Preparation and use of a carbocyclic nucleoside derivative containing an ethylene group

Abstract

The present application relates to the technical field of carbon ring nucleosides containing ethylidene, liver-targeting prodrugs thereof and pharmaceutical compositions containing these nucleosides, and discloses preparation and application of carbon ring nucleoside derivatives containing ethylidene, including hepatitis B virus (HBV), HIV and other retroviruses, secondary disease states and conditions (cirrhosis and liver cancer) thereof, and hepatitis D virus (HDV), herpes simplex virus I and II (HSV-I and HSV-2), cytomegalovirus (CMV), varicella-zoster virus (VZV) and Epstein-Barr virus (EBV) and secondary cancers caused by them.

Description

Technical Field

[0001] This invention relates to carbocyclic nucleosides containing ethyl subunits, their liver-targeting prodrugs, and pharmaceutical compositions containing these nucleosides, as well as their application in the treatment or prevention of various viral infections and their secondary disease states and symptoms. Specifically, it relates to the preparation and application of a carbocyclic nucleoside derivative containing ethyl subunits. Background Technology

[0002] When humans are infected with the hepatitis B virus (HBV), they may develop acute or fulminant hepatitis, which can be fatal in severe cases; they may also develop chronic hepatitis, progressing to cirrhosis, and in some cases, even hepatocellular carcinoma. The estimated number of people infected with HBV worldwide is approximately 260 million, primarily concentrated in parts of Asia, Africa, and Eastern Europe. China, Southeast Asia, and sub-Saharan Africa are high-prevalence areas for hepatitis B.

[0003] HBV is an incomplete double-stranded DNA virus. During its life cycle, viral DNA replication requires reverse transcription, specifically the synthesis of rcDNA from pgRNA. However, human hepatocytes, the host of HBV, do not perform reverse transcription. Therefore, by inhibiting this stage of reverse transcription, compounds that inhibit HBV replication can be found, leading to the development of drugs for treating hepatitis B. Similarly, the human immunodeficiency virus (HIV), which causes AIDS, also replicates via reverse transcription. Many nucleoside derivatives that inhibit HBV or HIV replication have been developed.

[0004] Currently, many nucleoside derivative preparations are toxic to host cells and suffer from side effects or drug resistance due to long-term use. Therefore, effective therapies for viral infections such as HBV have not yet been established. In view of these circumstances, the present invention aims to provide nucleoside derivatives with antiviral activity and low toxicity to host cells. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides a method for preparing and applying a carbocyclic nucleoside derivative containing an ethyl subunit, thus solving the problems mentioned above.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution: preparation and application of a carbocyclic nucleoside derivative containing a ethyl subunit. This type of compound exhibits excellent anti-HBV virus activity and is not cytotoxic to HBV virus host cells.

[0009] ;

[0010] In general formula (I), the bases are selected from R is selected from H, methyl, F, Cl, Br, I, or R' is selected from H, C1-C6 alkyl, cyclopropyl, benzyl, C2-C18 fatty acyl or benzoyl; R" is selected from H, F, Cl, Br, I, OH, OMe, OEt, OPri, OBn, NH2 or NHR'.

[0011] R1 and R2 are independent and can be selected from H, C2-C17 fatty acyl group, C1-C18 alkyl or ether group, O-linked amino acid residue (D or L), or aminophosphate group. Alternatively, R1 and R2 together with their bonded oxygen atoms form a carbonate diester or phosphate diester group; R3 is an α-amino acid side chain; R4 is benzyl, C1-C20 straight-chain alkyl or ether, C3-C20 branched alkyl or ether, C3-C6 cycloalkyl, or C4-C6 heterocyclic alkyl.

[0012] In one embodiment, the compound of formula (I) has the structure of formula (II) and is a carbocyclic nucleoside containing an ethyl subunit:

[0013] ;

[0014] In another embodiment, the compound of formula (I) has the structure of formula (III) and is an aminophosphate containing an ethyl subunit carbocyclic nucleoside:

[0015] ;

[0016] R3 is an α-amino acid side chain, such as glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine; R4 is benzyl, isopropyl, tert-butyl, or cyclobutyl ether.

[0017] In another embodiment, the carbocyclic nucleoside compound containing an ethyl subunit, its salt, or a solvate of the compound or its salt, wherein the base is selected from uracil or thymine analogs:

[0018] ;

[0019] In another embodiment, the base of the carbocyclic nucleoside compound containing an ethyl subunit, its salt, or a solvate of the compound or its salt is selected from the following cytosine analogs:

[0020] ;

[0021] In another embodiment, the bases of the carbocyclic nucleoside compound containing the ethyl subunit, its salt, or a solvate of the compound or its salt are selected from the following adenine analogs:

[0022] ;

[0023] In another embodiment, the base of the carbocyclic nucleoside compound containing the ethyl subunit, its salt, or a solvate of the compound or its salt is selected from the following guanine analogs:

[0024] ;

[0025] In another embodiment, the carbocyclic nucleoside compound containing an ethyl subunit, its salt, or a solvate of the compound or its salt is selected from, but not limited to, the following carbocyclic nucleoside compounds containing an ethyl subunit:

[0026] ;

[0027] ; ;

[0028] ; ; ; ; ;

[0029] ;

[0030] ;

[0031] ;

[0032] ;

[0033] ; ; ; ; ;

[0034] Another aspect of the application relates to a pharmaceutical composition comprising a carbocyclic nucleoside compound of formula (I) containing an ethyl subunit, a deuterated thereof, a salt thereof, a solvate of said compound or a salt thereof.

[0035] Another aspect of this application relates to a therapeutic agent for treating or preventing viral infections, useful in a variety of viral infections and their secondary disease states and conditions, including HIV, hepatitis B virus (HBV), human T-cell cytotoxic virus types I, II, and V (HTLV-I, II, V), human foamy virus, and other retroviruses and their secondary disease states and conditions (cirrhosis and liver cancer), as well as hepatitis D virus (HDV), herpes simplex virus I and II (HSV-I and HSV-2), cytomegalovirus (CMV), varicella-zoster virus (VZV), and Epstein-Barr virus (EBV), including drug-resistant viruses and other mutant forms of these viruses.

[0036] Another aspect of this application relates to a method for treating or preventing hepatitis B. The method comprises administering to a subject requiring treatment a therapeutically effective amount of a compound of formula (I)-(III), a deuterated form thereof, a salt thereof, or a solvate of said compound or a salt thereof.

[0037] (III) Beneficial Effects

[0038] Compared with the prior art, the present invention provides a method for preparing and applying a carbocyclic nucleoside derivative containing an ethyl subunit, which has the following beneficial effects:

[0039] The preparation and application of carbocyclic nucleoside derivatives containing ethyl subunits: Carbocyclic nucleoside derivatives with ethyl subunits have a good ability to inhibit the replication of hepatitis B virus. Some compounds have better in vitro antiviral activity than the clinical drug lamivudine, and have further development prospects. Detailed Implementation

[0040] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0041] Preparation and application of a carbocyclic nucleoside derivative containing an ethyl subunit: This type of compound exhibits excellent anti-HBV virus activity and is not cytotoxic to HBV virus host cells.

[0042] ;

[0043] In general formula (I), the bases are selected from R is selected from H, methyl, F, Cl, Br, I, or R' is selected from H, C1-C6 alkyl, cyclopropyl, benzyl, C2-C18 fatty acyl or benzoyl; R" is selected from H, F, Cl, Br, I, OH, OMe, OEt, OPri, OBn, NH2 or NHR'.

[0044] R1 and R2 are independent and can be selected from H, C2-C17 fatty acyl group, C1-C18 alkyl or ether group, O-linked amino acid residue (D or L), or aminophosphate group. Alternatively, R1 and R2 together with their bonded oxygen atoms form a carbonate diester or phosphate diester group; R3 is an α-amino acid side chain; R4 is benzyl, C1-C20 straight-chain alkyl or ether, C3-C20 branched alkyl or ether, C3-C6 cycloalkyl, or C4-C6 heterocyclic alkyl.

[0045] In one embodiment, the compound of formula (I) has the structure of formula (II) and is a carbocyclic nucleoside containing an ethyl subunit:

[0046] ;

[0047] In another embodiment, the compound of formula (I) has the structure of formula (III) and is an aminophosphate containing an ethyl subunit carbocyclic nucleoside:

[0048] ;

[0049] R3 is an α-amino acid side chain, such as glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine; R4 is benzyl, isopropyl, tert-butyl, or cyclobutyl ether.

[0050] In another embodiment, the carbocyclic nucleoside compound containing an ethyl subunit, its salt, or a solvate of the compound or its salt, wherein the base is selected from uracil or thymine analogs:

[0051] ;

[0052] In another embodiment, the carbocyclic nucleoside compound containing an ethyl subunit, its salt, or a solvate of the compound or its salt, wherein the base is selected from the following cytosine analogs:

[0053] ;

[0054] In another embodiment, the carbocyclic nucleoside compound containing an ethyl subunit, its salt, or a solvate of the compound or its salt, wherein the base is selected from the following adenine analogs:

[0055] ;

[0056] In another embodiment, the carbocyclic nucleoside compound containing an ethyl subunit, its salt, or a solvate of the compound or its salt, wherein the base is selected from the following guanine analogs:

[0057] ;

[0058] In another embodiment, the carbocyclic nucleoside compound containing an ethyl subunit, its salt, or a solvate thereof, is selected from, but not limited to, the following carbocyclic nucleoside compounds containing an ethyl subunit:

[0059] ;

[0060] ; ;

[0061] ; ; ; ; ;

[0062] ;

[0063] ;

[0064] ;

[0065] ;

[0066] ; ; ; ; ;

[0067] Another aspect of the application relates to a pharmaceutical composition comprising a carbocyclic nucleoside compound of formula (I) containing an ethyl subunit, a deuterated thereof, a salt thereof, a solvate of the compound or a salt thereof.

[0068] Another aspect of this application relates to a therapeutic agent for treating or preventing viral infections, useful in a variety of viral infections and their secondary disease states and conditions, including HIV, hepatitis B virus (HBV), human T-cell cytotoxic virus types I, II, and V (HTLV-I, II, V), human foamy virus, and other retroviruses and their secondary disease states and conditions (cirrhosis and liver cancer), as well as hepatitis D virus (HDV), herpes simplex virus I and II (HSV-I and HSV-2), cytomegalovirus (CMV), varicella-zoster virus (VZV), and Epstein-Barr virus (EBV), including drug-resistant viruses and other mutant forms of these viruses.

[0069] Another aspect of this application relates to a method for treating or preventing hepatitis B. The method comprises administering to a subject requiring treatment a therapeutically effective amount of a compound of formula (I)-(III), a deuterated form thereof, a salt thereof, a compound, or a solvate of a salt thereof.

[0070] Example 1

[0071] ;

[0072] Intermediate 1

[0073] D-(+)-ribonucleolone (20.0 g, 0.151 mol) and dried pyridine (200 mL) were added to a 500 mL round-bottom flask. 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (53.2 mL, 0.166 mol) was slowly added dropwise at -10 °C. The mixture was heated to 25 °C and stirred overnight. After TLC detection showed complete reaction, methanol (20 mL) was added to terminate the reaction. The solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate (300 mL). The organic phase was washed with 5% citric acid aqueous solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was subjected to silica gel column chromatography (ethyl acetate: petroleum ether = 1:20) to give a colorless oily product 1 (47.6 g, 85%). 1H NMR (400 MHz, Chloroform-d) δ 4.66-4.60 (m, 1 H), 4.23-4.19 (m, 1 H), 4.14 (dd, J = 12.3, 3.6 Hz, 1 H), 3.93 (dd, J = 12.3, 6.6Hz, 1 H), 2.86 (dd, J = 17.3, 8.0 Hz, 1 H), 2.71 (dd, J = 17.3, 9.2 Hz, 1 H), 1.06 (m, 28 H).

[0074] Intermediate 2b

[0075] Compound 1 (16.0 g, 42.7 mmol) was dissolved in dry tetrahydrofuran (200 mL), and diisobutylaluminum hydride (1.5 M, 31.3 mL, 46.9 mmol) was slowly added dropwise at -78 °C. After the addition was complete, the mixture was stirred at low temperature for 1.5 hours. After the reaction was confirmed to be complete by TLC, propynyl magnesium bromide Grignard reagent (0.5 M, 213 mL, 107 mmol) was slowly added dropwise. The mixture was then naturally heated to 25 °C and stirred overnight. After the reaction was confirmed to be complete by TLC, saturated sodium potassium tartrate (200 mL) was slowly added to terminate the reaction. After stirring the mixture vigorously overnight, it was filtered under reduced pressure with diatomaceous earth and washed with ethyl acetate (50 mL x 3). The aqueous phases were extracted with ethyl acetate (100 mL x 2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was subjected to silica gel column chromatography (ethyl acetate: petroleum ether = 1:3) to give a pale yellow oil 2b (9.92 g, 56%). 1 H NMR (400 MHz, Chloroform-d) δ 4.58 (d, J = 6.3 Hz, 1 H), 4.19 (d, J = 11.6Hz, 1 H), 4.10 (ddd, J = 9.2, 6.8, 4.4 Hz, 2 H), 3.80 (dd, J = 11.7, 2.2 Hz,1 H), 3.48 (t, J = 8.5 Hz, 1 H), 2.74 (d, J = 9.4 Hz, 1 H), 2.18-2.06 (m, 2H), 1.79 (d, J = 2.1 Hz, 3 H), 1.13-0.96 (m, 28 H); 13 C NMR (101 MHz, Chloroform-d) δ 81.51, 80.20, 75.52, 68.04, 62.39, 59.99, 44.28, 17.75,17.49, 17.41, 17.35, 13.50, 13.36, 12.68, 12.55, 3.64.

[0076] Intermediate 3

[0077] Compound 2b (3.78 g, 9.07 mmol), EDC•HCl (2.09 g, 10.9 mmol), benzoic acid (1.22 g, 10.0 mmol), and DMAP (111 mg, 0.91 mmol) were dissolved in 40 mL of dichloromethane. Triethylamine (1.64 mL, 11.8 mmol) was slowly added dropwise at -20 °C. After the addition was complete, the reaction was stirred at room temperature for 16 hours. The reaction was quenched with water, and the aqueous phase was extracted twice with dichloromethane (20 mL x 2), dried over anhydrous sodium sulfate, and concentrated by filtration. The residue was dried and dissolved in 20 mL of dichloromethane. DMAP (94 mg, 0.768 mmol) was added, followed by the addition of thiocarbonyldiimidazole (2.73 g, 15.4 mmol) under an ice-water bath. The reaction mixture was stirred at room temperature for 12 hours. The reaction was monitored by TLC until it was complete. After concentration, the residue was separated by silica gel column chromatography (ethyl acetate: petroleum ether = 1:3) to give product 3 (4.53 g, 82%), a yellowish-white solid. 1 H NMR (400 MHz, Chloroform-d) δ 8.35 (s, 1H), 8.02 (d, J = 7.7 Hz, 2H), 7.67 (s, 1H), 7.55(t, J = 7.4 Hz, 1H), 7.41 (t, J = 7.7 Hz, 2H), 7.07 (s, 1H), 5.79 (t, J = 7.7Hz, 1H), 5.38 (d, J = 9.6 Hz, 1H), 4.56 (dq, J = 9.9, 4.9, 3.9 Hz, 1H), 4.19(s, 2H), 2.16 (q, J = 5.2, 3.7 Hz, 2H), 1.84 (s, 3H), 1.21–1.01 (m, 28H); 13 CNMR (101 MHz, Chloroform-d) δ 183.06, 165.54, 136.80, 133.30, 131.09, 129.96,129.85, 128.45, 118.31, 85.09, 83.21, 64.66, 62.24, 58.94, 40.09, 17.90,17.48, 17.45, 17.31, 17.29, 17.27, 17.21, 13.77, 13.37, 12.78, 12.51, 3.78.

[0078] Intermediate 4

[0079] Under nitrogen protection, a toluene solution of compound 3 (1.31 g, 3.10 mmol), tri-n-butyltin hydrogen (1.00 mL, 3.72 mmol), and AIBN (254 mg, 1.55 mmol) was bubbled to remove oxygen for 20 minutes and reacted at 110 °C for 6 hours. The reaction was monitored by TLC until complete. After concentration under reduced pressure, the residue was separated by silica gel column chromatography (ethyl acetate: petroleum ether = 1:20) to give a colorless oily product 4 (850 mg, 81%). 1 H NMR (400 MHz, Chloroform-d) δ 7.94-7.89 (m,2H), 7.44-7.38 (m, 1H), 7.32-7.26 (m, 2H), 5.37-5.49 (m, 1H), 5.40 (t, J =7.2 Hz, 1H), 5.42-5.38 (m, 1H), 3.97- 3.93 (m, 1H), 3.92- 3.81 (m, 1H), 2.64-2.46 (m, 1H), 1.72-1.57 (m, 2H), 1.56-1.49 (m, 3H), 0.97-0.87 (m, 28H).

[0080] Intermediate 5

[0081] Compound 4 (500 mg, 0.99 mmol) was dissolved in anhydrous methanol (5.0 mL), and potassium carbonate (273 mg, 1.98 mmol) was added. The mixture was stirred at room temperature for 5 hours. The solvent was removed under reduced pressure, and the residue was separated by silica gel column chromatography (ethyl acetate: petroleum ether = 1:4) to give a colorless oily product 5 (350 mg, 88%). Two-dimensional NMR analysis of the obtained product revealed the following:

[0082] 5a (minor component, double bond E type) 1 H NMR (400 MHz, Chloroform-d) δ 5.57 (d, J = 7.4Hz, 1H), 4.32-4.29 (m, 1H), 4.05-4.03 (m, 1H), 3.48-3.45 (m, 1H), 2.88 (d, J= 10.9 Hz, 1H), 2.47-2.41 (m, 1H), 1.67 (d, J = 7.8 Hz, 3H), 1.57-1.54 (m,1H), 1.09-1.07 (s, 28H).

[0083] 5b (major component, Z-type double bond) 1H NMR (400 MHz, Chloroform-d) δ 5.47 (d, J = 7.4Hz, 1H), 4.66 (d, J = 6.6 Hz, 1H), 4.12 (q, J = 7.2 Hz, 1H), 4.04-3.98 (m,1H), 3.75 (dd, J = 12.1, 6.0 Hz, 1H), 2.63 (d, J = 8.7 Hz, 1H), 2.33-2.27 (m,1H), 1.81-1.78 (m, 1H), 1.77 (d, J = 7.0 Hz, 3H), 1.03-1.01 (m, 28H).

[0084] Intermediate 7

[0085] At 0 °C, diisopropyl azodicarbonate (120 µL, 0.60 mmol) was slowly added dropwise to a solution of triphenylphosphine (158 mg, 0.60 mmol), compound 5 (120 mg, 0.30 mmol), and N,N-bisBoc protected 6-chloro-2-aminopurine 6 (166 mg, 0.45 mmol) in THF (3.0 mL). The reaction mixture was stirred overnight at room temperature after removing the ice bath. The solvent was removed under reduced pressure, and the residue was separated by silica gel column chromatography (ethyl acetate: petroleum ether = 1:3) to give a colorless oily product 7 (167 mg, 75%); MS m / e: 751 (M + H). + .

[0086] Intermediates 8a and 8b

[0087] Compound 7 (80 mg, 0.106 mmol) was dissolved in 50% formic acid aqueous solution (5.0 mL) and stirred at 80 °C for 5 hours. After cooling, the solvent was removed by vacuum distillation, and the residue was added to saturated ammonia-methanol solution (5.0 mL) and stirred overnight at room temperature. The reaction solution was concentrated and purified by CHIRALPAK® AD-RH (amylose tris-3,5-dimethyl phenyl-carbamate; 150 x 10 mm, particle size 5 µm) reversed-phase chiral column, gradient elution (MeCN / H2O: 20%-60%, 5 mL / min) chiral column and reversed-phase chromatography mode to give white solids 8a (2 mg, 6%) and 8b (25 mg, 80%).

[0088] 8a: 1H NMR (400 MHz, DMSO-d6) δ 7.50 (s, 1 H), 5.65 (d, J = 7.3 Hz, 1H), 5.15-5.02 (m, 1 H), 4.22-4.25 (m, 1 H), 3.48 (qd, J = 11.0, 5.2 Hz, 2 H),2.38 (s, 1 H), 2.01-2.10 (m, 1 H), 1.89-1.97 (m, 1 H), 1.30 (s, 3 H); HRMS(ESI) for C 12 H 18 N3O3 (M + H) + Theoretical value: 252.1348; Detected value: 252.1333.

[0089] 8b: 1 H NMR (400 MHz, DMSO-d6) δ 7.64 (s, 1 H), 5.68 (d, J = 7.3 Hz, 1H), 5.35-5.25 (m, 1 H), 4.12 (q, J = 5.1 Hz, 1 H), 3.63 (qd, J = 11.0, 5.2Hz, 2 H), 2.40 (s, 1 H), 2.01 (dq, J = 26.4, 7.6, 7.0 Hz, 2 H), 1.17 (d, J =7.0 Hz, 3 H); 13 C NMR (101 MHz, DMSO-d6) δ 156.81, 153.47, 150.76, 140.23,135.90, 122.98, 116.10, 70.38, 61.67, 54.72, 52.28, 41.60, 13.39.

[0090] Using the same method, intermediate 5 was coupled with cytosine, thymine, and adenine bases, respectively, to obtain the following compounds 9a, 9b, 10, and 11:

[0091] 1H NMR (400 MHz, Methanol-d4) δ 8.04 (br, 1 H), 7.64 (d,J = 6.8 Hz, 1 H), 5.83 (d, J = 7.4 Hz, 2 H), 5.37 (s, 1 H), 4.36-4.38 (m, 1H), 3.61-3.67 (m, 2 H), 2.83-2.85 (m, 1 H), 2.06-2.12 (m, 1 H), 1.89-1.96 (m,1 H), 1.30 (d, J = 6.9 Hz, 3 H); HRMS (ESI) for C 13 H 18 N5O3 (M + H) + Theoretical value: 292.1410; Detected value: 292.1413.

[0092] 1 H NMR (400 MHz, Methanol-d4) δ 8.39 (br, 1 H), 7.79 (d,J = 6.8 Hz, 1 H), 5.83 (d, J = 7.4 Hz, 2 H), 5.62-5.59 (m, 1 H), 4.25-4.22(m, 1 H), 3.80-3.78 (m, 2 H), 2.49 (s, 1 H), 2.23-2.16 (m, 1 H), 2.01-1.96 (m, 1 H), 1.46 (d, J = 6.9 Hz, 3 H); 13 HRMS (ESI) for C 13 H 18 N5O3 (M + H) + Theoretical value: 292.1410; Detected value: 292.1400.

[0093] 1H NMR (400 MHz, Methanol-d4) δ 8.29 (d, J = 2.9 Hz, 1H), 8.23 ​​(d, J = 2.8 Hz, 1H), 5.93-5.82 (m, 1H), 5.69 (d, J = 7.0 Hz, 1H), 4.39-4.30 (m, 1H), 4.01-3.83 (m, 2H), 2.59 (s, 1H), 2.32-2.24 (m, 2H), 1.29 (d, J= 6.9 Hz, 3H); 13 C NMR (101 MHz, Methanol-d4) δ 156.54, 152.20, 149.92, 142.33,139.82, 126.49, 120.01, 72.75, 63.09, 55.83, 55.31, 42.63, 14.02; HRMS (ESI) for C 13 H 18 N5O2 (M + H) + Theoretical value: 276.1460; Detected value: 276.1461.

[0094] 1 H NMR (400 MHz, Methanol-d4) δ 7.64 (s, 1H), 5.85-5.73(m, 1H), 5.54-5.50 (m, 1H), 4.25-4.22 (m, 1H), 3.79 (s, 2H), 2.44 (s, 1H),2.11-2.00 (m, 2H), 1.79 (s, 3H), 1.45 (d, J = 7.0 Hz, 3H); 13 HRMS (ESI) for C 13 H 19 N₂O₄ (M + H) + Theoretical value: 267.1345; Detected value: 267.1342.

[0095] Example 2

[0096] ;

[0097] Nucleoside 9b (58 mg, 0.20 mmol) and 1.0 mL of anhydrous THF were added to a 10 mL single-necked flask, and the mixture was cooled to 0°C in an ice-water bath. Tert-butylmagnesium chloride Grignard reagent (1 M, 0.6 mL, 0.6 mmol) was added dropwise, and the reaction mixture was stirred at 0°C for 30 min. Then, a solution of phosphorus reagent 12 (145 mg, 0.32 mmol) in 1 mL of THF was added dropwise. The resulting clear reaction solution was heated to 35°C and stirred for 1 day. Saturated NH4Cl (3 mL) was added and stirred for 5 min. The mixture was diluted with ethyl acetate (60 mL). The organic phase was separated, and the aqueous layer was extracted with ethyl acetate (10 mL). The combined organic layers were washed with water (10 mL), saturated NaHCO3 (2 x 10 mL), and brine (10 mL), and dried over Na2SO4. The solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography (0-5% methanol in dichloromethane) to give a white solid product 13 (42 mg, 41%). 1 H NMR (400MHz, CDCl3) δ 9.85 (s, 1 H), 8.36 (br, 1 H), 7.75 (d, J = 6.8 Hz, 1 H), 7.15-7.34 (m, 5 H), 5.83 (d, J = 7.2 Hz, 1 H), 5.62-5.59 (m, 1 H), 4.25-4.22 (m, 3H), 3.80-3.78 (m, 2 H), 2.49 (s, 1 H), 2.23-2.16 (m, 1 H), 2.01-1.96 (m, 1H), 1.46 (d, J = 6.9 Hz, 3 H), 1.37 (d, J = 7.0 Hz, 3 H), 1.23 (d, J = 6.2Hz, 6 H); HRMS (ESI) for C 24 H 34 N4O7P (M + H) + Theoretical value: 521.2087; Detected value: 521.2091.

[0098] Example 3: Quantitative Real-Time PCR Detection of HBV DNA

[0099] According to 6.0×10 4HepG2.2.15 cells were seeded into 24-well plates and cultured overnight for adherent growth. The culture medium was then aspirated, and 600 μl of DMEM containing the drug was added to each well at final concentrations of 0, 0.0064, 0.032, 0.16, 0.8, 4, and 20 μM, with three replicates per treatment. After 3 days, the medium was replaced with fresh medium containing the same concentration of drug. HBV DNA was extracted from the collected medium using a hepatitis B virus nucleic acid extraction kit and stored at -20°C for later use. After 6 days of drug treatment, the culture supernatant and cells were collected, and HBV DNA was extracted using the hepatitis B virus nucleic acid extraction kit. The copy number of HBV DNA at each concentration was detected by qPCR, and the inhibition rate and EC50 were calculated. 50 .

[0100] HBV DNA inhibition rate = (number of copies in control group - number of copies in treatment group) / number of copies in control group × 100%

[0101] EC of each drug 50 The values ​​are shown in the table below:

[0102]

[0103] Experimental results confirmed that compounds 8 and 9 exhibited superior antiviral activity compared to the positive control lamivudine. Furthermore, the conversion of nucleoside compound 10 into its corresponding aminophosphate prodrug 13 significantly enhanced its anti-hepatitis B virus activity, indicating that nucleoside 10 was insensitive to mononucleotide kinase, thus affecting its ability to be further activated into a trinucleotide. In summary, these carbocyclic nucleoside derivatives containing ethyl subunits that we invented possess excellent inhibitory capabilities against hepatitis B virus replication. Some compounds exhibit superior in vitro antiviral activity compared to the clinical drug lamivudine, demonstrating promising potential for further development.

[0104] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A carbocyclic nucleoside derivative containing an ethyl subunit and its salt, characterized in that, As shown in general formula (I): ； Among them, the bases are selected from ; R is selected from H, methyl, F, Cl, Br or I; R' is selected from H, C1-C6 alkyl or cyclopropyl; R" is selected from H, F, Cl, Br or I; Where R1 and R2 are H, and have the structure shown in equation (II): ； Alternatively, it can have the structure shown in equation (III): ； R3 is an alanine side chain; R4 is isopropyl.

2. The carbocyclic nucleoside derivative containing an ethyl subunit and its salt according to claim 1, characterized in that, The bases are selected from the following uracil or thymine analogues: 。 3. The carbocyclic nucleoside derivative containing an ethyl subunit and its salt according to claim 1, characterized in that: The bases are selected from the following cytosine analogs: 。 4. A carbocyclic nucleoside derivative containing an ethyl subunit and its salt according to claim 1, characterized in that, The bases are selected from the following adenine analogues: 。 5. A carbocyclic nucleoside derivative containing an ethyl subunit and its salt according to claims 1-4, characterized in that, Selected from the following carbocyclic nucleoside compounds containing ethyl subunits: ； ； ； ； ； ； 。 6. A pharmaceutical composition comprising a carbocyclic nucleoside derivative containing an ethyl subunit as described in claims 1-5 and its salt.

7. Use of the carbocyclic nucleoside derivative containing an ethyl subunit as described in claims 1-5 and its salt, and the pharmaceutical composition as described in claim 6, in the preparation of a drug for treating or preventing hepatitis B virus.

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