Anti-HBVtetrahydroisoxazolo[4,3-c]pyridine compounds

Abstract

Disclosed are a class of anti-HBV tetrahydroisoxazolo[4,3-c]pyridine compounds and pharmaceutically acceptable salts thereof or isomers thereof, the compounds being represented by the formula (I). ##STR00001##

Claims

1. A compound of Formula (I), a pharmaceutically acceptable salt or an isomer thereof, ##STR00096## wherein ring A is 6- to 12-membered aryl or 5- to 6-membered heteroaryl; each R is independently F; Cl; Br; I; —CN; —OH; —NR.sup.aR.sup.b; —S(═O).sub.2NR.sup.aR.sup.b; —S(═O).sub.2R.sup.c; —C(═O)OR.sup.d; C.sub.1-6 alkoxy; or C.sub.1-6 alkyl optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of F, Cl, Br, I, —OH, —NH.sub.2 and —CN; n is 0, 1, 2 or 3; T is N or CR.sub.3; R.sub.1 and R.sub.5 are each independently H; F; Cl; Br; I; —CN; —OH; —NR.sup.aR.sup.b; C.sub.1-3 alkoxy; or C.sub.1-3 alkyl optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of F, Cl, Br, I, —OH, —NH.sub.2, —CN and —NO.sub.2; R.sub.2 and R.sub.4 are each independently H; F; Cl; Br; I; —CN; —OH; —NR.sup.aR.sup.b; C.sub.1-6 alkoxy; or C.sub.1-6 alkyl optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of F, Cl, Br, I, —OH, —NH.sub.2, —CN and —NO.sub.2; R.sub.3 is F, Br or —CN; R.sup.a, R.sup.b, R.sup.c and R.sup.d are each independently H or C.sub.1-6 alkyl; the 5- to 6-membered heteroaryl contains 1, 2 or 3 heteroatoms or heteroatom groups independently selected from the group consisting of —O—, —S—, N and —NH—.

2. The compound or a pharmaceutically acceptable salt or an isomer thereof according to claim 1, wherein, said ring A is phenyl or 5- to 6-membered heteroaryl; or said ring A is phenyl, thiazolyl, isothiazolyl, pyrazolyl, pyrrolyl, pyrazinyl, pyrimidinyl, or pyridyl.

3. The compound or a pharmaceutically acceptable salt or an isomer thereof according to claim 1, wherein, said R.sup.a, R.sup.b, R.sup.c and R.sup.d are each independently H, —CH.sub.3, —CH.sub.2CH.sub.3, —CH.sub.2CH.sub.2CH.sub.3 or —CH.sub.2(CH.sub.3).sub.2.

4. The compound or a pharmaceutically acceptable salt or an isomer thereof according to claim 1, wherein, said each R is independently F, Cl, Br, I, —CN, —OH, —OCH.sub.3, —OCH.sub.2CH.sub.3, —NH.sub.2, —N(CH.sub.3).sub.2, —S(═O).sub.2NH.sub.2, —S(═O).sub.2CH.sub.3, —C(═O)OCH.sub.3, —C(═O)OCH.sub.2CH.sub.3, —CH.sub.3, —CH.sub.2CH.sub.3, CF.sub.3 or —CH.sub.2OH.

5. The compound or a pharmaceutically acceptable salt or an isomer thereof according to claim 1, wherein, said R.sub.1 and R.sub.5 are each independently H, F, Cl, Br, I, —CN, —OH, —OCH.sub.3, —NH.sub.2, —CH.sub.3, —CH.sub.2F, —CHF.sub.2 or —CF.sub.3.

6. The compound or a pharmaceutically acceptable salt or an isomer thereof according to claim 1, wherein, said R.sub.2 and R.sub.4 are each independently H, F, Cl, Br, I, —CN, —OH, —OCH.sub.3, —OCH.sub.2CH.sub.3, —NH.sub.2, —CH.sub.3, —CH.sub.2CH.sub.3, —CH.sub.2F, —CHF.sub.2 or —CF.sub.3.

7. The compound or a pharmaceutically acceptable salt or an isomer thereof according to claim 1, wherein, said structural unit ##STR00097## is ##STR00098## or said structural unit ##STR00099## ##STR00100## or said structural unit is ##STR00101## is ##STR00102## ##STR00103##

8. The compound or a pharmaceutically acceptable salt or an isomer thereof according to claim 1, wherein, said structural unit ##STR00104## is ##STR00105## or said structural unit ##STR00106## is ##STR00107## or said structural unit ##STR00108## is ##STR00109##

9. The compound or a pharmaceutically acceptable salt or an isomer thereof according to claim 1, having a structure represented by Formula (II-a) or (III-a): ##STR00110## wherein, ring A, R.sub.2, R.sub.4, R and n are as defined in claim 1.

10. The compound or a pharmaceutically acceptable salt or an isomer thereof according to claim 7, having a structure represented by Formula (II-a-1) or (III-a-1): ##STR00111##

11. The compound or a pharmaceutically acceptable salt or an isomer thereof according to claim 1, having a structure represented by Formula (II-b), (II-c), (III-b) or (III-c): ##STR00112## wherein, T.sub.1 and T.sub.2 are each independently N or CH; R.sub.2, R.sub.4, R and n are as defined in claim 1.

12. The compound or a pharmaceutically acceptable salt or an isomer thereof according to claim 8, having a structure represented by Formula (II-b-1), (II-c-1), (III-b-1) or (III-c-1): ##STR00113## wherein, T.sub.1 and T.sub.2 are each independently N or CH.

13. The compound or a pharmaceutically acceptable salt or an isomer thereof according to claim 1, wherein the compound has a structure represented by Formula (II-d), (II-e), (II-f), (II-k), (III-d), (III-e), (III-f) or (III-k): ##STR00114## ##STR00115## ##STR00116## or the compound has a structure represented by Formulae (II-g) to (II-i) or Formulae (III-g) to (III-i): ##STR00117## ##STR00118## wherein, R.sub.2 and R.sub.4, R and n are as defined in claim 1.

14. The compound or a pharmaceutically acceptable salt or an isomer thereof according to claim 13, wherein the compound has a structure represented by Formula (II-d-1), (II-e-1), (II-f-1), (II-k-1), (III-d-1), (III-e-1), (III-f-1) or (III-k-1): ##STR00119## ##STR00120## or the compound has a structure represented by Formulae (II-g-1) to (II-i-1) or Formulae (III-g-1) to (III-i-1): ##STR00121## ##STR00122##

15. The compound or a pharmaceutically acceptable salt or an isomer thereof according to claim 13, having a structure represented by Formula (II-m) or (III-m): ##STR00123##

16. The compound or a pharmaceutically acceptable salt or an isomer thereof according to claim 15, having a structure represented by Formula (II-m-1) or (III-m-1): ##STR00124##

17. A compound or a pharmaceutically acceptable salt or an isomer thereof as follows: ##STR00125## ##STR00126## ##STR00127## ##STR00128## ##STR00129## ##STR00130## ##STR00131##

18. A compound or a pharmaceutically acceptable salt or an isomer thereof as follows: ##STR00132## ##STR00133## ##STR00134## ##STR00135## ##STR00136## ##STR00137## ##STR00138## ##STR00139##

19. A pharmaceutical composition comprising a therapeutically effective amount of a compound, a pharmaceutically acceptable salt or an isomer thereof according to claim 1 as an active ingredient, and a pharmaceutically acceptable carrier.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph showing changes in Log (HBV DNA copy number) per unit volume of plasma from mice in each test group after administration with the compound of Example 6 for 1 to 7 days.

(2) FIG. 2 shows the level of Log (HBV DNA copy number) per unit weight of liver tissue from mice in each test group on day 7 after administration with the compound of Example 6.

(3) FIG. 3 is a graph showing changes in Log (HBV DNA copy number) per unit volume of plasma from mice in each test group after administration with the compound of Example 23 for 1 to 7 days.

(4) FIG. 4 shows the level of Log (HBV DNA copy number) per unit weight of liver tissue mice in each test group on day 7 after administration with the compound of Example 23.

DETAIL DESCRIPTION OF THE PREFERRED EMBODIMENTS

(5) The present invention will be described below in detail with reference to the examples, but the present invention is not limited thereto. While the present invention has been described in detail and the specific embodiments thereof are disclosed herein, it will be apparent to those skilled in the art that various changes and modifications to the specific embodiments in the present invention can be made without departing from the spirit and scope of the present invention.

(6) Preparation of Intermediates

(7) ##STR00061##
Intermediate A-1 was Prepared by the Following Method:

(8) ##STR00062##

(9) To a solution of 3,4,5-trifluoroaniline (50.00 g) and pyridine (29.58 g, 30.18 mL) in dichloromethane (300 mL) was slowly added drop wise phenyl chloroformate (58.54 g, 46.83 mL) at 0° C. The reaction mixture was stirred at 25° C. for 3 hours, and then thereto was added 250 mL of water to quench the reaction, and a white precipitate was precipitated out. The precipitate was filtered and then dried under reduced pressure to afford the intermediate A-1.

(10) .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 7.46-7.39 (m, 2H), 7.32-7.27 (m, 1H), 7.21-7.11 (m, 4H), 4.82 (br. s., 1H); MS(ESI) m/z: 268 [M+H.sup.+].

(11) ##STR00063##

(12) The preparation of intermediate A-2 can be carried out with reference to the preparation method of intermediate A-1, except that 3,4,5-trifluoroaniline was replaced with 3-cyano-4-fluoroaniline.

(13) .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 7.85-7.74 (m, 1H), 7.67 (dd, J=3.8, 8.3 Hz, 1H), 7.46-7.41 (m, 2H), 7.31-7.18 (m, 5H); MS(ESI) m/z: 257 [M+H.sup.+].

(14) ##STR00064##

(15) The preparation of intermediate A-3 can be carried out with reference to the preparation method of intermediate A-1, except that 3,4,5-trifluoroaniline was replaced with 3-chloro-4-fluoroaniline.

(16) .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 7.64 (d, J=4.3 Hz, 1H), 7.51-7.38 (m, 3H), 7.32-7.29 (m, 1H), 7.22-7.10 (m, 3H), 6.95 (br. s., 1H); MS(ESI) m/z: 266 [M+H.sup.+].

(17) ##STR00065##

(18) The preparation of intermediate A-4 can be carried out with reference to the preparation method of intermediate A-1, except that 3,4,5-trifluoroaniline was replaced with 3-methyl-4-fluoroaniline.

(19) .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 7.49-7.33 (m, 3H), 7.28-7.15 (m, 4H), 7.03-6.93 (m, 1H), 6.96 (br.s., 1H), 2.29 (s, 3H). MS(ESI) m/z: 246 [M+H.sup.+].

(20) ##STR00066##

(21) The preparation of intermediate A-5 can be carried out with reference to the preparation method of intermediate A-1, except that 3,4,5-trifluoroaniline was replaced with 2-chloro-4-aminopyridine.

(22) .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 8.19 (d, J=5.6 Hz, 1H), 7.46 (d, J=1.6 Hz, 2H), 7.38-7.31 (m, 2H), 7.25-7.20 (m, 2H), 7.10 (d, J=7.7 Hz, 2H). MS(ESI) m/z: 249 [M+H.sup.+].

(23) ##STR00067##

(24) The preparation of intermediate A-6 can be carried out with reference to the preparation method of intermediate A-1, except that 3,4,5-trifluoroaniline was replaced with 3,4-difluoroaniline.

(25) .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 7.55-7.47 (m, 1H), 7.45-7.40 (m, 2H), 7.32-7.26 (m, 2H), 7.21-7.20 (m, 1H), 7.17-7.01 (m, 3H). MS(ESI) m/z: 250 [M+H.sup.+].

Example 1

(26) ##STR00068##

Preparation of Example 1

(27) ##STR00069##
Step A: Synthesis of Compound 1-2

(28) To a solution of Compound 1-1 (3.00 g) in dichloromethane (30 mL) was added oxalyl chloride (5.40 g, 42.52 mol) at 0° C., and then 1 to 2 drops of N,N-dimethylformamide was added. The reaction mixture was naturally warmed to room temperature, and continuously stirred for 14 hours. Then the resulting mixture was concentrated under reduced pressure to afford a crude product of compound 1-2.

(29) Step B: Synthesis of Compound 1-3

(30) N-Boc-(S)-2-methyl-4-piperidinone (0.50 g) was dissolved in diethyl ether (8 mL) at −70° C. under nitrogen gas protection, and then thereto was added lithium hexamethyldisilazide (2.34 mL, 1 mol/L). The reaction mixture was stirred at −70° C. for 0.5 h, and then to the system was added dropwise a solution of Compound 1-2 (374.04 mg) in diethyl ether (2 mL). The resulting reaction mixture was naturally warmed to room temperature and continuously stirred for 3 hours. The reaction solution was then poured into 1 mol/L hydrochloric acid (40 mL) and extracted with ethyl acetate (25 mL×2). The combined organic phase was washed with a saturated saline solution (40 mL), dried over anhydrous sodium sulfate and filtered, and then the filtrate was concentrated under reduced pressure to afford a crude product of compound 1-3.

(31) Step C: Synthesis of Compound 1-4

(32) To 2.5 mL of ethanol were added sequentially compound 1-3 (300.00 mg, crude), hydroxylamine hydrochloride (334.69 mg) and pyridine (2.5 mL) under stirring. The reaction mixture was gradually warmed to 100° C. and stirred at this temperature for 1 hour. After natural cooling, the mixture was distilled under reduced pressure, and the residue was diluted with ethyl acetate (30 mL), and then washed sequentially with 1 mol/L hydrochloric acid (20 mL) and a saturated saline solution (20 mL). The organic phase was dried over anhydrous sodium sulfate and filtered, and then the filtrate was concentrated under reduced pressure. The residue was separated by preparative TLC plate (developer: petroleum ether:ethyl acetate=2:1) to afford compound 1-4.

(33) .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 8.63-8.48 (m, 1H), 7.92 (dd, J=4.2, 8.6 Hz, 1H), 7.60-7.45 (m, 1H), 5.96-4.79 (m, 1H), 4.58-4.21 (m, 1H), 3.29-3.05 (m, 1H), 3.02-2.72 (m, 2H), 1.59-1.35 (m, 12H). MS(ESI) m/z: 334 [M+H.sup.+].

(34) Step D: Synthesis of Compound 1-5

(35) To a solution of hydrochloric acid in 1,4-dioxane (4 mol/L, 5 mL) was added compound 1-4 (93.00 mg) at room temperature. The reaction mixture was stirred at room temperature for 0.5 h, and then distilled under reduced pressure to afford a crude product of compound 1-5 (hydrochloride salt), which was used directly in the next step.

(36) MS(ESI) m/z: 234 [M+H+].

(37) Step E: Synthesis of Example 1

(38) Compound 1-5 (hydrochloride salt, 75.00 mg) was dissolved in 2 mL of N, N-dimethylformamide at room temperature, and then thereto were sequentially added N,N-diisopropylethylamine (107.82 mg) and intermediate A-1 (74.31 mg). The reaction mixture was stirred at 70° C. for 1 hour, and then slowly poured into 30 mL of water and then extracted with ethyl acetate (20 mL of ×2). The combined organic phase was washed with a saturated saline solution (30 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative high performance liquid chromatography (separation column: Phenomenex Synergi C18, 150×30 mm×4 μm, mobile phase: [water (0.225% trifluoroacetic acid)-acetonitrile]; B %: 50%-80%, 10.5 min) to afford Example 1.

(39) .sup.1HNMR (400 MHz, CD.sub.3OD) δ: 8.63 (d, J=2.8 Hz, 1H), 8.00-7.96 (m, 1H), 7.78-7.75 (m, 1H), 7.34-7.14 (m, 2H), 5.29 (d, J=17.5 Hz, 1H), 4.95 (quin, J=6.4 Hz, 1H), 4.50 (d, J=17.4 Hz, 1H), 3.06 (dd, J=5.7, 16.4 Hz, 1H), 2.87 (dd, J=1.2, 16.3 Hz, 1H), 1.22 (d, J=7.0 Hz, 3H).

(40) MS(ESI) m/z: 407 [M+H.sup.+].

Example 2

(41) ##STR00070##

(42) The preparation of Example 2 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with benzoic acid. The crude product was separated by preparative SEC (SEC separation method: separation column: DAICEL CHIRALPAK AS-H (dimension: 250 mm×30 mm, particle size: 5 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, EtOH]; B %: 30%-30%, 7.8 min) to afford Example 2.

(43) .sup.1HNMR (400 MHz, CD.sub.3OD) δ: 7.86-7.77 (m, 2H), 7.69-7.48 (m, 3H), 7.36-7.21 (m, 2H), 5.21 (d, J=15.94 Hz, 1H), 4.65-4.47 (m, 2H), 3.17-3.02 (m, 1H), 2.90 (dd, J=1.44, 16.38 Hz, 1H), 1.27 (d, J=6.90 Hz, 3H). MS(ESI) m/z: 388 [M+H.sup.+].

Example 3

(44) ##STR00071##

(45) The preparation of Example 3 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with 2-fluorobenzoic acid. The crude product was separated by preparative SEC (SEC separation method: separation column: DAICEL CHIRALPAK AD (dimension: 250 mm×30 mm, particle size: 10 μm); mobile phase: [0.1% NH.sub.3H.sub.2O, MeOH]; B %: 20%-20%, 4.0 min) to afford Example 3.

(46) .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 7.81 (dt, J=1.63, 7.53 Hz, 1H), 7.67-7.54 (m, 1H), 7.43-7.20 (m, 4H), 5.10 (d, J=16.69 Hz, 1H), 4.92-4.78 (m, 1H), 4.38 (d, J=16.81 Hz, 1H), 3.10 (dd, J=5.90, 16.56 Hz, 1H), 2.91 (d, J=16.31 Hz, 1H), 1.29 (d, J=6.90 Hz, 3H).

(47) MS(ESI) m/z: 406 [M+H.sup.+].

Example 4

(48) ##STR00072##

(49) The preparation of Example 4 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with 3-fluorobenzoic acid. The crude product was separated by preparative SEC (SEC separation method: separation column: DAICEL CHIRALPAK AS (dimension: 250 mm×50 mm, particle size: 10 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, IPA]; B %: 50%-50%, 1.9 min) to afford Example 4.

(50) .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 7.62-7.56 (m, 2H), 7.52 (dd, J=1.3, 8.7 Hz, 1H), 7.31-7.21 (m, 3H), 5.16 (d, J=16.1 Hz, 1H), 4.99 (quin, J=6.3 Hz, 1H), 4.49 (d, J=16.1 Hz, 1H), 3.09-3.00 (m, 1H), 2.92-2.83 (m, 1H), 1.23 (d, J=6.8 Hz, 3H). MS(ESI) m/z: 406 [M+H.sup.+].

Example 5

(51) ##STR00073##

(52) The preparation of Example 5 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with 4-fluorobenzoic acid. The crude product was separated by preparative SEC (SEC separation method: separation column: DAICEL CHIRALPAK AS-H (dimension: 250 mm×30 mm, particle size: 5 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, EtOH]; B %: 30%-30%, 4.2 min) to afford Example 5.

(53) .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 7.90-7.80 (m, 2H), 7.42-7.23 (m, 4H), 5.24-5.14 (m, 1H), 5.01 (quin, J=6.3 Hz, 1H), 4.50 (d, J=16.1 Hz, 1H), 3.08 (dd, J=5.8, 16.3 Hz, 1H), 2.89 (dd, j=1.2, 16.4 Hz, 1H), 1.26 (d, J=6.9 Hz, 3H). MS(ESI) m/z: 406 [M+H.sup.+].

Example 6

(54) ##STR00074##

(55) The preparation of Example 6 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with 2,4-difluorobenzoic acid. The crude product was separated by preparative SFC (SFC separation method: separation column: DAICEL CHIRALPAK AS (dimension: 250 mm×30 mm, particle size: 10 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, IPA]; B %: 30%-30%, 9 min) to afford Example 6.

(56) .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 7.86-7.75 (m, 1H), 7.26-7.14 (m, 4H), 5.05 (d, J=16.4 Hz, 1H), 4.93 (quin, J=6.4 Hz, 1H), 4.38-4.27 (m, 1H), 3.06 (dd, J=5.9, 16.4 Hz, 1H), 2.91-2.82 (m, 1H), 1.25 (d, J=6.9 Hz, 3H). MS(ESI) m/z: 424 [M+H.sup.+].

Example 7

(57) ##STR00075##

(58) The preparation of Example 7 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with 2-methoxybenzoic acid. The crude product was separated by preparative SFC (SFC separation method: separation column: DAICEL CHIRALPAK AD (dimension: 250 mm×30 mm, particle size: 10 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, MeOH]; B %: 30%-30%, 2.5 min) to afford Example 7.

(59) .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 7.60 (dd, J=1.6, 7.6 Hz, 1H), 7.53-7.45 (m, 1H), 7.25-7.12 (m, 3H), 7.07 (t, J=7.5 Hz, 1H), 4.99 (d, J=16.6 Hz, 1H), 4.94-4.88 (m, 1H), 4.24 (d, J=16.6 Hz, 1H), 3.94 (s, 3H), 3.04 (dd, J=5.9, 16.4 Hz, 1H), 2.83 (dd, J=1.0, 16.3 Hz, 1H), 1.26 (d, J=6.8 Hz, 3H). MS(ESI) m/z: 418 [M+H.sup.+].

Example 8

(60) ##STR00076##

(61) The preparation of Example 8 can be earned out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with 3-methoxybenzoic acid. The crude product was separated by preparative SFC (SFC separation method: separation column: DAICEL CHIRALPAK AD (dimension: 250 mm×30 mm, particle size: 10 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, EtOH]; B %: 30%-30%, 3.0 min) to afford Example 8.

(62) .sup.1H NMR (400 MHz, CD.sub.3OD): δ 7.51-7.43 (m, 1H), 7.34-7.23 (m, 4H), 7.07 (dd, J=2.1, 8.2 Hz, 1H), 5.15 (d, J=16.1 Hz, 1H), 4.97 (quin, J=6.4 Hz, 1H), 4.47 (d, J=16.0 Hz, 1H), 3.88 (s, 3H), 3.05 (dd, J=5.7, 16.3 Hz, 1H), 2.86 (dd, 7=1.1, 16.3 Hz, 1H), 1.24 (d, J=6.8 Hz, 3H). MS(ESI) m/z: 418 [M+H.sup.+].

Example 9

(63) ##STR00077##

(64) The preparation of Example 9 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with 4-methoxybenzoic acid. The crude product was separated by preparative SFC (SFC separation method: separation column: DAICEL CHIRALCEL OD (dimension: 250 mm×50 mm, particle size: 10 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, IPA]; B %: 30%-30%, 4.2 min) to afford Example 9.

(65) .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 7.75 (d, J=8.9 Hz, 2H), 7.33-7.25 (m, 2H), 7.14 (d, J=9.0 Hz, 2H), 5.16 (d, J=15.8 Hz, 1H), 5.05-4.97 (m, 1H), 4.48 (d, J=15.9 Hz, 1H), 3.90 (s, 3H), 3.06 (dd, J=5.6, 16.4 Hz, 1H), 2.87 (d, J=16.3 Hz, 1H), 1.26 (d, J=6.9 Hz, 3H).

(66) MS(ESI) m/z: 418 [M+H.sup.+].

Example 10

(67) ##STR00078##

(68) The preparation of Example 10 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with 3-cyanobenzoic acid. The crude product was separated by preparative SFC (SFC separation method: separation column: DAICEL CHIRALPAK AD (dimension: 250 mm×30 mm, particle size: 10 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, IPA]; B %: 30%-30%, 3.1 min) to afford Example 10.

(69) .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 8.17-8.06 (m, 2H), 7.88 (d, J=7.8 Hz, 1H), 7.81-7.72 (m, 1H), 7.33-7.22 (m, 2H), 5.23 (d, J=16.3 Hz, 1H), 5.08-4.96 (m, 1H), 4.62-4.58 (m, 1H), 3.09 (dd, J=5.7, 16.4 Hz, 1H), 2.91 (dd, J=1.3, 16.3 Hz, 1H), 1.26 (d, J=6.9 Hz, 3H).

(70) MS(ESI) m/z: 413 [M+H.sup.+].

Example 11

(71) ##STR00079##

(72) The preparation of Example 11 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with 3-chlorobenzoic acid. The crude product was separated by preparative SFC (SFC separation method: separation column: DAICEL CHIRALPAK AS-H (dimension: 250 mm×30 mm, particle size: 5 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, MeOH]; B %: 35%-35%, 5 min) to afford Example 11.

(73) .sup.1HNMR (400 MHz, CD.sub.3OD) δ: 7.71-7.55 (m, 2H), 7.51-7.38 (m, 2H), 7.16 (dd, J=6.3, 10.3 Hz, 2H), 5.08 (d, J=16.0 Hz, 1H), 4.94-4.82 (m, 1H), 4.50-4.37 (m, 1H), 3.04-2.91 (m, 1H), 2.78 (br. d, J=16.4 Hz, 1H), 1.14 (d, J=6.8 Hz, 3H). MS(ESI) m/z: 422 [M+H.sup.+].

Example 12

(74) ##STR00080##

(75) The preparation of Example 12 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with 4-chlorobenzoic acid. The crude product was separated by preparative SFC (SFC separation method: separation column: DAICEL CHIRALPAK AS-H (dimension: 250 mm×30 mm, particle size: 5 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, EtOH]; B %: 30%-30%, 8.3 min) to afford Example 12.

(76) .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 7.73-7.63 (m, 2H), 7.52-7.45 (m, 2H), 7.17 (dd, J=6.36, 10.27 Hz, 2H), 5.08 (d, J=16.14 Hz, 1H), 4.94-4.84 (m, 1H), 4.39 (d, J=16.02 Hz, 1H), 2.96 (dd, J=5.75, 16.38 Hz, 1H), 2.78 (dd, J=1.28, 16.32 Hz, 1H), 1.14 (d, J=6.97 Hz, 3H).

(77) MS(ESI) m/z: 422 [M+H.sup.+].

Example 13

(78) ##STR00081##

(79) The preparation of Example 13 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with pyrazine-2-carboxylic acid. The crude product was separated by preparative SFC (SFC separation method: separation column: DAICEL CHIRALPAK AS-H (dimension: 250 mm×30 mm, particle size: 5 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, MeOH]; B %: 60%-60%, 4.5 min) to afford Example 13.

(80) .sup.1HNMR (400 MHz, CD.sub.3OD) δ: 9.17 (d, J=1.5 Hz, 1H), 8.81 (dd, J=1.6, 2.4 Hz, 1H), 8.67 (d, J 20=2.5 Hz, 1H), 7.32-7.22 (m, 2H), 5.37 (d, J=17.6 Hz, 1H), 5.03-4.96 (m, 1H), 4.57 (d, J=17.7 Hz, 1H), 3.12 (dd, J=5.8, 16.4 Hz, 1H), 2.94 (dd, J=1.4, 16.4 Hz, 1H), 1.26 (d, J=6.9 Hz, 3H). MS(ESI) m/z: 390 [M+H.sup.+].

Example 14

(81) ##STR00082##

(82) The preparation of Example 14 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with thiazole-5-carboxylic acid. The crude product was separated by preparative SFC (SFC separation method: separation column: DAICEL CHIRALCEL OD (dimension: 250 mm×30 mm, particle size: 10 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, MeOH]; B %: 30%-30%, 2.3 min) to afford Example 14.

(83) .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 9.12 (s, 1H), 8.26 (s, 1H), 7.24-7.11 (m, 2H), 5.03 (d, J=16.38 Hz, 1H), 4.90 (t, J=6.72 Hz, 1H), 4.33 (d, J=16.38 Hz, 1H), 3.02-2.92 (m, 1H), 2.79 (dd, J=1.41, 16.44 Hz, 1H), 1.14 (d, J=6.85 Hz, 3H). MS(ESI) m/z: 395 [M+H.sup.+].

Example 15

(84) ##STR00083##

(85) The preparation of Example 15 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with thiazole-4-carboxylic acid. The crude product was separated by preparative SFC (SFC separation method: separation column: DAICEL CHIRALPAK AD (dimension: 250 mm×30 mm, particle size: 10 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, IPA]; B %: 30%-30%, 3.0 min) to afford Example 15.

(86) .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 9.20 (d, J=2.0 Hz, 1H), 8.18 (d, J=1.9 Hz, 1H), 7.37-7.21 (m, 2H), 5.30 (d, J=16.8 Hz, 1H), 5.05-4.95 (m, 1H), 4.52 (d, J=16.8 Hz, 1H), 3.09 (dd, J=5.7, 16.4 Hz, 1H), 2.90 (d, J=16.3 Hz, 1H), 1.26 (d, J=6.9 Hz, 3H). MS(ESI) m/z: 395 [M+H.sup.+].

Example 16

(87) ##STR00084##

(88) The preparation of Example 16 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with thiazole-2-carboxylic acid. The crude product was separated by preparative SFC (SFC separation method: separation column: DAICEL CHIRALPAK AS-H (dimension: 250 mm×30 mm, particle size: 5 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, MeOH]; B %: 35%-35%, 6.3 min) to afford Example 16.

(89) .sup.1HNMR (400 MHz, CD.sub.3OD) δ: 9.01 (s, 1H), 7.75 (d, J=4.8 Hz, 1H), 7.16 (dd, J=6.4, 10.3 Hz, 2H), 5.21 (d, J=17.2 Hz, 1H), 4.93-4.84 (m, 1H), 4.50-4.41 (m, 1H), 3.05-2.95 (m, 1H), 2.80 (d, j=16.5 Hz, 1H), 1.14 (d, J=6.8 Hz, 3H). MS(ESI) m/z: 395 [M+H.sup.+].

Example 17

(90) ##STR00085##

(91) The preparation of Example 17 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with isothiazole-4-carboxylic acid. The crude product was separated by preparative SFC (SFC separation method: separation column: DAICEL CHIRALPAK AD (dimension: 250 mm×30 mm, particle size: 10 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, IPA]; B %: 30%-30%, 30 min) to afford Example 17.

(92) .sup.1HNMR (400 MHz, CD.sub.3OD) δ: 9.40 (s, 1H), 9.00 (s, 1H), 7.33-7.27 (m, 2H), 5.18 (d, J=16.0 Hz, 1H), 5.08-5.00 (m, 1H), 4.50 (d, J=16.0 Hz, 1H), 3.08 (dd, J=5.7, 16.4 Hz, 1H), 2.94-2.88 (m, 1H), 1.25 (d, J=7.0 Hz, 3H). MS(ESI) m/z: 395 [M+H.sup.+].

Example 18

(93) ##STR00086##

(94) The preparation of Example 18 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with isothiazole-3-carboxylic acid. The crude product was separated by preparative SFC (SFC separation method: separation column: DAICEL CHIRALPAK AD-H (dimension: 250 mm×30 mm, particle size: 5 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, MeOH]; B %: 35%-35%, 2.1 min) to afford Example 18.

(95) .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 8.00 (d, J=3.2 Hz, 1H), 7.75 (d, J=3.2 Hz, 1H), 7.20-7.13 (m, 2H), 5.18 (d, J=17.2 Hz, 1H), 4.92-4.85 (m, 1H), 4.51-4.37 (m, 1H), 3.04-2.96 (m, 1H), 2.85-2.78 (m, 1H), 1.14 (d, J=7.0 Hz, 3H). MS(ESI) m/z: 395 [M+H.sup.+].

Example 19

(96) ##STR00087##

(97) The preparation process of Example 19 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with 2,4-difluorobenzoic acid; and intermediate A-1 was replaced with intermediate A-2. The crude product was separated by preparative SEC (SEC separation method: separation column: DAICEL CHIRALCEL OJ (dimension: 250 mm×30 mm, particle size: 10 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, EtOH]; B %: 25%-25%, 2.4 min) to afford Example 19.

(98) .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 7.89-7.76 (m, 2H), 7.72-7.64 (m, 1H), 7.29-7.16 (m, 3H), 5.08 (d, J=16.4 Hz, 1H), 4.96 (quin, J=6.4 Hz, 1H), 4.36 (d, J=16.6 Hz, 1H), 3.08 (dd, J=5.8, 16.4 Hz, 1H), 2.93-2.85 (m, 1H), 1.27 (d, J=6.8 Hz, 3H). MS(ESI) m/z: 413 [M+H.sup.+].

Example 20

(99) ##STR00088##

(100) The preparation process of Example 20 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with 2,4-difluorobenzoic acid; and intermediate A-1 was replaced with intermediate A-3. The crude product was separated by preparative SEC (SEC separation method: separation column: DAICEL CHIRALPAK AS (dimension: 250 mm×50 mm, particle size: 10 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, MeOH]; B %: 40%-40%, 1.9 min) to afford Example 20.

(101) .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 7.85-7.79 (m, 1H), 7.59 (dd, J=2.6, 6.8 Hz, 1H), 7.36-7.08 (m, 4H), 5.09 (dd, 7=1.1, 16.6 Hz, 1H), 4.97-4.94 (m, 1H), 4.36 (dd, 7=1.5, 16.6 Hz, 1H), 3.10 (dd, J=5.9, 16.4 Hz, 1H), 2.90 (dd, J=1.3, 16.4 Hz, 1H), 1.28 (d, J=6.9 Hz, 3H).

(102) MS(ESI) m/z: 422 [M+H.sup.+].

Example 21

(103) ##STR00089##

(104) The preparation of Example 21 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with 2,4-difluorobenzoic acid; and intermediate A-1 was replaced with intermediate A-4. The crude product was separated by preparative SEC (SEC separation method: separation column: DAICEL CHIRALPAK AD (dimension: 250 mm×30 mm, particle size: 10 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, EtOH]; B %: 20%-20%, 5.5 min) to afford Example 21.

(105) .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 7.87-7.83 (m, 1H), 7.30-7.10 (m, 4H), 6.96-6.94 (m, 1H), 5.09 (d, J=16.6 Hz, 1H), 5.02-4.94 (m, 1H), 4.35 (d, J=16.4 Hz, 1H), 3.16-3.06 (m, 1H), 2.89 (d, J=16.4 Hz, 1H), 2.24 (s, 3H), 1.28 (d, J=6.9 Hz, 3H). MS(ESI) m/z: 402 [M+H.sup.+].

Example 22

(106) ##STR00090##

(107) The preparation of Example 22 can be carried out with reference to the preparation procedures A to E of Example 1, except that 5-fluoropyridine-2-carboxylic acid was replaced with 2,4-difluorobenzoic acid; and intermediate A-1 was replaced with intermediate A-5. The crude product was separated by preparative SEC (SEC separation method: separation column: DAICEL CHIRALPAK AD (dimension: 250 mm×50 mm, particle size: 10 μm); mobile phase: [0.1% NH.sub.3.H.sub.2O, MeOH]; B %: 30%-30%, 1.8 min) to afford Example 22.

(108) .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 8.00 (d, J=5.9 Hz, 1H), 7.77-7.69 (m, 1H), 7.53 (d, 7=1.7 Hz, 1H), 7.31 (dd, 7=1.8, 5.7 Hz, 1H), 7.16-7.06 (m, 2H), 4.99 (d, 7=16.5 Hz, 1H), 4.90-4.83 (m, 1H), 4.27 (d, 7=16.6 Hz, 1H), 2.99 (dd, 7=5.9, 16.5 Hz, 1H), 2.79 (d, 7=16.5 Hz, 1H), 1.17 (d, 7=6.8 Hz, 3H). MS(ESI) m/z: 405 [M+H.sup.+].

Example 23

(109) ##STR00091##

Preparation of Example 23

(110) ##STR00092##
Step A: Synthesis of Compound 23-2

(111) To a solution of compound 23-1 (50.0 g) and potassium carbonate (77.53 g) in acetonitrile (500 mL) was added dropwise 3-bromopropyne (66.73 g) at 0° C. The reaction mixture was naturally warmed to room temperature, and continuously stirred for 12 hours. Then the resulting mixture was concentrated under reduced pressure to give a residue. The residue was diluted with water and extracted with ethyl acetate. The combined organic phase was washed with a saturated saline solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to afford a crude product of compound 23-2. MS(ESI) m/z: 128 [M+H.sup.+].

(112) Step B: Synthesis of Compound 23-3

(113) To a solution of compound 23-2 (52.0 g) in tetrahydrofuran (500 mL) were added potassium carbonate (26.56 g) and Boc.sub.2O (41.94 g) at room temperature. The reaction mixture was stirred at 18° C. for 12 hours, and then filtered to remove the solid. The filtrate was collected and concentrated under reduced pressure. The obtained residue was dissolved in ethyl acetate (800 mL), and the organic phase was washed sequentially with water, a saturated citric acid solution and a saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product of compound 23-3. MS(ESI) m/z: 228 [M+H.sup.+].

(114) Step C: Synthesis of Compound 23-4

(115) To a solution of compound 23-3 (40.0 g) in dichloromethane (400 mL) was added in batches Dess-Martin reagent (82.10 g) at 0° C. The reaction mixture was naturally warmed to room temperature and stirred for 2 hours. Then the mixture was washed twice with a saturated sodium bicarbonate solution and a saturated sodium thiosulfate solution in a volume ratio of 1:1. The organic phase was washed sequentially with a saturated sodium bicarbonate solution and a saturated saline solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford a crude product of compound 23-4. MS(ESI) m/z: 226 [M+H.sup.+].

(116) Step D: Synthesis of Compound 23-5

(117) To a mixture solution of compound 23-4 (42.0 g) in ethanol (400 mL) and water (40 mL) was added sodium acetate (22.94 g) and hydroxylamine hydrochloride (16.84 g) at room temperature. The reaction mixture was stirred at room temperature for 12 hours, and then concentrated under reduced pressure to remove the ethanol, thereby obtaining a residue. The residue was dissolved in ethyl acetate, and then the resulting mixture was washed sequentially with water and a saturated saline solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to afford a crude product of compound 23-5. MS(ESI) m/z: 241 [M+H.sup.+].

(118) Step E: Synthesis of Compound 23-6

(119) To a solution of compound 23-5 (300 mg) and 2-iodopyrimidine (257.16 mg) in N, N-dimethylformamide (8 mL) were added copper iodide (11.89 mg), triethylamine (252.66 mg) and his-triphenylphosphine palladium dichloride (43.81 mg) at room temperature. The reaction mixture was stirred at 12° C. for 12 hours under nitrogen gas protection. The resulting reaction solution was diluted with ethyl acetate, and then filtered. The filtrate was concentrated under reduced pressure, and the residue was separated by preparative TEC plates to afford compound 23-6. MS(ESI) m/z: 319 [M+H.sup.+].

(120) Step F: Synthesis of Compound 23-7

(121) To a mixture solution of compound 23-6 (180 mg) in methanol (4 mL) and water (0.8 mL) was added in batches bis(trifluoroacetoxy)iodobenzene (291.76 mg) at room temperature. The reaction mixture was stirred at 10-20° C. for 0.5 h, diluted with 20 mL of water, and then extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the residue was separated by preparative TLC plate to afford compound 23-7. MS(ESI) m/z: 317 [M+H.sup.+].

(122) Step G: Synthesis of Compound 23-8

(123) Compound 23-7 (90 mg) was dissolved in a solution of hydrochloric acid in dioxane (4 mol/L, 4 mL). The reaction mixture was stirred at 20° C. for 0.5 hour, and then concentrated under reduced pressure to afford a crude product of compound 23-8. MS(ESI) m/z: 217 [M+H.sup.+].

(124) Step H: Synthesis of Example 23

(125) To a solution of compound 23-8 (61.52 mg) in N,N-dimethylformamide (2 mL) were added A, TV-diisopropyl ethylamine (110.31 mg) and intermediate A-2 (72.90 mg) at room temperature. The reaction mixture was stirred at 70° C. for 1 hour. The resulting reaction solution was diluted with 20 mL of water, and then extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by preparative high performance liquid chromatography (separation column: Phenomenex Synergi C18, 150×30 mm×4 μm, mobile phase: [water (0.225% trifluoroacetic acid)-acetonitrile]; B %: 35%-65%, 10 min) to afford Example 23.

(126) .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 8.97 (d, J=5.0 Hz, 2H), 7.83 (dd, J=2.8, 5.6 Hz, 1H), 7.75-7.71 (m, 1H), 7.52 (t, J=5.0 Hz, 1H), 7.30 (t, J=9.0 Hz, 1H), 5.42 (d, J=17.9 Hz, 1H), 5.01 (quin, J=6.4 Hz, 1H), 4.59 (d, J=17.7 Hz, 1H), 3.14 (dd, J=5.7, 16.4 Hz, 1H), 2.95 (dd, j=1.2, 16.4 Hz, 1H), 1.27 (d, J=6.8 Hz, 3H). MS(ESI) m/z: 379 [M+H.sup.+].

Example 24

(127) ##STR00093##

(128) The preparation of Example 24 can be carried out with reference to the preparation procedures A to H of Example 23, except that intermediate A-2 was replaced with intermediate A-3.

(129) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ: 9.04 (d, J=4.9 Hz, 2H), 9.01 (s, 1H), 7.74 (dd, J=2.4, 6.8 Hz, 1H), 7.60 (t, J=4.9 Hz, 1H), 7.44-7.40 (m, 1H), 7.35-7.27 (m, 1H), 5.36 (d, J=18.0 Hz, 1H), 4.98-4.83 (m, 1H), 4.36 (d, J=18.0 Hz, 1H), 3.12-2.99 (m, 1H), 2.94-2.83 (m, 1H), 1.16 (d, J=6.8 Hz, 3H). MS(ESI) m/z: 388 [M+H.sup.+].

Example 25

(130) ##STR00094##

(131) The preparation of Example 25 can be carried out with reference to the preparation procedures A to H of Example 23, except that intermediate A-2 was replaced with intermediate A-1.

(132) .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 8.86 (d, J=4.89 Hz, 2H), 7.29 (t, J=4.89 Hz, 1H), 7.08 (dd, J=6.05, 9.48 Hz, 2H), 6.45 (s, 1H), 5.07 (d, J=16.75 Hz, 1H), 5.05-4.94 (m, 1H), 4.55 (d, J=16.87 Hz, 1H), 3.10-3.00 (m, 1H), 2.94-2.85 (m, 1H), 1.17 (d, J=6.97 Hz, 3H). MS(ESI) m/z: 390 [M+H.sup.+].

Example 26

(133) ##STR00095##

(134) The preparation of Example 26 can be carried out with reference to the preparation procedures A to H of Example 23, except that intermediate A-2 was replaced with intermediate A-6.

(135) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ: 9.03 (s, 3H), 7.60 (s, 2H), 7.43-7.09 (m, 2H), 5.36 (d, J=17.9 Hz, 1H), 4.90 (s, 1H), 4.36 (d, J=18.1 Hz, 1H), 3.06 (d, J=12.1 Hz, 1H), 2.89 (d, J=16.4 Hz, 1H), 1.15 (d, J=5.4 Hz, 3H). MS(ESI) m/z: 372 [M+H.sup.+].

(136) Activity Assay

(137) 1. In Vitro Anti-HBV Activity Assay

(138) 1) 100 μL of HepG2.2.15 cells were seeded at a number of 1.2×10.sup.5 cells per well into a 96-well cell culture plate, and then the cells were cultured in a 37° C. incubator with 5% carbon dioxide (CO.sub.2) overnight. On day 2, the compounds to be tested were diluted with DMSO in 3-fold gradient to eight concentrations in total. Then the compounds were diluted 100-fold with culture medium, and 100 μL of the diluted compounds were taken and added to the cell-containing plate in a final volume of 200 μL, and the final concentration of DMSO was 0.5%, and two duplicate wells were used. The cells were cultured in a 37° C. incubator with 5% CO.sub.2 for 3 days. On day 5, the solutions in the cell culture plate were replaced with fresh culture medium containing the same concentrations of compounds. When culture was performed for 8 days, cell culture plate supernatant was collected for the extraction of HBV DNA.

(139) 2) Detection of HBV DNA by real-time quantitative PCR: the total DNA in the supernatant was extracted with QIAamp 96 DNA Blood Kit, and the content of HBV DNA was detected by quantitative PCR using HBV specific primers and probes. 20 μL PCR pre-mixed solution and 5 μL HBV DNA sample or HBV plasmid standard sample were added to a quantitative PCR plate for reaction. The HBV plasmid standard sample was diluted in 10-fold gradient to seven concentrations from 10.sup.7 to 10 copies/μL. The quantitative PCR reaction procedure was as follows: pre-denaturation at 95° C. for 10 minutes; denaturation at 95° C. for 15 seconds, reaction at 60° C. for 1 minute, and this cycle was repeated 40 times. The inhibition rate of each well against HBV DNA was calculated according to the following equation, and the inhibition rate data of the compounds were subjected to a non-linear fitting analysis using GraphPad Prism software to obtain EC.sub.50 values of the compounds.
HBV DNA inhibition rate %=(1−copy number of HBV DNA of the sample/copy number of HBV DNA of DMSO control)*100%
The assay results are shown in Table 1 below.

(140) TABLE-US-00001 TABLE 1 Ex- HepG2.2.15EC.sub.50 Ex- HepG2.2.15 Ex- HepG2.2.15 ample (nM) amples EC.sub.50 (nM) amples EC.sub.50 (nM) 1 5.3 12 68.8 21 5.58 2 29.3 13 58.9 22 35.18 3 9.0 15 6.7 23 25.35 5 24.7 16 24.57 24 15.57 6 4.9 18 12.15 25 14.92 8 37.3 19 10.37 26 19.08 9 10.5 20 8.66
2. Pharmacokinetic Study for Mouse

(141) This experiment aims to evaluate the pharmacokinetic behavior of the compounds after a single intravenous or gavage administration in mice. As for the intravenous administration, the compounds were formulated into 0.5 mg/mL clear solutions, vehicle: 5% DMSO/5% 12-hydroxy stearate (solutol)/90% water; as for the gavage administration, the compounds were formulated into 2 mg/mL suspensions, vehicle: 0.5% sodium carboxymethylcellulose/0.2% Tween 80/99.3% water.

(142) The concentrations of the compounds in plasma were determined by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The retention time, chromatogram acquisition and integration of the chromatograms of the compounds and the internal standard (diclofenac) were processed using the software Analyst (Applied Biosystems), and data statistics were processed using the software Watson LIMS (Thermo Fisher Scientific) or Analyst (Applied Biosystems). The concentration unit of the analyte in the sample is ng/mL, 3 significant digits were retained, and all the values represented by percentages (e.g., % deviation and % coefficient of variation, etc.) have one decimal place. Each calibration curve contains at least 6 concentration levels. The calibration standard samples were formulated using stock solutions from different sources from the quality control samples. If the deviation between the calculated concentration of a calibration standard sample and the indicated value exceeds ±15.0% (the lower limit of quantitation exceeds ±20.0%), such standard sample should be excluded in regression analysis. The excluded calibration standard samples should be less than 25%, and each calibration curve contains at least 6 calibration standard samples that meet the acceptable standard. If the calibration standard samples at the lower and upper limits of quantification need to be excluded, the upper and lower limits of quantification of this batch of analytes will be increased and decreased accordingly.

(143) Plasma concentrations were processed using a non-compartmental model of the WinNonlin™ Version 6.3 (Pharsight, Mountain View, Calif.) pharmacokinetic software, and pharmacokinetic parameters were calculated using the linear logarithmic trapezoidal method. The pharmacokinetic parameters to be calculated include, but are not limited to (if the data is not allowed), T.sub.1/2, Vdss, CL, AUC.sub.0-24h of the intravenous group; C.sub.max, T.sub.max, AUC.sub.0-24h and bioavailability (F %) of the oral gavage group.

(144) The relevant pharmacokinetic parameters of the examples of the present invention in mice are shown in Table 2 below.

(145) TABLE-US-00002 TABLE 2 Intravenous injection CL Oral gavage dose (mL/ dose (mg/ T.sub.1/2 Vd.sub.ss mm/ AUC.sub.0-24 h (mg/ C.sub.max T.sub.max AUC.sub.0-24 h F Example Kg) (h) (L/Kg) Kg) (nM .Math. h) Kg) (nM) (h) (nM .Math. h) (%) 1 1 5.62 2.77 5.80 6709 10 4260 1.00 47167 76.1 6 1 9.31 2.33 2.84 11421 10 4935 0.50 66683 58.4 23 1 3.03 1.09 4.36 10047 3 3395 0.50 16737 55.3
3. In Vivo Activity Assay of HBV DNA in Mouse Plasmid Model of Tail Vein High-Pressure Injection with Water

(146) The purpose of this study was to test the inhibitory effect of the compound (Example 6) against HBV in mice through a mouse model of high-pressure tail vein injection. In this experiment, female BABL/c mice were used, and the mice were 6-7 weeks old. The HBV plasmid DNA was extracted by using pAAV2-HBV 1.3 mer with Qiagen EndoFree Plasmid Giga kit at a concentration of 1000 ng/μL. The resulting solution was diluted with normal saline before use and stored at 4° C. until use.

(147) 3.1 Grouping of Animals

(148) The grouping of experimental animals is shown in Table 3 below:

(149) TABLE-US-00003 TABLE 3 Time to start grouping Number of Time to start dosing is defined as animals per day 0 of the experiment Group group Dosing and handling of each group 1 5 Solvent 0.5% sodium carboxymethylcellulose/0.2% Tween 80/99.3% water, gavage, twice a day (8 hr/16 hr), from day 1 to day 7 * 2 5 Example 6, 30 mg/Kg, gavage, twice a day (8 hr/16 hr), from day 1 to day 7 * *: dosing only once on day 7.

(150) 3.2 Pharmaceutical Formulation

(151) The Pharmaceutical Formulation is shown in Table 4 below:

(152) TABLE-US-00004 TABLE 4 Pharmaceutical formulation and vehicle used Test compound (administration volume is 10 mL/Kg) Control: solvent An appropriate amount of sodium (0.5% sodium carboxymethylcellulose powder was weighed, carboxymethylcellulose/ and dissolved in pure water to formulate a 0.2% Tween 80/ solution at a concentration of 0.5%, and then 99.3% water) thereto was added Tween 80 in a ratio of v:v = 99.8:0.2, and mixed uniformly; it was formulated before the first administration and then stored at 4° C. until use. Example 6 An appropriate amount of Example 6 powder was weighed and dissolved in the administration vehicle to formulate a solution at a concentration of 3 mg/mL. The solution was sonicated and stirred uniformly. It is formulated every afternoon for the second administration of the same day and the first administration of the next day. After formulation, it should be stored in the dark at 4° C., and vortex-agitated before administration to make it as dispersed as possible.

(153) 3.3 Administration Schedule

(154) TABLE-US-00005 TABLE 5 Test Dosing regimen (dose/administration mode/ Group compound frequency/total duration) 1 Solvent control 10 mL/Kg, gavage, twice a day (8 hr/16 hr), group from day 1 to day 7 * 2 Example 6 30 mg/Kg, gavage, twice a day (8 hr/16 hr), from day 1 to day 7 * *: dosing only once on day 7.

(155) 3.4 Non-Endpoint Blood Collection and Transport

(156) 100 μL of whole blood was taken from each mouse approximately 4 hours after the first administration On days 1, 3, and 5, and the whole blood was collected into a tube containing sodium heparin, centrifuged at 4° C. at 7000 rpm for 10 minutes, and the supernatant was taken to obtain plasma. The plasma was stored in a refrigerator at −80° C. and transported to an analytical laboratory for testing under dry ice freezing condition.

(157) 3.5 Quantitative PCR Detection of HBV DNA Content in Mouse Plasma

(158) 1) DNA in plasma was extracted, and the experimental procedure was carried out with reference to the instruction for QIAamp 96 DNA Blood Kit.

(159) 2) Quantitative PCR detection of HBV DNA content in mouse plasma

(160) A qPCR reaction mixture was formulated (see Table 6). The qPCR reaction mixture, sample and standard sample were added to a 96-well reaction plate. The standard sample was plasmid DNA containing the full-length sequence of type D HBV. The standard sample was diluted from 10.sup.7 copies/μL in 10-fold gradient to successively obtain 10.sup.6˜10.sup.1 copies/μL of DNA standard samples. PCR reaction: 95° C., 10 min; 95° C., 15 seconds; 60° C., 1 minute; 40 cycles.

(161) TABLE-US-00006 TABLE 6 qPCR Reaction composition table components of PCR volume required for one reaction solution reaction system FastStart Universal Probe Master 12.5 μL   Forward primer (10 μM) 1 μL Reverse primer (10 μM) 1 μL Probe (10 μM) 0.5 μL   Water 5 μL HBV DNA content = DNA content detected by HBV primer − DNA content detected by pAAV2 primer.

(162) 3.6 Quantitative PCR Detection of HBV DNA in Mouse Liver

(163) 1) Extraction of Total Liver DNA

(164) Liver tissue was taken and homogenized with a tissue grinder. After centrifugation, the supernatant was transferred to a new centrifuge tube and digested with proteinase K digestion solution for 3 hours. The resulting mixture was cooled, and then RNAse A was added thereto to incubate for 30 minutes. The RNase A-treated mixture was extracted twice with an equal volume of phenol chloroform isoamyl alcohol to remove residual protein. The supernatant was transferred to a new centrifuge tube, and isopropanol was added thereto to precipitate the DNA. The DNA precipitation was washed twice with 70% ethanol. The precipitation was then air-dried and TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) was added thereto to dissolve the DNA.

(165) 2) Quantitative PCR Detection of HBV DNA in Mouse Liver

(166) The DNA concentrations were measured with Nanodrop and adjusted to 10 ng/μL for all the samples. 5 μL samples were added to the quantitative PCR reaction system for quantitative PCR.

(167) HBV DNA content=DNA content detected by HBV primer-DNA content detected by pAAV2 primer.

(168) 3.7 Assay Results

(169) 1) Blood samples were taken from mice of each test group on days 1, 3, 5, and 7 after administration, and the measured values of HBV DNA concentration (Log HBV DNA) are shown in Table 7 below:

(170) TABLE-US-00007 TABLE 7 Blood sampling Log HBV DNA mean value (copy number/μL) time Solvent control group Example 6 Day 1 2.22 2.23 Day 3 4.51 1.74 Day 5 5.01 2.02 Day 7 3.30 1.85 LLOQ = 40 copies/μL, Log(LLOQ) = 1.60 (LLOQ denotes the lowest detection limit)

(171) This result is shown in FIG. 1.

(172) 2) After 7 days of administration, the measured values of HBV DNA concentration (Log HBV DNA) in the liver tissues of the animals in each test group are shown in Table 8 below:

(173) TABLE-US-00008 TABLE 8 Log HBV DNA mean value (copy number/μL) Solvent control group Example 6 5.67 3.09

(174) This result is shown in FIG. 2.

(175) 4. In Vivo Activity Assay of HBV DNA in Mouse Plasmid Model of Tail Vein High-Pressure Injection with Water

(176) The purpose of this study was to test the inhibitory effect of the compound (Example 23) against HBV in mice through a mouse model of high-pressure tail vein injection. In this experiment, female BABL/c mice were used, and the mice were 6-7 weeks old. The HBV plasmid DNA was extracted by using pAAV2-HBV 1.3 mer with Qiagen EndoFree Plasmid Giga kit at a concentration of 1000 ng/μL. The resulting solution was diluted with normal saline before use and stored at 4° C. until use.

(177) 4.1 Grouping of Animals

(178) The grouping of experimental animals is shown in Table 9 below:

(179) TABLE-US-00009 TABLE 9 Time to start grouping Number of Time to start dosing is defined as animals per day 0 of the experiment Group group Dosing and handling of each group 1 5 Solvent 0.5% sodium carboxymethylcellulose/0.2% Tween 80/99.3% water, gavage, once a day, from day 1 to day 7 * 2 5 Example 23, 60 mg/Kg, gavage, once a day, from day 1 to day 7 * 3 5 Example 23, 60 mg/Kg, gavage, twice a day (8 hr/16 hr), from day 1 to day 7 * *: dosing only once on day 7.
4.2 Pharmaceutical Formulation

(180) The Pharmaceutical Formulation is shown in Table 10 below:

(181) TABLE-US-00010 TABLE 10 Test compound Pharmaceutical formulation and vehicle used Solvent control: Solutol HS 15 and deionized water were mixed at a 10% solutol volume ratio of 1:9 and stored at 4° C. until use. It was HS 15 formulated before the first administration and used for 7 days of administration. Example 23 An appropriate amount of Example 23 was weighed, dissolved in 10% solutol HS 15, and sonicated to a uniform suspension. The formulations were respectively prepared into 6 mg/mL, and stored at 4° C. until use. They were formulated before the first administration and used for 7 days of administration, and sonicated again to a uniform suspension before each administration.
4.3 Administration Schedule

(182) TABLE-US-00011 TABLE 11 Dosing regimen (dose/administration mode/ Group Test compound frequency/total duration) 1 Solvent control 10 mL/Kg, gavage, once a day, from day group 1 to day 7 * 2 Example 23 60 mg/Kg, gavage, once a day, from day 1 to day 7 * 3 Example 23 60 mg/Kg, gavage, twice a day (8 hr/16 hr), from day 1 to day 7 * *: dosing only once on day 7.
4.4 Assay Results

(183) 1) Blood samples were taken from mice of each test group on days 1, 3, 5, and 7 after administration, and the measured values of HBV DNA concentration (Log HBV DNA) are shown in Table 12 below:

(184) TABLE-US-00012 TABLE 12 Blood Log HBV DNA mean value (copy number/μL) sampling Solvent Example 23 Example 23 time control group (60 mg/Kg, QD) (60 mg/Kg, BID) Day 1 2.03 2.10 1.94 Day 3 4.47 3.15 3.02 Day 5 4.76 3.92 2.94 Day 7 3.53 3.19 2.14

(185) This result is shown in FIG. 3.

(186) 2) After 7 days of administration, the measured values of HBV DNA concentration (Log HBV DNA) in the liver tissues of the animals in each test group are shown in Table 13 below:

(187) TABLE-US-00013 TABLE 13 Log HBV DNA mean value (copy number/μL) Example 23 Example 23 Solvent control group (60 mg/Kg, QD) (60 mg/Kg, BID) 6.01 5.18 4.50
This result is shown in FIG. 4.