EA037576B1 - Combinations and methods comprising a capsid assembly inhibitor - Google Patents

Abstract

The invention relates to a combination product and a pharmaceutical composition comprising a core protein allosteric modulator (CpAM), which is a compound of formula IA, and a reverse transcriptase inhibitor, as well as the use thereof in the treatment of hepatitis B virus infection.

Description

Cross-reference to related applications

The application for this invention claims the benefit of US Provisional Application No. 62/323251, filed April 15, 2016, and US Provisional Application No. 62/421035, filed November 11, 2016. The contents of each of these applications are incorporated herein in their entirety. by reference.

Scope of the invention

The present disclosure relates to methods of using a capsid assembly inhibitor for the treatment of hepatitis B viral infection.

Background of the invention

Chronic hepatitis B virus (HBV) infection is an intractable, potentially progressive necroinflammatory liver disease associated with chronic HBV infection. Worldwide, about 240-400 million people are chronically infected with HBV, and chronic HBV infection is the leading global cause of severe liver disease and liver-related mortality (Hepatitis B Factsheet, World Health Organization, 2013; Hoofnagle JH, et al., Management of Hepatitis B: Summary of a Clinical Research Workshop, Hepatology, 2007, 45 (4): 1056-1075; EASL Clinical Practice Guidelines: Management of chronic hepatitis B virus infection, J. Hepatology, 2012, 57: 167-185 (EASL 2012 ); Lesmana LA, et al. Hepatitis B: overview of the burden of disease in the Asia-Pacific region, Liver International, 2006, 26: 3-10; Lok ASF and McMahon BJ, Chronic Hepatitis B: Update 2009, Hepatology. September 2009: 1-36 (Lok 2009)).

The chronic state of HBV infection in individual subjects has traditionally been diagnosed by confirming the stable detectability of hepatitis B surface antigen (HBsAg) in the subject's serum for 6 months or more. The US Centers for Disease Control (CDC) considers the following serologic profile to be consistent with chronic HBV infection: HBsAg positive and HBsAb negative with detectable anti-hepatitis B capsid antigen IgG (HBcAb IgG) and undetectable anti-hepatitis B capsid antigen IgM (HBcAb IgM). In such subjects, hepatitis B serum antigen-e (HBeAg) may or may not be detectable and is more likely to be undetectable in the later stages of chronic HBV infection.

Current, regulatory approved treatment regimens for chronic HBV infection include parenterally administered alpha interferons (non-pegylated or pegylated) and various orally administered nucleoside / nucleotide (nucleos (t )ide) HBV polymerase / reverse transcriptase inhibitors (HBV Pol-RT)) ... Each of these agents suppressed HBV replication and induced HBeAg reduction / seroconversion in only about 20-35% of HBeAg-positive patients after a year of treatment (EASL 2012; Lok 2009; Sorrell MF et al., National Institutes of Health Consensus Development Conference Statement: Management of Hepatitis B, Ann Intern Med, 2009, 150 (2): 104-110; Woo G. et al., Tenofovir and Entecavir Are the Most Effective Antiviral Agents for Chronic Hepatitis B: A Systematic Review and Bayesian Meta-Analyses, Gastroenterology. 2010: 1-17). Although patients with chronic HBV infection who were HBeAg-positive have experienced up to 30-35% reductions in HBeAg with current 48-week pegylated interferon (PegIFN) regimens, within 2-5 years after treatment, 20 -50% of patients experienced a rebound to baseline HBeAg levels (Perillo R., Benefits and Risks of Interferon Therapy for Hepatitis B, Hepatology, 2009, 49: S103-S111). Thus, current HBV regimens can provide long-term suppression of HBV replication, but most patients fail to achieve treatment responses that are stable long after treatment.

In contrast, suppression of HBV replication to low or undetectable levels can be maintained for longer periods in most patients who consistently receive potent HBV nucleos (t) ides with or without HBeAg reduction or seroconversion, but such extended periods of nucleos (t) ides are associated with the risk of tolerance, resistance of the virus and problems of adherence to treatment by patients (Chotiyaputta W. et al., Persistence and adherence to nucleos (t) ide analogue treatment for chronic hepatitis B, J. Hepatology, 2011, 54:12 -18; Lee M. and Keeffe EB, Study of adherence comes to the treatment of chronic hepatitis B, J. Hepatology, January 2011, 54 (1): 12-18; Scaglione SJ and Lok ASF, Effectiveness of Hepatitis B Treatment in Clinical Practice, Gastroenterology, 2012, 142: 1360-1368).

With the continued worldwide spread of HBV-related mortality and severe morbidity, there is still a need for improved antiviral HBV regimens that can provide a sustained viral response during and after treatment.

Summary of the invention

In one aspect provided herein, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an allosteric capsid protein modulator (CpAM) and a reverse transcriptase inhibitor. In one embodiment, CpAM is an allosteric modulator of a capsid protein that causes aberrant, defective, or incomplete assembly of HBV capsids. In another version

- 1 037576 implementation of CpAM is an allosteric modulator of capsid protein, causing the assembly of capsids, which are essentially empty in terms of their viral content.

In another aspect provided herein, there is provided a method for treating an infection

HBV in a patient requiring it, comprising administering to the patient a therapeutically effective amount of a compound of Formula I

or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof and a reverse transcriptase inhibitor, or a pharmaceutically acceptable salt thereof, or a prodrug thereof.

In yet another embodiment of this method, the compound of formula I is a compound of formula IA

or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof.

In another embodiment of this method, the compound of Formula I is a compound of Formula IB

or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof.

In one embodiment of this method, the compound of Formula I or Formula IB is a compound of Formula II

or a pharmaceutically acceptable form thereof.

salt, its hydrate, its solvate, or its crystalline

In another embodiment of the present method, the compound of Formula I or Formula IB is a compound of Formula IIIA

or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof.

In yet another embodiment of the present method, the compound of Formula I or Formula IB is a compound of Formula IIIB

- 2 037576 or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof.

In one aspect provided herein, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of Compound 1:

4-fluoro-3 - ((4-hydroxypiperidin-1-yl) sulfonyl) -N- (3,4,5-trifluorophenyl) benzamide or its pharmaceutically acceptable salt, its hydrate, its solvate, or its crystalline form, and an inhibitor reverse transcriptase, or a pharmaceutically acceptable salt or prodrug thereof.

In another aspect provided herein, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of Compound 2:

(S) -N- (3-cyano-4-fluorophenyl) -1 -methyl-4- (N- (1,1,1-trifluoropropan-2-yl) sulfamoyl) -1 N-pyrrole-2carboxamide or pharmaceutically an acceptable salt, a hydrate thereof, a solvate thereof, or a crystalline form thereof; and a reverse transcriptase inhibitor, or a pharmaceutically acceptable salt thereof, or a prodrug thereof.

In another aspect provided herein, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of Compound 3:

(R) -N- (2-chloropyridin-4-yl) -1 -methyl-4- (N- (1,1,1-trifluoropropan-2-yl) sulfamoyl) -1 N-pyrrole-2carboxamide or pharmaceutically an acceptable salt, a hydrate thereof, a solvate thereof, or a crystalline form thereof; and a reverse transcriptase inhibitor, or a pharmaceutically acceptable salt thereof, or a prodrug thereof.

In one embodiment, the reverse transcriptase inhibitor is a purine reverse transcriptase inhibitor such as entecavir or tenofovir.

In another embodiment, the reverse transcriptase inhibitor is selected from the group consisting of entecavir, tenofovir, lamivudine, telbivudine, adefovir, clevudine, CMX157, AGX-1009, zidovudine, didanosine, zalcitabine, stavudine, emtricitabine, D4 amacavir, D- , elvucitabine, delavirdine, efavirenz, nevirapine, capravirin, calanolide A, TMC278, BMS-561390 and DPC-083, or their prodrugs and their pharmaceutically acceptable salts. Pharmaceutically acceptable prodrugs of tenofovir include, for example, tenofovir disoproxil fumarate and tenofovir alafenamide fumarate.

In another embodiment, the reverse transcriptase inhibitor is selected from the group consisting of entecavir, tenofovir, and lamivudine, or pharmaceutically acceptable salts or prodrugs thereof.

In one embodiment, the CpAM and the reverse transcriptase inhibitor are part of the same formulation. In another embodiment, the CpAM and the reverse transcriptase inhibitor are part of different formulations.

In another embodiment, Compound 1, Compound 2, or Compound 3 and the reverse transcriptase inhibitor are part of the same composition. In another embodiment, Compound 1, Compound 2, or Compound 3 and the reverse transcriptase inhibitor are part of different

- 3 037576 trains.

In one embodiment, the patient is refractory to or refractory to treatment with a reverse transcriptase inhibitor. In another embodiment, the patient is refractory to, or refractory to, treatment with a nucleoside agent. In yet another embodiment, the patient is a previously untreated patient.

In one embodiment, Compound 1 is administered in an amount of 50 to 3000 mg per day. In one embodiment, Compound 1 is administered at about 2000 mg per day. In an additional embodiment, Compound 1 is administered at about 1000 mg twice a day. In one embodiment, Compound 1 is administered at 1200 mg per day. In one embodiment, Compound 1 is administered at 600 mg twice daily. In one embodiment, Compound 1 is administered at 600 mg per day. In one embodiment, Compound 1 is administered at 400 mg per day. In one embodiment, Compound 1 is administered at 200 mg per day. In one embodiment, Compound 1 is administered at 100 mg per day.

In one embodiment, Compound 2 or Compound 3 is administered in an amount of 5 to 600 mg per day. In another embodiment, Compound 2 or Compound 3 is administered in an amount of 10 to 50 mg per day. In a specific embodiment, Compound 2 or Compound 3 is administered at about 25 mg per day. In another embodiment, Compound 2 or Compound 3 is administered at about 25 mg four times a day. In yet another embodiment, Compound 2 or Compound 3 is administered at about 100 mg on the first day, followed by 25 mg four times a day. In an additional embodiment, Compound 2 or Compound 3 is administered in an amount of 10 to 200 mg once a day.

In one embodiment, Compound 1 is in crystalline form. In a further embodiment, the crystalline form is characterized by a powder X-ray diffraction structure having peaks expressed in degrees-2-theta at angles (± 0.2 °) equal to 17.1, 20.8, 22.2, 24.9 and 26 , 6 ° (Form XVI).

In another embodiment, the administration of the described compounds and the reverse transcriptase inhibitor is carried out over a period of less than 48 weeks.

In one embodiment, the patient is a patient chronically infected with HBV.

In one aspect provided herein, a combination product is provided comprising CpAM and a reverse transcriptase inhibitor. In one embodiment of the combination product, CpAM is an allosteric modulator of a capsid protein that causes aberrant, defective, or incomplete assembly of HBV capsids. In another embodiment of the combination product, CpAM is an allosteric modulator of a capsid protein, causing the assembly of capsids that are substantially empty in terms of their viral content. In one aspect provided herein, there is provided a combination product comprising CpAM, a compound of Formula I, Formula IA, Formula IB, Formula IIA, Formula IIIA, or Formula IIIB, or pharmaceutically acceptable salts, hydrates thereof, solvates thereof, or crystalline forms and a reverse transcriptase inhibitor, or a pharmaceutically acceptable salt or prodrug thereof.

In one aspect provided herein, there is provided a combination product comprising Compound 1, Compound 2, or Compound 3, or pharmaceutically acceptable salts, hydrates, solvates or crystalline forms thereof, and a reverse transcriptase inhibitor or pharmaceutically acceptable salt thereof , or a prodrug thereof. In one embodiment of the combination product, the reverse transcriptase inhibitor is selected from the group consisting of entecavir, tenofovir, and lamivudine, or pharmaceutically acceptable salts or prodrugs thereof.

In another aspect provided herein, there is provided a combination product comprising a compound of Formula IB, or pharmaceutically acceptable salts, hydrates, solvates or crystalline forms thereof, and a reverse transcriptase inhibitor, or a pharmaceutically acceptable salt or prodrug thereof. In one embodiment of the combination product, the reverse transcriptase inhibitor is selected from the group consisting of entecavir, tenofovir, and lamivudine, or pharmaceutically acceptable salts or prodrugs thereof.

In one embodiment of the combination product, the reverse transcriptase inhibitor is a purine reverse transcriptase inhibitor. In another embodiment of the combination product, the reverse transcriptase inhibitor is selected from the group consisting of entecavir, tenofovir, lamivudine, telbivudine, adefovir, clevudine, CMX157, AGX-1009, zidovudine, didanosine, zalcitabine, stavudine, ddItricitabine, D-D4 , amdoxovir, elvucitabine, delavirdine, efavirenz, nevirapine, capvirin, calanolide A, TMC278, BMS-561390 and DPC-083, or their prodrugs and their pharmaceutically acceptable salts. Pharmaceutically acceptable prodrugs of tenofovir include, for example, tenofovir disoproxil fumarate and tenofovir alafenamide fumarate.

In one embodiment of the combination product, the compound of Formula I, Formula IA, Formula IB, Formula IIA, Formula IIIA, or Formula IIIB and a reverse transcriptase inhibitor are part of the same formulation. In another embodiment of the combination product, the compound of Formula I, Formula IA, Formula IB, Formula IIA, Formula IIIA, or Formula IIIB and the reverse transcriptase inhibitor are part of different formulations. In a further embodiment of this embodiment, the compositions are for simultaneous or sequential administration.

In one embodiment of the combination product, Compound 1, Compound 2 and Compound 3 and the reverse transcriptase inhibitor are part of the same formulation. In another embodiment of the combination product, Compound 1, Compound 2 and Compound 3 and the reverse transcriptase inhibitor are part of different formulations. In a further embodiment of this embodiment, the compositions are for simultaneous or sequential administration.

In one embodiment of the combination product, the compound of Formula IB and the reverse transcriptase inhibitor are part of the same formulation. In another embodiment of the combination product, the compound of Formula IB and the reverse transcriptase inhibitor are part of different formulations. In a further embodiment of this embodiment, the compositions are for simultaneous or sequential administration.

In one embodiment, the combination product is for use in treating HBV infection in a patient.

In one embodiment, the combination product is for use in treating HBV infection in a patient, the patient being refractory to treatment with a reverse transcriptase inhibitor. In another embodiment, the combination product is for use in treating HBV infection in a patient, the patient being refractory to treatment with a nucleoside agent.

In one embodiment, the combination product is for use in treating HBV infection in a patient who has not previously received treatment. In one embodiment of the combination product, the amount of Compound 1 is 600 to 3000 mg. In another embodiment of the combination product, the amount of Compound 1 is about 2000 mg. In yet another embodiment of the combination product, the amount of Compound 1 is about 1000 mg.

In one embodiment of the combination product, the amount of Compound 2 or Compound 3 is 5 to 600 mg. In another embodiment of the combination product, the amount of Compound 2 or Compound 3 is 10 to 50 mg. In another embodiment of the combination product, the amount of Compound 2 or Compound 3 is about 25 mg. In yet another embodiment of the combination product, the amount of Compound 2 or Compound 3 is from about 10 mg to 200 mg. In one embodiment of the combination product, Compound 1 is in crystalline form. In an additional embodiment, the crystalline form is characterized by a powder X-ray diffraction structure having peaks expressed in degrees-2-theta at angles (± 0.2 °) equal to 17.1, 20.8, 22.2, 24.9 and 26 , 6 ° (Form XVI).

In one embodiment, the combination product is for use in treating HBV infection in a patient, the patient being chronically infected with HBV.

In another aspect provided herein, there is provided a pharmaceutical composition comprising an allosteric capsid protein modulator (CpAM) and a reverse transcriptase inhibitor. In one embodiment of the pharmaceutical composition, CpAM is an allosteric modulator of a capsid protein that causes aberrant, defective, or incomplete assembly of HBV capsids. In another embodiment of the pharmaceutical composition, CpAM is an allosteric modulator of a capsid protein, causing the assembly of capsids that are substantially empty in terms of their viral content.

In one aspect provided herein, there is provided a pharmaceutical composition comprising a compound of Formula I, Formula IA, Formula IB, Formula IIA, Formula IIIA, or Formula IIIB, or pharmaceutically acceptable salts, hydrates thereof, solvates thereof, or crystalline forms thereof, and a reverse transcriptase inhibitor; or a pharmaceutically acceptable salt or prodrug thereof.

In another aspect provided herein, there is provided a pharmaceutical composition comprising a compound of Formula IB, or pharmaceutically acceptable salts, hydrates, solvates or crystalline forms thereof, and a reverse transcriptase inhibitor, or a pharmaceutically acceptable salt, or prodrug thereof.

In one embodiment of the pharmaceutical composition, the reverse transcriptase inhibitor is selected from the group consisting of entecavir, tenofovir, and lamivudine, or pharmaceutically acceptable salts or prodrugs thereof. In another embodiment, the reverse transcriptase inhibitor is a purine reverse transcriptase inhibitor.

In another embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

In one aspect provided herein, there is provided a pharmaceutical composition comprising Compound 1, Compound 2, or Compound 3, or pharmaceutically acceptable salts, hydrates, solvates or crystalline forms thereof, and a reverse transcriptase inhibitor, or a pharmaceutically acceptable salt; or a prodrug thereof.

In one embodiment of the pharmaceutical composition, the reverse transcriptase inhibitor is selected from the group consisting of entecavir, tenofovir, and lamivudine, or pharmaceutically acceptable salts or prodrugs thereof. In embodiments of the pharmaceutical composition, the reverse transcriptase inhibitor is a purine reverse transcriptase inhibitor.

In one embodiment of the pharmaceutical composition, the reverse transcriptase inhibitor is selected from the group consisting of entecavir, tenofovir, lamivudine, telbivudine, adefovir, clevudine, CMX157, AGX-1009, zidovudine, didanosine, zalcitabine, stavudine, ddItricitabine, D-D-D4 , amdoxovir, elvucitabine, delavirdine, efavirenz, nevirapine, capvirin, calanolide A, TMC278, BMS-561390 and DPC-083, or their prodrugs and their pharmaceutically acceptable salts. Pharmaceutically acceptable prodrugs of tenofovir include, for example, tenofovir disoproxil fumarate and tenofovir alafenamide fumarate.

In another embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

In one aspect provided herein, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula I, Formula IA, Formula IB, Formula IIA, Formula IIIA, or Formula IIIB, or pharmaceutically acceptable salts thereof, their hydrates, their solvates, or their crystalline forms, and the patient is refractory to treatment with a reverse transcriptase inhibitor or does not respond to treatment.

In yet another aspect provided herein, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula I, Formula IA, Formula IB, Formula IIA, Formula IIIA, or Formula IIIB, or pharmaceutically acceptable salts thereof , their hydrates, their solvates, or their crystalline forms, and the patient is refractory to treatment with a nucleoside agent or does not respond to treatment.

In one aspect provided herein, there is provided a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of Compound 1, Compound 2, or Compound 3, or pharmaceutically acceptable salts thereof, their hydrates, their solvates, or their crystalline forms and the patient is refractory to or refractory to treatment with a reverse transcriptase inhibitor.

In yet another aspect provided herein, there is provided a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of Compound 1, Compound 2, or Compound 3, or pharmaceutically acceptable salts thereof, their hydrates, their solvates, or their crystalline forms, and the patient is refractory to treatment with a nucleoside agent or does not respond to treatment.

In one embodiment, for treating HBV infection, Compound 1 is administered in an amount of 600 to 3000 mg per day. In an additional embodiment, Compound 1 is administered at about 2000 mg per day. In one embodiment of this embodiment, Compound 1 is administered in an amount of about 1000 mg twice a day.

In one embodiment, for treating HBV infection, Compound 2 or Compound 3 is administered in an amount of 5 to 600 mg per day. In another embodiment, Compound 2 or Compound 3 is administered in an amount of 10 mg per day to 50 mg per day. In a specific embodiment, Compound 2 or Compound 3 is administered at about 25 mg per day. In an additional embodiment, Compound 2 or Compound 3 is administered in an amount of about 10 to 200 mg once a day.

In another embodiment of such methods, the administration of a compound of Formula I, Formula IA, Formula IB, Formula IIA, Formula IIIA, or Formula IIIB, Compound 1, Compound 2, or Compound 3 and a reverse transcriptase inhibitor occurs over a period of less than 48 weeks.

In another embodiment, the patient is a chronically infected HBV patient.

In one aspect provided herein, there is provided a method of inhibiting the replication of a nucleoside-resistant HBV variant comprising contacting said variant with an effective amount of a compound of Formula I, Formula IA, Formula IB, Formula IIA, Formula IIIA, or Formula IIIB, Compound 1, compound 2 or compound 3, or their pharmaceutically acceptable salts, their hydrates, their solvates, or their crystalline forms.

In another aspect provided herein, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula I, Formula IA, Formula IB, Formula IIA, Formula IIIA or Formula IIIB, Compound 1, Compound 2, or compound 3, or pharmaceutically acceptable salts, their hydrates, their solvates, or their crystalline forms and compound A

- 6 037576

F

A or a pharmaceutically acceptable salt thereof.

Brief description of graphic materials

FIG. 1 shows HepG2 cells transiently transfected with wild-type HBV (dashed circles) and variants containing amino acid changes rtL180M / M204V (dashed triangles) or rtL180M / M204V / N236T (dashed squares), which were incubated with increasing concentrations of LM. 1A), ETV (FIG. 1B), or TDF (FIG. 1C). Dose-response curves for treatment versus wild-type HBV are shown with dashed lines. Data points represent the means of at least three independent transfection studies, and standard deviations are shown as straight line segments.

FIG. 2 shows the effect of combining compound 1 with nucleoside analogs on cell viability for primary human hepatocytes taken from donor NiM4038. Dose-response curves depicting cell viability using Compound 1 alone (circles, solid line) or Compound 1 in combination with nucleoside analogs (squares, dashed line): (Fig.2A) 300 μM LMV, (Fig.2B ) 30 μM ETV or (Fig. 2C) 30 μM TFV. Primary human hepatocytes from donor HuM4038 were treated with test compounds for 6 days. Data points represent averages, and line segments represent standard deviations of three replicate measurements.

FIG. 3 shows the effect of combining compound 1 with nucleoside analogs on cell viability for primary human hepatocytes taken from donor HuM4055A. Dose-response curves depicting cell viability using Compound 1 alone (circles, solid line) or Compound 1 in combination with nucleoside analogs (squares, dashed line): (Fig.3A) 300 μM LMV, (Fig.3B ) 30 μM ETV or (Fig. 3C) 30 μM TFV. Primary human hepatocytes from donor HuM4055A were treated with test compounds for 6 days. Data points represent averages, and line segments represent standard deviations of three replicate measurements.

FIG. 4 shows the effect of combining compound 1 with nucleoside analogs on cell viability for primary human hepatocytes taken from donor HuM4059. Dose-response curves depicting cell viability using Compound 1 alone (circles, solid line) or Compound 1 in combination with nucleoside analogs (squares, dashed line): (Fig.4A) 300 μM LMV, (Fig.4B ) 30 μM ETV or (Fig. 4C) 30 μM TFV. Primary human hepatocytes from donor HUM4059 were treated with test compounds for 6 days. Data points represent averages, and line segments represent standard deviations of three replicate measurements.

FIG. 5 shows the results of an efficacy study in clinical trials for patients who received only Compound 1 or its combination with PegIFN.

FIG. 6 shows the decrease in serum HBV RNA in patients treated with placebo (RSV), compound 1 (600 mg 2 times / day), 180 μg / wk. pegylated interferon (PEG-IFN) and a combination of compound 1 and PEG-IFN at the same doses.

FIG. 7 shows the effect of Compound 1 in combination with nucleoside analogs. Compound 1 was used in combination with LMV (Fig. 7A), TFV (Fig. 7B) and ETV (Fig. 7C).

FIG. 8 shows the effect of Compound 1 in combination with Bay 41-4109, another capsid modulator. MacSynergy synergy plots with 95% confidence intervals for HepG2.2.15 cells treated with Compound 1 in combination with Bay 41-4109.

FIG. 9 shows the X-ray powder diffraction structure for Form XVI of Compound 1.

FIG. 10 shows the X-ray powder diffraction structure for Form XVI of Compound 1.

FIG. 11A-11C contain plots of synergy for the combination of compound 2 with ETV in HepG2.2.15 cells. Synergy plot calculations are based on the lower limit values for 95% CI (y-axis). Compound concentrations are shown on the X-axis and Z-axis in nM. FIG. 11A corresponds to Experiment 1. FIG. 11B corresponds to Experiment 2. FIG. 11C corresponds to experiment 3.

FIG. 12A-12C contain plots of synergy for the combination of compound 2 with TFV in HepG2.2.15 cells. Synergy plot calculations are based on the lower limit values for 95% CI (y-axis). Compound concentrations are shown on the X-axis and Z-axis in nM. FIG. 12A corresponds to Experiment 1. FIG. 12B corresponds to Experiment 2. FIG. 12C corresponds to experiment 3.

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FIG. 13 shows a synergy graph for the combination of Compound 3 with ETV in HepG2.2.15 cells. Synergy plot calculations are based on the lower limit values for 95% CI (y-axis). Compound concentrations are shown on the X-axis and Z-axis in nM.

FIG. 14 contains a synergy graph for the combination of Compound 3 with TFV in HepG2.2.15 cells. Synergy plot calculations are based on the lower limit values for 95% CI (y-axis). Compound concentrations are shown on the X-axis and Z-axis in nM.

FIG. 15A-15D reflect the percentage inhibition of HBV replication with Compound 2 and ETV or TFV in a concentration range. Each graph represents the percent inhibition of each compound when the concentration of the other compound was zero.

Detailed description of the invention

The present disclosure relates to methods of using a capsid assembly inhibitor for the treatment of hepatitis B virus (HBV) infection. In particular, provided herein is a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of an allosteric capsid protein modulator (CpAM) and a reverse transcriptase inhibitor. In one embodiment, CpAM is an allosteric modulator of a capsid protein that causes aberrant, defective, or incomplete assembly of HBV capsids. In another embodiment, CpAM is an allosteric modulator of a capsid protein that causes the assembly of capsids that are substantially empty in terms of their viral content. In addition, provided herein is a method of treating HBV infection in a patient requiring it, comprising administering CpAM (e.g., a compound of Formula I, Formula IA, Formula IB, Formula IIA, Formula IIIA or Formula IIIB, Compound 1, Compound 2, or Compound 3) and a reverse transcriptase inhibitor. Additionally, provided herein are combination products and pharmaceutical compositions comprising CpAM (e.g., a compound of Formula I, Formula IA, Formula IB, Formula IIA, Formula IIIA, or Formula IIIB, Compound 1, Compound 2, or Compound 3) and a reverse transcriptase inhibitor. In addition, provided herein are combination products and pharmaceutical compositions comprising a CpAM of Formula IB and a reverse transcriptase inhibitor. Also provided herein are methods of treating HBV infection in a patient refractory to treatment with a reverse transcriptase inhibitor or nucleos (t) id agent or refractory to treatment, comprising administering a compound of Formula I, Formula IA, Formula IB, Formula IIA, Formula IIIA, or Formula IIIB , Compound 1, Compound 2 or Compound 3. Also provided herein are methods of treating HBV infection in a patient refractory to treatment with a reverse transcriptase inhibitor or nucleos (t) id agent or refractory to treatment, comprising administering a compound of Formula IB.

I. Definitions.

As used in the description and in the claims, the term including may encompass embodiments consisting of and consisting essentially of.

The terms contain (it), include (e), has, has, may, consist (it) and variations thereof as used herein are intended to be used as non-limiting intermediate phrases, terms or words that require the mentioned ingredients / stages and admit the presence of other ingredients / stages. However, such disclosure should also be construed as describing compositions or methods as consisting of or consisting essentially of the recited compounds, which permits only the recited compounds along with any pharmaceutically acceptable carriers, but excludes other compounds.

All ranges of values described herein include the referenced endpoint and can be independently combined (eg, the 600 to 3000 mg range includes the 600 and 3000 mg endpoints and all intermediate values, eg 2000 mg). The endpoints of the ranges and any values described herein are not limited to an exact range or value; they are inaccurate enough to include values that approximate such ranges and / or values.

Used in this document, the approximate wording can be used to modify any quantitative representation that can be changed without changing the main function to which it refers. Accordingly, the meaning modified by the term or terms, such as about or substantially, in some cases may not be limited to the exact specified meaning. In at least some cases, the approximate wording may correspond to the accuracy of the instrument used to measure the value. The about modifier can also be thought of as describing the range defined by the absolute values of the two endpoints. For example, the expression about 600 to about 3000 also describes the range from 600 to 3000. The term about can refer to the range of plus or minus 10% of the indicated number. For example, about 10% could indicate a range of 9 to 11%, and about 1 could mean 0.9 to 1.1.

Other meanings of about may be obvious from context, such as rounding, so, for example, about 1 could also mean 0.5 to 1.4.

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As used herein, the expression allosteric capsid protein modulator (CpAM) refers to a compound that alters the assembly or activity of the capsid protein (Zlotnick,

Antiviral Research 121 (2015) 82-93). At least two classes of CpAM have been identified that alter the assembly of the capsid protein in two different ways.

It was shown that CpAM of the first class changes the direction of capsid assembly with the formation of aberrant non-capsid polymers. This first class is hereinafter referred to as the allosteric modulator of the capsid protein, which causes aberrant, defective or incomplete assembly of HBV capsids. An example of this class are compounds based on heteroaryldihydropyrimidine (HAP). At high concentrations, HAP leads to a change in the direction of the capsid assembly with the formation of aberrant non-capsid polymers. Compared to the Cp concentration at substoichiometric concentrations, HAP increases the capsid assembly rate. The crystalline structures between the HBV capsid and the HAP show changes in the quaternary structure of the capsid, which lead to the formation of bonded rigid components with minor changes in the tertiary structure. An example of HAP includes, without limitation, methyl 4- (2-chloro-4-fluorophenyl) -6-methyl-2- (pyridin-2-yl) -1,4 dihydropyrimidine-5-carboxylate (HAP-1).

One other type of CpAM that increases the rate of capsid assembly without affecting the morphology of the capsid is hereinafter referred to as an allosteric capsid protein modulator, causing the assembly of capsids that are essentially empty in terms of their viral content.

An example of this class are non-nucleoside phenylpropenamide compounds (PPA). PPA increases the speed of capsid assembly without affecting the morphology of the capsid. Cell culture studies show that capsids formed by the presence of multiple PPA are empty in terms of their viral content, which is a consequence of blocking the packaging of viral RNA. Crystal structures during the interaction of HBV and PPA show changes in the quaternary as well as in the tertiary structure. Other examples of such empty capsid-forming CpAM include, without limitation, compounds of Formula I, Formula IA, Formula IB, Formula IIA, Formula IIIA, or Formula IIIB, Compound 1, Compound 2, and Compound 3 described herein.

Formula IB is also an example of an allosteric capsid protein modulator that causes the assembly of substantially empty capsids.

As used herein, the term reverse transcriptase inhibitor refers to nucleosides or nucleotides and their analogs that inhibit HBV reverse transcriptase activity. Examples include, but are not limited to, for example, entecavir, tenofovir, lamivudine, telbivudine, adefovir, clevudine, CMX157, AGX-1009, zidovudine, didanosine, zalcitabine, stavudine, emtricitabine, abacavir, D-D4FC, elvocitabine, delavirdine, efavirenz, nevirapine, capvirine, calanolide A, TMC278, BMS-561390 and DPC-083 or their prodrugs and their pharmaceutically acceptable salts. Pharmaceutically acceptable prodrugs of tenofovir include, for example, tenofovir disoproxil fumarate and tenofovir alafenamide fumarate.

As used herein, a reverse transcriptase inhibitor may be a purine reverse transcriptase inhibitor, which is a reverse transcriptase inhibitor having a purine ring, such as, without limitation, entecavir and tenofovir.

As used herein, the term method of treatment or treatment means the use or administration of a therapeutic agent, i. E. a compound of the invention (one compound or compound in combination with another pharmaceutical agent), to a patient, or the use or administration of a therapeutic agent to an isolated tissue or cell line obtained from a patient (for example, for diagnosis or ex vivo use) who has HBV infection, symptom HBV infection or the potential for developing an HBV infection, with the aim of curing, healing, weakening, soothing, modifying, eradicating, ameliorating HBV infection, symptoms of HBV infection, or the potential for developing or influencing HBV infection. Such treatment options can be specially adapted or modified based on knowledge gained in the field of pharmacogenomics.

As used herein, the term “patient or subject” means a human or non-human mammal. Non-human mammals include, for example, livestock and domestic animals such as sheep, cows, pigs, dogs, cats, and mice. The patient or subject is preferably a human.

As used herein, the term pharmaceutically acceptable refers to a material, such as a carrier or diluent, which does not inhibit the biological activity or properties of the compound and is relatively non-toxic, i.e., the material can be administered to a subject without causing undesirable biological effects or interactions in an undesirable manner with any from the components of the composition in which it is contained.

In this document, the term pharmaceutically acceptable salt refers to derivatives of the described compounds, in which the parent compound is modified by converting an existing acidic or alkaline functional group into its salt form. Examples of pharmaceutically acceptable salts include, without limitation, inorganic or organic acid salts of basic residues such as amines; alkaline or organic salts of acidic residues,

- 9 037576 such as carboxylic acids; etc. The pharmaceutically acceptable salts of the present invention include the conventional non-toxic salts of the parent compound, derived, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound that contains a basic or acidic functional group by standard chemical methods. Essentially, such salts can be obtained by reacting the free acid or base forms of these compounds with a stoichiometric amount of the corresponding base or acid in water or in an organic solvent, or in a mixture of the two; essentially a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile is preferred. List acceptable salts is given in Remington's Pharmaceutical Sciences, 17 ^{th ed., Mack Publishing Company,} Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

As used herein, the term composition or pharmaceutical composition means a mixture of at least one compound used in the framework of the invention with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. There are many techniques for administering a compound in the art, including, but not limited to, intravenous, oral, aerosol, parenteral, ocular, pulmonary, and topical administration.

As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickener, diluent, or encapsulating material involved in the transport or transport of the compound used. within the scope of the invention, inside or onto a patient in such a way that he can perform the intended function. Typically, such constructs are transferred or transported from one organ or body site to another organ or body site. Each carrier must be acceptable in terms of compatibility with the other ingredients of the formulation, including a compound used in the framework of the invention, and harmless to the patient. Some examples of materials that can act as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth gum; malt; gelatin; talc; excipients such as cocoa butter and candle wax; oils such as vegetable peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions; and other non-toxic compatible substances used in pharmaceutical formulations. As used herein, the term pharmaceutically acceptable carrier also includes any coatings, antibacterial and antifungal agents and absorption delaying agents, etc., which are compatible with the effect of the compound used in the framework of the invention and physiologically acceptable to the patient. Ancillary active compounds can also be included in the composition. The term pharmaceutically acceptable carrier may further include a pharmaceutically acceptable salt of a compound used in the framework of the invention. Other additional ingredients that may be included in pharmaceutical compositions useful in the practice of the invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which includes to this document by reference.

The terms combination, therapeutic combination, pharmaceutical combination, or combination product as used herein refers to either a fixed combination of a unit dosage form, or a non-fixed combination, or a set of components for combined administration, in which two or more therapeutic agents can be administered independently, simultaneously or separately for certain periods of time, especially in which such periods of time allow the components of the combination to exhibit a cooperative, for example, synergistic effect.

The term combination therapy refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described herein. Such administration includes co-administration of therapeutic agents in a substantially simultaneous manner, for example, as part of the contents of one formulation having a fixed ratio of active ingredients, or as part of the contents of different (eg, capsules and / or intravenous preparations) for each active ingredient. In addition, such administration also includes the use of each type of therapeutic agent in sequential and separate administration modes, both at about the same time and at different points in time. Whether the active ingredients are administered as part of the content of the same formulation or as part of the content of different formulations,

- 10 037576 drugs are administered to the same patient within the same course of therapy. In either case, the treatment regimen will provide beneficial effects in treating the conditions or disorders described herein.

The term synergistic effect refers to the action of two agents, such as a capsid assembly inhibitor and a reverse transcriptase inhibitor, that produce an effect, for example, to slow the symptomatic progression of HBV infection or its symptoms, which is more effective than combining the effects of two separately administered drugs. ... The synergistic effect can be calculated, for example, using suitable methods such as the Sigmoid-Emax equation (Holford, NHG and Scheiner, LB, Clin. Pharmacokinet. 6: 429-453 (1981)), Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114: 313-326 (1926)) and the median effect equation (Chou, T.S. and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984) ) and Chou, Pharmacol Rev. 58: 621-681 (2006) Each of the above equations can be applied to manipulate experimental data to produce an appropriate graph that helps evaluate the effects of a drug combination. equations include a concentration-effect curve, an isobologram, and an additivity curve, respectively. In some embodiments, the combination of compounds exhibits a synergistic effect (i.e., more additive effect) in treating HBV infection. In certain embodiments, the combination of compounds shows a synergistic effect (i.e., more additive effect) in the treatment of HBV infection.

Synergy level <-100, -100 to -50, -50 to -25, -25 to 25, 25 to 50, 50 to 100 and> 100 indicates strong antagonism, moderate antagonism, weak antagonism, negligible synergism / antagonism (additivity), weak synergism, moderate synergism and strong synergy, respectively.

As used herein, the term refractory or refractory to a therapeutic agent in relation to a patient with HBV means that the patient with HBV has congenital or acquired resistance to the effects of exposure to the therapeutic agent as a result of contact with the therapeutic agent. In other words, the HBV patient is unresponsive to the usual treatment standards associated with a particular therapeutic agent.

As used herein, the term previously untreated refers to a patient who has not previously received treatment with an investigational or approved drug for HBV infection, in particular with a nucleos (t) id drug.

In an alternative embodiment, patients treated in accordance with the methods of the present disclosure may be previously treated. As used herein, the term previously treated refers to a patient who has received at least one prior course of HBV antiviral therapy, in particular with the use of nucleos (t) ide. In some embodiments, the last dose in this previous course was administered at least three months prior to performing a method as described herein.

HBV infections that can be treated in accordance with the described methods include genotypes of HBV infections A, B, C and / or D. However, in one embodiment, the described methods can be used to treat any HBV genotype (pan-genotypic treatment). HBV genotyping can be performed using methods known to those skilled in the art, for example INNO-LIPA® HBV Genotyping, Innogenetics N.V., Ghent, Belgium).

As used herein, the term alkyl, alone or as part of another substituent, means, unless otherwise indicated, a straight or branched hydrocarbon chain having a specified number of designated carbon atoms (i.e., Cl-C ₆ alkyl means alkyl having from one to six atoms carbon), and includes straight and branched chains. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, and hexyl. Other examples of Cl-C ₆ alkyl include ethyl, methyl, isopropyl, isobutyl, n-pentyl, and n-hexyl.

As used herein, the term alkenyl means a monovalent group derived from a hydrocarbon functional group containing at least two carbon atoms and at least one carbon double bond. The double bond may or may not be the attachment point of another group. Alkenyl groups (eg, C ₂ -C ₈ alkenyl) include, but are not limited to, for example, ethenyl, propenyl, prop-1-en-2-yl, butenyl, 1-methyl-2-buten-1-yl, heptenyl, octenyl and the like.

As used herein, the term alkynyl means a linear or branched hydrocarbon radical containing up to 6 carbon atoms and having at least one carbon triple bond. Examples of alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like.

As used herein, the term alkoxy denotes an —O-alkyl group, where alkyl is defined as defined herein. As an example, alkoxy includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, t-butoxy, and the like.

As used herein, the term halo or halogen alone or as part of another substituent

- 11 037576 means, unless otherwise indicated, a fluorine, chlorine, bromine or iodine atom, preferably fluorine, chlorine or bromine, more preferably fluorine or chlorine.

As used herein, the term cycloalkyl means a non-aromatic carbocyclic system that is partially or fully saturated with 1, 2 or 3 rings, and such rings may be fused.

The term fused means that the presence of the second ring in the structure (for example, attached or formed) is due to the presence of two adjacent atoms in common with the first ring (for example, joint). Cycloalkyl also includes bicyclic structures, which may be bridged or spirocyclic in nature, with each individual ring within the bicyclic ring containing 3-8 atoms.

The term cycloalkyl includes, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo [3.1.0] hexyl, spiro [3.3] heptanyl, and bicyclo [1.1.1] pentyl.

As used herein, the term heterocycloalkyl means a non-aromatic carbocyclic system containing 1, 2, 3 or 4 heteroatoms selected independently from N, O and S, and having 1, 2 or 3 rings, and such rings may be fused, the term fused defined above. Heterocycloalkyl also includes bicyclic structures, which may be bridged or spirocyclic in nature, but each individual ring within the bicycle contains 3-8 atoms and contains 0, 1, or 2 N, O, or S atoms.

The term heterocycloalkyl includes cyclic esters (e.g. lactones) and cyclic amides (e.g. lactams), and also specifically includes, without limitation, epoxidil, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl (e.g. oxanyl), pyranyl, dioxanyl, aziridinyl, azetildinyl, pyrrolidinyl, 2,5-dihydro-1H-pyrrolyl, oxazolidinyl, thiazolidinyl, piperidinyl, morpholinyl, piperazinyl, thiomorpholinyl, 1,3-oxazinanyl, 1,3-thiazinanyl, 2-azabicyclic hexanyl, 5-azabicyclo [2.1.1] hexanyl, 6-azabicyclo [3.1.1] heptanyl, 2-azabicyclo [2.2.1] heptanyl, 3-azabicyclo [3.1.1] heptanyl, 2-azabicyclo [3.1.1] heptanyl, 3-azabicyclo [3.1.0] hexanyl, 2-azabicyclo [3.1.0] hexanyl, 3-azabicyclo [3.2.1] octanyl, 8-azabicyclo [3.2.1] octanyl, 3oxa-7-azabicyclo [3.3. 1] nonanyl, 3-oxa-9 -azabicyclo [3.3.1] nonanyl, 2-oxa-5 -azabicyclo [2.2.1] heptanyl, 6-oxa-3 -azabicyclo [3.1.1] heptanyl, 2-azaspiro [ 3.3] heptanyl, 2-oxa-6-azaspiro [3.3] heptanyl, 2-oxaspiro [3.3] heptanyl, 2-ok saspiro [3.5] nonanyl, 3-oxaspiro [5.3] nonanyl, and 8-oxabicyclo [3.2.1] octanyl.

As used herein, the term aromatic means a carbon ring or heterocycle with one or more polyunsaturated rings and is aromatic in character, i. E. having (4n + 2) delocalized π (pi) electrons, where n is an integer.

As used herein, the term aryl means an aromatic carbocyclic system containing 1, 2, or 3 rings, and such rings may be fused, with the term fused as defined above. If the rings are condensed, one of the rings must be completely unsaturated, and the condensed ring (s) may be fully saturated, partially unsaturated, or fully unsaturated. The term aryl includes, without limitation, phenyl, naphthyl, indanyl, and 1,2,3,4-tetrahydronaphthalenyl.

As used herein, the term heteroaryl means an aromatic carbocyclic system containing 1, 2, 3 or 4 heteroatoms selected independently from N, O and S, and having 1, 2 or 3 rings, and such rings can be condensed, the term fused defined above. The term heteroaryl includes, without limitation, furanyl, thiophenyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyriminyl, imidinyl, pyrazinyl , pyrazolo [1,5-a] pyridinyl, 5,6,7,8-tetrahydroisoquinolinyl, 5,6,7,8-tetrahydroquinolinyl, 6,7-dihydro-5Ncyclopenta [b] pyridinyl, 6,7-dihydro-5H -cyclopenta [c] pyridinyl, 1,4,5,6-tetrahydrocyclopenta [c] pyrazolyl, 2,4,5,6-tetrahydrocyclopenta [c] pyrazolyl, 5,6-dihydro-4H-pyrrolo [1,2-b ] pyrazolyl, 6,7-dihydro-5H-pyrrolo [1,2-b] [1,2,4] triazolyl, 5,6,7,8-tetrahydro- [1,2,4] triazolo [1,5 -а] pyridinyl, 4,5,6,7-tetrahydropyrazolo [1,5-a] pyridinyl, 4,5,6,7-tetrahydro-1H-indazolyl and 4,5,6,7-tetrahydro-2Nindazolyl.

It should be understood that if an aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group is bonded or otherwise bonded to an isolated functional group via various ring atoms (e.g., shown or described without designating a specific attachment point), then all possible points are meant, such as a carbon atom and, for example, through a trivalent nitrogen atom. For example, the term pyridinyl means 2-, 3- or 4-pyridinyl, the term thiophenyl means 2- or 3-thiophenyl, etc.

As used herein, the term substituted means that an atom or group of atoms has replaced a hydrogen as a substituent attached to another group.

II. Connections.

Provided herein is a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of an allosteric modulator

- 12 037576 capsid protein (CpAM) and reverse transcriptase inhibitor. In one embodiment, CpAM is an allosteric modulator of a capsid protein that causes aberrant, defective, or incomplete assembly of HBV capsids. In another embodiment, CpAM is an allosteric modulator of a capsid protein that causes the assembly of capsids that are substantially empty in terms of their viral content. The methods, combination product, and compositions provided herein include a compound of Formula I

I or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof and a reverse transcriptase inhibitor, or a pharmaceutically acceptable salt, or a prodrug thereof, wherein A is phenyl or pyridinyl;

B is a monocyclic aromatic or heteroaromatic 5- or 6-membered ring, such aromatic or heteroaromatic ring optionally containing one or more substituents, each independently selected from halogen or C ₁ -C ₆ -αalkyl;

R ¹ is H or C ₁ -C ₆ -αalkyl;

R ² is C ₁ -C ₆ -alkyl, said C ₁ -C ₆ -alkyl optionally having one or more substituents, each independently selected from the group consisting of halogen, C ₁ -C ₆ alkoxy, oxo, C ₁ -C ₆ alkyl, OH, CN, CFH ₂ , CF2H and CF3; or

R ¹ and R ² are grouped together to form a C ₂ -C ₇ heterocycloalkyl ring, said C ₂ -C ₇ heterocycloalkyl ring optionally containing one or more substituents, each independently selected from the group consisting of halogen, C ₁ - C ₆ alkoxy, oxo, C ₁ -C ₆ alkyl, OH, CN, CFH2, CF2H and CF3;

each R ^{3 is} independently selected from the group consisting of halogen, C ₁ -C ₆ -αalkyl, C ₁ -C ₆ -α alkoxy, cyano, C ₁ -C ₆ alkenyl, C ₁ -C ₆ -alkynyl, and OH; and n is 0, 1, 2, or 3.

In one embodiment of Formula I, B is a 5 membered heteroaromatic ring that optionally and independently contains one or more halogen or C ₁ -C ₆ alkyl substituents.

In one embodiment, the compound of formula I is a compound of formula IA

IA or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof, wherein A is phenyl or pyridinyl;

R ¹ represents H or C ₁ -C ₆ -alkyl;

R ² is C ₁ -C ₆ -alkyl, optionally and independently containing one or more substituents in the form of halogen, C ₁ -C ₆ -alkoxy, oxo, C ₁ -C ₆ -alkyl, OH, CN, CFH ₂ , CF2H or CF3;

R ³ is independently for each occurrence halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, cyano, C ₁ -C ₆ alkenyl, C ₁ -C ₆ alkynyl, or OH;

X is CR4;

G ³ represents H or C ₁ -C ₆ -alkyl;

R4 is independently for each occurrence H, halogen, C ₁ -C ₃ alkyl, or cyano;

n is 0, 1, 2, or 3.

In one embodiment, the compound of Formula I is a compound of Formula IB

IB or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline

- 13 037576 form, where X is CR or N;

B is C ₅ -C ₆ aryl, C ₅ -C ₆ cycloalkyl, 5-6 membered heteroaryl or 5-6 membered heterocyclyl, all of which may optionally have a substituent in the form

C ₁ -C ₄ -alkyl or halogen;

R ¹ is H or C ₁ -C ₆ alkyl;

R ² is C ₁ -C ₆ alkyl, optionally and independently containing one or more halogen substituents, C ₁ -C ₆ alkoxy, oxo, C ₁ -C ₆ alkyl, OH, CN, CFH ₂ , CF2H or CF3;

R3 is independently for each occurrence halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, cyano, C ₁ -C ₆ α-alkenyl, C ₁ -C ₆ alkynyl, or OH;

R represents C ₁ -C ₄ -alkyl or halogen;

n is 0, 1, 2, or 3.

In one embodiment, the compound of formula I is a compound of formula II η ₃ o ^^ (R ³ ) n / —NH ^G V ~ A Ji / = \ I νλ

IS HE

II or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof, wherein R ³ is halogen;

G ¹ represents H, C ₁ -C ₄ -alkyl or halogen;

G ² is selected from the group consisting of H, halogen, _C1-C4 alkyl and OH;

n is 0, 1, 2, or 3;

m is 0, 1, or 2.

In another embodiment, the compound of formula I is a compound of formula IIIA

IIIA or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof, wherein R ¹ is H or C ₁ -C ₆ -alkyl;

R ² is C ₁ -C ₆ alkyl optionally and independently containing one or more halogen substituents, C ₁ -C ₆ alkoxy, oxo, C ₁ -C ₆ alkyl, OH, CN, CFH ₂ , CF2H or CF3;

R ³ is independently for each occurrence halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, cyano, C ₁ -C ₆ α-alkenyl, C ₁ -C ₆ alkynyl, or OH;

R ⁴ is H, halogen, C ₁ -C ₃ alkyl, or cyano.

In yet another embodiment, the compound of formula I is a compound of formula IIIB

IIIB or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof, wherein R ¹ is H or C ₁ -C ₆ alkyl;

R ² is C ₁ -C ₆ -alkyl, said C ₁ -C ₆ -alkyl optionally containing one or more substituents, each independently selected from the group consisting of halogen, C1-C6-alkoxy, oxo, ^^ -alkyl, OH, CN, CFH2, CF2H and CF3;

each R ^{3 is} independently selected from the group consisting of halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, cyano, C ₁ -C ₆ alkenyl, C ₁ -C ₆ alkynyl, and OH;

G ³ represents H or C ₁ -C ₆ -αalkyl;

X is CR ⁴ ;

- 14 037576

R4 is selected from the group consisting of H, halogen, _C1-C3 alkyl, and cyano;

n is 0, 1, 2, or 3.

Methods, combination product, and compositions provided herein may include Compound 1:

Compound 1

In this document, compound 1 is also referred to as compound I or comp. (I). Compound 1, including its synthesis, is described by PCT Publication No. WO 2013/096744, which is incorporated herein by reference in its entirety.

In addition, compound 1 can exist in a crystalline form, preferably one that is stable against high temperatures and humidity. The various crystalline forms of compound 1 are described in WO 2017/059059, which is incorporated herein by reference in its entirety.

For example, Form XVI of Compound 1 has the X-ray powder diffraction structure shown in FIG. 9. The corresponding values of ° 2-theta are given in table. one.

- 15 037576

Table 1

Powder X-ray diffraction structure for Form XVI of compound 1

No. Floor. [° 2-theta] Peak intensity [cps] one 8.3325 177.19 2 10.9344 1935.52 3 14.3722 3710.48 four 14.9241 373.24 five 15.8427 2224.43 6 16.4561 2064.13 7 17.0677 5116.86 eight 18.5296 4972.27 nine 18.9049 872.76 10 20.0163 3381.98 eleven 20.7658 13446.21 12 21.5994 1648.19 13 22.1592 5552.43 fourteen 22.8341 878.36 fifteen 23.4421 2910.94 sixteen 23.6338 2169.37 17 24.9292 12671.51 eighteen 26.5972 15673.37 nineteen 27.9963 3230.31 twenty 28.3825 1934.34 21 29.5627 1788.4 22 29,766 1697.44 23 30.4527 1526, 62 24 31.1958 954.79 25 31.7034 1030.38 26 32.9259 1755.93 27 34.1563 1312 28 34.5404 2059.7 29 35.6022 1008.97 thirty 36.3734 2480.94 31 36,753 1575.29 32 38.3689 1684.63 33 39.7099 915.35 34 40.1675 1190.9 35 41,707 685.21 36 43.6419 800.32 37 44.6892 1534.39

Thus, in one embodiment, Compound 1 is present in crystalline form, which is characterized by a powder X-ray diffraction structure having peaks expressed in degrees-2-theta at angles (± 0.2 °) equal to 17.1, 20.8, 22.2, 24.9 and 26.6 ° (Form XVI).

In a further embodiment, the crystalline form is characterized by a powder X-ray diffraction structure having peaks expressed in degrees-2-theta at angles (± 0.2 °) equal to 14.4, 17.1, 18.5, 20.0, 20 , 8, 22.2, 23.4, 24.9, 26.6, 28.0 and 36.4 ° (Form XVI).

In yet another further embodiment, the crystalline form is characterized by a powder X-ray diffraction structure having peaks expressed in degrees-2-theta at angles (± 0.2 °) of 8.3, 10.9, 14.4, 14.9 , 15.8, 16.5, 17.1, 18.5, 18.9, 20.0, 20.8, 21.6, 22.2, 22.8, 23.4, 23.6, 24 , 9, 26.6, 28.0, 28.4, 29.6, 29.8, 30.5, 31.2, 31.7, 32.9, 34.2, 34.5, 35.6 , 36.4, 36.8, 38.4, 39.7, 40.2, 41.7, 43.6 and 44.7 ° (Form XVI).

In another embodiment, the crystalline form is characterized by a powder X-ray diffraction structure having peaks expressed in degrees-2-theta at angles (± 0.2 °) equal to 8.33, 10.93, 14.37, 14.92, 15 , 84, 16.46, 17.07, 18.53, 18.90, 20.02, 20.77, 21.60, 22.16, 22.83, 23.44, 23.63, 24.93 , 26.60, 28.00, 28.38, 29.56, 29.77, 30.45, 31.20, 31.70, 32.93, 34.16, 34.54, 35.60, 36 , 37, 36.75, 38.37, 39.71, 40.17, 41.71, 43.64 and 44.69 ° (Form XVI).

In one embodiment, the crystalline form is characterized by a powder X-ray diffraction structure that is substantially identical to that shown in FIG. nine.

- 16 037576

Powder structure X-ray diffraction for Form III of compound 1 is shown in fig. 10. The corresponding values of ° 2-theta are given in table. 2. table 2 Powder X-ray diffraction structure of compound 1 (form III, acetone solvate) No. Floor. [° 2-theta] Height [cm] one 6.1533 339.26 2 9.0816 1104.7 3 9.9483 1907.02 four 10.0321 1552.49 five 12.1685 3556.97 6 12.9616 383.96 7 14.2397 315.01 eight 15.1483 2480.83 nine 16.2048 1828.9 10 16.8775 256.66 eleven 18,269 953.62 12 18.6378 3776.85 13 19.9348 205.82 fourteen 21.1993 1960.44 fifteen 21.9332 550.39 sixteen 22.2455 479.41 17 23.1308 548.36 eighteen 24.4803 948.12 nineteen 25.4636 170.21 twenty 25.8397 586.56 21 26, 139 787.4 22 26.7489 173.31 23 27.404 149.44 24 28.053 307.13 25 28.9464 155, 2 26 30.0145 564.17 27 31.9986 284.25 28 33.0882 659.21 29 34.0244 203.24 thirty 34.3991 227.63 31 37,0076 210.03 32 38.3419 102.07 33 40.4682 165.35 34 42.4278 144.39

In another aspect, the methods, combination product, and compositions provided herein include Compound 2:

or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof.

In this document, compound 2 is also referred to as compound II or comp. II. Compound 2, including its synthesis, is described in PCT Publication No. WO 2014/184350, which is incorporated herein by reference in its entirety.

In yet another aspect, the methods, combination product, and compositions provided herein include Compound 3:

- 17 037576

Compound 3 or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof. In this document, compound 3 is also referred to as compound III or comp. (III).

Compound 3, including its synthesis, is described in PCT Publication No. WO2015 / 118057, which is incorporated herein by reference in its entirety.

III. Ways.

In one aspect provided herein, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an allosteric capsid protein modulator (CpAM) and a therapeutically effective amount of a reverse transcriptase inhibitor. In one embodiment of the method, CpAM is an allosteric modulator of a capsid protein that causes aberrant, defective, or incomplete assembly of HBV capsids. In another embodiment of the method, CpAM is an allosteric modulator of a capsid protein that causes the assembly of capsids that are substantially empty in terms of their viral content. In one aspect provided herein, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof, and a reverse transcriptase inhibitor, or a pharmaceutically acceptable salt thereof, or a prodrug thereof.

In another aspect provided herein, there is provided a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of a compound of Formula IA, or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof, and a reverse transcriptase inhibitor, or a pharmaceutically acceptable salt thereof; or a prodrug thereof.

In yet another aspect provided herein, there is provided a method for treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of a compound of Formula IB, or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof, and a reverse transcriptase inhibitor, or a pharmaceutically acceptable salt thereof, or a prodrug thereof.

In yet another aspect provided herein, there is provided a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof, and a reverse transcriptase inhibitor, or a pharmaceutically acceptable salt thereof, or a prodrug thereof.

In yet another aspect provided herein, there is provided a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of a compound of Formula IIIA, or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof, and a reverse transcriptase inhibitor, or a pharmaceutically acceptable salt thereof, or a prodrug thereof.

In yet another aspect provided herein, there is provided a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of a compound of formula IIIB, or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof, and a reverse transcriptase inhibitor, or a pharmaceutically acceptable salt thereof, or a prodrug thereof.

In yet another embodiment provided by the present invention, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of Compound 1:

Compound 1

- 18 037576 or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof and a reverse transcriptase inhibitor or a pharmaceutically acceptable salt thereof, or a prodrug thereof.

In another aspect provided herein, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of Compound 2:

Compound 2, or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof and a reverse transcriptase inhibitor, or a pharmaceutically acceptable salt thereof, or a prodrug thereof.

In another aspect provided herein, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of Compound 3:

ci ^CF 'N ^Me _n ί ⁰

Compound 3 or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof and a reverse transcriptase inhibitor, or a pharmaceutically acceptable salt thereof, or a prodrug thereof.

Patients who can receive treatment using the described methods are humans in some embodiments. Other warm-blooded animals can also receive treatment.

In one embodiment of the method, the reverse transcriptase inhibitor is selected from the group consisting of entecavir, tenofovir, and lamivudine, or pharmaceutically acceptable salts or prodrugs thereof. Pharmaceutically acceptable prodrugs of tenofovir include tenofovir disoproxil fumarate and tenofovir alafenamide fumarate.

In another embodiment, the reverse transcriptase inhibitor is a purine reverse transcriptase inhibitor.

In one embodiment, a compound of Formula I, Formula IA, Formula IB, Formula II, Formula IIIA, or Formula IIIB and a reverse transcriptase inhibitor are part of the same formulation. In another embodiment, the compound of Formula I, Formula IA, Formula IB, Formula II, Formula IIIA, or Formula IIIB and a reverse transcriptase inhibitor are part of different formulations.

In another embodiment, the compound of formula IA and the reverse transcriptase inhibitor are part of the same formulation. In another embodiment, the compound of formula IA and the reverse transcriptase inhibitor are part of different formulations.

In another embodiment, the compound of Formula IB and the reverse transcriptase inhibitor are part of the same formulation. In another embodiment, the compound of Formula IB and the reverse transcriptase inhibitor are part of different formulations.

In one embodiment, Formula II and the reverse transcriptase inhibitor are part of the same formulation. In another embodiment, Formula II and the reverse transcriptase inhibitor are part of different formulations.

In one embodiment, Formula IIIA and the reverse transcriptase inhibitor are part of the same formulation. In another embodiment, Formula IIIA and the reverse transcriptase inhibitor are part of different formulations.

In one embodiment, Compound 1 and the reverse transcriptase inhibitor are part of the same formulation. In another embodiment, Compound 1 and the reverse transcriptase inhibitor are part of different formulations.

In one embodiment, Compound 2 or Compound 3 and a reverse transcriptase inhibitor are part of the same formulation. In another embodiment, Compound 2 or Compound 3 and the reverse transcriptase inhibitor are part of different formulations.

In one embodiment of the method, the patient is refractory to or refractory to treatment with a reverse transcriptase inhibitor. In another embodiment, the patient is refractory to, or refractory to, treatment with a nucleoside agent. In yet another embodiment, the patient is a previously untreated patient.

- 19 037576

In one embodiment, Compound 1 is administered in an amount of 600 mg per day to 3000 mg per day (including, for example, about 600, about 800, about 1000, about 1200, about 1400, about 1600, about 1800, about 2000 mg ). In one embodiment, Compound 1 is administered in an amount ranging from 600 mg per day to 3000 mg per day. In a specific embodiment, Compound 1 is administered at about 2000 mg per day. In an additional embodiment, Compound 1 is administered at about 1000 mg twice a day.

In one embodiment, Compound 2 or Compound 3 is administered in an amount of 5 mg per day to 600 mg per day (including, for example, about 5, about 25, about 50, about 100, about 200, about 300, about 400, about 500, about 600 mg). In one embodiment, Compound 2 or Compound 3 is administered in an amount of 5 to 600 mg per day. In a specific embodiment, Compound 2 or Compound 3 is administered at about 25 mg per day. In an additional embodiment, Compound 2 or Compound 3 is administered in an amount of about 10 to 200 mg once a day.

In one embodiment provided herein, Compound 1 is in crystalline form. In an additional embodiment, the crystalline form is characterized by a powder X-ray diffraction structure having peaks expressed in degrees-2-theta at angles (± 0.2 °) equal to 17.1, 20.8, 22.2, 24.9 and 26 , 6 ° (Form XVI).

In another embodiment of the method described herein, the administration of a compound of Formula I, Formula IA, Formula IB, Formula II, Formula IIIA, or Formula IIIB and a reverse transcriptase inhibitor occurs over a period of less than 48 weeks.

In another embodiment of the method provided herein, Compound 1 and the reverse transcriptase inhibitor are administered over a period of less than 48 weeks.

In another embodiment of the method described herein, the administration of Compound 2 or Compound 3 and the reverse transcriptase inhibitor occurs over a period of less than 48 weeks.

In one embodiment, the patient is a chronically infected HBV patient (with or without symptoms of underlying liver inflammation).

In one embodiment, the method further comprises administering an additional anti-HBV agent. In a specific embodiment, the additional anti-HBV agent is pegylated interferon alpha-2a.

In one aspect, provided herein is a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of a compound of Formula I, Formula IA, Formula IB, Formula II, Formula IIIA, or Formula IIIB, or a pharmaceutically acceptable salt thereof, a hydrate thereof, its solvate, or its crystalline form, and the patient is refractory to treatment with a reverse transcriptase inhibitor or does not respond to treatment.

In one aspect provided herein, there is provided a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of Compound 1, or a pharmaceutically acceptable salt, hydrate, solvate thereof, or crystalline form thereof, the patient being refractory to treatment with an inhibitor reverse transcriptase or refractory to treatment.

In another aspect provided herein, there is provided a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of Compound 2 or Compound 3, or pharmaceutically acceptable salts, their hydrates, their solvates, or their crystalline forms, wherein the patient refractory to treatment with a reverse transcriptase inhibitor or refractory to treatment.

In yet another aspect provided herein, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula I, Formula IA, Formula IB, Formula II, Formula IIIA, or Formula IIIB, or a pharmaceutically acceptable salt thereof , its hydrate, its solvate, or its crystalline form, and the patient is refractory to treatment with a nucleoside agent or does not respond to treatment.

In yet another aspect provided herein, there is provided a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of Compound 1, or a pharmaceutically acceptable salt, hydrate, solvate thereof, or crystalline form thereof, the patient being refractory to treatment nucleoside agent or refractory to treatment.

In an embodiment provided by the present invention, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of Compound 1 and entecavir.

In another embodiment provided by the present invention, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of Compound 1 and tenofovir.

In another embodiment provided by the present invention, there is provided a method of treating HBV infection in a patient requiring it, comprising administering to the patient therapeutically

- 20,037576 an effective amount of a compound of formula I and entecavir.

In another embodiment provided by the present invention, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula I and tenofovir.

In another embodiment provided by the present invention, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of formula! IA and entecavir.

In another embodiment provided by the present invention, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula IA and tenofovir.

In another embodiment provided by the present invention, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula IB and entecavir.

In another embodiment provided by the present invention, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula IB and tenofovir.

In another embodiment provided by the present invention, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula II and entecavir.

In another embodiment provided by the present invention, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula II and tenofovir.

In another embodiment provided by the present invention, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula IIIA and entecavir.

In another embodiment provided by the present invention, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula IIIA and tenofovir.

In another embodiment provided by the present invention, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of formula IIIB and entecavir.

In another embodiment provided by the present invention, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula IIIB and tenofovir.

In another embodiment provided by the present invention, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of Compound 2 and entecavir.

In another embodiment provided by the present invention, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of Compound 2 and tenofovir.

In another embodiment provided by the present invention, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of Compound 3 and entecavir.

In another embodiment provided by the present invention, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of Compound 3 and tenofovir.

In one embodiment for treating HBV infection, Compound 1 is administered in an amount of 600 to 3000 mg per day (e.g., about 600, about 800, about 1000, about 1200, about 1400, about 1600, about 1800, about 2000 mg). In an additional embodiment, Compound 1 is administered in an amount of 600 to 2000 mg per day. In yet another additional embodiment, Compound 1 is administered at about 2000 mg per day. In one embodiment of this embodiment, Compound 1 is administered in an amount of about 1000 mg twice a day.

In one aspect provided herein, there is provided a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of Compound 2 or Compound 3, or pharmaceutically acceptable salts, hydrates, solvates, or crystalline forms thereof, wherein the patient refractory to treatment with a nucleoside agent or refractory to treatment.

In one embodiment for treating HBV infection, Compound 2 or Compound 3 is administered in an amount of 5 to 600 mg per day (e.g., about 5, about 25, about 50, about 100, about 200, about 300, about 400, about 500, about 600 mg).

In an additional embodiment, Compound 2 or Compound 3 is administered in an amount of 5 mg per day to 600 mg per day. In yet another additional embodiment, compound 2 or

- 21 037576 Compound 3 is administered in an amount of about 25 mg per day. In one embodiment, Compound 2 or Compound 3 is administered in an amount of about 10 to 200 mg once a day.

In another embodiment of these methods, the administration of a compound of Formula I, Formula IA, Formula IB, Formula II, Formula IIIA, or Formula IIIB and a reverse transcriptase inhibitor occurs over a period of less than 48 weeks.

In another embodiment of these methods, the administration of Compound 1 and the reverse transcriptase inhibitor occurs over a period of less than 48 weeks.

In another embodiment of these methods, Compound 2 or Compound 3 and the reverse transcriptase inhibitor are administered over a period of less than 48 weeks.

In another embodiment, the patient is a chronically infected HBV patient (with or without symptoms of underlying liver inflammation).

In one aspect provided herein, there is provided a method of inhibiting the replication of a nucleoside-resistant HBV variant comprising contacting said variant with an effective amount of a compound of Formula I, Formula IA, Formula IB, Formula II, Formula IIIA, or Formula IIIB, or pharmaceutically an acceptable salt, its hydrate, its solvate, or its crystalline form.

In one aspect provided herein, there is provided a method of inhibiting the replication of a nucleoside-resistant HBV variant comprising contacting said variant with an effective amount of Compound 1, or a pharmaceutically acceptable salt, hydrate, solvate thereof, or crystalline form thereof. In another aspect provided herein, there is provided a method of inhibiting the replication of a nucleoside-resistant HBV variant, comprising contacting said variant with an effective amount of Compound 2 or Compound 3, or pharmaceutically acceptable salts thereof, their hydrates, their solvates, or their crystalline forms.

In another aspect provided herein, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula I, Formula IA, Formula IB, Formula II, Formula IIIA, or Formula IIIB, or a pharmaceutically acceptable salt thereof, its hydrate, its solvate, or its crystalline form and compound A

F

A or a pharmaceutically acceptable salt thereof.

In another aspect provided herein, there is provided a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of Compound 1, or a pharmaceutically acceptable salt, hydrate, solvate thereof, or crystalline form thereof, and Compound A or pharmaceutically acceptable salt.

In yet another aspect provided herein, there is provided a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of Compound 2 or Compound 3, or pharmaceutically acceptable salts thereof, hydrates thereof, solvates or crystalline forms thereof, and Compound A , or a pharmaceutically acceptable salt thereof.

In another aspect provided herein, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula I, Formula IA, Formula IB, Formula II, Formula IIIA, or Formula IIIB, or a pharmaceutically acceptable salt thereof, its hydrate, its solvate, or its crystalline form; and entecavir or its pharmaceutically acceptable salt. In one embodiment, a compound of Formula I, Formula IA, Formula IB, Formula II, Formula IIIA, or Formula IIIB and entecavir are administered at specific doses and for specific periods of time to produce a synergistic effect.

In another aspect provided herein, there is provided a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of Compound 1, or a pharmaceutically acceptable salt, a hydrate thereof, a solvate thereof, or a crystalline form thereof and entecavir, or a pharmaceutically acceptable salt. In one embodiment, Compound 1 and entecavir are administered at specific doses and for specific periods of time to produce a synergistic effect.

In another aspect provided herein, there is provided a method for treating an infection

- 22 037576

HBV in a patient requiring it, comprising administering to the patient a therapeutically effective amount of Compound 2, or pharmaceutically acceptable salts thereof, hydrates thereof, solvates or crystalline forms thereof, and entecavir, or pharmaceutically acceptable salt thereof. In one embodiment, Compound 2 and entecavir are administered at specific doses and for specific periods of time to produce a synergistic effect.

In yet another aspect provided herein, there is provided a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of Compound 3, or its pharmaceutically acceptable salts, its hydrates, its solvates, or its crystalline forms, and entecavir or its pharmaceutically acceptable salt. In one embodiment, Compound 3 and entecavir are administered at specific doses and for specific periods of time to produce a synergistic effect.

In another aspect provided herein, there is provided a method of treating HBV infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula I, Formula IA, Formula IB, Formula II, Formula IIIA, or Formula IIIB, or pharmaceutically acceptable salts thereof, their hydrates, their solvates, or their crystalline forms; and tenofovir, or a pharmaceutically acceptable salt or prodrug thereof. In one embodiment, tenofovir is tenofovir disoproxil fumarate or tenofovir alafenamide fumarate. In a further embodiment, the compound of Formula I, Formula IA, Formula IB, Formula II, Formula IIIA, or Formula IIIB and tenofovir are administered at specific doses and for specific periods of time to produce a synergistic effect.

In another aspect provided herein, there is provided a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of Compound 1, or a pharmaceutically acceptable salt, hydrate, solvate thereof, or crystalline form thereof, and tenofovir, or pharmaceutically an acceptable salt; or a prodrug thereof. In one embodiment, tenofovir is tenofovir disoproxil fumarate or tenofovir alafenamide fumarate. In an additional embodiment, Compound 1 and tenofovir are administered at specific doses and for specific periods of time to produce a synergistic effect.

In another aspect provided herein, there is provided a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of Compound 2, or its pharmaceutically acceptable salts, its hydrates, its solvates, or its crystalline forms, and tenofovir or pharmaceutically an acceptable salt; or a prodrug thereof. In one embodiment, tenofovir is tenofovir disoproxil fumarate or tenofovir alafenamide fumarate. In an additional embodiment, Compound 2 and tenofovir are administered at specific doses and for specific periods of time to produce a synergistic effect.

In yet another aspect provided herein, there is provided a method of treating HBV infection in a patient requiring it, comprising administering to the patient a therapeutically effective amount of Compound 3, or its pharmaceutically acceptable salts, its hydrates, its solvates, or its crystalline forms, and tenofovir or its a pharmaceutically acceptable salt; or a prodrug thereof. In one embodiment, tenofovir is tenofovir disoproxil fumarate or tenofovir alafenamide fumarate. In an additional embodiment, Compound 3 and tenofovir are administered at specific doses and for specific periods of time to produce a synergistic effect.

In one embodiment provided herein, an allosteric capsid protein modulator (CpAM) and a reverse transcriptase inhibitor for use in therapy are provided.

In one embodiment provided herein, there is provided a compound of Formula I and a reverse transcriptase inhibitor for use in therapy.

In one embodiment provided herein, a compound of Formula IB and a reverse transcriptase inhibitor for use in therapy is provided.

In one embodiment provided herein, Compound 1 and a reverse transcriptase inhibitor for use in therapy are provided.

In another embodiment provided herein, Compound 2 and a reverse transcriptase inhibitor for use in therapy are provided.

In a further embodiment provided herein, Compound 3 and a reverse transcriptase inhibitor for use in therapy are provided.

In one embodiment provided herein, an allosteric capsid protein modulator (CpAM) and a reverse transcriptase inhibitor are provided for use in treating HBV infection in a patient requiring it.

In one embodiment provided herein, there is provided a compound of Formula I and a reverse transcriptase inhibitor for use in the treatment of HBV infection in a subject patient.

In one embodiment provided herein, there is provided a compound of formula IA and a reverse transcriptase inhibitor for use in the treatment of HBV infection in a patient requiring it.

In one embodiment provided herein, there is provided a compound of Formula IB and a reverse transcriptase inhibitor for use in the treatment of HBV infection in an afflicted patient.

In one embodiment provided herein, there is provided a compound of Formula II and a reverse transcriptase inhibitor for use in the treatment of HBV infection in an afflicted patient.

In one embodiment provided herein, there is provided a compound of Formula IIIA and a reverse transcriptase inhibitor for use in the treatment of HBV infection in a patient requiring it.

In one embodiment provided herein, there is provided a compound of Formula IIIB and a reverse transcriptase inhibitor for use in the treatment of HBV infection in an afflicted patient.

In one embodiment provided herein, there is provided Compound 1 and a reverse transcriptase inhibitor for use in the treatment of HBV infection in a patient requiring it.

In another embodiment provided herein, there is provided Compound 2 and a reverse transcriptase inhibitor for use in treating HBV infection in a patient requiring it.

In a further embodiment provided herein, Compound 3 and a reverse transcriptase inhibitor are provided for use in treating HBV infection in a patient requiring it.

In one embodiment provided herein, there is provided an allosteric capsid protein modulator (CpAM) for use in treating HBV infection in a patient requiring it, wherein CpAM is intended for use in combination with a reverse transcriptase inhibitor.

In one embodiment provided herein, there is provided a compound of Formula I for use in treating HBV infection in a patient requiring it, wherein the compound of Formula I is for use in combination with a reverse transcriptase inhibitor.

In one embodiment provided herein, there is provided a compound of Formula IA for use in treating HBV infection in a patient requiring it, wherein the compound of Formula IA is for use in combination with a reverse transcriptase inhibitor.

In one embodiment provided herein, there is provided a compound of Formula IB for use in treating HBV infection in a patient requiring it, wherein the compound of Formula IB is for use in combination with a reverse transcriptase inhibitor.

In one embodiment provided herein, there is provided a compound of Formula II for use in treating HBV infection in a patient requiring it, wherein the compound of Formula II is for use in combination with a reverse transcriptase inhibitor.

In one embodiment provided herein, there is provided a compound of Formula IIIA for use in treating HBV infection in a patient requiring it, wherein the compound of Formula IIIA is for use in combination with a reverse transcriptase inhibitor.

In one embodiment provided herein, there is provided a compound of Formula IIIB for use in treating HBV infection in a patient requiring it, wherein the compound of Formula IIIB is for use in combination with a reverse transcriptase inhibitor.

In one embodiment provided herein, there is provided Compound 1 for use in treating HBV infection in a patient requiring it, wherein Compound 1 is for use in combination with a reverse transcriptase inhibitor.

In another embodiment provided herein, there is provided Compound 2 for use in treating HBV infection in a patient requiring it, wherein Compound 2 is for use in combination with a reverse transcriptase inhibitor.

In a further embodiment provided herein, there is provided Compound 3 for use in treating HBV infection in a patient requiring it, wherein Compound 3 is for use in combination with a reverse transcriptase inhibitor.

In one embodiment provided herein, a reverse transcriptase inhibitor is provided for use in treating HBV infection in a patient requiring it, the reverse transcriptase inhibitor for use in combination with an allosteric capsid protein modulator (CpAM).

In one embodiment provided herein, a reverse transcriptase inhibitor is provided for use in the treatment of HBV infection in a patient requiring it, wherein the reverse transcriptase inhibitor is intended for use in combination with a compound of formula I.

In one embodiment provided herein, there is provided a reverse transcriptase inhibitor for use in treating HBV infection in a patient requiring it, the reverse transcriptase inhibitor for use in combination with a compound of formula IA.

In one embodiment provided herein, a reverse transcriptase inhibitor is provided for use in treating HBV infection in a patient requiring it, the reverse transcriptase inhibitor for use in combination with Compound 1.

In one embodiment provided herein, a reverse transcriptase inhibitor is provided for use in treating HBV infection in a patient requiring it, the reverse transcriptase inhibitor for use in combination with an allosteric capsid protein modulator (CpAM).

In one embodiment provided herein, there is provided a reverse transcriptase inhibitor for use in treating HBV infection in a patient requiring it, the reverse transcriptase inhibitor for use in combination with a compound of formula I.

In one embodiment provided herein, there is provided a reverse transcriptase inhibitor for use in treating HBV infection in a patient requiring it, the reverse transcriptase inhibitor for use in combination with a compound of formula IA.

In one embodiment provided herein, there is provided a reverse transcriptase inhibitor for use in treating HBV infection in a patient requiring it, the reverse transcriptase inhibitor for use in combination with a compound of formula IB.

In one embodiment provided herein, there is provided a reverse transcriptase inhibitor for use in treating HBV infection in a patient requiring it, the reverse transcriptase inhibitor being for use in combination with a compound of formula II.

In one embodiment provided herein, there is provided a reverse transcriptase inhibitor for use in treating HBV infection in a patient requiring it, the reverse transcriptase inhibitor for use in combination with a compound of formula IIIA.

In one embodiment provided herein, there is provided a reverse transcriptase inhibitor for use in treating HBV infection in a patient requiring it, the reverse transcriptase inhibitor for use in combination with a compound of formula IIIB.

In one embodiment provided herein, a reverse transcriptase inhibitor is provided for use in treating HBV infection in a patient requiring it, the reverse transcriptase inhibitor for use in combination with Compound 1.

In another embodiment provided herein, a reverse transcriptase inhibitor is provided for use in treating HBV infection in a patient requiring it, the reverse transcriptase inhibitor for use in combination with Compound 2.

In a further embodiment provided herein, a reverse transcriptase inhibitor is provided for use in treating HBV infection in a patient requiring it, the reverse transcriptase inhibitor being for use in combination with Compound 3.

In one embodiment provided herein, an allosteric capsid protein modulator (CpAM) and a reverse transcriptase inhibitor are provided for use in combination therapy for treating HBV infection in a patient requiring it, wherein the CpAM and the reverse transcriptase inhibitor are intended for simultaneous, sequential, or separate administration ...

In one embodiment provided herein, there is provided a compound of Formula I and a reverse transcriptase inhibitor for use in combination therapy for treating HBV infection in an afflicted patient, wherein the compound of Formula I and the reverse transcriptase inhibitor are intended to be administered concurrently, sequentially, or separately.

In one embodiment provided herein, a compound of formula IA and a reverse transcriptase inhibitor is provided for use in combination therapy for treating HBV infection in a requiring patient, wherein the compound of formula IA and the reverse transcriptase inhibitor are intended for simultaneous, sequential, or separate administration.

- 25 037576

In one embodiment provided herein, a compound of formula IB and a reverse transcriptase inhibitor are provided for use in combination therapy for treating HBV infection in a requiring patient, wherein the compound of formula IB and the reverse transcriptase inhibitor are intended for simultaneous, sequential, or separate administration.

In one embodiment provided herein, there is provided a compound of Formula II and a reverse transcriptase inhibitor for use in combination therapy for treating HBV infection in a requiring patient, wherein the compound of Formula II and the reverse transcriptase inhibitor are intended to be administered concurrently, sequentially, or separately.

In one embodiment provided herein, a compound of Formula IIIA and a reverse transcriptase inhibitor is provided for use in combination therapy for treating HBV infection in a patient requiring it, wherein the compound of Formula IIIA and the reverse transcriptase inhibitor are intended to be administered concurrently, sequentially, or separately.

In one embodiment provided herein, a compound of Formula IIIB and a reverse transcriptase inhibitor is provided for use in combination therapy for treating HBV infection in a requiring patient, wherein the compound of Formula IIIB and the reverse transcriptase inhibitor are intended to be administered concurrently, sequentially, or separately.

In one embodiment provided herein, Compound 1 and a reverse transcriptase inhibitor are provided for use in combination therapy for treating HBV infection in a requiring patient, wherein Compound 1 and the reverse transcriptase inhibitor are intended to be administered simultaneously, sequentially, or separately.

In another embodiment provided herein, Compound 2 and a reverse transcriptase inhibitor are provided for use in combination therapy for treating HBV infection in a requiring patient, wherein Compound 2 and the reverse transcriptase inhibitor are intended to be administered concurrently, sequentially, or separately.

In another embodiment provided herein, Compound 3 and a reverse transcriptase inhibitor are provided for use in combination therapy for treating HBV infection in a requiring patient, wherein Compound 3 and the reverse transcriptase inhibitor are intended to be administered simultaneously, sequentially, or separately.

The daily doses described herein are calculated for an average body weight of about 60 to about 70 kg and should be recalculated for pediatric applications or when used for patients with substantially deviating body weights.

IV. Combined products and compositions.

In one aspect provided herein, a combination product is provided comprising an allosteric capsid protein modulator (CpAM) and a reverse transcriptase inhibitor. In one embodiment of the combination product, CpAM is an allosteric modulator of a capsid protein that causes aberrant, defective, or incomplete assembly of HBV capsids. In another embodiment of the combination product, CpAM is an allosteric modulator of a capsid protein, causing the assembly of capsids that are substantially empty in terms of their viral content. In one embodiment of the combination product of Formula I, Formula IA, Formula II, Formula IIIA, or Formula IIIB, the reverse transcriptase inhibitor is selected from the group consisting of entecavir, tenofovir, lamivudine, telbivudine, adefovir, clevudine, CMX157, AGX-1009, zidovudine, didanosine , zalcitabine, stavudine, emtricitabine, abacavir, D-D4FC, alovudine, amdoxovir, elvucitabine, delavirdine, efavirenz, nevirapine, capvirin, calanolide A, TMC278, BMS-561390 and DPC-083, or their pharmaceutically acceptable prodrugs and their salts. Pharmaceutically acceptable prodrugs of tenofovir include, for example, tenofovir disoproxil fumarate and tenofovir alafenamide fumarate.

In some embodiments, the combination product comprises at least Compound 1 or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, and entecavir.

In some embodiments, the combination product comprises at least Compound 1 or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, and tenofovir.

In some embodiments, the combination product contains at least one compound of Formula I and entecavir.

In some embodiments, the combination product contains at least one compound of Formula I and tenofovir.

In some embodiments, the combination product contains at least one compound of Formula IA and entecavir.

In some embodiments, the combination product contains at least one compound of Formula IA and tenofovir.

- 26 037576

In some embodiments, the combination product contains at least one compound of Formula IB and entecavir.

In some embodiments, the combination product contains at least one compound of Formula IB and tenofovir.

In some embodiments, the combination product contains at least one compound of Formula II and entecavir.

In some embodiments, the combination product contains at least one compound of Formula II and tenofovir.

In some embodiments, the combination product contains at least one compound of Formula IIIA and entecavir.

In some embodiments, the combination product contains at least one compound of Formula IIIA and tenofovir.

In some embodiments, the combination product contains at least one compound of Formula IIIB and entecavir.

In some embodiments, the combination product contains at least one compound of Formula IIIB and tenofovir.

In some embodiments, the combination product comprises at least Compound 1 or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, and entecavir.

In some embodiments, the combination product comprises at least Compound 1 or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, and tenofovir.

In some embodiments, the combination product comprises at least Compound 2 or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, and entecavir.

In some embodiments, the combination product comprises at least Compound 2 or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, and tenofovir.

In some embodiments, the combination product comprises at least Compound 3 or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, and entecavir.

In some embodiments, the combination product comprises at least Compound 3 or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, and tenofovir.

In one aspect provided herein, a combination product is provided comprising Compound 1, or a pharmaceutically acceptable salt thereof, hydrates thereof, solvates thereof, or crystalline forms thereof, and a reverse transcriptase inhibitor or a pharmaceutically acceptable salt thereof.

In one embodiment of the combination product, the reverse transcriptase inhibitor is selected from the group consisting of entecavir, tenofovir, lamivudine, telbivudine, adefovir, clevudine, CMX157, AGX-1009, zidovudine, didanosine, zalcitabine, stavudine, ddItricitabine, D-D4 , amdoxovir, elvucitabine, delavirdine, efavirenz, nevirapine, capvirin, calanolide A, TMC278, BMS-561390 and DPC-083, or their prodrugs and their pharmaceutically acceptable salts. Pharmaceutically acceptable prodrugs of tenofovir include, for example, tenofovir disoproxil fumarate and tenofovir alafenamide fumarate.

In one embodiment of the combination product, the reverse transcriptase inhibitor is selected from the group consisting of entecavir, tenofovir, and lamivudine, or pharmaceutically acceptable salts thereof. Pharmaceutically acceptable prodrugs of tenofovir include tenofovir disoproxil fumarate and tenofovir alafenamide fumarate.

In another aspect provided herein, there is provided a combination product comprising Compound 2, or a pharmaceutically acceptable salt thereof, hydrates thereof, solvates thereof, or crystalline forms thereof, and a reverse transcriptase inhibitor or a pharmaceutically acceptable salt thereof.

In one embodiment of the combination product of Compound 2, the reverse transcriptase inhibitor is selected from the group consisting of entecavir, tenofovir, lamivudine, telbivudine, adefovir, clevudine, CMX157, AGX-1009, zidovudine, didanosine, zalcitabine, stavudine, emtaviracitabine, D-abacitabine, abacitabine , alovudine, amdoxovir, elvucitabine, delavirdine, efavirenz, nevirapine, capvirin, calanolide A, TMC278, BMS-561390 and DPC-083, or their prodrugs and their pharmaceutically acceptable salts. Pharmaceutically acceptable prodrugs of tenofovir include, for example, tenofovir disoproxil fumarate and tenofovir alafenamide fumarate.

In yet another aspect provided herein, there is provided a combination product comprising Compound 3, or pharmaceutically acceptable salts, hydrates, solvates or crystalline forms thereof, and a reverse transcriptase inhibitor or pharmaceutically acceptable salt thereof.

In one embodiment of the combination product of Compound 3, the reverse transcriptase inhibitor is selected from the group consisting of entecavir, tenofovir, lamivudine, telbivudine, adefovir, clevudine, CMX157, AGX-1009, zidovudine, didanosine, zalcitabine, stavudine, emtricaviratin-27 037576 , D-D4FC, alovudine, amdoxovir, elvucitabine, delavirdine, efavirenz, nevirapine, capravirin, calanolide A, TMC278, BMS-561390 and DPC-083, or their prodrugs and their pharmaceutically acceptable salts. Pharmaceutically acceptable prodrugs of tenofovir include, for example, tenofovir disoproxil fumarate and tenofovir alafenamide fumarate.

In one embodiment of the combination product, Compound 1 and the reverse transcriptase inhibitor are part of the same formulation. In another embodiment of the combination product, Compound 1 and the reverse transcriptase inhibitor are part of different formulations. In a further embodiment of this embodiment, the compositions are for simultaneous or sequential administration.

In one embodiment of the combination product, Compound 2 or Compound 3 and the reverse transcriptase inhibitor are part of the same formulation. In another embodiment of the combination product, Compound 2 or Compound 3 and the reverse transcriptase inhibitor are part of different formulations. In a further embodiment of this embodiment, the compositions are for simultaneous or sequential administration.

In one embodiment, the combination product is for use in treating HBV infection in a patient.

In one embodiment, the combination product is for use in treating HBV infection in a patient, the patient being refractory to treatment with a reverse transcriptase inhibitor. In another embodiment, the combination product is for use in treating HBV infection in a patient, the patient being refractory to treatment with a nucleoside agent.

In one embodiment, the combination product is for use in treating HBV infection in a patient who has not previously received treatment.

In one embodiment, the combination product of Compound 2 or Compound 3 is for use in the treatment of HBV infection in a patient.

In one embodiment, the combination product of Compound 2 or Compound 3 is for use in treating HBV infection in a patient, the patient being refractory to treatment with a reverse transcriptase inhibitor. In another embodiment, the combination product is for use in treating HBV infection in a patient, the patient being refractory to treatment with a nucleoside agent.

In one embodiment, the combination product of Compound 2 or Compound 3 is for use in treating HBV infection in a patient who has not previously received treatment.

In one embodiment, the combination product is for use in treating HBV infection in a patient, the patient being chronically infected with HBV (with or without symptoms of underlying liver inflammation).

In one embodiment of the combination product, Compound 1 is used in an amount of 600 to 3000 mg (e.g., about 600, about 800, about 1000, about 1200, about 1400, about 1600, about 1800, about 2000 mg). In a further embodiment of the combination product, the amount of Compound 1 is 600 to 2000 mg. In another embodiment of the combination product, the amount of Compound 1 is about 2000 mg. In yet another embodiment of the combination product, the amount of Compound 1 is about 1000 mg.

In one embodiment of the combination product, Compound 2 or Compound 3 is used in an amount of 5 to 600 mg per day (e.g., about 5, about 25, about 50, about 100, about 200, about 300, about 400, about 500, about 600 mg). In a further embodiment of the combination product, the amount of Compound 2 or Compound 3 is 5 to 600 mg. In another embodiment of the combination product, the amount of Compound 2 or Compound 3 is about 25 mg. In yet another embodiment of the combination product, the amount of Compound 2 or Compound 3 is 10 to 200 mg.

In one embodiment of the combination product, Compound 1 is in crystalline form. In an additional embodiment, the crystalline form is characterized by a powder X-ray diffraction structure having peaks expressed in degrees-2-theta at angles (± 0.2 °) equal to 17.1, 20.8, 22.2, 24.9 and 26 , 6 ° (Form XVI).

In one embodiment, the combination product further comprises an additional anti-HBV agent. In one embodiment, the additional anti-HBV agent is pegylated interferon alpha-2a.

In one aspect provided herein, there is provided a combination product comprising Compound 1, or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof, and a reverse transcriptase inhibitor or a pharmaceutically acceptable salt thereof.

In one embodiment of the combination product, the ratio between CpAM and reverse transcriptase inhibitor is in the range of 700: 1-1: 40. In another embodiment, the ratio between CpAM and reverse transcriptase inhibitor is in the range of 2: 1-1: 2, eg 2: 1.1: 1 or 1: 2; 170: 1 to 150: 1, for example 170: 1, 160: 1, or 150: 1; from 3: 1 to 1: 1, for example 3: 1, 2: 1 or 1: 1; or from 30: 1 to 10: 1, for example 30: 1, 20: 1, or 10: 1.

In one aspect provided herein, there is provided a combination product comprising a compound of Formula I, Formula IA, Formula IB, Formula II, Formula IIIA, or Formula IIIB, or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof, and a reverse transcriptase inhibitor or a pharmaceutically acceptable salt thereof.

In one embodiment of the combination product, the ratio between Formula I and reverse transcriptase inhibitor is in the range of 700: 1-1: 40. In another embodiment of the combination product, the ratio between Formula I and reverse transcriptase inhibitor is in the range of 2: 1-1: 2, for example 2: 1, 1: 1 or 1: 2; 170: 1 to 150: 1, for example 170: 1, 160: 1, or 150: 1; from 3: 1 to 1: 1, for example 3: 1, 2: 1 or 1: 1; or from 30: 1 to 10: 1, for example 30: 1, 20: 1, or 10: 1.

In one embodiment of the combination product, the ratio between Formula IA and reverse transcriptase inhibitor is in the range of 700: 1-1: 40. In another embodiment of the combination product, the ratio between Formula I and reverse transcriptase inhibitor is in the range of 2: 1-1: 2, for example 2: 1, 1: 1 or 1: 2; 170: 1 to 150: 1, for example 170: 1, 160: 1, or 150: 1; from 3: 1 to 1: 1, for example 3: 1, 2: 1 or 1: 1; or from 30: 1 to 10: 1, for example 30: 1, 20: 1, or 10: 1.

In one embodiment of the combination product, the ratio between Formula IB and reverse transcriptase inhibitor is in the range of 700: 1-1: 40. In another embodiment of the combination product, the ratio between Formula I and reverse transcriptase inhibitor is in the range of 2: 1-1: 2, for example 2: 1, 1: 1 or 1: 2; 170: 1 to 150: 1, for example 170: 1, 160: 1, or 150: 1; from 3: 1 to 1: 1, for example 3: 1, 2: 1 or 1: 1; or from 30: 1 to 10: 1, for example 30: 1, 20: 1, or 10: 1.

In one embodiment of the combination product, the ratio between Formula II and reverse transcriptase inhibitor is in the range 700: 1-1: 40. In another embodiment of the combination product, the ratio between Formula I and reverse transcriptase inhibitor is in the range 2: 1-1: 2, for example 2: 1, 1: 1 or 1: 2; 170: 1 to 150: 1, for example 170: 1, 160: 1, or 150: 1; from 3: 1 to 1: 1, for example 3: 1, 2: 1 or 1: 1; or from 30: 1 to 10: 1, for example 30: 1, 20: 1, or 10: 1.

In one embodiment of the combination product, the ratio between Formula IIIA and reverse transcriptase inhibitor is in the range of 700: 1-1: 40. In another embodiment of the combination product, the ratio between Formula I and reverse transcriptase inhibitor is in the range of 2: 1-1: 2, for example 2: 1, 1: 1 or 1: 2; 170: 1 to 150: 1, for example 170: 1, 160: 1, or 150: 1; from 3: 1 to 1: 1, for example 3: 1, 2: 1 or 1: 1; or from 30: 1 to 10: 1, for example 30: 1, 20: 1, or 10: 1.

In one embodiment of the combination product, the ratio between Formula IIIB and reverse transcriptase inhibitor is in the range of 700: 1-1: 40. In another embodiment of the combination product, the ratio between Formula I and reverse transcriptase inhibitor is in the range of 2: 1-1: 2, for example 2: 1, 1: 1 or 1: 2; 170: 1 to 150: 1, for example 170: 1, 160: 1, or 150: 1; from 3: 1 to 1: 1, for example 3: 1, 2: 1 or 1: 1; or from 30: 1 to 10: 1, for example 30: 1, 20: 1, or 10: 1.

In one embodiment of the combination product, the ratio between Compound 1 and entecavir is in the range of 2: 1-1: 2, for example 2: 1, 1: 1, or 1: 2; from 17 0: 1 to 150: 1, for example 170: 1, 160: 1 or 150: 1; from 3: 1 to 1: 1, for example 3: 1, 2: 1 or 1: 1; or from 30: 1 to 10: 1, for example 30: 1, 20: 1, or 10: 1.

In one embodiment of the combination product, the ratio between Compound 1 and tenofovir is in the range of 2: 1-1: 2, such as 2: 1, 1: 1, or 1: 2; 170: 1 to 150: 1, for example 170: 1, 160: 1, or 150: 1; from 3: 1 to 1: 1, for example 3: 1, 2: 1 or 1: 1; or from 30: 1 to 10: 1, for example 30: 1, 20: 1, or 10: 1.

In one embodiment of the combination product, the ratio between compound 2 and entecavir ranges from 200: 1 to 1:40, for example 200: 1, 175: 1, 150: 1, 125: 1, 100: 1, 90: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30: 1, 20: 1, 15: 1, 10: 1, 8: 1, 5: 1, 2: 1, 1: 1, 1: 2, 1: 5, 1: 8, 1:10, 1:15, 1:20, 1:30 or 1:40. In another embodiment of the combination product, the ratio between compound 2 and entecavir is in the range of 40: 1 to 1: 1, for example 40: 1, 30: 1, 20: 1, 10: 1, 8: 1, 6: 1, 4: 1, 2: 1, or 1: 1. In a further embodiment of the combination product, the ratio between Compound 2 and entecavir is in the range of 30: 1-10: 1, for example 30: 1, 20: 1, or 10: 1.

In yet another embodiment of the combination product, the ratio between Compound 2 and entecavir is 20: 1.

In one embodiment of the combination product, the ratio between Compound 2 and tenofovir is in the range of 40: 1 to 1:40, for example 40: 1, 30: 1, 20: 1, 15: 1, 10: 1,

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8: 1, 5: 1, 2: 1, 1: 1, 1: 2, 1: 5, 1: 8, 1:10, 1:15, 1:20, 1:30 or 1:40. In another embodiment of the combination product, the ratio between Compound 2 and tenofovir is in the range of 10: 1 to 1: 1, for example 10: 1, 8: 1, 6: 1, 4: 1, 2: 1, or 1: 1. In a further embodiment of the combination product, the ratio between Compound 2 and tenofovir is in the range of 3: 1 to 1: 1, such as 3: 1, 2: 1, or 1: 1. In yet another embodiment of the combination product, the ratio between Compound 2 and tenofovir is 2: 1.

In one embodiment of the combination product, the ratio between compound 3 and entecavir ranges from 700: 1 to 1:30, for example 700: 1, 600: 1, 500: 1, 400: 1, 300: 1, 200: 1, 190: 1, 180: 1, 170: 1, 160: 1, 150: 1, 140: 1, 130: 1, 120: 1, 110: 1, 100: 1, 20: 1, 15: 1, 10: 1, 8: 1, 5: 1, 2: 1, 1: 1, 1: 2, 1: 5, 1: 8, 1:10, 1:15 or 1:20. In another embodiment of the combination product, the ratio between compound 3 and entecavir ranges from 180: 1 to 1: 2, for example 180: 1, 170: 1, 160: 1, 150: 1, 140: 1, 130: 1, 120: 1, 110: 1, 100: 1, 50: 1, 20: 1, 10: 1, 1: 1 or 1: 2. In a further embodiment of the combination product, the ratio between compound 3 and entecavir is in the range 170: 1-150: 1, for example 170: 1, 160: 1, or 150: 1. In yet another embodiment of the combination product, the ratio between Compound 3 and entecavir is 160: 1.

In one embodiment of the combination product, the ratio between Compound 3 and tenofovir ranges from 80: 1 to 1:10, for example 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30: 1, 20: 1, 15: 1, 10: 1, 1: 1, or 1:10. In another embodiment of the combination product, the ratio between Compound 3 and tenofovir is in the range of 10: 1 to 1:10, for example 10: 1, 8: 1, 6: 1, 4: 1, 2: 1, 1: 1, 1: 2, 1: 4, 1: 6, 1: 8 and 1:10. In a further embodiment of the combination product, the ratio between Compound 3 and tenofovir is in the range of 2: 1 to 1: 2, for example 2: 1, 1: 1, or 1: 2. In yet another embodiment of the combination product, the ratio between Compound 3 and tenofovir is 1: 1.

In another aspect provided herein, there is provided a pharmaceutical composition comprising Compound 1, or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a crystalline form thereof, and a reverse transcriptase inhibitor or a pharmaceutically acceptable salt thereof.

In one embodiment of the pharmaceutical composition, the reverse transcriptase inhibitor is selected from the group consisting of entecavir, tenofovir (including prodrugs thereof, eg, tenofovir disoproxil and tenofovir alafenamide), and lamivudine, or pharmaceutically acceptable salts thereof.

In another embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

In another aspect provided herein, there is provided a pharmaceutical composition comprising a compound of Formula I, Formula IA, Formula IB, Formula II, Formula IIIA, or Formula IIIB, or pharmaceutically acceptable salts, hydrates thereof, solvates thereof, or crystalline forms thereof, and a reverse transcriptase inhibitor or a pharmaceutically acceptable salt thereof.

In one embodiment of a pharmaceutical composition of Formula I, Formula IA, Formula II, Formula IIIA, or Formula IIIB, the reverse transcriptase inhibitor is selected from the group consisting of entecavir, tenofovir, lamivudine, telbivudine, adefovir, clevudine, CMX157, AGX-1009, zidovudine, didanosine , zalcitabine, stavudine, emtricitabine, abacavir, D-D4FC, alovudine, amdoxovir, elvucitabine, delavirdine, efavirenz, nevirapine, capvirin, calanolide A, TMC278, BMS-561390 and DPC-083, or their pharmaceutically acceptable prodrugs and their salts. Pharmaceutically acceptable prodrugs of tenofovir include, for example, tenofovir disoproxil fumarate and tenofovir alafenamide fumarate.

In another embodiment, the pharmaceutical composition of Formula I, Formula IA, Formula IB, Formula II, Formula IIIA, or Formula IIIB further comprises one or more pharmaceutically acceptable carriers.

In another aspect provided herein, there is provided a pharmaceutical composition comprising Compound 1, or a pharmaceutically acceptable salt thereof, hydrates thereof, solvates thereof, or crystalline forms thereof, and a reverse transcriptase inhibitor or a pharmaceutically acceptable salt thereof.

In one embodiment of the pharmaceutical composition of Compound 1, the reverse transcriptase inhibitor is selected from the group consisting of entecavir, tenofovir, lamivudine, telbivudine, adefovir, clevudine, CMX157, AGX-1009, zidovudine, didanosine, zalcitabine, stavudine, emtaviracitabine, D-abacitabine, abacitabine , alovudine, amdoxovir, elvucitabine, delavirdine, efavirenz, nevirapine, capvirin, calanolide A, TMC278, BMS-561390 and DPC-083, or their prodrugs and their pharmaceutically acceptable salts. Pharmaceutically acceptable prodrugs of tenofovir include, for example, tenofovir disoproxil fumarate and tenofovir alafenamide fumarate.

In another embodiment, the pharmaceutical composition of Compound 1 further comprises one or more pharmaceutically acceptable carriers.

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In another aspect provided herein, there is provided a pharmaceutical composition comprising Compound 2, or pharmaceutically acceptable salts, hydrates, solvates or crystalline forms thereof, and a reverse transcriptase inhibitor or pharmaceutically acceptable salt thereof.

In one embodiment of the pharmaceutical composition of Compound 2, the reverse transcriptase inhibitor is selected from the group consisting of entecavir, tenofovir, lamivudine, telbivudine, adefovir, clevudine, CMX157, AGX-1009, zidovudine, didanosine, zalcitabine, stavudine, emtaviracitabine, D-abacitabine, abacitabine , alovudine, amdoxovir, elvucitabine, delavirdine, efavirenz, nevirapine, capvirin, calanolide A, TMC278, BMS-561390 and DPC-083, or their prodrugs and their pharmaceutically acceptable salts. Pharmaceutically acceptable prodrugs of tenofovir include, for example, tenofovir disoproxil fumarate and tenofovir alafenamide fumarate.

In another embodiment, the pharmaceutical composition of Compound 2 further comprises one or more pharmaceutically acceptable carriers.

In yet another aspect provided herein, there is provided a pharmaceutical composition comprising Compound 3, or pharmaceutically acceptable salts, hydrates, solvates or crystalline forms thereof, and a reverse transcriptase inhibitor or pharmaceutically acceptable salt thereof.

In one embodiment of the pharmaceutical composition of Compound 3, the reverse transcriptase inhibitor is selected from the group consisting of entecavir, tenofovir, lamivudine, telbivudine, adefovir, clevudine, CMX157, AGX-1009, zidovudine, didanosine, zalcitabine, stavudine, emtricitabine, D-abacitabine, abacitabine , alovudine, amdoxovir, elvucitabine, delavirdine, efavirenz, nevirapine, capvirin, calanolide A, TMC278, BMS-561390 and DPC-083, or their prodrugs and their pharmaceutically acceptable salts. Pharmaceutically acceptable prodrugs of tenofovir include, for example, tenofovir disoproxil fumarate and tenofovir alafenamide fumarate.

In another embodiment, the pharmaceutical composition of Compound 3 further comprises one or more pharmaceutically acceptable carriers.

In another aspect, the present description provides a kit for the treatment of HBV infections, containing CpAM, compound 1, compound 2 or compound 3, or their pharmaceutically acceptable salts, their hydrates, their solvates, or their crystalline forms in an amount of from 600 to 3000 mg per day , and a reverse transcriptase inhibitor. In another embodiment, provided herein is a kit for the treatment of HBV infections comprising at least two or more of the group consisting of CpAM, Compound 1, Compound 2, or Compound 3, or pharmaceutically acceptable salts thereof, hydrates thereof, solvates thereof, or their crystalline forms, in an amount from 600 to 3000 mg per day; a reverse transcriptase inhibitor; and an additional agent against HBV virus. In some embodiments, the kit further comprises packaging and instructions. In some embodiments, the kit includes a pharmaceutical product comprising a pharmaceutical composition comprising CpAM, Compound 1, Compound 2, or Compound 3, or pharmaceutically acceptable salts, hydrates, solvates or crystalline forms thereof, and a pharmaceutically acceptable carrier or diluent ; and a pharmaceutical composition comprising a reverse transcriptase inhibitor and a pharmaceutically acceptable carrier or diluent.

In some embodiments, the kit contains a pharmaceutical composition comprising CpAM, Compound 1, Compound 2, or Compound 3, or their pharmaceutically acceptable salts, their hydrates, their solvates, or their crystalline forms; an additional agent against the HBV virus; and a pharmaceutically acceptable carrier or diluent. In another embodiment, the kit contains a pharmaceutical product comprising:

a pharmaceutical composition containing CpAM, compound 1, compound 2 or compound 3, or their pharmaceutically acceptable salts, their hydrates, their solvates, or their crystalline forms, in an amount from 600 mg to 3000 mg, and a pharmaceutically acceptable carrier or diluent;

a reverse transcriptase inhibitor;

a sealed container for packaging a pharmaceutical composition;

a sealed container for holding a reverse transcriptase inhibitor; instructions for use.

In another embodiment, the kit contains a pharmaceutical preparation containing at least two components from the group consisting of a pharmaceutical composition containing CpAM, compound 1, compound 2 or compound 3, or their pharmaceutically acceptable salts, their hydrates, their solvates, or their crystalline forms, in an amount of 600 to 3000 mg, and a pharmaceutically acceptable carrier or diluent;

a reverse transcriptase inhibitor;

an additional agent against the HBV virus;

additionally containing a sealed container for packaging the pharmaceutical composition;

sealed container for interferon;

- 31 037576 instructions for use.

In additional embodiments, pharmaceutical kits are provided. The kit includes a sealed container approved for storing pharmaceutical compositions, the container containing one of the pharmaceutical compositions described above. In some embodiments, the sealed container minimizes air contact with the ingredients, such as an evacuated bottle. In other embodiments, the sealed container is a sealed tube. The kit should include instructions for using the composition and information about the composition.

Examples of

Example 1. Antiviral activity of compound 1 against HBV variants resistant to lamivudine, tenofovir and entecavir.

In this example, the antiviral activity of Compound 1 was determined using HepG2 liver cells transiently transfected with replicable HBV DNA expression plasmids, and the intracellular encapsulated HBV DNA was quantified. The antiviral activity of compound 1 was measured against nucleoside-sensitive wild-type HBV inhibitors, as well as against HBV variants resistant to nucleoside analogs that contain formed amino acid changes in the coding sequence of the reverse transcriptase protein: rtL180M / M204V, rtN236T, rtA181V, rtA / N236T and rtL180M / M204V / N236T.

Example 1.1. Materials and methods.

Connections.

Compound 1 was obtained by synthesis. Lamivudine (LMV), entecavir (ETV) and tenofovir disoproxil fumarate (TDF) were purchased from Toronto Research Chemicals (Toronto, Canada) and their chemical structures are shown below with the designation compounds (IV), (V) and (VI ), respectively:

Plasmids HBV.

Plasmid DNA constructs containing the 1.1x HBV genome, under the control of the cytomegalovirus (CMV) promoter, were pre-cloned from the serum of an HBV-infected patient prior to LMV treatment (Genbank AY220698, Fudan University, China; SEQ ID NO: 1, see Table 1) and after the formation of resistance to LMV (Genbank AY220697, Fudan University, China; SEQ ID NO: 2, see Table 1) (Zhang JM et al. 2005. J. Med. Virol. 77: 203-208). Genotyping analysis confirmed that both isolates were HBV genotype B and that the isolate harvested after LMV resistance had developed contained two amino acid changes in the polymerase gene (L180M / M204V). The HBV variant was designated rtL180M / M204V to indicate that amino acid changes were present in the reverse transcriptase (rt) protein. Two additional plasmids were constructed by introducing changes in coding sequences leading to amino acid changes in N236T and A181V in HBV polymerase, respectively. Nucleotide changes were introduced into the wild-type genotype B plasmid by site-directed mutagenesis according to the manufacturer's recommendations (Agilent Technologies; Santa Clara, CA, USA; Cat # 200519) using the following primers and their corresponding reverse complement sequence (nucleotide change underlined): 5 '- CTT TGG GTA TAC ATT TAA CCC CTC ACA AAA C - 3' (rtN236T; SEQ ID NO: 3), 5'-GTC CGT TTC TCT TGG TTC AGT TTA CTA GTG - 3 '( rtA181V; SEQ ID NO: 4). In two additional plasmid constructs, the amino acid change rtN236T was also introduced into plasmids rtA181V and rtL180M / M204V to create HBV variants of the double mutant rtA181V / N236T and triple mutant rtL180M / M204V / N236T, respectively. The full length HBV genome was sequenced for all plasmids to confirm that only the putative nucleotide change (s) were present in the final HBV expression constructs.

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Table 3

HBV genome sequences for plasmid constructs

SEQ ID NO: 1 aactccacca ctttccacca aactcttcaa gatcccagag tcagggccct gtactttcct60 gctggtggct ccagttcagg aacagtgagc cctgctcaaa atactgtctc tgccatatcg120 tcaatcttat cgaaaactgg ggaccctgta ccgaacatgg agaacatcgc atcaggactc180 ctaggacccc tgctcgtgtt acaggcgggg tttttcttgt tgacaaaaat cctcacaata240 ccacagagtc tagactcgtg gtggacttct ctcaattttc tagggggaac acccgtgtgt300 cttggccaaa attcgcagtc ccaaatctcc agtcactcac caacctgttg tcctccaatt360 tgtcctggtt atcgctggat gtatctgcgg cgttttatca tattcctctg catcctgctg420 ctatgcctca tcttcttgtt ggttcttctg gactatcaag gtatgttgcc cgtttgtcct480 ctaattccag gatcatcaac aaccagcacc ggaccatgca aaacctgcac gactcctgct540 caaggaacct ctatgtttcc ctcatgttgc tgtacaaaac ctacggacgg aaactgcacc600 tgtattccca tcccatcatc ttgggctttc gcaaaattcc tatgggagtg ggcctcagtc660 cgtttctctt ggctcagttt actagtgcca tttgttcagt ggttcgtagg gctttccccc720 actgtctggc tttcagttat atggatgatt tggttttggg ggccaagtct gtacaacatc780 ttgagtccct ttatgccgct gttaccaatt ttcttttgtc tttgggtata catttaaacc840 ctcacaaaac aaaaagatgg ggatattccc ttaactttat gggatatgta attgggagtt900 ggggcacatt gccacaggaa catattgtac aaaaaatcaa aatatgtttt aggaaacttc960 ctgtaaacag gcctattgat tggaaagtct gtcaacgaat tgtgggtctt ttggggtttg1020 ccgccccttt cacgcaatgt ggatatcctg ctttaatgcc tttatatgca tgtatacaag1080 caaaacaggc ttttattttc tcgccaactt acaaggcctt tctgagtaaa cagtatttga1140 acctttaccc cgttgctcgg caacggcctg gtctgtgcca agtgtttgct gacgcaaccc1200 ccactggttg gggcttggcc ataggccatc agcgcatgcg tggcaccttt gtgtctcctc1260 tgccgatcca tactgcggaa ctcctagccg cttgttttgc tcgcagcagg tctggggcaa1320 aactcatcgg gactgacaat tctgtcgtgc tctcccgcaa gtatacatca tttccatggc1380 tgctaggctg tgctgccaac tggatcctgc gcgggacgtc ctttgtttac gtcccgtcgg1440 cgctgaatcc cgcggacgac ccctcccggg gccgcttggg gctctaccgc ccgcttctcc1500 gcctgttgta ccgaccgacc acggggcgca cctctcttta cgcggactcc ccgtctgtgc1560 cttctcatct gccggaccgt gtgcacttcg cttcacctct gcacgtcgca tggaaaccac1620 cgtgaacgcc cacaggaacc tgcccaaggt cttgcataag aggactcttg gactttcagc1680 aatgtcaacg accgaccttg aggcatactt caaagactgt gtgtttactg agtgggagga1740

- 33 037576 gttgggggag gaggttaggt taatgatctt tgtactagga ggctgtaggc ataaattggt1800 gtgttcacca gcaccatgca actttttcac ctctgcctaa tcatctcatg ttcatgtcct1860 actgttcaag cctccaagct gtgccttggg tggctttggg gcatggacat tgacccgtat1920 aaagaatttg gagcttctgt ggagttactc tcttttttgc cttctgactt ctttccttct1980 attcgagatc tcctcgacac cgcctctgct ctgtatcggg aggccttaga gtctccggaa2040 cattgttcac ctcaccatac ggcactcagg caagctattc tgtgttgggg tgagttaatg2100 aatctagcca cctgggtggg aagtaatttg gaagatccag catccaggga attagtagtc2160 agctatgtca acgttaatat gggcctaaaa atcagacaac tattgtggtt tcacatttcc2220 tgtcttactt ttgggagaga aactgttctt gaatatttgg tgtcttttgg agtgtggatt2280 cgcactcctc ccgcatatag accgccaaat gcccctatct tatcaacact tccggaaact2340 actgttgtta gacgaagagg caggtcccct agaagaagaa ctccctcgcc tcgcagacga2400 aggtctcaat cgccgcgtcg cagaagatct caatctcggg aatctcaatg ttagtattcc2460 ttggacacac aaggtgggaa actttacggg gctttattct tctacggtac cttgctttaa2520 tcctaaatgg caaactcctt cttttcctga cattcatttg caggaggaca ttgttgatag2580 atgtaagc aa tttgtggggc cccttacagt aaatgaaaac aggagactta aattaattat2640 gcctgctagg ttttatccca atgttactaa atatttgccc ttagataaag ggatcaaacc2700 gtattatcca gagtatgtag ttaatcatta cttccagacg cgacattatt tacacactct2760 ttggaaggcg gggatcttat ataaaagaga gtccacacgt agcgcctcat tttgcgggtc2820 accatattct tgggaacaag atctacagca tgggaggttg gtcttccaaa cctcgaaaag2880 gcatggggac aaatctttct gtccccaatc ccctgggatt cttccccgat catcagttgg2940 accctgcatt caaagccaac tcagaaaatc cagattggga cctcaacccg cacaaggaca3000 actggccgga cgccaacaag gtgggagtgg gagcattcgg gccagggttc acccctcccc3060 atgggggact gttggggtgg agccctcagg ctcagggcct actcacaact gtgccagcag3120 ctcctcctcc tgcctccacc aatcggcagt taggaaggca gcctactccc ttatctccac3180 ctctaaggga cactcatcct caggccatgc agtgg 3215

SEQ ID NO: 2 aactccacca ctttccacca aactcttcaa gatcccagag tcagggccct gtactttcct60 gctggtggct ccagttcagg aacagtgagc cctgctcaga atactgtctc tgccatatcg120 tcaatcttat cgaagactgg ggaccctgta ccgaacatgg agaacatcgc atcaggactc180 ctaggacccc tgctcgtgtt accggcgggg ttttccttgt tgacaaaaat cctcacaata240 ccacagagtc tagactcgtg gtggacttct ctcagttttc tagggggaac acccgtgtgt300 cgtggccaaa attcgcagtc ccaaatctcc agtcactcac caacctgttg tcctccaatt360 tgtcctggtt atcgctggat gtgtctgcgg cgttttatca tattcctctg catcctgctg420 ctatgcctca tcttcttgtt ggttcttctg gactatcaag gtatgttgcc cgtttgtcct480 ctaattccag gatcatcaac aaccagcacc ggaccatgca aaacctgcac gactcctgct540 caaggaacct ctatgtttcc ctcatgttgc tgtacaaaac ctacggacgg aaactgcacc600 tgtattccca tcccatcatc ttgggctttc gcaaaattcc tatgggagtg ggcctcagtc660 cgtttctcat ggctcagttt actagtgcca tttgttcagt ggttcgtagg gctttccccc720 actgtctggc tttcagttat gtggatgatt tggttttggg ggccaagtct gtacaacatc780 ttgagtccct ttatgccgct gttaccaatt ttcttttgtc tttgggtata catttaaacc840 ctcacaaaac aaaaagatgg ggatattccc ttaacttcat gggatatgta attgggagtt900 ggggcacatt gccacaggaa catattgtac aaaaaatcaa aatgtgtttt aggaaacttc960

- 34 037576 ctgtaaacag gcctattgat tggaaagtct gtcaacgaat tgtgggtctt ttggggtttg1020 ccgccccttt cacgcaatgt ggatatcctg ctttaatgcc tttatatgca tgtatacaag1080 caaaacaggc ttttattttc tcgccaactt acaaggcctt tctgagtaaa cagtatctga1140 acctttaccc cgttgctcgg caacggcctg gtctgtgcca agtgtttgct gacgcaaccc1200 ccactggttg gggcttggcc ataggccatc agcgcatgcg tggaaccttt gtgtctcctc1260 tgccgatcca tactgcggaa ctcctagccg cttgttttgc tcgcagcagg tctggggcaa1320 aactcatcgg gactgacaat tctgtcgtgc tctcccgcaa gtatacatca tttccatggc1380 tgctaggctg tgctgccaac tggatcctgc gcgggacgtc ctttgtttac gtcccgtcgg1440 cgctgaatcc cgcggacgac ccctcccggg gccgcttggg gctctaccgc ccgcttctcc1500 gcctgttgta ccgaccgacc acggggcgca cctctcttta cgcggactcc ccgtctgtgc1560 cttctcatct gccggaccgt gtgcacttcg cttcacctct gcacgtcgca tggaaaccac1620 cgtgaacgcc cactggaacc tgcccaaggt cttgcataag aggactcttg gactttcagc1680 aatgtcaacg accgaccttg aggcatactt caaagactgt gtgttcaatg agtgggagga1740 gttgggggag gagtttaagt taatgatctt tgtactagga ggctgtaggc ataaattggt1800 gtgttcac ca gcaccatgca actttttcac ctctgcctaa tcatctcttg ttcatgtcct1860 actgttcaag cctccaagct gtgccttggg tggctttagg gcatggacat tgacacgtat1920 aaagaatttg gagcttctgt ggaattactc tcttttttgc cttctgactt ctttccttct1980 attcgagatc tcctcgacac cgccactgct ctgtatcggg aggccttaga gtctccggaa2040 cattgttcac ctcaccatac ggcactcagg caagctattc tgtgttgggg tgagttaatg2100 aatctagcca cctgggtggg aagtaatttg gaagatcaag catccaggga tttagtagtc2160 ggctatgtca acgttaatat gggcctaaaa ctcagacaac tattgtggtt tcacatttcc2220 tgtcttactt ttggaagaga aactgttctt gaatatttgg tgtcttttgg agtgtggatt2280 cgcactcctc ccgcatatag accgccaaat gcccctatct tatcaacact tccggaaact2340 actgttgtta gacgaagagg caggtcccct agaagaagaa ctccctcgcc tcgcagacga2400 aggtctcaat cgccgcgtcg cagaagatct aaatctcggg aatctcaatg ttagtattcc2460 ttggacacac aaggtgggaa actttacggg gctttattct tctacggtac cttgctttaa2520 tcctaaatgg caaactcctt cttttcctga cattcatttg caggaggaca ttgttgatag2580 atgtaagcaa tttgtggggc cccttacagt aaatgaaaat aggagactta aattaattat2640 gcctgctagg ttttatccca atgtta ctaa atatttgccc ttagataaag ggatcaaacc2700 gtattatcca gagtatgtag ttgatcatta cttccagacg cgacattatt tacacactct2760 ttggaaggcg gggatcttat ataaaagaga gtccacacgt agcgcctcat tttgcgggtc2820 accatattct tgggaacaag atctacagca tgggaggttg gtcttccaaa cctcgaaaag2880 gcatggggac aaatctttct gtccccaatc ccctgggatt cttccccgat catcagttgg2940 accctgcatt caaagccaac tcagaaaatc cagattggga cctcaacccg tacaaggaca3000 actggccgga cgccaacaag gtgggagtgg gagcattcgg gccagggttc acccctcccc3060 atgggggact gttgggttgg agccctcagg ctcagggtct actcacaact gtgccagcag3120 ctcctcctcc tgcctccacc aatcggcagt taggaaggca gcctactccc ttatctccac3180 ctctaaggga cactcatcct caggccatac agtgg 3215

SEQ ID NO: 5 aactccacca ctttccacca aactcttcaa gatcccagag tcagggccct gtactttcct60 gctggtggct ccagttcagg aacagtgagc cctgctcaga atactgtctc tgccatatcg120 tcaatcttat cacatagct1 agcga

- 35 037576 ctaggacccc tgctcgtgtt accggcgggg ttttccttgt tgacaaaaat cctcacaata 240 ccacagagtc tagactcgtg gtggacttct ctcagttttc tagggggaac acccgtgtgt300 cgtggccaaa attcgcagtc ccaaatctcc agtcactcac caacctgttg tcctccaatt360 tgtcctggtt atcgctggat gtgtctgcgg cgttttatca tattcctctg catcctgctg420 ctatgcctca tcttcttgtt ggttcttctg gactatcaag gtatgttgcc cgtttgtcct480 ctaattccag gatcatcaac aaccagcacc ggaccatgca aaacctgcac gactcctgct540 caaggaacct ctatgtttcc ctcatgttgc tgtacaaaac ctacggacgg aaactgcacc600 tgtattccca tcccatcatc ttgggctttc gcaaaattcc tatgggagtg ggcctcagtc660 cgtttctcat ggctcagttt actagtgcca tttgttcagt ggttcgtagg gctttccccc720 actgtctggc tttcagttat gtggatgatt tggttttggg ggccaagtct gtacaacatc780 ttgagtccct ttatgccgct gttaccaatt ttcttttgtc tttgggtata catttaaacc840 ctcacaaaac aaaaagatgg ggatattccc ttaacttcat gggatatgta attgggagtt900 ggggcacatt gccacaggaa catattgtac aaaaaatcaa aatgtgtttt aggaaacttc960 ctgtaaacag gcctattgat tggaaagtct gtcaacgaat tgtgggtctt ttggggtttg1020 ccgccccttt cacgcaatg t ggatatcctg ctttaatgcc tttatatgca tgtatacaag1080 caaaacaggc ttttattttc tcgccaactt acaaggcctt tctgagtaaa cagtatctga1140 acctttaccc cgttgctcgg caacggcctg gtctgtgcca agtgtttgct gacgcaaccc1200 ccactggttg gggcttggcc ataggccatc agcgcatgcg tggaaccttt gtgtctcctc1260 tgccgatcca tactgcggaa ctcctagccg cttgttttgc tcgcagcagg tctggggcaa1320 aactcatcgg gactgacaat tctgtcgtgc tctcccgcaa gtatacatca tttccatggc1380 tgctaggctg tgctgccaac tggatcctgc gcgggacgtc ctttgtttac gtcccgtcgg1440 cgctgaatcc cgcggacgac ccctcccggg gccgcttggg gctctaccgc ccgcttctcc1500 gcctgttgta ccgaccgacc acggggcgca cctctcttta cgcggactcc ccgtctgtgc1560 cttctcatct gccggaccgt gtgcacttcg cttcacctct gcacgtcgca tggaaaccac1620 cgtgaacgcc cactggaacc tgcccaaggt cttgcataag aggactcttg gactttcagc1680 aatgtcaacg accgaccttg aggcatactt caaagactgt gtgttcaatg agtgggagga1740 gttgggggag gagtttaagt taatgatctt tgtactagga ggctgtaggc ataaattggt1800 gtgttcacca gcaccatgca actttttcac ctctgcctaa tcatctcttg ttcatgtcct1860 actgttcaag cctccaagct gtgccttggg tggcttt agg gcatggacat tgacacgtat1920 aaagaatttg gagcttctgt ggaattactc tcttttttgc cttctgactt ctttccttct1980 attcgagatc tcctcgacac cgccactgct ctgtatcggg aggccttaga gtctccggaa2040 cattgttcac ctcaccatac ggcactcagg caagctattc tgtgttgggg tgagttaatg2100 aatctagcca cctgggtggg aagtaatttg gaagatcaag catccaggga tttagtagtc2160 ggctatgtca acgttaatat gggcctaaaa ctcagacaac tattgtggtt tcacatttcc2220 tgtcttactt ttggaagaga aactgttctt gaatatttgg tgtcttttgg agtgtggatt2280 cgcactcctc ccgcatatag accgccaaat gcccctatct tatcaacact tccggaaact2340 actgttgtta gacgaagagg caggtcccct agaagaagaa ctccctcgcc tcgcagacga2400 aggtctcaat cgccgcgtcg cagaagatct aaatctcggg aatctcaatg ttagtattcc2460 ttggacacac aaggtgggaa actttacggg gctttattct tctacggtac cttgctttaa2520 tcctaaatgg caaactcctt cttttcctga cattcatttg caggaggaca ttgttgatag2580 atgtaagcaa tttgtggggc cccttacagt aaatgaaaat aggagactta aattaattat2640 gcctgctagg ttttatccca atgttactaa atatttgccc ttagataaag ggatcaaacc2700 gtattatcca gagtatgtag ttgatcatta cttccagacg cgacattatt tacac actct2760 ttggaaggcg gggatcttat ataaaagaga gtccacacgt agcgcctcat tttgcgggtc2820 accatattct tgggaacaag atctacagca tgggaggttg gtcttccaaa cctcgaaaag2880 gcatggggac aaatctttct gtccccaatc ccctgggatt cttccccgat catcagttgg2940 accctgcatt caaagccaac tcagaaaatc cagattggga cctcaacccg tacaaggaca3000 actggccgga cgccaacaag gtgggagtgg gagcattcgg gccagggttc acccctcccc3060 atgggggact gttgggttgg agccctcagg ctcagggtct actcacaact gtgccagcag3120 ctcctcctcc tgcctccacc aatcggcagt taggaaggca gcctactccc ttatctccac3180 ctctaaggga cactcatcct caggccatac agtgg 3215

Cell culture.

HepG2 cells were obtained from the American Type Structure Collection (Manassas, VA, USA; ATCC, Cat # HB-8065) and stored in humidified incubators at 37 ° C and 5% CO ₂ in complete medium containing Dulbecco's modified Eagle's Medium (DMEM) (Fisher Scientific (Life Technologies); Waltham, MA, USA; Cat # 11995-065), 10% Fetal Bovine Serum (FBS) (Life Technologies, Cat # 10082-147), 100 U / ml penicillin, 10 μg / ml streptomycin and 0.25 μg / ml fungizone (Life Technologies Cat # 15240-062).

Transient transfection.

HepG2 cells were seeded in collagen-coated 96-well plates (BIOCOAT ™; Fisher Scientific, Cat # 354407) at 20,000 cells / well and fixed overnight at 37 ° C and 5% CO ₂ . Cells were co-transfected with HBV plasmids (100 ng / well)

- 36 037576 and Gaussia Plasmid Expression (10 ng / well) (THERMO SCIENTIFIC ™, Fisher Scientific, Cat # 16148) using Lipofectamine LTX Plus transfection reagent according to the manufacturer's recommendations (Life Technologies, Cat # 15338-100). The transfected mixtures were separated the next day, the cells were washed twice with complete medium and treated with serially diluted compounds at a final concentration of dimethyl sulfoxide (DMSO) of 0.5%. Cells were incubated with compounds for three days, after which intracellular HBV DNA was extracted from the cells and levels of secreted Gaussian luciferase from the medium were determined using the Gaussia Flash Luciferase assay kit (THERMO SCIENTIFIC ™, Fisher Scientific, Cat # 16158). To extract HBV intracellular DNA, cells were washed once with 100 μl Dulbecco's PBS (Life Technologies, Cat # 14190-144) and lysed with 0.33% NP-40 (THERMO SCIENTIFIC ™, Fisher Scientific, Cat # 85124) with incubation for 30 min at room temperature (110 μl / well). Turbo DNase was prepared by 5-fold dilution in Turbo DNase buffer (Life Technologies, Cat # AM2238), and S7 nuclease (Roche Cat # 10-107-921-001, available from Sigma-Aldrich; St. Louis, State Missouri, USA) was prepared by 50-fold dilution in CUTSMART® buffer solution (New England Bioloabs; Ipswich, MA, USA; Cat # B7204S) containing 25 μM CaCl ₂ (GBiosciences; St. Louis, MO , USA; Cat. No. R033). The nuclei were granulated by centrifugation and the supernatant (35 μl) was transferred to a fresh 96-well plate and treated with 2 units of Turbo DNase and 10 units of S7 nuclease at 37 ° C for 60 min, after which the enzyme was inactivated at 75 ° C for 15 min. Encapsulated HBV DNA was diluted with 60 μl molecular biology water (GBiosciences Cat # 786-293) and extracted by incubation in 50 μl lysis buffer (Affymetrix, Cat # QS0010) containing 2.5 μg protease K (Affymetrix ; Santa Clara, California, USA; Cat. No. 14600) at 50 ° C for 40 minutes. HBV DNA was denatured for 30 min at 25 ° C with the addition of 2.5 M NaOH (Sigma, Cat # S5881) to a final concentration of 0.2 M in the presence of 1 μL of HBV DNA probes (Affymetrix, Cat # SF-10326 ). Denatured DNA was neutralized by the addition of 2 M HEPES (Sigma, Cat # H3375) to a final concentration of 0.3 M and recorded using a QuantiGene assay kit (Affymetrix, Cat # QS0010). The average background signal from the wells containing only culture medium was subtracted from all other samples and the percentage inhibition at each concentration of the compound was calculated by normalization from the signals from cells treated with DMSO 0.5% using equation E1:

(E1):% inhibition = (DMSO _cf - X ₁ ) / DMSO _cfx 100%, where DMSO _cf is the mean signal calculated in wells treated with DMSO control (control with 0% inhibition);

Xi is the signal measured in individual wells.

EC ₅₀ values, i.e. the effective concentrations that provided an inhibitory effect in 50% of cases were determined by nonlinear fitting using Graphpad Prism software (San Diego, California, USA) and equation E2:

(E2): Y = Ym _m + (Ymax - Ymln) / (1 + 10 ⁽ ^ ^Ε “ ^{0-Χ) χΗ3ΚΛΟΗ} ), where Y represents the percent inhibition values;

X is the logarithm of the compound concentrations.

To determine replication-competent HBV variants, background corrected HBV DNA values from the QuantiGene assay were normalized using Gaussian luciferase activity values to account for any differences in transfection efficiency. The normalized HBV DNA values obtained from the analysis of cells transfected with HBV variants were then compared with the values obtained from the wild type HBV transfection, with the replication competence of the wild type HBV being set to 100%.

Example 1.2. Relative replication competence of nucleoside-resistant HBV variants.

Five HBV expression plasmids were constructed, representing a panel of nucleoside-resistant HBV variants most commonly observed in HBV-infected patients receiving nucleoside drugs. The nucleoside resistant HBV panel includes five HBV variants with the following single, double or triple mutations in the HBV polymerase protein: (1) L180M / M204V, (2) N236T, (3) A181V, (4) A181V / N236T and (5 ) L180M / M204V / N236T. A181V, N236T and A181V / N236T were introduced into the HBV DNA backbone obtained from a clinical isolate of genotype B (Genbank AY220698). Sequencing of the A181V, N236T and A181V / N236T variants confirmed the proposed amino acid changes in the wild-type HBV B genome construct. To introduce a triple mutation, N236T was introduced into the HBV DNA backbone obtained from an LMV-resistant clinical isolate (Genbank AY220697; SEQ ID NO: 5 (see Table 3)). It has previously been reported that the LMV-resistant clinical isolate contained a number of additional amino acid changes within the HBV genome as compared to the isolate obtained prior to LMV treatment (Zhang J.M. et al. 2005. J. Med. Virol 77: 203-208). Sequencing of L180M / M204V variants and

- 37 037576

L180M / M204V / N236T confirmed an additional amino acid change at position 271 within the reverse transcriptase domain, which was consistent with a previously published sequence from an LMV resistant clinical isolate (Genbank AY220697; SEQ ID NO: 5).

The listed plasmids were used to transfect HepG2 cells, and the amount of intracellular encapsulated HBV DNA that was generated by HBV replication was determined 3 days after transfection. The Gaussia luciferase expression plasmid was co-transfected with HBV (Gaussia-Luc: HBV, 1:10) to allow any differences in transfection efficiency to be normalized. The normalized HBV DNA signal obtained in cells transfected with wild-type HBV corresponded to 100% relative replication competence. The N236T variant showed close replication competence to wild-type HBV, while the replication competence of the other four variants showed changes from close to twice as high as that of the wild-type. The replication competence of all HBV variants was acceptable for antiviral assay using nucleoside analogs and Compound 1.

Example 1.3. The HBV variants were resistant to nucleoside analogs, but remained susceptible to compound 1.

HBV variants with nucleoside resistance mutations were first evaluated in terms of their sensitivity to the inhibitory effect of nucleoside analogs. As expected from published data (Yang et al. 2005. Antivir Ther 10: 625-633; Brunelle et al. 2005. Hep 41: 1391-1398), both HBV variants rtL180M / M204V and rtL180M / M204V / N236T were resistant with respect to inhibition by lamivudine (LMV) and entecavir (ETV): LMV inhibited wild-type HBV with a mean EC50 of 0.53 μM, but did not inhibit replication of either of the two variants up to the highest LMV concentrations tested (100 μM), whereas the antiviral activity of ETV was reduced by 31 and 14 times compared to the rtL180M / M204V and rtL180M / M204V / N236T variants, respectively (Fig. 1 and Table 4).

Table 4

Antiviral Activity of Compound 1, LMV, ETV, and TDF in HepG2 Cells Transiently Transfected with Nucleoside Resistant Variants

Multiplicity of change Compound WT EC ₅₀ [μM] rtL 180M / M204V rtL 180M / M204V / N236T rtA181V rtN236T rtA181V / N236T LMV 0.53 ± 0.12 > 190 > 190 1.7 ± 0.9 ^{ns 1.0} ± 0.5 ns 4, 8 ± 2, Z ^a ETV 0.0014 ± 0.0004 31 ± 16 ^a 14 ± 4 ^a 2.2 ± 0.5 ^a 0.67 ± 0.22 ^ns 1.8 ± ^{0.6 ns} TDF 0.032 ± 0.015 1.1 ± 0.3 ^ns 2.9 ± 1.5 ^b 1.4 ± 0.05 ^ns 2.2 ± 1.0 ^b 2.8 ± 1.4 ^b Conn. one 0.31 ± 0.10 1.3 ± 0.6 ^ns 1.4 ± 0.5 ^ns 0.82 ± ^{0.19 ns} 0.85 ± ^{0.40 ns} 0.85 ± 0.2 6 ^ns

EC ₅₀ and fold are given as mean ± standard deviation (SD) from at least three independent studies.

* The mean fold change and SD fold change were calculated from the individual fold change values of the mutant variants compared to the mean EC ₅₀ for the wild-type variant.

^a Compared to wild-type, p-value in the t-test <0.01.

^b P-value in the t-test <0.05.

^ns The p-value in the t-test> 0.05.

Tenofovir disoproxil fumarate (TDF) showed similar antiviral activity against wild-type HBV and rtL180M / M204V and rtA181V variants (mean EC ₅₀ values of 0.032, 0.034 and 0.043 μM, respectively), but showed a mean decrease in antiviral activity in the range of 2.2 up to 2.9 times compared to HBV variants containing the rtN236T mutation both in single form and in combination with rtL180M / M204V or rtA181V (Fig. 1; Table 4). Such relative fold changes associated with the N236T mutation were similar to previously published fold change values (Delaney et al. 2006. Antimicrob Agents Chemother 50: 2471-2477).

HBV containing the rtN236T mutation retained sensitivity to inhibition by LMV and ETV, similar to wild-type HBV (Table 4). A slight increase in the mean EC ₅₀ values (about 2 times) was observed for LMV and ETV when tested in relation to the rtA181V variant (Table 4). The combination of mutations from rtN236T to rtA181V in the double mutant variant resulted, on average, in increases in

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4.8 and 1.8 times the EC50 values for LMV and ETV, respectively (Table 4).

All five nucleoside resistant HBV variants were susceptible to inhibition by Compound 1 with antiviral EC ₅₀ values close to wild type HBV. Mean fold EC50 changes ranged from 0.82 to 1.4 fold, indicating that no cross resistance to the HBV capsid inhibitor Compound 1 occurred in the nucleoside resistance mutations tested herein (Table 4).

As shown in this example, phenotyping assays using HepG2 cells transiently transfected with nucleoside analogs demonstrated the potential for cross-resistance between different classes of nucleoside analogs, including LMV, ETV, and TDF. In contrast, compound 1 retained activity against nucleoside-resistant variants, and therefore did not show cross-resistance with other nucleoside analogs.

Example 2. Viability of primary human hepatocytes in the presence of compound 1 and separate nucleoside analogs or their combination.

In this example, the viability of primary human hepatocytes (PHN) was determined in the presence of compound 1 and individual nucleoside analogs or a combination thereof. Cell viability was determined by the relative concentration of intracellular adenosine triphosphate (ATP) in cells treated with one or another compound compared with untreated cells.

PHN viability was similar in untreated cells and cells treated with either 300 μM lamivudine (LMV), 30 μM tenofovir (TFV), or 30 μM entecavir (ETV). In cells treated with Compound 1, a dose-dependent decrease in cell viability was noted; mean CC50 values ranged from 16 to 82 μM. The CC ₅₀ values obtained for compound 1 in the presence of 300 μM lamivudine (LMV), 30 μM tenofovir (TFV) or 30 μM entecavir (ETV) were similar to those obtained for cells treated with compound 1 only.

Example 2.1. Materials and methods.

Connections.

Compound 1 was obtained by synthesis. Lamivudine, tenofovir and entecavir were purchased from Toronto Research Chemicals (Toronto, Canada).

Cell culture.

Cryopreserved primary human hepatocytes from individual donors (primary hepatocyte identifiers HuM4038, HuM4055A and HuM4059) were purchased from Triangle Research Labs (TRL; Research Triangle Park, NC, USA). Cells were thawed using Hepatocyte Thawing Medium (TRL, Cat # MCHT50) according to the manufacturer's recommendations. After centrifugation, the cells were resuspended in an enriched hepatocyte seeding medium (TRL, cat. No. MP250). Cells were plated on 96-well collagen type I plates (Corning; Corning, NY, USA; Cat # 356407) at a density of 40,000 cells per well and kept in humidified incubators at 37 ° C and 5% CO ₂ overnight before adding test compounds.

Cell Viability Assays.

Three 96-well plates were prepared for each donor to evaluate the effect of increasing the concentration of Compound 1 on hepatocyte viability, either alone or in combination with nucleoside analogs. In each plate, cells were incubated with compound 1 separately in duplicate or with compound 1 in the presence of LMV, TFV, or ETV in triplicate. The individual effect of nucleoside analogs on the viability of hepatocytes was also determined in triplicate for each donor. Compound 1 was semi-log diluted in DMSO (Sigma, Cat # D2650) and added to primary human hepatocytes individually or in combination with LMV (30 and 300 μM), TFV (30 μM) or ETV (30 μM). Individual preparations or their combinations were added to primary human hepatocytes at a final concentration of DMSO (Sigma D2650) of 0.5% for the entire concentration range. The cells were incubated with compounds for three days, after which the medium was removed and fresh medium containing the compounds was added and incubated for another three days. Primary human hepatocytes treated with 0.5% DMSO were used as a control without added compounds, and the values obtained were then used to determine the 0% inhibition level. The background signal was determined as the average value for wells containing only culture medium. Cell viability was monitored using the CellTiter-Glo Cell Viability Reagent according to the manufacturer's protocol (Promega; Madison, Wis., USA; G7573). A chemiluminescence signal proportional to the amount of cellular ATP was measured using a Victor X4 plate reader (Perkin Elmer; Waltham, MA, USA). The average background signal from the wells containing only the medium was subtracted from all other samples and the percentage inhibition was calculated using the equation E1 (as in example 1):

(E1):% inhibition = (DMSO _cf - X ₁ ) / DMSO _cf x 100%, where DMSO _cf is the mean signal calculated in the wells that were treated

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0.5% DMSO control (control with 0% inhibition);

Xi is the signal measured in individual wells.

_{CC 50} values were determined from% inhibition data obtained at various concentrations of compounds with non-linear processing using Graphpad Prism software and equation E3 in cases where the% inhibition values exceeded 50% at the highest concentration tested:

(E3): Y = Ym, + (Y _max -Ymi „) / (1 + 10 g ').

where Y represents the percent inhibition values;

X is the logarithm of the compound concentrations.

Example 2.2. Effect of Compound 1 or Nucleoside Analogs on Cell Viability Using Primary Human Hepatocytes.

Primary human hepatocytes from three different donors (TRL HuM4038, HuM4055A and HuM4059) were incubated with increasing concentrations of Compound 1. Cells from the same three donors were also incubated with LMV (30 and 300 μM), TFV (30 μM) or ETV (30 μM) ... Cell viability assays were based on intracellular ATP levels 6 days after drug treatment. A concentration-dependent decrease in cell viability was observed upon incubation of hepatocytes with compound 1: CC ₅₀ values for compound 1 ranged from 16 to 82 μM (Table 3). The previously noted CC50 values for compound 1 using fresh (Bioreclamation IVT) and cryopreserved primary hepatocytes (TRL donors HuM4038 and Invitrogen Hu1457) ranged from 14 to 27 μM. There was no decrease in cell viability when hepatocytes were treated with 30 or 300 µM LMV, 30 µM TFV, or 30 µM ETV (Table 5).

Table 5

Effect of Compound 1, LMV, TFV, or ETV on Cell Viability for Primary Human Hepatocytes

Compound HuM4038 CC50 [μM] NiM4038 CC50 [μM] NiM4055A CC50 [μM] HuM4059 CC50 [μM] Conn. one sixteen 26 nineteen 82 LMV > 30 > 300 > 300 > 300 TFV > 30 > 30 > 30 > 30 ETV > 30 > 30 > 30 > 30

Example 2.3. Combined effect of the action of compound 1 with nucleoside analogs on cell viability using primary human hepatocytes.

To determine the effect of combining Compound 1 and nucleoside analogs on cell viability, primary human hepatocytes were treated with increasing concentrations of Compound 1 in combination with fixed concentrations of LMV (300 μM), TFV (30 μM), or ETV (30 μM). As shown in FIG. 2-4, the presence of LMV, TFV, or ETV did not affect the dose response profiles for Compound 1 when tested for three different donors. The corresponding CC50 values for compound 1 were similar in the presence and absence of nucleoside analogs (Tables 6-8).

Table 6

Effect of Compound 1 alone and in combination with 300 μM LMV on the viability of primary human hepatocytes

Identifier donor Conn. (I) CC50 [μM] Conn. (D + LMV CC50 [μM] Change rate CC50 mono / combination NiM4038 26 22 1/2 HUM4055A nineteen nineteen 1 / o HUM4059 83 83 1 / o

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Table 7

Effect of Compound 1 alone and in combination with 30 μM TFV on the viability of primary human hepatocytes

Multiplicity

Identifier Conn. (I) CC50 Conn. (I) + TFV change CC50

donor [μM] CC50 [μM] mono / combination NiM4038 sixteen 23 0.7 HUM4055A nineteen nineteen 1.0 NiM4059 83 80 1.0

Table 8

Effect of Compound 1 alone and in combination with 30 μM ETV on the viability of primary human hepatocytes

Identifier donor Conn. (I) CC50 [μM] Conn. (D + ETV CC50 [μM] Change rate CC50 mono / combination HUM4038 sixteen fourteen 1.1 HUM4055A nineteen 21 0, 9 HUM4059 83 81 10

In this example, the viability of primary human hepatocytes (PHN) was determined in the presence of compound 1 and individual nucleoside analogs or a combination thereof. Cell viability was determined by the relative concentration of intracellular ATP in cells treated with one or another compound compared to untreated cells. PHN viability was similar in untreated cells and cells treated with either 300 μM lamivudine (LMV), 30 μM tenofovir (TFV), or 30 μM entecavir (ETV). In cells treated with Compound 1, a dose-dependent decrease in cell viability was noted; average values CC ₅₀ ranged from 16 to 82 .mu.M. The CC50 values obtained for Compound 1 in the presence of 300 μM lamivudine (LMV), 30 μM tenofovir (TFV), or 30 μM entecavir (ETV) were similar to those obtained for cells treated with Compound 1 alone.

Example 3. Effect of a combination of HBV capsid inhibitor compound 1 with nucleoside analogs or other HBV capsid inhibitors on inhibition of HBV DNA replication in HepG2.2.15 cells.

In the present example, the additivity of the combination of the HBV capsid modulator Compound 1 with LMV was demonstrated by the analysis using MacSynergy and CalcuSyn. The combination of Compound 1 with TFV or ETV showed an additive effect as shown by MacSynergy, as well as minor to moderate synergy as measured by CalcuSyn analysis. The combination of two different HBV capsid inhibitors showed overall additive antiviral activity. Cell viability was maintained above 85% in all samples treated with the highest concentrations of the compound, both individually and in combination.

Example 3.1. Materials and methods.

HepG2.2.15 cells were treated with increasing concentrations of Compound 1 (0.05-5 μM) in combination with increasing concentrations of either lamivudine (LMV), tenofovir (TFV), entecavir (ETV), or Bay 41-4109 (0.01-5 μM) within six days. Secreted HBV DNA was determined by Quantigene assay and cell viability was assessed by CellTiter-glo assay. The synergistic effect was measured using the MacSyngergy assay and the Calcusyn assay.

Example 3.2. Results.

Figure 7 shows the effect of Compound 1 in combination with nucleoside analogs. It also shows synergistic plots with 95% confidence intervals for MacSynergy assay using three different assay plates for HepG2.2.15 cells treated with Compound 1 in combination with LMV (FIG. 7A), TFV (FIG. 7B), or ETV of FIG. 7C).

Table 9 shows the volumes of synergy / antagonism for compound 1 in combination with nucleoside analogs with predicted MacSynergy effect. Synergy / antagonism volumes with a confidence interval of 95% <25 μM 2% were considered insignificant, in the range from 25 to 50 μM 2% - small, from 50 to 100 μM 2% - moderate and> 100 μM 2% were considered as significant synergy / antagonism ... Compound 1 has been shown to show additive effects in combination with LMV, TFV, or ETV using MacSynergy.

- 41 037576

Table 9

Combination of compound 1 and Synergy (μM ² %) Antagonism (μM ² %) MacSynergy predicted effect LMV 5, 1 -14.3 Additive TFV 18.5 -7.3 Additive ETV 10 -16.5 Additive

Table 10 shows the values of the additivity index (CI) for compound 1 in combination with nucleoside analogs. The results show that Compound 1 in combination with LMV shows an additive effect when using CalcuSyn, as well as little to moderate synergy when Compound 1 is combined with TFV or ETV.

Table 10

CI values Combination of compound 1 and ED50 ED75 ED90 CI Cumulative CalcuSyn predicted effect LMV 10 0, 8 0, 8 0.9 ± 0.1 Additive TFV 0, 8 0, 8 0, 8 0.8 + 0.06 Minor to moderate synergy ETV 1.0 0.5 0.5 0.7 ± 0.4 Minor to moderate synergy

FIG. 8 shows the effect of Compound 1 in combination with other capsid modulators. MacSynergy synergistic plots with 95% confidence intervals for three different plates of HepG2.2.15 cells treated with Compound 1 in combination with Bay 41-4109 (FIG. 8).

Table 11 shows the amounts of synergy / antagonism for compound 1 in combination with another capsid modulator. Compound 1 showed an additive effect in combination with Bay 41-4109 (Formula B) when using MacSynergy.

Table 11

Combination of compound 1 and Synergy (μM ² %) Antagonism (μM ² %) MacSynergy predicted effect Wow 41-4109 2,3 -3, 9 Additive

Table 10 shows the CI values for Compound 1 in combination with another capsid modulator Bay 41-4109, which shows that the combination exhibits an additive effect as predicted using CalcuSyn.

Table 12

CI values Combination of compound 1 and ED50 ED75 ED90 CI Cumulative CalcuSyn predicted effect Wow 41-4109 eleven eleven eleven 1.1 + 0.1 Additive

Example 4. Effect of a combination of HBV capsid inhibitor compound 2 and compound 3 with nucleoside analogs on the inhibition of HBV DNA replication in HepG2.2.15 cells.

In the present example, an additive to synergistic effect was demonstrated for the combination of the HBV capsid modulator Compound 2 or Compound 3 with TFV or ETV in a MacSynergy assay.

Example 4.1. Materials and methods.

The anti-HBV activity of combinations of Compound 2 and Compound 3 with nucleos (t) ide analogs ETV or TFV was assessed in a 6-day anti-HBV antiviral assay using quantitative PCR to detect HBV DNA in cell culture supernatant as an indicator. ... The effect of the combination was analyzed using Mac Synergy II software. In addition, the activity of anti-HBV ETV and TFV was determined when tested as individual agents in HepG2.2.15 cells (Table 16).

In the process of testing the antiviral activity, HepG2.2.15 cells were cultured in RPMI1640 medium and the amount of FBS was reduced to 2%. Cells were plated at a density of 50,000 cells / well in a 96-well plate.

One day after plating HepG2.2.15 cells, the supernatant was removed and added to the cells with 200 μl of medium with test compounds diluted in a checkerboard pattern. After three days, the test compound medium was renewed and the cells were incubated with the compound for an additional three days. Upon completion of compound treatment, 150 μl of cell culture supernatant and 50 μl of PBS were added to a 96-well DNA extraction block using MagNA Pure 96 DNA and Viral NA Small Volume kits. HBV DNA was determined using quantitative real-time PCR (quantitative PCR). HBV DNA was quantified by real-time PCR analysis using LightCycler480 Probes Master kit (Roche) with primers 5'-GTGTCTGCGGCGTTTTATCA-3 '(sens) and 5'-GACAAACGGGCAACATACCT-3' (antisense, SEQ ID NO: 7). The HBV probe 5'-CCTCTKCATCCTGCTGCTATGCCTCATC-3 '(SEQ ID NO: 8) contains a fluorescent reporter dye (FAM) at the 5' end of the probe and a quencher (TAMRA) at the 3 'end. PCR was performed as follows: by denaturation at 95 ° C for 10 min followed by 40 cycles of amplification at 95 ° C for 15 seconds and at 60 ° C for 1 min. Compound cytotoxicity testing was performed for HepG2.2.15 in parallel using the same experimental design as for the antiviral activity assay. The ATP lite kit from Perkin Elmer was used to detect ATP as a marker of the cytotoxicity of the compound used in the treatment.

The percent inhibition values obtained for each combination of compound concentration were calculated as the average of 3 to 5 replication plates for the same combination in each experiment. To reliably identify statistical outliers, the deviation from the mean for the remaining four replications was calculated for each data point. As expected, the distribution of such deviations for all data points was approximately normal and centered at zero, but with long distribution tails, indicating significant outliers. To determine cutoffs to exclude them, Tukey's outlier test was used, which sets the limits at the first quartile minus the 1.5-fold interquartile range (Q1-1.5 * (Q3-Q1)) and at the second quartile plus 1.5- multiple interquartile range (Q3 + 1.5 * (Q3-Q1)). For symmetric filtering of emissions, the maximum absolute values of such limits were used as cutoffs. Any data point for which the absolute deviation from the mean in the other 4 replications exceeded this cutoff was considered an outlier and excluded from the calculations. Removal of outliers was carried out only in experiments 2 and 3. The anti-HBV activity of various combinations of anti-HBV agents was evaluated using the Bliss-Independence model based on the algorithm proposed by Prichard and Shipman (Prichard MN, Shipman C. Jr. A three- dimensional model to analyze drug-drug interactions, Antiviral Res. 1990; 14 (4-5): 181-205), using MacSynergy ™ II software. In this model, the theoretical additive effect is calculated from the dose response curves for individual compounds according to the equation Z = X + Y x (1 - X), where X and Y reflect the inhibition achieved by drug 1 only and only drug 2, respectively, and Z reflects the effect achieved with a combination of drug 1 and drug 2. The theoretical additive surface is subtracted from the actual experimental surface, which appears as a horizontal plane at 0% inhibition if the combination was additive. Any peak above such a plane indicates synergy, and any deflection below such a surface indicates antagonism. The lower limits of the 95% confidence intervals (CI) for the experimental dose response surface were used to statistically evaluate the data. The peak or sag volume was calculated to quantify the total generated synergy or antagonism. To determine the effect of the combination, synergy and antagonism values were analyzed at 95% CI according to the Mac Synergy II manual (which can be found at http://www.uab.edu/images/pediatrics/ID/MacSynergy.pdf).

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Table 15

Concentration Ranges of Various Compounds Used in Each Experiment

Experiment No. Concentration range (nM) Compound 2 Compound 3 ETV TFV one 1000-0.24 1000-0.24 25-0.024 250-0.24 2 250-3.9 N / A 25-0.10 250-1.0 3 250-3.9 N / A 25-0.10 250-1.0

Table 16

Summary of Synergy Results for Combining Compound 2 or Compound 3 with ETV or TFV Compounds

Anti-HBV agent formulas and combinations Repeated measurements for each experiment ³ Synergy volumes (lower limit 95% CI) (μM ² %) ^b Antagonism volumes (lower limit 95% CI) (μM ² %) ^b Combination effect ⁰ Compound 2 + ETV (experiment 1) 3 0 -21.92 Minor synergy / antagonism (additivity) Compound 2 + TFV (experiment 1) 3 1.13 -10.98 Minor synergy / antagonism (additivity) Compound 2 + ETV (experiment 2) five 14.66 -2.7 Minor synergy / antagonism (additivity) Compound 2 + TFV (experiment 2) five 273.83 -0.55 Strong synergy Compound 2 + ETV five 332.45 -15.1 Strong synergy (experiment 3) Compound 2 + TFV (experiment 3) five 413.62 -6, 19 Strong synergy Compound 3 + ETV (Experiment 1) 3 79.1 -13.75 Moderate synergy Compound 3 + TFV (Experiment 1) 3 144.51 -2.61 Strong synergy

Table 17

Activity of anti-HBV ETV and TFV when tested as _______ individual agents in HepG2 cells. 2.1.15 _________

Compound Median EC50, nM No. of repeated experiment Inhibitor class ETV 0.07 2 Nucleoside analogue TFV fifteen one Nucleoside analogue

The above ATP cytotoxicity assay was performed during Experiment 1. Based on raw data (not shown), the compounds do not show toxicity in any combination.

FIG. 11A-11C and FIG. 12A-12C show the effect of Compound 2 in combination with the nucleoside analogs ETV and TFV. The graphs of synergistic effects with a confidence interval of 95% are given.

44 037576 by MacSynergy assay using three different assay plates for HepG2.2.15 cells treated with Compound 2 in combination with ETV (FIGS. 11A-11C) and TFV (FIG. 12A12C).

FIG. 13 and 14 show the effect of Compound 3 in combination with the nucleoside analogs ETV and TFV. Synergistic plots are shown with 95% confidence intervals for MacSynergy assay using three different assay plates for HepG2.2.15 cells treated with Compound 3 in combination with ETV (FIG. 13) and TFV (FIG. 14).

FIG. 15A-15D show the% Inhibition of HBV by Compound 2 in combination with the nucleoside analogs ETV and TFV. In the graph in FIG. 15A shows the% inhibition of HBV over the described concentration ranges of Compound 2 at zero ETV concentration. In the graph in FIG. 15B shows the% inhibition of HBV over the described ranges of ETV concentrations at zero concentration of compound 2. In the graph in FIG. 15C shows the% inhibition of HBV over the described concentration ranges of Compound 2 at a TFV concentration of zero. In the graph in FIG. 15D shows the% inhibition of HBV over the described TFV concentration ranges at zero compound 2 concentration.

The scope of the invention is not limited to the specific embodiments and examples described herein. In fact, various modifications of the invention will be apparent to those skilled in the art in addition to the described modifications from the foregoing description and accompanying figures. Such modifications are intended to be included within the scope of the appended claims.

All references (e.g., publications, or patents, or patent applications) cited herein are hereby incorporated by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g. publication, or patent, or patent application) has been specifically and individually indicated as being incorporated herein by reference in its entirety for all purposes. Other embodiments are included in the appended claims.

Claims (25)

CLAIM 1. A combination product having anti-HBV activity, comprising a therapeutically effective amount of an allosteric basic protein modulator (CpAM) and a therapeutically effective amount of a reverse transcriptase inhibitor, in which the allosteric basic protein modulator is a compound of formula IA or a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof, where A is phenyl; R ¹ is H or C ₁ -C ₆ alkyl; R ² is C ₁ -C ₆ alkyl optionally and independently containing one or more halogen substituents, C ₁ -C ₆ alkoxy, oxo, C ₁ -C ₆ alkyl, OH, CN, CFH ₂ , CF ₂ H or CF ₃ ; R ³ is independently for each occurrence halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, cyano, C ₁ -C ₆ alkenyl, C ₁ -C ₆ alkynyl, or OH; X is CR ⁴ ; G ³ represents H or C ₁ -C ₆ -alkyl; R ⁴ is independently for each occurrence H, halogen, C ₁ -C ₃ alkyl, or cyano; n is 0, 1, 2, or 3; an allosteric basic protein modulator is an allosteric basic protein modulator that causes the assembly of capsids that are substantially empty in terms of their viral content; wherein the reverse transcriptase inhibitor is entecavir or tenofovir, or pharmaceutically acceptable salts thereof. 2. A combination product according to claim 1, wherein the compound of formula IA is compound 2 Compound 2 or a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof. - 45 037576 3. A combination product according to claim 1 or 2, wherein the reverse transcriptase inhibitor is entecavir or a pharmaceutically acceptable salt thereof. 4. The combination product of claim 1, wherein the reverse transcriptase inhibitor is tenofovir. 5. A combination product according to any one of claims 1 to 4, wherein the CpAM of formula IA and the reverse transcriptase inhibitor are in the same formulation. 6. A combination product according to any one of claims 1 to 4, wherein the CpAM of formula IA and the reverse transcriptase inhibitor are in different formulations. 7. Use of a combination product according to any one of claims 1 to 4 for the treatment of HBV infection in a patient. 8. A pharmaceutical composition having anti-HBV activity, comprising an allosteric basic protein modulator (CpAM) and a reverse transcriptase inhibitor, wherein the allosteric basic protein modulator is a compound of formula IA or its pharmaceutically acceptable salt, hydrate or solvate thereof, where A is phenyl; R ¹ is H or C ₁ -C ₆ -αalkyl; R ² is C ₁ -C ₆ -alkyl, optionally and independently containing one or more substituents in the form of halogen, C ₁ -C ₆ -alkoxy, oxo, C ₁ -C ₆ -alkyl, OH, CN, CFH ₂ , CF2H or CF ₃ ; R ³ is independently for each occurrence halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, cyano, C ₁ -C ₆ alkenyl, C ₁ -C ₆ alkynyl, or OH; X is CR ⁴ ; G ³ represents H or C ₁ -C ₆ -alkyl; R ⁴ is independently for each occurrence H, halogen, C ₁ -C ₃ alkyl, or cyano; n is 0, 1, 2, or 3; an allosteric basic protein modulator is an allosteric basic protein modulator that causes the assembly of capsids, which are essentially empty in terms of their viral content, and a pharmaceutically acceptable carrier; wherein the reverse transcriptase inhibitor is entecavir or tenofovir, or pharmaceutically acceptable salts thereof. 9. A pharmaceutical composition according to claim 8, wherein the compound of formula IA is compound 2 or a pharmaceutically acceptable salt, hydrate or solvate thereof. 10. A pharmaceutical composition according to claim 8 or 9, wherein the reverse transcriptase inhibitor is entecavir or a pharmaceutically acceptable salt thereof. 11. The composition of claim 8, wherein the reverse transcriptase inhibitor is tenofovir. 12. Use of a pharmaceutical composition according to any one of claims 8 to 11 for the treatment of HBV infection in a patient. 13. A method of treating HBV infection in a patient in need thereof, comprising administering to the patient a combination product according to any one of claims 1-7 or a pharmaceutical composition according to any one of claims 8-12. 14. The method of claim 13, comprising administering to the patient a therapeutically effective amount of the combination product of claim 1 or the pharmaceutical composition of claim 8. 15. The method of claim 14, wherein the compound of formula IA is compound 2 or a pharmaceutically acceptable salt, hydrate or solvate thereof. 16. The method according to any one of claims 13-15, wherein the reverse transcriptase inhibitor is enteca - 46 037576 virom or a pharmaceutically acceptable salt thereof. 17. The method according to any one of claims 13-16, wherein the CpAM of formula IA and the reverse transcriptase inhibitor are in the same formulation. 18. A method according to any one of claims 13-16, wherein the CpAM of formula IA and the reverse transcriptase inhibitor are in different formulations. 19. A method according to any one of claims 13-18, wherein the patient is refractory to treatment with a nucleoside agent or refractory to treatment. 20. A method according to any one of claims 13-18, wherein the patient is a untreated patient. 21. A method according to any one of claims 13-18, wherein the administration of the CpAM of formula IA and the reverse transcriptase inhibitor occurs over a period of less than 48 weeks. 22. A method according to any one of claims 13-18, wherein the patient is a patient chronically infected with HBV. 23. A method according to any one of claims 13-18, further comprising administering an additional anti-HBV agent. 24. The method of claim 23, wherein the additional anti-HBV agent is pegylated interferon alpha-2a. 25. A method according to any one of claims 13-15, wherein the reverse transcriptase inhibitor is tenofovir or a pharmaceutically acceptable salt thereof.