COMBINATION COMPRISING TRICYCLE COMPOUND AND USE THEREOF IN PREPARATION OF MEDICAMENT FOR TREATING HBV

Abstract

A combination comprising a tricycle compound and use thereof in the preparation of a medicament for treating HBV. The combination is a combination of a compound of formula (I) or a pharmaceutically acceptable salt thereof and any one drug in the following groups a-c: a. a hepatitis B surface antigen inhibitor, b. a reverse transcriptase inhibitor, and c. a hepatitis B surface antigen inhibitor and a reverse transcriptase inhibitor.

##STR00001##

Claims

1. A pharmaceutical combination, wherein the combination is a compound of formula (I) or a pharmaceutically acceptable salt thereof ##STR00051## and a combination of any one of the following groups a to c of drugs: a. a hepatitis B surface antigen inhibitor, b. a reverse transcriptase inhibitor, and c. a hepatitis B surface antigen inhibitor and a reverse transcriptase inhibitor, wherein, in formula (I), L.sub.1 is a single bond or —C.sub.1-6 alkyl; R.sub.1 is H, Cl, F, Br, I, or C.sub.1-3 alkyl optionally substituted with 1, 2 or 3 R.sub.a; ring A is 4-8 membered heterocycloalkyl or C.sub.3-8 cycloalkyl; R.sub.2 is H and C.sub.1-3 alkyl; R.sub.3 is H and C.sub.1-3 alkyl; R.sub.a is each independently H, F, Cl, Br, I, NH.sub.2, OH, CN, C.sub.1-6 alkyl or C.sub.1-6 heteroalkyl, or C.sub.1-6 alkyl and C.sub.1-6 heteroalkyl optionally substituted with 1, 2 or 3 R′; R′ is each independently selected from: Cl, F, Br, I, NH.sub.2, CH.sub.3, CN and —N(CH.sub.3).sub.2; the 4-8 membered heterocycloalkyl and C.sub.1-6 heteroalkyl each comprise 1, 2, 3 or 4 heteroatoms or heteroatom groups independently selected from —O—, —NH—, —S— and N.

2. The combination according to claim 1, wherein R.sub.a is each independently H, F, Cl, Br, I, NH.sub.2, OH or CN, or, ring A is 5-6 membered heterocycloalkyl, or, R.sub.1 is H, Cl, F, Br, I, or CH.sub.3 optionally substituted with 1, 2 or 3 R.sub.a; or, R.sub.2 is H or CH.sub.3; or, R.sub.3 is H or CH.sub.3; or, L.sub.1 is —CH.sub.2— or —CH.sub.2CH.sub.2—, or, the compound of formula (I) has a structure of formula (I-1): ##STR00052## wherein R.sub.1, R.sub.2, R.sub.3 and L.sub.1 are as defined in claim 1.

3. (canceled)

4. The combination according to claim 2, wherein ring A is tetrahydrofuryl, tetrahydropyranyl or dioxanyl; or, R.sub.1 is H, Cl or CH.sub.3.

5. The combination according to claim 4, wherein ring A is ##STR00053##

6-11. (canceled)

12. The combination according to claim 1, wherein the compound of formula (I) is selected from the following specific compounds: ##STR00054## ##STR00055##

13. The combination according to claim 1, wherein the hepatitis B surface antigen inhibitor is selected from one of a compound of formula (II) or a pharmaceutically acceptable salt thereof: ##STR00056## wherein, the carbon atom with “*” is a chiral carbon atom present in a form of a single (R) or (S) enantiomer or in a form enriched with one enantiomer; R.sub.1 is selected from: H, OH, CN, NH.sub.2, or from the following groups optionally substituted with 1, 2 or 3 R: C.sub.1-6 alkyl, C.sub.1-6 heteroalkyl, C.sub.2-5 alkenyl, C.sub.2-5 heteroalkenyl, C.sub.3-6 cycloalkyl and 3-6 membered heterocycloalkyl; R.sub.2 is selected from: H, OH, CN, NH.sub.2 and halogen, or from the following groups optionally substituted with 1, 2 or 3 R: C.sub.1-3 alkyl, C.sub.1-3 heteroalkyl, C.sub.3-6 cycloalkyl and 3-6 membered heterocycloalkyl; R.sub.3 is selected from the following groups optionally substituted with 1, 2 or 3 R: C.sub.1-6 alkyl and C.sub.3-6 cycloalkyl; m is selected from: 0, 1, 2, 3, 4 and 5; when m is 0, R.sub.1 is not selected from: OH, CN and NH.sub.2; R is selected from: H, halogen, OH, CN, NH.sub.2, or from the following groups optionally substituted with 1, 2 or 3 R′: C.sub.1-3 alkyl and C.sub.1-3 heteroalkyl; R′ is selected from: F, Cl, Br, I, OH, CN, NH.sub.2, CH.sub.3, CH.sub.3CH.sub.2, CH.sub.3O, CF.sub.3, CHF.sub.2 and CH.sub.2F; “hetero” represents a heteroatom or a heteroatom group, and the “hetero” in C.sub.1-6 heteroalkyl, C.sub.2-5 heteroalkenyl, 3-6 membered heterocycloalkyl and C.sub.1-3 heteroalkyl is each independently selected from: —C(═O)N(R)—, —N(R)—, —C(═NR)—, —(R)C═N—, —S(═O).sub.2N(R)—, —S(═O)N(R)—, N, —O—, —S—, ═O, ═S, —C(═O)O—, —C(═O)—, —C(═S)—, —S(═O)—, —S(═O).sub.2— and —N(R)C(═O)N(R)—; in any one of the above cases, the number of heteroatoms or heteroatom groups is each independently selected from 1, 2 or 3; or the hepatitis B surface antigen inhibitor is selected from one of a compound of formula (III) or a pharmaceutically acceptable salt thereof, ##STR00057## wherein, R.sub.1 is selected from H, OH, CN, NH.sub.2, or from the following groups optionally substituted with 1, 2 or 3 R: C.sub.1-5 alkyl, C.sub.1-5 heteroalkyl, C.sub.2—S alkynyl, C.sub.3-6 cycloalkyl and 3-6 membered heterocycloalkyl; R.sub.2 is selected from H, halogen, or from the following groups optionally substituted with 1, 2 or 3 R: C.sub.1-3 alkyl and C.sub.1-3 heteroalkyl; m is selected from 0, 1, 2, 3, 4 and 5; A is selected from the following groups optionally substituted with 1, 2 or 3 R: phenyl and 5-6 membered heteroaryl; R is selected from H, halogen, OH, CN, NH.sub.2, ═O, CH.sub.3, CH.sub.3CH.sub.2, CH.sub.3O, CF.sub.3, CHF.sub.2 and CH.sub.2F: the “hetero” in C.sub.1-5 heteroalkyl, 3-6 membered heterocycloalkyl, C.sub.1-3 heteroalkyl and 5-6 membered heteroaryl is each independently selected from: N, —O—, ═O, —S—, —NH—, —(C═O)—, —(S═O)— and —(S═O)—; in any one of the above cases, the number of heteroatoms or heteroatom groups is each independently selected from 1, 2 or 3.

14. The combination according to claim 13, wherein the hepatitis B surface antigen inhibitor is selected from one of the following compounds: ##STR00058## ##STR00059## ##STR00060## ##STR00061## ##STR00062## ##STR00063## ##STR00064## ##STR00065## ##STR00066## ##STR00067## ##STR00068## ##STR00069## ##STR00070##

15. The combination according to claim 13, wherein the hepatitis B surface antigen inhibitor is selected from one of the following compounds: ##STR00071## ##STR00072## ##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077## ##STR00078##

16. The combination according to claim 14, wherein the hepatitis B surface antigen inhibitor is selected from the following compounds: ##STR00079##

17. The combination according to claim 15, wherein the hepatitis B surface antigen inhibitor is selected from the following compounds: ##STR00080##

18-22. (canceled)

23. The combination according to claim 1, wherein the reverse transcriptase inhibitor is selected from: lamivudine, adefovir dipivoxil, entecavir, tenofovir disoproxil fumarate and tenofovir alafenamide fumarate.

24. The combination according to claim 23, wherein the reverse transcriptase inhibitor is selected from: entecavir and tenofovir disoproxil fumarate.

25. The combination according to claim 1, wherein a pharmaceutical composition is prepared by mixing the compound of formula (I) or the pharmaceutically acceptable salt thereof and any one of the groups a to c of drugs as pharmaceutically active ingredients.

26. The combination according to claim 25, wherein the preparation of the pharmaceutical composition by mixing the pharmaceutically active ingredients is that a compound pharmaceutical composition is prepared by mixing two or three different drugs as pharmaceutically active ingredients.

27. The combination according to claim 1, wherein the compound of formula (I) or the pharmaceutically acceptable salt thereof and any one of the groups a to c of drugs, as pharmaceutically active ingredients, are separately prepared into pharmaceutical compositions, and the pharmaceutical compositions are further packaged separately and administered separately at the time of administration.

28. The combination according to claim 27, wherein the separate administrations comprise the administration of one or two of the pharmaceutical compositions, followed by the administration of another one or two of the pharmaceutical compositions, and the administration of two or three of the pharmaceutical compositions simultaneously.

29. A method for treating hepatitis B virus infection in a subject in need thereof, comprising: administering an effective amount of the combination according to claim 1 to the subject.

30. A pharmaceutical composition comprising the combination according to claim 1 and at least one pharmaceutically acceptable carrier and/or excipient.

31. A kit comprising the combination according to claim 1.

32. A method for treating hepatitis B in a subject in need thereof, comprising: administering an effective amount of the pharmaceutical composition according to claim 30 to the subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0120] FIG. 1: in vitro combination efficacy profile of compound 1 and TDF;

[0121] FIG. 2: the effect of the treatment of test compound for 28 days on AAV/HBV mouse serum HBV DNA;

[0122] FIG. 3: the effect of the treatment of test compound on AAV/HBV mouse liver HBV DNA;

[0123] FIG. 4: the effect of the treatment of test compound on AAV/HBV mouse serum HBV RNA;

[0124] FIG. 5: concentration of test compound in mouse serum in AAV/HBV model; and

[0125] FIG. 6: weight change of mice in each group.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0126] The present disclosure is described in detail below by way of examples. However, this is by no means disadvantageously limiting the scope of the present disclosure. Although the present disclosure has been described in detail herein and specific examples have also been disclosed, it will be apparent to those skilled in the art that various changes and modifications can be made to the specific examples without departing from the spirit and scope of the present disclosure.

Example 1 Preparation of the Compound 1

[0127] ##STR00046##

[0128] Synthetic Route

##STR00047##

[0129] Step 1: Synthesis of Compound 1-A

[0130] Anhydrous dichloromethane (5 L) was added into a dry three-necked flask (10 L) and stirred, then compound 1-SMA (500.00 g) and nitromethane were added into the three-necked flask successively to obtain a mixture. The mixture was placed in a dry ice ethanol bath, and cooled to −10° C. The temperature was controlled between −10° C.-0° C. Aluminum trichloride (1.15 kg) was slowly added into the reaction flask and the temperature was controlled to less than −0° C. Next, α,α-dichlorodimethyl methyl ether (495.00 g) was slowly added into the reaction kettle to obtain a reaction solution, which was slowly heated to room temperature and stirred for 18 hours. TLC (PE:EA 3:1) monitoring revealed the disappearance of the raw material spot and the appearance of a new spot with high polarity. Potassium bisulfate solution (3 L) was slowly added dropwise to the extracted reaction solution to a concentration of 10%, stirred for 20 minutes while crushed ice was added to prevent overheating. The mixed solution was transferred to a 25 L separatory funnel and allowed to stand for stratification to separate the dichloromethane layer, and the aqueous phase was extracted with dichloromethane (2 L*2). After washing with 10% potassium bisulfate solution (5 L*2), the organic phase was separated and dried with anhydrous sodium sulfate (1 kg). The organic phase was concentrated under reduced pressure to obtain compound 1-A as a dark-green solid.

[0131] .sup.1H NMR (400 MHz, deuterated chloroform) 6=9.97 (br s, 1H), 9.87-9.82 (m, 1H), 7.58 (dd, J=1.5, 3.3 Hz, 1H), 7.36-7.29 (m, 1H), 4.37 (q, J=7.1 Hz, 2H), 1.38 (t, J=7.2 Hz, 3H).

[0132] Step 2: Synthesis of Compound 1-B

[0133] p-toluenesulfonyl hydrazide (2.23 kg, 11.96 mol) was added into the THF (20 L) solution containing compound 1-A (2 kg, 11.96 mol). The solution was stirred at 20° C. for about 1 h. When the disappearance of the raw material was monitored by TLC, the reaction system was heated to 60° C., and sodium cyanoborohydride (902 g, 14.36 mol) was added in batches. After the addition, the reaction solution was heated to 70° C. and stirred for 3 h. After the heating was stopped and cooled down to room temperature, 5 L of water was added to quench the reaction, followed by removing most of the THF under reduced pressure, and the residue was extracted with a large amount of EA (1.5 L*3). The organic phases were pooled, washed with saturated sodium chloride and dried with anhydrous sodium sulfate. Then the organic phases were filtered, and the solvent was removed under reduced pressure. Finally, the crude product was column chromatographed to obtain compound 1-B as a pale-yellow solid.

[0134] Step 3: Synthesis of Compound 1-C

[0135] Methanol (32 L) was added into a 50 L jacketed kettle and stirred, then compound 1-SMB (4000.00 g) and diisopropylethylamine (5.25 L) were added successively, and the internal temperature was reduced to 5-10° C. Benzyl mercaptan (2490.00 g) was slowly added dropwise, and the internal temperature was maintained at 5-15° C. After the addition was complete, the cooling system was turned off to let the temperature rise naturally, and stirring was continued for 2.5 h. Next, stirring was stopped, the speed was adjusted to 100 rpm, and the reaction liquid was released and filtered through a desktop filter to obtain a filter cake. The filter cake was washed three times with water (5 L), followed by once with EtOH (3 L). The filter cake was filtered by suction filtration until it was no longer viscous, thereby obtaining compound 1-C as a pale-yellow solid.

[0136] Step 4: Synthesis of Compound 1-D

[0137] Dichloromethane (7.5 L) was added into a 50 L kettle and stirred, then compound 1-C (1500 g) was added. The internal temperature was reduced to 0-10° C., and HCl solution (6 M, 4.12 L) was added. The sodium hypochlorite solution (commercially available 8% solution, 23.0 kg) was added dropwise under 0-10° C. with the lid open. After dropwise addition, the cooling system was turned off and stirring was continued for about 17 hours with the lid open. Then sodium bisulfite was added for heat capacity (1000 g, 5 L aqueous solution), and starch potassium iodide test paper was used to detect if there is no oxidant remaining in the water phase. Next, stirring was stopped, the solution was allowed to stand for stratification. The dichloromethane layer was collected while the aqueous layer was extracted with dichloromethane (2.5 L), and the dichloromethane layers were pooled. The organic phase was dried with anhydrous sodium sulfate and filtered, then the solvent was removed under reduced pressure to obtain compound 1-D as a white solid.

[0138] .sup.1H NMR (400 MHz, deuterated chloroform) δ=8.50-8.43 (m, 2H), 8.34 (d, J=8.2 Hz, 1H), 4.04 (s, 3H).

[0139] Step 5: Synthesis of Compound 1-E

[0140] Tetrahydrofuran (10 L) was added into a dry 50 L jacketed kettle and stirred, then compound 1-B (2000 g) was added. The internal temperature was reduced to 0-10° C. The temperature was maintained at 0-15° C. in about 1.5 hours, and potassium tert-butoxide (1 M THF solution, 15.67 L) was added. After addition, the temperature was raised to about 20° C. and the stirring was continued for 1 hour. Next, the temperature was reduced to 0-10° C., and the THF (10 L) solution containing compound 1-D (4380 g) was slowly added. After addition, the temperature was slowly raised to 15° C. and the stirring was continued for about 16 hours. Ethyl acetate (10 L) was added for extraction, and the organic phase was washed with saturated sodium chloride solution (10 L) twice. The aqueous phases were combined and extracted with EA (5 L), and the organic phases were combined. Finally, the solvent was removed from the organic phase under reduced pressure to obtain compound 1-E as a pale-yellow solid.

[0141] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=8.55 (d, J=1.4 Hz, 1H), 8.37 (dd, J=1.5, 8.3 Hz, 1H), 7.91 (d, J=8.3 Hz, 1H), 7.60 (s, 1H), 7.13 (d, J=1.8 Hz, 1H), 4.02 (q, J=7.0 Hz, 2H), 3.93 (s, 3H), 2.10 (s, 3H), 1.08 (t, J=7.1 Hz, 3H).

[0142] Step 6: Synthesis of Compound 1-SM1

[0143] Compound 1-E (1000.0 g) was added into a dry 10 L three-necked flask and stirred, then glacial acetic acid (5 L) was added and the internal temperature of the reaction was controlled at 25-30° C. Iron powder (1 eq, 140.9 g) was slowly added. After stirring for 30 minutes, the second batch of iron powder (0.5 eq, 70.44 g) was slowly added. After continuing stirring for 30 minutes, the third batch of iron powder (0.5 eq, 70.44 g) was added. After another 30 min of stirring, the fourth batch of iron powder (0.5 eq, 70.44 g) was added, and the reaction continued to be stirred until the disappearance of raw material and the appearance of anew point with high polarity were monitored. Next, stirring was stopped, and the reaction solution was transferred to 25 L dispenser for liquid separation. 10 L of ethyl acetate was added to the solution, which was then washed with 5 L saturated sodium bisulfate aqueous solution twice. After the liquid separation, the aqueous phase was back-extracted with 5 L of ethyl acetate. The organic phases were then combined and washed with 10% NaOH aqueous solution until pH>8, and the organic phase was collected by liquid separation. The organic phase was concentrated under reduced pressure to obtain compound 1-SM1 as a white solid.

[0144] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=7.79-7.71 (m, 2H), 7.50 (d, J=1.8 Hz, 1H), 7.14 (dd, J=1.7, 8.5 Hz, 1H), 6.96 (d, J=2.0 Hz, 1H), 6.42 (s, 2H), 4.11 (q, J=7.2 Hz, 2H), 3.84 (s, 3H), 2.04 (s, 3H), 1.16 (t, J=7.1 Hz, 3H).

[0145] Step 7: Synthesis of Compound 1-F

[0146] Toluene (12 L) was added to a dry 50 L jacketed kettle and stirred, then 2-bromoethanol (9930 g) was added, followed by boron trifluoride ether (268 g). The reaction was heated to 30-35° C. Compound 1-SMC (3500 g) was slowly added dropwise, and the addition was completed in about 1.5 hours. After addition, the internal temperature of the reaction was raised to about 55-65° C. and the temperature setting of heater was adjusted to 60° C. to keep the internal temperature at 55-65° C. for 1 hour. Then the internal temperature of the system was reduced to about 10° C., and sodium hydroxide aqueous solution (3783 g, water 17.5 L) at about 20° C. was slowly added into the reaction system while the internal temperature was maintained at 10-20° C. After the addition of NaOH solution, the temperature control of the heater was turned off, and the reaction continued to stir for about 16 h. Then the stirring was stop that the reaction solution was allowed to stand for stratification. The aqueous layer was extracted with 2-methyltetrahydrofuran (10 L) and the organic phases were combined, washed with water (10 L) and allowed to stand for stratification to collect organic phase. The organic phase was finally concentrated under reduced pressure to obtain compound 1-F as a colorless oil. .sup.1H NMR (400 MHz, deuterated chloroform) δ=3.87-3.71 (m, 4H), 3.66-3.59 (m, 3H), 3.42 (dd, J=6.0, 11.7 Hz, 1H), 3.20-3.13 (m, 1H), 2.79 (t, J=4.6 Hz, 1H), 2.65-2.59 (m, 1H).

[0147] Step 8: Synthesis of compound 1-G

[0148] An aqueous solution of sodium hydroxide (3240 g, 15 L of water) was added into a 50 L jacketed kettle, compound 1-F (4430 g) was then added and the heating was turned on. After the reaction was heated to 90° C., the stirring was continued for 1 h. The cooling was turned on to reduce the reaction to about 15° C., then THF solution (6180 g, THF: 15 L) containing p-toluenesulfonyl chloride was added. The temperature control of the heater was turned off, and the reaction was further stirred at about 15° C. for about 16 h. Next, the stirring was stopped to let the reaction allowed to stand for stratification. The aqueous phase was extracted with 2-methyltetrahydrofuran (10 L), and the 2-methyltetrahydrofuran phase (there was white insoluble matter, which disappeared after the washing) was washed with water (5 L), and the organic phases were combined. DMAP (500 g) and triethylamine (2.5 L) were added to the organic phase, followed by stirring the organic phase for 30 minutes. Then the organic phase was washed with saturated sodium chloride solution (10 L) and allowed to stand for stratification, and the aqueous phase was discarded. The organic phase was washed with potassium hydrogen sulfate solution (3800 g, 15 L of water) and saturated sodium chloride solution (5 L*twice) successively, and then allowed to stand for stratification, and the organic phase was collected. The organic phase was finally concentrated under reduced pressure to remove the solvent to obtain the crude product compound 1-G. .sup.1H NMR (400 MHz, deuterated chloroform) δ=7.77 (d, J=8.3 Hz, 2H), 7.34 (d, J=8.2 Hz, 2H), 4.03-3.91 (m, 2H), 3.80-3.49 (m, 8H), 3.33 (dd, J=9.9, 11.4 Hz, 1H), 2.43 (s, 3H).

[0149] Step 9: Synthesis of Compound 1-H

[0150] Acetone (30 L) was added into a clean 50 L jacketed kettle and stirred, then compound 1-G (4500 g) was added, followed by sodium iodide (6190 g). The heating was turned on, and after the reaction was heated to 75° C., the stirring was continued for 16 hours. After being cooled to room temperature, the reaction was filtered, and the filtrate was concentrated under reduced pressure at 50° C. Ethyl acetate (15 L) and water (10 L) were added to the concentrated crude product, and the mixture was stirred and then allowed to stand for stratification. The organic phase was washed with 0.5 M sodium thiosulfate (10 L). The aqueous phase and sodium thiosulfate solution were combined and extracted with EtOAc (5 L). The organic phases were combined and washed with saturated sodium chloride solution (10 L), then allowed to stand for stratification, and the organic phase was collected. The organic phase was finally concentrated under reduced pressure to remove the solvent to obtain the crude product compound 1-H. .sup.1H NMR (400 MHz, deuterated chloroform) δ=3.90-3.83 (m, 2H), 3.81-3.75 (m, 2H), 3.74-3.65 (m, 5H), 3.63-3.49 (m, 7H), 3.31-3.18 (m, 3H), 3.06-3.04 (m, 2H).

[0151] Step 10: Synthesis of Compound 1-I

[0152] DMSO (20 L) was added into a clean 50 L jacketed kettle and stirred, then compound 1-H (4700 g) was added and the temperature was raised to 35° C., followed by adding sodium cyanide (1010 g). The internal temperature of the reaction was raised to about 60° C. within 20 minutes, then the temperature was gradually reduced to 35° C., and the stirring was continued for about 16 hours. Sodium bicarbonate solution (2000 g sodium bicarbonate, 10 L of water) was added to the reaction system, which was then stirred for about 5 minutes. EtOAc: MeOH (20 L, 2 L) was added and the reaction system was further stirred for 2 minutes, followed by standing for about 1 hour. Next, the reaction system was partitioned and about 30 L of the lower layer solution was separated. The lower layer solution was extracted twice with EtOAc:MeOH (15 L:1.5 L for the first time, and 5 L:0.5 L for the second time). After the extraction, the upper organic phase and the upper layer of the remaining reaction liquid were combined, washed three times with saturated sodium chloride solution (10 L each), and allowed to stand for stratification. The aqueous phase was discarded while the organic phase was collected. Finally, the solvent was removed from the organic phase under reduced pressure, and the crude product was column chromatographed to obtain compound 1-I as a colorless oil. Docket No. 10022.0029 .sup.1H NMR (400 MHz, deuterated chloroform) δ=3.84-3.65 (m, 6H), 3.61-3.53 (m, 2H), 3.35 (t, J=10.5 Hz, 1H), 2.49-2.44 (m, 2H).

[0153] Step 11: Synthesis of Compound 1-J

[0154] Under the protection of argon, Raney nickel (10.00 g, 116.73 mmol) and EtOH (150 mL) were added into the dry hydrogenation flask, and then 1-I (20 g, 157.31 mmol) and NH.sub.3.Math.H.sub.2O (13.65 g, 97.36 mmol, 15.00 mL, 25% purity) were added, followed by replacement, and the reaction was stirred at 50 psi and 50° C. for 3.5 h. The reaction solution was filtered using diatomite, and the filtrate was concentrated under reduced pressure to obtain compound 1-J as a yellow oil. .sup.1H NMR (400 MHz, deuterated chloroform) δ=3.82-3.57 (m, 6H), 3.34-3.18 (m, 1H), 2.86-2.72 (m, 2H), 1.60-1.38 (m, 2H).

[0155] Step 12: Synthesis of Compound 1-SM2

[0156] 1-J (800.00 g) was added into a 5 L three-necked flask and stirred, then ethyl acetate (800 mL) was added within 0.5 h, and 4M HCl/EtOAc (1.6 L) was slowly added dropwise till the pH of the system was smaller than 5 while the internal temperature was maintained at 5-15° C. Next, the cooling system was turned off, and the reaction was heated to room temperature and further stirred for 1 hour. After stopping the stirring, the reaction was filtered through a desktop filter to obtain a filter cake, which was then concentrated under reduced pressure (40-45° C.) to obtain a crude product. Acetonitrile (2 mL/g) was added to the preceding product and the mixture was slurried for 1 hour. After the slurried product was filtered, the filter cake was collected separately, and the organic solution was removed under reduced pressure to obtain a white solid compound 1-SM2. .sup.1H NMR (399 MHz, METHANOL-d.sub.4) δ=3.88-3.72 (m, 5H), 3.67-3.59 (m, 1H), 3.36-3.31 (m, 1H), 3.14 (t, J=6.7 Hz, 2H), 1.87-1.67 (m, 2H).

[0157] Step 13: Synthesis of Compound 1-1-A and Compound 1-1-B

[0158] Toluene (20 L) was added into a dry 50 L jacketed kettle and stirred, then compound 1-SM1 (2500 g), and the internal temperature was raised to 30-35° C. The inert gas environment in the kettle was maintained by nitrogen purging. Then trimethylaluminum (3.0 L, the temperature in the kettle rises slowly with the addition of Al(CH.sub.3).sub.3) was added dropwise. After addition, the nitrogen purging was turned off. The temperature was raised to 80-85° C. and the reaction was further stirred about 16 hours. Next, the cooling was turned on to reduce the temperature of the reaction to 20-30° C. Half of the reaction solution (about 12 L), to which EtOAc (10 L) was added, was transferred and mixed well. The mixed solution was added to 10% KHSO.sub.4 solution (10 L) while stirring, stirred for 2 minutes and then allowed to stand for stratification. The organic layer was washed with 10% KHSO.sub.4 solution (10 L), the water phases were combined and extracted twice with DCM (each 7.5 L). The other half of the reaction solution (about 12 L) was transferred out, which was treated in the same way as defined above. Then the organic phases were combined and concentrated under reduced pressure to obtain a crude product. Two times the volume of n-heptane was added and beaten for 1 hour to form a slurry. The slurry was filtered, and vacuum-dried for >12 hours at 40° C., P≤−0.1 MPa. A mixture of compound 1-1-A and compound 1-1-B was obtained.

[0159] Step 14: Synthesis of Compound 1-2

[0160] Tetrahydrofuran (3840 mL) was added into a 10 L three-necked flask and stirred, and the mixture of compound 1-1-A and compound 1-1-B (480.00 g) was added slowly, then H.sub.2O (960 mL) solution of LiOH.Math.H.sub.2O (118.84 g) was added dropwise slowly. After addition, the temperature was raised to 60° C. and the reaction was stirred for 1 hour. Then concentrated HCl was added to the reaction solution to adjust the pH of the system to 2, and stop stirring. Next, the solution was left standing for liquid stratification. The aqueous phase was extracted twice with THF (600 mL), and the organic phases were combined and concentrated under reduced pressure (40-45° C.). The solid was slurried with pure water (2 mL/g) for 0.5 hours and filtered, and the filter cake was vacuum-dried for over 12 hours at 40° C. and P≤−0.1 MPa to obtain compound 1-2. .sup.1H NMR (399 MHz, DMSO-d.sub.6) δ=11.19 (s, 1H), 8.09 (d, J=8.2 Hz, 1H), 8.00 (d, J=1.6 Hz, 1H), 7.88 (dd, J=1.5, 8.3 Hz, 1H), 7.39 (dd, J=1.2, 2.0 Hz, 1H), 7.01 (d, J=2.0 Hz, 1H), 2.05 (s, 3H).

[0161] Step 15: Synthesis of Compound 1

[0162] DMF (2.25 L) was added into a 5 L three-necked flask and stirred, then compound 1-2 (400.00 g) and HATU (744.83 g) were added successively and stirred for 30 min, followed by compound 1-SM2 (229.86 g). DIPEA (568.68 mL) was slowly added dropwise at room temperature within 1 hour. After addition, the reaction was further stirred at room temperature for 16 hours. Next, the reaction solution was transferred to a separatory funnel, ethyl acetate (2 L) and pure water (1 L) were added, and the solution was stirred for 2 min and then left standing for stratification to separate the water phase. Then pure water (1 L) was added to wash the organic phase which was then stirred and allowed to stand for stratification. The combined aqueous phase was extracted three times with EtOAc (500 mL), and the organic phases were combined. The organic phase was washed twice with sodium carbonate solution (1.5 L), twice with potassium hydrogen sulfate solution (1 L) and twice with pure water (1 L), successively. The organic phase was concentrated under reduced pressure (40-45° C.) to obtain a crude product. Ethyl acetate (2 mL/g) was added to the crude product and the mixture was slurried for 1 hour. Finally, the slurried mixture was filtered and the filter cake was collected to obtain compound 1. .sup.1H NMR (400 MHz, DMSO-d.sub.6) (=11.13 (br s, 1H), 8.73 (br t, J=5.5 Hz, 1H), 8.05 (d, J=8.2 Hz, 1H), 7.83 (d, J=1.3 Hz, 1H), 7.74 (dd, J=1.5, 8.4 Hz, 1H), 7.36 (s, 1H), 6.98 (d, J=2.0 Hz, 1H), 3.71-3.48 (m, 5H), 3.45-3.31 (m, 1H), 3.45-3.30 (m, 1H), 3.27-3.21 (m, 1H), 3.14 (dd, J=9.9, 11.2 Hz, 1H), 2.03 (s, 3H), 1.53 (q, J=7.0 Hz, 2H).

Biological Test Experiment

Experimental Example 1: Compound 1 and Tenofovir Disoproxil Fumarate (Tenofovir Difumarate Fumarate, TDF) Combined Drug In Vitro on HBV Inhibitory Activity

[0163] 1. Experimental Method

[0164] 1.1 Day 1, HepG2.2.15 cells were seeded into a 96-well cell culture plate at a density of 40,000 cells/well, and then the cells were cultured overnight at 5% CO.sub.2 and 37° C.

[0165] 1.2 Day 2, the compound 1 and TDF were combined with 7 different concentrations (selected about 8×, 4×, 2×, 1×, 0.5×, 0.25×, 0.125×EC.sub.50 concentration gradient) for orthogonal proportioning, and added to 96-well plate, each combination is 3 double plates, and the final concentration of DMSO is 0.5%. The detection concentration of each compound is shown in Table 1.

[0166] 1.3 Day 5, the cell supernatant was discarded, a fresh medium containing the compound was added, and the cells were cultured under 5% CO.sub.2, 37° C. for 3 days. On the 8th day, the supernatant from the cell plate treated with the compound was extracted according to the QIAamp 96 DNA Blood Kit (12) instruction.

[0167] 1.4 HBV DNA was quantified by qPCR method. HBV plasmid DNA is used as the standard. The concentration of standard HBV plasmid DNA starts from 107 copies/μL with 10-fold dilution at 7 points. Fit the standard curve with the HBV DNA copy number and CT value of each standard, and calculate the HBV DNA copy number in each test sample.

[0168] MacSynegy software (Prichard et al., 1990) was used to process the HBV DNA copy number test data and analyze the effect parameters of compound 1 and TDF in combination. The CellTiter-Glo kit was used to detect the cytotoxicity of the compounds to HepG2.2.15 cells. According to the kit instructions, use a multifunctional microplate reader to detect the chemiluminescence intensity (RLU) of each cell well.

TABLE-US-00001 TABLE 1 The detected concentrations of compounds in the combination test Compound Detection of final concentration (nM) Compound 1 34.09 17.04 8.522 4.261 2.131 1.065 0.533 TDF 64 32 16 8 4 2 1

[0169] 2. Result

[0170] HepG2.2.15 cells were used to evaluate the inhibitory activity of compound 1 and TDF in vitro on HBV. The inhibitory activity and cytotoxicity results of the combined drugs on HBV are summarized in Table 2, and the combined drug effect diagram is shown in FIG. 1.

[0171] The experimental results showed that the synergy index and antagonism index of the combination of compound 1 and TDF in vitro in 95% confidence space were 13.27 and −1.74, respectively, showing an additive effect. None of the compounds showed cytotoxicity in the tested concentrations (see Table 3).

TABLE-US-00002 TABLE 2 Inhibitory activity of compound 1 and TDF in vitro on HBV Synergy index Antagonism (95% index Compound Compound confidence (95% confidence Combined a b interval) interval) drug effect Compound TDF 13.27 −1.74 Additive 1 effect Note: Description of drug combination index: if the index is positive, it is synergistic, and if it is negative, it is antagonistic; the absolute value of the index is less than 25, that is, the additive effect; the absolute value of the index is in the range of 25-50, that is, mild but clear synergy or Antagonistic effect; the absolute value of the index is in the range of 50-100, that is, moderate synergistic or antagonistic effect, which may have important significance for in vivo effects. The absolute value of the index is in the range of > 100, that is, a highly synergistic or antagonistic effect, which is likely to have important meanings in vivo.

TABLE-US-00003 TABLE 3 The average cell viability percentage (%) of the cytotoxicity test of the combination drug Compound 1 Compound Concentration (nM) 34.09 17.04 8.522 4.261 2.131 1.065 0.533 0 TDF 64 125.9 ± 129.7 ± 130.7 ± 132.7 ± 131 ± 133.6 ± 130.4 ± 127 ±14 24.5 27.6 24.6 24.6 26.9 26.2 17.9 32 113.2 ± 112.4 ± 108.4 ± 112.1 ± 111.7 ± 112.8 ± 111.7 ± 111 ± 7.3 18.7 18.4 15.8 18.8 18.3 16.2 13.1 16 109.2 ± 112.5 ± 113 ± 117.2 ± 114.9 ± 113.8 ± 115.5 ± 111.4 ± 18.3 19.4 19 21.7 16.6 15.3 10.6 7.7 8 113.3 ± 114.8 ± 119 ± 119.4 ± 124.3 ± 122.6 ± 123.5 ± 116.6 ± 20.2 18.5 21.7 19.4 15.7 13.6 10.4 10.9 4 102.5 ± 102.5 ± 104.6 ± 106.9 ± 110.2 ± 111.2 ± 108.1 ± 103.8 ± 15.8 15.4 15.5 12.6 9.6 8 4.7 6 2 102. l ± 101.9 ± 105 ± 107.3 ± 105.7 ± 102.2 ± 103.2 ± 101.1 ± 13.6 16.3 15.9 15 10.4 8.7 2.8 2.9 1 105.5 ± 106.9 ± 106.1 ± 107.2 ± 109.8 ± 108.3 ± 105.2 ± 103.1 ± 16.7 17 19.4 14.9 12.3 7.3 6.3 7.8 0 115.2 ± 123.1 ± 124.3 ± 125.1 ± 124.5 ± 128.1 ± 121.5 ± 120.9 ± 17.1 20.4 14.8 11.9 8.9 3.7 7.1 5.2

[0172] 3. Conclusion

[0173] The combination of compound 1 and TDF in vitro showed an additive effect on the inhibitory activity of HBV, and the compound showed no cytotoxicity in the tested concentration. The test results support the combined application of compound 1 and TDF in the clinical treatment of patients with chronic HBV infection.

Experimental Example 2: Using AAV/HBV Mouse Model to Evaluate the Anti-Hepatitis B Virus Efficacy of the Test Compound In Vivo

[0174] Experimental Materials

[0175] Animals

[0176] 5-week-old male C57BL/6 mice without specific pathogen level, purchased from Shanghai Slack Laboratory Animal Co., Ltd.

[0177] 2. Solvents and Compounds

[0178] Solvent: 10% solutol aqueous solution

[0179] Test Compound:

[0180] Compound 1 was added with an appropriate amount of the above-mentioned solvent, and a uniform particle suspension was obtained after vortexing, and the preparation concentration was 1.0, 3.0 and 10.0 mg/mL. Store at 4° C. until used. Compound 1 is calculated according to the salt coefficient of 1.0 and the purity of 100%.

[0181] Tenofovir disoproxil (TDF) was purchased from Shanghai Panhong Chemical Technology Co., Ltd. Weigh an appropriate amount of TDF and dissolve it in physiological saline to prepare a 1 mg/mL mother liquor, vortex until TDF is fully dissolved, dispense into 1 mL size and store at −20° C. Take 1 mL of mother liquor before each administration, and dilute the working solution 10 times to 0.1 mg/mL with normal saline for the day's administration.

[0182] Recombinant Virus rAAV8-1.3HBV

[0183] rAAV8-1.3HBV (Type D, ayw) was purchased from Beijing Wujiahe Institute of Molecular Medicine, batch number x2018032301, 1×10.sup.12 viral genome (v.g.)/mL. Dilute with sterile PBS to 5×10.sup.11 v.g./mL before the experiment. Each mouse was injected with 200 μL, that is, each mouse was injected with 1×10.sup.11 v.g.

[0184] 3. Test Method

[0185] Establishment of AAV/HBV Mouse Model

[0186] AAV/HBV injection: rAAV8-1.3HBV is pre-prepared with sterile PBS to a concentration of 1×10.sup.11 v.g./200 μL solution before injection.

[0187] Infection level detection: On days 14 and 21 after virus injection, about 120 μL of blood was collected from the submandibular vein for all infected mice to collect serum. After the whole blood was incubated in a 37° C. incubator for 30 minutes, the blood was centrifuged at 13,200×g for 3 minutes at 4° C. to collect about 30 μL of serum. The serum is stored at −80° C. for the detection of HBV DNA, HBeAg and HBsAg.

[0188] Grouping: On the 28th day after virus injection, according to the levels of HBV DNA, HBsAg and HBeAg in serum samples on the 14th and 21st days after virus injection, and the body weight of the mice.

[0189] Definition of experimental days: the day of the first dosing is set to day 0 of the experiment.

[0190] 4. In Vivo Experiment Design

[0191] In vivo experimental dosing and sampling plan are shown in Table 4

TABLE-US-00004 TABLE 4 In vivo experimental dosing and sampling plan Sample collection Dosing Non-end point Number Dosing Frequency Drug of Compound Dosage (mL/kg) Dosage Virology metabolism Group animals name (mg/kg) volume of oral testing testing End point 1 8 Solvent 0 10 2 times a day Serum was Group 2: On On the 28th (BID), days collected the day, serum 0-27 on day 27th day and 2 8 Compound 1 10 10 2 times a 0 0 hours liver day, days (before (before samples 0-27 dosage), 3, dosage), were 3 8 Compound 1 30 10 2 times a 7, and 14 1.4, 8, 9, collected. day, days days. 12 and 24 0-27 hours 4 8 Compound 1 100 10 2 times a after the first day, days dosage, 0-27 blood was 5 8 TDF 1 10 1 time a day, collected to days collect 0-27 plasma. 6 8 Compound 1 10 10 2 times a day, days 0-27 TDF 1 10 1 time a day, days 0-27

[0192] Serum sample preparation: After the blood sample was incubated at 37° C. for about 30 minutes, centrifuged at 4° C., 13,200 g for 3 minutes, and the separated supernatant was quickly frozen in dry ice.

[0193] Plasma sample preparation and pretreatment: After the blood sample was anticoagulated with K2EDTA, the supernatant was separated by centrifugation at 4° C. and 7,000 g for 10 minutes. The separated plasma was added with precipitant, [methanol: acetonitrile (v: v, 50:50) solution] at a ratio of 1:20, vortexed to mix, and quickly frozen in dry ice.

[0194] Body weight recording. During the in vivo experiment, the status of the mice was regularly observed, and the weight of the mice was recorded on the day of infection, dosage, blood sampling and the end of the experiment.

[0195] Blood was collected from the heart to collect serum for HBV DNA detection and detection of HBV RNA levels in groups 1 and 4.

[0196] 5. Sample Analysis

[0197] 1. HBsAg ELISA (Antu Biological, CL 0310) kit instructions for detecting HBsAg content in mouse serum;

[0198] 2. Instructions for HBeAg ELISA kit (Antu Biotech, CL 0312), to detect HBeAg content in mouse serum;

[0199] 3. Quantitative PCR detection of HBV DNA content in mouse serum and liver:

TABLE-US-00005 TABLE 5 qPCR reaction component list PCR reaction Required volume of 1 solution composition reaction system (μL) Taqman Universal Master Mix (2X) 5 Forward primer (10 μM) 0.4 Reverse primer (10 μM) 0.4 Probe (10 μM) 0.2 AE buffer 2 Sample 2

[0200] Data analysis: The data is expressed as the average value standard error of each group of samples. Unless otherwise specified, groups 1-6: n=8. Use Student's t test for statistical analysis.

[0201] 6. Results

[0202] 1) The Effect of Test Compound on Serum HBV DNA in AAV/HBV Mouse Experiment

[0203] The content of serum HBV DNA in each group of mice is summarized in FIG. 2:

[0204] The serum HBV DNA content of mice in the solvent group (Group 1) remained stable after dosage. Compound 1 single-drug group: Compared with the solvent group (Group 1), compound 1 was low (10 mpk, Group 2) and medium (30 mpk). The HBV DNA content in the serum of mice in the high (100 mpk) three dose groups began to show a dose-dependent trend after 3 days of dosage, and the HBV DNA content of the low and medium dose groups remained stable after 7 days of dosage; the serum HBV DNA content in the dose group continued to decrease for 7-28 days after dosage, which was significantly lower than that in the solvent group (p<0.01).

[0205] Compound 1 and TDF combined dosage group (Group 6, 10+1 mpk): Compared with the solvent group (Group 1), the HBV DNA content in the serum of mice decreased after 3 days of dosage, and the level of serum was 7-28 days after dosage. The HBV DNA content remained stable. Compared with compound 1 (Group 2, 10 mpk) and TDF (Group 5, 1 mpk) single-agent groups, the combined dosage showed a significant reduction in serum HBV DNA content in mice (p<0.01).

[0206] 2) The Effect of Test Compound AAV/HBV on HBV DNA in Liver in Mice

[0207] The content of HBV DNA in the liver of mice in each group is summarized in FIG. 3:

[0208] Compared with the solvent group (Group 1), after 28 days of dosage, the HBV DNA content in the liver of mice in the low (10 mpk), medium (30 mpk), and high (100 mpk) dose groups of Compound 1 all decreased. In the compound 1 and TDF combined dosage group (Group 6, 10+1 mpk) 28 days after dosage, the HBV DNA content in the liver was significantly lower than that in the compound 1 10 mpk single drug group (p<0.001).

[0209] 3) The Effect of Test Compound on Serum HBV RNA in AAV/HBV Mouse Experiment

[0210] The content of HBV RNA in the serum of each group of mice is summarized in FIG. 4:

[0211] Compared with the solvent group (Group 1), after 28 days of dosage, the HBV RNA content in the serum of mice in the high dose (100 mpk) group of compound 1 decreased.

[0212] 4) Pharmacokinetic Analysis of Test Compounds in AAV/HBV Model Mice

[0213] The concentration of drugs in the serum of mice in group 2 is summarized in FIG. 5 and Table 6

[0214] On day 27, 0 hours after the first dosage (before dosage), 1, 4, 8, 9, 12, and 24, the average drug concentration in the mouse serum was 78, 122200, 1630, 605, 10300, 1280 and 66 nM, respectively. The drug absorption reached its peak 1 hour after dosage, the plasma half-life was 2.23 hours, and the AUC.sub.0-inf was 47600 nM.Math.h.

TABLE-US-00006 TABLE 6 Metabolic analysis of test compounds in AAV/HBV model mice PK parameters 10 mg/kg, BID T .sub.1/2 calculation time range 9-24 C.sub.max (nM) 12200 T.sub.max (h) 1.00 T.sub.1/2 (h) 2.23 T.sub.last (h) 24.0 AUC.sub.0-last (nM .Math. h) 47300 AUC.sub.0-8 (nM .Math. h) 26000 AUC.sub.0-inf (nM .Math. h) 47600 MRT.sub.0-last (h) 6.27 MRT.sub.0-inf (h) 6.36 AUC.sub.0-inf/AUC.sub.0-last (%) 101

[0215] 5) Mouse Health and Weight Monitoring

[0216] During the experiment, the health and weight of the mice were monitored regularly.

[0217] Comparing with body weight on day 0, the results of mouse body weight changes are summarized in FIG. 6:

[0218] The body weight of all mice remained stable during the expected period, and no significant drop was seen, and the mice were in good condition.

[0219] Note: From the −28th day to the 28th day, the weight change of the mice was recorded (the first day is changed to the day 0). The body weight on day 0 is used as a benchmark for comparison. According to IACUC regulations, a 20% weight loss is used as the humane endpoint. If a mouse loses more than 20% weight, it must be removed from the experiment.

[0220] 7. Conclusion

[0221] Compared with the solvent control group, mouse serum and liver HBV DNA were significantly reduced after compound 1 treatment, and showed a dose-dependent trend in serum. After compound 1 and TDF are used in combination, the effect is significantly better than compound 1 single-drug therapy, showing a good combination drug effect. The body weight of the mice did not drop significantly during the entire experiment, indicating that the mice tolerated the test compound well.

[0222] 8. Description

[0223] TDF in the experiment is replaced with ETV (entecavir), so that the in vivo antiviral effect of compound 1 and ETV is obtained, and the mouse serum and liver HBV DNA are significantly reduced and are superior to the single-drug therapy of compound 1.

Experimental Example 3: Compound 1 Combined with the Following Formula (IIb) Compound and Formula (IIIb) Compound on HBV Inhibitory Activity In Vitro

[0224] ##STR00048##

[0225] 1. Experimental Materials

[0226] Cell line: HepG2.2.15 cells are constructed and provided by WuXi AppTec. The cell culture medium is DMEM/F12 medium supplemented with 2% fetal bovine serum, 2 mM glutamine, 1×non-essential amino acids, 100 U/mL penicillin and 100 g/mL streptomycin.

[0227] Reagents: The main reagents used in this study include FastStart Universal Probe Master (Roche, article number 04914058001), DNA extraction kit (Qiagen, article number 51162), CellTiter-Glo (Promega-G7573);

[0228] Instruments: The main instruments used in this study are 7900 real-time fluorescent quantitative PCR instrument (Applied Biosystems), multifunctional microplate reader (BioTek, Synergy2), QuantStudio™ 6 Flex System (Applied Biosystems)

[0229] Test compound: compound 1, compound of formula (IIb), compound of formula (IIIb).

[0230] 2. Experimental Method

[0231] 2.1 Anti-HBV Activity of Compound Single Agent

[0232] 2.1.1 On the first day, HepG2.2.15 cells were seeded into a 96-well cell culture plate at a density of 60,000 cells/well, and then the cells were cultured overnight at 5% CO2 and 37° C. On the second day, the compound 1 and the compound of formula (IIb) and the compound of formula (IIIb) were diluted and added to a 96-well plate. Each combination was a three-fold plate. The cells were cultured under 5% CO2 and 37° C. for 3 days. The detection concentration of each compound is shown in Table 7. On the 5th day, a new culture medium containing the compound was replaced, and the supernatant was collected on the 8th day. The HBsAg in the supernatant was detected by ELISA, and the DNA in the supernatant was extracted at the same time, and the content of HBV DNA in the supernatant was detected by quantitative PCR. After collecting the cell supernatant, add CellTiter-Glo to detect cell viability.

TABLE-US-00007 TABLE 7 The concentration setting of test compound alone Compound (nM) 1 2 3 4 5 6 7 8 Compound 1 1000.00 333.33 111.11 37.04 12.35 4.12 1.37 0.46 Compound of 1000.00 333.33 111.11 37.04 12.35 4.12 1.37 0.46 formula (IIb) Compound of 50.00 16.67 5.56 1.85 0.62 0.21 0.07 0.02 formula (IIIb)

[0233] 2.2 Anti-HBV Activity of the Compounds in Combination

[0234] 2.2.1 On the first day, HepG2.2.15 cells were seeded into a 96-well cell culture plate at a density of 60,000 cells/well, and then the cells were cultured overnight at 5% CO.sub.2 and 37° C. On the second day, different concentrations of compounds were added to treat the cells. Compounds were diluted in gradients, 7×7 concentration combinations, and 3 replicate plates were tested in parallel. The compound concentration tested for joint activity is based on the EC.sub.50 value of each compound. The 7 concentration gradients are approximately: 2×, 1×, ½×, ¼×, ⅛×, 1/16× and 1/32×EC.sub.50. The compound concentration of the combined activity test experiment is shown in Table 8, and the combination drug arrangement is shown in Table 9. On the 5th day, replace the fresh culture medium containing the compound. On the 8th day, the culture supernatant was collected, DNA was extracted, and the content of HBV DNA was detected by quantitative PCR. At the same time, CellTiter Glo reagent was used to detect cell viability and MacSynergy software was used to analyze the combined effect of the two drugs.

TABLE-US-00008 TABLE 8 Concentration setting of test compound combination Concentrations (nM) Compounds 7 6 5 4 3 2 1 Compound 1 12 6 3 1.5 0.75 0.375 0.188 Compound of 6 3 1.5 0.75 0.375 0.188 0.094 formula (IIb) Compound of 1.2 0.6 0.3 0.15 0.075 0.038 0.018 formula (IIIb)

TABLE-US-00009 TABLE 9 Combination medication arrangement of test compounds Row 1 2 3 4 5 6 7 8 9 10 11 12 A a0; b7 a7; b7 a6; b7 a5; b7 a4; b7 a3; b7 a2; b7 a1; b7 a0; b7 a7; b0 a0; b0 BG B a0; b6 a7; b6 a6; b6 a5; b6 a4; b6 a3; b6 a2; b6 a1; b6 a0; b6 a6; b0 a0; b0 BG C a0; b5 a7; b5 a6; b5 a5; b5 a4; b5 a3; b5 a2; b5 a1; b5 a0; b5 a5; b0 a0; b0 BG D a0; b4 a7; b4 a6; b4 a5; b4 a4; b4 a3; b4 a2; b4 a1; b4 a0; b4 a4; b0 a0; b0 BG E a0; b3 a7; b3 a6; b3 a5; b3 a4; b3 a3; b3 a2; b3 a1; b3 a0; b3 a3; b0 a0; b0 BG F a0; b2 a7; b2 a6; b2 a5; b2 a4; b2 a3; b2 a2; b2 a1; b2 a0; b2 a2; b0 a0; b0 BG G a0; b1 a7; b1 a6; b1 a5; b1 a4; b1 a3; b1 a2; b1 a1; b1 a0; b1 a1; b0 a0; b0 BG H a0; b0 a7; b0 a6; b0 a5; b0 a4; b0 a3; b0 a2; b0 a1; b0 a0; b0 a0; b0 a0; b0 BG Note: a = compound 1, b = compound of formula (IIb) or compound of formula (IIIb); 0 means no compound, 1-7 means 7 concentrations.

[0235] 3. Experimental Results

[0236] HepG2.2.15 cells were used to evaluate the inhibitory activity of compound 1 and compound of formula (IIb), compound 1 and compound of formula (IIb) in vitro against HBV. The inhibitory activity and cytotoxicity of the compound single drugs on HBV are summarized in Table 10. The inhibitory activity and cytotoxicity of the combined drugs on HBV are summarized in Tables 11-13.

TABLE-US-00010 TABLE 10 Single-agent anti-HBV activity and cytotoxicity of compounds HBV DNA HBsAg Cytotoxicity Test compound EC.sub.50 (nM) EC.sub.50 (nM) CC.sub.50 (nM) Compound 1 6.03 >1000 >1000 Compound of formula (IIb) 1.52 4.90 >1000 Compound of formula (IIIb) 0.78 0.91 >50

TABLE-US-00011 TABLE 11 Co-administration results of compound 1 and compound of formula (IIb) and compound of formula (IIIb) (99% confidence interval) Synergy Antagonism Index Joint Index Index Cytotoxicity Result HBV DNA Compound 1 + Compound of 7.39 0 NA Additive formula (IIb) effect HBV DNA Compound 1 + Compound of 2.77 −7.31 NA Additive formula (IIIb) effect Note: Description of drug combination index: the absolute value of the index is less than 25, that is, the additive effect; the absolute value of the index is in the range of 25-50, that is, mild but clear synergy or antagonism; the absolute value of the index is in the range of 50-100, that is, moderate Synergistic or antagonistic effects may have important implications for in vivo effects. The absolute value of the index is in the range of > 100, that is, a highly synergistic or antagonistic effect, which is likely to have important meanings in vivo.

TABLE-US-00012 TABLE 12 Cytotoxicity test average cell viability percentage (%) of Compound 1 combined with compound of formula (IIb) compound 1 Compound Concentration (nM) 12 6 3 1.5 0.75 0.375 0.1875 0 Compound 6 104.60 ± 110.65 ± 111.24 ± 108.30 ± 104.72 ± 107.32 ± 110.60 ± 111.27 ± of 3.29 4.09 2.77 5.24 10.16 6.73 1.62 2.19 formula 3 96.23 ± 97.35 ± 99.53 ± 100.30 ± 100.39 ± 101.63 ± 101.72 ± 102.61 ± (IIb) 5.87 1.48 0.62 2.29 1.38 2.84 3.39 4.65 1.5 96.78 ± 100.15 ± 100.73 ± 99.76 ± 100.36 ± 98.83 ± 103.22 ± 104.15 ± 2.78 2.32 3.01 0.99 2.65 2.03 3.95 2.94 0.75 100.70 ± 100.30 ± 101.31 ± 101.51 ± 106.55 ± 105.83 ± 104.84 ± 105.36 ± 2.42 0.58 4.83 2.28 2.91 3.84 2.86 1.65 0.375 100.63 ± 100.83 ± 102.04 ± 102.87 ± 105.16 ± 103.29 ± 101.57 ± 102.39 ± 3.24 1.50 1.76 1.67 2.52 2.78 2.93 0.92 0.1875 99.83 ± 100.86 ± 102.44 ± 101.94 ± 103.76 ± 102.48 ± 104.81 ± 101.98 ± 5.27 2.67 2.66 4.75 3.85 3.02 3.98 2.11 0.09375 96.90 ± 95.50 ± 97.47 ± 96.64 ± 100.23 ± 97.60 ± 102.21 ± 101.23 ± 4.27 4.11 2.36 2.22 2.47 2.59 0.84 3.41 0 102.08 ± 103.50 ± 104.39 ± 105.37 ± 106.91 ± 109.88 ± 109.29 ± 109.76 ± 8.49 6.49 7.05 8.80 6.98 3.82 2.25 1.63

TABLE-US-00013 TABLE 13 Cytotoxicity test average cell viability percentage (%) of Compound 1 combined with compound of formula (IIIb) Compound 1 Compound Concentration (nM) 12 6 3 1.5 0.75 0.375 0.1875 0 Compound 1.2 110.95 ± 112.23 ± 113.70 ± 112.03 ± 110.25 ± 112.48 ± 112.88 ± 112.02 ± of 5.45 4.72 3.93 1.06 2.28 2.80 2.98 2.99 formula 0.6 102.44 ± 103.05 ± 102.51 ± 103.62 ± 102.75 ± 105.55 ± 104.66 ± 103.50 ± (IIIb) 4.62 4.11 6.12 3.13 3.52 5.15 4.40 5.95 0.3 99.66 ± 104.61 ± 103.85 ± 101.30 ± 101.01 ± 100.77 ± 105.16 ± 103.14 ± 5.31 4.31 3.26 5.94 4.42 5.22 3.30 6.14 0.15 103.81 ± 108.76 ± 106.66 ± 107.26 ± 109.23 ± 110.09 ± 105.10 ± 105.68 ± 5.15 4.93 4.55 3.21 3.36 3.89 6.11 4.30 0.075 103.12 ± 104.90 ± 105.13 ± 104.24 ± 108.04 ± 109.61 ± 105.89 ± 105.64 ± 4.19 3.36 6.65 3.69 2.39 3.02 3.68 3.61 0.0375 104.13 ± 103.87 ± 104.20 ± 102.52 ± 102.13 ± 103.94 ± 104.85 ± 103.81 ± 4.37 5.20 5.09 3.80 3.02 1.76 1.22 4.25 0.01875 100.86 ± 102.05 ± 102.06 ± 101.19 ± 101.90 ± 100.34 ± 101.23 ± 103.45 ± 3.97 2.76 1.87 2.25 2.14 4.33 3.54 4.84 0 111.17 ± 112.45 ± 112.58 ± 111.96 ± 111.36 ± 112.42 ± 110.07 ± 112.83 ± 2.41 3.65 5.37 2.92 5.07 2.82 5.45 4.86

[0237] 4. Experimental Results

[0238] In the HepG2.2.15 in vitro HBV infection model, the test compound inhibited HBV DNA in a dose-dependent manner; compound 1 and compound of formula (IIb), compound 1 and compound of formula (IIIb) showed the combined effect of additive effect, and the combination was in use No cytotoxicity was shown in the tested concentration. The test results support the combined application of compound 1 and the compound of formula (IIb), compound of formula (IIIb) and other hepatitis B surface antigen inhibitors in clinical treatment of chronic HBV infection.

[0239] 5. Description

[0240] In accordance with the above embodiment, compound 1 is replaced with compound 2

##STR00049##

or compound 3

##STR00050##

to obtain the results of the co-administration of compound 2 with the compound of formula (IIb) and with the compound of formula (IIIb) and the results of the co-administration of compound 3 with the compound of formula (IIb) and with the compound of formula (IIIb), both showing data results with additive effects and showing no cytotoxicity in the concentrations tested in combination use. The test results support the clinical combination use of the compounds such as compound 2 and compound 3 and the hepatitis B surface antigen inhibitors such as the compound of formula (IIb) and the compound of formula (IIIb) in the treatment of chronic HBV infection.