WO2017108630A1 - Polythérapie à inhibiteur hbsag et inhibiteur d'assemblage de capside du vhb - Google Patents

Polythérapie à inhibiteur hbsag et inhibiteur d'assemblage de capside du vhb Download PDF

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WO2017108630A1
WO2017108630A1 PCT/EP2016/081556 EP2016081556W WO2017108630A1 WO 2017108630 A1 WO2017108630 A1 WO 2017108630A1 EP 2016081556 W EP2016081556 W EP 2016081556W WO 2017108630 A1 WO2017108630 A1 WO 2017108630A1
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oxo
carboxylic acid
chloro
methoxy
methoxypropoxy
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PCT/EP2016/081556
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English (en)
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Hassan JAVANBAKHT
Isabel Najera
Steffen Wildum
Guang Yang
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F. Hoffmann-La Roche Ag
Hoffmann-La Roche Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses

Definitions

  • the present invention is directed to compositions and methods for treating hepatitis B virus infection.
  • the present invention is directed to a combination therapy comprising administration of an HBsAg inhibitor and an HBV capsid assembly inhibitor for use in the treatment or prophylaxis of chronic hepatitis B patient.
  • HBV Hepatitis B virus
  • HBV belongs to the Hepadnaviridae family of viruses. Following entry into hepatocyte, its viral genome is delivered into nucleus where a covalently closed circular DNA (cccDNA) is formed through DNA repair of partially double- stranded viral genome cccDNA serves as the template for transcription of viral RNAs. Viral pre-genomic RNA interacts with other two viral components, capsid protein and polymerase to form capsid particles where viral DNA replication occurs. HBV has an icosahedral core comprising of 240 copies of the capsid (or core) protein.
  • capsid protein The predominant biological function of capsid protein is to act as a structural protein to encapsidate pre-genomic RNA and form immature capsid particles in the cytoplasm. This step is prerequisite for viral DNA replication. When a near full-length relaxed circular DNA is formed through reverse-transcription of viral pregenomic RNA, an immature capsid becomes a mature capsid. Most copies of the encapsidated genome efficiently associate with cellular lipids and viral envelope proteins (S, M, and L) for virion assembly and secretion. However, non-infectious particles are also produced that greatly outnumber the infectious virions. These empty, enveloped particles are referred to as subviral particles (SVPs).
  • SVPs subviral particles
  • the S, M, and L envelope proteins are expressed from a single ORF (open reading frame) that contains three different start codons. All three proteins share a 226aa sequence, the S -domain, at their C-termini. S-domain contains the HBsAg epitope (Lambert, C. & R. Prange. Virol J, 2007, 4, 45). Viral proteins expressed from the HBV genome include HBsAg, HBV polymerase, HBV
  • HBV empty subviral particles may participate to the maintenance of the
  • HBsAg immunological tolerant state observed in chronically infected patients
  • CHB chronically infected patients
  • HBsAg may also play a role in suppressing host innate immune responses.
  • the persistent exposure to HBsAg and other viral antigens can lead to HBV-specific T-cell deletion or to progressive functional impairment (Kondo et al. Journal of Immunology 1993, 150, 4659- 4671 ; Kondo et al. Journal of Medical Virology 2004, 74, 425-433; Fisicaro et al.
  • HBsAg has been reported to suppress the function of immune cells such as monocytes, dendritic cells (DCs) and natural killer (NK) cells by direct interaction (Op den Brouw et al. Immunology, 2009b, 126, 280-9; Woltman et al. PLoS One, 2011, 6, el 5324; Shi et al. J Viral Hepat. 2012, 19, e26-33; Kondo et al. ISRN
  • HBV capsid protein plays essential roles in HBV replication.
  • HAP Heteroaryldihydropyrimidines or HAP, including compounds named Bay 41 -4109, Bay 38-7690 and Bay 39-5493, were discovered in a tissue culture-based screening (Deres K. et al. Science 2003, 893). These HAP analogs act as synthetic allosteric activators and are able to induce aberrant capsid formation that leads to degradation of the core protein.
  • HAP analogs also reorganized core protein from preassembled capsids into noncapsid polymers, presumably by interaction of HAP with dimers freed during capsid 'breathing ', the transitory breaking of individual intersubunit bonds.
  • Bay 41-4109 was administered to HBV infected transgenic mouse or humanized mouse models and demonstrated in vivo efficacy with HBV DNA reduction (Deres K. et al. Science 2003, 893; Brezillon N. et al. PLoS ONE 2011, e25096). It was also shown that bis-ANS, a small molecule that acts as a molecular 'wedge' and interferes with normal capsid- protein geometry and capsid formation (Zlotnick A. et al. J. Virol. 2002, 4848-4854).
  • HBsAg is a biomarker for prognosis and treatment response in CHB.
  • the standard of clinic cure of HBV infection is the loss and/or seroconversion of HBsAg.
  • PEG- IFN-a are available to HBV patients, the majority (around or more than 90%) of treated patients fail to achieve this goal.
  • the Hepatitis B virus (HBV) infection remains a major health problem worldwide which concerns an estimated 240 million chronic carriers who have a higher risk of liver cirrhosis and hepatocellular carcinoma.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an HBsAg inhibitor and an HBV capsid assembly inhibitor, in a pharmaceutically acceptable carrier.
  • the "HBsAg inhibitor” is a compound of formula (I) or (II), or any one of the compounds disclosed in patent application WO 2015113990, or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • the "HBsAg inhibitor” herein is (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3- carboxylic acid, (-)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid, (+)-10-chloro-6-isopropyl-9-(3- methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid, (-)- 10-chloro-6- isopropyl-9-(3-(3-
  • the "HBV capsid assembly inhibitor” herein is a compound of formula (III), (IV) or (V), or any one of the compounds disclosed in patent WO2014/037480, WO 2014/184328 and WO2015/132276; particularly the "HBV capsid assembly inhibitor” herein is ( l S , )-4-[(R)-6-(2-chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2-thiazol-2-yl-3,6-dihydro- pyrimidin-4-ylmethyl]-morpholine-3-carboxylic acid, (R)-6-((5')-2-carboxy-4,4-difluoro- pyrrolidin-l-ylmethyl)-4-(2-chloro-4-fluoro-phenyl)-2-thiazol-2-yl-l,4-dihydro-pyrimidine-5- carboxylic acid methyl ester, (2R,35')-4-[(R,35'
  • Figure 1 Isobologram of FIC for the pair-wise checkerboard combination of Compound 1A and Compound 7 (at the 50% effect level).
  • Data points below this lane show synergism, data points above show antagonism. Shown are mean values from 3 independent experiments.
  • Figure 2 Isobologram of FIC for the pair-wise checkerboard combination of Compound 1A and Compound 10 (at the 50% effect level).
  • Data points below this lane show synergism, data points above show antagonism. Shown are mean values from 3 independent experiments.
  • Ci_ 6 alkyl refers to a monovalent linear or branched saturated hydrocarbon group of 1 to 6 carbon atoms.
  • Ci_ 6 alkyl has 1 to 6 carbon atoms, and in more particular embodiments 1 to 4 carbon atoms.
  • Examples of Ci_ 6 alkyl include methyl, ethyl, propyl, isopropyl, n-butyl, iso -butyl, sec-butyl or tert-butyl.
  • halo or halogen are used interchangeably herein and refer to fluoro, chloro, bromo, or iodo. Halogen is particularly fluorine, chlorine or bromine.
  • haloCi_ 6 alkyl refers to a Ci_ 6 alkyl group wherein at least one of the hydrogen atoms of the Ci_ 6 alkyl group has been replaced by same or different halogen atoms, particularly fluoro atoms.
  • haloCi_ 6 alkyl include monofluoro-, difluoro- or trifluoro-methyl, - ethyl or -propyl, for example 3,3,3-trifluoropropyl, 2-fluoroethyl, trifluoroethyl, fluoromethyl, difluoromethyl, difluoroethyl or trifluoromethyl.
  • Ci_ 6 alkoxy refers to a group of Ci_ 6 alkyl-0-, wherein the "Ci_ 6alkyl” is as defined above; for example methoxy, ethoxy, propoxy, zsopropoxy, w-butoxy, iso- butoxy, 2-butoxy, ie/t-butoxy and the like.
  • Particular "Ci_ 6 alkoxy” groups are methoxy and ethoxy and more particularly methoxy.
  • C 3 _ 7 Cycloalkyl refers to a saturated carbon ring containing from
  • C 3 to 7 carbon atoms particularly from 3 to 6 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like.
  • Particular "C 3 _ 7 Cycloalkyl” groups are cyclopropyl, cyclopentyl and cyclohexyl.
  • the term "C 2 - 6 alkenyl” refers to an unsaturated, linear or branched chain alkenyl group containing 2 to 6, particularly 2 to 4 carbon atoms, for example vinyl, propenyl, allyl, butenyl and the like.
  • Particular "C 2 - 6 alkenyl” group is allyl.
  • C 2 - 6 alkynyl refers to an unsaturated, linear or branched chain alkynyl group containing 2 to 6, particularly 2 to 4 carbon atoms, for example ethynyl, 1 - propynyl, propargyl, butynyl and the like.
  • Particular "C 2 - 6 alkynyl” groups are ethynyl, 1 - propynyl and propargyl.
  • C X H 2X alone or in combination signifies a saturated, linear- or branched chain alkyl group containing 1 to 6, particularly 1 to 4 carbon atoms.
  • monocyclic heteroaryl denotes a monovalent aromatic heterocyclic mono- ring system of 5 to 8 ring atoms, comprising 1, 2, 3 or 4 heteroatoms selected from N, O and S, the remaining ring atoms being carbon.
  • Examples of monocyclic heteroaryl moieties include pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, triazinyl, azepinyl, diazepinyl, isoxazolyl, isothiazolyl and the like.
  • the term 'W-containing monocyclic heteroaryl refers to a monocyclic heteroaryl wherein at least one of the heteroatoms is N.
  • N-containing monocyclic heteroaryl are pyrrolyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, triazinyl, azepinyl, diazepinyl, isoxazolyl, isothiazolyl and the like.
  • Particular 'W-containing monocyclic heteroaryl” groups are imidazolyl, pyrazolyl and triazolyl.
  • monocyclic heterocycloalkyl is a monovalent saturated or partly unsaturated monocyclic ring system of 3 to 7 ring atoms, comprising 1, 2, or 3 ring heteroatoms selected from N, O and S, the remaining ring atoms being carbon.
  • Examples for monocyclic heterocycloalkyl are aziridinyl, oxiranyl, azetidinyl, oxetanyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, tetrahydrofuranyl, tetrahydro -thienyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl, morpholinyl, thiomorpholinyl, l, l-dioxo-thiomorpholin-4-yl, azepanyl, diazepanyl, homopiperazinyl, or oxazepanyl.
  • heterocyclic heterocycloalkyl groups are morpholinyl, 2-oxo- pyrrolidinyl, pyrrolidinyl, tetrahydropyranyl.
  • heterocyclic ring or heterocyclyl refers to a saturated or partly unsaturated monocyclic or bicyclic ring containing from 3 to 10 ring atoms which can comprise one, two or three atoms selected from nitrogen, oxygen and/or sulfur.
  • Examples of monocyclic heterocyclyl rings containing in particular from 3 to 7 ring atoms include, but not limited to, aziridinyl, azetidinyl, oxetanyl, piperidinyl, piperazinyl, azepinyl, diazepanyl, pyrrolidinyl, morpholinyl, dihydrofuryl, tetrahydrofuryl, tetrahydropyranyl, tetrahydrothiopyranyl and thiomorpholinyl.
  • Bicyclic heterocyclyl can be bicyclic fused ring or bicyclic bridged ring.
  • bicyclic heterocyclyl examples include 8-aza-bicyclo[3.2.1]octyl, quinuclidinyl, 8-oxa-3-aza- bicyclo[3.2.1]octyl, 9-aza-bicyclo[3.3.1]nonyl, 3-oxa-9-aza-bicyclo[3.3.1]nonyl, 3-thia-9-aza- bicyclo[3.3. l]nonyl, or difluoroazabicyclo[3.2. l]octyl.
  • Monocyclic and bicyclic heterocyclyl can be further substituted by halogen, Ci_ 6 alkyl, cyano, carboxy, carboxyCi_ 6 alkyl.
  • diastereomer refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties, activities and reactivities.
  • enantiomers refers to two stereoisomers of a compound which are non-superimpo sable mirror images of one another.
  • the term “pharmaceutically acceptable salts” refers to salts which are not biologically or otherwise undesirable. Pharmaceutically acceptable salts include both acid and base addition salts.
  • the term “prodrug” refers to a form or derivative of a compound which is metabolized in vivo, e.g., by biological fluids or enzymes by a subject after administration, into a pharmacologically active form of the compound in order to produce the desired pharmacological effect. Prodrugs are described e.g. in the Organic Chemistry of Drug Design and Drug Action by Richard B. Silverman, Academic Press, San Diego, 2004, Chapter 8 Prodrugs and Drug Delivery Systems, pp. 497-558.
  • pharmaceutically acceptable acid addition salt refers to those pharmaceutically acceptable salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and organic acids selected from aliphatic, cyclo aliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, maloneic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonic acid, ethane sulfonic acid, p-tolu
  • pharmaceutically acceptable base addition salt refers to those pharmaceutically acceptable salts formed with an organic or inorganic base.
  • acceptable inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts.
  • Salts derived from pharmaceutically acceptable organic nontoxic bases includes salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperizine, piperidine, N-ethylpiperidine, and polyamine resins.
  • substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, trieth
  • hepatitis B virus or “HBV” refers to a member of the Hepadnaviridae family having a small double- stranded DNA genome of approximately 3,200 base pairs and a tropism for liver cells.
  • HBV includes hepatitis B virus that infects any of a variety of mammalian (e.g., human, non-human primate, etc.) and avian (duck, etc.) hosts.
  • HBV includes any known HBV genotype, e.g., serotype A, B, C, D, E, F, and G; any HBV serotype or HBV subtype; any HBV isolate; HBV variants, e.g., HBeAg-negative variants, drug-resistant HBV variants (e.g., lamivudine-resistant variants; adefovir-resistant mutants; tenofovir-resistant mutants; entecavir-resistant mutants; etc.); and the like.
  • HBV genotype e.g., serotype A, B, C, D, E, F, and G
  • HBV serotype or HBV subtype e.g., HBeAg-negative variants
  • drug-resistant HBV variants e.g., lamivudine-resistant variants; adefovir-resistant mutants; tenofovir-resistant mutants; entecavir-resistant mutants; etc.
  • HBV DNA refers to DNA material of HBV.
  • HBsAg refers to hepatitis B surface antigen.
  • HBeAg refers to hepatitis B e antigen.
  • HBsAg inhibitor refers to a compound that inhibits expression of the hepatitis B virus surface antigen. Unless otherwise indicated, an HBsAg inhibitor can include the compound in any pharmaceutically acceptable form, including any isomer (e.g., diastereomer or enantiomer), salt, solvate, polymorph, and the like.
  • HBV capsid assembly inhibitor refers to a compound that inhibits and/or disrupt and/or accelerates and/or hinders and/or delays and or reduces and/or modifies normal HBV capsid assembly (e.g., during maturation) and/or normal capsid disassembly (e.g., during infectivity) and/or perturbs capsid stability, thereby inducing aberrant capsid morphology and function.
  • terapéuticaally effective amount refers to an amount of a compound or molecule of the present invention that, when administered to a subject, (i) treats or prevents the particular disease, condition or disorder, (ii) attenuates, ameliorates or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition or disorder described herein.
  • the therapeutically effective amount will vary depending on the compound, the disease state being treated, the severity of the disease treated, the age and relative health of the subject, the route and form of administration, the judgment of the attending medical or veterinary practitioner, and other factors.
  • the present invention relates to a pharmaceutical composition comprising an HBsAg inhibitor and an HBV capsid assembly inhibitor, in a pharmaceutically acceptable carrier.
  • diastereomeric salts which can be separated by crystallization are formed from the racemic mixtures by reaction with an optically active acid such as e.g. D- or L- tartaric acid, mandelic acid, malic acid, lactic acid or camphorsulfonic acid.
  • an optically active acid such as e.g. D- or L- tartaric acid, mandelic acid, malic acid, lactic acid or camphorsulfonic acid.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an HBsAg inhibitor and an HBV capsid assembly inhibitor, in a pharmaceutically acceptable carrier.
  • the "HBsAg inhibitor” is a compound of formula (I), wherein
  • R 1 is hydrogen, halogen, C 1-6 alkyl, Ci_ 6 alkylamino or
  • R is hydrogen; halogen; C 1-6 alkyl, which is unsubstituted or once, twice or three times
  • Ci- 6 alkoxy which is unsubstituted or once, twice or three times substituted by fluoro; cyano; C 3-7 cycloalkyl; hydroxy or phenyl- C x H 2X -0-;
  • R is hydrogen
  • C 1-6 alkyl which is unsubstituted or once, twice or three times substituted by fluoro;
  • R 7 is hydrogen; C 1-6 alkyl, which is unsubstituted or substituted with one to three substituents independently selected from fluoro, hydroxy and C 2-6 alkenyl; Ci_ 6 alkoxyCi- 6alkyl; C 1-6 alkoxyC 1-6 alkoxyC 1-6 alkyl; aminoCi-salkyl; Ci_ 6 alkylcarbonylaminoCi- 8 alkyl; Ci_ 6alkylsulfonylaminoCi-8alkyl; C 1-6 alkylsulfanylC 1-6 alkyl; C 1-6 alkylsulfonylC 1-6 alkyl; cyanoCi- 6alkyl; C 3- 7cycloalkylC 1-6 alkyl; cyanoC 3- 7cycloalkylC 1-6 alkyl; phenylC 1-6 alkyl;
  • pynOlidinylcarbonylC 1-6 alkyl C 2-6 alkynyl; hydroxyC 1-6 alkylC 2-6 alkynyl; aminoCi_6alkoxyCi- 6alkyl; C 1-6 alkylaminoC 1-6 alkoxyC 1-6 alkyl; diC 1-6 alkylaminoC 1-6 alkoxyC 1-6 alkyl; carboxyCi- 6alkyl; or Ci- 6 alkoxycarbonylaminoCi_ 8 alkyl; heteroarylC 1-6 alkyl, wherein heteroaryl is N- containing monocyclic heteroaryl; or heterocycloalkylC 1-6 alkyl, wherein heterocycloalkyl is monocyclic heterocycloalkyl;
  • R 4 is hydrogen, halogen, C 1-6 alkyl, cyano or C 1-6 alkoxy
  • R 5 is hydrogen or C 1-6 alkyl
  • R 6 is hydrogen
  • Ci_ 6 alkyl which is unsubstituted or once, twice or three times substituted by fluoro
  • C3_ 7 cycloalkyl which is unsubstituted or once, twice or three times substituted by fluoro or Ci_ 6 alkyl
  • phenyl-C x H2 X - is hydrogen or C 1-6 alkyl
  • R 6 is hydrogen
  • Ci_ 6 alkyl which is unsubstituted or once, twice or three times substituted by fluoro
  • C3_ 7 cycloalkyl which is unsubstituted or once, twice or three times substituted by fluoro or Ci_ 6 alkyl
  • phenyl-C x H2 X -
  • x is 1-6;
  • the "HBsAg inhibitor" of the present invention relates to
  • the "HBsAg inhibitor” is a compound of formula (II):
  • R 8 , R 9 , R 10 and R 11 are independently selected from hydrogen, halogen, Ci- 6 alkyl, diCi_ 6alkylamino, cyano, N-containing monocyclic heterocycloalkyl and OR 14 , wherein R 14 is hydrogen; C 1-6 alkyl; or Ci- 6 alkyl which is substituted once or more times by fluoro, C3_ 7 cycloalkyl, phenyl, hydroxyl, amino, Ci_ 6 alkoxy, Ci- 6 alkylsulfanyl, Ci_ 6 alkylsulfonyl, diCi_ 6 alkylamino, Ci_ 6 alkoxycarbonylamino, monocyclic heterocycloalkyl, pyrazoyl or imidazolyl;
  • R is hydrogen or C h alky
  • R is hydrogen, Ci- 6 alkyl, phenyl-C x H2 X -, Ci_ 6 alkylcarbonyl, Ci_ 6 alkylsulfonyl, benzoyl or monocyclic heterocycloalkyl, wherein
  • x is 1-6;
  • W is a bond, C y H 2y C(R 15 )(R 16 )C z H 2z or C y H 2y CH(R 15 )CH(R 16 )C z H 2z , wherein
  • R 15 and R 16 are independently selected from hydrogen, fluoro, hydroxy and Ci_ 6 alkyl, y is 0-6;
  • z is 0-6;
  • X is a bond; O; S; S(0) 2 ; or NR 17 , wherein R 17 is hydrogen, Ci_ 6 alkyl;
  • the "HBsAg inhibitor" of the present invention relates to
  • an HBsAg inhibitor can include any one of the compounds of formula (I) and (II), in any pharmaceutically acceptable form, including any isomer (e.g., diastereomer or enantiomer), salt, solvate, polymorph, and the like.
  • the HBV capsid assembly inhibitor is a compound of formula (III):
  • R is Ci-6alkyl or trifluoromethyl-C 3 H2 S -, wherein s is 1, 2, 3, 4, 5 or 6;
  • R 19 and R 20 is phenyl, which is once or twice or three times substituted by Ci_ 6 alkyl, cyano or halogen; and the other one is hydrogen or deuterium;
  • R 21 is phenyl, thiazolyl, oxazolyl, imidazolyl, thienyl or pyridinyl, which is unsubstituted or substituted by C h alky!, Ci_ 6 alkylsulfanyl, halogen or cycloalkyl, wherein Ci- 6 alkyl can be further o tionally substituted with halogen;
  • HBV capsid assembly inhibitor of the present invention relates to (5)-4-[(R)-6-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2-thiazol-2-yl-3,6-dihydro- pyrimidin-4- ylmethyl] - morpho line- 3 -carboxylic acid ;
  • the HBV capsid assembly inhibitor is a compound of formula (IV):
  • R 22 is Ci_ 6 alkyl
  • R 23 is phenyl, which is once or twice or three times substituted by halogen or Ci- 6 alkyl;
  • R 24 is hydrogen or Ci- 6 alkyl
  • R 25 is bicyclic bridged heterocyclyl
  • HBV capsid assembly inhibitor of the present invention relates to 2-[(lR,35,55)-8-[[(4R)-4-(2-chloro-3-fluoro-phenyl)-5-methoxycarbonyl-2-thiazol-2-yl- l,4-dihydropyrimidin-6-yl]methyl]-6,6-difluoro-8-azabicyclo[3.2.1]octan-3-yl]acetic acid;
  • the HBV capsid assembly inhibitor is a compound of formula (V):
  • R 26 is hydrogen, halogen or Ci- 6 alkyl
  • R 27 is hydrogen or halogen
  • R 28 is hydrogen or halogen
  • R 29 is Ci_ 6 alkyl
  • R 30 is hydrogen, hydroxyCi_ 6 alkyl, aminocarbonyl, Ci_ 6 alkoxycarbonyl or carboxy;
  • R is hydrogen, Ci_6alkoxycarbonyl or carboxy-C m H2 m -;
  • U is carbonyl or sulfonyl
  • Y is -CH 2 -, -O- or -N(R 32 )-,
  • R is hydrogen, Chalky!, haloCi_6alkyl, C3_7cycloalkyl-C m H2m-, Ci_
  • Q is -CH 2 -, -C(Ci_ 6 alkyl) 2 -, -O- or carbonyl;
  • n 0 or 1 ;
  • n 0-7;
  • t 1-7;
  • HBV capsid assembly inhibitor of the present invention relates to 3-[(8a5')-7-[[(4 l S , )-5-ethoxycarbonyl-4-(3-fluoro-2-methyl-phenyl)-2-thiazol-2-yl-l,4- dihydropyrimidin-6-yl]methyl]-3-oxo-5,6,8,8a-tetrahydro-lH-imidazo[l,5-a]pyrazin-2-yl]-2,2- dimethyl-propanoic acid;
  • HBV capsid assembly inhibitor is anyone of the compounds disclosed in WO2008154817, WO2008154819, WO2014029193, WO2015074546, CN 103664897 and CN 103664925.
  • the pharmaceutical composition comprises an HBsAg inhibitor and HBV capsid assembly inhibitor, wherein the HBsAg inhibitor and the HBV capsid assembly inhibitor are independently selected from Table 1.
  • Compounds 1A, IB, 2A, 2B, 3A and 3B were first disclosed in WO 2015/113990; Compounds 7, 8 and 9 were first disclosed in WO2014/037480; Compounds 16 and 17 were disclosed in WO2014/184328; Compounds 10- 15 were disclosed in WO2015/132276.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an HBsAg inhibitor and an HBV capsid assembly inhibitor which is selected from any one of the following combinations:
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an HBsAg inhibitor and an HBV capsid assembly inhibitor which is selected from any one of the following combinations:
  • the pharmaceutical composition consists of an HBsAg inhibitor and an HBV capsid assembly inhibitor, in a pharmaceutically acceptable carrier. More particularly, the composition consists of:
  • HBV capsid assembly inhibitors can also be used in the pharmaceutical composition including small molecules or large molecules.
  • HBV capsid assembly inhibitors include, but not limited to, Bay 41-4109, Bay 38-7690, Bay 39-5493, GLS4, AT-61 and AT-130.
  • Typical dosages of an HBsAg inhibitor and/or an HBV capsid assembly inhibitor can be in various ranges, and where indicated by in vitro responses in an animal model, can be reduced by up to about one order of magnitude concentration or amount.
  • the actual dosage will depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based on the in vitro responsiveness of the appropriate animal models.
  • Another embodiment of the present invention relates to a method for manufacturing a medicament for treatment or prophylaxis of hepatitis B virus infection, characterized in that an HBsAg inhibitor and an HBV capsid assembly inhibitor are used in the medicament.
  • a further embodiment of the present invention relates to the method for manufacturing a medicament for treatment or prophylaxis of hepatitis B virus infection, characterized in that an HBsAg inhibitor and an HBV capsid assembly inhibitor are co- administered in the same formulation or different formulation.
  • co -administer refers to any administration of an HBsAg inhibitor and an HBV capsid assembly inhibitor as the two active agents, either separately or together, where the two active agents are administered as part of an appropriate dose regimen designed to obtain the benefit of the combination therapy.
  • the two active agents can be administered either as part of the same pharmaceutical composition or in separate pharmaceutical compositions.
  • the two active agents can be administered either at the same time, or sequentially.
  • the pharmaceutical composition of the HBsAg inhibitor and the HBV capsid assembly inhibitor can be administered with various pharmaceutically acceptable inert carriers in the form of tablets, capsules, lozengens, troches, hard candies, powders, sprays, creams, salves, suppositories, jellies, gels, pastes, lotions, ointments, elixirs, syrups, and the like. Administration of such dosage forms can be carried out in single or multiple doses. Carries include solid diluents of fillers, sterile aqueous media and various non-toxic organic solvents. Administration of such dosage forms can be carried out through, but not limited to, oral administration, parenteral administration, veterinary administration.
  • a further embodiment of the present invention relates to the method for manufacturing a medicament for treatment or prophylaxis of hepatitis B virus infection, characterized in that an HBsAg inhibitor and an HBV capsid assembly inhibitor are intended for administration to a subject by the same route or different routes.
  • a further embodiment of the present invention relates to the method for manufacturing a medicament for treatment or prophylaxis of hepatitis B virus infection, characterized in that an HBsAg inhibitor and an HBV capsid assembly inhibitor are intended for administration to a subject by parenteral or oral administration.
  • a further embodiment of the present invention relates to the method for manufacturing a medicament for treatment or prophylaxis of hepatitis B virus infection, characterized in that the administration of an HBsAg inhibitor and an HBV capsid assembly inhibitor to a subject is simultaneous or sequential.
  • the administration of agents simultaneously can be performed by separately or sequentially administering agents at the same time, or together as a fixed combination.
  • the administration of agents separately or sequentially can be in any order.
  • Another embodiment of the present invention relates to the method for manufacturing a medicament for treatment or prophylaxis of hepatitis B virus infection, characterized in that the HBsAg inhibitor is a compound of formula (I) or formula (II), or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • the HBsAg inhibitor is a compound of formula (I) or formula (II), or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • the HBsAg inhibitor is a compound of formula (I) or formula (II), or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • the HBsAg inhibitor is a compound of formula (I) or formula (II), or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • the HBsAg inhibitor is
  • HBV capsid assembly inhibitor is a compound of formula (III), formula (IV) or formula (V), or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • the HBV capsid assembly inhibitor is a compound of formula (III), formula (IV) or formula (V), or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • the HBV capsid assembly inhibitor is a compound of formula (III), formula (IV) or formula (V), or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • the HBV capsid assembly inhibitor is
  • Another embodiment of the present invention relates to the method for manufacturing a medicament for treatment or prophylaxis of hepatitis B virus infection, characterized in that the HBsAg inhibitor and the HBV capsid assembly inhibitor used in the medicament are (+)- 10- methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3- carboxylic acid and ( l S')-4-[(R)-6-(2-chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2-thiazol-2-yl- 3,6-dihydro-pyrimidin-4-ylmethyl]-morpholine-3-carboxylic acid; or (+)- 10-methoxy-6- isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid and 3-[(8a5')-7-[
  • kits comprising a container comprising an HBsAg inhibitor and an HBV capsid assembly inhibitor, said kit can further comprise a sterile diluent.
  • kit can further comprise a package insert comprising printed instructions directing the use of a combined treatment of an HBsAg inhibitor and an HBV capsid assembly inhibitor as a method for treatment or prophylaxis of hepatitis B virus infection.
  • Another embodiment of the present invention relates to the said kit, wherein the HBsAg inhibitor is
  • Another embodiment of the present invention relates to the said kit, wherein the HBV capsid assembly inhibitor is
  • kits characterized in that the HBsAg inhibitor and the HBV capsid assembly inhibitor used in the container are (+)-10- methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3- carboxylic acid and ( l S')-4-[(R)-6-(2-chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2-thiazol-2-yl- 3,6-dihydro-pyrimidin-4-ylmethyl]-morpholine-3-carboxylic acid; or (+)-10-methoxy-6- isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid and 3-[(8a5')-7-[[[
  • Another embodiment of the present invention relates to a method for the treatment or prophylaxis of hepatitis B virus infection, comprising administration to a subject with an effective first amount of an HBsAg inhibitor, or pharmaceutically acceptable salt, enantiomer or diastereomer thereof; and a second amount of an HBV capsid assembly inhibitor; or vice versa; wherein the HBsAg inhibitor is
  • Another embodiment of the present invention relates to a method for the treatment or prophylaxis of hepatitis B virus infection, comprising administration to a subject with an effective first amount of an HBsAg inhibitor, or pharmaceutically acceptable salt, enantiomer or diastereomer thereof; and a second amount of an HBV capsid assembly inhibitor; or vice versa; wherein the HBV capsid assembly inhibitor is
  • Another embodiment of the present invention relates to a method for the treatment or prophylaxis of hepatitis B virus infection, characterized in that the HBsAg inhibitor and the HBV capsid assembly inhibitor used are (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo- 6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid and (5 , )-4-[(R)-6-(2-chloro-4-fluoro-phenyl)-5- methoxycarbonyl-2-thiazol-2-yl-3,6-dihydro-pyrimidin-4-ylmethyl]-morpholine-3-carboxylic acid; or (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 3-[(8a5 , )-7-[[(45 ,
  • Another embodiment of present invention relates to the use of an HBsAg inhibitor and an HBV capsid assembly inhibitor for the manufacture of pharmaceutical composition herein mentioned above as an antiviral medicament, in particular the medicament for treatment or prophylaxis of hepatitis B virus infection.
  • the combined organic layers were washed with water (200 mL) 2 times and brine, dried over anhydrous Na 2 S0 4 and concentrated to give 4-methoxy- l-[4-methoxy-3-(3- methoxypropoxy)phenyl]-3,3-dimethyl-butan-2-amine (120 g).
  • Step 5 Preparation of N-[3-methoxy-l-[[4-methoxy-3-(3-methoxypropoxy)phenyl]methyl]- 2,2-dimethyl-propyl] formamide
  • Step 7 Preparation of ethyl 10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3- methoxypropoxy)-2-oxo-l,6,7,llb-tetrahydrobenzo[a]quinolizine-3-carboxylate
  • Step 8 Preparation of ethyl 10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3- methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylate
  • Step 9 Preparation of 10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3- methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid
  • Step 10 preparation of (+)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3- methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (Compound 4A) and ( )-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid (Compound 4B)
  • Step 3 Preparation of 4-benzyloxy-l-[4-methoxy-3-(3-methoxypropoxy)phenyl]-3,3- dimethyl-butan-2-amine
  • Step 4 Preparation of N-[3-benzyloxy-l-[[4-methoxy-3-(3-methoxypropoxy)phenyl]methyl] -2,2-dimethyl-propyl]formamide
  • Step 5 Preparation of 3-(2-benzyloxy-l,l-dimethyl-ethyl)-7-methoxy-6-(3- methoxypropoxy)-3,4-dih droisoquinoline
  • Step 6 Preparation of ethyl 6-(2-benzyloxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3- methoxypropoxy)-2-oxo-l,6,7,llb-tetrahydrobenzo[a]quinolizine-3-carboxylate
  • Step 7 Preparation of ethyl 6-(2-benzyloxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3- methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylate
  • Step 8 Preparation of 6-(2-benzyloxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3- methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid
  • Step 9 Preparation of 6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3- methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid
  • Step 10 Preparation of (+)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3- methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (Compound 5A) and (-)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid (Compound 5B)
  • Step 1 Preparation of 4-chloro-l-[4-methoxy-3-(3-methoxypropoxy)phenyl]-3,3-dimethyl- butan-2-one
  • Step 2 Preparation of 4-chloro-l-[4-methoxy-3-(3-methoxypropoxy)phenyl]-3,3-dimethyl- butan-2-amine
  • Step 3 Preparation of N-[l-[[4-chloro-3-(3-methoxypropoxy)phenyl]methyl]-3-methoxy- 2,2-dimethyl-propyl] formamide
  • Step 4 Preparation of 7-chloro-3-(2-methoxy-l,l-dimethyl-ethyl)-6-(3-methoxypropoxy)- 3,4-dihydroisoquinoline
  • Step 5 Preparation of ethyl 10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3- methoxypropoxy)-2-oxo-l,6,7,llb-tetrahydrobenzo[a]quinolizine-3-carboxylate
  • Step 6 Preparation of ethyl 10-chloro-6-(2-methoxy- l, l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylate
  • Step 7 Preparation of 10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)- 2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid
  • Step 8 Preparation of (+)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3- methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid (Compound 6A) and (-)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2- dihydrobenzo[a]quinolizine-3-carboxylic acid (Compound 6B)
  • HBsAg Assay to test capacity of HBsAg inhibitor to inhibit HBsAg
  • HepG2.2.15 cells (Acs et al. Proc Natl Acad Sci U S A, 84, (1987), 4641-4), a
  • constitutively HBV-expressing cell line were cultured in DMEM+Glutamax-I medium
  • HepG2.2.15 cells were seeded in duplicate into white, 96-well plates at 1.5 x 10 4 cells/well. The cells were treated with a three-fold serial dilution series of the compounds in DMSO. The final DMSO concentration in all wells was 1% and DMSO was used as no drug control.
  • the HBsAg chemiluminescence immunoassay (CLIA) kit (Autobio Diagnostics Co., Zhengzhou, China, Catalog number: CL0310-2) was used to measure the levels of secreted HBV antigens semi-quantitatively.
  • CLIA HBsAg chemiluminescence immunoassay
  • 50 ⁇ ⁇ culture supernatant was used and HBsAg was quantified using HBsAg chemiluminescence immunoassay (CLIA) kit (Autobio Diagnostics Co., Zhengzhou, China, Catalog number: CL0310-2), 50 ⁇ ⁇ of the supernatant was transferred to the CLIA assay plate and 50 ⁇ ⁇ of enzyme conjugate reagent was added into each well. The plates were sealed and gently agitated for 1 hour at room temperature. The
  • Luminance was measured using a lumino meter (Mithras LB 940 Multimode Microplate Reader) after 10 minutes incubation. Dose-response curves were generated and the IC 50 value was extrapolated by using the E- WorkBook Suite (ID Business Solutions Ltd., Guildford, UK). The IC 50 was defined as the compound concentration (or conditioned media log dilution) at which HBsAg secretion was reduced by 50% compared to the no drug control.
  • Example 5 Combination of an HBsAg inhibitor and an HBV capsid assembly inhibitor in HBV infected HepaRG cells
  • HepG2.2.15 cells were cultured in DMEM+Glutamax I (Gibco, #21885) supplemented with 10% FBS, 1% Pen/Strep (Gibco, #15140) and G-418 (250 ⁇ ) and used for production of infectious HBV (genotype D). 90% confluent cells from three T175 flasks were trypsinized and transferred into one collagen coated hyperflask (550 mL). Once the cells are confluent, medium was changed to DMEM+Glutamax I with 1% DMSO and 2.5% FBS.
  • HepaRG cells (Biopredic International, Saint-Gregoire, France) were cultured in working growth medium (500 mL Willams E Medium with 50 mL HepaRG Growth supplement from Biopredic, 5 mL Glutamax-I (Gibco, #35050) and 5 mL Pen/Strep) for 2 weeks. After 2 weeks medium was changed to differentiation medium containing 1.8% DMSO (500 mL Willams E Medium with 50 mL HepaRG Growth supplement from Biopredic, 5 mL Pen/Strep, 5 mL Glutamax-I and 9 mL DMSO). Medium was changed twice a week up to 2 weeks. Once fully differentiated, cells were trypsinized and seeded into collagenated 96 well plates (50,000 cells/well in 100 ⁇ ) in differentiation medium. Cells were cultured at least 5 days in the 96 well plates before they were infected with HBV.
  • working growth medium 500 mL Willams E Medium with 50 mL HepaRG
  • Compound 1A The concentration ranges tested were 300 nM to 0.4 nM for Compound 7, 150 nM to 0.2 nM for Compound 10, and 30 nM to 0.37 nM for Compound 1A.
  • Medium was replaced by new medium with compound at day 7 post infection and at day 11 post infection cell supernatants were harvested and directly used for HBV DNA extraction or stored at -20°C. Cell viability of the cells was determined using the cell viability assay described below.
  • A1+B7 example of combination of drug A and B at different ratios DNA extraction
  • HBV DNA from HepaRG cell supernatants was extracted using the MagNA Pure 96 (Roche) robot. 100 ⁇ ⁇ of the supernatants were mixed in a processing cartridge with 200 ⁇ ⁇ MagNA Pure 96 external lysis buffer (Roche, Cat. No. 06374913001) and incubated for 10 minutes. DNA was then extracted using the "MagNA Pure 96 DNA and Viral Nucleic Acid Small Volume Kit” (Roche, Cat. No. 06543588001) and the "Viral NA Plasma SV external lysis 2.0" protocol. DNA elution volume was 50 ⁇ ⁇ . qPCR
  • Forward core primer (F3_core): CTG TGC CTT GGG TGG CTT T
  • Reverse primer (R3_core): AAG GAA AGA AGT CAG AAG GCA AAA
  • Taqman probe (P3_core): 56-FAM/AGC TCC AAA /ZEN/TTC TTT ATA AGG GTC
  • FIC ratio [EC50 combination : EC50 alone]
  • a CI ⁇ 1 means synergism, a CI 1 means additivity and a CI > 1 means antagonism.
  • Combination effect assessment was based on overall CI values as follows: CI value ⁇ 0.7 as synergy, 0.7 to 0.9 as slight to moderate synergy, 0.9 to 1.1 as additive, 1.1 to 1.5 as slight to moderate antagonism and >1.5 as antagonism (Chou TC (2006). Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol. Rev., 58:621-681). Drug combinations were analyzed at three different fixed drug ratios spanning and including the approximate ratio of their EC 5 os.
  • Combination of Compound 1A with Compound 7, and combination of Compound 1A with Compound 10 were tested for anti-HBV activity in HBV infected differentiated HepaRG cells.
  • Combination of Lamivudine with Lamivudine was set as the control combination.
  • the single compound inhibitory activites (EC 50 ) obtained in the combination studies were determined (Table 4). The concentration ranges chosen were also confirmed below the cytotoxic

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Abstract

L'invention concerne des compositions et des méthodes pour traiter une infection par le virus de l'hépatite B. En particulier, la présente invention porte sur une polythérapie comprenant l'administration d'un inhibiteur HBsAG et d'un inhibiteur d'assemblage de capside du VHB à utiliser dans le traitement ou la prophylaxie d'un patient infecté par l'hépatite B chronique.
PCT/EP2016/081556 2015-12-21 2016-12-16 Polythérapie à inhibiteur hbsag et inhibiteur d'assemblage de capside du vhb WO2017108630A1 (fr)

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US11053235B2 (en) 2018-08-09 2021-07-06 Janssen Sciences Ireland Unlimited Company Substituted 1,4-dihydropyrimidines for the treatment of HBV infection or HBV-induced diseases
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US11234977B2 (en) 2017-12-20 2022-02-01 Novartis Ag Fused tricyclic pyrazolo-dihydropyrazinyl-pyridone compounds as antivirals
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WO2022052923A1 (fr) * 2020-09-08 2022-03-17 和博医药有限公司 Composé dihydropyrimidine et son utilisation
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