COMBINATION FOR TREATING HEPATITIS B

Abstract

The present invention relates to a combination of a compound represented by formula (I) and other drugs for treating hepatitis B that is used for treating hepatitis B, and use of the combination in the preparation of drugs for treating hepatitis B.

##STR00001##

Claims

1. A combination, comprising a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, ##STR00206## wherein, R.sub.1 is selected from H, OH, CN, NH.sub.2, C.sub.1-5 alkyl, C.sub.1-5 heteroalkyl, C.sub.2-5 alkynyl, C.sub.3-6 cycloalkyl and 3- to 6-membered heterocycloalkyl, and the C.sub.1-5 alkyl, C.sub.1-5 heteroalkyl, C.sub.2-5 alkynyl, C.sub.3-6 cycloalkyl and 3- to 6-membered heterocycloalkyl are optionally substituted by 1, 2 or 3 R; R.sub.2 is selected from H, halogen, C.sub.1-3 alkyl and C.sub.1-3 heteroalkyl, and the C.sub.1-3 alkyl and C.sub.1-3 heteroalkyl are optionally substituted by 1, 2 or 3 R; m is selected from 0, 1, 2, 3, 4 and 5; A is selected from phenyl and 5- to 6-membered heteroaryl, and the phenyl and 5- to 6-membered heteroaryl are optionally substituted by 1, 2 or 3 R; 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, CH.sub.2F; the C.sub.1-5 heteroalkyl, 3- to 6-membered heterocycloalkyl, C.sub.1-3 heteroalkyl and 5- to 6-membered heteroaryl each independently comprise 1, 2 or 3 heteroatoms or heteroatom groups each independently selected from N, -O-, =O, -S-, -NH-, -(C=O)-, -(S=O)- and -(S=O).sub.2-, wherein, the combination further comprises an immunomodulator.

2. The combination according to claim 1, wherein, the immunomodulator is selected from interferon, or, the combination further comprises a nucleotide reverse transcriptase inhibitor.

3. (canceled)

4. The combination according to claim 2, wherein, the nucleotide reverse transcriptase inhibitor is selected from tenofovir disoproxil fumarate and tenofovir alafenamide fumarate; or, the immunomodulator is peginterferon α-2a or interferon α-2b.

5. The combination according to claim 4, wherein, the immunomodulator is selected from interferon.

6. A combination, comprising the compound represented by formula (I) as defined in claim 1, or the pharmaceutically acceptable salt thereof, wherein the combination further comprises a nucleotide reverse transcriptase inhibitor or a nucleoside reverse transcriptase inhibitor.

7. The combination according to claim 6, wherein, the nucleotide reverse transcriptase inhibitor is selected from tenofovir disoproxil fumarate and tenofovir alafenamide fumarate; or, the nucleoside reverse transcriptase inhibitor is selected from Entecavir.

8. (canceled)

9. The combination according to claim 1, wherein, R is selected from H, F, Cl, Br, OH, CH.sub.3, CH.sub.3O, CF.sub.3, CHF.sub.2, CH.sub.2F; or, R.sub.1 is selected from H, OH, CN, NH.sub.2, CH.sub.3, CH.sub.3CH.sub.2, CH.sub.3CH.sub.2CH.sub.2, CH.sub.3CH.sub.2CH.sub.2CH.sub.2, CH.sub.3O, CH.sub.3CH.sub.2O, CH.sub.3S, CH.sub.3S(=O), CH.sub.3S(=O).sub.2, CH.sub.3SCH.sub.2, CH.sub.3CH.sub.2S, CH.sub.3NH, ##STR00207## ##STR00208## ##STR00209## ##STR00210## ##STR00211## ##STR00212## ##STR00213## ##STR00214## ##STR00215## , pyrrolidinyl, piperidinyl, tetrahydropyranyl, morpholinyl, 2-pyrrolidonyl and 3-pyrrolidonyl, and the CH.sub.3, CH.sub.3CH.sub.2, CH.sub.3CH.sub.2CH.sub.2, CH.sub.3CH.sub.2CH.sub.2CH.sub.2, CH.sub.3O, CH.sub.3CH.sub.2O, CH.sub.3S, CH.sub.3S(=O), CH.sub.3S(=O).sub.2, CH.sub.3SCH.sub.2, CH.sub.3CH.sub.2S, CH.sub.3NH, ##STR00216## ##STR00217## ##STR00218## ##STR00219## ##STR00220## ##STR00221## ##STR00222## ##STR00223## ##STR00224## pyrrolidinyl, piperidinyl, tetrahydropyranyl, morpholinyl, 2-pyrrolidonyl and 3-pyrrolidonyl are optionally substituted by 1, 2 or 3 R; or, R.sub.2 is selected from H, F, Cl, Br, CH.sub.3, CH.sub.3CH.sub.2, CH.sub.3O, CH.sub.3CH.sub.2O and ##STR00225## and the CH.sub.3, CH.sub.3CH.sub.2, CH.sub.3O, CH.sub.3CH.sub.2O and ##STR00226## are optionally substituted by 1, 2 or 3 R; or, A is selected from: phenyl, thienyl, thiazolyl, isothiazolyl, oxazolyl and isoxazolyl, each of which is optionally substituted by 1, 2 or 3 R; or, m is 3; or, m is 1.

10. (canceled)

11. The combination according to claim 9, wherein, R.sub.1 is selected from H, OH, CN, NH.sub.2, CH.sub.3, CH.sub.3CH.sub.2, CH.sub.3CH.sub.2CH.sub.2, CH.sub.3CH.sub.2CH.sub.2CH.sub.2, CH.sub.3O, CH.sub.3CH.sub.2O, CH.sub.3S, CH.sub.3S(=O), CH.sub.3S(=O).sub.2, CH.sub.3SCH.sub.2, CH.sub.3CH.sub.2S, CH.sub.3NH, ##STR00227## ##STR00228## ##STR00229## ##STR00230## ##STR00231## ##STR00232## ##STR00233## ##STR00234## ##STR00235## ##STR00236## ##STR00237## ##STR00238## ##STR00239## ##STR00240## and the CH.sub.3, CH.sub.3CH.sub.2, CH.sub.3CH.sub.2CH.sub.2, CH.sub.3CH.sub.2CH.sub.2CH.sub.2, CH.sub.3O, CH.sub.3CH.sub.2O, CH.sub.3S, CH.sub.3S(=O), CH.sub.3S(=O).sub.2, CH.sub.3SCH.sub.2, CH.sub.3CH.sub.2S, CH.sub.3NH, ##STR00241## ##STR00242## ##STR00243## ##STR00244## ##STR00245## ##STR00246## ##STR00247## ##STR00248## ##STR00249## ##STR00250## ##STR00251## ##STR00252## ##STR00253## ##STR00254## optionally substituted by 1, 2 or 3 R; or, R.sub.2 is selected from Cl and CH.sub.3O or, A is selected from: ##STR00255## ##STR00256## ##STR00257## ##STR00258## ##STR00259## ##STR00260## ##STR00261## ##STR00262## ##STR00263## ##STR00264## each of which is optionally substituted by 1, 2 or 3 R; or, when m is 3, R.sub.2 is selected from Cl and CH.sub.3O or, when m is 1, R.sub.2 is Cl.

12. The combination according to claim 11, wherein, R.sub.1 is selected from H, OH, CH.sub.3, CHF.sub.2, CH.sub.3O, ##STR00265## ##STR00266## ##STR00267## ##STR00268## ##STR00269## ##STR00270## ##STR00271## or, A is selected from ##STR00272## ##STR00273## ##STR00274## ##STR00275## ##STR00276## ##STR00277## ##STR00278## ##STR00279## or, when m is 3, R.sub.2 is selected from Cl and CH.sub.3O, R.sub.1 is CH.sub.3O; or, when m is 1, R.sub.2 is Cl, R.sub.1 is ##STR00280## .

13. (canceled)

14. (canceled)

15. (canceled)

16. (canceled)

17. (canceled)

18. The combination according to claim 12, wherein, A is selected from ##STR00281## ##STR00282## ##STR00283## ##STR00284## ##STR00285## ##STR00286## ##STR00287## ##STR00288## ##STR00289## ##STR00290## ##STR00291## ##STR00292## ##STR00293## ##STR00294## ##STR00295## ##STR00296## ##STR00297## ##STR00298## ##STR00299## ##STR00300## ##STR00301## ##STR00302## ##STR00303## ##STR00304## ##STR00305## ##STR00306## ##STR00307## ##STR00308## ##STR00309## ##STR00310## ##STR00311## ##STR00312## ##STR00313## ##STR00314## ##STR00315## or, when m is 3, R.sub.2 is selected from Cl and CH.sub.3O, R.sub.1 is CH.sub.3O,A is selected from: ##STR00316## ##STR00317## ##STR00318## ##STR00319## ##STR00320## ##STR00321## ##STR00322## each of which is optionally substituted by 1, 2 or 3 R; or, when m is 1, R.sub.2 is Cl, R.sub.1 is ##STR00323## A is selected from: ##STR00324## ##STR00325## ##STR00326## ##STR00327## ##STR00328## ##STR00329## ##STR00330## each of which is optionally substituted by 1, 2 or 3 R.

19. (canceled)

20. (canceled)

21. (canceled)

22. (canceled)

23. (canceled)

24. (canceled)

25. (canceled)

26. The combination according to claim 1, wherein, the compound is selected from: ##STR00331## ##STR00332## ##STR00333## ##STR00334## ##STR00335## ##STR00336## ##STR00337## ##STR00338## wherein, R.sub.1, R.sub.2, R and m are as defined above.

27. The combination according to claim 1, wherein, the compound is selected from: ##STR00339## ##STR00340## ##STR00341## ##STR00342## ##STR00343## ##STR00344## ##STR00345## ##STR00346## ##STR00347## ##STR00348## ##STR00349## ##STR00350## ##STR00351## ##STR00352## ##STR00353## ##STR00354## ##STR00355## ##STR00356## ##STR00357## ##STR00358## ##STR00359## ##STR00360## ##STR00361## ##STR00362## ##STR00363## ##STR00364## ##STR00365## ##STR00366## ##STR00367## ##STR00368## ##STR00369## ##STR00370## ##STR00371## ##STR00372## ##STR00373## ##STR00374## ##STR00375## ##STR00376## ##STR00377## ##STR00378## ##STR00379## ##STR00380## ##STR00381## ##STR00382## ##STR00383## ##STR00384## ##STR00385## ##STR00386## ##STR00387## ##STR00388## ##STR00389## .

28. The combination according to claim 27, wherein, the compound is selected from: ##STR00390## ##STR00391## ##STR00392## ##STR00393## ##STR00394## ##STR00395## ##STR00396## ##STR00397## ##STR00398## ##STR00399## ##STR00400## ##STR00401## ##STR00402## ##STR00403## ##STR00404## ##STR00405## ##STR00406## ##STR00407## .

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

30. A 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 the treatment of hepatitis B in a subject in need thereof, comprising: administering an effective amount of the composition according to claim 30 to the subject.

33. The combination according to claim 5, wherein, the immunomodulator is peginterferon α-2a or interferon α-2b.

34. The combination according to claim 6, wherein, R is selected from H, F, Cl, Br, OH, CH.sub.3, CH.sub.3O, CF.sub.3, CHF.sub.2, CH.sub.2F; or, R.sub.1 is selected from H, OH, CN, NH.sub.2, CH.sub.3, CH.sub.3CH.sub.2, CH.sub.3CH.sub.2CH.sub.2, CH.sub.3CH.sub.2CH.sub.2CH.sub.2, CH.sub.3O, CH.sub.3CH.sub.2O, CH.sub.3S, CH.sub.3S(=O), CH.sub.3S(=O).sub.2, CH.sub.3SCH.sub.2, CH.sub.3CH.sub.2S, CH.sub.3NH, ##STR00408## ##STR00409## ##STR00410## ##STR00411## ##STR00412## ##STR00413## ##STR00414## ##STR00415## ##STR00416## pyrrolidinyl, piperidinyl, tetrahydropyranyl, morpholinyl, 2-pyrrolidonyl and 3-pyrrolidonyl, and the CH.sub.3, CH.sub.3CH.sub.2, CH.sub.3CH.sub.2CH.sub.2, CH.sub.3CH.sub.2CH.sub.2CH.sub.2, CH.sub.3O, CH.sub.3CH.sub.2O, CH.sub.3S, CH.sub.3S(=O), CH.sub.3S(=O).sub.2, CH.sub.3SCH.sub.2, CH.sub.3CH.sub.2S, CH.sub.3NH, ##STR00417## ##STR00418## ##STR00419## ##STR00420## ##STR00421## ##STR00422## ##STR00423## ##STR00424## ##STR00425## pyrrolidinyl, piperidinyl, tetrahydropyranyl, morpholinyl, 2-pyrrolidonyl and 3-pyrrolidonyl are optionally substituted by 1, 2 or 3 R; or, R.sub.2 is selected from H, F, Cl, Br, CH.sub.3, CH.sub.3CH.sub.2, CH.sub.3O, CH.sub.3CH.sub.2O and ##STR00426## and the CH.sub.3, CH.sub.3CH.sub.2, CH.sub.3O, CH.sub.3CH.sub.2O and ##STR00427## are optionally substituted by 1, 2 or 3 R; or, A is selected from: phenyl, thienyl, thiazolyl, isothiazolyl, oxazolyl and isoxazolyl, each of which is optionally substituted by 1, 2 or 3 R; or, m is 3; or, m is 1.

35. The combination according to claim 34, wherein, R.sub.1 is selected from H, OH, CN, NH.sub.2, CH.sub.3, CH.sub.3CH.sub.2, CH.sub.3CH.sub.2CH.sub.2, CH.sub.3CH.sub.2CH.sub.2CH.sub.2, CH.sub.3O, CH.sub.3CH.sub.2O, CH.sub.3S, CH.sub.3S(=O), CH.sub.3S(=O).sub.2, CH.sub.3SCH.sub.2, CH.sub.3CH.sub.2S, CH.sub.3NH, ##STR00428## ##STR00429## ##STR00430## ##STR00431## ##STR00432## ##STR00433## ##STR00434## ##STR00435## ##STR00436## ##STR00437## ##STR00438## ##STR00439## ##STR00440## ##STR00441## and the CH.sub.3, CH.sub.3CH.sub.2, CH.sub.3CH.sub.2CH.sub.2, CH.sub.3CH.sub.2CH.sub.2CH.sub.2, CH.sub.3O, CH.sub.3CH.sub.2O, CH.sub.3S, CH.sub.3S(=O), CH.sub.3S(=O).sub.2, CH.sub.3SCH.sub.2, CH.sub.3CH.sub.2S, CH.sub.3NH, ##STR00442## ##STR00443## ##STR00444## ##STR00445## ##STR00446## ##STR00447## ##STR00448## ##STR00449## ##STR00450## ##STR00451## ##STR00452## ##STR00453## ##STR00454## ##STR00455## are optionally substituted by 1, 2 or 3 R; or, R.sub.2 is selected from Cl and CH.sub.3O; or, A is selected from: ##STR00456## ##STR00457## ##STR00458## ##STR00459## ##STR00460## ##STR00461## ##STR00462## ##STR00463## ##STR00464## ##STR00465## each of which is optionally substituted by 1, 2 or 3 R; or, when m is 3, R.sub.2 is selected from Cl and CH.sub.3O; or, when m is 1, R.sub.2 is Cl.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0107] The present disclosure is described in detail by the embodiments below, but it does not mean that there are any adverse restrictions on the present disclosure. The present disclosure has been described in detail herein, wherein specific embodiments thereof are also disclosed, and it will be apparent to those skilled in the art that various variations and improvements can be made to specific embodiments of the present disclosure without departing from the spirit and scope of the present disclosure.

Embodiment 1

[0108] Experimental objective: The purpose of this study was to evaluate the in vitro anti-hepatitis B virus efficacy of the test compounds in combination with TAF (tenofovir alafenamide fumarate) and Peg-IFN-α2a (peginterferon α 2a) using human primary hepatocytes (PHH) isolated from a humanized FRG mouse model.

[0109] Experimental materials: [0110] (1) Main instrument: fluorescence qPCR instrument (Applied Biosystems, model 7500). Microplate reader (Bio Tek, model Synergy 2). Chemiluminescence imaging system (GE, model LAS4010) [0111] (2) Main reagents and consumables: FastStart Universal Probe Master (Roche), DNA extraction kit (Qiagen), 96-well plate (Costar 3599) and 96-well V-type plate (Axygen WIPP02280), HBsAg ELISA kit (Autobio) and HBeAg ELISA kit (Autobio), Cell Counting Kit-8 (CCK-8) (Biolite). [0112] (3) Others: FRG mice, D-type HBV, test compound WWSO

[0113] Experimental method:

[0114] 1. The PHH model was used to evaluate the single drug in vitro antiviral activity of the test compound

[0115] On day 0, primary human hepatocytes were resuscitated and plated into two 48-well plates (1.32×10.sup.5 cells/well).

[0116] On day 1, HBV was added to infect PHH (2000 GE/cell). Two kinds of compound treatment methods were set up: One cell plate was treated with the test compound and Peg-IFNα-2a for four hours on day 1, and then the medium was then replaced with an HBV-containing medium to infect cells (at the same time, compounds were added to continue treatment); another plate was treated with test compound and Peg-IFNα-2a started on day 2. Fresh medium with compound was replaced every 1 to 2 days.

[0117] The final concentration of DMSO in the medium was 2%. The initial concentration of the test compound was 1000 nM, and the initial concentration of the control compound Peg-IFNα-2a was 500 lU/mL with 5-fold dilution, 7 concentration points, and three duplicate wells (see Table 3 for compound concentration settings).

[0118] The medium was replaced with the fresh medium containing the compound on days 4, 6, 8, 10, 12, 14, 16, 18 and 20. On day 22, the culture supernatant was collected and cell viability was detected using CellTiter-Glo.

[0119] Cell culture supernatants on days 8, 14 and 22 were collected for HBV DNA, HBeAg and HBsAg detection.

TABLE-US-00001 Individual administered concentration settings of the test compound Compound Concentration Solvent 7 6 5 4 3 2 1 Unit a Test compound 1000 200 40 8 1.6 0.32 0.064 nM DMSO b Peg-IFNα-2a 500 100 20 4 0.8 0.16 0.032 lU/mL Medium

[0120] 2. The PHH model was used to evaluate the in vitro anti-HBV activity of the test compound in combination with TAF and Peg-IFNα-2a

[0121] On day 0, primary human hepatocytes were resuscitated and plated into six 48-well plates (1.32×10.sup.5 cells/well).

[0122] On day 1, PHH was added to infect HBV (2000 GE/cell). On the second day, different groups of combined drugs were added for treatment:

[0123] (1) Test compound administered in combination with Peg-IFNα-2a: Both the test compound and Peg-IFN-α2a were diluted 3-fold at 5 concentration points, tested in a 5×5 matrix, and three duplicate plates. The starting concentrations of the test compound and Peg-IFNα-2a were 400 nM and 100 IU/mL, respectively. The arrangement of the compounds in the combined action is shown in Table 2, and the test concentrations of the compounds are shown in Table 3.

[0124] The final concentration of DMSO in the cell medium was 2%. The medium was replaced with the medium containing the compound on days 4 and 6. On day 8, the culture supernatant was collected for detecting HBV DNA, HBeAg and HBsAg, and then the collected supernatant was added with CellTiter-Glo to measure cell viability.

TABLE-US-00002 Administered arrangement of the test compound in combination with Peg-IFNα-2a 1 2 3 4 5 6 7 8 A a0;b5 a5;b5 a4;b5 a3;b5 a2;b5 a1;b5 DMSO Uninf B a0;b4 a5;b4 a4;b4 a3;b4 a2;b4 a1;b4 DMSO Uninf C a0;b3 a5;b3 a4;b3 a3;b3 a2;b3 a1;b3 DMSO Uninf D a0;b2 a5;b2 a4;b2 a3;b2 a2;b2 a1;b2 DMSO Uninf E a0;b1 a5;b1 a4;b1 a3;b1 a2;b1 a1;b1 DMSO Uninf F DMSO a5;b0 a4;b0 a3;b0 a2;b0 a1;b0 DMSO Uninf a=test compound, b=Peg-IFNα-2a; 0 means no compound, 1-5 means 5 concentrations

TABLE-US-00003 Administered concentration settings of the test compound in combination with Peg-IFNα-2a Compound Concentration Solvent 5 4 3 2 1 Unit a Test compound 400 133.33 44.44 14.81 4.94 nM DMSO b Peg-IFNα-2a 100 33.33 11.11 3.70 1.23 IU/mL Medium

[0125] (2) Test compound administered in combination with TAF and Peg-IFNα-2a: Both the test compound and Peg-IFNα-2a were diluted 3-fold at 5 concentration points, tested in a 5×5 matrix, and three duplicate plates. The starting concentrations of the test compound and Peg-IFNα-2a were 400 nM and 100 IU/mL, respectively, and TAF was set a concentration point of 0.3 nM. The arrangement of the compounds in the combined action is shown in Table 4, and the test concentrations of the compounds are shown in Table 5.

[0126] The final concentration of DMSO in the medium was 2%. The medium was replaced with the medium containing the compound on days 4 and 6. On day 8, the culture supernatant was collected for detecting HBV DNA, HBeAg and HBsAg, and then the collected cell supernatant was added with CellTiter-Glo to measure cell viability.

TABLE-US-00004 Administered arrangement of the test compound in combination with TAF and Peg-IFNα-2a 1 2 3 4 5 6 7 8 A a0;b5;c a5;b5;c a4;b5;c a3;b5;c a2;b5;c a1;b5;c DMSO Uninf B a0;b4;c a5;b4;c a4;b4;c a3;b4;c a2;b4;c a1;b4;c DMSO Uninf C a0;b3;c a5;b3;c a4;b3;c a3;b3;c a2;b3;c a1;b3;c DMSO Uninf D a0;b2;c a5;b2;c a4;b2;c a3;b2;c a2;b2;c a1;b2;c DMSO Uninf E a0;b1;c a5;b1;c a4;b1;c a3;b1;c a2;bl;c a1;b1;c DMSO Uninf F DMSO a5;b0;c a4;b0;c a3;b0;c a2;b0;c a1;b0;c DMSO Uninf a=test compound, b=Peg-IFNα-2a, c=TAF; 0 means no compound, 1 to 5 means 5 concentrations, Uninf means uninfected control group

TABLE-US-00005 Administered concentration settings of the test compound in combination with TAF and Peg-IFNα-2a Compound Concentration Solvent 5 4 3 2 1 Unit a The subject compound 400 133.33 44.44 14.81 4.94 nM DMSO b Peg-IFNα-2a 100 33.33 11.11 3.70 1.23 IU/mL Medium c TAF 0.3 nM DMSO

[0127] 3. Sample detection

[0128] Cell viability detection: After collecting the cell culture supernatant, CellTiter-Glo was diluted with medium at a ratio of 1:1, and added to the cell plate, and left at room temperature for 10 minutes to detect the luminescence value with a microplate reader.

[0129] HBV DNA detection: DNA in the supernatant was extracted according to the DNA extraction kit (Qiagen-51162), and HBV DNA in the sample was quantified by qPCR method, and the qPCR reaction system is shown in Table 6. HBV plasmid DNA was used as a standard for 10-fold serial dilution, and the standard range was from 1.0× 10.sup.7 copies/.Math.L to 10 copies/.Math.L. The PCR reaction program was: Heating at 95° C. for 10 minutes, then cycle mode was entered, and heating at 95° C. for 15 seconds, then at 60° C. for 1 minute for a total of 40 cycles. The HBV DNA content in the samples was calculated based on the standard curve and the Ct value of each sample.

TABLE-US-00006 qPCR reaction system Reagent Volume required to configure 1 well (.Math.L) Quantitative and rapid initiation of universal probe reagents 5 Forward primer (10 .Math.M) 0.4 Reverse primer (10 .Math.M) 0.4 Probe (10 .Math.M) 0.2 AE solvent 2 DNA in Sample 2

[0130] Detection of HBeAg and HBsAg: According to the instructions of ELISA kit, the levels of HBeAg and HBsAg in supernatant were detected. The method was briefly described as follows: 50 .Math.L, of standard was taken, and sample and control sample were added into the detection plate, then 50 .Math.L of enzyme conjugate was added to each well, incubated at 37° C. for 60 minutes, and washed with a washing solution and then dried, and then 50 .Math.L of pre-mixed luminescent substrate was added and the mixture was incubated at room temperature for 10 minutes in the dark, and finally the luminescence value was measured by a microplate reader. The content of HBeAg and HBsAg in the sample was calculated according to the standard curve and the chemiluminescence value of each sample.

[0131] 4. Data processing and statistical analysis

[0132] Cell activity% = (luminescence value of sample-average value of blank control well)/(luminescence value of DMSO control group-average value of blank control well) × 100

[0133] Inhibition rate %=(1- HBV DNA content or HBsAg content or HBeAg content in the sample/HBV DNA content or HBsAg content or HBeAg content in DMSO control group) × 100

[0134] CC.sub.50 and EC.sub.50 were calculated by GraphPad Prism software, and the inhibition curve fitting method was sigmoidal dose-response (variable slope).

[0135] Combined efficacy was analyzed by MacSynergy™ II software. The combined efficacy was calculated according to a confidence interval of 95% according to the synergism/antagonism, and the results were explained as follows according to MacSynergy criteria: [0136] <25 = Insignificant synergism/antagonism (additive); [0137] 25-50 = Minor but significant synergism/antagonism; [0138] 50-100 = Moderate synergism/antagonism - may be important in vivo; [0139] >100 = Strong synergism/antagonism - probably important in vivo; [0140] about 1000 = Possible errors - check data and repeat experiment;

[0141] 5. Experimental results: See Tables 7 and 8.

TABLE-US-00007 Anti-HBV activity and cytotoxicity of single drug in PHH (48-well culture plate) Compound Administration after infection Administration before infection Unit EC .sub.50 (Day 8) CC.sub.50 Day 22 EC .sub.50 (Day 8) CC.sub.50 Day 22 HBV DNA HBeAg HBsAg HBV DNA HBeAg HBsAg WWS0 45.52 32.35 47.34 >1000 13.47 25.99 47.15 >1000 nM Peg-IFNα-2a 0.81 13.5 42.76 >500 3.59 8.53 23.49 77.56 IU/m L

TABLE-US-00008 Results of combined administration of the test compound in PHH (95% confidence interval) Combination HBsAg HBeAg Synergistic index Antagonistic index Result Synergistic index Antagonistic index Result WWS0+ Peg-IFNα-2a 37.76 0 Moder ate coordi nation 22.43 0 additio n WWSO + Peg-IFNα-2a + TAF 36.47 0 Moder ate coordi nation 0.78 -0.8 additio n

[0142] 6. Experimental conclusion: In the PHH in vitro infection model of HBV infection, the test compound WWSO inhibits HBV DNA, HBeAg and HBsAg in a dose-dependent manner, and the replication activity of the inhibition of HBV by WWSO is basically the same in the two modes of administration before and after HBV infection.

[0143] The combined administration result of the test compound shows that the combined administration of WWSO and Peg-IFNα-2a has a moderate synergism on the inhibition of HBsAg, and additive effect on the inhibition of HBeAg. The three-drug combination of test compound WWSO with TAF and Peg-IFNα-2a, has a moderate synergism on the inhibition of HBsAg and additive effect on the inhibition of HBeAg.

[0144] In the test compound WWSO single drug and combined action experiment, no obvious cytotoxicity is displayed within the test concentration range.

Embodiment 2

[0145] In this study, HepG2-NTCP cell in vitro infection model was used to evaluate the inhibitory activity of a combined administration of compound WWSO with Entecavir (ETV) on HBV The content of HBV DNA in cell supernatant was detected by real-time quantitative PCR (qPCR) method, and the content of HBeAg and HBsAg was detected by ELISA, and the anti-HBV activity of the combined action of the compound was determined, while CellTiter-Glo was used to detect the effect of the test compound on cell viability.

1. Experimental Materials

[0146] Cell line: HepG2-NTCP cell line was constructed and provided by WuXi AppTec. HepG2-NTCP cells were HepG2 cells stably transfected and continued to highly express the human NTCP gene, and were susceptible to be infected by HBV The cell medium was DMEM medium added with 10% fetal bovine serum, 1% Penicillin-Streptomycin, 1% glutamine, 1% non-essential amino acids and G418 (500 .Math.g/mL).

[0147] Virus: D-type HBV was concentrated from HepG2.2.15 cell culture supernatants.

[0148] Reagents: Main reagents used in this study included: FastStart Universal Probe Master (Roche), DNA extraction kit (Qiagen), 96-well plate (Costar), HBsAg ELISA kit (Autobio) and HBeAg ELISA kit (Autobio).

[0149] Instruments: The main instruments used in this study were real-time fluorescence quantitative PCR instrument (Thermo, QuantStudio™ 6 Flex) and multi-functional microplate reader (BioTek, Synergy2).

[0150] Test compound: WWSO and ETV

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[0151] ETV was a drug that had been marketed for the treatment of HBV

2. Experimental Method

[0152] 2.1 On day 0, HepG2-NTCP cells were seeded into a 48-well cell culture plate at a density of 75,000 cells/well, and then the cells were cultured at 5% CO.sub.2 and 37° C. overnight.

[0153] 2.2 On day 2, HBV was added to infect HepG2-NTCP cells.

[0154] 2.3 On day 3, WWSO and ETV single drugs were administered at seven different concentrations, respectively, or WWSO in combination with ETV was administered at 5 different concentrations in orthorhombic proportion, added to 48-well plates with each combination having three duplicate plates.

TABLE-US-00009 Individual administered concentration settings of the test compound Test compound Test concentration (nM) Solvent ETV 20 4 0.8 0.16 0.032 0.0064 0.00128 DMSO WWS0 100 20 4 0.8 0.16 0.032 0.0064 DMSO

TABLE-US-00010 Administered arrangement of WWSO in combination with ETV 1 2 3 4 5 6 7 8 A a0;b5 a5;b5 a4;b5 a3;b5 a2;b5 a1;b5 DMSO Uninf B a0;b4 a5;b4 a4;b4 a3;b4 a2;b4 a1;b4 DMSO Uninf C a0;b3 a5;b3 a4;b3 a3;b3 a2;b3 a1;b3 DMSO Uninf D a0;b2 a5;b2 a4;b2 a3;b2 a2;b2 a1;b2 DMSO Uninf E a0;b1 a5;b1 a4;b1 a3;b1 a2;b1 a1;b1 DMSO Uninf F DMSO a5;b0 a4;b0 a3;b0 a2;b0 a1;b0 DMSO Uninf a=WWS0, b=ETV; 0 means no compound, and 1-5 means five concentrations.

TABLE-US-00011 Administered concentration settings of WWSO in combination with ETV Test compound Concentration (nM) 5 4 3 2 1 a WWS0 12 6 3 1.5 0.75 b ETV 0.2 0.1 0.05 0.025 0.0125

[0155] 2.4 On day 6, the cell supernatant was discarded and fresh medium containing the compounds was added, and the cells were cultured at 5% CO.sub.2 and 37° C. for 3 days.

[0156] 2.5 On day 9, cell plate supernatant treated by compound was collected and DNA was extracted according to the QIAamp 96 DNA Blood Kit (12) instructions. HBeAg and HBsAg were detected according to the instructions of ELISA detection kit, meanwhile, the cytotoxicity of compound pairs was detected by CellTiter-Glo kit, and the chemiluminescence intensity (RLU) of each cell well was detected by multifunctional microplate reader according to the method of kit instructions. HBV DNA was quantified by qPCR method. HBV plasmid DNA was used as the standard, and standard HBV plasmid DNA concentration was diluted 7 points starting from 10.sup.7 copies/.Math.L at a 10-fold gradient. The HBV DNA copy number was calculated in each test sample by fitting the standard curve using the HBV DNA copy number and CT values of each standard.

[0157] 2.6 MacSynegy software (Prichard et al., 1990) was used to process the test data and analyze the effect parameters of combined administration of compound. Description of combined administration index:

[0158] A positive index is synergism, while a negative index is antagonism; and the absolute value of index < 25, that is, additive effect; the absolute value of the index is in the range of 25 to 50, that is, a mild but definite synergism or antagonism; the absolute value of the index is in the range of 50 to 100, that is, a moderate synergism or antagonism, which may be of great significance to the in vivo effect. The absolute value of the index is in the range of >100, that is, a high degree of synergism or antagonism, which may be of great significance to the in vivo effect.

[0159] 3. Experimental results: See Table 12 and Table 13.

TABLE-US-00012 Anti-HBV activity and cytotoxicity of single drug Test compound HBeAg EC.sub.50 (nM) HBsAg EC.sub.50 (nM) HBV DNA EC.sub.50 (nM) Cytotoxicity CC.sub.50 (nM) ETV >20 >20 0.06 >20 WWS0 2.51 3.19 1.01 >100

TABLE-US-00013 Results of combined administration of WWSO and ETV (95% confidence interval) Index Combination Synergistic index Antagonistic Index Cytotoxicity Result HBeAg WWSO + ETV 33.82 -6.03 none Mild coordination HBsAg WWSO + ETV 136.04 0 none Strong coordination

[0160] 4. Experimental conclusion: In the in vitro model of HepG2-NTCP infected by HBV, the test compound WWSO inhibited HBV DNA, HBeAg and HBsAg in a dose-dependent manner; the combination of WWSO and ETV shows that HBeAg and HBsAg have synergistic or additive anti-HBV activities; in the test compound WWWS0 single drug and combined action experiments, no obvious cytotoxicity is displayed within the test concentration range.

Embodiment 3

[0161] According to the method of embodiment 1, the test compound WWSO

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was replaced by the compounds WWS01

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WWS02

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and WWS03

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[0162] the results showed that the two-drug combination of the test compounds WWS01, WWS02, WWS03 with Peg-IFNα-2a had a synergistic or additive effects on the inhibition of HBsAg and HBeAg, in which the synergistic index was in the range of 20.00 to 50.00 at the 95% confidence interval, and the antagonistic index was 0.

Embodiment 4

[0163] According to the method of embodiment 2, ETV (Entecavir) was replaced by TDF (tenofovir disoproxil fumarate) to obtain a combined administration result of the test compound WWSO in a HepG2-NTCP in vitro infection HBV model: The combination of WWSO with TDF had synergism on the inhibition of HBsAg and HBeAg, with the synergistic indexes being 86.55 and 25.49 respectively in the 95% confidence interval and the antagonistic indexes being 0 and 1.06.

Embodiment 5

[0164] According to the method of embodiment 1, interferon Peg-IFNα-2a was replaced by IFNα-2b, and the results of the two drug-combination of the test compound WWS0 and IFNα-2b and three drug-combination of WWSO and TAF and IFNα-2b were obtained. The results showed that the synergistic indexes of both the two combinations for HBsAg and HBeAg were greater than 25, and had a synergism on the inhibition of HBsAg and HBeAg.