WO2017211791A1 - Combination therapy of an hbsag inhibitor and a tlr7 agonist - Google Patents

Combination therapy of an hbsag inhibitor and a tlr7 agonist Download PDF

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Publication number
WO2017211791A1
WO2017211791A1 PCT/EP2017/063665 EP2017063665W WO2017211791A1 WO 2017211791 A1 WO2017211791 A1 WO 2017211791A1 EP 2017063665 W EP2017063665 W EP 2017063665W WO 2017211791 A1 WO2017211791 A1 WO 2017211791A1
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Prior art keywords
oxo
methoxy
methoxypropoxy
dihydrobenzo
quinolizine
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PCT/EP2017/063665
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French (fr)
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Hassan JAVANBAKHT
Isabel Najera
Lu Gao
Steffen Wildum
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F. Hoffmann-La Roche Ag
Hoffmann-La Roche Inc.
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Publication of WO2017211791A1 publication Critical patent/WO2017211791A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • 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

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 a TLR7 agonist for use in the treatment or prophylaxis of chronic hepatitis B virus infection.
  • 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
  • TLRs Toll- like receptors detect a wide range of conserved pathogen-associated molecular patterns (PAMPs). They play an important role of sensing invading pathogens and subsequent initiation of innate immune responses.
  • PAMPs pathogen-associated molecular patterns
  • TLR3 TLR7, TLR8, and TLR9 are located within endosomes.
  • TLR7 can be activated by binding to a specific small molecule ligand (i.e., TLR7 agonist) or its native ligand (i.e., single- stranded RNA, ssRNA). Following binding of ssRNA to TLR7, the receptor in its dimerized form is believed to undergo a structural change leading to the subsequent recruitment of adapter proteins at its cytoplasmic domain, including the myeloid differentiation primary response gene 88 (MyD88). Following the initiation of the receptor signalling cascade via the MyD88 pathway, cytoplasmic transcription factors such as interferon regulatory factor 7 (IRF-7) and nuclear factor kappa B (NF- ⁇ ) are activated.
  • IRF-7 interferon regulatory factor 7
  • NF- ⁇ nuclear factor kappa B
  • TLR7 is predominately expressed on plasmacytoid cells, and also on B -cells. Altered responsiveness of immune cells might contribute to the reduced innate immune responses during chronic viral infections. Agonist-induced activation of TLR7 might therefore represent a novel approach for the treatment of chronic viral infections.
  • D. J Connolly and L. AJ O'Neill, Current Opinion in Pharmacology 2012, 12:510-518, P. A. Roethle et al, J. Med. Chem. 2013, 56, 7324-7333 Treatment with an oral, small- molecule TLR7 agonist is a promising approach that has the potential to provide greater efficacy and better tolerability (T. Asselah et al, Clin Liver Dis 2007, 11, 839-849).
  • 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 and nucleos(t)ide analogues 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.
  • HBV Hepatitis B virus
  • HBV Hepatitis B virus
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an HBsAg inhibitor and a TLR7 agonist, in a pharmaceutically acceptable carrier, for use in the treatment or prophylaxis of chronic hepatitis B virus infection.
  • the "HBsAg inhibitor” is a compound of formula (I), (II) or any one of the compounds disclosed in patent applications WO2015/113990 and WO2016/071215, particularly 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-methoxypropoxy)-2-oxo-6,7
  • the "TLR7 agonist” herein is a compound of formula (III), (IV) or any one of the compounds disclosed in patent WO2006/066080 and patent application
  • WO2016/055553 particularly the "TLR7 agonist" herein is [(lS)-l-[(2S,4R,5R)-5-(5-amino-2- oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl] acetate; [(S)- [(2S,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]-cyclopropyl- methyl] acetate; 5-amino-3-(3'-deoxy-P-D-ribomranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one; 5- amino-3-(2'-0-acetyl-3'-deoxy-P-D-ribomranosyl)-3H-thiazolo[4,5
  • Figure 1 Isobologram of FIC for the pair-wise checkerboard combination of Compound 1A and Compound 11 hPBMC conditioned medium (at the 50% effect level).
  • Data points below this lane show synergism, data points above show antagonism.
  • the results shown are mean values from 3 independent experiments.
  • Figure 2 Isobologram of FIC for the pair-wise checkerboard combination of Compound 1A and Compound 12 hPBMC conditioned medium (at the 50% effect level).
  • Data points below this lane show synergism, data points above show antagonism.
  • the results 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.
  • 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 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.
  • 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.
  • 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” containing in particular from 3 to 7 ring atoms include, but not limited to, aziridinyl, oxiranyl, azetidinyl, oxetanyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, tetrahydro -thienyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl, morpholinyl, thio morpholinyl, l, l-dioxo-thiomorpholin-4-yl, azepanyl, diazepanyl, homopiperazinyl, or oxazepanyl.
  • Bicyclic heterocyclyl can be bicyclic fused ring or bicyclic bridged ring.
  • Examples for bicyclic heterocyclyl are 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[3.3.1]nonyl, or difluoroazabicyclo[3.2.1]octyl.
  • Monocyclic and bicyclic heterocyclyl can be further substituted by halogen, Ci_ 6 alkyl, cyano, carboxy, carboxyCi_ 6 alkyl.
  • heterocyclic amino refers to an amino group with the nitrogen atom on the heterocyclic ring, wherein “heterocyclic” ring is as defined above.
  • 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. As used herein, the term “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.
  • anti-HBs refers to antibodies against HBsAg.
  • HBsAg inhibitor refers to a compound that inhibits 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.
  • TLR7 refers to the Toll-like receptor 7 of any species of origin (e.g., human, murine, woodchuck etc.).
  • TLR7 agonist refers to a compound that acts as an agonist of TLR7.
  • a TLR7 agonist can include the compound in any pharmaceutically acceptable form, including any isomer (e.g., diastereomer or enantiomer), salt, solvate, polymorph, and the like.
  • the TLR agonism for a particular compound may be determined in any suitable manner. For example, assays for detecting TLR agonism of test compounds are described, for example, in U.S. Provisional Patent Application Ser. No. 60/432,650, filed Dec. 11, 2002, and recombinant cell lines suitable for use in such assays are described, for example, in U.S. Provisional Patent Application Ser. No.
  • 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 a nucleos(t)ide analogue, 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.
  • One embodiment of the present invention relates to (1) a pharmaceutical composition comprising an HBsAg inhibitor and a TLR7 agonist, in a pharmaceutically acceptable carrier.
  • Another embodiment of the present invention is (2) the pharmaceutical composition according to embodiment (1), wherein the "HBsAg inhibitor” is a compound of formula (I):
  • R 1 is hydrogen, halogen, C 1-6 alkyl, Ci_ 6 alkylamino or Ci ⁇ alkoxy;
  • R is hydrogen; halogen; Ci_ 6 alkyl, which is unsubstituted or once, twice or three times substituted by fluoro;
  • Ci- 6 alkoxy which is unsubstituted or once, twice or three times substituted by fluoro;
  • cyano C3_ 7 cycloalkyl; hydroxy or phenyl- C x H2 X -0-;
  • R is hydrogen
  • Ci_ 6 alkyl which is unsubstituted or once, twice or three times substituted by fluoro;
  • R 7 is hydrogen; Ci_ 6 alkyl, which is unsubstituted or substituted with one to three substituents independently selected from fluoro, hydroxy and C 2 - 6 alkenyl; Ci_ 6alkoxyCi_ 6 alkyl; Ci_ 6 alkoxyCi- 6 alkoxyCi_ 6 alkyl; aminoCi-salkyl; Ci_
  • R 4 is hydrogen, halogen, Ci- 6 alkyl, cyano or
  • R 5 is hydrogen or Ci_ 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; or phenyl-C x H 2x -;
  • x is 1-6;
  • a further embodiment of the present invention is (3) the pharmaceutical composition according to embodiment (2), wherein the "HBsAg inhibitor” is (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
  • Another embodiment of the present invention is (4) the pharmaceutical composition according to embodiment (1), wherein the "HBsAg inhibitor” is a compound of formula (II):
  • R 9 , R 10 and R u are independently selected from hydrogen, halogen, Ci- 6 alkyl, diCi
  • R is hydrogen; Ci_ 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 heterocyclyl, 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 heterocyclyl, 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, C 1-6 alkyl;
  • a further embodiment of the present invention is (5) the pharmaceutical composition according to embodiment (4), wherein the "HBsAg inhibitor" is
  • an HBsAg inhibitor is 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.
  • Another embodiment of the present invention is (6) the pharmaceutical composition according to any one of embodiments (1) to (5), wherein the "TLR7 agonist" is a compound of formula (III):
  • R 18 is hydroxy, Ci- 6 alkyl, haloCi_ 6 alkyl, Ci_ 6 alkylcarbonyl-0-, Ci- 6 alkyl-S-, azido, cyano, C2- 6 alkenyl, Ci_ 6 alkylsulfonyl-NH-, (C 1-6 alkyl) 2 N-, Ci_ 6 alkylcarbonyl-NH- or heterocyclic amino;
  • R 19 is hydrogen, C 1-6 alkyl, Ci- 6 alkoxyCi_ 6 alkyl, C3_ 7 cycloalkyl, C 2 - 6 alkynyl, C 2 - 6 alkenyl, benzyl and thiophenyl;
  • R 20 is hydrogen or Ci_ 6 alkylcarbonyl
  • R 21 is hydrogen or hydroxy
  • a further embodiment of the present invention is (7) the pharmaceutical composition according to embodiment (6), wherein the "TLR7 agonist" is
  • Another embodiment of the present invention is (8) the pharmaceutical composition according to any one of embodiments (1) to (5), wherein the "TLR7 agonist” is a compound of formula (IV):
  • R 2"2 and 23 are independently selected from hydrogen, C 2 - 6 alkenyl and Ci_ 6 alkyl;
  • R 2"5 J are independently selected from hydrogen, Ci_ 6 alkyl, C3_ 7 cycloalkyl, C 3 _ 7 cycloalkylC 2 - 6 alkynyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl and 2-thiophenyl;
  • R 26 is hydrogen or Ci_ 6 alkylcarbonyl
  • R 2"7 is hydro gen or hydroxy
  • a further embodiment of the present invention is (9) the pharmaceutical composition according to embodiment (8), wherein the "TLR7 agonist" is
  • Another embodiment of the present invention is (10) the pharmaceutical composition according to any one of embodiments (1) to (5), wherein the TLR7 agonist is [(lS)-l-[(2S,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy- tetrahydrofuran-2- yl] propyl] acetate ;
  • Another embodiment of the present invention is (11) the pharmaceutical composition according to any one of embodiments (1) to (5), wherein the "TLR7 agonist” is any one of the compounds disclosed in patent WO2006/066080 and patent application WO2016/055553.
  • Another embodiment of the present invention is (12) the pharmaceutical composition according to embodiment (1), wherein the composition consists of an HBsAg inhibitor and a TLR7 agonist, in a pharmaceutically acceptable carrier.
  • Another embodiment of the present invention is (13) the pharmaceutical composition according to embodiment (1) or (12), wherein the HBsAg inhibitor and the TLR7 agonist are independently selected from Table 1.
  • Table 1 List of HBsAg inhibitors and TLR7 agonists
  • a further embodiment of the present invention is (14) the pharmaceutical composition according to embodiment (1) or (12), wherein the composition is selected from any one of the following combinations:
  • Another further embodiment of the present invention is (16) the pharmaceutical composition according to embodiment (1) or (12), consisting of
  • Another embodiment of the present invention is (17) a pharmaceutical composition, which is
  • Another embodiment of the present invention is (18) the pharmaceutical composition according to any one of embodiments (1), (12), (16) and (17), wherein the HBsAg inhibitor and the TLR7 agonist can also selected from other HBsAg inhibitors and TLR7 agonists including small molecules or large molecules.
  • TLR7 agonists include, but not limited to, Imiquimod, Resiquimod, PF-4878691, SM-276001, ANA975, ANA773 and GS9620.
  • Another embodiment of the present invention is (19) a pharmaceutical composition according to any one of embodiments (1), (12), (16), (17) and (18), wherein the composition additionally comprising one or more other antiviral agents, which include, but not limited to nucleos(t)ide analogues such as Lamivudine , Adefovir dipivoxil, Entecavir, Telbivudine, Clevudine, Tenofovir disoproxil and Tenofovir disoproxil fumarate; interferons such as Roferon A ® , Intron A ® , Pegasys ® , Pegintron ® and Pai Ge Bin.
  • nucleos(t)ide analogues such as Lamivudine , Adefovir dipivoxil, Entecavir, Telbivudine, Clevudine, Tenofovir disoproxil and Tenofovir disoproxil fumarate
  • interferons such as Roferon A
  • Typical dosages of an HBsAg inhibitor and/or a TLR7 agonist can be in the ranges recommended by the manufacturer, 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 v tro responsiveness of the
  • Another embodiment of the present invention is (20) a method for manufacturing a medicament for treatment or prophylaxis of hepatitis B virus infection, characterized in that an HBsAg inhibitor and a TLR7 agonist are used in the medicament.
  • a further embodiment of the present invention is (21) the method according to
  • co -administer refers to any administration of the HBsAg inhibitor and interferon 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 TLR7 agonist 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.
  • Another further embodiment of the present invention is (22) the method according to embodiment (xx) or (xxi), wherein the HBsAg inhibitor and the TLR7 agonist are intended for administration to a subject by the same route or different routes.
  • Another further embodiment of the present invention is (23) the method for according to any one of embodiments (20) to (22), wherein the HBsAg inhibitor and the TLR7 agonist are intended for administration to a subject by parenteral or oral administration.
  • Another further embodiment of the present invention is (24) the method according to any one of embodiments (20) to (23), wherein the administration of the HBsAg inhibitor and the TLR7 agonist 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 is (25) the method according to any one of embodiments (20) to (24), wherein HBsAg inhibitor is a compound of formula (I), or formula (II), or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • a further embodiment of the present invention is (26) the method according to
  • HBsAg inhibitor is (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo [a] quino lizine- 3 -carboxylic acid ;
  • the HBsAg inhibitor is (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • Another embodiment of the present invention is (27) the method according to any one of embodiments (20) to (26), wherein the TLR7 agonist is a compound of formula (III) or formula (IV), or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • a further embodiment of the present invention is (28) the method according to embodiment (27), wherein the TLR7 agonist is
  • TLR7 agonist is any pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • TLR7 agonist is any pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • the TLR7 agonist is any pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • the TLR7 agonist is any pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • the TLR7 agonist is
  • Another further embodiment of the present invention is (29) a method for manufacturing a medicament for treatment or prophylaxis of hepatitis B virus infection, characterized in that the medicament is
  • a further embodiment of the present invention (30) is the method according to any one of embodiments (20) to (29), wherein the medicament additionally comprises one or more other antiviral agents, which include, but not limited to nucleos(t)ide analogues such as Lamivudine , Adefovir dipivoxil, Entecavir, Telbivudine, Clevudine, Tenofovir disoproxil and Tenofovir disoproxil fumarate; interferons such as Roferon A®, Intron A®, Pegasys®, Pegintron® and Pai Ge Bin.
  • nucleos(t)ide analogues such as Lamivudine , Adefovir dipivoxil, Entecavir, Telbivudine, Clevudine, Tenofovir disoproxil and Tenofovir disoproxil fumarate
  • interferons such as Roferon A®, Intron A®, Pegasys®, Peg
  • kits comprising a container comprising an HBsAg inhibitor and a TLR7 agonist, said kit can further comprise a sterile diluent.
  • a further embodiment of the present invention is (32) the kit according to embodiment
  • Another embodiment of the present invention is (33) the kit according to embodiment (31) or (32), wherein the HBsAg inhibitor is
  • the HBsAg inhibitor is (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid; or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • Another embodiment of the present invention is (34) the kit according to any one of embodiments (31) to (33), wherein the TLR7 agonist is
  • Another embodiment of the present invention relates to (35) a kit containing a combination of (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-
  • Another embodiment of the present invention relates to (36) 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 a TLR7 agonist; or vice versa.
  • a further embodiment of the present invention relates to (37) the method according to embodiment (36),wherein the HBsAg inhibitor is
  • the HBsAg inhibitor is (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • Another embodiment of the present invention relates to (38) the method according to embodiment (36) or (37), wherein the TLR7 agonist is
  • TLR7 agonist is any pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • TLR7 agonist is any pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • the TLR7 agonist is any pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • the TLR7 agonist is any pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
  • the TLR7 agonist is
  • a further embodiment of the present invention relates to (39) the method according to any one of embodiments (36) to (38), wherein the HBsAg inhibitor and the TLR7 agonist used in the subject are
  • Another embodiment of the present invention relates to (40) a method for the treatment or prophylaxis of hepatitis B virus infection, comprising administration to a subject with an effective amount of composition of
  • Another embodiment of the present invention relates to (41) the use of pharmaceutical composition according to any one of embodiments (1) to (19) as an antiviral medicament, in particular as the medicament for treatment or prophylaxis of hepatitis B virus infection.
  • Another embodiment of the present invention relates to (42) the use of an HBsAg inhibitor and a TLR7 agonist for the manufacture of pharmaceutical composition according to any one of embodiments (1) to (19) as an antiviral medicament, in particular the medicament for treatment or prophylaxis of hepatitis B virus infection.
  • Step 1 preparation of [(2R)-2-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a- tetrahydrofuro[2,3- ⁇ i] [l,3]dioxol-5-yl] -2-hydroxy-ethyl] 4-methylbenzenesulfonate
  • Step 2 preparation of (3aR,5S,6aR)-2,2-dimethyl-5-[(2R)-oxiran-2-yl]-3a,5,6,6a- tetrahydrofuro[2,3- ⁇ i] [1,3] dioxole
  • Step 3 preparation of (lR)-l-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a- tetrahydrofuro[2,3-rf][l,3]dioxol-5-yl]propan-l-ol
  • Step 4 preparation of [(lS)-l-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a- tetrahydrofuro[2,3-rf][l,3]dioxol-5-yl]propyl] 4-nitrobenzoate
  • Step 5 preparation of (lS)-l-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a- tetrahydrofuro[2,3-rf][l,3]dioxol-5-yl]propan-l-ol
  • Step 6 preparation of [(lS)-l-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a- tetrahydrofuro[2,3-rf][l,3]dioxol-5-yl]propyl] acetate
  • Step 7 preparation of [(3R,5S)-2-acetoxy-5-[(lS)-l-acetoxypropyl]tetrahydrofuran-3- yl] acetate OAc
  • Step 8 preparation of [(2R,3R,5S)-5-[(lS)-l-acetoxypropyl]-2-(5-amino-2-oxo- thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl] acetate
  • Step 9 preparation of [(15)-l-[(25,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5- ⁇ i]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl] acetate
  • Step 1 preparation of [(2R,3R,55)-5-[(15)-l-acetoxypropyl]-2-(5-amino-2,7-dioxo-6H- thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl] acetate
  • Step 2 preparation of 5-amino-3-[(2/i,3/i,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione
  • a stable HEK293-Blue-hTLR-7 cell line was purchased from InvivoGen (Cat.#: hkb-htlr7, San Diego, California, USA). These cells were designed for studying the stimulation of human TLR7 by monitoring the activation of NF- ⁇ .
  • a SEAP (secreted embryonic alkaline phosphatase) reporter gene was placed under the control of the IFN- ⁇ minimal promoter fused to five NF-KB and AP-1 -binding sites. The SEAP was induced by activating NF- ⁇ and AP-1 via stimulating HEK-Blue hTLR7 cells with TLR7 ligands. Therefore the reporter expression was regulated by the NF-KB promoter upon stimulation of human TLR7 for 20 hours.
  • the cell culture supernatant SEAP reporter activity was determined using QUANTI-BlueTM kit (Cat.#: rep-qbl, Invivogen, San Diego, Ca, USA) at a wavelength of 640 nm, a detection medium that turns purple or blue in the presence of alkaline phosphatase.
  • HEK293-Blue-hTLR7 cells were incubated at a density of 250,000-450,000 cells/mL in a volume of 180 ⁇ ⁇ in a 96- well plate in Dulbecco's Modified Eagle's medium (DMEM) containing 4.5 g/L glucose, 50 U/mL penicillin, 50 mg/mL streptomycin, 100 mg/mL Normocin, 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum for 24 h.
  • DMEM Dulbecco's Modified Eagle's medium
  • the HEK293- Blue-hTLR-7 cells were incubated with addition of 20 ⁇ ⁇ test compound in a serial dilution in the presence of final DMSO at 1% and perform incubation under 37 °C in a C0 2 incubator for 20 hours. Then 20 ⁇ ⁇ of the supernatant from each well was incubated with 180 ⁇ ⁇ Quanti-blue substrate solution at 37°C for 2 hours and the absorbance was read at 620-655 nm using a spectrophotometer.
  • TLR7 activation leads to downstream NF-KB activation has been widely accepted, and therefore similar reporter assay was also widely used for evaluating TLR7 agonist (Tsuneyasu Kaisho and Takashi Tanaka, Trends in Immunology, Volume 29, Issue 7, July 2008, Pages 329. sci; Hiroaki Hemmi et al, Nature Immunology 3, 196 - 200 (2002).
  • the TLR7 agonism activity in HEK293- hTLR-7 assay of compound 11 was 446 ⁇
  • the TLR7 agonism activity in HEK293- hTLR-7 assay of compound 12 was 72 ⁇
  • the TLR7 agonism activity in HEK293- hTLR-7 assay of compound 14 was 7.9 ⁇ .
  • Cryopreserved hepatocytes plating medium (Cat.#: PY-HMD-01) was purchased from RILD Research Institute for Liver Diseases (Shanghai) Co. Ltd.
  • Cryopreserved human hepatocyte (Cat.#: X008005, Lot#:VRR) was purchased from In Vitro Technologies (Baltimore, MD).
  • the stock hepatocyte suspension was prepared from cryopreserved hepatocytes in plating medium at the concentration of 1.8x10 6 cells/mL.
  • Working solutions of compounds Compounds were dissolved in DMSO to make 50 mM stock solutions. 10 ⁇ ⁇ of the stock solution was diluted to 5 mL plating medium to get a 100 ⁇ working solution.
  • Reaction suspensions were prepared in 24-well cell culture plate by mixing 200 ⁇ ⁇ of hepatocytes suspension (Cyno or human) and 200 ⁇ ⁇ of working solution. The final incubation contained 0.9xl0 6 cells/ mL and 50 ⁇ compound. The above mixtures were incubated at 37 °C in a humidified 5% C0 2 atmosphere, with a 150 rpm shaking.
  • the calibration curves were prepared in the following way. To a 200 ⁇ ⁇ of cell suspension (cell density of 1.8 million cells/ mL), 198 ⁇ ⁇ of hepatocyte plating medium and 2 ⁇ L ⁇ of the appropriate concentration of the compound in DMSO were added. Samples were mixed thoroughly and 200 ⁇ ⁇ of the mixture was transferred to 400 uL of the stop solution (see above). The standard curve range is from 1 ⁇ to 25 ⁇ .
  • Table 2 Concentration of the metabolites formed in human hepatocytes after 3 -hour incubation of 50 ⁇ of prodrugs.
  • Blood from healthy in-house volunteers was obtained through Employee Health and Services from Roche Basel. Whole blood from 6 donors was used for this study. All volunteers were properly consented before becoming an active donor. Whole blood was collected in sodium heparin green-top tubes (Becton Dickinson, cat #367874).
  • hPBMCs from whole blood were purified by ficoll gradient centrifugation using 50 mL Accuspin tubes (Sigma, cat# A2055) specially designed with two chambers separated by a porous high-density polyethylene barrier ("frit").
  • the lower chamber contains 15mL of Ficoll- Paque Plus (GE Healthcare Life Sciences, cat#17-1440-02).
  • hPBMCs were seeded at 5xl0 6 cells/mL in RPMI 1640 medium supplemented with 10% FBS and 100 units/mL penicillin and 100 ⁇ g/mL streptomycin in a 6 well plate (2 mL of cell suspension per well).
  • hPBMCs were incubated for 3 h at 37°C and subsequently treated with 100 ⁇ of TLR7 agonist Compound 11 or 12.5 ⁇ of TLR7 agonist Compound 12 at a final DMSO concentration of 0.2% each. The treated cells were incubated for 16h at 37°C before supernatants were harvested.
  • TLR7 agonist hPBMC conditioned medium from all 6 blood donors was pooled, aliquoted and stored at -80°C, which was used later in Part 1.6 "Infection and compound treatment of hepaRG cells" of this Example 6.
  • HepG2.2.15 cells were cultured in DMEM+Glutamax I (Gibco, #21885) supplemented with 10% FBS, 1% Pen/Strep (Gibco, #15140) and 250 ⁇ ⁇ , G-418 (Gibco, #10131027) 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 became confluent, medium was changed to DMEM+Glutamax I with 1% DMSO and 2.5% FBS.
  • the medium was changed to DMEM/F12+Glutamax I (Gibco, #31331) supplemented with MEM non-essential amino acids (6 mL, Gibco, #11140), Pen/Strep (6 mL), sodium pyruvate (6 mL), DMSO (9 mL) and FBS (10 mL) (all per 500 mL medium).
  • the medium was changed every 3 days and the supernatants were harvested for 2 weeks. Virus was concentrated from the supernatants by PEG precipitation and the titer (genome equivalent (GE) / mL) was determined by qPCR.
  • the supernatants were mixed with 40% PEG solution at a ratio of 4: 1, incubated on a shaker at 4°C overnight and then centrifuged using 50 mL falcon tubes at 4°C for one hour at 3724 g (RCF). The supernatant was discarded and the centrifugation step was repeated with new supernatant reusing the tubes until all PEG- precipitated supernatant was processed. The pellets were re-suspended in William's E Medium
  • HepaRG cells Biopredic International, Saint-Gregoire, France
  • the medium was changed to differentiation medium.
  • the medium was changed twice a week up to 2 weeks.
  • the cells were trypsinized and seeded into collagenated 96 well plates (50,000 cells/well in 100 ⁇ ) in differentiation medium.
  • the cells were cultured at least 5 days in the 96 well plates before they were infected with HBV.
  • the medium was removed and differentiation medium (120 ⁇ ) containing 4% PEG-8000 and virus stock (20 to 30 GE/cell) was added.
  • the cells were cultured at 37 °C for 16 to 20 h before the medium was removed, the cells were washed 4 times with PBS and differentiation medium (120 ⁇ ) was added.
  • the medium was removed and 100 ⁇ ⁇ differentiation medium was added to each well.
  • 3-fold serial dilutions (30 ⁇ ⁇ Drug A to 60 ⁇ ⁇ DMSO) of Drug A (HBsAg inhibitor) were prepared in 100% DMSO starting with 90 ⁇ , undiluted Drug A (400- fold concentration of highest test concentration).
  • Drug A tested is Compound 1A; Drug B tested is Compound 11 hPBMC conditioned medium or Compound 12 hPBMC conditioned medium.
  • the concentration ranges tested were 30 nM to 0.37 nM for Compound 1A, and 0.008 to 0.000001 DF for Compound 11 hPBMC conditioned medium or Compound 12 hPBMC conditioned medium.
  • the medium was replaced by drug medium at day 6 and 8 post infection and at day 11 post infection the 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 1.7 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 ⁇ ⁇ .
  • Quantification of extracted HBV DNA was performed using a Taqman qPCR machine (ViiA7, life technologies). Each DNA sample was tested in duplicate in the PCR. 5 ⁇ ⁇ of DNA sample were added to 15 ⁇ ⁇ of PCR mastermix containing 10 ⁇ ⁇ TaqMan Gene Expression Master Mix (Applied Biosystems, Cat. no.
  • Forward core primer (F3_core): CTG TGC CTT GGG TGG CTT T
  • Reverse primer AAG GAA AG A AGT CAG AAG GCA AAA
  • Taqman probe (P3_core): 56-FAM/AGC TCC AAA /ZEN/TTC TTT ATA AGG GTC GAT GTC CAT G/3IABkFQ
  • this program converts the dose-effect curves for each drug or drug combination to median effect plots.
  • a combination index (CI) for each experimental combination was then calculated by the following equation (for mutually nonexclusive interactions): [(D) 1 /(Dx) 1 ]+[(D) 2 /(Dx) 2 ]+[(D) 1 (D) 2 /(Dx) 1 (Dx) 2 ] where (Dx)i and (Dx) 2 are the doses of drug 1 and drug 2 that have x effect when each drug is used alone, and (D)i and (D) 2 are the doses of drug 1 and drug 2 that have the same x effect when they are used in combination, respectively.
  • the software calculates the CIs at 50%, 75% and 90% antiviral effect of combinations.
  • 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 ECsos. 2 Results
  • Combination of Compound 1A with Compound 11 hPBMC conditioned medium, and combination of Compound 1A with Compound 12 hPBMC conditioned medium were tested for anti-HBV activity in HBV infected differentiated HepaRG cells.
  • Lamivudine with Lamivudine was set as the control combination.
  • the single compound inhibitory activities (EC 50 ) obtained in the combination studies were determined (Table 5).
  • the data from the combination study of Compound 1A and Compound 12 was analyzed using the CalcuSyn software (Table 6). The overall CI for three different concentration ratios was between 0.30 and 0.72, thus the combination of Compound 1A and Compound 12 was synergistic.
  • the data from the combination study of Compound 1A and Compound 12 was also analyzed using the Isobologram model ( Figure 2). The FIC values for the combination of Compound 1A and Compound 12 were plotted one against the other for each of the experiments. The analysis showed that most of the CI values were ⁇ 1. Therefore, the 5 combination of Compound 1A and Compound 12 was synergistic.

Abstract

The present invention is directed to compositions and methods for treating hepatitis B virus infection. In particular, the present invention is directed to a combination therapy comprising administration of an HBs Ag inhibitor and a TLR7 agonist for use in the treatment of chronic hepatitis B patient.

Description

Combination therapy of an HBsAg inhibitor and a TLR7 agonist
The present invention is directed to compositions and methods for treating hepatitis B virus infection. In particular, the present invention is directed to a combination therapy comprising administration of an HBsAg inhibitor and a TLR7 agonist for use in the treatment or prophylaxis of chronic hepatitis B virus infection. FIELD OF THE INVENTION
Chronic infection of Hepatitis B virus (HBV) is a serious public health problem
worldwide, with more than 240 million people chronically infected worldwide. 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 genomecccDNA 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. 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). 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
X protein and core protein, and are involved in the multiple steps of the viral life cycle. Many observations showed that several HBV viral proteins could counteract the initial host cellular response by interfering with the viral recognition signaling system. Among these, the excessive secretion of HBV empty subviral particles may participate to the maintenance of the
immunological tolerant state observed in chronically infected patients (CHB). HBsAg, as well as other HBV antigens, 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.
Gastroenterology, 2010, 138, 682-93;). Moreover 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
Gasteroenterology, 2013, Article ID 935295). Therefore, targeting HBsAg together with HBV DNA levels in CHB patients may significantly improve CHB patient immune reactivation and remission (Wieland, S. F. & F. V. Chisari. / Virol, (2005), 79, 9369-80; Kumar et al. / Virol, (2011), 85, 987-95; Woltman et al. PLoS One, (2011), 6, el5324; Op den Brouw et al.
Immunology, (2009b), 126, 280-9).
Toll- like receptors (TLRs) detect a wide range of conserved pathogen-associated molecular patterns (PAMPs). They play an important role of sensing invading pathogens and subsequent initiation of innate immune responses. There are 10 known members of the TLR family in human, which are type I transmembrane proteins featuring an extracellular leucine-rich domain and a cytoplasmic tail that contains a conserved Toll/ interleukin (IL)-l receptor (TIR) domain. Within this family, TLR3, TLR7, TLR8, and TLR9 are located within endosomes. TLR7 can be activated by binding to a specific small molecule ligand (i.e., TLR7 agonist) or its native ligand (i.e., single- stranded RNA, ssRNA). Following binding of ssRNA to TLR7, the receptor in its dimerized form is believed to undergo a structural change leading to the subsequent recruitment of adapter proteins at its cytoplasmic domain, including the myeloid differentiation primary response gene 88 (MyD88). Following the initiation of the receptor signalling cascade via the MyD88 pathway, cytoplasmic transcription factors such as interferon regulatory factor 7 (IRF-7) and nuclear factor kappa B (NF-κΒ) are activated. These transcription factors then translocate to the nucleus and initiate the transcription of various genes, e.g., IFN-a and other antiviral cytokine genes. TLR7 is predominately expressed on plasmacytoid cells, and also on B -cells. Altered responsiveness of immune cells might contribute to the reduced innate immune responses during chronic viral infections. Agonist-induced activation of TLR7 might therefore represent a novel approach for the treatment of chronic viral infections. (D. J Connolly and L. AJ O'Neill, Current Opinion in Pharmacology 2012, 12:510-518, P. A. Roethle et al, J. Med. Chem. 2013, 56, 7324-7333). Treatment with an oral, small- molecule TLR7 agonist is a promising approach that has the potential to provide greater efficacy and better tolerability (T. Asselah et al, Clin Liver Dis 2007, 11, 839-849).
HBsAg is a biomarker for prognosis and treatment response in CHB. Now, the standard of clinic cure of HBV infection is the loss and/or seroconversion of HBsAg. Even though PEG- IFN-a and nucleos(t)ide analogues 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. Hence, there is certainly an unmet medical need for treatments with improved success rate of inducing HBsAg loss, and/or HBeAg loss, and/or HBV DNA reduction, and/or HBV clearance and/or
seroconversion, and/or normalization of ALT, and/or promoting the production of anti-HBs to address the Hepatitis B virus (HBV) infections.
SUMMARY OF THE INVENTION
The present invention relates to a pharmaceutical composition comprising an HBsAg inhibitor and a TLR7 agonist, in a pharmaceutically acceptable carrier, for use in the treatment or prophylaxis of chronic hepatitis B virus infection.
In one embodiment, the "HBsAg inhibitor" is a compound of formula (I), (II) or any one of the compounds disclosed in patent applications WO2015/113990 and WO2016/071215, particularly 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-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid, (+)-6-ie/t-butyl- 10-methoxy-9-(3- methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid, (-)-6-ie/t-butyl- 10- methoxy-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid, (+)- 10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid, (-)-10-methoxy-6-(2-methoxy-l,l-dimethyl- ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid, (+)-6-(2- hydroxy- 1, 1 -dimethyl-ethyl)- 10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid, (-)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10- methoxy-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid, (+)- 10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid, (-)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)- 9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid, or
pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
In one embodiment, the "TLR7 agonist" herein is a compound of formula (III), (IV) or any one of the compounds disclosed in patent WO2006/066080 and patent application
WO2016/055553, particularly the "TLR7 agonist" herein is [(lS)-l-[(2S,4R,5R)-5-(5-amino-2- oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl] acetate; [(S)- [(2S,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]-cyclopropyl- methyl] acetate; 5-amino-3-(3'-deoxy-P-D-ribomranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one; 5- amino-3-(2'-0-acetyl-3'-deoxy-P-D-ribomranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one; 5- amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; 5-amino-3- [(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5- d]pyrimidine-2,7-dione ; 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one; or 5-amino-3-[(2S,5R)-2- [(lS)-cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7- dione or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
BRIEF DESCRIPTION OF THE FIGURE(S)
Figure 1: Isobologram of FIC for the pair-wise checkerboard combination of Compound 1A and Compound 11 hPBMC conditioned medium (at the 50% effect level). The diagonal lane connecting points (0, 1) and (1, 0) represents additivity (CI =1). Data points below this lane show synergism, data points above show antagonism. The results shown are mean values from 3 independent experiments.
Figure 2: Isobologram of FIC for the pair-wise checkerboard combination of Compound 1A and Compound 12 hPBMC conditioned medium (at the 50% effect level). The diagonal lane connecting points (0, 1) and (1, 0) represents additivity (CI =1). Data points below this lane show synergism, data points above show antagonism. The results shown are mean values from 3 independent experiments. DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
As used herein, the term "Ci_6alkyl" refers to a monovalent linear or branched saturated hydrocarbon group of 1 to 6 carbon atoms. In particular embodiments, Ci_6alkyl has 1 to 6 carbon atoms, and in more particular embodiments 1 to 4 carbon atoms. Examples of Ci_6alkyl include methyl, ethyl, propyl, isopropyl, n-butyl, iso -butyl, sec-butyl or tert-butyl.
As used herein, the term "halo" or "halogen" are used interchangeably herein and refer to fluoro, chloro, bromo, or iodo. Halogen is particularly fluorine, chlorine or bromine.
As used herein, the term "Ci_6alkoxy" refers to a group of Ci_6alkyl-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_6alkoxy" groups are methoxy and ethoxy and more particularly methoxy.
As used herein, the term "C3_7Cycloalkyl" refers to a saturated carbon ring containing from 3 to 7 carbon atoms, particularly from 3 to 6 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like. Particular "C3_7Cycloalkyl" groups are cyclopropyl, cyclopentyl and cyclohexyl. As used herein, the term "C2-6alkenyl" 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 "C2-6alkenyl" group is allyl.
As used herein, the term "C2-6alkynyl" 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 "C2-6alkynyl" groups are ethynyl, 1- propynyl and propargyl.
As used herein, the term "CXH2X" alone or in combination signifies a saturated, linear- or branched chain alkyl group containing 1 to 6, particularly 1 to 4 carbon atoms. As used herein, the term "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.
As used herein, the term 'W-containing monocyclic heteroaryl" refers to a monocyclic heteroaryl wherein at least one of the heteroatoms is N. Examples for 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.
As used herein, the term "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" containing in particular from 3 to 7 ring atoms include, but not limited to, aziridinyl, oxiranyl, azetidinyl, oxetanyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, tetrahydro -thienyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl, morpholinyl, thio morpholinyl, l, l-dioxo-thiomorpholin-4-yl, azepanyl, diazepanyl, homopiperazinyl, or oxazepanyl. Bicyclic heterocyclyl can be bicyclic fused ring or bicyclic bridged ring. Examples for bicyclic heterocyclyl are 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[3.3.1]nonyl, or difluoroazabicyclo[3.2.1]octyl. Monocyclic and bicyclic heterocyclyl can be further substituted by halogen, Ci_6alkyl, cyano, carboxy, carboxyCi_6alkyl.
The term "heterocyclic amino" refers to an amino group with the nitrogen atom on the heterocyclic ring, wherein "heterocyclic" ring is as defined above.
As used herein, the term "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. As used herein, the term "enantiomers" refers to two stereoisomers of a compound which are non-superimpo sable mirror images of one another.
As used herein, 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. As used herein, 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.
The term "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-toluenesulfonic acid, and salicyclic acid.
The term "pharmaceutically acceptable base addition salt" refers to those pharmaceutically acceptable salts formed with an organic or inorganic base. Examples of 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. As used herein, "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.
As used herein, "combo" refers to combination. As used herein, "HBV DNA" refers to DNA material of HBV.
As used herein, "HBsAg" refers to hepatitis B surface antigen.
As used herein, "HBeAg" refers to hepatitis B e antigen.
As used herein, "anti-HBs" refers to antibodies against HBsAg.
As used herein, "HBsAg inhibitor" refers to a compound that inhibits 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.
As used herein, "TLR7" refers to the Toll-like receptor 7 of any species of origin (e.g., human, murine, woodchuck etc.).
As used herein, "TLR7 agonist" refers to a compound that acts as an agonist of TLR7. Unless otherwise indicated, a TLR7 agonist can include the compound in any pharmaceutically acceptable form, including any isomer (e.g., diastereomer or enantiomer), salt, solvate, polymorph, and the like. The TLR agonism for a particular compound may be determined in any suitable manner. For example, assays for detecting TLR agonism of test compounds are described, for example, in U.S. Provisional Patent Application Ser. No. 60/432,650, filed Dec. 11, 2002, and recombinant cell lines suitable for use in such assays are described, for example, in U.S. Provisional Patent Application Ser. No. 60/432,651, filed Dec. 11, 2002. The term "therapeutically 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 a nucleos(t)ide analogue, in a pharmaceutically acceptable carrier.
Compounds of the general formula (I), (II), (III) and (IV) which contain one or several chiral centers can either be present as racemates, diastereomeric mixtures, or optically active single isomers. The racemates can be separated according to known methods into the
enantiomers. Particularly, 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.
One embodiment of the present invention relates to (1) a pharmaceutical composition comprising an HBsAg inhibitor and a TLR7 agonist, in a pharmaceutically acceptable carrier.
Another embodiment of the present invention is (2) the pharmaceutical composition according to embodiment (1), wherein the "HBsAg inhibitor" is a compound of formula (I):
Figure imgf000011_0001
wherein
R1 is hydrogen, halogen, C1-6alkyl, Ci_6alkylamino or Ci^alkoxy; R is hydrogen; halogen; Ci_6alkyl, which is unsubstituted or once, twice or three times substituted by fluoro; Ci-6alkoxy, which is unsubstituted or once, twice or three times substituted by fluoro; cyano; C3_7cycloalkyl; hydroxy or phenyl- CxH2X-0-;
R is hydrogen;
halogen;
Ci_6alkyl, which is unsubstituted or once, twice or three times substituted by fluoro;
cyano;
pyrrolidinyl;
amino;
phenyl-CxH2x-N(Ci_6alkyl)- ;
Ci_6alkoxycarbonylpiperazinyl;
or R 7 -O- , wherein R 7 is hydrogen; Ci_6alkyl, which is unsubstituted or substituted with one to three substituents independently selected from fluoro, hydroxy and C2-6alkenyl; Ci_ 6alkoxyCi_6alkyl; Ci_6alkoxyCi-6alkoxyCi_6alkyl; aminoCi-salkyl; Ci_
6alkylcarbonylaminoCi-8alkyl; Ci_6alkylsulfonylaminoCi_8alkyl; Ci_6alkylsulfanylCi_6alkyl; Ci-6alkylsulfonylCi_6alkyl; cyanoCi_6alkyl; C3_7cycloalkylCi_6alkyl; cyanoC3_7cycloalkylCi_ 6alkyl; phenylCi-6alkyl; pyrrolidinylcarbonylCi_6alkyl; C2-6alkynyl; hydroxyCi_6alkylC2- 6alkynyl; aminoCi_6alkoxyCi_6alkyl; Ci_6alkylaminoCi-6alkoxyCi_6alkyl; diCi_
6alkylaminoCi-6alkoxyCi-6alkyl; carboxyCi_6alkyl; or Ci_6alkoxycarbonylaminoCi_8alkyl; heteroarylCi_6alkyl, wherein heteroaryl is N-containing monocyclic heteroaryl; or heterocyclylCi_6alkyl, wherein heterocyclyl is monocyclic heterocyclyl;
R4 is hydrogen, halogen, Ci-6alkyl, cyano or
Figure imgf000012_0001
R5 is hydrogen or Ci_6alkyl;
R6 is hydrogen; Ci_6alkyl, which is unsubstituted or once, twice or three times substituted by fluoro; C3_7cycloalkyl, which is unsubstituted or once, twice or three times substituted by fluoro or Ci_6alkyl; or phenyl-CxH2x-;
x is 1-6;
or pharmaceutically acceptable salts, or enantiomers thereof.
Compounds of formula (I), Compounds 1A to 3A and Compounds IB to 3B can be obtained according to the synthetic procedures disclosed in WO2015/113990.
A further embodiment of the present invention is (3) the pharmaceutical composition according to embodiment (2), wherein the "HBsAg inhibitor" 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- 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-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3- carboxylic acid;
(+)-6-ieri-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-6-ieri-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
or any other compound disclosed in WO2015/113990;
or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
Another embodiment of the present invention is (4) the pharmaceutical composition according to embodiment (1), wherein the "HBsAg inhibitor" is a compound of formula (II):
Figure imgf000013_0001
wherein
, R9, R10 and Ru are independently selected from hydrogen, halogen, Ci-6alkyl, diCi
6alkylamino, cyano, N-containing monocyclic heterocyclyl and OR14, wherein
R is hydrogen; Ci_6alkyl; or Ci-6alkyl which is substituted once or more times by fluoro, C3_7cycloalkyl, phenyl, hydroxyl, amino, Ci_6alkoxy, Ci_6alkylsulfanyl, Ci_6alkylsulfonyl, diCi_6alkylamino, Ci_6alkoxycarbonylamino, monocyclic heterocyclyl, pyrazoyl or imidazolyl;
R is hydrogen or Chalky; 13
R is hydrogen, Ci_6alkyl, phenyl-CxH2X-, Ci_6alkylcarbonyl, Ci_6alkylsulfonyl, benzoyl or monocyclic heterocyclyl, wherein
x is 1-6;
W is a bond, CyH2yC(R15)(R16)CzH2z or CyH2yCH(R15)CH(R16)CzH2z, wherein
R15 and R16 are independently selected from hydrogen, fluoro, hydroxy and Ci-6alkyl, y is 0-6;
z is 0-6;
X is a bond; O; S; S(0)2; or NR17, wherein R17 is hydrogen, C1-6alkyl;
or R 13 and R 17 , together with the nitrogen to which they are attached, form monocyclic
heterocyclyl;
or pharmaceutically acceptable salts, or enantiomers thereof.
Compounds of formula (II), Compounds 4 A to 6 A and Compounds 4B to 6B can be obtained according to the synthetic procedures disclosed in WO2016/071215.
A further embodiment of the present invention is (5) the pharmaceutical composition according to embodiment (4), wherein the "HBsAg inhibitor" is
(+)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(+)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(+)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
or any other compound disclosed in WO2016/071215;
or pharmaceutically acceptable salt, enantiomer or diastereomer thereof. Unless otherwise indicated, an HBsAg inhibitor is 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. Another embodiment of the present invention is (6) the pharmaceutical composition according to any one of embodiments (1) to (5), wherein the "TLR7 agonist" is a compound of formula (III):
Figure imgf000015_0001
wherein
R 18 is hydroxy, Ci-6alkyl, haloCi_6alkyl, Ci_6alkylcarbonyl-0-, Ci-6alkyl-S-, azido, cyano, C2-6alkenyl, Ci_6alkylsulfonyl-NH-, (C1-6alkyl)2N-, Ci_6alkylcarbonyl-NH- or heterocyclic amino;
R19 is hydrogen, C1-6alkyl, Ci-6alkoxyCi_6alkyl, C3_7cycloalkyl, C2-6alkynyl, C2-6alkenyl, benzyl and thiophenyl;
R 20 is hydrogen or Ci_6alkylcarbonyl;
R 21 is hydrogen or hydroxy;
or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
A further embodiment of the present invention is (7) the pharmaceutical composition according to embodiment (6), wherein the "TLR7 agonist" is
[(lS)-l-[(2S,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy- tetrahydrofuran-2- yl] propyl] acetate ;
5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one;
5-amino-3-(2'-0-acetyl-3'-deoxy-P-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one;
5-amino-3-(3'-deoxy-P-D-ribomranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione;
5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione; or
5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2- yl]thiazolo[4,5-d]pyrimidin-2-one;
or pharmaceutically acceptable salt, enantiomer or diastereomer thereof. Another embodiment of the present invention is (8) the pharmaceutical composition according to any one of embodiments (1) to (5), wherein the "TLR7 agonist" is a compound of formula (IV):
Figure imgf000016_0001
wherein
R 2"2 and 23 are independently selected from hydrogen, C2-6alkenyl and Ci_6alkyl;
24 and R 2"5J are independently selected from hydrogen, Ci_6alkyl, C3_7cycloalkyl, C3_ 7cycloalkylC2-6alkynyl, C2-6alkenyl, C2-6alkynyl and 2-thiophenyl;
R26 is hydrogen or Ci_6alkylcarbonyl;
R 2"7 is hydro gen or hydroxy;
or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
Compounds of formula (IV), Compound 8 and Compound 14 can be obtained according to the synthetic procedures disclosed in WO2016/055553.
A further embodiment of the present invention is (9) the pharmaceutical composition according to embodiment (8), wherein the "TLR7 agonist" is
[(S)-[(2S,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]- cyclopropyl-methyl] acetate;
5-amino-3-[(2S,5R)-2-[(lS)-cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione;
or any other compound disclosed in WO2016/055553;
or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
Another embodiment of the present invention is (10) the pharmaceutical composition according to any one of embodiments (1) to (5), wherein the TLR7 agonist is [(lS)-l-[(2S,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy- tetrahydrofuran-2- yl] propyl] acetate ;
[(S)-[(2S,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]- cyclopropyl-methyl] acetate;
5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one;
5-amino-3-(2'-0-acetyl-3'-deoxy-P-D-ribomranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one; 5-amino-3-(3'-deoxy-P-D-ribomranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione;
5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2- yl]thiazolo[4,5-d]pyrimidin-2-one; or
5-amino-3-[(2S,5R)-2-[(lS)-cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione;
or pharmaceutically acceptable salt, enantiomer or diastereomer thereof. Another embodiment of the present invention is (11) the pharmaceutical composition according to any one of embodiments (1) to (5), wherein the "TLR7 agonist" is any one of the compounds disclosed in patent WO2006/066080 and patent application WO2016/055553.
After administration, compounds of formula (III) or formula (IV) or compounds in patent WO2006/066080 and patent application WO2016/055553 are metabolized into their active forms as TLR7 agonists.
Another embodiment of the present invention is (12) the pharmaceutical composition according to embodiment (1), wherein the composition consists of an HBsAg inhibitor and a TLR7 agonist, in a pharmaceutically acceptable carrier.
Another embodiment of the present invention is (13) the pharmaceutical composition according to embodiment (1) or (12), wherein the HBsAg inhibitor and the TLR7 agonist are independently selected from Table 1.
Table 1: List of HBsAg inhibitors and TLR7 agonists
Compoun
Class Compound Name Structure
d Number
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
d]pyrimidine-2,7-dione
A further embodiment of the present invention is (14) the pharmaceutical composition according to embodiment (1) or (12), wherein the composition is selected from any one of the following combinations:
Compound 1A and Compound 7; Compound 1 A and Compound 8; Compound 1A and Compound 9; Compound 1 A and Compound 10; Compound 1A and Compound 11 ; Compound 1A and Compound 12; Compound 1A and Compound 13; Compound 1A and Compound 14; Compound IB and Compound 7; Compound IB and Compound 8; Compound IB and Compound 9; Compound IB and Compound 10; Compound IB and Compound 11 ; Compound IB and Compound 12; Compound IB and Compound 13; Compound IB and Compound 14; Compound 2A and Compound 7; Compound 2A and Compound 8; Compound 2A and Compound 9; Compound 2A and Compound 10; Compound 2A and Compound 11 ; Compound 2A and Compound 12; Compound 2A and Compound 13; Compound 2A and Compound 14; Compound 2B and Compound 7; Compound 2B and Compound 8; Compound 2B and Compound 9; Compound 2B and Compound 10; Compound 2B and Compound 11 ; Compound 2B and Compound 12; Compound 2B and Compound 13; Compound 2B and Compound 14; Compound 3 A and Compound 7; Compound 3 A and Compound 8; Compound 3A and Compound 9; Compound 3A and Compound 10; Compound 3A and Compound 11 ; Compound 3A and Compound 12; Compound 3A and Compound 13; Compound 3A and Compound 14; Compound 3B and Compound 7; Compound 3B and Compound 8; Compound 3B and Compound 9; Compound 3B and Compound 10; Compound 3B and Compound 11 ; Compound 3B and Compound 12; Compound 3B and Compound 13; Compound 3B and Compound 14; Compound 4A and Compound 7; Compound 4A and Compound 8; Compound 4A and Compound 9; Compound 4A and Compound 10; Compound 4A and Compound 11 ; Compound 4A and Compound 12; Compound 4A and Compound 13; Compound 4A and Compound 14; Compound 4B and Compound 7; Compound 4B and Compound 8; Compound 4B and Compound 9; Compound 4B and Compound 10; Compound 4B and Compound 11 ; Compound 4B and Compound 12; Compound 4B and Compound 13; Compound 4B and Compound 14; Compound 5 A and Compound 7; Compound 5 A and Compound 8; Compound 5A and Compound 9; Compound 5A and Compound 10; Compound 5A and Compound 11 ; Compound 5A and Compound 12;
Compound 5A and Compound 13; Compound 5A and Compound 14;
Compound 5B and Compound 7; Compound 5B and Compound 8;
Compound 5B and Compound 9; Compound 5B and Compound 10;
Compound 5B and Compound 11 ; Compound 5B and Compound 12;
Compound 5B and Compound 13; Compound 5B and Compound 14;
Compound 6 A and Compound 7; Compound 6 A and Compound 8;
Compound 6A and Compound 9; Compound 6A and Compound 10;
Compound 6A and Compound 11 ; Compound 6A and Compound 12;
Compound 6A and Compound 13; Compound 6A and Compound 14;
Compound 6B and Compound 7; Compound 6B and Compound 8;
Compound 6B and Compound 9; Compound 6B and Compound 10;
Compound 6B and Compound 11 ; Compound 6B and Compound 12;
Compound 6B and Compound 13; and Compound 6B and Compound 14;
in a pharmaceutically acceptable carrier.
Any one of Compounds 1A to 6A, IB to 6B and 7 to 14 of the aforementioned
combinations can be replaced by its corresponding pharmaceutically acceptable salt, enantiomer or diastereomer, which is another aspect of this invention.
Another further embodiment of the present invention is (15) the pharmaceutical composition according to embodiment (1) or (12), wherein the composition is selected from any one of the following combinations:
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and [(lS)-l-[(2S,4R,5R)-5-(5-amino-2-oxo- thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl] acetate;
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and [(S)-[(2S,5R)-5-(5-amino-2-oxo-thiazolo[4,5- d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]-cyclopropyl-methyl] acetate;
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H- thiazolo[4,5-d]pyrimidin-2-one; (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- di ydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(2'-0-acetyl-3'-deoxy-P-D- ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one;
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)- 3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione;
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)- l-hydroxypropyl]tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one;
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2S,5R)-2-[(lS)- cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(-)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and [(lS)-l-[(2S,4R,5R)-5-(5-amino-2-oxo- thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl] acetate;
(-)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and [(S)-[(2S,5R)-5-(5-amino-2-oxo-thiazolo[4,5- d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]-cyclopropyl-methyl] acetate;
(-)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H- thiazolo[4,5-d]pyrimidin-2-one;
(-)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(2'-0-acetyl-3'-deoxy-P-D- ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one;
(-)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)- 3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione;
(-)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione; (-)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- di ydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one;
(-)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2S,5R)-2-[(lS)- cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(+)-10-chloro-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine- 3-carboxylic acid and [(lS)-l-[(2S,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4- hydroxy-tetrahydrofuran-2-yl]propyl] acetate;
(+)-10-chloro-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine- 3-carboxylic acid and [(S)-[(2S,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-l,3- oxathiolan-2-yl] -cyclopropyl- methyl] acetate;
(+)-10-chloro-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine- 3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2- one;
(+)-10-chloro-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine- 3-carboxylic acid and 5-amino-3-(2'-0-acetyl-3'-deoxy-P-D-ribofuranosyl)-3H-thiazolo[4,5- d]pyrimidin-2-one;
(+)-10-chloro-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine- 3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin- 2,7-dione;
(+)-10-chloro-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine- 3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(+)-10-chloro-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine- 3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one;
(+)-10-chloro-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine- 3-carboxylic acid and 5-amino-3-[(2S,5R)-2-[(lS)-cyclopropyl(hydroxy)methyl]-l,3-oxathiolan- 5-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(-)-10-chloro-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3- carboxylic acid and [(lS)-l-[(2S,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4- hydroxy-tetrahydrofuran-2-yl]propyl] acetate; (-)-10-chloro-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3- carboxylic acid and [(S)-[(2S,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-l,3- oxathiolan-2-yl] -cyclopropyl- methyl] acetate;
(-)-10-chloro-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3- carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one;
(-)-10-chloro-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3- carboxylic acid and 5-amino-3-(2'-0-acetyl-3'-deoxy-P-D-ribofuranosyl)-3H-thiazolo[4,5- d]pyrimidin-2-one;
(-)-10-chloro-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3- carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin- 2,7-dione;
(-)-10-chloro-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3- carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydroi iran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(-)-10-chloro-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3- carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one;
(-)-10-chloro-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3- carboxylic acid and 5-amino-3-[(2S,5R)-2-[(lS)-cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5- yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(+)-6-tert-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and [(lS)-l-[(2S,4R,5R)-5-(5-amino-2-oxo- thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl] acetate;
(+)-6-tert-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and [(S)-[(2S,5R)-5-(5-amino-2-oxo-thiazolo[4,5- d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]-cyclopropyl-methyl] acetate;
(+)-6-tert-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H- thiazolo[4,5-d]pyrimidin-2-one;
(+)-6-tert-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(2'-0-acetyl-3'-deoxy-P-D- ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one; (+)-6-tert-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- di ydrobenzo[a]quinolizine-3-carboxylic acid and 5-airrino-3-(3'-deoxy-P-D-ribofuranosyl)- 3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione;
(+)-6-tert-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(+)-6-tert-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one;
(+)-6-tert-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2S,5R)-2-[(lS)- cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(-)-6-tert-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and [(lS)-l-[(2S,4R,5R)-5-(5-amino-2-oxo- thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl] acetate;
(-)-6-tert-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine- 3-carboxylic acid and [(S)-[(2S,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-l,3- oxathiolan-2-yl] -cyclopropyl- methyl] acetate;
(-)-6-tert-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine- 3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2- one;
(-)-6-tert-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine- 3-carboxylic acid and 5-amino-3-(2'-0-acetyl-3'-deoxy-P-D-ribofuranosyl)-3H-thiazolo[4,5- d]pyrimidin-2-one;
(-)-6-tert-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine- 3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin- 2,7-dione;
(-)-6-tert-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine- 3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydro xypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(-)-6-tert-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine- 3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one; (-)-6-tert-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7-di ydrobenzo[a]quinolizine- 3-carboxylic acid and 5-amino-3-[(2S,5R)-2-[(lS)-cyclopropyl(hydroxy)methyl]-l,3-oxathiolan- 5-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(+)- 10-methoxy-6-(2-methoxy- 1 , 1 -dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and [(lS)-l-[(2S,4R,5R)-5-(5-amino-2-oxo- thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl] acetate;
(+)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and [(S)-[(2S,5R)-5-(5-amino-2-oxo-thiazolo[4,5- d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]-cyclopropyl-methyl] acetate;
(+)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H- thiazolo[4,5-d]pyrimidin-2-one;
(+)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(2'-0-acetyl-3 '-deoxy-P-D- ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one;
(+)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)- 3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione;
(+)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(+)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one;
(+)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2S,5R)-2-[(lS)- cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(-)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and [(lS)-l-[(2S,4R,5R)-5-(5-amino-2-oxo- thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl] acetate;
(-)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and [(S)-[(2S,5R)-5-(5-amino-2-oxo-thiazolo[4,5- d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]-cyclopropyl-methyl] acetate; (-)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H- thiazolo[4,5-d]pyrimidin-2-one;
(-)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(2'-0-acetyl-3 '-deoxy-P-D- ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one;
(-)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)- 3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione;
(-)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(-)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydro xypropyl]tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one;
(-)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2S,5R)-2-[(lS)- cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(+)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and [(lS)-l-[(2S,4R,5R)-5-(5-amino-2-oxo- thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl] acetate;
(+)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and [(S)-[(2S,5R)-5-(5-amino-2-oxo-thiazolo[4,5- d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]-cyclopropyl-methyl] acetate;
(+)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H- thiazolo[4,5-d]pyrimidin-2-one;
(+)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(2'-0-acetyl-3'-deoxy-P-D- ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one;
(+)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)- 3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; (+)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(+)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one;
(+)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2S,5R)-2-[(lS)- cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(-)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and [(lS)-l-[(2S,4R,5R)-5-(5-amino-2-oxo- thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl] acetate;
(-)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and [(S)-[(2S,5R)-5-(5-amino-2-oxo-thiazolo[4,5- d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]-cyclopropyl-methyl] acetate;
(-)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H- thiazolo[4,5-d]pyrimidin-2-one;
(-)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(2'-0-acetyl-3 '-deoxy-P-D- ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one;
(-)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)- 3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione;
(-)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(-)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydro xypropyl]tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one;
(-)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2S,5R)-2-[(lS)- cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione; (+)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and [(lS)-l-[(2S,4R,5R)-5-(5-amino-2-oxo- thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl] acetate;
(+)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and [(S)-[(2S,5R)-5-(5-amino-2-oxo-thiazolo[4,5- d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]-cyclopropyl-methyl] acetate;
(+)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H- thiazolo[4,5-d]pyrimidin-2-one;
(+)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(2'-0-acetyl-3'-deoxy-P-D- ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one;
(+)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)- 3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione;
(+)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(+)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one;
(+)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2S,5R)-2-[(lS)- cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(-)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and [(lS)-l-[(2S,4R,5R)-5-(5-amino-2-oxo- thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl] acetate;
(-)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and [(S)-[(2S,5R)-5-(5-amino-2-oxo-thiazolo[4,5- d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]-cyclopropyl-methyl] acetate;
(-)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H- thiazolo[4,5-d]pyrimidin-2-one; (-)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(2'-0-acetyl-3'-deoxy-P-D- ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one;
(-)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-airrino-3-(3'-deoxy-P-D-ribofuranosyl)- 3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione;
(-)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
(-)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one; and
(-)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2S,5R)-2-[(lS)- cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione; in a pharmaceutically acceptable carrier.
Another further embodiment of the present invention is (16) the pharmaceutical composition according to embodiment (1) or (12), consisting of
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)- 3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; or
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
in a pharmaceutically acceptable carrier.
Another embodiment of the present invention is (17) a pharmaceutical composition, which is
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)- 3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; or (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
in a pharmaceutically acceptable carrier. Another embodiment of the present invention is (18) the pharmaceutical composition according to any one of embodiments (1), (12), (16) and (17), wherein the HBsAg inhibitor and the TLR7 agonist can also selected from other HBsAg inhibitors and TLR7 agonists including small molecules or large molecules. Examples of other TLR7 agonists include, but not limited to, Imiquimod, Resiquimod, PF-4878691, SM-276001, ANA975, ANA773 and GS9620. Another embodiment of the present invention is (19) a pharmaceutical composition according to any one of embodiments (1), (12), (16), (17) and (18), wherein the composition additionally comprising one or more other antiviral agents, which include, but not limited to nucleos(t)ide analogues such as Lamivudine , Adefovir dipivoxil, Entecavir, Telbivudine, Clevudine, Tenofovir disoproxil and Tenofovir disoproxil fumarate; interferons such as Roferon A®, Intron A®, Pegasys®, Pegintron® and Pai Ge Bin.
Typical dosages of an HBsAg inhibitor and/or a TLR7 agonist can be in the ranges recommended by the manufacturer, 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. Thus, 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 v tro responsiveness of the
appropriate animal models.
Another embodiment of the present invention is (20) a method for manufacturing a medicament for treatment or prophylaxis of hepatitis B virus infection, characterized in that an HBsAg inhibitor and a TLR7 agonist are used in the medicament. A further embodiment of the present invention is (21) the method according to
embodiment (20), wherein the HBsAg inhibitor and the TLR7 agonist are co-administered in the same formulation or different formulation.
For purposes of the present invention, "co -administer" refers to any administration of the HBsAg inhibitor and interferon 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. Thus, the two active agents can be administered either as part of the same pharmaceutical composition or in separate pharmaceutical compositions. Also, the two active agents can be administered either at the same time, or sequentially.
The pharmaceutical composition of the HBsAg inhibitor and the TLR7 agonist 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.
Another further embodiment of the present invention is (22) the method according to embodiment (xx) or (xxi), wherein the HBsAg inhibitor and the TLR7 agonist are intended for administration to a subject by the same route or different routes. Another further embodiment of the present invention is (23) the method for according to any one of embodiments (20) to (22), wherein the HBsAg inhibitor and the TLR7 agonist are intended for administration to a subject by parenteral or oral administration.
Another further embodiment of the present invention is (24) the method according to any one of embodiments (20) to (23), wherein the administration of the HBsAg inhibitor and the TLR7 agonist to a subject is simultaneous or sequential. In any of the methods of the present invention, the administration of agents simultaneously can be performed by separately or sequentially administering agents at the same time, or together as a fixed combination. Also, in any of the methods of the present invention, the administration of agents separately or sequentially can be in any order. Another embodiment of the present invention is (25) the method according to any one of embodiments (20) to (24), wherein HBsAg inhibitor is a compound of formula (I), or formula (II), or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
A further embodiment of the present invention is (26) the method according to
embodiment (25), wherein the HBsAg inhibitor is (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo [a] quino lizine- 3 -carboxylic acid ;
(-)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo [a] quino lizine- 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-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine- 3-carboxylic acid;
(+)-6-ieri-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-6-ieri-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(+)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(+)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(+)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid; or
(-)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof. Particularly, the HBsAg inhibitor is (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
Another embodiment of the present invention is (27) the method according to any one of embodiments (20) to (26), wherein the TLR7 agonist is a compound of formula (III) or formula (IV), or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof. A further embodiment of the present invention is (28) the method according to embodiment (27), wherein the TLR7 agonist is
[(15)-l-[(25,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy- tetrahydrofuran-2- yl] propyl] acetate ;
[(5)-[(25,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]- cyclopropyl-methyl] acetate;
5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-JH-thiazolo[4,5-d]pyrimidin-2-one;
5-amino-3-(2'-0-acetyl-3'-deoxy-P-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one;
5-amino-3-(3'-deoxy-P-D-ribomranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione;
5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2- yl]thiazolo[4,5-d]pyrimidin-2-one; or
5-amino-3-[(2S,5R)-2-[(lS)-cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione;
or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof. Particularly the TLR7 agonist is
5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; or
5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione;
or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
Another further embodiment of the present invention is (29) a method for manufacturing a medicament for treatment or prophylaxis of hepatitis B virus infection, characterized in that the medicament is
a combination of (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-
3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; or
a combination of (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydro xypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione ;
in a pharmaceutically acceptable carrier. A further embodiment of the present invention (30) is the method according to any one of embodiments (20) to (29), wherein the medicament additionally comprises one or more other antiviral agents, which include, but not limited to nucleos(t)ide analogues such as Lamivudine , Adefovir dipivoxil, Entecavir, Telbivudine, Clevudine, Tenofovir disoproxil and Tenofovir disoproxil fumarate; interferons such as Roferon A®, Intron A®, Pegasys®, Pegintron® and Pai Ge Bin.
Another embodiment of the present invention is (31) a kit comprising a container comprising an HBsAg inhibitor and a TLR7 agonist, said kit can further comprise a sterile diluent. A further embodiment of the present invention is (32) the kit according to embodiment
(31), further comprising a package insert comprising printed instructions directing the use of a combined treatment of an HBsAg inhibitor and a TLR7 agonist as a method for treatment or prophylaxis of hepatitis B virus infection.
Another embodiment of the present invention is (33) the kit according to embodiment (31) or (32), wherein the HBsAg inhibitor is
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo [a] quino lizine- 3 -carboxylic acid ;
(-)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo [a] quino lizine- 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-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine- 3-carboxylic acid;
(+)-6-ieri-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-6-ieri-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(+)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid; (+)-6-(2-hydroxy-l, 1-dimethyl-ethyl)- 10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-6-(2-hydroxy-l, 1-dimethyl-ethyl)- 10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(+)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid; or
(-)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof. Particularly, the HBsAg inhibitor is (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid; or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
Another embodiment of the present invention is (34) the kit according to any one of embodiments (31) to (33), wherein the TLR7 agonist is
[(15)-l-[(25,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy- tetrahydrofuran-2- yl] propyl] acetate ;
[(5)-[(25,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]- cyclopropyl-methyl] acetate;
5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-JH-thiazolo[4,5-d]pyrimidin-2-one;
5-amino-3-(2'-0-acetyl-3'-deoxy-P-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one; 5-amino-3-(3'-deoxy-P-D-ribomranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione;
5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2- yl]thiazolo[4,5-d]pyrimidin-2-one; or
5-amino-3-[(2S,5R)-2-[(lS)-cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione; or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof. Particularly the TLR7 agonist is
5-amino-3-(3'-deoxy-P-D-ribomranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; or 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione;
or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof. Another embodiment of the present invention relates to (35) a kit containing a combination of (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-
3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; or
a combination of (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione; in a pharmaceutically acceptable carrier.
Another embodiment of the present invention relates to (36) 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 a TLR7 agonist; or vice versa.
A further embodiment of the present invention relates to (37) the method according to embodiment (36),wherein the HBsAg inhibitor is
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo [a] quino lizine- 3 -carboxylic acid ;
(-)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo [a] quino lizine- 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-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine- 3-carboxylic acid;
(+)-6-ieri-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-6-ieri-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(+)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(+)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid; (-)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(+)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid; or
(-)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof. Particularly, the HBsAg inhibitor is (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
Another embodiment of the present invention relates to (38) the method according to embodiment (36) or (37), wherein the TLR7 agonist is
[(15)-l-[(25,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy- tetrahydrofuran-2- yl] propyl] acetate ;
[(5)-[(25,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]- cyclopropyl-methyl] acetate;
5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-JH-thiazolo[4,5-d]pyrimidin-2-one;
5-amino-3-(2'-0-acetyl-3'-deoxy-P-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one;
5-amino-3-(3'-deoxy-P-D-ribomranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione;
5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2- yl]thiazolo[4,5-d]pyrimidin-2-one; or
5-amino-3-[(2S,5R)-2-[(lS)-cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione;
or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof. Particularly the TLR7 agonist is
5-amino-3-(3'-deoxy-P-D-ribomranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; or 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione;
or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof. A further embodiment of the present invention relates to (39) the method according to any one of embodiments (36) to (38), wherein the HBsAg inhibitor and the TLR7 agonist used in the subject are
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)- 3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione;
or (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
in a pharmaceutically acceptable carrier.
Another embodiment of the present invention relates to (40) a method for the treatment or prophylaxis of hepatitis B virus infection, comprising administration to a subject with an effective amount of composition of
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)- 3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; or
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
in a pharmaceutically acceptable carrier.
Another embodiment of the present invention relates to (41) the use of pharmaceutical composition according to any one of embodiments (1) to (19) as an antiviral medicament, in particular as the medicament for treatment or prophylaxis of hepatitis B virus infection.
Another embodiment of the present invention relates to (42) the use of an HBsAg inhibitor and a TLR7 agonist for the manufacture of pharmaceutical composition according to any one of embodiments (1) to (19) as an antiviral medicament, in particular the medicament for treatment or prophylaxis of hepatitis B virus infection.
EXAMPLES
The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. ABBREVIATIONS
CI Combination index
CL Confidence level
Combo Combination
DF Dilution factor
DMSO Dimethyl sulfoxide
ETV Entecavir
FBS Fetal Bovine Serum
FIC Fractional inhibitory concentration
GE Genome equivalent
HBsAg Hepatitis B surface antigen
HBV Hepatitis B virus
μΜ Micromolar
min Minute
nM Nanomolar
hPBMC human Peripheral blood mononuclear cell
PBS Phosphate buffered saline
PEG Polyethyleneglycol
Pen/Strep Penicillin/Streptomycin
qPCR Real-time quantitative polymerase chain reaction
RCF Relative centrifugal force
RLU Relative light unit
SD Standard deviation
sec Second
TLR7 Toll like receptor 7
PREPARATIVE EXAMPLES
Example 1
Preparation of [(15)-l-[(25,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-<i]pyrimidin-3-yl)-4- hydroxy-tetrahydrofuran-2-yl]propyl] acetate (Compound 7)
Figure imgf000044_0001
OH
Compound 7 was prepared through the following scheme:
Figure imgf000044_0002
7G 7H
Figure imgf000044_0003
7J Step 1: preparation of [(2R)-2-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a- tetrahydrofuro[2,3-<i] [l,3]dioxol-5-yl] -2-hydroxy-ethyl] 4-methylbenzenesulfonate
Figure imgf000045_0001
To a solution of (lR)-l-[(3aR,55,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3- JJ[l,3]dioxol-5-yl]ethane-l,2-diol (Compound 7A, 100 g, 490 mmol) in dry pyridine (1000 mL) was added /7-toluenesulfonyl chloride (139 g, 735 mmol) at 0°C. After being stirred at room temperature for 12 hours, the resulted solution was quenched by water (100 mL) and
concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 1: 10 to 1:3 EtOAc in petroleum ether) to afford 130 g of [(2R)-2-[(3aR,5S,6aR)-2,2- dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-(i][l,3]dioxol-5-yl]-2-hydroxy-ethyl] 4- methylbenzenesulfonate (Compound 7B) as a slight yellow oil.
Compound 7B: 1H NMR (400 MHz, CDC13) ppm: 7.82 (d, / = 8.00 Hz, 2H), 7.38 (d, / = 8.00 Hz, 2H), 5.78 (d, / = 3.76 Hz, 1H), 4.75 (t, /= 4.00 Hz, 1H), 4.20- 4.12 (m, 2H), 4.03- 3.97 (m, 2H), 2.48 (s, 3H), 2.39 (d, / = 3.51 Hz, 1H), 2.08-2.15 (m, 1 H), 1.75-1.80 (m, 1 H), 1.51 (s, 3 H), 1.33 (s, 3 H).
Step 2: preparation of (3aR,5S,6aR)-2,2-dimethyl-5-[(2R)-oxiran-2-yl]-3a,5,6,6a- tetrahydrofuro[2,3-<i] [1,3] dioxole
Figure imgf000045_0002
To a solution of [(2R)-2-[(3aR,55,,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3- JJ[l,3]dioxol-5-yl]-2-hydroxy-ethyl] 4-methylbenzenesulfonate (Compound 7B, 100 g, 280 mmol) in anhydrous THF (1500 mL) cooled at -70 °C was added potassium
bis(trimethylsilyl)amide (340 mL, 340 mmol, 1 M in THF) under N2 atmosphere. After being stirred at -70 °C for 1 hour, the reaction mixture was poured into saturated NH4C1 solution. The organic layer was separated and the aqueous phase was extracted with EtOAc. The combined organic layers were dried over Na2S04 and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 1:3 EtOAc in petroleum ether) to afford 40.5 g of (3aR,55,6aR)-2,2-dimethyl-5-[(2R)-oxkan-2-yl]-3a,5,6,6a-tetrahydrofuro[2,3-(i][l,3]dioxole (Compound 7C) as a slight yellow oil.
Compound 7C: 1H NMR: (400 MHz, CDC13) ppm: 5.87 (d, / = 3.76 Hz, 1H), 4.77 (t, / = 4.00, 1H), 4.20-4.28 (m, 1H), 3.14-3.20 (m, 1H), 2.83-2.88 (m, 1H), 2.63 (dd, / = 5.00, 2.80 Hz, 1H), 2.09 (dd, / = 12.00, 4.00 Hz, 1H), 1.69-1.79 (m, 1H), 1.52 (s, 3H), 1.34 (s, 3H).
Step 3: preparation of (lR)-l-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a- tetrahydrofuro[2,3-rf][l,3]dioxol-5-yl]propan-l-ol
Figure imgf000046_0001
To a suspension of Cul (19.3 g, 107 mmol) in dry THF (2000 mL) under N2 atmosphere was added methyl magnesium bromide (3 M in diethyl ether, 537 mL, 1.61 mol) at -70 °C. After being stirred at the same temperature for 1 hour, a solution of (3aR,5S,6aR)-2,2-dimethyl-5- [(2R)-oxiran-2-yl]-3a,5,6,6a-tetrahydrofuro[2,3-(i][l,3]dioxole (Compound 7C, 100 g, 537 mmol, dissolved in anhydrous THF 200 mL) was added to reaction mixture dropwise. After being stirred at -70 °C for additional 2 hours, the reaction mixture was poured into saturated NH4C1 solution. The organic layer was separated and the aqueous phase was extracted with EtOAc twice. The combined organic layers were dried over Na2S04 and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 1:3 EtOAc in petroleum ether) to afford 82 g of (lR)- l-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a- tetrahydrofuro[2,3-JJ[l,3]dioxol-5-yl]propan-l-ol (Compound 7D) as a slight yellow solid.
Compound 7D: 1H NMR (400 MHz, CDC13) Sppm: 5.83 (d, / = 3.76 Hz, 1H), 4.81 - 4.73 (m, 1H), 4.26-4.19 (m, 1H), 3.91-3.82 (m, 1H), 2.08-2.02 (m, 1H), 1.93 - 1.89 (m, 1H), 1.54 (s, 3H), 1.49-1.39 (m, 2H), 1.34 (s, 3H), 1.02 (t, 7 = 7.53 Hz, 3H). Step 4: preparation of [(lS)-l-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a- tetrahydrofuro[2,3-rf][l,3]dioxol-5-yl]propyl] 4-nitrobenzoate
Figure imgf000047_0001
To a stirred solution of (lR)-l-[(3aR,55,,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3- JJ[l,3]dioxol-5-yl]propan-l-ol (Compound 7D, 50 g, 245 mmol), triphenylphosphine (195 g, 743 mmol), 4-nitrobenzoic acid (124 g, 743 mmol) in THF (1200 mL) was added diethyl azodicarboxylate (130 g, 743 mmol) dropwise at 0 °C under N2. After being stirred at 18 °C for 10 hours, the mixture was quenched by addition of saturated NaHC03 solution and extracted with EtOAc. The organic layers were combined, dried over Na2S04 and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 1:3 EtOAc in petroleum ether) to afford 61 g of [(lS)- l-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a- tetrahydrofuro[2,3-JJ[l,3]dioxol-5-yl]propyl] 4-nitrobenzoate (Compound 7E) as a slight yellow solid.
Compound 7E: 1H NMR (400 MHz, CDC13) ppm: 8.34- 8.22 (m, 4 H), 5.85 (d, / = 3.76 Hz, 1H), 5.23- 5.17 (m, 1H), 4.76 (t, / = 4.27 Hz, 1H), 4.48- 4.39 (m, 1H), 2.12 (dd, / = 13.30, 4.52 Hz, 1H), 1.88- 1.78 (m, 2H), 1.71- 1.62 (m, 1H), 1.55 (s, 3 H), 1.34 (s, 3 H), 1.01 (t, / = 7.40 Hz, 3 H).
Step 5: preparation of (lS)-l-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a- tetrahydrofuro[2,3-rf][l,3]dioxol-5-yl]propan-l-ol
O H
Figure imgf000047_0002
To a solution of [(15)-l-[(3aR,55,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3- JJ[l,3]dioxol-5-yl]propyi] 4-nitrobenzoate (Compound 7E, 100 g, 285 mmol) in methanol (1200 iriL) was added K2CO3 (78.7 g, 570 mmol). After being stirred at room temperature for 10 minutes, the resulted mixture was filtered. The filtrate was concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 1:8 EtOAc in petroleum ether) to afford 54.7 g of (15)-l-[(3aR,55,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3- JJ[l,3]dioxol-5-yl]propan-l-ol (Compound 7F) as a slight yellow solid.
Compound 7F: 1H NMR (400 MHz, CDC13) ppm: 5.81 (d, / = 3.64 Hz, 1H), 4.75 (t, / = 4.20 Hz, 1H), 4.18- 4.11 (m, 1H), 3.49-3.40 (m, 1H), 2.07-2.00 (m, 2H), 1.84-1.75 (m, 1H), 1.59- 1.47 (m, 5H), 1.32 (s, 3H), 1.01 (t, 7 = 7.40 Hz, 3H).
Step 6: preparation of [(lS)-l-[(3aR,5S,6aR)-2,2-dimethyl-3a,5,6,6a- tetrahydrofuro[2,3-rf][l,3]dioxol-5-yl]propyl] acetate
OAc
Figure imgf000048_0001
To a stirred solution of (llS,)-l-[(3aR,55,,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3- JJ[l,3]dioxol-5-yl]propan-l-ol (Compound 7F,13.5 g, 67 mmol), TEA (81 g, 804 mmol), DMAP (1.6 g, 13 mmol) in anhydrous DCM (150 mL) was added acetic anhydride (62 g, 603 mmol). After being stirred at 22 °C for 10 hours, the reaction was quenched by the saturated NaHC03 solution. The organic layer was separated and the aqueous phase was extracted with EtOAc. The combined organic layers were dried over Na2S04, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 1:8 EtOAc in petroleum ether) to afford 13 g of [(15)-l-[(3aR,55,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-JJ[l,3]dioxol-5- yl]propyl] acetate (Compound 7G) as a colourless oil.
Compound 7G: 1H NMR (400 MHz, CDC13) ppm: 5.83 (d, / = 3.76 Hz, 1H), 4.92 (dt, / = 7.97, 5.18 Hz, 1H), 4.74 (t, / = 4.00 Hz, 1H), 4.35- 4.27 (m, 1H), 2.12 (s, 3H), 2.08 - 1.99 (m, 1H), 1.74- 1.56 (m, 3H), 1.53 (s, 3H), 1.34 (s, 3H), 0.95 (t, / = 7.40 Hz, 3H).
Step 7: preparation of [(3R,5S)-2-acetoxy-5-[(lS)-l-acetoxypropyl]tetrahydrofuran-3- yl] acetate OAc
Figure imgf000049_0001
To a solution of [(15)-l-[(3aR,55,6aR)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3- JJ[l,3]dioxol-5-yl]propyi] acetate (Compound 7G, 4.8 g, 20 mmol), acetic acid (12.2 g, 200 mmol) and acetic anhydride (10.2 g, 100 mmol) in anhydrous DCM (100 mL) was added concentrated H2SO4 (0.5 mL) at 0 °C. After being stirred at 22 °C for 3 hours, the reaction was quenched by addition of saturated NaHC03 solution. The organic layer was separated and the aqueous phase was extracted with EtOAc. The combined organic layers were dried over Na2S04, filtered, and concentrated in vacuo. The residue was purified by column on silica gel (eluting with 1:8 EtOAc in petroleum ether) to afford 2.3 g of [(3R,55,)-2-acetoxy-5-[(15,)-l- acetoxypropyl]tetrahydrofuran-3-yl] acetate (Compound 7H) as a colourless oil.
Compound 7H: 1H NMR (400 MHz, CDC13) ppm: 6.12 (s, 1H), 5.19 (d, / = 4.52 Hz, 1H), 4.83- 4.91 (m, 1H), 4.34- 4.44 (m, 1H), 2.09- 2.19 (m, 9H), 1.51- 1.74 (m, 4H), 0.94 (t, / = 7.40 Hz, 3H).
Step 8: preparation of [(2R,3R,5S)-5-[(lS)-l-acetoxypropyl]-2-(5-amino-2-oxo- thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl] acetate
Figure imgf000049_0002
To a suspension of 5-amino-3H-thiazolo[4,5-<i]pyrimidin-2-one (3.5 g, 20.8 mmol) in xylene (160 mL) was added BSA (21.2 g, 104 mmol). The reaction mixture was stirred at 70 °C for 1 hour under argon to form a clear solution. After some of xylene and excrescent BSA were evaporated, [(3R,5S)-2-acetoxy-5-[(lS)- l-acetoxypropyl]tetrahydrofuran-3-yl] acetate
(Compound 7Η, 3.0 g, 10.4 mmol) and TMSOTf (2.6 g, 11.6 mmol) were added in sequence at 0 °C. After being heated with stirring at 65 °C for 2 hours, the reaction was quenched with water (30 mL), extracted with EA (30 mL) three times. The combined organic layers were dried over Na2S04 and concentrated in vacuo. The residue was purified by column on silica gel (eluting with 1: 10 to 1: 1 EtOAc in petroleum ether) to give 2.0 g of [(2R,3R,5S)-5-[(lS)-l- acetoxypropyl]-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl] acetate (Compound 7J) as a white solid. Compound 7J: 1H NMR (400 MHz, CDC13) ppm: 8.15 (s, 1 H), 6.04 (d, / = 1.51 Hz, 1
H), 5.80 (d, / = 7.03 Hz, 1 H), 5.27 (br. s., 2 H), 4.98- 5.04 (m, 1 H), 4.32- 4.39 (m, 1 H), 2.63 - 2.77 (m, 1 H), 2.13 (s, 3 H), 2.09 (s, 3 H), 2.00 - 2.07 (m, 1 H), 1.61- 1.75 (m, 2 H), 0.94 (t, / = 7.40 Hz, 3 H).
Step 9: preparation of [(15)-l-[(25,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5- <i]pyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl] acetate
Figure imgf000050_0001
O H
[(2R,3R,55)-5-[(15)-l-acetoxypropyl]-2-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3- yl)tetrahydrofuran-3-yl] acetate (Compound 7J, 3.2 g, 8.0 mmol) and K2C03 (2.2 g, 16.0 mmol) were suspended in anhydrous ethanol (85 mL) at room temperature. Methanol (85 mL) was added dropwise under N2 atmosphere. After the addition, the mixture was stirred at room temperature for 10 minutes (monitored by TLC). After the reaction, the mixture was poured into saturate NH4C1, extracted with EA (150 mL) four times. The combined organic layers were dried over Na2S04 concentrated in vacuo. The residue was purified by column on silica gel (eluting with 1: 100 to 1:70 MeOH in DCM) to give the crude product, which was further purified by flash column (eluting with acetonitrile and water) to give 1.64 g of [(15,)-l-[(2lS',4R,5R)-5-(5- amino-2-oxo-thiazolo[4,5-JJpyrimidin-3-yl)-4-hydroxy-tetrahydrofuran-2-yl]propyl] acetate (Compound 7) as a white power.
Compound 7: 1H NMR (400 MHz, Methanol- d4) ppm: 8.19 (s, 1 H), 6.02- 6.05 (m, 1 H), 4.94- 5.00 (m, 2 H), 4.33- 4.40 (m, 1 H), 2.58- 2.68 (m, 1 H), 2.03 (s, 3 H), 1.86- 1.96 (m, 1 H), 1.56 - 1.77 (m, 2 H), 0.93 (t, / = 7.40 Hz, 3 H). MS obsd. (EST) [(M+H)+]: 355.0. Example 2
Preparation of 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione (Compound 12)
Figure imgf000051_0001
Compound 12 was prepared according to the following Scheme:
Figure imgf000051_0002
7H 12A
Figure imgf000051_0003
12
Step 1: preparation of [(2R,3R,55)-5-[(15)-l-acetoxypropyl]-2-(5-amino-2,7-dioxo-6H- thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl] acetate
Figure imgf000052_0001
To a suspension of 5-amino-3,6-dihydrothiazolo[4,5-d]pyrimidine-2,7-dione (3.83 g, 20.8 mmol) and [(3R,55,)-2-acetoxy-5-[(llS,)-l-acetoxypropyl]tetrahydrofuran-3-yl] acetate (compound 7H, 2.0 g, 6.9 mmol) in acetonitrile (70 mL) was added BSA (12.7 g, 62.4 mmol). The reaction mixture was stirred at 80 °C for 3 hour under argon to form a clear solution. Then to the solution was added TMSOTf (2.6 g, 11.6 mmol) and the mixture was stirred at 80 °C for another 4 hours, The reaction was quenched with saturated NaHC03 solution (100 mL) and extracted with EtOAc (200 mL) three times. The combined organic layers were washed by brine, dried over Na2S04 and concentrated in vacuo. The residue was purified by column on silica gel (eluting with 1:30 MeOH in DCM) to give 2.0 g of [(2R,3R,55)-5-[(15)-l-acetoxypropyl]-2-(5-amino-2,7-dioxo- 6H-thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl] acetate (compound 12A) as a yellow solid. MS obsd. (ESI ) [(M+H)+]: 413.1.
Step 2: preparation of 5-amino-3-[(2/i,3/i,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione
Figure imgf000052_0002
O H
To a solution of [(2R,3R,5lS,)-5-[(llS,)-l-acetoxypropyl]-2-(5-amino-2,7-dioxo-6H- thiazolo[4,5-d]pyrimidin-3-yl)tetrahydrofuran-3-yl] acetate (compound 12A, 20 g, 48.5 mmol) in methanol (150 mL) was added sodium methoxide (13.1 g, 242 mmol). After the addition, the mixture was stirred at room temperature for 3 hours (monitored by TLC). After the reaction was complete, the mixture was adjusted to PH-7.0 by addition of HOAc and concentrated in vacuo. The residue was purified by preparative HPLC to afford 620 mg of 5-amino-3-[(2R,3R,55,)-3- hydroxy-5-[(15)- l-hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione (Compound 12) as a white power.
Compound 12: 1H NMR (400 MHz, DMSO- ) ^ppm: 6.97 (br s, 2H), 5.70-5.74 (m, IH), 5.39-5.44 (m, IH), 4.69-4.76 (m, IH), 4.51-4.56 (m, IH), 3.91-4.01 (m, IH), 3.25-3.32 (m, IH), 2.27-2.37 (m, IH), 1.65- 1.73 (m, IH), 1.33- 1.46 (m, IH), 1.21- 1.29 (m, IH), 0.88 ppm (s, 3H). MS obsd. (ESI ) [(M+H)+] : 329.0.
Example 3
Preparation of 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin-2-one (Compound 13)
Figure imgf000053_0001
Compound 13 was prepared according to the following Scheme:
Figure imgf000053_0002
To a solution of [(15)- l-[(25,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-<i]pyrirnidin-3-yl)-4- hydroxy-tetrahydrofuran-2-yl]propyl] acetate (10.0 g, 28.2 mmol) in MeOH (250 mL) was added sodium methoxide (7.62 g, 141 mmol) in one portion. After the mixture was stirred at room temperature for 3 hr, the reaction was quenched with saturated aqueous NH4C1 (150 mL). Then the mixture was extracted with EtOAc (200 mL) 5 times. The combined organic layer was dried over Na2S04 and concentrated in vacuo. The residue was purified by silica gel column eluted with DCM/MeOH =19/1 to give a white solid. The solid was triturated in MeOH/n-hexane (100 mL, v/v=l/20) and then filtered. The filter cake was dried in vacuo to give 5-amino-3- [(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin- 2-one as a white powder (3.8 g, 43.1%). The filtrate was concentrated in vacuo and the residue was purified by silica gel column eluted with DCM/MeOH = 19/1 to give 1.1 g of 5-amino-3- [(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2-yl]thiazolo[4,5-d]pyrimidin- 2-one as a white solid.
Compound 13: 1H NMR (400 MHz, DMSO- ) Sppm: 8.36 (s, 1H), 6.87 (s, 2H), 5.84 (d, 7=2.51 Hz, 1H), 5.45 (d, 7=4.52 Hz, 1H), 4.77 (dt, 7=2.26, 4.39 Hz, 1H), 4.55 (d, 7=5.77 Hz, 1H), 4.00 (td, 7=6.02, 10.04 Hz, 1H), 3.27-3.35 (m, 1H), 2.30-2.48 (m, 1H), 1.73 (ddd, 7=1.51, 5.90, 12.67 Hz, 1H), 1.33-1.49 (m, 1H), 1.26 (ddd, 7=7.28, 8.72, 13.87 Hz, 1H), 0.88
(t, 7=7.40 Hz, 3H).
Example 4
HEK293-Blue-hTLR-7 cells assay:
A stable HEK293-Blue-hTLR-7 cell line was purchased from InvivoGen (Cat.#: hkb-htlr7, San Diego, California, USA). These cells were designed for studying the stimulation of human TLR7 by monitoring the activation of NF-κΒ. A SEAP (secreted embryonic alkaline phosphatase) reporter gene was placed under the control of the IFN-βϋ minimal promoter fused to five NF-KB and AP-1 -binding sites. The SEAP was induced by activating NF-κΒ and AP-1 via stimulating HEK-Blue hTLR7 cells with TLR7 ligands. Therefore the reporter expression was regulated by the NF-KB promoter upon stimulation of human TLR7 for 20 hours. The cell culture supernatant SEAP reporter activity was determined using QUANTI-Blue™ kit (Cat.#: rep-qbl, Invivogen, San Diego, Ca, USA) at a wavelength of 640 nm, a detection medium that turns purple or blue in the presence of alkaline phosphatase.
HEK293-Blue-hTLR7 cells were incubated at a density of 250,000-450,000 cells/mL in a volume of 180 μΐ^ in a 96- well plate in Dulbecco's Modified Eagle's medium (DMEM) containing 4.5 g/L glucose, 50 U/mL penicillin, 50 mg/mL streptomycin, 100 mg/mL Normocin, 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum for 24 h. Then the HEK293- Blue-hTLR-7 cells were incubated with addition of 20 μΐ^ test compound in a serial dilution in the presence of final DMSO at 1% and perform incubation under 37 °C in a C02 incubator for 20 hours. Then 20 μΐ^ of the supernatant from each well was incubated with 180 μΐ^ Quanti-blue substrate solution at 37°C for 2 hours and the absorbance was read at 620-655 nm using a spectrophotometer. The signalling pathway that TLR7 activation leads to downstream NF-KB activation has been widely accepted, and therefore similar reporter assay was also widely used for evaluating TLR7 agonist (Tsuneyasu Kaisho and Takashi Tanaka, Trends in Immunology, Volume 29, Issue 7, July 2008, Pages 329. sci; Hiroaki Hemmi et al, Nature Immunology 3, 196 - 200 (2002).
The TLR7 agonism activity in HEK293- hTLR-7 assay of compound 11 was 446 μΜ, the TLR7 agonism activity in HEK293- hTLR-7 assay of compound 12 was 72 μΜ; and the TLR7 agonism activity in HEK293- hTLR-7 assay of compound 14 was 7.9 μΜ.
Example 5
Metabolism of Compound 7, Compound 8 and Compound 13
A study was undertaken to evaluate the metabolic conversion of formula (III) or formula (IV) of the present invention. Compounds of formula (III) or formula (IV) can be metabolized to their active form.
A study was undertaken to evaluate the metabolic conversion of Compound 7, Compound 13 and Compound 8, to the corresponding active forms, Compound 12 and Compound 14, in the presence of human hepatocytes. The formation of active forms, Compound 12 and Compound 14, were monitored in the study. For comparison, the metabolic conversion of famciclovir to penciclovir was also assessed.
Hepatocytes Suspension
Cryopreserved hepatocytes plating medium (Cat.#: PY-HMD-01) was purchased from RILD Research Institute for Liver Diseases (Shanghai) Co. Ltd. Cryopreserved human hepatocyte (Cat.#: X008005, Lot#:VRR) was purchased from In Vitro Technologies (Baltimore, MD).
The stock hepatocyte suspension was prepared from cryopreserved hepatocytes in plating medium at the concentration of 1.8x106 cells/mL.
Working solutions of compounds Compounds were dissolved in DMSO to make 50 mM stock solutions. 10 μΐ^ of the stock solution was diluted to 5 mL plating medium to get a 100 μΜ working solution.
Incubations
Reaction suspensions were prepared in 24-well cell culture plate by mixing 200 μΐ^ of hepatocytes suspension (Cyno or human) and 200 μΐ^ of working solution. The final incubation contained 0.9xl06 cells/ mL and 50 μΜ compound. The above mixtures were incubated at 37 °C in a humidified 5% C02 atmosphere, with a 150 rpm shaking.
Preparation of Samples for Analysis
After 180 min of incubation, 200 μΐ^ of the incubation mixture was transferred to 1.5 mL tube and quenched with 400 μΐ^ stop solution (ice-cold acetonitrile with 0.2 μΜ Tolbutamide as internal standard). The samples were centrifuged at 12000 rpm for 10 minutes and the resultant supernatants were subjected to LC-MS/MS analysis.
The calibration curves were prepared in the following way. To a 200 μΐ^ of cell suspension (cell density of 1.8 million cells/ mL), 198 μΐ^ of hepatocyte plating medium and 2 μL· of the appropriate concentration of the compound in DMSO were added. Samples were mixed thoroughly and 200 μΐ^ of the mixture was transferred to 400 uL of the stop solution (see above). The standard curve range is from 1 μΜ to 25 μΜ.
Bioanalysis
The compounds were quantified on an API5500 LC-MC/MC instrument in the ESI- Positive MRM mode. The results of prodrug conversion and metabolite generation are summarized in Table 2.
Table 2: Concentration of the metabolites formed in human hepatocytes after 3 -hour incubation of 50 μΜ of prodrugs.
Figure imgf000056_0001
In human hepatocytes, compounds of Compound 7, Compound 13, Compound 8, as well as famciclovir were metabolized to yield the corresponding active metabolites of Compound 12, Compound 14 and penciclovir, respectively.
Example 6
Combination studies: Compound 1A and Compound 11, Compound 1A and
Compound 12
1 Material and Methods
1.1 Mediums used in this Example 6 are listed in Table 3
Table 3: Mediums
Figure imgf000057_0001
1.2 Blood from healthy human donors
Blood from healthy in-house volunteers was obtained through Employee Health and Services from Roche Basel. Whole blood from 6 donors was used for this study. All volunteers were properly consented before becoming an active donor. Whole blood was collected in sodium heparin green-top tubes (Becton Dickinson, cat #367874).
1.3 hPBMC purification
hPBMCs from whole blood were purified by ficoll gradient centrifugation using 50 mL Accuspin tubes (Sigma, cat# A2055) specially designed with two chambers separated by a porous high-density polyethylene barrier ("frit"). The lower chamber contains 15mL of Ficoll- Paque Plus (GE Healthcare Life Sciences, cat#17-1440-02).
50 mL of blood were divided into 2 separate 50 mL Accuspin tubes (25 mL of blood per tube) and 10 mL of RPMI 1640 medium containing 2% Fetal Bovine Serum (FBS) were added to each tube. Tubes were centrifuged at 797 RCF for 25 min at room temperature without brake in an Allegra X-30R centrifuge (Beckman Coulter). The top RPMI/plasma layer from each tube was removed and discarded, and the buffy coat layer (cream layer) was transferred to a fresh tube. The total volume was made up to 50 mL with RPMI 1640 medium containing 2% FBS and the tubes were then centrifuged for 10 min at 559 RCF at room temperature. The cells were washed 3 times with RPMI 1640 medium containing 2% FBS at 489 RCF for 10 min. Before the final wash, 100 μΐ^ of cells suspension was transfer to a micro fuge tube, diluted 1: 1
(volume/volume) with trypan blue. Cells/trypan blue solution (10 μί) was loaded into the chamber slide of a cell counter (Countess, Invitrogen) to determine cell count and viability. High cell viability (>91%) was obtained for all blood donors. The cell pellet (containing hPBMCs) was then resuspended in RPMI 1640 medium supplemented with 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin.
1.4 Preparation of TLR7 agonist hPBMC conditioned medium hPBMCs were seeded at 5xl06 cells/mL in RPMI 1640 medium supplemented with 10% FBS and 100 units/mL penicillin and 100 μg/mL streptomycin in a 6 well plate (2 mL of cell suspension per well). hPBMCs were incubated for 3 h at 37°C and subsequently treated with 100 μΜ of TLR7 agonist Compound 11 or 12.5μΜ of TLR7 agonist Compound 12 at a final DMSO concentration of 0.2% each. The treated cells were incubated for 16h at 37°C before supernatants were harvested. Cultures were centrifuged for 10 min at 931 RCF in an Allegra X- 30R centrifuge (Beckman Coulter) at room temperature and the supernatants were harvested to obtain TLR7 agonist hPBMC conditioned medium. TLR7 agonist hPBMC conditioned medium from all 6 blood donors was pooled, aliquoted and stored at -80°C, which was used later in Part 1.6 "Infection and compound treatment of hepaRG cells" of this Example 6.
1.5 Virus and cells
HepG2.2.15 cells were cultured in DMEM+Glutamax I (Gibco, #21885) supplemented with 10% FBS, 1% Pen/Strep (Gibco, #15140) and 250 μ ηύ, G-418 (Gibco, #10131027) 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 became confluent, medium was changed to DMEM+Glutamax I with 1% DMSO and 2.5% FBS. Once the cells were slightly over confluent, the medium was changed to DMEM/F12+Glutamax I (Gibco, #31331) supplemented with MEM non-essential amino acids (6 mL, Gibco, #11140), Pen/Strep (6 mL), sodium pyruvate (6 mL), DMSO (9 mL) and FBS (10 mL) (all per 500 mL medium). The medium was changed every 3 days and the supernatants were harvested for 2 weeks. Virus was concentrated from the supernatants by PEG precipitation and the titer (genome equivalent (GE) / mL) was determined by qPCR. Briefly, the supernatants were mixed with 40% PEG solution at a ratio of 4: 1, incubated on a shaker at 4°C overnight and then centrifuged using 50 mL falcon tubes at 4°C for one hour at 3724 g (RCF). The supernatant was discarded and the centrifugation step was repeated with new supernatant reusing the tubes until all PEG- precipitated supernatant was processed. The pellets were re-suspended in William's E Medium
(Gibco, #22551) at a concentration of 10 7 -109 genome equivalents (GE) per mL and frozen at - 80°C. DNA copy number calculation was based on a standard curve generated from HBV plasmid dilutions with known concentrations.
HepaRG cells (Biopredic International, Saint-Gregoire, France) were cultured in working growth medium for 2 weeks. After 2 weeks the medium was changed to differentiation medium. The medium was changed twice a week up to 2 weeks. Once fully differentiated, the cells were trypsinized and seeded into collagenated 96 well plates (50,000 cells/well in 100 μί) in differentiation medium. The cells were cultured at least 5 days in the 96 well plates before they were infected with HBV.
1.6 Infection and compound treatment of hepaRG cells
For HBV infection of differentiated HepaRG cells, the medium was removed and differentiation medium (120 μίΛνεΙΙ) containing 4% PEG-8000 and virus stock (20 to 30 GE/cell) was added. The cells were cultured at 37 °C for 16 to 20 h before the medium was removed, the cells were washed 4 times with PBS and differentiation medium (120 μίΛνεΙΙ) was added. At day 4 post infection, the medium was removed and 100 μΐ^ differentiation medium was added to each well. 3-fold serial dilutions (30 μΐ^ Drug A to 60 μΐ^ DMSO) of Drug A (HBsAg inhibitor) were prepared in 100% DMSO starting with 90 μΐ, undiluted Drug A (400- fold concentration of highest test concentration). 5-fold serial dilutions (12 μL· Drug B hPBMC conditioned medium to 48μί working growth medium) of Drug B (TLR7 agonist) were prepared in working growth medium starting with 60 μΐ^ undiluted Drug B hPBMC conditioned medium (125-fold concentration of highest test concentration). 2.5 μΐ^ of drug A and 8 μΐ^ of Drug B dilutions were then added to 489.5 μΐ^ differentiation medium (containing 1.3% DMSO) in a 96 deep well plate in a fashion according to the design shown in Table 4 to give drug medium. 100 μL· drug medium thereof were added to the HepaRG cells with a final DMSO concentration of 1.8%. Drug A tested is Compound 1A; Drug B tested is Compound 11 hPBMC conditioned medium or Compound 12 hPBMC conditioned medium. The concentration ranges tested were 30 nM to 0.37 nM for Compound 1A, and 0.008 to 0.000001 DF for Compound 11 hPBMC conditioned medium or Compound 12 hPBMC conditioned medium. The medium was replaced by drug medium at day 6 and 8 post infection and at day 11 post infection the 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.
Table 4: Plate layout for combinations of Drug A and Drug B
Figure imgf000060_0001
X: PBS
CC: cell control (uninfected)
VC: virus control
ETV: reference control (200nM Entecavir)
Al-5: serial dilution of drug A
Bl-7: serial dilution of drug B
A1+B7: example of combination of drug A and B at different ratios 1.7 DNA extraction
HBV DNA from HepaRG cell supernatants was extracted using the MagNA Pure 96 (Roche) robot. 100 μΐ^ of the supernatants were mixed in a processing cartridge with 200 μΐ^ 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 μΐ^.
1.8 qPCR
Quantification of extracted HBV DNA was performed using a Taqman qPCR machine (ViiA7, life technologies). Each DNA sample was tested in duplicate in the PCR. 5 μΐ^ of DNA sample were added to 15 μΐ^ of PCR mastermix containing 10 μΐ^ TaqMan Gene Expression Master Mix (Applied Biosystems, Cat. no. 4369016), 0.5 PrimeTime XL qPCR Primer/Probe (IDT, Leuven, Belgium) and 4.5 μΐ^ distilled water in a 384 well plate and the PCR was performed using the following settings: UDG Incubation (2 min, 50°C), Enzyme Activation (lOmin, 95°C) and PCR (40 cycles with 15sec, 95° for Denaturing and lmin, 60°C for annealing and extension). DNA copy numbers were calculated from Ct values based on a HBV plasmid DNA standard curve by the ViiA7 software.
Sequences for TaqMan primers and probes (IDT):
Forward core primer (F3_core): CTG TGC CTT GGG TGG CTT T
Reverse primer (R3_core): AAG GAA AG A AGT CAG AAG GCA AAA
Taqman probe (P3_core): 56-FAM/AGC TCC AAA /ZEN/TTC TTT ATA AGG GTC GAT GTC CAT G/3IABkFQ
1.9 Data Analysis
Isobologram model
The combination experimental results were analyzed using the model described by Craig et al. (Craig J, Duncan I, Whittaker L and Roberts N. (1990), Antiviral synergy between inhibitors of HIV proteinase and reverse transcriptase. Antiviral Chem. Chemother, 4: 161-166). EC50 values were obtained for compounds used alone and in combination with others. To relate these two values and describe the degree of synergy/additivity/antagonism between them, the Fractional Inhibitory Concentration (FIC) was first calculated and used to generate isobolograms. Briefly, the FIC is the ratio of the EC50 of the drug in combination to the EC50 of the drug on its own:
FIC = ratio [EC50 combination : EC50 alone] The Combination Index (CI), obtained by adding the FICs of the two compounds, was then used to describe the effect between compounds used in the combinations. A CI < 1 means synergism, a CI = 1 means additivity and a CI > 1 means antagonism.
CalcuSyn model
Each experiment was performed in at least triplicate and performed independently 3 times. Mean percent inhibition of HBV replication based on DNA copy number was calculated from all experiments and analyzed using the Calcusyn software (CalcuSyn Version 2.11, Biosoft, Cambridge, UK) based on the Loewe additivity model described by Chou and Talalay (Chou TC (2006), Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies, Pharmacol. Rev., 58:621-681). For the CalcuSyn evaluation, data from three diagonal lanes with a constant concentration ratio for the drug combinations of each lane from the checkerboard plate were used (marked in bold in Table 4). In a first step, this program converts the dose-effect curves for each drug or drug combination to median effect plots. A combination index (CI) for each experimental combination was then calculated by the following equation (for mutually nonexclusive interactions): [(D)1/(Dx)1]+[(D)2/(Dx)2]+[(D)1(D)2/(Dx)1(Dx)2] where (Dx)i and (Dx)2 are the doses of drug 1 and drug 2 that have x effect when each drug is used alone, and (D)i and (D)2 are the doses of drug 1 and drug 2 that have the same x effect when they are used in combination, respectively. The software calculates the CIs at 50%, 75% and 90% antiviral effect of combinations. 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 ECsos. 2 Results
Combination of Compound 1A with Compound 11 hPBMC conditioned medium, and combination of Compound 1A with Compound 12 hPBMC conditioned medium 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 activities (EC50) obtained in the combination studies were determined (Table 5).
Table 5: Mean EC50 + SD values for the individual compounds used in the combination studies
Figure imgf000063_0001
a} number of independent experiments
The data from the combination study of Compound 1A and Compound 11 was analyzed using the CalcuSyn software (Table 6). The overall CI for three different concentration ratios was between 0.27 and 0.34, thus the combination of Compound 1A and Compound 11 was synergistic.
The data from the combination study of Compound 1A and Compound 11 was analyzed using the Isobologram model (Figure 1). The FIC values for the combination of Compound 1A and Compound 11 were plotted one against the other for each of the experiments. The analysis showed that most of the CI values were < 1. Therefore, the combination of Compound 1A and Compound 11 was synergistic.
The data from the combination study of Compound 1A and Compound 12 was analyzed using the CalcuSyn software (Table 6). The overall CI for three different concentration ratios was between 0.30 and 0.72, thus the combination of Compound 1A and Compound 12 was synergistic. The data from the combination study of Compound 1A and Compound 12 was also analyzed using the Isobologram model (Figure 2). The FIC values for the combination of Compound 1A and Compound 12 were plotted one against the other for each of the experiments. The analysis showed that most of the CI values were < 1. Therefore, the 5 combination of Compound 1A and Compound 12 was synergistic.
CalcuSyn based combination indices for the pair-wise checkerboard combinations (HBV DNA)
Figure imgf000064_0001
Number of independent experiments
Assessment was based on CI values determined by the CalcuSyn software.

Claims

1. A pharmaceutical composition comprising an HBsAg inhibitor and a TLR7 agonist, in a pharmaceutically acceptable carrier.
2. The pharmaceutical composition according to claim 1, wherein the HBsAg inhibitor is compound of formula (I):
Figure imgf000065_0001
wherein
R1 is hydrogen, halogen, Chalky!, Ci_6alkylamino or
Figure imgf000065_0002
R is hydrogen; halogen; Ci_6alkyl, which is unsubstituted or once, twice or three times
substituted by fluoro; Ci_6alkoxy, which is unsubstituted or once, twice or three times substituted by fluoro; cyano; C3_7cycloalkyl; hydroxy or phenyl- CxH2X-0-;
R is hydrogen;
halogen;
Ci_6alkyl, which is unsubstituted or once, twice or three times substituted by fluoro;
cyano;
pyrrolidinyl;
amino;
phenyl-CxH2x-N(Ci_6alkyl)-;
Ci_6alkoxycarbonylpiperazinyl;
or R 7 -O- , wherein R 7 is hydrogen; C1-6alkyl, which is unsubstituted or substituted with one to three substituents independently selected from fluoro, hydroxy and C2-6alkenyl; Ci_ 6alkoxyCi-6alkyl; Ci_6alkoxyCi_6alkoxyCi-6alkyl; aminoCi-salkyl; Ci_
6alkylcarbonylaminoCi-8alkyl; Ci_6alkylsulfonylaminoCi_8alkyl; Ci_6alkylsulfanylCi_6alkyl; Ci-6alkylsulfonylCi-6alkyl; cyanoCi_6alkyl; C3_7cycloalkylCi-6alkyl; cyanoC3_7cycloalkylCi_ 6alkyl; phenylCi_6alkyl; pyrrolidinylcarbonylCi_6alkyl; C2-6 lkynyl; hydroxyCi_6alkylC2- 6alkynyl; aminoCi_6alkoxyCi_6alkyl; Ci-6alkylaminoCi-6alkoxyCi_6alkyl; diCi_
6alkylaminoCi-6alkoxyCi-6alkyl; carboxyCi_6alkyl; or Ci_6alkoxycarbonylaminoCi_8alkyl; heteroarylCi_6alkyl, wherein heteroaryl is N-containing monocyclic heteroaryl; or heterocyclylCi_6alkyl, wherein heterocyclyl is monocyclic heterocyclyl;
R4 is hydrogen, halogen, Chalky!, cyano or Ci_6alkoxy;
R5 is hydrogen or Ci_6alkyl;
R6 is hydrogen; Ci-6alkyl, which is unsubstituted or once, twice or three times substituted by fluoro; C3_7cycloalkyl, which is unsubstituted or once, twice or three times substituted by fluoro or Ci_6alkyl; or phenyl-CxH2X-;
x is 1-6;
or a pharmaceutically acceptable salt, or enantiomer thereof.
3. The pharmaceutical composition according to claim 2, wherein the HBsAg inhibitor 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-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3- carboxylic acid;
(+)-6-ieri-butyl- 10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid; or
(-)-6-ieri-butyl- 10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
4. The pharmaceutical composition according to claim 1, wherein the HBsAg inhibitor is a compound of formula (II):
Figure imgf000067_0001
R8, R9, R10 and R11 are independently selected from hydrogen, halogen, Ci_6alkyl, diCi_ 6alkylamino, cyano, N-containing monocyclic heterocyclyl and OR14, wherein
R14 is hydrogen; Ci_6alkyl; or Ci_6alkyl which is substituted once or more times by fluoro,
C3_7cycloalkyl, phenyl, hydroxyl, amino, Ci_6alkoxy, Ci_6alkylsulfanyl, Ci_6alkylsulfonyl, diCi_6alkylamino, Ci_6alkoxycarbonylamino, monocyclic heterocyclyl, pyrazoyl or imidazolyl;
12
R is hydrogen or Chalky;
13
R is hydrogen, Ci_6alkyl, phenyl-CxH2X-, Ci_6alkylcarbonyl, Ci_6alkylsulfonyl, benzoyl or monocyclic heterocyclyl, wherein
x is 1-6;
W is a bond, CyH2yC(R15)(R16)CzH2z or CyH2yCH(R15)CH(R16)CzH2z, wherein
R15 and R16 are independently selected from hydrogen, fluoro, hydroxy and Ci_6alkyl, y is 0-6;
z is 0-6;
X is a bond; O; S; S(0)2; or NR17, wherein R17 is hydrogen, Ci_6alkyl;
13 17
or R and R , together with the nitrogen to which they are attached, form monocyclic heterocyclyl;
or a pharmaceutically acceptable salts, or enantiomers thereof.
5. The pharmaceutical composition according to claim 4, wherein the HBsAg inhibitor is (+)- 10-methoxy-6-(2-methoxy- l, l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)- 10-methoxy-6-(2-methoxy- l, l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(+)-6-(2-hydroxy- l, l-dimethyl-ethyl)- 10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid; (-)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(+)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid; or
(-)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the TLR7 agonist is a compound of formula (III):
Figure imgf000068_0001
R 18 is hydroxy, Ci-6alkyl, haloCi_6alkyl, Ci_6alkylcarbonyl-0-, Ci_6alkyl-S-, azido, cyano, C2_ 6alkenyl, Ci_6alkylsulfonyl-NH-, (C1-6alkyl)2N-, Ci-6alkylcarbonyl-NH- or heterocyclic amino;
R19 is hydrogen, Ci-6alkyl, Ci_6alkoxyCi-6alkyl, C3_7cycloalkyl, C2-6alkynyl, C2_6alkenyl, benzyl and thiophenyl;
R 20 is hydrogen or Ci_6alkylcarbonyl;
R 21 is hydrogen or hydroxy;
or pharmaceutically acceptable salt, enantiomer or diastereomer thereof. 7. The pharmaceutical composition according to claim 6, wherein the TLR7 agonist is
[(lS)-l-[(2S,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy- tetrahydrofuran-2- yl] propyl] acetate ;
5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one;
5-amino-3-(2'-0-acetyl-3'-deoxy-P-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one; 5-amino-3-(3'-deoxy-P-D-ribomranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dion^ 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2-yl]-6H- thiazolo[4,5-d]pyrimidine-2,
7-dione; or
5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2- yl]thiazolo[4,5-d]pyrimidin-2-one;
or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
8. The pharmaceutical composition according to any one of claim 1 to 5, wherein the TLR7 agonist is a compound of formula (IV):
Figure imgf000069_0001
wherein
R 2"2 and R 2"3J are independently selected from hydrogen, C2-6alkenyl and C1-6alkyl;
24 and R 2"5J are independently selected from hydrogen, Ci_6alkyl, C3_7cycloalkyl, C3_
7cycloalkylC2-6alkynyl, C2_6alkenyl, C2_6alkynyl and 2-thiophenyl;
R26 is hydro gen or Ci_6alkylcarbonyl;
R 27 is hydrogen or hydroxy;
or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
9. The pharmaceutical composition according to claim 8, wherein the TLR7 agonist is [(S)-[(2S,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]- cyclopropyl-methyl] acetate; or
5-amino-3-[(2S,5R)-2-[(lS)-cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione;
or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
10. The pharmaceutical composition according to any one of claims 1 to 5, wherein the TLR7 agonist is
5-amino-3-(3'-deoxy-P-D-ribomranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; or 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione;
or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
11. The pharmaceutical composition according to claim 1, wherein the composition consists of an HBsAg inhibitor and a TLR7 agonist, in a pharmaceutically acceptable carrier.
12. The pharmaceutical composition according to claim 1 or 11, consisting of
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)- 3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; or
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3- carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
in a pharmaceutically acceptable carrier.
13. A pharmaceutical composition, which is
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)- 3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; or
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
in a pharmaceutically acceptable carrier.
14. The pharmaceutical composition according to any one of claims 1, 11, 12 and 13, wherein the composition additionally comprising one or more other antiviral agents.
15. The pharmaceutical composition according to claim 14, wherein said other antiviral agents are selected from Lamivudine, Adefovir dipivoxil, Entecavir, Telbivudine, Clevudine, Tenofovir disoproxil, Tenofovir disoproxil fumarate, Roferon A®, Intron A®, Pegasys®, Pegintron® and Pai Ge Bin.
16. A method for manufacturing a medicament for treatment or prophylaxis of hepatitis B virus infection, characterized in that an HBsAg inhibitor and a TLR7 agonist are used in the medicament.
17. The method according to claim 16, wherein the HBsAg inhibitor and the TLR7 agonist are co -administered in the same formulation or different formulation.
18. The method according to claim 16, wherein the HBsAg inhibitor and the TLR7 agonist are intended for administration to a subject by the same route or different routes.
19. The method according to claim 16, wherein the HBsAg inhibitor and the TLR7 agonist are intended for administration to a subject by parenteral or oral administration.
20. The method according to any one of claims 16, wherein the administration is simultaneous or sequential.
21. The method according to claim 16, wherein the HBsAg inhibitor is a compound of formula (I) or formula (II), or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
22. The method according to claim 21, wherein the HBsAg inhibitor 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-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3- carboxylic acid;
(+)-6-ieri-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-6-ieri-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid; (+)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(+)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(+)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid); or
(-)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid);
or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
23. The method according to claim 16, wherein the TLR7 agonist is a compound of formula (III) or formula (IV), or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
24. The method according to claim 23, wherein the TLR7 agonist is
[(15)-l-[(25,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy- tetrahydrofuran-2- yl] propyl] acetate ;
[(5)-[(25,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]- cyclopropyl-methyl] acetate;
5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-JH-thiazolo[4,5-d]pyrimidin-2-one;
5-amino-3-(2'-0-acetyl-3'-deoxy-P-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one; 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione;
5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione;
5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2- yl]thiazolo[4,5-d]pyrimidin-2-one; or
5-amino-3-[(2S,5R)-2-[(lS)-cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione;
or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
25. A method for manufacturing a medicament for treatment or prophylaxis of hepatitis B virus infection, characterized in that the medicament is a combination of (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)- 3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; or
a combination of (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
in a pharmaceutically acceptable carrier.
26. The method according to any one of claims 16 to 25, wherein the medicament additionally comprises one or more other antiviral agents.
27. The method according to claim 26, wherein said other antiviral agents are selected from Lamivudine , Adefovir dipivoxil, Entecavir, Telbivudine, Clevudine, Tenofovir disoproxil, Tenofovir disoproxil fumarate, Roferon A®, Intron A®, Pegasys®, Pegintron® and Pai Ge Bin.
28. A kit comprising a container comprising an HBsAg inhibitor and a TLR7 agonist.
29. The kit according to claim 28, further comprising a sterile diluent.
30. The kit according to claim 28, further comprising a package insert comprising printed instructions directing the use of a combined treatment of an HBsAg inhibitor and a TLR7 agonist as a method for treatment or prophylaxis of hepatitis B virus infection.
31. The kit according claim 28, wherein the HBsAg inhibitor 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-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3- carboxylic acid;
(+)-6-ieri-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-6-ieri-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid; (+)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(+)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(+)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid); or
(-)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
32. The kit according to claim 28, wherein the TLR7 agonist is
[(15)-l-[(25,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy- tetrahydrofuran-2- yl] propyl] acetate ;
[(5)-[(25,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]- cyclopropyl-methyl] acetate;
5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-JH-thiazolo[4,5-d]pyrimidin-2-one;
5-amino-3-(2'-0-acetyl-3'-deoxy-P-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one; 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione;
5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione;
5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2- yl]thiazolo[4,5-d]pyrimidin-2-one; or
5-amino-3-[(2S,5R)-2-[(lS)-cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione;
or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
33. A kit containing
a combination of (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)- 3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; or a combination of (+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
in a pharmaceutically acceptable carrier.
34. 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 a
pharmaceutically acceptable salt, enantiomer or diastereomer thereof; and a second amount of a TLR7 agonist; or vice versa.
35. The method according to claim 34, wherein the HBsAg inhibitor 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-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3- carboxylic acid;
(+)-6-ieri-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-6-ieri-butyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(+)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-10-methoxy-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(+)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(-)-6-(2-hydroxy-l,l-dimethyl-ethyl)-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
(+)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid); or (-)-10-chloro-6-(2-methoxy-l,l-dimethyl-ethyl)-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid;
or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
36. The method according to claim 34, wherein the TLR7 agonist is
[(15)-l-[(25,4R,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-4-hydroxy- tetrahydrofuran-2- yl] propyl] acetate ;
[(5)-[(25,5R)-5-(5-amino-2-oxo-thiazolo[4,5-d]pyrimidin-3-yl)-l,3-oxathiolan-2-yl]- cyclopropyl-methyl] acetate;
5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-JH-thiazolo[4,5-d]pyrimidin-2-one;
5-amino-3-(2'-0-acetyl-3'-deoxy-P-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one; 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)-3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione;
5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione;
5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l-hydroxypropyl]tetrahydrofuran-2- yl]thiazolo[4,5-d]pyrimidin-2-one; or
5-amino-3-[(2S,5R)-2-[(lS)-cyclopropyl(hydroxy)methyl]-l,3-oxathiolan-5-yl]-6H- thiazolo[4,5-d]pyrimidine-2,7-dione;
or pharmaceutically acceptable salt, enantiomer or diastereomer thereof.
37. A method for the treatment or prophylaxis of hepatitis B virus infection, comprising administration to a subject with an effective amount of composition of
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-(3'-deoxy-P-D-ribofuranosyl)- 3H,6H-thiazolo[4,5-d]pyrimidin-2,7-dione; or
(+)-10-methoxy-6-isopropyl-9-(3-methoxypropoxy)-2-oxo-6,7- dihydrobenzo[a]quinolizine-3-carboxylic acid and 5-amino-3-[(2R,3R,5S)-3-hydroxy-5-[(lS)-l- hydroxypropyl]tetrahydrofuran-2-yl]-6H-thiazolo[4,5-d]pyrimidine-2,7-dione;
in a pharmaceutically acceptable carrier.
38. The use of pharmaceutical composition according to any one of claims 1 to 15 as an antiviral medicament, in particular as the medicament for treatment or prophylaxis of hepatitis B virus infection.
39. The use of an HBsAg inhibitor and a TLR7 agonist for the manufacture of pharmaceutical composition according to any one of claims 1 to 15 as an antiviral medicament, in particular the medicament for treatment or prophylaxis of hepatitis B virus infection.
40. The invention as hereinbefore described.
***
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