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). Research results show that in tests for resisting hand and foot EV-A71 virus and CVA16 virus, the five-membered sulfur-containing heterocyclic nucleoside lead compounds 22d and 23d have relatively strong activity for resisting the EV-A71 virus and the CVA16 virus, and a structural reference is provided for antiviral candidate drug molecules.

Description

Technical Field

[0001] The present invention relates to a five-membered sulfur-containing heterocyclic nucleoside compound applied to drugs with anti-hand, foot and mouth virus activity, belonging to the field of pharmaceutical chemistry. Background Art

[0002] Due to their unique molecular structures, nucleoside drugs are currently commonly used in the fields of antiviral and anti-tumor. Antiviral nucleoside drugs include acyclovir, ribavirin, penciclovir, desciclovir, entecavir, sofosbuvir, zidovudine, lamivudine, etc., and anti-tumor nucleoside drugs include cytarabine, floxuridine, decitabine, etc.

[0003] Hand, foot and mouth disease is a kind of enterovirus, an infectious disease caused by enterovirus, mostly occurring in children under 5 years old, which can cause herpes in hands, feet, mouth and other parts. A small number of children may cause complications such as myocarditis, pulmonary edema, aseptic meningoencephalitis, etc. Enteroviruses EV71 and CVA16 are the most common, and other respiratory viruses such as adenovirus and syncytial virus can also be combined.

[0004] At present, five-membered sulfur heterocyclic nucleoside compounds are an important class of antiviral drugs that have been marketed. In the past, five-membered sulfur heterocyclic nucleosides were mainly used for anti-HIV and anti-HBV, but there are relatively few studies on the anti-hand, foot and mouth virus (EV-A71 and CVA16) activity of five-membered sulfur heterocyclic nucleoside compounds. The innovative research on five-membered sulfur heterocyclic nucleoside drugs for anti-hand, foot and mouth virus (EV-A71 and CVA16) 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, on the basis of the previous research of the research group, the present invention has developed nucleoside compounds with more functional structures and disclosed five-membered sulfur heterocyclic nucleoside lead compounds with anti-hand, foot and mouth virus (EV-A71 and CVA16) activity. The nucleoside compounds of the present invention synthesized a series of five-membered sulfur heterocyclic nucleoside compounds through reactions such as two-step Michael addition reaction. This type of structure includes three structural types: racemate, 3S,4R-configuration and 3R,4S-configuration.

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

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

[0008] Further, in the above technical solution, R 1 、R 2Each independently selected from hydrogen, halogen, C1-C4 alkoxy, benzyl, amino, C1-C4 alkylamino, bis-C1-C4 alkylamino; R 3 Selected from alkyl formates or alkyl acetates.

[0009] Furthermore, in the above technical solution, R 1 Is hydrogen, halogen, C1-C4 alkoxy, benzyl, amino, C1-C4 alkylamino, bis-C1-C4 alkylamino; R 2 Is hydrogen, chlorine; R 3 Is ethyl formate group, methyl formate group, isopropyl formate group, ethyl acetate group, methyl acetate group, isopropyl acetate group.

[0010] Furthermore, in the above technical solution, R 1 Is dimethylamino, R 2 Is chlorine, R 3 Is ethyl formate or ethyl acetate group.

[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] Furthermore, under the most preferred conditions, the structure of the five-membered thiaheterocyclic nucleoside compound is

[0013] The present invention also provides the use of the five-membered thiaheterocyclic nucleoside compound in the preparation of antiviral drugs.

[0014] Furthermore, in the above technical solution, the antiviral is against hand, foot and mouth disease viruses (EV-A71 and CVA16).

[0015] The present invention also provides the use of the five-membered thiaheterocyclic nucleoside compound in the preparation of drugs for the treatment of hand, foot and mouth disease.

[0016] The present invention also provides a pharmaceutical composition containing the above five-membered thiaheterocyclic nucleoside compound, and its active ingredients include the above chiral five-membered thiaheterocyclic nucleoside compound and its pharmaceutically acceptable salts.

[0017] Furthermore, in the above technical solution, the pharmaceutically acceptable salts include salts formed by acyclic nucleoside compounds and organic acids or inorganic acids. The organic acids are selected from one or more of malic acid, lactic acid, camphorsulfonic acid, citric acid, fumaric acid or oxalic acid, and the inorganic acids are selected from one or more of phosphoric acid, hydrohalic acid, sulfuric acid or nitric acid.

[0018] Furthermore, in the above technical solution, the antiviral activity is against hand, foot and mouth disease viruses (EV-A71 and CVA16) activity.

[0019] To study the antiviral activity of five-membered sulfur heterocyclic nucleoside compounds, a series of five-membered sulfur heterocyclic nucleoside compounds with different substituents at the 2-position / 6-position of purine and the 1'-position of the sugar ring were synthesized through reactions such as two-step Michael addition reaction.

[0020] The experimental results showed that the chiral five-membered sulfur-containing heterocyclic nucleoside compound 22d had in vitro cell IC 50 values of 0.2877 μM and less than 1.6 nM against hand, foot and mouth disease viruses (EV-A71 and CVA16), respectively. The above results provided the basic structural unit of chiral five-membered sulfur heterocyclic nucleoside compounds for the design of drugs with anti-hand, foot and mouth disease virus (EV-A71 and CVA16) activity. On this basis, further fine structural modification and transformation could be carried out.

[0021] Through the research on the synthesis and in vitro and in vivo antitumor activity evaluation of chiral five-membered sulfur heterocyclic nucleosides, lead compounds or preferred drug molecules with good activity against hand, foot and mouth disease viruses (EV-A71 and CVA16) were obtained, providing reasonable synthetic strategies and methods for five-membered sulfur heterocyclic nucleoside drugs, and laying a good foundation for the development of antiviral five-membered sulfur heterocyclic nucleoside drugs with independent intellectual property rights. Description of the Drawings

[0022] Figure 1 It is the anti-EV-A71 virus activity diagram of 22d (the first time);

[0023] Figure 2 It is the anti-EV-A71 virus activity diagram of 22d (the second time);

[0024] Figure 3 It is the anti-EV-A71 virus activity diagram of 23d;

[0025] Figure 4 It is the anti-CVA16 virus activity diagram of 22d. Detailed Description of the Invention

[0026] Example 1 Example 1:

[0027]

[0028] (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 °C, Reflux, N2. (c) 19, NaH, DCM, RT, N2.

[0029] Synthesis of Compounds 22s, 22t, 23s and 23t:

[0030]

[0031] Reagents and Conditions: (a) Pd / C, H2, MeOH, Rt.

[0032] Synthesis of Compounds 22u and 22v:

[0033]

[0034] Reagents and Conditions: (a) m-CPBA (1 equiv.), DCM, Rt. (b) m-CPBA (2 equiv.). DCM, Rt.

[0035] Representative experimental procedure: In a reaction flask, 1,4-dithio-2,5-diol 18 (20 mmol, 3.0448 g), (ethoxycarbonylmethylene)triphenylphosphorane (24 mmol, 8.3609 g) and dichloromethane (100 mL) were successively added, and the reaction was stirred at room temperature for 4 h. After the reaction was completed as detected by TLC, it was concentrated and purified to obtain Compound 19 (colorless oil).

[0036] When there are different substituents at the 6-position of purine, the synthesis method is similar. Taking 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 successively added to a round-bottom flask. After stirring evenly, glacial acetic acid (4 mmol, 0.6 mL) and ethyl propiolate (24 mmol, 2.42 mL) were then added, and the reaction was stirred at room temperature for 12 h. After the reaction was completed as detected by TLC, it was extracted, dried, concentrated, and separated and purified to obtain Compound 21 (white solid).

[0037] 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. Taking 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 flask 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. The solution was quenched with ammonium chloride aqueous solution, 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 series are racemic. Characterization data of representative compounds are as follows:

[0038] (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),2.9 4-2.90(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: 1HNMR(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=16.2,2.4 Hz,1H),2.47(t,J=10.8 Hz,1H),1.60 - 1.54(m,1H),1.19(t,J=7.2 Hz,3H),1.12(t,J=7.2 Hz,3H); 13 C NMR(150 MHz,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)calcd C 16 H 19 ClN4NaO4S(M + Na + ),421.0708;found,421.0707.

[0039] (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); 1313C NMR (150 MHz, CDCl3) δ 171.0, 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 for C 16 H 19 BrN4NaO4S (M + Na + ), 465.0203; found, 465.0200. 23b: 1 1H NMR (600 MHz, CDCl3) δ 8.80 (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, 3.0 Hz, 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 13C NMR (150 MHz, CDCl3) δ 171.0, 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 for C 16 H 20 BrN4O4S (M + H + ), 443.0383; found, 443.0390.

[0040] (Rac) ethyl-4-(2-ethoxy-2-oxoethyl)-3-(6-(propylthio)-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate (22c, 23c). Prepared analogously to compounds 22a and 23a. Colorless oil. 77.2 mg, 88% yield. 3a: 11H NMR (600 MHz, CDCl3) δ 8.66 (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); 13 13C NMR (150 MHz, CDCl3) δ 171.2, 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) calcd for C 19 H 26 N4NaO4S2 (M + Na + ), 461.1, 288; found, 461.1282. 3b: 1 1H NMR (600 MHz, CDCl3) δ 8.62 (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 = 13.2 Hz, 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 13C NMR (150 MHz, CDCl3) δ 171.3, 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 for C 19 H 26 N4NaO4S2 (M + Na +), 461.1288; found, 461.1285.

[0041] (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 °C. Prepared analogously to compounds 22a and 23a. 22d: 1 1H NMR (600 MHz, CDCl3) δ 8.27 (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, 6.0 Hz, 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 = 7.2 Hz, 3H), 1.09 (t, J = 7.2 Hz, 3H); 13 13C NMR (150 MHz, CDCl3) δ 171.4, 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 1H NMR (400 MHz, CDCl3) δ 8.36 (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 = 10.8 Hz, 1H), 1.64 - 1.55 (m, 1H), 1.19 (t, J = 7.2 Hz, 3H), 1.11 (t, J = 7.2 Hz, 3H); 13CNMR(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.

[0042] (Rac)ethyl-3-(6-(diethylamino)-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)tetrahydro-othiophene-3-carboxylate(22e,23e). Prepared analogously to compounds 22a and 23a. 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: 1HNMR(600 MHz, CDCl3) δ 8.36 (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 = 7.2 Hz, 6H), 1.21 - 1.18 (m, 3H), 1.14 (t, J = 7.2 Hz, 3H); 13 C NMR(150 MHz, CDCl3) δ 171.8, 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) calcd C 20 H 30 N5O4S (M + H + ), 436.2013; found, 436.2019.

[0043] (Rac) ethyl - 4 - (2 - ethoxy - 2 - oxoethyl) - 3 - (6 - methoxy - 9H - purin - 9 - yl) tetrahydrothioph - ene - 3 - carboxylate (22f, 23f). Prepared analogously to compounds 22a and 23a. Colorless oil. 66.3 mg, 84% yield. 22f: 1 H NMR(600 MHz, CDCl3) δ 8.48 (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, 10.2 Hz, 1H), 1.25 (t, J = 7.2 Hz, 3H), 1.07 (t, J = 7.2 Hz, 3H); 1313C NMR (150 MHz, CDCl3) δ 171.2, 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 for C 17 H 22 N4NaO5S (M + Na + ), 417.1203; found, 417.1202. 23f: 1 1H NMR (600 MHz, CDCl3) δ 8.55 (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 = 10.8 Hz, 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 13C NMR (150 MHz, CDCl3) δ 171.4, 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) calcd for C 17 H 22 N4NaO5S (M + Na + ), 417.1203; found, 417.1203.

[0044] (Rac) ethyl-4-(2-ethoxy-2-oxoethyl)-3-(6-ethoxy-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate (22g, 23g). Prepared analogously to compounds 22a and 23a. Colorless oil. 67.8 mg, 83% yield. 22g: 11H NMR (600 MHz, CDCl3) δ 8.45 (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); 13 13C NMR (150 MHz, CDCl3) δ 171.2, 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) calcd for C 18 H 24 N4NaO5S (M + Na + ), 431.1360; found, 431.1364. 23 g: 1 1H NMR (600 MHz, CDCl3) δ 8.54 (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 13C NMR (150 MHz, CDCl3) δ 171.4, 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 for C 18 H 24 N4NaO5S (M + Na + ), 431.1360; found, 431.1363.

[0045] (Rac)ethyl-3-(6-(benzyloxy)-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)tetrahydrothiophene-3-carboxylate (22h, 23h). Prepared analogously to compounds 22a and 23a. Colorless oil. 81.9 mg, 87% yield. 22h: 1 1H NMR (600 MHz, CDCl3) δ 8.49 (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=7.2 Hz, 3H), 1.09 (t, J=7.2 Hz, 3H); 13 13C NMR (150 MHz, CDCl3) δ 171.2, 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: 1HNMR(600 MHz, CDCl3) δ 8.55(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); 13 C NMR(150 MHz, CDCl3) δ 171.4, 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.

[0046] (Rac)ethyl - 4 - (2 - ethoxy - 2 - oxoethyl) - 3 - (6 - (pyrrolidin - 1 - yl) - 9H - purin - 9 - yl)tetrahyd - rothiophene - 3 - carboxylate(22i, 23i). Prepared analogously to compounds 22a and 23a. Colorless oil. 77.2 mg, 89% yield. 22i: 11H NMR (600 MHz, CDCl3) δ 8.29 (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 13C NMR (150 MHz, CDCl3) δ 171.4, 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) calcd for C 20 H 28 N5O4S (M + H + ), 434.1857; found, 434.1855. 23i: 1 1H NMR (400 MHz, CDCl3) δ 8.35 (s, 1H), 8.26 (s, 1H), 4.32 - 4.00 (m, 6H), 3.82 - 3.52 (m, 4H), 3.48 (d, J = 12.8 Hz, 1H), 3.42 - 3.35 (m, 1H), 3.20 (dd, J = 16.4, 2.4 Hz, 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 13C NMR (150 MHz, CDCl3) δ 171.7, 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) calcd for C 20 H 28 N5O4S (M + H + ), 434.1857; found, 434.1859.

[0047] (Rac)ethyl-4-(2-ethoxy-2-oxoethyl)-3-(6-(piperidin-1-yl)-9H-purin-9-yl)tetrahydro-thiophene-3-carboxylate(22j,23j). Prepared analogously to compounds 22a and 23a. Colorless oil. 75.2 mg, 84% yield. 22j: 1 1H NMR(400 MHz, CDCl3) δ 8.25(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); 13 13C NMR(150 MHz, CDCl3) δ 171.4, 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 1H NMR(600 MHz, CDCl3) δ 8.36(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); 1313C NMR (150 MHz, CDCl3) δ 171.7, 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.

[0048] (Rac) ethyl-4-(2-ethoxy-2-oxoethyl)-3-(6-morpholino-9H-purin-9-yl)tetrahydrothio-phene-3-caroxylate (22k, 23k). Prepared analogously to compounds 22a and 23a. Colorless oil. 81.8 mg, 91% yield. 22k: 1 1H NMR (600 MHz, CDCl3) δ 8.28 (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 13C NMR (150 MHz, CDCl3) δ 171.3, 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: 11H NMR (600 MHz, CDCl3) δ 8.39 (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 = 12.6 Hz, 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 13C NMR (150 MHz, CDCl3) δ 171.6, 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.

[0049] (Rac) ethyl-4-(2-ethoxy-2-oxoethyl)-3-(6-phenyl-9H-purin-9-yl)tetrahydrothiophene-3-carboxylate (22l, 23l). Prepared analogously to compounds 22a and 23a. Colorless oil. 57.2 mg, 65% yield. 22l: 1 1H NMR (600 MHz, CDCl3) δ 8.98 (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); 1313C NMR (150 MHz, CDCl3) δ 171.3, 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 for C 22 H 25 N4O4S (M + H + ), 441.1591; found, 441.1586. 23l: 1 1H NMR (600 MHz, CDCl3) δ 8.95 (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 = 7.2 Hz, 3H); 13 13C NMR (150 MHz, CDCl3) δ 171.4, 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 for C 22 H 25 N4O4S (M + H + ), 441.1591; found, 441.1590.

[0050] (Rac) ethyl-3-(2-chloro-6-(pyrrolidin-1-yl)-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)-tetrahydrothiophene-3-carboxylate (22m, 23m). Prepared analogously to compounds 22a and 23a. Colorless oil. 81.4 mg, 87% yield. 22m: 11H NMR (600 MHz, CDCl3) δ 7.86 (s, 1H), 4.23 - 4.06 (m, 2H), 4.16 - 4.07 (m, 4H), 3.86 (d, J = 12.0 Hz, 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, 3.0 Hz, 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 13C NMR (150 MHz, CDCl3) δ 171.2, 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: 1 1H NMR (600 MHz, CDCl3) δ 8.33 (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 13C NMR (150 MHz, CDCl3) δ 171.6, 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) calcd C 20 H 27 ClN5O4S (M + H+ ), 468.1467; found, 468.1466.

[0051] (Rac)ethyl-3-(2-chloro-6-(piperidin-1-yl)-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)-tetrahydrothiophene-3-carboxylate (22n, 23n). Prepared analogously to compounds 22a and 23a. Colorless oil. 85.8 mg, 89% yield. 22n: 1 H NMR (600 MHz, CDCl3) δ 7.88 (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) δ 171.3, 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) calcd C 21 H 28 ClN5NaO4S (M + Na + ), 504.1443; found, 504.1449. 23n: 1 H NMR (600 MHz, CDCl3) δ 8.35 (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); 1313C NMR (150 MHz, CDCl3) δ 171.6, 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) calcd for C 21 H 28 ClN5NaO4S (M + Na + ), 504.1443; found, 504.1446.

[0052] (Rac) ethyl-3-(2,6-dichloro-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)tetrahydrothiophene-3-carboxylate (22o, 23o). Prepared analogously to compounds 22a and 23a. Colorless oil. 37.3 mg, 43% yield. 22o: 1 1H NMR (400 MHz, CDCl3) δ 8.33 (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 13C NMR (150 MHz, CDCl3) δ 171.0, 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 for C 16 H 18 Cl2N4NaO4S (M + Na + ), 455.0318; found, 455.0314. 23o: 11H NMR (600 MHz, CDCl3) δ 8.77 (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 = 10.8 Hz, 1H), 1.63 - 1.53 (m, 1H), 1.21 (dd, J = 16.2, 7.2 Hz, 6H); 13 13C NMR (150 MHz, CDCl3) δ 170.9, 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 C 16 H 18 Cl2N4NaO4S (M + Na + ), 455.0318; found, 455.0323.

[0053] (Rac) methyl-3-(6-chloro-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)tetrahydrothioph-ene-3-carboxylate (22p, 23p). Prepared analogously to compounds 22a and 23a. Colorless oil. 57.7 mg, 75% yield. 22p: 1 1H NMR (600 MHz, CDCl3) δ 8.72 (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 13C NMR (150 MHz, CDCl3) δ 171.1, 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) calcd C15 H 17 ClN4NaO4S(M+Na + ),407.0551;found,407.0551.23p: 1 1H NMR(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); 13 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)calcd for C 15 H 17 ClN4NaO4S(M+Na + ),407.0551;found,407.0557.

[0054] (Rac)isopropyl - 3-(6 - chloro - 9H - purin - 9 - yl)-4-(2 - ethoxy - 2 - oxoethyl)tetrahydrothiophene - 3 - carboxylate(22q,23q). Prepared analogously to compounds 22a and 23a. Colorless oil. 74.3 mg, 90% yield. 22q: 11H NMR (600 MHz, CDCl3) δ 8.69 (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 13C NMR (150 MHz, CDCl3) δ 171.1, 166.9, 152.0 151.8, 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) calcd C 17 H 21 ClN4NaO4S (M + Na + ), 435.0864; found, 435.0858. 23q: 1 1H NMR (600 MHz, CDCl3) δ 8.77 (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 = 13.2 Hz, 1H), 3.43 (dd, J = 10.8, 6.6 Hz, 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 13C NMR (150 MHz, CDCl3) δ 171.1, 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.

[0055] (Rac)ethyl-3-(6-chloro-2-fluoro-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)tetrahydr-othiophene-3-carboxylate(22r,23r). Prepared analogously to compounds 22a and 23a. Colorless oil. 35.0 mg, 42% yield. 22r: 1 1H NMR(400 MHz, CDCl3) δ 8.33(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 13C NMR(100 MHz, CDCl3) δ 171.0, 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 19F NMR(565 MHz, CDCl3) δ - 48.32; ESI - HRMS(m / z) calcd C 16 19 18 H + ClF19N4NaO4S(M + Na 1 ), 439.0614; found, 439.0607. 23r: 13 1H NMR(600 MHz, CDCl3) δ 8.76(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); C-F 13C NMR(150 MHz, CDCl3) δ 170.9, 167.0, 156.9(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 19F NMR (565 MHz, CDCl3) δ -48.52. ESI-HRMS (m / z) calcd C 16 H 18 ClFN4NaO4S (M + Na + ), 439.0614; found, 439.0612.

[0056] (Rac) ethyl-4-(2-ethoxy-2-oxoethyl)-3-(9H-purin-9-yl)tetrahydrothiophene-3-carboxylate (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) at room temperature. When compound 22s 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 22s as a colorless oil in 23.7 mg, 65% yield. 23s was prepared analogously to 22s. Colorless oil. 22.9 mg, 63% yield. 22s: 1 1H NMR (400 MHz, CDCl3) δ 9.18 (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); 1313C NMR (100 MHz, CDCl3) δ 171.2, 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 for C 16 H 20 N4NaO4S (M + Na + ), 387.1097; found, 387.1091. 23s: 1 1H NMR (400 MHz, CDCl3) δ 9.07 (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 13C NMR (100 MHz, CDCl3) δ 171.0, 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 for C 16 H 20 N4NaO4S (M + Na + ), 387.1097; found, 387.1090.

[0057] (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) at room temperature. When compound 22o 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 22t as a colorless oil in 14.8 mg, 37% yield. 23t was prepared analogously to 22t. Colorless oil. 12.7 mg, 32% yield. 22t: 1 1H NMR(400 MHz, CDCl3) δ 9.03(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 13C NMR(100 MHz, CDCl3) δ 171.1, 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) calcd C 16 H 19 ClN4NaO4S(M + Na + ), 421.0708; found, 421.0708. 23t: 11H NMR (400 MHz, CDCl3) δ 9.00 (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 = 10.8 Hz, 1H), 1.59 (q, J = 11.2, 16.0 Hz, 1H), 1.23 - 1.15 (m, 6H); 13 13C NMR (100 MHz, CDCl3) δ 171.1, 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.

[0058] (Rac) ethyl - 3 - (2,6 - dichloro - 9H - purin - 9 - yl) - 4 - (2 - ethoxy - 2 - oxoethyl)tetrahydrothiophene - 3 - carboxylate 1 - oxide (22u). Compounds 22a (86.4 mg, 0.2 mmol), m - chloroperbenzoic acid (34.5 mg, 0.2 mmol) and DCM (2 ml) 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 colorless oil in 51.2 mg, 57% yield. 2u: 11H NMR (400 MHz, CDCl3) δ 8.46 (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, 16.4 Hz, 1H), 1.32 (t, J = 7.2 Hz, 3H), 1.16 (t, J = 7.2 Hz, 3H); 13 13C NMR (100 MHz, CDCl3) δ 177.7, 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 12 18 12Cl2N4NaO5S (M + Na + +), 471.0267; found, 471.0258.

[0059] (Rac) ethyl-3-(2,6-dichloro-9H-purin-9-yl)-4-(2-ethoxy-2-oxoethyl)tetrahydrothiophene-3-carboxylate 1,1-dioxide (22v). Compounds 22a (86.4 mg, 0.2 mmol), m-chloroperbenzoic acid (69.0 mg, 0.4 mmol) and DCM (2 ml) 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 63.3 mg, 68% yield. mp: 118.7 - 120.1 °C. 11H NMR(400 MHz, CDCl3) δ 8.36 (s, 1H), 4.52 (d, J = 14.4 Hz, 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, 16.4 Hz, 1H), 1.31 - 1.19 (m, 6H); 13 13C NMR(100 MHz, 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.

[0060] Example 2

[0061] 2.1 Experimental materials

[0062] 1. Virus Enterovirus 71 (EV-A71 Zhenjiang isolate).

[0063] 2. Cells 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. Preliminary screening of the activity of compounds against EV-A71

[0069] (1) Take Vero cells in good growth condition and free of contamination, inoculate them into a 12-well plate with complete medium, 4 × 10 5 cells per well, and culture them in a constant temperature incubator at 37 °C with 5% CO2 for 12 - 16 h until they grow to confluence.

[0070] (2) Discard the original medium, dilute the compound with complete medium, add 1 mL of 10% FBS medium to the cells to dilute the compound, incubate at 37 °C in 5% CO₂ for 4 h, discard the medium, infect with EV-A71 at an MOI of 0.001, and add 1 mL of FBS-free medium to dilute the compound; incubate at 37 °C for 2 h, wash away the free virus, and add 1 mL of 2% FBS medium to dilute the compound; then set up a virus control without compound treatment. Incubate in a 37 °C constant temperature incubator with 5% CO₂ for 48 h.

[0071] (3) RT-qPCR: After culturing the cells for 48 h, discard the supernatant, use a nucleic acid extraction kit to extract the total RNA in the cells, reverse transcribe it, and then perform RT-qPCR to simultaneously detect the intracellular viral load and the expression level of the GAPDH gene, and calculate the relative viral expression level of the sample wells according to relative quantification. Amplification region (EV-A71 5’UTR).

[0072] qPCR primers:

[0073]

[0074] 2. Concentration-dependent anti-EV-A71 activity of compound 22d

[0075] (1) Take Vero cells in good growth condition and free of contamination, inoculate them with complete medium in a 12-well plate, with 4×10 5 cells per well, and incubate in a 37 °C constant temperature incubator with 5% CO₂ for 12 - 16 h until they grow to a monolayer.

[0076] (2) Discard the original medium, dilute compound 22d to 5 concentrations with complete medium in a 2-fold dilution manner, add 1 mL of 10% FBS medium to the cells to dilute compound 22d, incubate at 37 °C in 5% CO₂ for 4 h, discard the medium, infect with EV-A71 at an MOI of 0.001, and add 1 mL of FBS-free medium to dilute compound 22d (at the same concentration as above); incubate at 37 °C for 2 h, wash away the free virus, and add 1 mL of 2% FBS medium to dilute compound 22d (at the same concentration as above); then set up a virus control without compound treatment. Incubate in a 37 °C constant temperature incubator with 5% CO₂ for 48 h.

[0077] (3) RT-qPCR: The experimental method is the same as described above.

[0078] (4) Detect the cytotoxicity of compound 22d to cells by the CCK-8 method: Take Vero cells in good growth condition, inoculate them with complete medium in a 96-well plate, with 1×10 4Count them, culture in a 37°C constant temperature incubator containing 5% CO₂ for 12 - 16 h; discard the old medium, dilute compound 22d with complete medium by a 5-fold dilution method, add 100 μL to each well, and at the same time set normal growing cells as the cell control and the cell-free group as the blank zeroing well; treat with compound 22d for 48 h, add 10 μL of CCK-8 to each well and incubate in the dark for 4 h, detect the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the cell survival rate of the compound 22d treatment group relative to the cell control group.

[0079] 2.3 Detection results: Preliminary screening of compounds for inhibiting EV-A71 activity

[0080]

[0081]

[0082] Results of rescreening of compounds at 5 μM (compounds with an inhibition rate of EV-A71 greater than 50% at a concentration of 25 μM and compounds with high cytotoxicity at a concentration of 25 μM):

[0083] Compound Name Concentration Inhibition Rate against EV-A71 22d 5 μM 89.46％ 22m 5 μM 55.52％ 22n 5 μM 60.34％ 22t 5 μM 75.72％ 22v 5 μM 50.45％ 23a 5 μM 84.54％ 23i 5 μM 87.21％ 23l 5 μM 76.59％ 23s 5 μM 57.15％

[0084] The compound with the best inhibitory effect

[0085] Compound concentration-dependent anti-EV-A71 activity:

[0086]

[0087] Example 3

[0088] 3.1 Experimental materials

[0089] 1. Coxsackievirus type 16 (CVA16).

[0090] 2. Cells African green monkey kidney cells (Vero), culture conditions: DMEM + 10% FBS + 1% Pen-Strep; 37°C, 5% CO₂.

[0091] 3. Main reagents

[0092] DMEM medium (GIBCO, cat: C11875500BT);

[0093] Fetal bovine serum FBS (Gibco, cat: 10099-141);

[0094] Dual antibody Pen-Strep (10,000 U / Ml) (Gibco, cat: 15140122);

[0095] Nucleic acid extraction (Magnetic bead method tissue / cell / blood total RNA extraction kit, cat: DP661) Reverse transcriptase (HiScript II Q RT SuperMix for qPCR, cat: R223-01:)

[0096] qPCR enzyme (ChamQ SYBR Color qPCR MasterMix, cat: Q411-02 / 03) 3.2 Research methods

[0097] 1. Compound 22d concentration-dependent anti-CVA16 activity

[0098] (1) Take Vero cells in good growth condition and free of contamination, inoculate them in a 12-well plate with complete medium, 4×10 5 cells per well, and culture them in a constant temperature incubator at 37 °C with 5% CO2 for 12 - 16 h until they grow to confluence as a monolayer.

[0099] (2) Discard the original medium, dilute compound 22d into 5 concentrations with complete medium in a 2-fold dilution manner, add 1 mL of compound 22d diluted with 10% FBS medium to the cells, incubate at 37 °C with 5% CO2 for 4 h, discard the medium, infect with EV-A71 at MOI = 0.001, add 1 mL of compound 22d diluted with serum-free medium (the same concentration as above); incubate at 37 °C for 2 h, wash away the free virus, add 1 mL of compound 22d diluted with 2% FBS medium (the same concentration as above); then set up a virus control without compound treatment. Culture in a constant temperature incubator at 37 °C with 5% CO2 for 48 h.

[0100] (3) RT-qPCR: After culturing the cells for 48 h, discard the supernatant, use a nucleic acid extraction kit to extract the total RNA in the cells, reverse transcribe it and then perform RT-qPCR to simultaneously detect the viral load in the cells and the expression level of the GAPDH gene, and calculate the relative viral expression level of the sample wells according to relative quantification. Amplification region (CVA16 VP1).

[0101] qPCR primers:

[0102]

[0103]

[0104] 3.3 Detection results: Concentration-dependent anti-CVA16 activity

[0105]

[0106] It can be seen from Figure 1 that the anti-EV-A71 EC 50 of the (±)-22d compound is 0.2877 μM, CC50 >200 μM. The selectivity coefficient of the two is SI = CC 50 / EC 50 >695.16 (the first time).

[0107] From Figure 2 it can be seen that the anti-EV-A71 EC of the (±)-22d compound 50 = 0.1922 μM, CC 50 > 500 μM. The selectivity coefficient of the two is SI = CC 50 / EC 50 > 2601.45 (the second time).

[0108] From Figure 3 it can be seen that the anti-EV-A71 EC of the (±)-23d compound 50 = 0.2893 μM, CC 50 > 500 μM. The selectivity coefficient of the two is SI = CC 50 / EC 50 > 1728.30.

[0109] From Figure 4 it can be seen that the anti-CVA16 EC of the (±)-22d compound 50 < 1.6 nM, CC 50 > 500 μM. The selectivity coefficient of the two is SI = CC 50 / EC 50 > 312500.

[0110] Through antiviral activity experiments, it was found that the compounds 22d and 23d of the present invention have good anti-EV-A71 and anti-CVA16 activities, and have good prospects in the preparation of drugs for treating hand, foot and mouth disease and related symptoms.

[0111] 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. The descriptions in the above embodiments and the specification only illustrate the principles of the present invention. Without departing from the principles of the present invention, the present invention will have various changes and improvements. Any structural changes of the five-membered thiaheterocyclic nucleoside, and 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 formula structure is as follows: Wherein: * represents a chiral center, and each general formula structure includes three structures: a racemate, a 3S,4R-configuration, and a 3R,4S-configuration; R 1 , R 2 are each independently selected from hydrogen, halogen, C1-C4 alkoxy, benzyl, amino, C1-C4 alkylamino, and bis-C1-C4 alkylamino; R 3 is selected from alkyl formate or alkyl acetate.

2. The five-membered sulfur heterocyclic nucleoside compound according to claim 1, wherein: R 1 is hydrogen, halogen, C1-C4 alkoxy, benzyl, amino, C1-C4 alkylamino, bis-C1-C4 alkylamino; R 2 is hydrogen, chlorine; R 3 is ethyl formate group, methyl formate group, isopropyl formate group, ethyl acetate group, methyl acetate group, isopropyl acetate group.

3. The five-membered sulfur heterocyclic nucleoside compound according to claim 2, wherein: R 1 is dimethylamino, R 2 is hydrogen, R 3 is ethyl formate or ethyl acetate.

4. Use of the five-membered thiaheterocyclic nucleoside compound according to any one of claims 1-4 in the preparation of an antiviral drug.

5. Use of the chiral five-membered thioheterocyclic nucleoside lead compound according to claim 4 in the preparation of antiviral drugs, characterized in that: The antiviral activity is anti-hand, foot and mouth disease virus activity.

6. Use of the chiral five-membered sulfur heterocyclic nucleoside lead compound according to claim 5 in the preparation of antiviral drugs, characterized in that: The anti-hand, foot and mouth disease virus activity is anti-EV-A71 and CVA16 activity.

7. An active drug against hand, foot and mouth disease viruses EV-A71 and CVA16, characterized in that: Its active ingredient includes the compound according to any one of claims 1-4 and its corresponding pharmaceutically acceptable salts.

8. The active drug against hand, foot and mouth disease EV-A71 and CVA16 according to claim 7, wherein: The pharmaceutically acceptable salts include salts formed by acyclic nucleoside compounds and organic acids or inorganic acids; the organic acids are selected from one or more of malic acid, lactic acid, camphorsulfonic acid, citric acid, fumaric acid or oxalic acid, and the inorganic acids are selected from one or more of phosphoric acid, hydrohalic acid, sulfuric acid or nitric acid.

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

10. A pharmaceutical composition containing the compound according to any one of claims 1-4.

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