1H),1.19(t,J=7.2 Hz,3H),1.11(t,J=7.2 Hz,3H); 13 C NMR(150 MHz,CDCl3)δ171.4,168.1,161.4,152.4,152.0,140.8,121.3,72.9,63.0,61.0,54.4,46.8,40.1,34.6,33.6,14.3,13.9;ESI-HRMS(m / z)calcdC 17 H 22 N4NaO5S(M+Na + ),417.1203;found,417.1203.

[0042] (Rac)ethyl-4-(2-ethoxy-2-oxoethyl)-3-(6-ethoxy-9H-purin-9-yl)tetrahydrothiophe-ne-3-carboxylate(22g,23g).Prepared analogously to compounds22a and 23a.Colorless oil.67.8 mg,83%yield.22g: 1 H NMR(600 MHz,CDCl3)δ8.45(s,1H),8.12(s,1H),4.64(q,J=7.2 Hz,2H),4.18-4.11(m,4H),3.89-3.82(m,3H),3.17-3.12(m,1H),2.91-2.87(m,1H),2.78(dd,J=16.8,3.0 Hz,1H),2.54(dd,J=16.8,10.2 Hz,1H),1.49(t,J=7.2 Hz,3H),1.27-1.21(m,3H),1.10-1.06(m,3H); 13CNMR(150 MHz,CDCl3)δ171.2,168.0,161.0,152.2,152.1,139.5,122.0,74.5,63.3,62.9,61.3,45.6,37.1,34.7,34.4,14.6,14.2,13.9;ESI-HRMS(m / z)calcdC 18 H 24 N4NaO5S(M+Na + ),431.1360;found,431.1364.23g: 1 H NMR(600 MHz,CDCl3)δ8.54(s,1H),8.44(s,1H),4.70-4.63(m,2H),4.30-4.24(m,1H),4.23-4.16(m,1H),4.10-4.04(m,2H),3.70-3.58(m,2H),3.50(d,J=12.6 Hz,1H),3.40(dd,J=11.4,6.6 Hz,1H),3.15(dd,J=16.2,2.4 Hz,1H),2.48(t,J=10.8 Hz,1H),1.61-1.49(m,4H),1.19(t,J=7.2 Hz,3H),1.11(t,J=7.2 Hz,3H); 13 C NMR(150 MHz,CDCl3)δ171.4,168.1,161.1,152.4,152.0,140.6,121.3,72.9,63.4,63.0,61.0,46.8,40.1,34.6,33.6,14.6,14.3,14.0;ESI-HRMS(m / z)calcd C 18 H 24 N4NaO5S(M+Na + ),431.1360;found,431.1363.

[0043] (Rac)ethyl-3-(6-(benzyloxy)-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)tetrahydrothio-phene-3-carboxylate(22h,23h).Prepared analogously to compounds22a and 23a.Colorless oil.81.9 mg,87%yield.22h: 1H NMR(600 MHz,CDCl3)δ8.49(s,1H),8.14(s,1H),7.54-7.51(m,2H),7.37-7.33(m,2H),7.31-7.28(m,1H),5.65(s,2H),4.19-4.12(m,4H),3.90-3.83(m,3H),3.17-3.13(m,1H),2.90(dd,J=11.4,5.4 Hz,1H),2.79(dd,J=16.8,3.0 Hz,1H),2.55(dd,J=16.8,10.2 Hz,1H),1.25(t,J=7.2Hz,3H),1.09(t,J=7.2 Hz,3H); 13 C NMR(150 MHz,CDCl3)δ171.2,167.9,160.7,152.4,151.9,139.7,136.1,128.5,128.4,128.2,122.1,74.5,68.6,62.9,61.3,45.5,37.1,34.6,34.4,14.2,13.9;ESI-HRMS(m / z)calcd C 23 H 27 N4O5S(M+H + ),471.1697;found,471.1695.23h: 1 HNMR(600 MHz,CDCl3)δ8.55(s,1H),8.46(s,1H),7.56-7.53(m,2H),7.39-7.35(m,2H),7.34-7.30(m,1H),5.70(d,J=12.0 Hz,1H),5.67(dd,J=30.6,12.0 Hz,1H),4.31-4.24(m,1H),4.22-4.16(m,1H),4.10-4.04(m,2H),3.68-3.59(m,2H),3.50(d,J=13.2 Hz,1H),3.42-3.38(m,1H),3.17-3.12(m,1H),2.48(t,J=10.8 Hz,1H),1.58(dd,J=16.2,11.4Hz,1H),1.21-1.17(m,3H),1.11(t,J=7.2 Hz,3H); 13C NMR(150 MHz,CDCl3)δ171.4,168.0,160.8,152.6,151.9,140.8,136.1,128.6,128.5,128.3,121.3,72.9,68.7,62.9,60.9,46.8,40.1,34.6,33.6,14.2,13.9;ESI-HRMS(m / z)calcd C 23 H 26 N4NaO5S(M+Na + ),493.1516;found,493.1513.

[0044] (Rac)ethyl-4-(2-ethoxy-2-oxoethyl)-3-(6-(pyrrolidin-1-yl)-9H-purin-9-yl)tetrahyd-rothiophene-3-carboxylate(22i,23i).Prepared analogously tocompounds 22a and23a.Colorless oil.77.2 mg,89%yield.22i: 1 H NMR(600 MHz,CDCl3)δ8.29(s,1H),7.93(s,1H),4.22-4.13(m,6H),3.88(d,J=12.6 Hz,1H),3.82-3.70(m,4H),3.18(dd,J=11.4,6.6 Hz,1H),2.90(dd,J=11.4,6 Hz,1H),2.84-2.79(m,1H),2.53(dd,J=16.8,10.8 Hz,1H),2.10-1.95(m,4H),1.28-1.24(m,3H),1.11(t,J=7.2 Hz,3H); 13 CNMR(150 MHz,CDCl3)δ171.4,168.4,153.2,152.8,150.5,135.9,120.7,73.9,62.7,61.3,45.6,37.4,34.8,34.4,14.3,14.0;ESI-HRMS(m / z)calcdC 20 H 28 N5O4S(M+H + ),434.1857;found,434.1855.23i: 1H NMR(400 MHz,CDCl3)δ8.35(s,1H),8.26(s,1H),4.32-4.00(m,6H),3.82-3.52(m,4H),3.48(d,J=12.8Hz,1H),3.42-3.35(m,1H),3.20(dd,J=16.4,2.4Hz,1H),2.53-2.45(m,1H),2.13-1.95(m,4H),1.66-1.56(m,1H),1.19(t,J=7.2 Hz,3H),1.12(t,J=7.2 Hz,3H); 13 CNMR(150 MHz,CDCl3)δ171.7,168.5,153.2,152.6,150.7,137.2,119.9,72.3,62.7,60.8,46.8,40.2,34.7,33.8,14.3,14.0;ESI-HRMS(m / z)calcdC 20 H 28 N5O4S(M+H + ),434.1857;found,434.1859.

[0045] (Rac)ethyl-4-(2-ethoxy-2-oxoethyl)-3-(6-(piperidin-1-yl)-9H-purin-9-yl)tetrahydro-thiophene-3-carboxylate(22j,23j).Prepared analogously tocompounds 22a and 23a.Colorless oil.75.2 mg,84%yield.22j: 1 H NMR(400 MHz,CDCl3)δ8.25(s,1H),7.93(s,1H),4.26-4.12(m,8H),3.89(d,J=18 Hz,1H),3.81-3.72(m,2H),3.18(dd,J=16.8,9.0 Hz,1H),2.93-2.79(m,2H),2.52(dd,J=25.2,15.6 Hz,1H),1.78-1.65(m,6H),1.26(t,J=10.8 Hz,3H),1.11(t,J=10.8 Hz,3H); 13C NMR(150 MHz,CDCl3)δ171.4,168.4,154.0,152.4,151.1,135.1,120.2,73.8,62.7,61.3,45.6,37.5,34.8,34.4,26.2,24.9,14.3,14.0;ESI-HRMS(m / z)calcd C 21 H 30 N5O4S(M+H + ),448.2013;found,448.2007.23j: 1 H NMR(600 MHz,CDCl3)δ8.36(s,1H),8.22(s,1H),4.31-4.03(m,8H),3.64-3.54(m,2H),3.47(d,J=12.6 Hz,1H),3.38(dd,J=11.4,6.6 Hz,1H),3.23-3.19(m,1H),2.49(t,J=10.8 Hz,1H),1.77-1.68(m,6H),1.62-1.58(m,1H),1.19(t,J=7.2 Hz,3H),1.12(t,J=7.1 Hz,3H); 13 C NMR(150MHz,CDCl3)δ171.7,168.5,154.0,152.3,151.3,136.4,119.4,72.3,62.7,60.8,46.8,40.2,34.7,33.8,26.3,24.9,14.3,14.0;ESI-HRMS(m / z)calcd C 21 H 30 N5O4S(M+H + ),448.2013;found,448.2015.

[0046] (Rac)ethyl-4-(2-ethoxy-2-oxoethyl)-3-(6-morpholino-9H-purin-9-yl)tetrahydrothio-phene-3-caroxylate(22k,23k).Prepared analogously to compounds22a and 23a.Colorless oil.81.8 mg,91%yield.22k: 1H NMR(600 MHz,CDCl3)δ8.28(s,1H),7.95(s,1H),4.34-4.12(m,7H),3.90-3.74(m,8H),3.17(dd,J=11.4,6.0 Hz,1H),2.90(dd,J=11.4,5.4 Hz,1H),2.81(dd,J=16.8,3.0 Hz,1H),2.55-2.49(m,1H),1.28-1.23(m,3H),1.11(t,J=7.2 Hz,3H); 13 C NMR(150 MHz,CDCl3)δ171.3,168.3,154.0,152.3,151.3,135.8,120.4,74.0,67.1,62.7,61.3,45.5,37.4,34.8,34.5,14.3,14.0;ESI-HRMS(m / z)calcd C 20 H 27 N5NaO5S(M+Na + ),472.1625;found,472.1618.23k: 1 H NMR(600MHz,CDCl3)δ8.39(s,1H),8.26(s,1H),4.36-4.17(m,6H),4.12-4.02(m,2H),3.85(t,J=4.8 Hz,4H),3.66-3.54(m,2H),3.47(d,J=12.6Hz,1H),3.39(dd,J=11.4,6.6 Hz,1H),3.23-3.19(m,1H),2.48(t,J=10.8 Hz,1H),1.60(dd,J=16.2,11.4 Hz,1H),1.20(t,J=7.2 Hz,3H),1.13(t,J=7.2 Hz,3H); 13 CNMR(150 MHz,CDCl3)δ171.6,168.4,154.0,152.2,151.5,137.1,119.6,72.4,67.2,62.8,60.9,46.8,40.2,34.7,33.7,14.3,13.9;ESI-HRMS(m / z)calcd C 20 H 28 N5O5S(M+H + ),450.1806;found,450.1803.

[0047] (Rac)ethyl-4-(2-ethoxy-2-oxoethyl)-3-(6-phenyl-9H-purin-9-yl)tetrahydrothiophe-ne-3-carboxylate(22l,23l).Prepared analogously to compounds22a and 23a.Colorless oil.57.2 mg,65%yield.22l: 1 H NMR(600 MHz,CDCl3)δ8.98(s,1H),8.78(d,J=7.2 Hz,2H),8.36(s,1H),7.60-7.51(m,3H),4.24-4.14(m,4H),3.97-3.89(m,3H),3.20(dd,J=11.4,6.0 Hz,1H),2.96(dd,J=11.4,6.0 Hz,1H),2.87(dd,J=16.8,3.0 Hz,1H),2.58(dd,J=16.8,10.2 Hz,1H),1.31-1.26(t,J=7.2 Hz,3H),1.13-1.09(m,3H); 13 C NMR(150 MHz,CDCl3)δ171.3,168.0,155.4,152.8,152.3,141.7,135.6,131.5,131.3,129.9,128.8,74.5,63.0,61.4,45.7,37.3,34.8,34.6,29.8,14.3,14.0;ESI-HRMS(m / z)calcd C 22 H 25 N4O4S(M+H + ),441.1591;found,441.1586.23l: 1 H NMR(600 MHz,CDCl3)δ8.95(s,1H),8.87-8.71(m,3H),7.60-7.52(m 3H),4.33-4.22(m,2H),4.12-4.03(m,2H),3.74-3.63(m,2H),3.55(d,J=12.6 Hz,1H),3.4(dd,J=11.4,6.6 Hz,1H),3.25-3.21(m,1H),2.53(t,J=10.8 Hz,1H),1.67-1.62(m,1H),1.19(d,J=7.2 Hz,3H),1.13(d,J=7.2Hz,3H); 13C NMR(150 MHz,CDCl3)δ171.4,168.1,155.5,152.9,152.3,142.9,135.5,131.4,130.7,129.9,128.9,72.8,63.0,61.0,46.9,40.2,34.7,33.6,29.8,14.3,13.9;ESI-HRMS(m / z)calcd C 22 H 25 N4O4S(M+H + ),441.1591;found,441.1590.

[0048] (Rac)ethyl-3-(2-chloro-6-(pyrrolidin-1-yl)-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)-tetrahydrothiophene-3-carboxylate(22m,23m).Preparedanalogously to compounds22a and 23a.Colorless oil.81.4 mg,87%yield.22m: 1 HNMR(600 MHz,CDCl3)δ7.86(s,1H),4.23-4.06(m,2H),4.16-4.07(m,4H),3.86(d,J=12.0Hz,1H),3.72-3.66(m,4H),3.19-3.15(m,1H),2.89-2.85(m,1H),2.75(dd,J=16.8,3.0Hz,1H),2.48(dd,J=16.8,10.2 Hz,1H),2.04(q,J=6.6 Hz,2H),1.94(q,J=6.6 Hz,2H),1.24(t,J=7.2 Hz,3H),1.15(t,J=7.2 Hz,3H); 13 C NMR(150 MHz,CDCl3)δ171.2,168.0,153.9,153.4,151.5,136.2,119.5,73.9,62.8,61.2,49.0,47.8,45.6,37.5,34.8,34.3,26.3,24.2,14.3,14.0;ESI-HRMS(m / z)calcd C 20 H 27 ClN5O4S(M+H + ),468.1467;found,468.1461.23m: 1H NMR(600 MHz,CDCl3)δ8.33(s,1H),4.31-4.22(m,2H),4.18-4.04(m,4H),3.73(t,J=6.6 Hz,2H),3.60(d,J=12.6 Hz,1H),3.56-3.50(m,1H),3.43(d,J=12.6 Hz,1H),3.34(dd,J=10.8,6.6 Hz,1H),3.18(dd,J=16.2,2.4 Hz,1H),2.47-2.42(m,1H),2.10-2.04(m,2H),1.99-1.93(m,2H),1.58(dd,J=16.8,11.4 Hz,1H),1.22-1.16(m,6H); 13 C NMR(150 MHz,CDCl3)δ171.6,168.0,153.9,153.5,151.6,137.5,118.7,72.5,63.0,60.9,49.1,47.9,46.9,40.2,34.6,33.6,26.4,24.3,14.3,14.0;ESI-HRMS(m / z)calcdC 20 H 27 ClN5O4S(M+H + ),468.1467;found,468.1466.

[0049] (Rac)ethyl-3-(2-chloro-6-(piperidin-1-yl)-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)-tetrahydrothiophene-3-carboxylate(22n,23n).Prepared analogouslyto compounds22a and 23a.Colorless oil.85.8 mg,89%yield.22n: 1H NMR(600 MHz,CDCl3)δ7.88(s,1H),4.52-4.38(m,2H),4.26-4.12(m,6H),3.90(d,J=12.0 Hz,1H),3.74-3.65(m,2H),3.19(dd,J=11.4,6.0 Hz,1H),2.89(dd,J=11.4,6.0 Hz,1H),2.81-2.76(m,1H),2.50(dd,J=16.8,10.2 Hz,1H),1.76-1.65(m,6H),1.27(t,J=7.2 Hz,3H),1.19(t,J=7.2 Hz,3H); 13 C NMR(150 MHz,CDCl3)δ171.3,168.1,154.0,153.8,152.3,135.4,119.1,74.0,62.9,61.4,45.6,37.7,34.9,34.4,26.2,24.7,14.3,14.1;ESI-HRMS(m / z)calcdC 21 H 28 ClN5NaO4S(M+Na + ),504.1443;found,504.1449.23n: 1 H NMR(600 MHz,CDCl3)δ8.35(s,1H),4.35-4.05(m,7H),3.61(d,J=12.6 Hz,1H),3.57-3.50(m,1H),3.43(d,J=12.6 Hz,1H),3.38-3.33(m,1H),3.23-3.18(m,1H),2.49-2.44(m,1H),1.77-1.68(m,6H),1.63-1.56(m,2H),1.24-1.18(m,6H); 13 C NMR(150 MHz,CDCl3)δ171.6,168.0,154.0,153.7,152.4,136.7,118.2,72.5,63.1,60.9,46.9,40.3,34.7,33.7,24.7,14.3,14.0;ESI-HRMS(m / z)calcdC 21 H 28 ClN5NaO4S(M+Na + ),504.1443;found,504.1446.

[0050] (Rac)ethyl-3-(2,6-dichloro-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)tetrahydrothi-ophene-3-carboxylate(22o,23o).Prepared analogously to compounds22a and 23a.Colorless oil.37.3 mg,43%yield.22o: 1 H NMR(400MHz,CDCl3)δ8.33(s,1H),4.27-4.15(m,4H),3.88-3.81(m,3H),3.15(dd,J=11.6,6.4 Hz,1H),2.94(dd,J=11.6,5.6 Hz,1H),2.80(dd,J=16.8,3.6 Hz,1H),2.94(dd,J=16.8,9.6 Hz,1H),1.29(t,J=7.2 Hz,3H),1.84(t,J=7.2 Hz,3H); 13 C NMR(150 MHz,CDCl3)δ171.0,167.3,153.3,153.0,152.4,143.6,131.3,75.4,63.5,61.6,45.9,37.3,34.9,34.7,14.3,14.1;ESI-HRMS(m / z)calcd C 16 H 18 Cl2N4NaO4S(M+Na + ),455.0318;found,455.0314.23o: 1 H NMR(600MHz,CDCl3)δ8.77(s,1H),4.3(q,J=7.2 Hz,2H),4.09(q,J=7.2 Hz,2H),3.70(d,J=13.2 Hz,1H),3.65-3.59(m,1H),3.49-3.39(m,2H),3.09(dd,J=16.2,2.4 Hz,1H),2.47(t,J=10.8Hz,1H),1.63-1.53(m,1H),1.21(dd,J=16.2,7.2 Hz,6H); 13 C NMR(150 MHz,CDCl3)δ170.9,167.1,153.3,153.0,152.6,144.6,130.5,73.7,63.6,61.2,46.9,40.2,34.5,33.4,14.3,14.0;ESI-HRMS(m / z)calcd C16 H 18 Cl2N4NaO4S(M+Na + ),455.0318;found,455.0323.

[0051] (Rac)methyl-3-(6-chloro-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)tetrahydrothioph-ene-3-carboxylate(22p,23p).Prepared analogously to compounds22a and 23a.Colorless oil.57.7 mg,75%yield.22p: 1 H NMR(600 MHz,CDCl3)δ8.72(s,1H),8.36(s,1H),4.18(q,J=7.2 Hz,2H),3.94-3.86(m,3H),3.71(s,3H),3.13(dd,J=11.4,6.0 Hz,1H),2.93(dd,J=11.4,5.4 Hz,1H),2.81(dd,J=16.8,3.6 Hz,1H),2.59-2.54(m,1H),1.30-1.26(m,3H); 13 C NMR(150 MHz,CDCl3)δ171.1,168.2,152.0,151.8,142.8,132.3,75.1,61.5,53.8,45.8,37.1,34.9,34.7,29.8,14.3;ESI-HRMS(m / z)calcdC 15 H 17 ClN4NaO4S(M+Na + ),407.0551;found,407.0551.23p: 1 HNMR(600 MHz,CDCl3)δ8.80(s,1H),8.71(s,1H),4.12-4.04(m,2H),3.75(s,3H),3.71(d,J=13.2 Hz,1H),3.66-3.60(m,1H),3.50(d,J=12.6 Hz,1H),3.46(dd,J=11.4,6.6 Hz,1H),3.15-3.11(m,1H),2.50-2.45(m,1H),1.59-1.52(m,1H),1.20(t,J=7.2 Hz,3H); 13C NMR(150 MHz,CDCl3)δ171.0,168.1,152.2,152.0,143.8,131.5,73.3,61.1,53.7,47.0,40.2,34.5,33.4,29.8,14.3;ESI-HRMS(m / z)calcdC 15 H 17 ClN4NaO4S(M+Na + ),407.0551;found,407.0557.

[0052] (Rac)isopropyl-3-(6-chloro-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)tetrahydrothio-phene-3-carboxylate(22q,23q).Prepared analogously to compounds22a and 23a.Colorless oil.74.3 mg,90%yield.22q: 1 H NMR(600 MHz,CDCl3)δ8.69(s,1H),8.35(s,1H),5.06-5.01(m,1H),4.18-4.12(m,2H),3.89-3.84(m,3H),3.13(dd,J=11.4,6.0 Hz,1H),2.92(dd,J=11.4,5.4 Hz,1H),2.80(dd,J=16.8,3.0 Hz,1H),2.54(dd,J=16.8,10.2 Hz,1H),1.26(t,J=7.2 Hz,3H),1.12(d,J=6.6 Hz,3H),1.03(d,J=6.6 Hz,3H); 13 C NMR(150 MHz,CDCl3)δ171.1,166.9,152.0151.8,151.6,142.9,132.1,75.1,71.5,61.4,45.6,37.1,34.7,34.5,21.5,21.4,14.3;ESI-HRMS(m / z)calcdC 17 H 21 ClN4NaO4S(M+Na + ),435.0864;found,435.0858.23q: 1HNMR(600 MHz,CDCl3)δ8.77(s,1H),8.69(s,1H),5.18-5.11(m,1H),4.13-4.03(m,2H),3.70-3.60(m,2H),3.49(d,J=13.2Hz,1H),3.43(dd,J=10.8,6.6Hz,1H),3.15(dd,J=16.2,2.4 Hz,1H),2.49-2.44(m,1H),1.61-1.55(m,1H),1.23-1.17(m,6H),1.03(d,J=6.6 Hz,3H); 13 C NMR(150 MHz,CDCl3)δ171.1,166.9,152.1,151.8,144.1,131.4,73.6,71.4,61.1,46.8,40.2,34.6,33.5,21.7,21.4,14.3;ESI-HRMS(m / z)calcd C 17 H 21 ClN4NaO4S(M+Na + ),435.0864;found,435.0871.

[0053] (Rac)ethyl-3-(6-chloro-2-fluoro-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)tetrahydr-othiophene-3-carboxylate(22r,23r).Prepared analogously tocompounds 22a and23a.Colorless oil.35.0 mg,42%yield.22r: 1 H NMR(400 MHz,CDCl3)δ8.33(s,1H),4.27-4.15(m,4H),4.82-4.90(m,3H),3.14(dd,J=6.4,3.6 Hz,1H),2.94(dd,J=5.2,11.6 Hz,1H),2.80(dd,J=9.6,16.8 Hz,1H),2.56(dd,J=9.6,16.8 Hz,1H),1.32-1.27(m,3H),1.21-1.16(m,3H); 13 C NMR(100 MHz,CDCl3)δ171.0,167.3,161.3,156.9(d,J C-F=22.0 Hz),143.6,75.4,63.5,61.6,45.9,37.1,34.9,34.7,29.9,14.3,14.1; 19 FNMR(565 MHz,CDCl3)δ-48.32;ESI-HRMS(m / z)calcdC 16 H 18 ClFN4NaO4S(M+Na + ),439.0614;found,439.0607.23r: 1 H NMR(600 MHz,CDCl3)δ8.76(s,1H),4.35-4.23(m,2H),4.12-4.07(m,2H),3.71(d,J=12.6 Hz,1H),3.65-3.59(m,1H),3.50-3.41(m,2H),3.11-3.07(m,1H),2.48(t,J=10.8 Hz,1H),1.63(dd,J=11.4,16.2 Hz,1H),1.17-1.23(m,6H); 13 C NMR(150MHz,CDCl3)δ170.9,167.0,156.9(d,J C-F =220.5 Hz),153.6(d,J C-F =18 Hz),144.6,73.8,63.5,61.2,46.9,40.1,34.5,33.4,29.8,14.3,14.0; 19 F NMR(565 MHz,CDCl3)δ-48.52.ESI-HRMS(m / z)calcd C 16 H 18 ClFN4NaO4S(M+Na + ),439.0614;found,439.0612.

[0054] (Rac)ethyl-4-(2-ethoxy-2-oxoethyl)-3-(9H-purin-9-yl)tetrahydrothiophene-3-carb-oxylate(22s,23s).Compounds 22s(39.9 mg,0.1 mmol),Pd / C(10 wt%),and DCM(1.0 mL)were added to a reaction tube under H2(1 atm)atroom temperature.Whencompound 22s was completely consumed,the residue wasconcentrated and purifiedby flash column chromatography on silica gel(PE / EA=2:1)to give the targetcompound 22s as a colorless oil in 23.7 mg,65%yield.23s was prepared analogouslyto 22s.Colorless oil.22.9 mg,63%yield.22s: 1 HNMR(400 MHz,CDCl3)δ9.18(s,1H),8.95(s,1H),8.34(s,1H),4.22-4.12(m,3H),3.96-3.86(m,3H),3.19-3.13(m,1H),2.93(dd,J=5.2,11.6 Hz,1H),2.86-2.79(m,1H),2.57(dd,J=10.0,16.8 Hz,1H),1.76(s,1H),1.27(t,J=7.2 Hz,3H),1.09(t,J=7.2 Hz,3H); 13 C NMR(100MHz,CDCl3)δ171.2,167.9,152.6,149.1,143.0,74.6,63.1,61.4,45.7,37.1,34.8,34.6,29.8,14.3,14.0,1.14;ESI-HRMS(m / z)calcd C 16 H 20 N4NaO4S(M+Na + ),387.1097;found,387.1091.23s: 1H NMR(400 MHz,CDCl3)δ9.07(s,1H),8.84(s,1H),8.32(s,1H),4.12-4.00(m,4H),3.90-3.76(m,3H),3.12-3.04(m,1H),2.84(dd,J=5.2,16.8 Hz,1H),2.77-2.70(m,1H),2.48(dd,J=2.8,16.8 Hz,1H),1.16(q,J=7.2 Hz,3H),1.02-0.94(m,3H); 13 C NMR(100 MHz,CDCl3)δ171.0,167.6,151.5,148.9,142.8,134.3,74.4,62.8,61.1,45.4,36.9,34.6,34.3,14.1,13.7;ESI-HRMS(m / z)calcd C 16 H 20 N4NaO4S(M+Na + ),387.1097;found,387.1090.

[0055] (Rac)ethyl-3-(2-chloro-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)tetrahydrothiophe-ne-3-carboxylate(22t,23t).Compounds 22o(43.3 mg,0.1 mmol),Pd / C(10 wt%),and DCM(1.0 mL)were added to a reaction tube under H2(1 atm)atroomtemperature.When compound 22o was completely consumed,the residuewasconcentrated and purified by flash column chromatography on silica gel(PE / EA=2:1)to give the target compound 22t as a colorless oil in 14.8 mg,37%yield.23t wasprepared analogously to 22t.Colorless oil.12.7 mg,32%yield.22t: 1HNMR(400MHz,CDCl3)δ9.03(s,1H),8.37(s,1H),4.26-4.14(m,4H),3.91-3.82(m,2H),3.20-3.12(m,1H),2.97-2.90(m,1H),2.84-2.76(m,1H),2.60-2.50(m,1H),1.63(s,1H),1.28(t,J=6.8 Hz,3H)1.17(t,J=7.2 Hz,3H); 13 C NMR(100 MHz,CDCl3)δ171.1,167.6,154.4,150.6,75.0,63.3,61.5,45.8,37.3,34.9,34.6,29.8.14.3,14.0;ESI-HRMS(m / z)calcdC 16 H 19 ClN4NaO4S(M+Na + ),421.0708;found,421.0708.23t: 1 H NMR(400 MHz,CDCl3)δ9.00(s,1H),8.76(s,1H),4.25-4.34(m,2H),4.09(q,J=7.2,2H),3.73-3.58(m,2H),3.52-3.38(m,2H),3.16-3.08(m,1H),2.49(q,J=10.8Hz,1H),1.59(q,J=11.2,16.0 Hz,1H),11.23-1.15(m,6H); 13 C NMR(100 MHz,CDCl3)δ171.1,167.4,154.4,153.4,150.8,144.7,132.8,73.2,63.4,61.2,47.0,40.2,34.6,33.5,14.3,14.0;ESI-HRMS(m / z)calcd C 16 H 19 ClN4NaO4S(M+Na + ),421.0708;found,421.0702.

[0056] (Rac)ethyl-3-(2,6-dichloro-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)tetrahydrothio-phene-3-carboxylate 1-oxide(22u).Compounds 22a(86.4 mg,0.2mmol),m-chloroperbenzoic acid(34.5 mg,0.2 mmol)and DCM(2 ml)were added to areactiontube and stirred at room temperature.When compound 22a was completelyconsumed,the residue was concentrated and purified by flash columnchromatography on silicagel(PE / EA=2:1)to give the target compound 22u as acolorless oil in 51.2 mg,57%yield.2u: 1 H NMR(400 MHz,CDCl3)δ8.46(s,1H),4.72-4.58(m,2H),4.35-4.12(m,4H),3.54-3.47(m,1H),3.22(dd,J=2.4,14.8 Hz,1H),3.02-2.90(m,2H),3.39(dd,J=10.4,16.4Hz,1H),1.32(t,J=7.2Hz,3H),1.16(t,J=7.2Hz,3H); 13 CNMR(100MHz,CDCl3)δ177.7,169.8,167.9,152.7,142.6,64.2,63.5,62.1,42.1,34.3,14.3,14.1;ESI-HRMS(m / z)calcd C 16 H 18 Cl2N4NaO5S(M+Na + ),471.0267;found,471.0258.

[0057] (Rac)ethyl-3-(2,6-dichloro-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)tetrahydrothiophe ne-3-carboxylate 1,1-dioxide(22v). Compounds 22a(86.4mg, 0.2mmol), m-chloroperbenzoic acid(69.0mg, 0.4mmol) and DCM(2ml) were added to a reaction tube and stirred at room temperature. When compound 22a was completely consumed, the residue was concentrated and purified by flash column chromatography on silica gel(PE / EA=2:1) to give the target compound 22u as a white solid in 63.3mg, 68% yield. mp: 118.7-120.1℃. 1 1H NMR(400MHz, CDCl3) δ 8.36(s, 1H), 4.52(d, J=14.4Hz, 1H), 4.40-4.05(m, 5H), 3.75-3.67(m, 2H), 3.36-3.28(m, 1H), 2.94-2.87(m, 1H), 2.44(q, J=10.4, 16.4Hz, 1H), 1.31-1.19(m, 6H); 13 13C NMR(100MHz, CDCl3) δ 169.7, 166.7, 153.5, 153.2, 152.8, 142.4, 131.2, 67.9, 64.7, 62.3, 59.5, 54.1, 38.2, 34.1, 14.2, 14.1; ESI-HRMS(m / z) calcd C 16 H 18 Cl2N4NaO6S(M+Na + ), 487.0216; found, 487.0206.

[0058] Example 2

[0059] 2.1 Experimental Materials

[0060] 1. Virus

[0061] Enterovirus type 71 (EV-A71 Zhenjiang isolate).

[0062] 2. Cells

[0063] African green monkey kidney cells (Vero), culture conditions: DMEM+10% FBS+1% Pen-Strep; 37°C 5% CO2.

[0064] 3. Main reagents

[0065]

[0066]

[0067] 2.2 Research Methods

[0068] 1. Concentration-dependent anti-EV-A71 activity of compound 22d

[0069] (1) Take well-growing, uncontaminated Vero cells and inoculate them in a 12-well plate with complete medium, 4 × 10 cells per well. 5 The cells were cultured in a 37°C incubator containing 5% CO2 for 12-16 hours until they grew to a monolayer.

[0070] (2) The original culture medium was discarded, and compound 22d was diluted to 5 concentrations with complete culture medium in a 2-fold dilution manner. 1 mL of compound 22d diluted with 5% FBS culture medium was added to the cells, and the cells were incubated at 37°C with 5% CO2 for 4 h. The culture medium was discarded, and EV-A71 was infected at an MOI of 0.001, and 1 mL of compound 22d diluted with FBS-free culture medium (concentration as above) was added; incubated at 37°C for 2 h, free viruses were washed away, and 1 mL of 2% FBS culture medium was added to dilute compound 22d (concentration as above); a virus control without compound treatment was set up. The cells were cultured in a constant temperature incubator at 37°C with 5% CO2 for 48 h.

[0071] (3) RT-qPCR: After 48 h of cell culture, the supernatant was discarded, and the total RNA in the cells was extracted using a nucleic acid extraction kit and then reversed for RT-qPCR to simultaneously detect the intracellular viral load and the expression of the GAPDH gene. The relative expression of the virus in the sample well was calculated based on the relative quantitative amplification region (EV-A715'UTR).

[0072] qPCR Primers:

[0073]

[0074]

[0075] (4) CCK-8 assay for cytotoxicity of compound 22d: Vero cells with good growth status were inoculated in a 96-well plate with complete medium, and 1×10 4 , cultured in a constant temperature incubator containing 5% CO2 at 37°C for 12-16h; discarded the old culture medium, diluted compound 22d with complete culture medium according to the 5-fold dilution method, and added 100μL to each well. At the same time, set normal growth cells as cell control and cell-free group as blank zero well; after compound 22d treatment for 48h, added 10μL CCK-8 to each well and protected from light for 4h, and used an ELISA reader to detect the absorbance at 450nm, and calculated the cell survival rate of the compound 22d treatment group relative to the cell control group. 2.3 Test results: concentration-dependent anti-EV-A71 activity

[0076] Compound Name concentration Inhibition rate of EV-A71 22a 25μM 40.07％ 22b 25μM 9.19％ 22d 25μM 93.76％ 22e 25μM 60.47％ 22f 25μM 57.09％ 22g 25μM -2.10％ 22h 25μM 55.44％ 22i 25μM 52.22％ 22j 25μM 7.32％ 22l 25μM 41.81％ 22m 25μM 93.04％ 22n 25μM 96.78％ 22o 25μM 47.72％ 22q 25μM 3.79％ 22r 25μM 53.84％ 22s 25μM 54.62％ 22t 25μM 73.11％ 22v 25μM 93.18％ 23a 25μM 60.35％ 23i 25μM 81.86％ 23l 25μM 67.17％ 23s 25μM 66.45％ 23t 25μM 31.66％

[0077] Compound 5μM rescreening results (compounds with an inhibition rate of EV-A71 greater than 50% at 25μM concentration and compounds with high cytotoxicity at 25μM concentration):

[0078] Compound Name concentration Inhibition rate of EV-A71 22d 5μM 89.46％ 22m 5μM 55.52％ 22n 5μM 60.34％ 22t 5μM 75.72％ 22v 5μM 50.45％ 23a 5μM 84.54％ 23i 5μM 87.21％ 23l 5μM 76.59％ 23s 5μM 57.15％

[0079] The compound with the best inhibitory effect at 5 μM concentration

[0080]

[0081]

[0082] from Figure 1 It can be seen that (±)-22d compound EC 50 =0.2877μM,CC 50 >200μM. The selectivity coefficient of the two is SI=CC 50 / EC 50 >695.16.

[0083] The antiviral activity experiment found that the compound 22d of the present invention has good anti-EV-A71 activity and has good prospects in the preparation of drugs for treating hand, foot and mouth disease and related symptoms.

[0084] 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 compound, characterized in that: The general structure is as follows: Where: * represents chiral center, each general structure includes racemic, 3S, 4R-configuration and 3R, 4S-configuration; R 1 , R 2 Each is independently selected from hydrogen, halogen, C1-C4 alkoxy, benzyl, amino, C1-C4 alkylamino, di-C1-C4 alkylamino; R 3 Selected from alkyl acetate.

2. The five-membered sulfur heterocyclic nucleoside compound according to claim 1, characterized in that: R 1 is hydrogen, halogen, C1-C4 alkoxy, benzyl, amino, C1-C4 alkylamino, or di-C1-C4 alkylamino; R 2 is hydrogen, chlorine; R 3 It is ethyl acetate, methyl acetate, and isopropyl acetate.

3. The five-membered sulfur heterocyclic nucleoside compound according to claim 2, characterized in that: R 1 is dimethylamino, R 2 is hydrogen, R 3 It is ethyl acetate.

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

5. Use of the five-membered sulfur heterocyclic nucleoside compound according to any one of claims 1 to 4 in the preparation of antiviral drugs.

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

7. An active drug against hand, foot and mouth virus (EV-A71), characterized in that: The active ingredients thereof include the compound according to any one of claims 1 to 4 and its corresponding pharmaceutically acceptable salt.

8. The active drug against hand, foot and mouth virus (EV-A71) according to claim 7, characterized in that: The pharmaceutically acceptable salts include salts formed by acyclic nucleoside compounds and organic or inorganic acids; the organic acid is selected from one or more of malic acid, lactic acid, camphorsulfonic acid, citric acid, fumaric acid or oxalic acid, and the inorganic acid is selected from one or more of phosphoric acid, hydrohalic acid, sulfuric acid or nitric acid.

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

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

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