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WO2020202091A1 - Chemical compounds - Google Patents

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Publication number
WO2020202091A1
WO2020202091A1 PCT/IB2020/053193 IB2020053193W WO2020202091A1 WO 2020202091 A1 WO2020202091 A1 WO 2020202091A1 IB 2020053193 W IB2020053193 W IB 2020053193W WO 2020202091 A1 WO2020202091 A1 WO 2020202091A1
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WO
WIPO (PCT)
Prior art keywords
methyl
ethyl
pyrazole
benzo
dihydro
Prior art date
Application number
PCT/IB2020/053193
Other languages
French (fr)
Inventor
Bryan Wayne KING
Andrew James Peat
Joseph E. Pero
Jared Troy SPLETSTOSER
Peng Li
Original Assignee
Glaxosmithkline Intellectual Property Development Limited
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Publication of WO2020202091A1 publication Critical patent/WO2020202091A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the present invention relates to compounds that modulate the activity of transmembrane protein 173 (TMEM173), also known as STING (STimulator of Interferon Genes), and methods of treating hepatitis B virus infections in a mammal by
  • Hepatitis B is a viral disease transmitted parenterally by contaminated material such as blood and blood products, contaminated needles, sexually and vertically from infected or carrier mothers to their offspring. In those areas of the world where the disease is common vertical transmission at an early age results in a high proportion of infected individuals becoming chronic carriers of hepatitis B. It is estimated by the World Health Organization that more than 2 billion people have been infected worldwide, with about 4 million acute cases per year, 1 million deaths per year, and 350-400 million chronic carriers. Approximately 25% of carriers die from chronic hepatitis, cirrhosis, or liver cancer and nearly 75% of chronic carriers are Asian. Hepatitis B virus (HBV) is the second most significant carcinogen behind tobacco, causing from 60% to 80% of all primary liver cancer. HBV is 100 times more contagious than HIV.
  • HBV Hepatitis B virus
  • Hepatitis B viral infections are a continuing medical problem because, like any rapidly-replicating infectious agent, there are continuing mutations that help some sub-populations of HBV become resistant to current treatment regimens. At the present time there are no effective therapeutic agents for treating humans infected with HBV infections which result in seroconversion to the virus in the body, or which effect a 90% reduction of antigen, compared to baseline numbers before treatment, in persons suffering from a hepatitis B viral infection.
  • nucleoside and nucleotide therapies entecavir and tenofovir are successful at reducing viral load, but the rates of HBeAg seroconversion and HBsAg loss are even lower than those obtained using IFNa therapy.
  • Other similar therapies including lamivudine (3TC), telbivudine (LdT), and adefovir are also used, but for
  • nucleoside/nucleotide therapies in general, the emergence of resistance limits therapeutic efficacy.
  • Vertebrates have evolved mechanisms of immune defense to eliminate infective pathogens.
  • this immune system comprises two branches; innate immunity and adaptive immunity.
  • the innate immune system is the first line of defense which is initiated by Pattern Recognition Receptors (PRRs) which detect ligands from the pathogens as well as damage associated molecular patterns (Takeuchi O. et al, Cell, 2010: 140, 805-820).
  • PRRs Pattern Recognition Receptors
  • TLRs Toll-like receptors
  • C-type lectin receptors C-type lectin receptors
  • RIG-I retinoic acid inducible gene I
  • NLRs NOD-like receptors
  • Activation of PRRs leads to up-regulation of genes involved in the inflammatory response including type 1 interferons, pro-inflammatory cytokines and chemokines which suppress pathogen replication and facilitate adaptive immunity.
  • Interferon was first described as a substance which could protect cells from viral infection (Isaacs & Lindemann, J. Virus Interference. Proc. R. Soc. Lon. Ser. B. Biol. Sci. 1957: 147, 258-267).
  • the type I interferons are a family of related proteins encoded by genes on chromosome 9 and encoding at least 13 isoforms of interferon alpha (IFNa) and one isoform of interferon beta (IFNp).
  • Recombinant IFNa was the first approved biological therapeutic and has become an important therapy in viral infections and in cancer.
  • interferons are known to be potent modulators of the immune response, acting on cells of the immune system.
  • the adaptor protein STING (STimulator of Interferon Genes), also known as TMEM 173, MPYS, MITA and ERIS, has been identified as a central signaling molecule in the innate immune response to cytosolic nucleic acids (Ishikawa H and Barber G N, Nature, 2008: 455, 674-678; WO2013/1666000). Activation of STING results in up-regulation of IRF3 and NFKB pathways leading to induction of lnterferon-b and other cytokines. STING is critical for responses to cytosolic DNA of pathogen or host origin, and of unusual nucleic acids called Cyclic Dinucleotides (CDNs)
  • CDNs Cyclic Dinucleotides
  • CDNs were first identified as bacterial secondary messengers responsible for controlling numerous responses in the prokaryotic cell.
  • Bacterial CDNs such as c-di- GMP are symmetrical molecules characterized by two 3’, 5’ phosphodiester linkages.
  • Direct activation of STING by bacterial CDNs has recently been confirmed through X-ray crystallography (Burdette D L and Vance R E, Nature Immunology, 2013: 14, 19-26).
  • Bacterial CDNs and their analogues have consequently attracted interest as potential vaccine adjuvants (Libanova R. et al, Microbial Biotechnology 2012: 5, 168-176; W02007/054279, W02005/087238).
  • cGAMP cyclic GMP-AMP synthase
  • hepatitis B viral infections in animals may be advantageously treated by administration of a first compound that modulates STING activity and administering a second compound that does not modulate STING activity.
  • STING is essential for antimicrobial host defense, and has been linked to protection against a range of DNA and RNA viruses and bacteria (reviewed in Barber et al. Nat. Rev. Immunol. 2015: 15(2): 87-103, Ma and Damania, Cell Host & Microbe, 2016: 19(2) 150-158).
  • Herpesviridae, Flaviviridae, Coronaviridae, Papillomaviridae, Adenoviridae, Hepadnaviridae, ortho- and paramyxoviridae and rhabdoviridae have evolved mechanisms to inhibit STING mediated Type I interferon production and evade host immune control (Holm et al., Nat Comm.
  • inhibitors of STING provide a treatment to patients with chronic type I interferon and proinflammatory cytokine production associated with infections or complex autoimmune diseases.
  • Allergic diseases are associated with a Th2-biased immune-response to allergens.
  • Th2 responses are associated with raised levels of IgE, which, via its effects on mast cells, promotes a hypersensitivity to allergens, resulting in the symptoms seen, for example, in allergic rhinitis and asthma.
  • the immune-response to allergens is more balanced with a mixed Th2/Th1 and regulatory T cell response.
  • Type 1 interferons have been shown to result in reduction of Th2-type cytokines in the local environment and promote Th1/Treg responses.
  • induction of type 1 interferons by, for example, activation of STING may offer benefit in treatment of allergic diseases such as asthma and allergic rhinitis (Huber J.P. et al J Immunol 2010: 185, 813-817).
  • WO2013/185052, U.S.2014/0341976, WO 2015/077354, PCT/EP2015/062281 and GB 1501462.4 disclose certain cyclic di-nucleotides and their use in inducing an immune response via activation of STING.
  • International Patent Applications WO2017/106740 describes the use of cyclic-di-nucleotide and related scaffold that measurably inhibit STING signaling and methods of identifying potent inhibitors of STING signaling.
  • TMEM173 transmembrane protein 173
  • STING Stimulator of Interferon Genes
  • STING-mediated disease or disorders including inflammation, allergic and autoimmune diseases, cancer, and pre-cancerous syndromes, infectious diseases, and for use as immunogenic composition or vaccine adjuvants.
  • targeting STING with small molecules may be a promising approach for treating diseases and conditions in which modulation for the type 1 IFN pathway is beneficial, including infectious diseases.
  • HBV serum markers HBeAg hepatitis B envelope antigen
  • HBsAg hepatitis B surface antigen
  • Targeting STING with small molecules may be a promising new approach for treating hepatitis B virus infections.
  • combination therapy of compounds which target STING with other therapies used to treat HBV has the potential to result in an effective cure of hepatitis B infections by combining multiple therapeutic agents with different modes of action in a combination therapy.
  • administering compounds that target STING, and administering small molecule inhibitors of the HBV antigens HBsAg and HBeAg can directly target the receptor involved in inflammation and the HBV antigens responsible for invoking an immunogenic response in a mammal, so there is the potential to reduce serum HBeAg and HBsAg levels.
  • the compounds of this invention are useful as moderators of STING, and accordingly, may provide a beneficial therapeutic impact in treatment of diseases, disorders and/or conditions in which modulation of STING (Stimulator of Interferon Genes) is beneficial, for example for infectious diseases.
  • STING Stimulator of Interferon Genes
  • One embodiment of the invention is directed to a compound as described herein, or a pharmaceutically acceptable salt thereof or prodrug thereof.
  • One embodiment of the invention is directed to a compound according to
  • C 2 -C 6 alkyl or said C 2 -C 6 alkenyl is each independently optionally substituted by 1 -2 substituents selected from -R c , -OH, and -OR c ;
  • R 1 and R 2 are independently absent or Ci-C 3 alkyl
  • W 1 , X 1 , Y 1 , and Z 1 are each independently -CR 3 , -CR 3 ', -CR 3 ", -CR 3 "' or N, with the proviso that no more than two of W 1 , X 1 , Y 1 and Z 1 may be N;
  • W 2 , X 2 , Y 2 , and Z 2 are each independently -CR 4 , -CR 4 ', -CR 4 ", -CR 4 "' or N, with the proviso that no more than two of W 2 , X 2 , Y 2 and Z 2 may be N;
  • R 3 , R 3 ', R 3 ", R 3 "', R 4 , R 4 ', R4", R 4 "', R 9 , R 10 , R 11 , and R 12 are each independently selected from H, Ci- 4 alkoxyl, Ci_ 4 alkyl, -COO-Ci- 4 alkyl, -C 4-7 heterocycloalkyl, -Ci- 4 alkylC 4. yheterocycloalkyl, and -OCi- 4 alkylC 4-7 heterocycloalkyl, wherein the -C 4.7 heterocycloalkyl, the
  • -OCi- 4 alkylC 4-7 heterocycloalkyl comprises one or more heteroatoms selected from O, and N, wherein the Ci_ 4 alkyl, Ci- 4 alkoxyl, the Ci_ 4 alkyl of the -Ci- 4 alkylC 4.
  • yheterocycloalkyl or the Ci_ 4 alkyl of the -OCi- 4 alkylC 4-7 heterocycloalkyl is optionally substituted by 1 -4 substituents independently selected from -R c , -OH and -OR c , and wherein the C 4 -7heterocycloalkyl, the C 4 -7heterocycloalkyl of the -Ci- 4 alkylC 4.
  • yheterocycloalkyl or the C 4-7 heterocycloalkyl of the -OCi- 4 alkylC 4-7 heterocycloalkyl is substituted by 1 -4 substituents R’, R”, R’”, and R””, wherein R’, R”, R’”, and R”” are each independently selected from -R c , -OH and -OR c , or any two of R’, R”, R’”, and R”” may combine with the ring atoms to which they are attached to form a 3-7 membered ring;
  • R 5 and R 6 are independently Ci-C 4 alkyl
  • R 7 and R 8 are each independently H or Ci-C 4 alkyl
  • each R c is independently Ci-C 4 alkyl optionally substituted by a substituent selected from -OH or Ci_ 4 alkoxyl;
  • this invention provides a compound of Formula (I) or a salt thereof, particularly a
  • This invention specifically provides for the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as an active therapeutic substance in the treatment of a STING-mediated disease or disorder, specifically, for use in the treatment of a disease mediated by agonism or antagonism of STING.
  • the invention also provides a compound of Formula (I), or a salt thereof, particularly a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for the treatment of a STING-mediated disease or disorder.
  • the invention is also directed to a method of modulating STING, which method comprises contacting a cell with a compound according to Formula (I), or a salt, particularly a pharmaceutically acceptable salt, thereof.
  • the invention is further directed to a method of treating a STING mediated disease or disorder which comprises administering a therapeutically effective amount of a compound according to Formula (I), or a salt, particularly a pharmaceutically acceptable salt thereof, to a patient (a human or other mammal, particularly, a human) in need thereof.
  • STING mediated diseases or disorders include inflammation, allergic and autoimmune diseases, infectious diseases including Hepatitis B virus (HBV) infection and/or Hepatitis D virus (HDV) infection, cancer, pre-cancerous syndromes, metabolic diseases, and cardiovascular diseases.
  • modulators of STING may be useful as immunogenic composition or vaccine adjuvants.
  • the present invention is further directed to a pharmaceutical composition comprising a compound according to Formula (I), or a salt, particularly a pharmaceutically acceptable salt, thereof and a pharmaceutically acceptable excipient.
  • this invention is directed to a pharmaceutical composition for the treatment of a STING mediated disease or disorder, where the composition comprises a compound according to Formula (I), or a salt, particularly a pharmaceutically acceptable salt, thereof and a pharmaceutically acceptable excipient.
  • each occurrence of R is independently H or any appropriate substituent group on nitrogen, for example alkyl.
  • alkyl represents a saturated, straight or branched hydrocarbon group having the specified number of carbon atoms.
  • Ci-C 4 alkyl refers to a straight or branched alkyl moiety containing from 1 to 4 carbon atoms.
  • Exemplary alkyls include, but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, f-butyl, pentyl and hexyl.
  • substituent term such as "alkyl”
  • another substituent term for example as in“hydroxy(Ci-C 4 alkyl)
  • the linking substituent term e.g., alkyl
  • alkyl is intended to encompass a divalent moiety, wherein the point of attachment is through that linking substituent.
  • “hydroxy(Ci-C 4 alkyl)” groups include, but are not limited to, hydroxymethyl, hydroxyethyl, and
  • Alkenyl refers to straight or branched hydrocarbon group having the specified number of carbon atoms and at least 1 and up to 3 carbon-carbon double bonds. Examples include ethenyl and propenyl.
  • Alkoxy- or“(alkyl)oxy- refers to an "alkyl-oxy-” group, containing an alkyl moiety, having the specified number of carbon atoms, attached through an oxygen linking atom.
  • the term "Ci-C 4 alkoxy-" represents a saturated, straight or branched hydrocarbon moiety having at least 1 and up to 4 carbon atoms attached through an oxygen linking atom.
  • Exemplary "Ci-C 4 alkoxy-" or“(Ci-C 4 alkyl)oxy-” groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, s- butoxy, and f-butoxy.
  • a carbocyclic group or moiety is a cyclic group or moiety in which the ring members are carbon atoms, which may be saturated, partially unsaturated
  • Cycloalkyl refers to a non-aromatic, saturated, hydrocarbon ring group containing the specified number of carbon atoms in the ring.
  • C3-C 6 cycloalkyl refers to a cyclic group having from three to six ring carbon atoms.
  • Exemplary "C3-C6cycloalkyl” groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
  • a heterocyclic group or moiety is a cyclic group or moiety having, as ring members, atoms of at least two different elements, which cyclic group or moiety may be saturated, partially unsaturated (non-aromatic) or fully unsaturated (aromatic).
  • Heteroatom refers to a nitrogen, sulfur, or oxygen atom, for example a nitrogen atom or an oxygen atom.
  • Heterocycloalkyl refers to a non-aromatic, monocyclic or bicyclic group containing from 3 to 10 ring atoms and containing one or more (generally one or two) heteroatom ring members independently selected from oxygen, sulfur, and nitrogen.
  • the point of attachment of a heterocycloalkyl group may be by any suitable carbon or nitrogen atom.
  • heterocycloalkyl groups include, but are not limited to, aziridinyl, thiiranyl, oxiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, 1 ,3-dioxolanyl, piperidinyl, piperazinyl, tetrahydropyranyl,
  • dihydropyranyl tetrahydrothiopyranyl, 1 ,3-dioxanyl, 1 ,4-dioxanyl, 1 ,3-oxathiolanyl, 1 ,3- oxathianyl, 1 ,3-dithianyl, 1 ,4-oxathiolanyl, 1 ,4-oxathianyl, 1 ,4-dithianyl, morpholinyl, thiomorpholinyl, and hexahydro-1 H-1 ,4-diazepinyl.
  • Examples of "4-7 membered heterocycloalkyl” groups include oxetanyl, thietanyl and azetidinyl.
  • Examples of“4-7 membered heterocycloalkyl” groups include those found in the compounds N-((E)-1 -((E)-4-((E)-7-((3-oxa-6-azabicyclo[3.1 .1 ]heptan-6-yl)methyl)-2- ((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H- benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2- ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide and N-((E)-4-((3-oxa-6- azabicyclo[3.1 .1 ]heptan-6-yl)methyl)-1 -((E)-4-((E)-2-((E)
  • 5-6 membered heterocycloalkyl represents a non-aromatic, monocyclic group, containing 5 or 6 ring atoms, which includes one or two heteroatoms selected independently from oxygen, sulfur, and nitrogen.
  • Illustrative examples of 5-6 membered heterocycloalkyl groups include, but are not limited to pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, piperazinyl, morpholinyl, and thiomorpholinyl.
  • Heteroaryl refers to an aromatic monocyclic or bicyclic group containing 5 to 10 ring atoms, including 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, wherein at least a portion of the group is aromatic.
  • this term encompasses bicyclic heterocyclic-aryl groups containing either a phenyl ring fused to a heterocyclic moiety or a heteroaryl ring moiety fused to a carbocyclic moiety.
  • the point of attachment of a“heteroaryl” group may be by any suitable carbon or nitrogen atom.
  • 5-6 membered heteroaryl represents an aromatic monocyclic group containing 5 or 6 ring atoms, including at least one carbon atom and 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur.
  • Selected 5- membered heteroaryl groups contain one nitrogen, oxygen, or sulfur ring heteroatom, and optionally contain 1 , 2, or 3 additional nitrogen ring atoms.
  • Selected 6-membered heteroaryl groups contain 1 , 2, or 3 nitrogen ring heteroatoms.
  • 5-membered heteroaryl groups include furyl (furanyl), thienyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, and oxadiazolyl.
  • Selected 6-membered heteroaryl groups include pyridinyl (pyridyl), pyrazinyl, pyrimidinyl, pyridazinyl and triazinyl.
  • 9-10 membered heteroaryl refers to an aromatic bicyclic group containing 9 or 10 ring atoms, including 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur.
  • 9-membered heteroaryl (6,5-fused heteroaryl) groups include benzothienyl, benzofuranyl, indolyl, indolinyl (dihydroindolyl), isoindolyl, isoindolinyl, indazolyl, isobenzofuryl, 2,3-dihydrobenzofuryl, benzoxazolyl,
  • benzoisoxazolyl benzothiazolyl, benzoisothiazolyl, benzimidazolyl, benzoxadiazolyl, benzothiadiazolyl, benzotriazolyl, purinyl, imidazopyridinyl, pyrazolopyridinyl, triazolopyridinyl and 1 ,3-benzodioxolyl.
  • 10-membered heteroaryl (6,6-fused heteroaryl) groups include quinolinyl (quinolyl), isoquinolyl, phthalazinyl, naphthridinyl (1 ,5-naphthyridinyl,
  • cyano refers to a nitrile group, -CoN.
  • the term "optionally substituted” indicates that a group (such as an alkyl, cycloalkyl, alkoxy, heterocycloalkyl, aryl, or heteroaryl group) or ring or moiety may be unsubstituted, or the group, ring or moiety may be substituted with one or more substituent(s) as defined in the substituent definitions (A, R 3 , etc,) provided herein.
  • groups may be selected from a number of alternative groups, the selected groups may be the same or different.
  • pharmaceutically acceptable refers to those compounds, materials, compositions, and dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • compound(s) of this invention mean a compound of Formula (I) as defined herein, in any form, i.e., any tautomeric/isomeric form, any salt or non-salt form (e.g., as a free acid or base form, or as a salt, particularly a pharmaceutically acceptable salt thereof) and any physical form thereof (e.g., including non-solid forms (e.g., liquid or semi-solid forms), and solid forms (e.g., amorphous or crystalline forms, specific polymorphic forms, solvate forms, including hydrate forms (e.g., mono-, di- and hemi- hydrates)), and mixtures of various forms.
  • any form i.e., any tautomeric/isomeric form, any salt or non-salt form (e.g., as a free acid or base form, or as a salt, particularly a pharmaceutically acceptable salt thereof) and any physical form thereof (e.g., including non-solid forms (e.g., liquid or semi
  • the compounds of this invention may contain one or more asymmetric centers (also referred to as a chiral center), such as a chiral carbon, or a chiral -SO- moiety.
  • asymmetric centers also referred to as a chiral center
  • Compounds of this invention containing one or more chiral centers may be present as racemic mixtures, diastereomeric mixtures, enantiomerically enriched mixtures, diastereomerically enriched mixtures, or as enantiomerically or
  • the stereochemistry of the chiral center present in compounds of this invention is generally represented in the compound names and/or in the chemical structures illustrated herein. Where the stereochemistry of a chiral center present in a compound of this invention, or in any chemical structure illustrated herein, is not specified, the structure is intended to encompass any stereoisomer and all mixtures thereof. Accordingly, the present invention encompasses all isomers of the compounds described herein or the compounds of Formula (I), and salts thereof, whether as individual isomers isolated such as to be substantially free of the other isomer (i.e. pure) or as mixtures (i.e. racemates and racemic mixtures). An individual isomer isolated such as to be substantially free of the other isomer (i.e. pure) may be isolated such that less than 10%, particularly less than about 1 %, for example less than about 0.1 % of the other isomer is present.
  • Individual stereoisomers of a compound of this invention may be resolved (or mixtures of stereoisomers may be enriched) using methods known to those skilled in the art. For example, such resolution may be carried out (1) by formation of
  • stereoisomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer to the other by asymmetric transformation.
  • “cleavable bond” means any chemical bond capable of being broken.
  • a cleavable bond is selected from among: an amide, a polyamide, an ester, an ether, a phosphodiester, a phosphate ester, a carbamate, a di-sulfide, or a peptide.
  • modulation means a change of amount or quality of a molecule, function, or activity when compared to the amount or quality of the molecule, function, or activity prior to modulation.
  • “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an animal.
  • a pharmaceutically acceptable carrier or diluent is sterile saline.
  • such sterile saline is pharmaceutical grade saline.
  • prodrug means an inactive or less active form of a compound which, when administered to a subject, undergoes some in vivo
  • a prodrug may include a cleavable moiety, wherein the cleavable moiety is removed or metabolized to form the active, or more active, compound.
  • the cleavable moiety may be connected to the active form of the compound by a cleavable bond, including but not limited to an ester, an amide, a sulfonamide, an amine, an imine, an ether, a thioether, a disulfide, a glycosidic, a phosphate, a carbamate, a carbonate or a boronic ester bond, or some other cleavable bond.
  • a cleavable bond including but not limited to an ester, an amide, a sulfonamide, an amine, an imine, an ether, a thioether, a disulfide, a glycosidic, a phosphate, a carbamate, a carbonate or a boronic ester bond, or some other cleavable bond.
  • the term "effective amount” means that amount of a drug or drug substance, or pharmaceutical agent, that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician.
  • therapeutically effective amount means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder.
  • the term also includes within its scope amounts effective to enhance normal
  • HBV means mammalian Hepatitis B virus, including human Hepatitis B virus.
  • the term encompasses geographical genotypes of Hepatitis B virus, particularly human Hepatitis B virus, as well as variant strains of geographical genotypes of Hepatitis B virus.
  • Hepatitis B-related condition or“HBV-related condition” means any disease, biological condition, medical condition, or event which is exacerbated, caused by, related to, associated with, or traceable to a Hepatitis B infection, exposure, or illness.
  • Hepatitis B-related condition includes jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, and conditions having symptoms which may include any or all of the following: flu-like illness, weakness, aches, headache, fever, loss of appetite, diarrhea, jaundice, nausea and vomiting, pain over the liver area of the body, clay- or grey-colored stool, itching all over, and dark-colored urine, when coupled with a positive test for presence of a Hepatitis B virus, a Hepatitis B viral antigen, or a positive test for the presence of an antibody specific for a Hepatitis B viral antigen.
  • One embodiment of the invention is directed to a compound according to Formula (I):
  • C 2 -C 6 alkyl or said C 2 -C 6 alkenyl is each independently optionally substituted by 1 -2 substituents selected from -R c , -OH, and -OR c ;
  • R 1 and R 2 are independently absent or Ci-C 3 alkyl
  • W 1 , X 1 , Y 1 , and Z 1 are each independently -CR 3 , -CR 3 ', -CR 3 ", -CR 3 "' or N, with the proviso that no more than two of W 1 , X 1 , Y 1 and Z 1 may be N;
  • W 2 , X 2 , Y 2 , and Z 2 are each independently -CR 4 , -CR 4 ', -CR 4 ", -CR 4 "' or N, with the proviso that no more than two of W 2 , X 2 , Y 2 and Z 2 may be N;
  • R 3 , R 3 ', R 3 ", R 3 "', R 4 , R 4 ', R4", R 4 "', R 9 , R 10 , R 11 , and R 12 are each independently selected from H, Ci- 4 alkoxyl, Ci_ 4 alkyl, -COO-Ci_ 4 alkyl, -C 4-7 heterocycloalkyl, -Ci- 4 alkylC 4.
  • -OCi- 4 alkylC 4-7 heterocycloalkyl comprises one or more heteroatoms selected from O, and N,
  • Ci_ 4 alkyl or the Ci_ 4 alkyl of the -OCi- 4 alkylC 4-7 heterocycloalkyl is optionally substituted by 1 -4 substituents independently selected from -R c , -OH and -OR c , and wherein the C 4-7 heterocycloalkyl, the C 4-7 heterocycloalkyl of the -Ci- 4 alkylC 4.
  • R 5 and R 6 are independently Ci-C 4 alkyl
  • R 7 and R 8 are each independently H or Ci-C 4 alkyl
  • each R c is independently Ci-C 4 alkyl optionally substituted by a substituent selected from -OH or Ci_ 4 alkoxyl;
  • One embodiment of the invention is directed to a compound according to Formula (I) wherein:
  • R 1 and R 2 are -CH 2 -;
  • Y 1 , Y 2 , X 1 and X 2 are -CH-;
  • W 1 , W 2 , Z 1 and Z 2 are each independently selected from: -CH-, -C(azepine- methyl)-,
  • R 5 and R 6 are -CH 3 ;
  • R 7 and R 8 are C 2 alkyl
  • R 9 , R 10 , R 11 , and R 12 are each independently selected from: H and CH 3 ;
  • One embodiment provides compounds of Formula (I), wherein R 1 and R 2 are -CH 2 - and R 5 and R 6 are -CH 3 .
  • One embodiment provides compounds of Formula (I), wherein W 1 , X 1 , Y 1 , and Z 1 are each independently -CR 3 , -CR 3 ', -CR 3 " or -CR 3 "' and W 2 , X 2 , Y 2 , and Z 2 are each independently -CR 4 , -CR 4 ', -CR 4 " or -CR 4 "'.
  • W 1 is CR 3 and W 2 is CR 4 , wherein CR 3 and CR 4 are each independently selected from -C 4.
  • yheterocycloalkyl -Ci- 4 alkylC 4 -7heterocycloalkyl, and -OCi- 4 alkylC 4-7 heterocycloalkyl.
  • One embodiment provides compounds of Formula (I), wherein W 1 is CR 3 and W 2 is CR 4 , wherein R 3 and CR 4 are each independently selected from Ci_ 4 alkoxyl or Ci- 4 alkyl, wherein the Ci_ 4 alkyl or Ci_ 4 alkoxyl is optionally substituted by 1 -4 substituents independently selected from -R c , -OH and -OR c .
  • One embodiment provides compounds of Formula (I), wherein A is C 2 -C 6 alkylene.
  • One embodiment provides compounds of Formula (I), wherein A is C2-C6alkenylene.
  • One embodiment provides compounds of Formula (I), wherein R 1 and R 2 are each -CH 2 - and A is C 2 -alkenylene.
  • R 7 and R 8 are each independently H or C 2 alkyl.
  • One embodiment provides compounds of Formula (I), wherein R 7 and R 8 are each C 2 alkyl.
  • One embodiment provides compounds of Formula (I), wherein R 9 , R 10 , R 11 , and R 12 are each independently H or -CH3.
  • One embodiment provides compounds of Formula (I), wherein Y 1 , Y 2 , X 1 and X 2 are -CH.
  • W 1 , W 2 , Z 1 and Z 2 are each independently selected from: H, azepine-methyl, 3-oxa-6- azabicyclo[3.1 1 ]heptane-methyl, methoxymethyl, 1 -hydroxyethyl, -C(0)0CH 3 , methoxy, 3-hydroxypropoxy, hydroxymethyl, morpholinomethyl, 1 ,2-dihydroxyethyl, and 1 -hydroxy- 2-morpholinoethyl.
  • a compound selected from the compounds of Examples 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13-1 , 13-2, 14, 15, 16, 17, 19, 32 and 33, or a salt thereof.
  • compounds of the invention may exist in both unsolvated and solvated forms.
  • solvate is used herein to describe a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, ethanol.
  • solvent molecules for example, ethanol.
  • hydrate is employed when said solvent is water.
  • Pharmaceutically acceptable solvates include hydrates and other solvates wherein the solvent of crystallization may be isotopically substituted, e.g. D 2 0, deacetone, d 6 -DMSO.
  • Suitable pharmaceutically acceptable salts of the compounds described herein can include acid addition salts or base addition salts.
  • suitable pharmaceutically acceptable salts see Berge et al., J. Pharm. Sci., 66:1 -19, (1977) and P. H. Stahl and C. G. Wermuth, Eds., Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim/ZOrich:Wiley-VCH/VHCA (2002).
  • Salts of the compounds described herein containing a basic amine or other basic functional group may be prepared by any suitable method known in the art, such as treatment of the free base with a suitable inorganic or organic acid.
  • suitable inorganic or organic acid examples include acetate, adipate, ascorbate, aspartate, benzenesulfonate, benzoate, camphorate, camphor-sulfonate (camsylate), caprate (decanoate), caproate (hexanoate), caprylate (octanoate), carbonate, bicarbonate, cinnamate, citrate, cyclamate, dodecylsulfate (estolate), ethane-1 ,2- disulfonate (edisylate), ethanesulfonate (esylate), formate, fumarate (hemi-fumarate, etc.), galactarate (mucate), gentisate (2,5-dihydroxybenzoate),
  • gluceptate gluconate, glucuronate, glutamate, glutarate, glycerophosphorate, glycolate, hippurate, hydrobromide, hydrochloride (dihydrochloride, etc.), hydroiodide, isobutyrate, lactate, lactobionate, laurate, maleate, malate, malonate, mandelate, methanesulfonate (mesylate), naphthalene-1 ,5-disulfonate (napadisylate), naphthalene-sulfonate
  • diphosphate, etc. proprionate, pyroglutamate, salicylate, sebacate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate (tosylate), undecylenate, 1 -hydroxy-2- naphthoate, 2,2-dichloroacetate, 2-hydroxyethanesulfonate (isethionate), 2-oxoglutarate, 4-acetamidobenzoate, and 4-aminosalicylate.
  • Salts of the disclosed compounds containing a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base.
  • a pharmaceutically acceptable salt may be made with a base which affords a
  • pharmaceutically acceptable cation which includes alkali metal salts (especially sodium and potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum salts and ammonium salts, as well as salts made from physiologically acceptable organic bases such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N,N’-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2- hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine,
  • alkali metal salts especially sodium and potassium
  • alkaline earth metal salts especially calcium and magnesium
  • aluminum salts and ammonium salts as well as salts made from physiologically acceptable organic bases such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N,N’-dibenzylethylenedi
  • dehydroabietylamine N,N’-bisdehydroabietylamine
  • glucamine N-methylglucamine
  • collidine choline
  • quinine quinine
  • quinoline and basic amino acids such as lysine and arginine.
  • the invention includes within its scope all possible stoichiometric and non- stoichiometric forms of the salts (e.g., hydrobromide, dihydrobromide, fumarate, hemi- fumarate, etc.) of the compounds described herein.
  • salts e.g., hydrobromide, dihydrobromide, fumarate, hemi- fumarate, etc.
  • the compounds described herein may be prepared and administered as a prodrug wherein the compound as described herein comprises a cleavable moiety connected to the compound of by a cleavable bond.
  • the cleavable moiety is cleaved after the compound has been administered to an animal only after being internalized by a targeted cell. Inside the cell the cleavable moiety is cleaved, thereby releasing the active compound. While not wanting to be bound by theory it is believed that the cleavable moiety is cleaved by one or more enzymes or chemical transformations that occur within the cell.
  • the one or more enzymes or chemical transformations cleave the cleavable bond, which bond may be an ester, an amide, a sulfonamide, an ether, a thioether, a disulfide, an amine, an imine, a phosphate, a carbamate, a carbonate, a boronic ester, a glycosidic bond, or some other cleavable bond.
  • the cleavable bond will typically be between the cleavable moiety and a linker connected to the compound as described herein.
  • One particular embodiment of the invention there is provided the use of a first compound as described herein, in combination with the use of a second compound, for preventing, ameliorating or treating liver disease, or symptoms thereof, in an animal.
  • the first compound is a compound of Formula (I)
  • the second compound is a compound of described herein or a pharmaceutically acceptable salt thereof.
  • One particular embodiment of the invention provides the use of a compound as described herein or a pharmaceutically acceptable salt thereof, for preventing, ameliorating or treating liver disease, or symptoms thereof, in an animal.
  • One embodiment of the invention provides a compound as disclosed herein, or a pharmaceutically acceptable salt thereof for use in therapy.
  • This invention provides a compound as described herein, or a pharmaceutically acceptable salt thereof, for use in therapy, specifically for use in the treatment of a hepatitis B virus-related disease, condition or disorder (for example, a hepatitis B virus-related disease, condition or disorder may be Hepatitis D virus, jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome or serum hepatitis).
  • this invention provides a compound as described in Formula (I), or a pharmaceutically acceptable salt thereof, for use in therapy.
  • this invention provides a compound of the invention as described herein, for use in the treatment of a hepatitis B virus-related disease, condition or disorder, specifically, a disease, condition or disorder recited herein.
  • This invention provides a compound of Formula (I) as described herein, including a compound of Table 1 , or a pharmaceutically acceptable salt thereof, for use in the treatment of a hepatitis B virus-related disease, condition or disorder.
  • This invention provides a compound of Formula (I) as described herein, including a compound of Table 1 , or a pharmaceutically acceptable salt thereof, for use in the treatment of a hepatitis B virus-related disease, condition or disorder, wherein the hepatitis B virus-related disease, condition or disorder may be Hepatitis D virus, jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome or serum hepatitis.
  • This invention provides a compound of Formula (I) as described herein, including a compound of Table 1 , or a pharmaceutically acceptable salt thereof, for use in a functional cure of a Hepatitis B virus infection in a human, as determined by an undetectable level of HBV DNA and sustained HBsAg seroclearance, with or without anti- HBs seroconversion after the cessation of finite anti-HBV treatments by the human.
  • This invention specifically provides for the use of a compound of Formula (I) as described herein, including a compound of Table 1 , or a pharmaceutically acceptable salt thereof. More specifically, this provides for the use of the compounds described herein for the treatment of a Hepatitis B virus-related disease, condition or disorder, specifically, a disease, condition or disorder recited herein. Accordingly, the invention provides for the use of a compound of Formula (I) as described including a compound of Table 1 , as an active therapeutic agent or pharmaceutically acceptable salt thereof, in the treatment of a human in need thereof having, or a risk of having, a Hepatitis B virus-related disease, condition or disorder, specifically, a disease condition or disorder recited herein.
  • the invention further provides for the use of a compound of Formula (I) as described herein, including a compound of Table 1 , or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment of a Hepatitis B virus-related disease condition or disorder, for example the diseases and disorders recited herein.
  • the invention also provides for the use of a compounds of Formula (I) as described herein, in the manufacture of a medicament for use in the treatment of a Hepatitis B virus-related disease condition or disorder, for example the diseases, conditions and disorders recited herein.
  • a compound as described herein that is a compound of Formula (I), or a salt, particularly a pharmaceutically acceptable salt, for preventing, ameliorating or treating liver disease, or symptoms thereof, in an animal.
  • the compound of Formula (I) may be a compound from Table 1.
  • Cyertain embodiments provide the use of a compound of Formula (I) as described herein, suitably a compound of Table 1 , or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating, ameliorating, delaying or preventing an HBV-related disease, disorder or condition in an animal.
  • Certain embodiments provide the use of a compound of Formula (I) as described herein, suitably a compound of Table 1 , or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating, ameliorating, delaying or preventing liver disease in an animal.
  • Another embodiment of the invention provides a method for treating a Hepatitis B virus infection, treating a Hepatitis B virus-related condition, or treating a Hepatitis B/Hepatitis D virus co-infection in a mammal, the method comprising administering a therapeutically effective amount of a STING compound as described herein, so as to treat the Hepatitis B virus infection, the Hepatitis B virus-related condition and/or the Hepatitis B/Hepatitis D virus co-infection.
  • the mammal is a human and the Hepatitis B virus infection, the Hepatitis B virus-related condition, and/or the hepatitis B/Hepatitis D virus co-infection is an infection of a Hepatitis B virus from a human Hepatitis B virus.
  • the human Hepatitis B virus may be any of the human geographical genotypes: A (Northwest Europe, North America, Central America); B (Indonesia, China, Vietnam); C (East Asia, Korea, China, Japan, Polynesia, Vietnam); D (Mediterranean area, Middle East, India); E (Africa); F (Native Americans, Polynesia); G (United States, France); or H (Central America).
  • Related embodiments provide a method for treating a Hepatitis B virus infection or a Hepatitis B virus-related condition in a mammal, the method comprising administering a therapeutically effective amount of any pharmaceutical composition as described herein to a mammal in need thereof, wherein the human Hepatitis B virus- related condition may be jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome or serum hepatitis.
  • a method of treating a Hepatitis B virus infection, a Hepatitis B virus-related condition or a Hepatitis B/Hepatitis D virus coinfection in a mammal in need of such treatment comprising administering a compound of Formula (I) of the invention or a pharmaceutically acceptable salt thereof as described herein to the mammal.
  • the method of treating an HBV infection comprises administering a first therapeutic agent that is a compound of Formula (I) or pharmaceutically acceptable salt thereof as described herein.
  • the methods comprise administering a first therapeutic agent that is a therapeutically effective amount of a compound of Formula (I) or pharmaceutically acceptable salt as described herein, or a prodrug or pharmaceutically acceptable salt thereof, and administering one or more second therapeutic agents.
  • the first therapeutic agent and one or more second therapeutic agents are co-administered.
  • the first therapeutic agent and the one or more second therapeutic agent are administered separately, in any order.
  • Certain embodiments provide the use of a compound as described herein, wherein the compound of Formula (I) as described herein is a conjugate or prodrug with a cleavable moiety connected to the compound by a cleavable bond.
  • the cleavable moiety is cleaved after the compound has been administered to an animal only after being internalized by a targeted cell. Inside the cell the cleavable moiety is cleaved, thereby releasing the active compound of Formula (I). While not wanting to be bound by theory it is believed that the cleavable moiety is cleaved by one or more enzymes or chemical transformations within the cell.
  • the prodrug may comprise a cleavable bond.
  • cleavable bonds include, but are not limited to, an ester, an amide, an amine, an imine, an ether, a thioether, a disulfide, a carbamate, a carbonate, a phosphate, a sulfonamide, a boronic ester or a glycosidic bond, or some other cleavable bond.
  • the cleavable bond may be between the cleavable moiety and a linker connected to the compound of Formula (I).
  • a first therapeutic agent that is a compound of Formula (I) as described herein, or a pharmaceutically acceptable salt thereof, and a second therapeutic agent.
  • the compound of Formula (I) is a compound of Table 1 .
  • the first therapeutic agent that is a compound of Formula (I) is co-administered sequentially or concomitantly with the second therapeutic agent.
  • the second therapeutic agent is selected from an anti-inflammatory agent, a chemotherapeutic agent, or an anti-infection agent.
  • the second therapeutic agent is an HBV agent, an HCV agent, a chemotherapeutic agent, an antibiotic, an analgesic, a non-steroidal antiinflammatory (NSAID) agent, an antifungal agent, an antiparasitic agent, an anti-nausea agent, an anti-diarrheal agent, an immunomodulatory, or an immunosuppressant agent.
  • a chemotherapeutic agent an antibiotic, an analgesic, a non-steroidal antiinflammatory (NSAID) agent, an antifungal agent, an antiparasitic agent, an anti-nausea agent, an anti-diarrheal agent, an immunomodulatory, or an immunosuppressant agent.
  • NSAID non-steroidal antiinflammatory
  • the second therapeutic agent is an HBV agent.
  • the HBV agent is any of a compound described in WO2019/069293, interferon alpha-2b, interferon alpha-2a, and interferon alphacon-1 (pegylated and unpegylated), ribavirin; an HBV RNA replication inhibitor; an HBV antigen production inhibitor; an HBV therapeutic vaccine; an HBV prophylactic vaccine; lamivudine (3TC); entecavir (ETV); tenofovir diisoproxil fumarate (TDF); telbivudine (LdT); adefovir; or an HBV antibody therapy (monoclonal or polyclonal).
  • the second therapeutic agent is an HCV agent.
  • the HCV agent is any of interferon alpha-2b, interferon alpha-2a, and interferon alphacon-1 (pegylated and unpegylated); ribavirin; an HCV RNA replication inhibitor (e.g., ViroPharma's VP50406 series); an HCV antisense agent; an HCV therapeutic vaccine; an HCV protease inhibitor; an HCV helicase inhibitor; or an HCV monoclonal or polyclonal antibody therapy.
  • the second therapeutic agent is an antiinflammatory agent (i.e., an inflammation lowering therapy).
  • the inflammation lowering therapy is any of a therapeutic lifestyle change, a steroid, a NSAID or a DMARD.
  • the steroid can be a corticosteroid.
  • the NSAID can be an aspirin, acetaminophen, ibuprofen, naproxen, COX inhibitors, indomethacin and the like.
  • the DMARD can be a TNF inhibitor, purine synthesis inhibitor, calcineurin inhibitor, pyrimidine synthesis inhibitor, a sulfasalazine, methotrexate and the like.
  • the second therapeutic agent is a
  • chemotherapeutic agent i.e., a cancer treating agent.
  • Chemotherapeutic agents can include, but are not limited to, daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan,
  • cyclophosphamide 6-mercaptopurine, 6-thioguanine, cytarabine (CA)
  • 5-azacytidine hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide
  • 5-fluorouracil 5- FU
  • 5-fluorodeoxyuridine 5-FUdR
  • methotrexate MTX
  • colchicine taxol, vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatin, gemcitabine and
  • the second therapeutic agent is an
  • immunomodulatory agent known as an innate immune activator, a check point inhibitor,
  • Immune-modulators includes, but are not limited to, antibodies or small molecules antagonizing CTLA-4 such as ipilimumab (YERVOY), PD-1 such as Opdivo/nivolumab and Keytruda/pembrolizumab), PDL1 such as TECENTRIQTM (atezolizumab), LAG3, TIM3, or IDO.
  • Immune-modulators includes, but are not limited to, antibodies or small molecules stimulating ICOS, OX-40, TLRs, IL7R or IL12R.
  • the second therapeutic agent is an anti-infection agent.
  • antiinfection agents include, but are not limited to, antibiotics, antifungal drugs and antiviral drugs.
  • the HBV agent may include, but is not limited to, a compound described in WO2019/069293 , thymosin a1 , interferon b-1 a, interferon b- 1 b, interferon g-1 b, interferon g1 , interferon g2, interferon g3, interferon a-2a, interferon a-2b, pegylated interferon p-2a, pegylated interferon p-2b, pegylated interferon g1 , pegylated interferon g2, GS-9620 (4-amino-2-butoxy-8-[[3-(pyrrolidin-1- ylmethyl)phenyl]methyl]-5,7-dihydropteridin
  • a method of treating an HBV infection in a human by administering to the human a therapeutically effective amount of a compound of Formula (I) as described herein or a pharmaceutically acceptable salt thereof.
  • a method of treating an HBV infection, in a human having or at risk of having the HBV infection by administering to the human a therapeutically effective amount of a compound of Formula (I) described herein such as a compound of Table 1 , or a pharmaceutically acceptable salt or prodrug thereof, and optionally administering a therapeutically effective amount of a second therapeutically active compound, wherein the second therapeutically active agent is selected from: an HBV agent, an HCV agent, a chemotherapeutic agent, an antibiotic, an analgesic, a nonsteroidal anti-inflammatory (NSAID) agent, an antifungal agent, an antiparasitic agent, an anti-nausea agent, an anti-diarrheal agent, or an immunosuppressant agent.
  • the second therapeutically active agent is selected from: an HBV agent,
  • a method of treating an HBV infection in a human by administering to the human a therapeutically effective amount of a compound of Formula (I) as described herein or a pharmaceutically acceptable salt thereof.
  • this invention is directed to a method of treating an HBV infection in a human having or at risk of having the HBV infection, comprising
  • Another embodiment provides a method for reducing an amount of HBV DNA and an amount of HBV antigen in a mammal infected with a Hepatitis B virus, the method comprising administering to the mammal a therapeutically effective amount of a pharmaceutical composition comprising a compound of Formula (I) as described herein or a pharmaceutically acceptable salt thereof, so as to reduce the Hepatitis B virus infection and the Hepatitis B antigen, compared to the amount of HBV DNA and the amount of HBV antigen in the mammal before treatment.
  • the mammal may be human
  • the Hepatitis B virus may be a human Hepatitis B virus.
  • the human Hepatitis B virus may be any of the human geographical genotypes: A (Northwest Europe, North America, Central America); B (Indonesia, China, Vietnam); C (East Asia, Korea, China, Japan, Polynesia, Vietnam); D (Mediterranean area, Middle East, India); E (Africa); F (Native Americans, Polynesia); G (United States, France); or H (Central America).
  • a method for reducing an amount of HBV DNA and an amount of HBV antigen in a mammal infected with a Hepatitis B virus, the method comprising administering a therapeutically effective amount of a therapeutic agent, wherein the therapeutic agent is a compound of Formula (I) as described herein or a pharmaceutically acceptable salt thereof, so as to reduce the hepatitis B virus infection and the hepatitis B antigen, compared to the amount of hepatitis B virus and the amount of HBV antigen in the mammal before treatment, and wherein the amount of hepatitis B virus is reduced 90% compared to the amount before administration of the therapeutic agent.
  • the therapeutic agent is a compound of Formula (I) as described herein or a pharmaceutically acceptable salt thereof
  • the HBV antigen may be HBsAg or may be HBeAg, and more particularly, the amount of HBV antigen may be sufficiently reduced to result in seroconversion, defined as serum HBeAg absence plus serum HBeAb presence if monitoring HBeAg as the determinant for seroconversion, or defined as serum HBsAg absence if monitoring HBsAg as the determinant for seroconversion, as determined by currently available detection limits of commercial ELISA systems.
  • a method for reducing an amount of HBV DNA and an amount of HBV antigen in a mammal infected with a hepatitis B virus by administering a therapeutically active amount of a first therapeutic agent that is a compound of Formula (I) as described, and administering a second therapeutic agent, wherein the first therapeutic agent, the second therapeutic agent are administered either together in a single formulation, or administered separately in different formulations, and wherein the administration of the first therapeutic agent and the second therapeutic agent is done concomitantly, or in series.
  • the method as described further comprises administering a therapeutically effective amount of an optional third therapeutic agent, wherein the optional third therapeutic agent is selected from an HBV agent, an HCV agent, a chemotherapeutic agent, an antibiotic, an analgesic, a non-steroidal anti-inflammatory (NSAID) agent, an antifungal agent, an antiparasitic agent, an anti-nausea agent, an anti-diarrheal agent, and an immunosuppressant agent.
  • the optional third therapeutic agent is selected from an HBV agent, an HCV agent, a chemotherapeutic agent, an antibiotic, an analgesic, a non-steroidal anti-inflammatory (NSAID) agent, an antifungal agent, an antiparasitic agent, an anti-nausea agent, an anti-diarrheal agent, and an immunosuppressant agent.
  • the optional third therapeutic agent is selected from an HBV agent, an HCV agent, a chemotherapeutic agent, an antibiotic, an analgesic, a non
  • the optional third therapeutic agent is selected from interferon alpha-2b; interferon alpha-2a; and interferon alphacon-1 (pegylated and unpegylated); ribavirin; an HBV RNA replication inhibitor; an HBV antigen production inhibitor; an HBV therapeutic vaccine; an HBV prophylactic vaccine; lamivudine (3TC); entecavir (ETV); tenofovir diisoproxil fumarate (TDF); telbivudine (LdT); adefovir; or an HBV antibody therapy (monoclonal or polyclonal); an HCV agent selected from an HCV RNA replication inhibitor (e.g., ViroPharma's VP50406 series); an HCV therapeutic vaccine; an HCV protease inhibitor; an HCV helicase inhibitor; or an HCV antibody therapy (monoclonal or polyclonal).
  • an HCV agent selected from an HCV RNA replication inhibitor (e.g., Viro
  • Another embodiment provides a method for promoting seroconversion of a hepatitis B virus in a mammal infected with HBV, the method comprising administering a therapeutically effective amount of a compound of Formula (I) as described herein to a mammal infected with hepatitis B; the method comprising monitoring for the presence of HBeAg plus HBeAb in a serum sample of the mammal, or monitoring for the presence of HBsAg in a serum sample of the mammal, such that the absence of HBeAg plus the presence of HBeAb in the serum sample if monitoring HBeAg as the determinant for seroconversion, or the absence of HBsAg in the serum sample if monitoring HBsAg as the determinant for seroconversion, as determined by current detection limits of commercial ELISA systems, is indication of seroconversion in the mammal.
  • the method comprises administering a first therapeutic agent that is a compound of Formula (I) as described herein or a pharmaceutically acceptable salt or prodrug thereof, and a second therapeutic agents, wherein the second therapeutic agents may be an anti-inflammatory agent; a chemotherapeutic agent or anti-infection agent; an HBV agent; an HCV agent; an antibiotic; an analgesic; a non-steroidal anti-inflammatory (NSAID) agent; an antifungal agent; an antiparasitic agent; an anti-nausea agent; an anti-diarrheal agent; an immunomodulatory; or an immunosuppressant agent.
  • a first therapeutic agent that is a compound of Formula (I) as described herein or a pharmaceutically acceptable salt or prodrug thereof
  • the second therapeutic agents may be an anti-inflammatory agent; a chemotherapeutic agent or anti-infection agent; an HBV agent; an HCV agent; an antibiotic; an analgesic; a non-steroidal anti-inflammatory (NSAID) agent; an antifung
  • compositions provide the use of a combination of a compound of Formula (I) as described herein, suitably a compound of Table 1 , or pharmaceutically acceptable salt or prodrug thereof and a second therapeutic agents or compositions as described herein in the manufacture of a medicament for treating, ameliorating, delaying or preventing an HBV-related disease, disorder or condition in an animal.
  • a compound of Formula (I) as described herein, suitably a compound of Table 1 , or pharmaceutically acceptable salt or prodrug thereof and a second therapeutic agents or compositions as described herein in the manufacture of a medicament for treating, ameliorating, delaying or preventing an HBV-related disease, disorder or condition in an animal.
  • a compound of Formula (I) as described herein including a compound of Table 1
  • other pharmaceutically active agents such as other HBV agents known in the field.
  • the following methods and excipients are merely exemplary and are in no way limiting.
  • the STING compounds of Formula (I) as described herein and in Table 1 , or pharmaceutically acceptable salt thereof, can be administered in combination with a conventional pharmaceutical carrier, excipient or the like (e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and the like).
  • the pharmaceutical composition can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like (e.g., sodium acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, and the like).
  • nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like (e.g., sodium acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, and the like).
  • the compounds described herein can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, corn starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
  • lubricants such as talc or magnesium stearate
  • the compounds described herein can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water- soluble bases, and can be administered rectally via a suppository.
  • bases such as emulsifying bases or water- soluble bases
  • the suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
  • the compounds of Formula (I) as described herein including the compounds of Table 1 , or a pharmaceutically acceptable salt or prodrug thereof, for use in combination with the therapeutic agents and HBV agents described herein, will be administered in a therapeutically effective amount by any of the accepted modes of administration for therapeutic agents that serve similar utilities.
  • compositions may be administered to the host using any convenient means capable of resulting in the desired reduction of target viral transcripts, viral genome amount or load in the target cell.
  • a compound as described herein or pharmaceutically acceptable salt or prodrug thereof can be incorporated into a variety of formulations for therapeutic administration. More particularly, the compounds that as described herein or a pharmaceutically acceptable salt or prodrug thereof, and combinations thereof described herein, can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents.
  • compounds described herein or a pharmaceutically acceptable salt of prodrug thereof, and combinations thereof described herein may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.
  • administration of a compound as described herein or a pharmaceutically acceptable salt or prodrug thereof, and combinations thereof described herein may be achieved in various ways, including, but not limited to, oral, buccal, rectal, parenteral, intraperitoneal, intravenous, intradermal, transdermal or intratracheal administration, and also administration may be via microneedle patch delivery or topical formulation.
  • pharmaceutically acceptable salt or prodrug thereof described herein can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, corn starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
  • lubricants such as talc or
  • the compounds of Formula (I) as described herein can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • aqueous or nonaqueous solvent such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol
  • solubilizers isotonic agents
  • suspending agents emulsifying agents
  • stabilizers and preservatives emulsifying agents
  • Liquid pharmaceutically administrable compositions of STING compounds as described herein can, for example, be prepared by dissolving, dispersing, etc.
  • compositions will comprise from about 0.2 to 2% of the active agent in solution.
  • the actual amount of the therapeutic compound(s) - i.e. the therapeutic ingredient(s), will depend upon numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compounds used, the route and form of administration, and other factors.
  • the pharmaceutical composition can be administered more than once a day, such as once or twice a day.
  • the HBV pharmaceutical formulation will be administered once or twice per week over the course of 24, 36, or 48 weeks or longer, as needed, to effectively treat the HBV infection, including to reduce viral load, to reduce viral antigens, produce seroconversion, bolster the subjects immune response and/or decrease HBV DNA levels and normalize alanine transferase (ALT) levels.
  • Unit dosage forms for oral or rectal administration of a compound as described herein or a pharmaceutically acceptable salt thereof, or combinations thereof described herein, such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors.
  • unit dosage forms for injection or intravenous administration of a compound as described herein or a pharmaceutically acceptable salt thereof, or combinations thereof described herein may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
  • compositions such as vehicles, adjuvants, carriers or diluents
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
  • dose levels can vary as a function of the specific therapeutic agent, the nature of the delivery vehicle, and the like.
  • Preferred dosages for a given therapeutic agent, including for a specific compound as described herein including in combination with a second therapeutic agent, are readily determinable by those of skill in the art by a variety of means.
  • the choice of formulation and dosage depends on various factors such as the mode of drug administration and bioavailability of the drug substance.
  • mammal cells of interest include, but are not limited to cells of: ungulates or hooved animals, e.g., cattle, goats, pigs, sheep, etc.; rodents, e.g., hamsters, mice, rats, etc.; lagomorphs, e.g., rabbits; primates, e.g., monkeys, baboons, humans, etc.; and the like.
  • Pharmaceutical formulations of compounds described herein for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal may comprises at least one STING compound of Formula (I) as described herein or pharmaceutically acceptable salt or prodrug thereof, including a compound from Table 1 or a pharmaceutically acceptable salt thereof, and a second therapeutic agent as described herein, as an active ingredient.
  • the pharmaceutical composition may include a diluent, and optionally may comprises a pharmaceutical carrier; and that the pharmaceutical composition optionally may comprise further compounds, such as chemotherapeutic compounds, anti-inflammatory compounds, antiviral compounds, analgesics, NSAIDs, narcotics, antibiotics, antifungal compounds, antiparasitic compounds, and/or immuno-modulating compounds.
  • chemotherapeutic compounds such as chemotherapeutic compounds, anti-inflammatory compounds, antiviral compounds, analgesics, NSAIDs, narcotics, antibiotics, antifungal compounds, antiparasitic compounds, and/or immuno-modulating compounds.
  • the compounds described herein for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal can be used "as is” or in the form of a variety of pharmaceutically acceptable salts or prodrugs.
  • pharmaceutically acceptable salts refers to salts that retain the desired biological activity of the herein-identified compounds and exhibit minimal undesired toxicological effects.
  • Non-limiting examples of such salts can be formed with organic amino acid and base addition salts formed with metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and the like, or with a cation formed from ammonia, N,N-dibenzylethylene-diamine, D-glucosamine, tetraethylammonium, or ethylenediamine.
  • metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and the like, or with a cation formed from ammonia, N,N-dibenzylethylene-diamine, D-glucosamine, tetraethylammonium, or ethylenediamine.
  • the compound that as described herein may be in the form of a pro-drug.
  • the prodrug may include a cleavable moiety connected to the compound, by a cleavable bond.
  • the cleavable moiety may be a moiety that directs the compound as described herein to the liver.
  • liver-targeting cleavable moieties include carbohydrate moieties, including mannose or galactose moieties, or N-acetylmannosamine or more particularly N-acetylgalactosamine (GalNAc) moieties.
  • a GalNAc cluster may include 2 GalNAc moieties, 3 GalNac moieties or 4 GalNac moieties.
  • [00145]0ther moieties and techniques that can be used for carrier-mediated delivery to target compounds that as described herein to the liver include the use of cholesterol, polyethyleneglycol (PEG), dextran, lipid nanoparticles, masked endosomal compounds that incorporate both targeting iigands and endosomal escape moieties, for exampie by conjugating poiy(vinyiether) to GaiNAc and PEG and then iinking the
  • GaiNAc-PEG to the compound to be delivered through a cleavable bond, such as a disulfide bond.
  • a cleavable bond such as a disulfide bond.
  • STING agonists of Formula (I) as described herein, suitably a compound of Table 1 , to tissue may also be enhanced by carrier-mediated delivery including, but not limited to, cationic liposomes, cyclodextrins, porphyrin derivatives, branched chain dendrimers, polyethylenimine polymers, nanoparticles and microspheres (Dass, C R. J Pharm Pharmacol 2002; 54(1 ):3-27), incorporated by reference herein.
  • Other discussions of targeting drug delivery to the liver can be found in“Efficient Hepatic Delivery of Drugs: Novel Strategies and Their Significance’ BioMed Research international vol. 2013 (2013), Article ID 328184, 20 pages, by N. ishra et a!., the entire contents of which are incorporated by reference herein.
  • compositions of the compounds described herein for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal include, but are not limited to, solutions, emulsions, and liposome-containing
  • compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self- emulsifying semisolids.
  • Pharmaceutical formulations of compounds described herein for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the compounds described herein for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels and suppositories.
  • the compounds described herein for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the compounds described herein for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal may also be conjugated to active drug substances, for example, aspirin, ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic, and an anticancer drug substance.
  • the compounds described herein for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal may be administered as a combination therapy together with, or separately in conjunction with, drug substances useful for treating cancer, inflammation, pain, bacterial, fungal and/or parasitic infections, alcoholism, substance abuse, diabetes, as well as with drug substances useful for treating other viral infections such as HIV, HCV and the like.
  • hepatitis B viral infections There are two general types of hepatitis B viral infections: acute, and chronic. Subjects who have experienced a HBV infection may also recover and become non-symptomatic carriers. Acute hepatitis B results when a person exposed to the hepatitis B virus begins to develop the signs and symptoms of viral hepatitis. This period of time, called the incubation period, is an average of 90 days, but could be as short as 45 days or as long as 6 months. For most people this infection will cause mild to moderate discomfort but will go away by itself because of the body's immune response succeeds in fighting the virus. However, some people, particularly those with
  • compromised immune systems such as persons suffering from AIDS, undergoing chemotherapy, taking immunosuppressant drugs, or taking steroids, have very serious problems as a result of the acute HBV infection, and go on to develop more severe conditions such as fulminant liver failure.
  • Chronic hepatitis B occurs when a person initially suffers from an acute infection but is then unable to fight off the infection. Whether the disease becomes chronic or completely resolves depends mostly on the age of the infected person. About 90% of infants infected at birth will progress to chronic disease. However, as a person ages, the risk of chronic infection decreases such that between 20%-50% of children and less than 10% of older children or adults will progress from acute to chronic infection.
  • Chronic HBV infections are the primary treatment goal for embodiments of the present invention, although compounds of the present invention are also capable of treating HBV- related conditions, such as inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, and more.
  • the first phase is the immune tolerant phase, where minimal fibrosis and inflammation of the liver is exhibited, coupled with high HBV DNA and normal alanine aminotransferase (ALT) levels.
  • the second phase is an immune clearance phase, where active inflammation of the liver is observable upon histology, which may also include fluctuating levels of ALT activity and fluctuating levels of HBV DNA.
  • a third inactive carrier state phase may follow, in which infected subjects may exhibit mild hepatitis and minimal fibrosis upon histology.
  • a fourth is also reported, termed reactivation of HBV. This last phase is characterized by active inflammation of the liver, as observed by biopsy, despite hepatitis B e antigen (HBeAg) negativity and anti-HBeAg positivity.
  • HBeAg positive, or“wild type” HBV infection is characterized as anti-HBeAg negative and exhibiting quantities of HBV DNA > 20,000 lU/mL (> 10 5 copies/mL).
  • HBeAg negative, or“mutant core” HBV infection is characterized as anti-HBeAg positive and exhibiting HBV DNA >2,000 lU/mL (> 10 4 copies/mL).
  • Hepatitis occurs when a weak HBV-specific immune response is activated that is sufficiently strong to destroy HBV-infected hepatocytes. Little is known regarding the mechanism responsible for T cell hyporesponsiveness or tolerance, but it is thought that it may involve the following mechanisms: negative selection (neonates); immunological ignorance; peripheral anergy or lack of co-stimulatory molecules, e.g. regulatory T cells (Tregs); exhaustion (up-regulation of PD-1). A common factor for all these suggested mechanisms is high levels of antigen persisting in the patient.
  • HBV antigen HBeAg is a secreted, non-particulate form of HBV core protein. HBV antigens HBeAg and HBeAg share primary amino acid sequences, so show cross-reactivity at the T cell level. HBeAg is not required for viral assembly or replication, although studies suggest they may be required for establishment of chronic infection.
  • HBeAg-negative mutant often results in fulminant acute rather than chronic HBV infection (Terezawa et al (1991) Pediatr. Res. 29:5), whereas infection of young woodchucks with WHeAg-negative mutant results in a much lower rate of chronic WHV infection (Cote et al (2000) Hepatology 31 :190).
  • HBeAg may possibly function as a toleragen by inactivating core specific T cells through deletion or clonal anergy (Milich et al (1998) J. Immunol. 160:8102).
  • HBV surface antigen is the envelope protein of infectious HBV viral particles but is also secreted as a non-infectious particle with serum levels 1000-fold higher than HBV viral particles.
  • the serum levels of HBsAg in an infected person or animal can be as high as 1000 pg/mL (Kann and Gehrlich (1998) Topley & Wilson’s Microbiology and Microbial Infections, 9 th ed. 745).
  • the half-life of HBsAg in the serum, or serum t 1 ⁇ 2 is 8.3 days (Chulanov et al (2003) J. Med. Virol. 69: 313).
  • Internalization of HBsAg by myeloid dendritic cells inhibits up-regulation of costimulatory molecules (i.e.
  • HBsAg specific CD8 cells from CHB patients show altered tetramer binding. These CD8 cells are not anergic but may have TCR topology that confers partial tolerance or ignorance (Reignat et al (2002) J. Exp. Med. 195:1089). Moreover, reduction in serum HBsAg > 1 log at week 24 has a high predictive value (92%) for sustained virological response (SVR - defined as nondetectable HBV DNA by PCR at 1 year after treatment) during Peg-IFNa2a therapy (Moucari et al (2009) Hepatology 49:1 151 ).
  • SVR sustained virological response
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • hepatitis C hepatitis C
  • interferons e.g., interferon alpha-2b, interferon alpha-2a, and interferon alphacon-1.
  • interferon alpha-2b pegylated and unpegylated
  • ribavirin ribavirin
  • HCV RNA replication inhibitors e.g., ViroPharma's VP50406 series
  • HCV antisense agents e.g., HCV antisense agents
  • HCV therapeutic vaccines e.g., HCV IL-12, HCV helicase inhibitors
  • HCV antibody therapy monoclonal or polyclonal.
  • a STING compound of Formula (I) as described herein suitably a compound of Table 1 , or a pharmaceutically acceptable salt or prodrug thereof as described herein for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal may be administered to a human subject infected with HBV, in combination with a second therapeutic agent, wherein the second therapeutic agent may be administered in the same drug formulation as the STING compound, or may be administered in a separate formulation.
  • the second therapeutic agent may also be administered simultaneously with an optional third therapeutic agent, or may be administered separately, so that a dose of each of the STING compound of Formula (I) as described herein, suitably a compound of Table 1 , or a pharmaceutically acceptable salt thereof, the second therapeutic agent, and the optional third therapeutic agent overlap, in time, within the subject’s body.
  • the second and optional third therapeutic agent may be therapeutic agent as described herein, including an HBV agent selected from a compound described in WO2019/069293, thymosin a1 , interferon b-1 a, interferon b-1 b, interferon g-1 b, interferon g1 , interferon g2, interferon g3, interferon a- 2a, interferon a-2b, pegylated interferon p-2a, pegylated interferon p-2b, pegylated interferon g1 , pegylated interferon g2, GS-9620 (4-amino-2-butoxy-8-[[3-(pyrrolidin-1- ylmethyl)phenyl]methyl]-5,7-dihydropteridin-6-one), dehydroepiandrosterone, androstenediol or androstenetrial; interferon alphacon-1 (pegylated and
  • HBV-related liver conditions such as cirrhosis, hepatocellular carcinoma (HCC), and fibrosis and inflammation, resulting in the need for fewer liver transplants and helping to prevent deaths from liver disease.
  • HCC hepatocellular carcinoma
  • HBeAg is believed to have an immunosuppressive role, and HBsAg is thought to contribute to T cell exhaustion.
  • treatment with a compound as described herein or a pharmaceutically acceptable salt or prodrug thereof in combination with a second therapeutic agent, in combination with an optional third therapeutic agent, in combination with an optional fourth therapeutic agent will bolster a patient’s immune response by reducing HBeAg and HBsAg levels, allowing the immune system to better control infection.
  • the second, third and fourth therapeutic agent may be an HBV agent, selected from a compound described in WO2019/069293, thymosin a1 , interferon b-1 a, interferon b-1 b, interferon g-1 b, interferon g1 , interferon g2, interferon g3, interferon a-2a, interferon a-2b, pegylated interferon p-2a, pegylated interferon p-2b, pegylated interferon g1 , pegylated interferon g2, GS-9620 (4-amino-2-butoxy-8-[[3-(pyrrolidin-1 -ylmethyl)phenyl]methyl]-5,7- dihydropteridin-6-one), dehydroepiandrosterone, androstenediol or androstenetrial;
  • interferon alphacon-1 pegylated and unpegylated
  • ribavirin an HBV RNA replication inhibitor
  • an HBV therapeutic vaccine an HBV prophylactic vaccine
  • lamivudine (3TC); entecavir (ETV); tenofovir diisoproxil fumarate (TDF); telbivudine (LdT); adefovir dipivoxil
  • ETV tenofovir diisoproxil fumarate
  • TDF tenofovir diisoproxil fumarate
  • LdT telbivudine
  • adefovir dipivoxil an HBV antibody therapy (monoclonal or polyclonal).
  • reduction of HBV antigen presentation in the liver will help to minimize the extent of HBV infection flare-ups, a result of the immune system restoration, all of which will increase the chances of a complete cure of chronic HBV infection.
  • an embodiment of the invention provides a method for treating hepatitis B virus infection and reducing hepatitis B viral genome in a target cell of a mammal, comprising administering a therapeutically effective amount of a STING compound as described herein or a pharmaceutically acceptable salt or prodrug thereof, administering a therapeutically effective amount of a second therapeutic agent, and administering a therapeutically effective amount of an optional third therapeutic agent and optional fourth therapeutic agent which may each be an HBV agent, thereby treating the hepatitis B virus infection.
  • pharmaceutical compositions for use in practicing the subject methods comprising administering a therapeutically effective amount of a STING compound as described herein or a pharmaceutically acceptable salt or prodrug thereof, administering a therapeutically effective amount of a second therapeutic agent, and administering a therapeutically effective amount of an optional third therapeutic agent and optional fourth therapeutic agent which may each be an HBV agent, thereby treating the hepatitis B virus infection.
  • Embodiments of the invention comprising use of the compounds as described herein or a pharmaceutically acceptable salt or prodrug thereof and a second, therapeutic agent, an optional third therapeutic agent and an optional fourth therapeutic agent as described herein finds use in a variety of applications, including the treatment of subjects suffering from a viral mediated disease condition, e.g., an HBV mediated disease condition.
  • a viral mediated disease condition e.g., an HBV mediated disease condition.
  • Such conditions include, but are not limited to, fibrosis and inflammation of the liver, cirrhosis of the liver, and hepatocellular cancer (liver cancer).
  • a first therapeutic agent that is a STING compound of Formula (I) as described herein, suitably a compound of Table 1 , or a pharmaceutically acceptable salt thereof, in combination with a second therapeutic agent that is an HBV agent , in combination with an optional third agent that is an HBV agent, in combination with an optional fourth therapeutic agent that is an HBV agent for use in combination for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal.
  • a method of treating an HBV infection in a mammal comprising administering a therapeutically effective dose of a first therapeutic agent that is a compound as described herein or a pharmaceutically acceptable salt or prodrug thereof, administering a therapeutically effective amount of a second therapeutic agent that is an HBV agent, and administering a therapeutically effective amount of an optional third therapeutic agent that is an HBV agent, and administering a therapeutically effective amount of an optional fourth therapeutic agent that is an HBV agent so as to treat the HBV infection and promote seroconversion in the mammal, including a human.
  • the effective dose or each therapeutic agent may range from 0.01 mg/kg body weight, to approximately 100 mg/kg body weight, administered once or twice weekly, for 24, 36 or 48 weeks, or longer, as determined by a physician.
  • Efficacy of treatment may be determined using quantification of viral load or other evidence of infection, such as through measurement of HBeAg, HBsAg, HBV DNA levels, ALT activity levels, serum HBV levels, and the like, thereby allowing adjustment of treatment dose, treatment frequency, and treatment length.
  • the compounds of this invention may be prepared using the synthetic procedures illustrated in the reaction schemes below, which can be readily adapted to prepare other compounds of the invention by drawing on the knowledge of a skilled organic chemist.
  • the syntheses provided in these schemes are applicable for producing compounds of the invention having a variety of R groups employing appropriate precursors, which are suitably protected if needed, to achieve compatibility with the reactions outline herein. Subsequent deprotection, where needed, affords compounds of the nature generally disclosed. Intermediately may also be present as salts.
  • the resulting reaction mixture was stirred at room temperature for 18 h. Following this duration, the reaction was warmed to 45 °C and stirred for 1 h. Following this duration, the reaction mixture was filtered through Celite and washed with EtOAc (2 x 20 mL). The filtrate was concentrated under reduced pressure. This was dissolved in 20 mL of 3:1 DCME:EtOH and washed with water (10 mL), brine (20 mL) and dried over anhydrous sodium sulfate. After filtration, the organic layer was concentrated under reduced pressure to afford the crude compound.
  • Example 6 1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en- 1 -yl)-1 -methyl-4-(morpholinomethyl)-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3- methyl-1 H-pyrazole-5-carboxamide
  • Example compounds 7-9 in Table 1 were prepared analogously to the synthetic sequence described for Example 6:
  • Example compounds 13-2, 14 and 15 in Table 1 were prepared analogously to the synthetic sequence described for Example 13-1 .
  • Example 16 (rac)-1 -Ethyl-N-((£)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1- yl)but-2-en-1 -yl)-4-(1 -hydroxyethyl)-3-methyl-1 ,3-dihydro-2H- benzo[d]imidazol-2-ylidene)-3-methyl-1H-pyrazole-5-carboxamide
  • Example 18 The compound of Example 18 is made generally according to the above scheme and the processes described in the Examples.
  • Example 19 1 -Ethyl-N-((E)-3-(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-1 -methyl-4- (morpholinomethyl)-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide, Formic acid salt
  • tert-Butyl (4-((2-(hydroxymethyl)-6-nitrophenyl)amino)butyl)carbamate A mixture of commercially-available (2-fluoro-3-nitrophenyl)methanol (1 .05 g, 6.14 mmol), commercially-available tert-butyl (4-aminobutyl)carbamate (1.271 g, 6.75 mmol), and DIPEA (3.21 mL, 18.41 mmol) in isopropanol (10 mL) was stirred for 20 hours at 70 °C. The mixture was cooled room temperature and was concentrated. The residue was diluted with water and was extracted with ethyl acetate.
  • Example 33 1 -Ethyl-N-((£)-3-((£)-4-((£)-2-((1 -Ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en- 1 -yl)-4-(1 -hydroxy-2 -morpholinoethyl)-1 -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2- ylidene)-3-methyl-1 H-pyrazole-5-carboxamide
  • the reaction mixture was stirred for 10 min at room temperature. The reaction was monitored by LC-MS. To the mixture 0.2 ml_ of TEA was added and the reaction was stirred for 5 min. The reaction was monitored by LC-MS again. To the mixture was added additional 0.2 mL of TEA and 0.05 mL of mesyl-CI and it was stirred for 5 min. The reaction was determined complete. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic extract was washed with water and dried over anhydrous MgS0 4 . It was filtered and the filtrate was concentrated to give the title compound (1 .0 g, 1 .189 mmol, 90% purity, 90% yield) as white solid used as the intermediate without further purification. LC-MS m/z 757.6 (M+H)+, 0.82 min (ret. time).
  • DMEM Dulbecco’s modified Eagle medium
  • FBS fetal bovine serum
  • Cryopreserved primary human hepatocytes (PHH) from Lonza are used in these experiments. Vials of cryopreserved PHH are placed in a 37°C water bath for approximately 70 seconds, just until thawed. The cells are pooled, resuspended gently in differentiation medium (Williams medium containing differentiation supplement, GlutaMax-1TM, and penicillin/streptomycin) and counted using a
  • the cells are pelleted by centrifugation at 100 X g for 10 min and resuspended to a density of 5.5 X 10 5 cells/mL in differentiation medium. 100 m ⁇ of cell suspension are plated in each well of collagen-coated 96-well plates. The plates are incubated at 37°C and 5% C0 2 for 2-4 days prior to infection.
  • STING compounds can be made as described herein and as disclosed in International Patent Application PCT/IB2017/051945 filed April 5, 2016, titled
  • Recombinant murine IFN-a, IL-1 , IL-6 and TNF-a can be purchased from PBL InterferonSource.
  • Antibody against carboxyl terminal 14 amino acid of HBV core protein can be prepared as described previously in Xu C, et al. Interferons accelerate decay of replication-competent nucleocapsids of hepatitis B virus. J Virol 2010; 84:9332- 40.
  • Antibodies against b-actin and mouse IFNAR-1 were obtained from Sigma-Aldrich and Santa Cruz Biotechnology, respectively.
  • Antibodies against Human and mouse STING, TBK1 , S 172 -phosphorylated TBK1 , IkBa p38, phosphorylated-p38, JNK, phosphorylated-JNK, ERK, phosphorylated-ERK can be purchased from Cell Signaling Technology.
  • Activation of STING in cells may be determined using a luciferase reporter assay in human embryonic kidney cells (HEK293T) co-transfected with plasmids expressing STING and the enzyme firefly luciferase driven by the interferon stimulated response element promoter (pISRE-Luc) (Agilent Technologies).
  • pISRE-Luc interferon stimulated response element promoter
  • Full-length human STING Gene ID 340061
  • cGAS full-length human cyclic guanine adenine synthase
  • Transfections can be prepared using a cell suspension with Fugene ® 6 following the manufacturer’s instructions (3:1
  • Fugene ® :DNA Fifty microliters of the transfection suspension are dispensed into wells of a 384-well plate containing 250 nl_ of a compound of Formula (I) as described herein, suitably a compound of Table 1 , or a pharmaceutically acceptable salt thereof.
  • the final well composition contains ⁇ 20,000 cells/well, 1 ng STING, 20 ng pISRE-Luc, and empty vector pcDNA3.1 (lnvitrogen) to bring the total DNA concentration to 125 ng.
  • Control wells expected to generate maximal activation of STING were cotransfected with a cGAS expression plasmid. Plates are sealed and incubated for 24 hours at 37 °C.
  • firefly luciferase is processed using Steady-Glo ® luciferase assay system (Promega) and can be analyzed using a standard laboratory luminescence plate reader. Data is normalized to luminescence response in the presence of cGAS, is plotted as a function of compound concentration, and can be fit using a standard model of receptor activation to derive the pEC 5 o.
  • HBeAg levels can be measured using the Abazyme HBV s Ag kit, catalog # EL10018, according to the manufacturer’s instructions.
  • HBeAg levels can be measured using the BioChain HBV e Ag kit, catalog #K031006096 according to the manufacturer’s instructions.
  • DNA is extracted from 50 pL of supernatant using the QiaAmp 96 DNA Blood Kit (Qiagen) according to the
  • RNA is extracted using TRIzol reagent or RNeasy kit.
  • cDNAs are synthesized using Superscript III (Invitrogen). Quantitative realtime PCR analysis is performed using a LightCycler 480 II (Roche).
  • the non-parenchymal cells such as liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC), natural killer (NK) and NK-T cells, dendritic cells, CD4+ and CD8+ T cells and hepatic stellate cells (HSC) are isolated from the liver as descried in Mohar et.al., Methods in Molecular Biology, 2015. This protocol permits the collection of peripheral blood, intact liver tissue, and hepatocytes, in addition to NPCs. In situ perfusion via the portal vein leads to efficient liver digestion. NPCs are enriched from the resulting single-cell suspension by differential and gradient centrifugation. The NPCs can be analyzed or sorted into highly enriched populations using flow cytometry.
  • the isolated cells are suitable for flow cytometry, protein, and mRNA analyses as well as primary culture.
  • epitopes of CD8a, CD4, CD11 b, NK1.1 , Tie2, F4/80, GR1 can be used for major liver NPC and leukocytes.
  • ICS assays can be performed on both splenocytes and liver mononuclear cells.
  • Cells are seeded in Ubottom 96-well plates. Plates with cells are incubated overnight at 37 °C either in complete medium alone as negative control or with the peptides of HLA-A2/DR1 restricted HBV epitopes at a concentration of 2 pg/ml.
  • HBV specific epitopes are described in Bertoletti et al., Gastroenterology 1997, Pajot et al., Microbes Infect 2006, Sette et al., J Immuno 1994, Rehermann et al., J Exp Med 1995, Nayersina et al., J Immunol 1993, Loirat et al., J Immunol 2000, and Mizukoshi et al., J Immunol 2004. Brefeldin A at 2pg/ml_ (Sigma, B6542) is added after one hour of incubation.
  • CD4 rat Mab anti-mouse CD4- PE-Cy7, BD Biosciences,
  • NK cells Rosbergenchymal cells 552775
  • NK cells Rosbergenchymal cells 552775
  • NK cells Rosbergenchymal cells 552775
  • Cells are fixed after several washes and permeabilized for 20 min in the dark at room temperature with Cytofix/Cytoperm, washed with Perm/Wash solution (BD Biosciences, 554714) at 4 'C.
  • Intracellular cytokine staining with antibodies against IFNy (rat Mab anti-mouse IFNy-APC, clone XMG1 .2, BD Biosciences, 554413) and tumor necrosis factor alpha (TNFa) (rat Mab antimouse TNFa— FITC, clone MP6-XT22; 1/250 (BD Biosciences 554418) is performed for 30 min in the dark at 4 'C.
  • TNFa tumor necrosis factor alpha
  • Live CD3+CD8+CD4- and cells CD3+CD8-CD4+ are gated and presented on dot-plot. Two regions can be defined to gate for positive cells for each cytokine. Numbers of events found in these gates can be divided by total number of events in parental population to yield percentages of responding T cells. For each mouse, the percentage is obtained in medium alone is considered as background and subtracted from the percentage obtained with peptide stimulations. Threshold of positivity is defined according to experiment background i.e. the mean percentage of stained cells is obtained for each group in medium alone condition plus two standard deviations. Only percentage of cytokine representing at least 5 events is considered as positive.
  • mice Male C57BL/6 mice (4-6 weeks old), specific pathogen free, are purchased from SLAG (Shanghai Laboratory Animal Center of Chinese Academy of Sciences) and housed in an animal care facility in individually ventilated cages.
  • SLAG Standhai Laboratory Animal Center of Chinese Academy of Sciences
  • mice are allowed to acclimate to the new environment for 3 days and are grouped according to the experimental design.
  • Recombinant AAV-HBV is diluted in PBS, 200 pL per injection. This recombinant virus carriesl .3 copies of the HBV genome (genotype D, serotype ayw).
  • genotyp D serotype ayw
  • days 6, 13 and 20 after AAV injection all mice in is
  • mice with stable viremia are ready for treatment.
  • mice are tested for the efficacy of STING agonist in vivo at 30 ug as bolus intravenously for one week every 3-4 days.
  • a group of mice are then sacrificed 24 h after treatment for detection of intrahepatic upregulation of cytokines (IFN-I, II, IL6, TNF, IL12, CXCL10) and interferon stimulated genes (OAS1 , IFH1) by measuring mRNA expression by real-time RT-PCR assay. Weight of individual mouse is monitored before and 24 h after the treatment. Liver tissues are collected from mice and fixed in 3.7% formaldehyde. Each liver is divided into six parts that are individually embedded in paraffin and sectioned.
  • HBcAg- positive hepatocytes are analyzed by using ImageJ software (version 1 .43; W. S.
  • HBsAg and HBeAg in mouse sera are assayed with commercial enzyme-linked immunosorbent assay (ELISA) kits.
  • the HBsAg concentration is calculated in ng/ml by reference to a standard curve established with known concentrations of HBsAg.
  • Serum HBeAg levels are determined in 10-fold-diluted sera.
  • Antibodies are quantified by ELISA as previously described in Mancini et.al., J. Med. Virol 1993.
  • Alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) activities in sera of AAV8-HBV- and PBS injected mice are measured.
  • PRRs Unlike RIG-l-like receptors that are ubiquitously expressed in many types of somatic cells, expression of other PRRs, such as TLRs, cGAS STING, is usually restricted to macrophages, dendritic cells and a few other cell types. Due to the lack of expression or expression in a low amount of PRRs, such as STING and TLRs, treatment of hepatocytes usually does not induce a robust cytokine response. For instance, direct treatment of hepatocytes with TLR agonists induces a negligible cytokine response. However, liver resident dendritic cells, macrophages (Kupffer cells) and other hepatic nonparenchymal cells (NPCs) express high levels of TLRs and thus respond to TLR agonists and produce inflammatory cytokines.
  • TLRs Unlike RIG-l-like receptors that are ubiquitously expressed in many types of somatic cells, expression of other PRRs, such as TLRs, cGAS STING
  • HBV infected primary human hepatocytes PHHs
  • testing compounds HBV infected PHHs
  • the conditioned media of treated macrophages are then applied to HBV infected PHHs to test the compound-induced antiviral cytokine response in macrophages.
  • STING compounds of Formula (I), suitably a compound of Table 1 , or a pharmaceutically acceptable salt thereof, are tested for indirect, but not direct, antiviral activity against HBV.
  • HBV infected PHHs can be treated for 2 days with the varying concentrations of STING compound (Direct Treatment), and 50% of the conditioned media can be harvested from isolated human liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC), or NK cells or isolated human macrophages (to be treated with each of the STING compounds for 12 h) (Indirect Treatment).
  • LSEC isolated human liver sinusoidal endothelial cells
  • KC Kupffer cells
  • NK cells isolated human macrophages
  • HBV infected PHH, RAW264.7 or macrophage cells can be mock-treated or treated with 0.5 mM to 125 mM of STING compound or 1 m9/hiI of LPS for 30 and 60 min.
  • Expression and activation of STING is determined by Western blot assay. A shift in gel mobility will be seen as a result of STING phosphorylation b-actin can serve as a loading control.
  • Intracellular HBV RNA is determined by qRT-PCR.
  • HBV core protein is determined by Western blot assay using antibody against carboxyl terminal 14 amino acid of HBV core protein (b actin serves as loading control).
  • HBV nucleocapsids are examined by a particle gel assay to detect both the intact nucleocapsids and capsid-associated HBV DNA.
  • Encapsidated HBV (core DNA) replication intermediates are determined by Southern blot assay.
  • Any antiviral response induced by STING compounds in nonparenchymal cell types such as LSEC, NK, KC is expected to post-transcriptionally reduce the amounts of HBV capsid protein and the assembled capsids. Consequentially, the amounts of HBV DNA replication intermediates are also expected to be decreased.
  • Cells are treated with STING compound in dose response for the varying times.
  • Total cellular proteins can be fractioned by SDS-PAGE and transferred onto PVDF membranes.
  • Total and phosphorylated STING, TBK1 , p38, JNK and Erk as well as IKBa can be detected by Western blot assays with their specific antibodies b-actin serves as a loading control.
  • STING compounds are expected to induce STING phosphorylation, which should be detectable in cells treated with STING compounds for more than 30 min.
  • STING compounds are expected to induce phosphorylation of TBK1 , a kinase essential for IRF3 phosphorylation and induction of IFN-b. in the STING pathways. Also, STING compounds are expected to induce degradation of IKBa.
  • qRT-PCR assays is expected to show whether STING compounds induce an IFN response, and/or an inflammatory cytokine response.
  • HBV-infected PHHs are incubated either with or without 10 pg/ml of a monoclonal antibody against type I interferon receptor IFNAR1 (Ab INFAR) at 37°C. for 1 h, followed by treatment for 2 days with varying concentrations of IFN-a. or 50% of conditioned media that can be harvested from macrophages (treated with the same varying concentrations of STING compounds for 12 h).
  • HBV-infected PHHs can be treated with the varying concentrations of IL-1 , IL-6 or TNF- a. for 4 days. Cytoplasmic HBV core DNA can then be analyzed by qPCR or Southern blot hybridization.
  • Blockade of type I IFN receptor in HBV-infected PHHs is expected to significantly reduced an antiviral response by IFN-a.
  • Treatment of HBV-infected PHHs with the type I IFN receptor antibody may also attenuate an antiviral response by conditioned media from STING compound-treated macrophages, which could then indicate that type I IFNs may be mediators of STING compound-induced antiviral response against HBV.
  • mice To determine the target engagement upon STING agonist treatment in vivo, pharmacokinetics and pharmacodynamic relationship upon administration of STING agonist intravenously or orally is examined in C57BL/6 mice. Varying doses of STING agonist is administered along with vehicle only arm in 3 mice per group. At time points of 0, 1 h, 3h, 4h, 6h, and 24h, the mice are euthanized and blood, sera, liver, spleen, and draining lymph node are collected for measuring drug concentration in the liver and sera as well as responses of type I and II IFNs, and various cytokines including IL6, TNFa, IL12, IL7, CXCL10 in collected tissues.
  • NPC nonparenchymal cells
  • STING activation of STING is expected to induce strong upregulation of type I responses at early time points upon administration of the compound and to decline over time by 24 hours.
  • the response is more prone to NPCs rather than hepatocytes, which is parenchymal cells in the liver.
  • mice can be infected with AAV8-HBV to establish HBV replication in mice. After a month after infection, mice can be either treated with 3 doses of STING compound at 30ug or 100 ug or treated with just vehicle, intravenously for a week every 3 days.
  • livers and sera are collected and analyzed for HBsAg, HBV DNA, and cytokine responses.
  • mice may be included in each of the control group and the STING compound treatment group.
  • the number of HBV DNA copies/ml from each animal after subtraction of the copies from input plasmid can be plotted.
  • Total RNA are extracted from livers and mRNA levels of cytokines can be analyzed by real-time RT-PCR assay.
  • Plots are prepared to represent mRNA levels from each animal. All the data are presented in boxplots to indicate medians, interquartiles as well as ranges (min, max), and can be statistically analyzed by t-test.
  • mice are maintained for 4 more weeks without further treatment. Sera are collected at scheduled times and a subset of mice in the group is euthanized at scheduled times and collected for tissues. All of the mice are euthanized at the end of the study and processed for HBV end points such as HBsAg, HBeAg, HBV DNA, intrahepatic HBV mRNA and cellular responses such as cytokine responses are measured by RT-PCR, ELISA, or qPCR. NPC are isolated and analyzed for the activation of STING pathway. The levels of HBV specific CD4 and CD8 T cells are determined using known HBV epitopes by tetramer staining. Anti-HBs are determined from collected sera.
  • HBV end points such as HBsAg, HBeAg, HBV DNA, intrahepatic HBV mRNA and cellular responses such as cytokine responses are measured by RT-PCR, ELISA, or qPCR.
  • NPC are isolated and analyzed for the activation of STING
  • HBV antiviral inhibitor known to suppress the level of HBsAg is expected to reduce the viral barrier so that the host immune response can be rejuvenated upon treatment of an immune modulator. After 8 week-long treatment of an antiviral followed by co-treatment with STING agonist, sustained response of
  • seroclearance and seroconversion is an indication of breaking immunotolerance against HBV. It can be confirmed by the increase of the frequency of IFNy producing CD4+ and CD8+ T cells in the liver and spleen that are specific to HBV epitopes.
  • [00363JFRET assay The binding potency of molecules to the C-terminal Domain (CTD) of human STING was determined using a competition binding assay as described in WO2017/175156. In this assay, STING recombinant protein with a C-terminal biotinylated Avi-tag was employed. When bound to STING, an Alexa488-labeled orthosteric site probe accepted the 490 nm emission from Tb-Streptavidin-Avi-STING and an increase in fluorescence was measured at 520 nm. Molecules that compete for the probe binding site resulted in a low 520nm signal.
  • the assay was run in Greiner black 384-well plates (Catalog # 784076) containing 125nL compounds in neat DMSO.
  • PBMC agonist assay Activation of STING by compounds was determined by measuring levels of IFNp secreted from human peripheral blood mononuclear cells (PBMC, homozygous WT-STING) treated with different doses of compounds.
  • Frozen PBMC cells were thawed, resuspended in media (RPMI-1640 with L-glutamine, 1 .5 g/L NaHC0 3 , 4.5 g/L glucose, 10 mM Hepes, and 1 mM NaPyruvate + 10% FBS + 1 % Glutamax Supplement) to a final concentration of 3X10 5 cells/mL and dispensed into a 384-well tissue culture plate (Griener 781073) at a density of 15,000 cells per well containing 250 nl_ of compound diluted in DMSO.
  • media RPMI-1640 with L-glutamine, 1 .5 g/L NaHC0 3 , 4.5 g/L glucose, 10 mM Hepes, and 1 mM NaPyruvate + 10% FBS + 1 % Glutamax Supplement
  • the level of IFNp protein secreted into the growth media was measured after four hours of incubation at 37 °C using a human IFNp electrochemiluminescence kit (Meso Scale Diagnostics) following the manufacturer’s instructions.
  • CMPD the unknown value
  • CTRL1 the average of the 100% activation control wells (using 750nM or a known full agonist)
  • C2 the average of the 0% activation control wells (DMSO).
  • Example 32 The compound of Example 32 was tested in the above assays and found to have an activity (EC 5 o) of 5 nM in the above PBMC agonist assay, and an activity (IC 5 o) of 1 nM in the above STING FRET assay.
  • Table 3 The compound of Example 32 was tested in the above assays and found to have an activity (EC 5 o) of 5 nM in the above PBMC agonist assay, and an activity (IC 5 o) of 1 nM in the above STING FRET assay.

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Abstract

Compounds that are useful as modulators of transmembrane protein 173 (TMEM173), also known as STING (Stimulator of Interferon Genes), and methods for treating Hepatitis B virus-related conditions in mammals by administering such a compound, are disclosed.

Description

CHEMICAL COMPOUNDS
FIELD OF THE INVENTION
[0001]The present invention relates to compounds that modulate the activity of transmembrane protein 173 (TMEM173), also known as STING (STimulator of Interferon Genes), and methods of treating hepatitis B virus infections in a mammal by
administering such compounds.
BACKGROUND ART
[0002] Hepatitis B is a viral disease transmitted parenterally by contaminated material such as blood and blood products, contaminated needles, sexually and vertically from infected or carrier mothers to their offspring. In those areas of the world where the disease is common vertical transmission at an early age results in a high proportion of infected individuals becoming chronic carriers of hepatitis B. It is estimated by the World Health Organization that more than 2 billion people have been infected worldwide, with about 4 million acute cases per year, 1 million deaths per year, and 350-400 million chronic carriers. Approximately 25% of carriers die from chronic hepatitis, cirrhosis, or liver cancer and nearly 75% of chronic carriers are Asian. Hepatitis B virus (HBV) is the second most significant carcinogen behind tobacco, causing from 60% to 80% of all primary liver cancer. HBV is 100 times more contagious than HIV.
[0003] Hepatitis B viral infections are a continuing medical problem because, like any rapidly-replicating infectious agent, there are continuing mutations that help some sub-populations of HBV become resistant to current treatment regimens. At the present time there are no effective therapeutic agents for treating humans infected with HBV infections which result in seroconversion to the virus in the body, or which effect a 90% reduction of antigen, compared to baseline numbers before treatment, in persons suffering from a hepatitis B viral infection. Currently the recommended therapies for chronic HBV infection by the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) include interferon alpha (INFa), pegylated interferon alpha-2a (Peg-IFN2a), entecavir, and tenofovir. However, typical interferon therapy is 48-weeks and results in serious and unpleasant side effects, and HBeAg seroconversion, 24 weeks after therapy has ceased, ranges from only 27-36%. Seroconversion of HBsAg is even lower - only 3% observed immediately after treatment ceases, with an increase to upwards of 12% after 5 years. [0004] The nucleoside and nucleotide therapies entecavir and tenofovir are successful at reducing viral load, but the rates of HBeAg seroconversion and HBsAg loss are even lower than those obtained using IFNa therapy. Other similar therapies, including lamivudine (3TC), telbivudine (LdT), and adefovir are also used, but for
nucleoside/nucleotide therapies in general, the emergence of resistance limits therapeutic efficacy.
[0005] Vertebrates have evolved mechanisms of immune defense to eliminate infective pathogens. In mammals, this immune system comprises two branches; innate immunity and adaptive immunity. The innate immune system is the first line of defense which is initiated by Pattern Recognition Receptors (PRRs) which detect ligands from the pathogens as well as damage associated molecular patterns (Takeuchi O. et al, Cell, 2010: 140, 805-820). A growing number of these receptors have been identified including Toll-like receptors (TLRs), C-type lectin receptors, retinoic acid inducible gene I (RIG-I)- like receptors and NOD-like receptors (NLRs) and also double stranded DNA sensors. Activation of PRRs leads to up-regulation of genes involved in the inflammatory response including type 1 interferons, pro-inflammatory cytokines and chemokines which suppress pathogen replication and facilitate adaptive immunity.
[0006] Interferon was first described as a substance which could protect cells from viral infection (Isaacs & Lindemann, J. Virus Interference. Proc. R. Soc. Lon. Ser. B. Biol. Sci. 1957: 147, 258-267). In man, the type I interferons are a family of related proteins encoded by genes on chromosome 9 and encoding at least 13 isoforms of interferon alpha (IFNa) and one isoform of interferon beta (IFNp). Recombinant IFNa was the first approved biological therapeutic and has become an important therapy in viral infections and in cancer. As well as direct antiviral activity on cells, interferons are known to be potent modulators of the immune response, acting on cells of the immune system.
[0007] The adaptor protein STING (STimulator of Interferon Genes), also known as TMEM 173, MPYS, MITA and ERIS, has been identified as a central signaling molecule in the innate immune response to cytosolic nucleic acids (Ishikawa H and Barber G N, Nature, 2008: 455, 674-678; WO2013/1666000). Activation of STING results in up-regulation of IRF3 and NFKB pathways leading to induction of lnterferon-b and other cytokines. STING is critical for responses to cytosolic DNA of pathogen or host origin, and of unusual nucleic acids called Cyclic Dinucleotides (CDNs)
[0008] CDNs were first identified as bacterial secondary messengers responsible for controlling numerous responses in the prokaryotic cell. Bacterial CDNs, such as c-di- GMP are symmetrical molecules characterized by two 3’, 5’ phosphodiester linkages. [0009] Direct activation of STING by bacterial CDNs has recently been confirmed through X-ray crystallography (Burdette D L and Vance R E, Nature Immunology, 2013: 14, 19-26). Bacterial CDNs and their analogues have consequently attracted interest as potential vaccine adjuvants (Libanova R. et al, Microbial Biotechnology 2012: 5, 168-176; W02007/054279, W02005/087238).
[0010] More recently, the response to cytosolic DNA has been elucidated and shown to involve generation, by an enzyme called cyclic GMP-AMP synthase (cGAS, previously known as C6orf150 or MB21 D1 ), of a novel mammalian CDN signaling molecule identified as cGAMP, which then activates STING. Unlike bacterial CDNs, cGAMP is an unsymmetrical molecule characterized by its mixed 2’, 5’ and 3’, 5’ phosphodiester linkages. (Gao P et al, Cell, 2013: 153, 1094-1 107). Interaction of cGAMP (II) with STING has also been demonstrated by X-ray crystallography (Cai X et al, Molecular Cell, 2014: 54, 289-296).
[0011] Administration of a small molecule compound which could modulate the innate immune response, including the activation or inhibition of type I interferon production and other cytokines, could become an important strategy for the treatment or prevention of human diseases including viral infections and autoimmune disease. In particular, hepatitis B viral infections in animals may be advantageously treated by administration of a first compound that modulates STING activity and administering a second compound that does not modulate STING activity.
[0012] STING is essential for antimicrobial host defense, and has been linked to protection against a range of DNA and RNA viruses and bacteria (reviewed in Barber et al. Nat. Rev. Immunol. 2015: 15(2): 87-103, Ma and Damania, Cell Host & Microbe, 2016: 19(2) 150-158). Herpesviridae, Flaviviridae, Coronaviridae, Papillomaviridae, Adenoviridae, Hepadnaviridae, ortho- and paramyxoviridae and rhabdoviridae have evolved mechanisms to inhibit STING mediated Type I interferon production and evade host immune control (Holm et al., Nat Comm. 2016: 7:10680; Ma et al, PNAS 2015: 1 12(31 ) E4306-E4315; Wu et al, Cell Host Microbe 2015: 18(3) 333-44; Liu et al, J Virol 2016: 90(20) 9406-19; Chen et al., Protein Cell 2014: 5(5) 369-81 ; Lau et al, Science 2013: 350(6260) 568-71 ; Ding et al, J Hepatol 2013: 59(1 ) 52-8; Nitta et al, Hepatology 2013 57(1) 46-58; Sun et al, PloS One 2012: 7(2) e30802; Aguirre et al, PloS Pathog 2012: 8(10) e1002934; Ishikawa et al, Nature 2009: 461 (7265) 788-92). Thus, small molecule activation of STING could be beneficial for treatment of these infectious diseases.
[0013] In contrast, increased and prolonged type I IFN production is associated with a variety of chronic infections, including Mycobacteria (Collins et al, Cell Host Microbe 2015: 17(6) 820-8); Wassermann et al., Cell Host Microbe 2015: 17(6) 799-810; Watson et al., Cell Host Microbe 2015: 17(6) 81 1-9), Franciscella (Storek et al., J Immunol. 2015: 194(7) 3236-45; Jin et al., J Immunol. 2011 : 187(5) 2595-601),
Chlamydia (Prantner et al., J Immunol 2010: 184(5) 2551 -60; , Plasmodium (Sharma et al., Immunity 2011 : 35(2) 194-207. and HIV (Herzner et al., Nat Immunol 2015 16(10) 1025-33; Gao et al., Science 2013: 341 (6148) 903-6. Similarly, excess type I interferon production is found among patients with complex forms of autoimmune disease. Genetic evidence in humans and support from studies in animal models support the hypothesis that inhibition of STING results in reduced type I interferon that drives autoimmune disease (Crow YJ, et al., Nat. Genet. 2006; 38(8) 38917-920, Stetson DB, et al., Cell 2008; 134 587-598). Therefore, inhibitors of STING provide a treatment to patients with chronic type I interferon and proinflammatory cytokine production associated with infections or complex autoimmune diseases. Allergic diseases are associated with a Th2-biased immune-response to allergens. Th2 responses are associated with raised levels of IgE, which, via its effects on mast cells, promotes a hypersensitivity to allergens, resulting in the symptoms seen, for example, in allergic rhinitis and asthma. In healthy individuals the immune-response to allergens is more balanced with a mixed Th2/Th1 and regulatory T cell response. Induction of Type 1 interferons have been shown to result in reduction of Th2-type cytokines in the local environment and promote Th1/Treg responses. In this context, induction of type 1 interferons by, for example, activation of STING, may offer benefit in treatment of allergic diseases such as asthma and allergic rhinitis (Huber J.P. et al J Immunol 2010: 185, 813-817).
[0014] Compounds that bind to STING and act as agonist have been shown to induce type 1 interferons and other cytokines on incubation with human PBMCs. For example, International Patent Applications WO2014/093936, WO2014/189805,
WO2013/185052, U.S.2014/0341976, WO 2015/077354, PCT/EP2015/062281 and GB 1501462.4 disclose certain cyclic di-nucleotides and their use in inducing an immune response via activation of STING. International Patent Applications WO2017/106740 describes the use of cyclic-di-nucleotide and related scaffold that measurably inhibit STING signaling and methods of identifying potent inhibitors of STING signaling.
International Patent Application WO 2017/175147 and WO 2017/175156 describes the use of heterocyclic amides and their analogues as STING modulators.
[0015] International Patent Application PCT/IB2017/051945 filed April 5, 2016, incorporated by reference herein in its entirety, discloses novel compounds that are useful as modulators of transmembrane protein 173 (TMEM173), also known as STING (Stimulator of Interferon Genes), for use in the treatment of STING-mediated disease or disorders, including inflammation, allergic and autoimmune diseases, cancer, and pre-cancerous syndromes, infectious diseases, and for use as immunogenic composition or vaccine adjuvants. It is envisaged that targeting STING with small molecules may be a promising approach for treating diseases and conditions in which modulation for the type 1 IFN pathway is beneficial, including infectious diseases.
[0016] The growing resistance of viruses to existing therapies, and the low seroconversion rates seen in serum of patients infected with certain viruses when treated with existing anti-viral therapies points to a need to discover and develop new anti-viral therapies. More particularly, there is a need for new anti-HBV therapies capable of increasing HBeAg and HBsAg seroconversion rates. HBV serum markers HBeAg (hepatitis B envelope antigen) and HBsAg (hepatitis B surface antigen) are indicative of immunological control of HBV infection, and reduction in serum antigen levels leads to an improved prognosis, i.e. prevention of liver disease and progression to cirrhosis, prevention of liver failure, prevention of hepatocellular cancer (HCC), prevention of liver disease-related transplantation, and prevention of death.
[0017] Clinical research has found a correlation between seroconversion and reductions in HBeAg (Fried et al (2008) Hepatology 47:428) and reductions in HBsAg (Moucari et al (2009) Hepatology 49:1 151). Reductions in antigen levels may have allowed immunological control of HBV infection because high levels of antigens are thought to induce immunological tolerance. Current nucleoside therapies for HBV are capable of dramatic reductions in serum levels of HBV but have little impact on HBeAg and HBsAg levels.
[0018] Targeting STING with small molecules may be a promising new approach for treating hepatitis B virus infections. In addition, combination therapy of compounds which target STING with other therapies used to treat HBV has the potential to result in an effective cure of hepatitis B infections by combining multiple therapeutic agents with different modes of action in a combination therapy. For example, administering compounds that target STING, and administering small molecule inhibitors of the HBV antigens HBsAg and HBeAg, can directly target the receptor involved in inflammation and the HBV antigens responsible for invoking an immunogenic response in a mammal, so there is the potential to reduce serum HBeAg and HBsAg levels.
[0019]The compounds of this invention are useful as moderators of STING, and accordingly, may provide a beneficial therapeutic impact in treatment of diseases, disorders and/or conditions in which modulation of STING (Stimulator of Interferon Genes) is beneficial, for example for infectious diseases. SUMMARY OF THE INVENTION
[0020] One embodiment of the invention is directed to a compound as described herein, or a pharmaceutically acceptable salt thereof or prodrug thereof.
[0021] One embodiment of the invention is directed to a compound according to
Formula (I):
when
Figure imgf000007_0001
wherein said C2-C6alkyl or said C2-C6alkenyl is each independently optionally substituted by 1 -2 substituents selected from -Rc, -OH, and -ORc;
R1 and R2 are independently absent or Ci-C3alkyl;
W1, X1, Y1, and Z1 are each independently -CR3, -CR3', -CR3", -CR3"' or N, with the proviso that no more than two of W1, X1, Y1 and Z1 may be N;
W2, X2, Y2, and Z2 are each independently -CR4, -CR4', -CR4", -CR4"' or N, with the proviso that no more than two of W2, X2, Y2 and Z2 may be N;
R3, R3', R3", R3"', R4, R4', R4", R4"', R9, R10, R11, and R12 are each independently selected from H, Ci-4 alkoxyl, Ci_4 alkyl, -COO-Ci-4alkyl, -C4-7heterocycloalkyl, -Ci-4alkylC4. yheterocycloalkyl, and -OCi-4alkylC4-7heterocycloalkyl, wherein the -C4.7heterocycloalkyl, the
-C4-7heterocycloalkyl of the -Ci-4alkylC4.7heterocycloalkyl, or the C4.7heterocycloalkyl of the
-OCi-4alkylC4-7heterocycloalkyl comprises one or more heteroatoms selected from O, and N, wherein the Ci_4 alkyl, Ci-4 alkoxyl, the Ci_4 alkyl of the -Ci-4alkylC4.
yheterocycloalkyl or the Ci_4 alkyl of the -OCi-4alkylC4-7heterocycloalkyl is optionally substituted by 1 -4 substituents independently selected from -Rc, -OH and -ORc, and wherein the C4-7heterocycloalkyl, the C4-7heterocycloalkyl of the -Ci-4alkylC4. yheterocycloalkyl, or the C4-7heterocycloalkyl of the -OCi-4alkylC4-7heterocycloalkyl is substituted by 1 -4 substituents R’, R”, R’”, and R””, wherein R’, R”, R’”, and R”” are each independently selected from -Rc, -OH and -ORc, or any two of R’, R”, R’”, and R”” may combine with the ring atoms to which they are attached to form a 3-7 membered ring;
R5 and R6 are independently Ci-C4alkyl;
R7 and R8 are each independently H or Ci-C4alkyl;
each Rc is independently Ci-C4alkyl optionally substituted by a substituent selected from -OH or Ci_4 alkoxyl;
or a salt thereof.
[0022] In one particular embodiment there is provided a compound selected from the group consisting of:
N,N'-((2E,2'E)-((E)-but-2-ene-1 ,4-diyl)bis(3-methyl-1 ,3-dihydro-2H- benzo[d]imidazole-1 -yl-2-ylidene))bis(1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide);
Methyl (E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-1 -((E)-4-((E)-2-((1 - ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol- 1 -yl)but-2-en-1 -yl)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazole-5-carboxylate;
1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2, 3-dihydro- 1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-methoxy-1 -methyl-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7- (3-hydroxypropoxy)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4- methoxy-1 -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
(E)-1 -Ethyl-N-(3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(hydroxymethyl)-1 -methyl- 1 H-benzo[d]imidazol-2(3H)-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-1 -methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2, 3-dihydro- 1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-1 -methyl-4-(piperidin-1 - ylmethyl)-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
N-((E)-1 -((E)-4-((E)-7-(azepan-1 -ylmethyl)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3-methyl-
1 .3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide;
N-((E)-1 -((E)-4-((E)-7-((3-oxa-6-azabicyclo[3.1 .1 ]heptan-6-yl)methyl)-2-((1 -ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but- 2-en-1 -yl)-3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H- pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(methoxymethyl)-1 -methyl-
1 .3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(1 -hydroxyethyl)-1 -methyl-
1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(hydroxymethyl)-3-methyl-
1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3-methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
1 -Ethyl-N-((E)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3-methyl-4-(piperidin-1 - ylmethyl)-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
N-((E)-4-((3-oxa-6-azabicyclo[3.1 .1 ]heptan-6-yl)methyl)-1 -((E)-4-((E)-2-((1 -ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but- 2-en-1 -yl)-3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H- pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(1 -hydroxyethyl)-3-methyl-
1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
N,N'-((2E,2'E)-Butane-1 ,4-diylbis(3-methyl-1 ,3-dihydro-2H-benzo[d]imidazole-1 -yl- 2-ylidene))bis(1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide); 1 -ethyl-N-((E)-3-(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-4-(hydroxymethyl)-1 -methyl-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-1 -methyl-4-(morpholinomethyl)-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-1 -methyl-4-(piperidin-1 -ylmethyl)-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
N-((E)-1 -(4-((E)-7-((3-oxa-6-azabicyclo[3.1 .1 ]heptan-6-yl)methyl)-2-((1 -ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 - yl)butyl)-3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H- pyrazole-5-carboxamide;
1 -ethyl-N-((E)-1 -(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-4-(hydroxymethyl)-3-methyl-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-1 -(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-3-methyl-4-(morpholinomethyl)-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((2R,5R)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-4- (hydroxymethyl)-l -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((2R,5R)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-1 -methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
1 -ethyl-N-((E)-3-((2S,5S)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-4- (hydroxymethyl)-l -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((2S,5S)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-1 -methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide; 1 -ethyl-N-((E)-3-((2S,5R)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-4- (hydroxymethyl)-l -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((2S,5R)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydrc>-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-1 -methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
1 -ethyl-N-((E)-3-((2R,5S)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-4- (hydroxymethyl)-l -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((2R,5S)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-1 -methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
N-((E)-1 -((E)-4-((E)-7-(1 ,2-dihydroxyethyl)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3-methyl- 1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(1 -hydroxy-2- morpholinoethyl)-1 -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-7-(morpholinomethyl)-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4- (hydroxymethyl)-l -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-1 - methyl-1 ,2-dihydrc>-3H-imidazo[4,5-b]pyridin-3-yl)but-2-en-1 -yl)-1 -methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-1 - methyl-1 ,2-dihydrc>-3H-imidazo[4,5-c]pyridin-3-yl)but-2-en-1 -yl)-1 -methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide; 1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-imidazo[4,5-c]pyridin-1-yl)but-2-en-1 -yl)-1-methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
1 -ethyl-N-((E)-3-((E)-4-((Z)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-imidazo[4,5-b]pyridin-1-yl)but-2-en-1-yl)-1 -methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-4-(morpholinomethyl)-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-1 - methyl-4-(morpholinomethyl)-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide;
or a salt thereof.
[0023] The compounds according to Formula (I), or salts, particularly
pharmaceutically acceptable salts, thereof, are modulators of STING. Accordingly, this invention provides a compound of Formula (I) or a salt thereof, particularly a
pharmaceutically acceptable salt thereof, for use in therapy. This invention specifically provides for the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as an active therapeutic substance in the treatment of a STING-mediated disease or disorder, specifically, for use in the treatment of a disease mediated by agonism or antagonism of STING. The invention also provides a compound of Formula (I), or a salt thereof, particularly a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for the treatment of a STING-mediated disease or disorder.
[0024] The invention is also directed to a method of modulating STING, which method comprises contacting a cell with a compound according to Formula (I), or a salt, particularly a pharmaceutically acceptable salt, thereof. The invention is further directed to a method of treating a STING mediated disease or disorder which comprises administering a therapeutically effective amount of a compound according to Formula (I), or a salt, particularly a pharmaceutically acceptable salt thereof, to a patient (a human or other mammal, particularly, a human) in need thereof. Such STING mediated diseases or disorders include inflammation, allergic and autoimmune diseases, infectious diseases including Hepatitis B virus (HBV) infection and/or Hepatitis D virus (HDV) infection, cancer, pre-cancerous syndromes, metabolic diseases, and cardiovascular diseases. In addition, modulators of STING may be useful as immunogenic composition or vaccine adjuvants. [0025] The present invention is further directed to a pharmaceutical composition comprising a compound according to Formula (I), or a salt, particularly a pharmaceutically acceptable salt, thereof and a pharmaceutically acceptable excipient. Particularly, this invention is directed to a pharmaceutical composition for the treatment of a STING mediated disease or disorder, where the composition comprises a compound according to Formula (I), or a salt, particularly a pharmaceutically acceptable salt, thereof and a pharmaceutically acceptable excipient.
Detailed Description of Specific Embodiments
[0026] Definitions.
[0027] As used in this description and the accompanying claims, the following terms shall have the meanings indicated, unless the context otherwise requires.
[0028] The alternative definitions for the various groups and substituent groups of Formula (I) provided throughout the specification are intended to particularly describe each compound species disclosed herein, individually, as well as groups of one or more compound species. The scope of this invention includes any combination of these group and substituent group definitions. The compounds of the invention are only those which are contemplated to be“chemically stable” as will be appreciated by those skilled in the art.
[0029] It will be appreciated by those skilled in the art that the compounds of this invention may exist in other tautomeric forms including zwitterionic forms, or isomeric forms. All tautomeric (including zwitterionic forms) and isomeric forms of the formulas and compounds described herein are intended to be encompassed within the scope of the present invention.
[0030] It will also be appreciated by those skilled in the art that the compounds of this invention may exist in tautomeric (or isomeric) forms including, but not limited to, Formula (A), Formula (B) and/or Formula (C) or zwitterionic forms including, but not limited to, Formula (D) or Formula (E). In Formula (B), (C), (D) or (E), each occurrence of R is independently H or any appropriate substituent group on nitrogen, for example alkyl.
Figure imgf000014_0001
Formula (D) Formula (E)
[0032] The chemical names provided for the intermediate compounds and/or the compounds of this invention described herein may refer to any one of the tautomeric/isomeric representations of such compounds (in some instances, such alternate names are provided with the experimental). It is to be understood that any reference to a named compound (an intermediate compound or a compound of the invention) or a structurally depicted compound (an intermediate compound or a compound of the invention) is intended to encompass all tautomeric/isomeric forms including zwitterionic forms of such compounds and any mixture thereof.
[0033] As used herein, the term "alkyl" represents a saturated, straight or branched hydrocarbon group having the specified number of carbon atoms. The term "Ci-C4alkyl" refers to a straight or branched alkyl moiety containing from 1 to 4 carbon atoms. Exemplary alkyls include, but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, f-butyl, pentyl and hexyl.
[0034] When a substituent term such as "alkyl" is used in combination with another substituent term, for example as in“hydroxy(Ci-C4alkyl)”, the linking substituent term (e.g., alkyl) is intended to encompass a divalent moiety, wherein the point of attachment is through that linking substituent. Examples of“hydroxy(Ci-C4alkyl)” groups include, but are not limited to, hydroxymethyl, hydroxyethyl, and
hydroxyisopropyl.
[0035] "Alkenyl" refers to straight or branched hydrocarbon group having the specified number of carbon atoms and at least 1 and up to 3 carbon-carbon double bonds. Examples include ethenyl and propenyl. [0036] "Alkoxy-" or“(alkyl)oxy-” refers to an "alkyl-oxy-" group, containing an alkyl moiety, having the specified number of carbon atoms, attached through an oxygen linking atom. For example, the term "Ci-C4alkoxy-" represents a saturated, straight or branched hydrocarbon moiety having at least 1 and up to 4 carbon atoms attached through an oxygen linking atom. Exemplary "Ci-C4alkoxy-" or“(Ci-C4alkyl)oxy-” groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, s- butoxy, and f-butoxy.
[0037] A carbocyclic group or moiety is a cyclic group or moiety in which the ring members are carbon atoms, which may be saturated, partially unsaturated
(non-aromatic) or fully unsaturated (aromatic).
[0038] "Cycloalkyl" refers to a non-aromatic, saturated, hydrocarbon ring group containing the specified number of carbon atoms in the ring. For example, the term "C3-C6cycloalkyl" refers to a cyclic group having from three to six ring carbon atoms. Exemplary "C3-C6cycloalkyl" groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
[0039] A heterocyclic group or moiety is a cyclic group or moiety having, as ring members, atoms of at least two different elements, which cyclic group or moiety may be saturated, partially unsaturated (non-aromatic) or fully unsaturated (aromatic).
[0040] "Heteroatom" refers to a nitrogen, sulfur, or oxygen atom, for example a nitrogen atom or an oxygen atom.
[0041] "Heterocycloalkyl" refers to a non-aromatic, monocyclic or bicyclic group containing from 3 to 10 ring atoms and containing one or more (generally one or two) heteroatom ring members independently selected from oxygen, sulfur, and nitrogen. The point of attachment of a heterocycloalkyl group may be by any suitable carbon or nitrogen atom.
[0042] Examples of "heterocycloalkyl" groups include, but are not limited to, aziridinyl, thiiranyl, oxiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, 1 ,3-dioxolanyl, piperidinyl, piperazinyl, tetrahydropyranyl,
dihydropyranyl, tetrahydrothiopyranyl, 1 ,3-dioxanyl, 1 ,4-dioxanyl, 1 ,3-oxathiolanyl, 1 ,3- oxathianyl, 1 ,3-dithianyl, 1 ,4-oxathiolanyl, 1 ,4-oxathianyl, 1 ,4-dithianyl, morpholinyl, thiomorpholinyl, and hexahydro-1 H-1 ,4-diazepinyl.
[0043] Examples of "4-7 membered heterocycloalkyl" groups include oxetanyl, thietanyl and azetidinyl.
[0044] Examples of“4-7 membered heterocycloalkyl” groups include those found in the compounds N-((E)-1 -((E)-4-((E)-7-((3-oxa-6-azabicyclo[3.1 .1 ]heptan-6-yl)methyl)-2- ((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H- benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2- ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide and N-((E)-4-((3-oxa-6- azabicyclo[3.1 .1 ]heptan-6-yl)methyl)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3-methyl- 1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide of Examples 9 and 15, respectively.
[0045] The term "5-6 membered heterocycloalkyl" represents a non-aromatic, monocyclic group, containing 5 or 6 ring atoms, which includes one or two heteroatoms selected independently from oxygen, sulfur, and nitrogen. Illustrative examples of 5-6 membered heterocycloalkyl groups include, but are not limited to pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, piperazinyl, morpholinyl, and thiomorpholinyl.
[0046] "Heteroaryl" refers to an aromatic monocyclic or bicyclic group containing 5 to 10 ring atoms, including 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, wherein at least a portion of the group is aromatic. For example, this term encompasses bicyclic heterocyclic-aryl groups containing either a phenyl ring fused to a heterocyclic moiety or a heteroaryl ring moiety fused to a carbocyclic moiety. The point of attachment of a“heteroaryl” group may be by any suitable carbon or nitrogen atom.
[0047] The term "5-6 membered heteroaryl" represents an aromatic monocyclic group containing 5 or 6 ring atoms, including at least one carbon atom and 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur. Selected 5- membered heteroaryl groups contain one nitrogen, oxygen, or sulfur ring heteroatom, and optionally contain 1 , 2, or 3 additional nitrogen ring atoms. Selected 6-membered heteroaryl groups contain 1 , 2, or 3 nitrogen ring heteroatoms. Examples of 5-membered heteroaryl groups include furyl (furanyl), thienyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, and oxadiazolyl.
Selected 6-membered heteroaryl groups include pyridinyl (pyridyl), pyrazinyl, pyrimidinyl, pyridazinyl and triazinyl.
[0048] The term "9-10 membered heteroaryl" refers to an aromatic bicyclic group containing 9 or 10 ring atoms, including 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur. Examples of 9-membered heteroaryl (6,5-fused heteroaryl) groups include benzothienyl, benzofuranyl, indolyl, indolinyl (dihydroindolyl), isoindolyl, isoindolinyl, indazolyl, isobenzofuryl, 2,3-dihydrobenzofuryl, benzoxazolyl,
benzoisoxazolyl, benzothiazolyl, benzoisothiazolyl, benzimidazolyl, benzoxadiazolyl, benzothiadiazolyl, benzotriazolyl, purinyl, imidazopyridinyl, pyrazolopyridinyl, triazolopyridinyl and 1 ,3-benzodioxolyl.
[0049] Examples of 10-membered heteroaryl (6,6-fused heteroaryl) groups include quinolinyl (quinolyl), isoquinolyl, phthalazinyl, naphthridinyl (1 ,5-naphthyridinyl,
1 ,6-naphthyridinyl, 1 ,7-naphthyridinyl, 1 ,8-naphthyridinyl), quinazolinyl, quinoxalinyl, 4H-quinolizinyl, 1 ,2,3,4-tetrahydroquinolinyl (tetrahydroquinolinyl), 1 ,2,3,4- tetrahydroisoquinolinyl (tetrahydroisoquinolinyl), cinnolinyl, pteridinyl, and 2,3- dihydrobenzo[b][1 ,4]dioxinyl.
[0050] "Oxo" represents a double-bonded oxygen moiety; for example, if attached directly to a carbon atom forms a carbonyl moiety (C = O).
[0051] "Hydroxy" or "hydroxyl" is intended to mean the radical -OH.
[0052] As used herein, the term "cyano" refers to a nitrile group, -CºN.
[0053] As used herein, the term "optionally substituted" indicates that a group (such as an alkyl, cycloalkyl, alkoxy, heterocycloalkyl, aryl, or heteroaryl group) or ring or moiety may be unsubstituted, or the group, ring or moiety may be substituted with one or more substituent(s) as defined in the substituent definitions (A, R3, etc,) provided herein. In the case where groups may be selected from a number of alternative groups, the selected groups may be the same or different.
[0054] The term "independently" means that where more than one substituent is selected from a number of possible substituents, those substituents may be the same or different.
[0055] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
[0056] As used herein, the terms "compound(s) of the invention" or
"compound(s) of this invention" mean a compound of Formula (I) as defined herein, in any form, i.e., any tautomeric/isomeric form, any salt or non-salt form (e.g., as a free acid or base form, or as a salt, particularly a pharmaceutically acceptable salt thereof) and any physical form thereof (e.g., including non-solid forms (e.g., liquid or semi-solid forms), and solid forms (e.g., amorphous or crystalline forms, specific polymorphic forms, solvate forms, including hydrate forms (e.g., mono-, di- and hemi- hydrates)), and mixtures of various forms.
[0057] Accordingly, included within the present invention are the compounds of Formula (I), as defined herein, in any salt or non-salt form and any physical form thereof, and mixtures of various forms. While such are included within the present invention, it will be understood that the compounds of Formula (I), as defined herein, in any salt or non-salt form, and in any physical form thereof, may have varying levels of activity, different bioavailabilities and different handling properties for formulation purposes.
[0058] The compounds of this invention may contain one or more asymmetric centers (also referred to as a chiral center), such as a chiral carbon, or a chiral -SO- moiety. Compounds of this invention containing one or more chiral centers may be present as racemic mixtures, diastereomeric mixtures, enantiomerically enriched mixtures, diastereomerically enriched mixtures, or as enantiomerically or
diastereomerically pure individual stereoisomers.
[0059] The stereochemistry of the chiral center present in compounds of this invention is generally represented in the compound names and/or in the chemical structures illustrated herein. Where the stereochemistry of a chiral center present in a compound of this invention, or in any chemical structure illustrated herein, is not specified, the structure is intended to encompass any stereoisomer and all mixtures thereof. Accordingly, the present invention encompasses all isomers of the compounds described herein or the compounds of Formula (I), and salts thereof, whether as individual isomers isolated such as to be substantially free of the other isomer (i.e. pure) or as mixtures (i.e. racemates and racemic mixtures). An individual isomer isolated such as to be substantially free of the other isomer (i.e. pure) may be isolated such that less than 10%, particularly less than about 1 %, for example less than about 0.1 % of the other isomer is present.
[0060] Individual stereoisomers of a compound of this invention may be resolved (or mixtures of stereoisomers may be enriched) using methods known to those skilled in the art. For example, such resolution may be carried out (1) by formation of
diastereoisomeric salts, complexes or other derivatives; (2) by selective reaction with a stereoisomer-specific reagent, for example by enzymatic oxidation or reduction; or (3) by gas-liquid or liquid chromatography in a chiral environment, for example, on a chiral support such as silica with a bound chiral ligand or in the presence of a chiral solvent. It will be appreciated that where the desired stereoisomer is converted into another chemical entity by one of the separation procedures described above, a further step is required to liberate the desired form. Alternatively, specific stereoisomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer to the other by asymmetric transformation. [0061] As used herein,“cleavable bond” means any chemical bond capable of being broken. In certain embodiments, a cleavable bond is selected from among: an amide, a polyamide, an ester, an ether, a phosphodiester, a phosphate ester, a carbamate, a di-sulfide, or a peptide.
[0062] As used herein, "modulation" means a change of amount or quality of a molecule, function, or activity when compared to the amount or quality of the molecule, function, or activity prior to modulation.
[0063] As used herein,“pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an animal. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline.
[0064] As used herein,“prodrug” means an inactive or less active form of a compound which, when administered to a subject, undergoes some in vivo
biotransformation through chemical or enzymatic cleavage, enabling the delivery of the active, or more active, compound (e.g., drug). A prodrug may include a cleavable moiety, wherein the cleavable moiety is removed or metabolized to form the active, or more active, compound. The cleavable moiety may be connected to the active form of the compound by a cleavable bond, including but not limited to an ester, an amide, a sulfonamide, an amine, an imine, an ether, a thioether, a disulfide, a glycosidic, a phosphate, a carbamate, a carbonate or a boronic ester bond, or some other cleavable bond.
[0065] As used herein, the term "effective amount" means that amount of a drug or drug substance, or pharmaceutical agent, that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician. Furthermore, the term“therapeutically effective amount” means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal
physiological function.
[0066] As used herein, the term“HBV” means mammalian Hepatitis B virus, including human Hepatitis B virus. The term encompasses geographical genotypes of Hepatitis B virus, particularly human Hepatitis B virus, as well as variant strains of geographical genotypes of Hepatitis B virus.
[0067] As used herein,“Hepatitis B-related condition” or“HBV-related condition” means any disease, biological condition, medical condition, or event which is exacerbated, caused by, related to, associated with, or traceable to a Hepatitis B infection, exposure, or illness. The term Hepatitis B-related condition includes jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, and conditions having symptoms which may include any or all of the following: flu-like illness, weakness, aches, headache, fever, loss of appetite, diarrhea, jaundice, nausea and vomiting, pain over the liver area of the body, clay- or grey-colored stool, itching all over, and dark-colored urine, when coupled with a positive test for presence of a Hepatitis B virus, a Hepatitis B viral antigen, or a positive test for the presence of an antibody specific for a Hepatitis B viral antigen.
[0068] One embodiment of the invention is directed to a compound according to Formula (I):
when
Figure imgf000020_0001
wherein said C2-C6alkyl or said C2-C6alkenyl is each independently optionally substituted by 1 -2 substituents selected from -Rc, -OH, and -ORc;
R1 and R2 are independently absent or Ci-C3alkyl;
W1, X1, Y1, and Z1 are each independently -CR3, -CR3', -CR3", -CR3"' or N, with the proviso that no more than two of W1, X1, Y1 and Z1 may be N;
W2, X2, Y2, and Z2 are each independently -CR4, -CR4', -CR4", -CR4"' or N, with the proviso that no more than two of W2, X2, Y2 and Z2 may be N;
R3, R3', R3", R3"', R4, R4', R4", R4"', R9, R10, R11, and R12 are each independently selected from H, Ci-4 alkoxyl, Ci_4 alkyl, -COO-Ci_4alkyl, -C4-7heterocycloalkyl, -Ci-4alkylC4. yheterocycloalkyl, and -OCi-4alkylC4-7heterocycloalkyl, wherein the -C4.7heterocycloalkyl, the -C4-7heterocycloalkyl of the -Ci-4alkylC4-7heterocycloalkyl, or the C4.7heterocycloalkyl of the
-OCi-4alkylC4-7heterocycloalkyl comprises one or more heteroatoms selected from O, and N,
wherein the Ci_4 alkyl, Ci_4 alkoxyl, the Ci_4 alkyl of the -Ci-4alkylC4.
7heterocycloalkyl or the Ci_4 alkyl of the -OCi-4alkylC4-7heterocycloalkyl is optionally substituted by 1 -4 substituents independently selected from -Rc, -OH and -ORc, and wherein the C4-7heterocycloalkyl, the C4-7heterocycloalkyl of the -Ci-4alkylC4. 7heterocycloalkyl, or the C4-7heterocycloalkyl of the -OCi-4alkylC4-7heterocycloalkyl is substituted by 1 -4 substituents R’, R”, R’”, and R””, wherein R’, R”, R’”, and R”” are each independently selected from -Rc, -OH and -ORc, or any two of R’, R”, R’”, and R”” may combine with the ring atoms to which they are attached to form a 3-7 membered ring;
R5 and R6 are independently Ci-C4alkyl;
R7 and R8 are each independently H or Ci-C4alkyl;
each Rc is independently Ci-C4alkyl optionally substituted by a substituent selected from -OH or Ci_4 alkoxyl;
or a salt thereof.
[0069] One embodiment of the invention is directed to a compound according to Formula (I) wherein:
when A is C2alkenylene;
R1 and R2 are -CH2-;
Y1 , Y2, X1 and X2 are -CH-;
W1, W2, Z1 and Z2 are each independently selected from: -CH-, -C(azepine- methyl)-,
-C(3-oxa-6-azabicyclo[3.1 1 ]heptane-methyl)-, -C(methoxymethyl)-, -C(l -hydroxyethyl)-, -C(C(0)0CH3)-, -C(methoxy)-, -C(3-hydroxypropoxy)-, -C(hydroxymethyl)-,
-C(morpholinomethyl)-, -C(1 ,2-dihydroxyethyl)-, and -C(1 -hydroxy-2-morpholinoethyl)-;
R5 and R6 are -CH3;
R7 and R8 are C2alkyl;
R9, R10, R11 , and R12 are each independently selected from: H and CH3;
or a salt thereof, suitably a pharmaceutically acceptable salt thereof.
[0070] One embodiment provides compounds of Formula (I), wherein R1 and R2 are -CH2- and R5 and R6 are -CH3.
[0071] One embodiment provides compounds of Formula (I), wherein W1 , X1 , Y1 , and Z1 are each independently -CR3, -CR3', -CR3" or -CR3"' and W2, X2, Y2, and Z2 are each independently -CR4, -CR4', -CR4" or -CR4"'. [0072] One embodiment provides compounds of Formula (I), wherein W1 is CR3 and W2 is CR4, wherein CR3 and CR4 are each independently selected from -C4.
yheterocycloalkyl, -Ci-4alkylC4-7heterocycloalkyl, and -OCi-4alkylC4-7heterocycloalkyl.
[0073] One embodiment provides compounds of Formula (I), wherein W1 is CR3 and W2 is CR4, wherein R3 and CR4 are each independently selected from Ci_4 alkoxyl or Ci-4 alkyl, wherein the Ci_4 alkyl or Ci_4 alkoxyl is optionally substituted by 1 -4 substituents independently selected from -Rc, -OH and -ORc.
[0074] One embodiment provides compounds of Formula (I), wherein A is C2-C6alkylene.
[0075] One embodiment provides compounds of Formula (I), wherein A is C2-C6alkenylene.
[0076] One embodiment provides compounds of Formula (I), wherein R1 and R2 are each -CH2- and A is C2-alkenylene.
[0077] One embodiment provides compounds of Formula (I), wherein R7 and R8 are each independently H or C2alkyl.
[0078] One embodiment provides compounds of Formula (I), wherein R7 and R8 are each C2alkyl.
[0079] One embodiment provides compounds of Formula (I), wherein R9, R10, R11 , and R12 are each independently H or -CH3.
[0080] One embodiment provides compounds of Formula (I), wherein Y1, Y2, X1 and X2 are -CH.
[0081] One embodiment provides compounds of Formula (I), wherein W1 , W2, Z1 and Z2 are each independently selected from: H, azepine-methyl, 3-oxa-6- azabicyclo[3.1 1 ]heptane-methyl, methoxymethyl, 1 -hydroxyethyl, -C(0)0CH3, methoxy, 3-hydroxypropoxy, hydroxymethyl, morpholinomethyl, 1 ,2-dihydroxyethyl, and 1 -hydroxy- 2-morpholinoethyl.
[0082] In one particular embodiment, there is provided a compound selected from the group consisting of:
N,N'-((2E,2'E)-((E)-but-2-ene-1 ,4-diyl)bis(3-methyl-1 ,3-dihydro-2H- benzo[d]imidazole-1 -yl-2-ylidene))bis(1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide);
Methyl (E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-1 -((E)-4-((E)-2-((1 - ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol- 1 -yl)but-2-en-1 -yl)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazole-5-carboxylate;
1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2, 3-dihydro- 1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-methoxy-1 -methyl-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide; 1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7- (3-hydroxypropoxy)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4- methoxy-1 -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
(E)-1 -Ethyl-N-(3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(hydroxymethyl)-1 -methyl- 1 H-benzo[d]imidazol-2(3H)-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2, 3-dihydro- 1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-1 -methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-1 -methyl-4-(piperidin-1 - ylmethyl)-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
N-((E)-1 -((E)-4-((E)-7-(azepan-1 -ylmethyl)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3-methyl-
1 .3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide;
N-((E)-1 -((E)-4-((E)-7-((3-oxa-6-azabicyclo[3.1 .1 ]heptan-6-yl)methyl)-2-((1 -ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but- 2-en-1 -yl)-3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H- pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(methoxymethyl)-1 -methyl-
1 .3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(1 -hydroxyethyl)-1 -methyl-
1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(hydroxymethyl)-3-methyl-
1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3-methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide; 1 -Ethyl-N-((E)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2, 3-dihydro- 1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3-methyl-4-(piperidin-1 - ylmethyl)-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
N-((E)-4-((3-oxa-6-azabicyclo[3.1 .1 ]heptan-6-yl)methyl)-1 -((E)-4-((E)-2-((1 -ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but- 2-en-1 -yl)-3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H- pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(1 -hydroxyethyl)-3-methyl- 1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
N,N'-((2E,2'E)-Butane-1 ,4-diylbis(3-methyl-1 ,3-dihydro-2H-benzo[d]imidazole-1 -yl- 2-ylidene))bis(1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide);
1 -ethyl-N-((E)-3-(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-4-(hydroxymethyl)-1 -methyl-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-1 -methyl-4-(morpholinomethyl)-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-1 -methyl-4-(piperidin-1 -ylmethyl)-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
N-((E)-1 -(4-((E)-7-((3-oxa-6-azabicyclo[3.1 .1 ]heptan-6-yl)methyl)-2-((1 -ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 - yl)butyl)-3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H- pyrazole-5-carboxamide;
1 -ethyl-N-((E)-1 -(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-4-(hydroxymethyl)-3-methyl-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-1 -(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-3-methyl-4-(morpholinomethyl)-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((2R,5R)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-4- (hydroxymethyl)-l -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide; 1 -ethyl-N-((E)-3-((2R,5R)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-1 -methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
1 -ethyl-N-((E)-3-((2S,5S)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-4- (hydroxymethyl)-l -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((2S,5S)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-1 -methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
1 -ethyl-N-((E)-3-((2S,5R)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-4- (hydroxymethyl)-l -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((2S,5R)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-1 -methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
1 -ethyl-N-((E)-3-((2R,5S)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-4- (hydroxymethyl)-l -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((2R,5S)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-1 -methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
N-((E)-1 -((E)-4-((E)-7-(1 ,2-dihydroxyethyl)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3-methyl- 1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(1 -hydroxy-2- morpholinoethyl)-1 -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide; 1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-7-(morpholinomethyl)-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4- (hydroxymethyl)-l -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-1 - methyl-1 ,2-dihydro-3H-imidazo[4,5-b]pyridin-3-yl)but-2-en-1 -yl)-1 -methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-1 - methyl-1 ,2-dihydro-3H-imidazo[4,5-c]pyridin-3-yl)but-2-en-1 -yl)-1 -methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-imidazo[4,5-c]pyridin-1 -yl)but-2-en-1 -yl)-1 -methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
1 -ethyl-N-((E)-3-((E)-4-((Z)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-imidazo[4,5-b]pyridin-1 -yl)but-2-en-1 -yl)-1 -methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-4-(morpholinomethyl)-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-1 - methyl-4-(morpholinomethyl)-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide;
or a salt thereof.
[0083] In one particular embodiment, there is provided a compound selected from the compounds of Examples 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13-1 , 13-2, 14, 15, 16, 17, 19, 32 and 33, or a salt thereof.
[0084] In one particular embodiment, there is provided a compound selected from the group consisting of:
(E)-1 -Ethyl-N-(3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(hydroxymethyl)-1 -methyl- 1 H-benzo[d]imidazol-2(3H)-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2, 3-dihydro- 1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(hydroxymethyl)-3-methyl- 1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide; N-((E)-4-((3-oxa-6-azabicyclo[3.1 .1 ]heptan-6-yl)methyl)-1 -((E)-4-((E)-2-((1 -ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but- 2-en-1 -yl)-3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H- pyrazole-5-carboxamide;
N-((E)-1 -((E)-4-((E)-7-(1 ,2-dihydroxyethyl)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3-methyl- 1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide; and
1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(1 -hydroxy-2- morpholinoethyl)-1 -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide;
or a pharmaceutically acceptable salt thereof.
[0085] compounds of the invention may exist in both unsolvated and solvated forms. The term‘solvate’ is used herein to describe a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term‘hydrate’ is employed when said solvent is water. Pharmaceutically acceptable solvates include hydrates and other solvates wherein the solvent of crystallization may be isotopically substituted, e.g. D20, deacetone, d6-DMSO.
[0086] Suitable pharmaceutically acceptable salts of the compounds described herein can include acid addition salts or base addition salts. For reviews of suitable pharmaceutically acceptable salts see Berge et al., J. Pharm. Sci., 66:1 -19, (1977) and P. H. Stahl and C. G. Wermuth, Eds., Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim/ZOrich:Wiley-VCH/VHCA (2002).
[0087] Salts of the compounds described herein containing a basic amine or other basic functional group may be prepared by any suitable method known in the art, such as treatment of the free base with a suitable inorganic or organic acid. Examples of pharmaceutically acceptable salts so formed include acetate, adipate, ascorbate, aspartate, benzenesulfonate, benzoate, camphorate, camphor-sulfonate (camsylate), caprate (decanoate), caproate (hexanoate), caprylate (octanoate), carbonate, bicarbonate, cinnamate, citrate, cyclamate, dodecylsulfate (estolate), ethane-1 ,2- disulfonate (edisylate), ethanesulfonate (esylate), formate, fumarate (hemi-fumarate, etc.), galactarate (mucate), gentisate (2,5-dihydroxybenzoate), glucoheptonate
(gluceptate), gluconate, glucuronate, glutamate, glutarate, glycerophosphorate, glycolate, hippurate, hydrobromide, hydrochloride (dihydrochloride, etc.), hydroiodide, isobutyrate, lactate, lactobionate, laurate, maleate, malate, malonate, mandelate, methanesulfonate (mesylate), naphthalene-1 ,5-disulfonate (napadisylate), naphthalene-sulfonate
(napsylate), nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, phosphate
(diphosphate, etc.), proprionate, pyroglutamate, salicylate, sebacate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate (tosylate), undecylenate, 1 -hydroxy-2- naphthoate, 2,2-dichloroacetate, 2-hydroxyethanesulfonate (isethionate), 2-oxoglutarate, 4-acetamidobenzoate, and 4-aminosalicylate.
[0088] Salts of the disclosed compounds containing a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base. Such a pharmaceutically acceptable salt may be made with a base which affords a
pharmaceutically acceptable cation, which includes alkali metal salts (especially sodium and potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum salts and ammonium salts, as well as salts made from physiologically acceptable organic bases such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N,N’-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2- hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine,
dehydroabietylamine, N,N’-bisdehydroabietylamine, glucamine, N-methylglucamine, collidine, choline, quinine, quinoline, and basic amino acids such as lysine and arginine.
[0089] The invention includes within its scope all possible stoichiometric and non- stoichiometric forms of the salts (e.g., hydrobromide, dihydrobromide, fumarate, hemi- fumarate, etc.) of the compounds described herein.
[0090] The compounds described herein may be prepared and administered as a prodrug wherein the compound as described herein comprises a cleavable moiety connected to the compound of by a cleavable bond. In certain embodiments, the cleavable moiety is cleaved after the compound has been administered to an animal only after being internalized by a targeted cell. Inside the cell the cleavable moiety is cleaved, thereby releasing the active compound. While not wanting to be bound by theory it is believed that the cleavable moiety is cleaved by one or more enzymes or chemical transformations that occur within the cell. In certain embodiments, the one or more enzymes or chemical transformations cleave the cleavable bond, which bond may be an ester, an amide, a sulfonamide, an ether, a thioether, a disulfide, an amine, an imine, a phosphate, a carbamate, a carbonate, a boronic ester, a glycosidic bond, or some other cleavable bond. The cleavable bond will typically be between the cleavable moiety and a linker connected to the compound as described herein.
[0091] Included within the scope of the claimed compounds of the present invention are all stereoisomers, geometric isomers and tautomeric forms of the compounds as described herein and compounds of Formula (I), including compounds exhibiting more than one type of isomerism, and mixtures of one or more thereof.
[0092] In a particular embodiment of the invention, there is provided the use of a compound as described herein, for preventing, ameliorating or treating liver disease, or symptoms thereof, in an animal.
[0093] One particular embodiment of the invention there is provided the use of a first compound as described herein, in combination with the use of a second compound, for preventing, ameliorating or treating liver disease, or symptoms thereof, in an animal.
In such embodiments, the first compound is a compound of Formula (I), and the second compound is a compound of described herein or a pharmaceutically acceptable salt thereof.
[0094] One particular embodiment of the invention provides the use of a compound as described herein or a pharmaceutically acceptable salt thereof, for preventing, ameliorating or treating liver disease, or symptoms thereof, in an animal.
[0095] One embodiment of the invention provides a compound as disclosed herein, or a pharmaceutically acceptable salt thereof for use in therapy. This invention provides a compound as described herein, or a pharmaceutically acceptable salt thereof, for use in therapy, specifically for use in the treatment of a hepatitis B virus-related disease, condition or disorder (for example, a hepatitis B virus-related disease, condition or disorder may be Hepatitis D virus, jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome or serum hepatitis). Specifically, this invention provides a compound as described in Formula (I), or a pharmaceutically acceptable salt thereof, for use in therapy.
[0096] In another embodiment, this invention provides a compound of the invention as described herein, for use in the treatment of a hepatitis B virus-related disease, condition or disorder, specifically, a disease, condition or disorder recited herein. This invention provides a compound of Formula (I) as described herein, including a compound of Table 1 , or a pharmaceutically acceptable salt thereof, for use in the treatment of a hepatitis B virus-related disease, condition or disorder. This invention provides a compound of Formula (I) as described herein, including a compound of Table 1 , or a pharmaceutically acceptable salt thereof, for use in the treatment of a hepatitis B virus-related disease, condition or disorder, wherein the hepatitis B virus-related disease, condition or disorder may be Hepatitis D virus, jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome or serum hepatitis. [0097] This invention provides a compound of Formula (I) as described herein, including a compound of Table 1 , or a pharmaceutically acceptable salt thereof, for use in a functional cure of a Hepatitis B virus infection in a human, as determined by an undetectable level of HBV DNA and sustained HBsAg seroclearance, with or without anti- HBs seroconversion after the cessation of finite anti-HBV treatments by the human.
[0098] This invention specifically provides for the use of a compound of Formula (I) as described herein, including a compound of Table 1 , or a pharmaceutically acceptable salt thereof. More specifically, this provides for the use of the compounds described herein for the treatment of a Hepatitis B virus-related disease, condition or disorder, specifically, a disease, condition or disorder recited herein. Accordingly, the invention provides for the use of a compound of Formula (I) as described including a compound of Table 1 , as an active therapeutic agent or pharmaceutically acceptable salt thereof, in the treatment of a human in need thereof having, or a risk of having, a Hepatitis B virus-related disease, condition or disorder, specifically, a disease condition or disorder recited herein.
[0099] The invention further provides for the use of a compound of Formula (I) as described herein, including a compound of Table 1 , or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment of a Hepatitis B virus-related disease condition or disorder, for example the diseases and disorders recited herein. Specifically, the invention also provides for the use of a compounds of Formula (I) as described herein, in the manufacture of a medicament for use in the treatment of a Hepatitis B virus-related disease condition or disorder, for example the diseases, conditions and disorders recited herein.
[00100]ln one particular embodiment of the invention there is provided the use of a compound as described herein that is a compound of Formula (I), or a salt, particularly a pharmaceutically acceptable salt, for preventing, ameliorating or treating liver disease, or symptoms thereof, in an animal.
[00101 ] In one particular embodiment of the invention there is provided the use of a compound that is a compound of Formula (I) as described herein, or a pharmaceutically acceptable salt thereof, for preventing, ameliorating or treating liver disease, or symptoms thereof, in an animal. In such embodiments, the compound of Formula (I) may be a compound from Table 1.
[00102]Certain embodiments provide the use of a compound of Formula (I) as described herein, suitably a compound of Table 1 , or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating, ameliorating, delaying or preventing an HBV-related disease, disorder or condition in an animal. [00103]Certain embodiments provide the use of a compound of Formula (I) as described herein, suitably a compound of Table 1 , or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating, ameliorating, delaying or preventing liver disease in an animal.
[00104]Another embodiment of the invention provides a method for treating a Hepatitis B virus infection, treating a Hepatitis B virus-related condition, or treating a Hepatitis B/Hepatitis D virus co-infection in a mammal, the method comprising administering a therapeutically effective amount of a STING compound as described herein, so as to treat the Hepatitis B virus infection, the Hepatitis B virus-related condition and/or the Hepatitis B/Hepatitis D virus co-infection. In related embodiments, the mammal is a human and the Hepatitis B virus infection, the Hepatitis B virus-related condition, and/or the hepatitis B/Hepatitis D virus co-infection is an infection of a Hepatitis B virus from a human Hepatitis B virus. The human Hepatitis B virus may be any of the human geographical genotypes: A (Northwest Europe, North America, Central America); B (Indonesia, China, Vietnam); C (East Asia, Korea, China, Japan, Polynesia, Vietnam); D (Mediterranean area, Middle East, India); E (Africa); F (Native Americans, Polynesia); G (United States, France); or H (Central America).
[00105] Related embodiments provide a method for treating a Hepatitis B virus infection or a Hepatitis B virus-related condition in a mammal, the method comprising administering a therapeutically effective amount of any pharmaceutical composition as described herein to a mammal in need thereof, wherein the human Hepatitis B virus- related condition may be jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome or serum hepatitis.
[00106] In particular embodiments, there is provided a method of treating a Hepatitis B virus infection, a Hepatitis B virus-related condition or a Hepatitis B/Hepatitis D virus coinfection in a mammal in need of such treatment, the method comprising administering a compound of Formula (I) of the invention or a pharmaceutically acceptable salt thereof as described herein to the mammal.
[00107] In certain embodiments, the method of treating an HBV infection comprises administering a first therapeutic agent that is a compound of Formula (I) or pharmaceutically acceptable salt thereof as described herein. In certain embodiments, the methods comprise administering a first therapeutic agent that is a therapeutically effective amount of a compound of Formula (I) or pharmaceutically acceptable salt as described herein, or a prodrug or pharmaceutically acceptable salt thereof, and administering one or more second therapeutic agents. In certain embodiments, the first therapeutic agent and one or more second therapeutic agents are co-administered. In certain embodiments, the first therapeutic agent and the one or more second therapeutic agent are administered separately, in any order.
[00108] Certain embodiments provide the use of a compound as described herein, wherein the compound of Formula (I) as described herein is a conjugate or prodrug with a cleavable moiety connected to the compound by a cleavable bond. In certain
embodiments, the cleavable moiety is cleaved after the compound has been administered to an animal only after being internalized by a targeted cell. Inside the cell the cleavable moiety is cleaved, thereby releasing the active compound of Formula (I). While not wanting to be bound by theory it is believed that the cleavable moiety is cleaved by one or more enzymes or chemical transformations within the cell. In certain embodiments, the prodrug may comprise a cleavable bond. Examples of cleavable bonds include, but are not limited to, an ester, an amide, an amine, an imine, an ether, a thioether, a disulfide, a carbamate, a carbonate, a phosphate, a sulfonamide, a boronic ester or a glycosidic bond, or some other cleavable bond. Typically, the cleavable bond may be between the cleavable moiety and a linker connected to the compound of Formula (I).
[00109] In certain embodiments there is provided a first therapeutic agent that is a compound of Formula (I) as described herein, or a pharmaceutically acceptable salt thereof, and a second therapeutic agent. In certain embodiments, the compound of Formula (I) is a compound of Table 1 . In certain embodiments, the first therapeutic agent that is a compound of Formula (I) is co-administered sequentially or concomitantly with the second therapeutic agent. In certain embodiments, the second therapeutic agent is selected from an anti-inflammatory agent, a chemotherapeutic agent, or an anti-infection agent. In certain embodiments, the second therapeutic agent is an HBV agent, an HCV agent, a chemotherapeutic agent, an antibiotic, an analgesic, a non-steroidal antiinflammatory (NSAID) agent, an antifungal agent, an antiparasitic agent, an anti-nausea agent, an anti-diarrheal agent, an immunomodulatory, or an immunosuppressant agent.
[00110] In certain embodiments, the second therapeutic agent is an HBV agent. In certain embodiments the HBV agent is any of a compound described in WO2019/069293, interferon alpha-2b, interferon alpha-2a, and interferon alphacon-1 (pegylated and unpegylated), ribavirin; an HBV RNA replication inhibitor; an HBV antigen production inhibitor; an HBV therapeutic vaccine; an HBV prophylactic vaccine; lamivudine (3TC); entecavir (ETV); tenofovir diisoproxil fumarate (TDF); telbivudine (LdT); adefovir; or an HBV antibody therapy (monoclonal or polyclonal).
[00111]ln certain embodiments, the second therapeutic agent is an HCV agent. In certain embodiments the HCV agent is any of interferon alpha-2b, interferon alpha-2a, and interferon alphacon-1 (pegylated and unpegylated); ribavirin; an HCV RNA replication inhibitor (e.g., ViroPharma's VP50406 series); an HCV antisense agent; an HCV therapeutic vaccine; an HCV protease inhibitor; an HCV helicase inhibitor; or an HCV monoclonal or polyclonal antibody therapy.
[00112] In certain embodiments, the second therapeutic agent is an antiinflammatory agent (i.e., an inflammation lowering therapy). In certain embodiments the inflammation lowering therapy is any of a therapeutic lifestyle change, a steroid, a NSAID or a DMARD. The steroid can be a corticosteroid. The NSAID can be an aspirin, acetaminophen, ibuprofen, naproxen, COX inhibitors, indomethacin and the like. The DMARD can be a TNF inhibitor, purine synthesis inhibitor, calcineurin inhibitor, pyrimidine synthesis inhibitor, a sulfasalazine, methotrexate and the like.
[00113] In certain embodiments, the second therapeutic agent is a
chemotherapeutic agent (i.e., a cancer treating agent). Chemotherapeutic agents can include, but are not limited to, daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan,
cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5- FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatin, gemcitabine and
diethylstilbestrol (DES).
[00114] In certain embodiments, the second therapeutic agent is an
immunomodulatory agent known as an innate immune activator, a check point inhibitor,
T- a cell stimulatory agent, or an agent that restores adaptive immune responses against HBV. Immune-modulators includes, but are not limited to, antibodies or small molecules antagonizing CTLA-4 such as ipilimumab (YERVOY), PD-1 such as Opdivo/nivolumab and Keytruda/pembrolizumab), PDL1 such as TECENTRIQ™ (atezolizumab), LAG3, TIM3, or IDO. Immune-modulators includes, but are not limited to, antibodies or small molecules stimulating ICOS, OX-40, TLRs, IL7R or IL12R.
[00115] In certain embodiments, the second therapeutic agent is an anti-infection agent. Examples of antiinfection agents include, but are not limited to, antibiotics, antifungal drugs and antiviral drugs. [00116] In particular embodiments, the HBV agent may include, but is not limited to, a compound described in WO2019/069293 , thymosin a1 , interferon b-1 a, interferon b- 1 b, interferon g-1 b, interferon g1 , interferon g2, interferon g3, interferon a-2a, interferon a-2b, pegylated interferon p-2a, pegylated interferon p-2b, pegylated interferon g1 , pegylated interferon g2, GS-9620 (4-amino-2-butoxy-8-[[3-(pyrrolidin-1- ylmethyl)phenyl]methyl]-5,7-dihydropteridin-6-one), dehydroepiandrosterone, androstenediol or androstenetrial; interferon alphacon-1 (pegylated and unpegylated), ribavirin; an HBV RNA replication inhibitor; an HBV therapeutic vaccine; an HBV prophylactic vaccine; lamivudine (3TC); entecavir (ETV); tenofovir diisoproxil fumarate (TDF); telbivudine (LdT); adefovir dipivoxil; and an HBV antibody therapy (monoclonal or polyclonal).
[00117] In one embodiment, there is provided a method of treating an HBV infection in a human by administering to the human a therapeutically effective amount of a compound of Formula (I) as described herein or a pharmaceutically acceptable salt thereof. In one embodiment, there is provided a method of treating an HBV infection, in a human having or at risk of having the HBV infection, by administering to the human a therapeutically effective amount of a compound of Formula (I) described herein such as a compound of Table 1 , or a pharmaceutically acceptable salt or prodrug thereof, and optionally administering a therapeutically effective amount of a second therapeutically active compound, wherein the second therapeutically active agent is selected from: an HBV agent, an HCV agent, a chemotherapeutic agent, an antibiotic, an analgesic, a nonsteroidal anti-inflammatory (NSAID) agent, an antifungal agent, an antiparasitic agent, an anti-nausea agent, an anti-diarrheal agent, or an immunosuppressant agent.
[00118] In one embodiment, there is provided a method of treating an HBV infection in a human by administering to the human a therapeutically effective amount of a compound of Formula (I) as described herein or a pharmaceutically acceptable salt thereof. In one embodiment, this invention is directed to a method of treating an HBV infection in a human having or at risk of having the HBV infection, comprising
administering to the human a therapeutically effective amount of a compound of Formula (I) as described herein, or a pharmaceutically acceptable salt thereof.
[00119]Another embodiment provides a method for reducing an amount of HBV DNA and an amount of HBV antigen in a mammal infected with a Hepatitis B virus, the method comprising administering to the mammal a therapeutically effective amount of a pharmaceutical composition comprising a compound of Formula (I) as described herein or a pharmaceutically acceptable salt thereof, so as to reduce the Hepatitis B virus infection and the Hepatitis B antigen, compared to the amount of HBV DNA and the amount of HBV antigen in the mammal before treatment. In some embodiments, the mammal may be human, and the Hepatitis B virus may be a human Hepatitis B virus. More particularly, the human Hepatitis B virus may be any of the human geographical genotypes: A (Northwest Europe, North America, Central America); B (Indonesia, China, Vietnam); C (East Asia, Korea, China, Japan, Polynesia, Vietnam); D (Mediterranean area, Middle East, India); E (Africa); F (Native Americans, Polynesia); G (United States, France); or H (Central America).
[00120] In particular embodiments, a method is provided for reducing an amount of HBV DNA and an amount of HBV antigen in a mammal infected with a Hepatitis B virus, the method comprising administering a therapeutically effective amount of a therapeutic agent, wherein the therapeutic agent is a compound of Formula (I) as described herein or a pharmaceutically acceptable salt thereof, so as to reduce the hepatitis B virus infection and the hepatitis B antigen, compared to the amount of hepatitis B virus and the amount of HBV antigen in the mammal before treatment, and wherein the amount of hepatitis B virus is reduced 90% compared to the amount before administration of the therapeutic agent. In related methods, the HBV antigen may be HBsAg or may be HBeAg, and more particularly, the amount of HBV antigen may be sufficiently reduced to result in seroconversion, defined as serum HBeAg absence plus serum HBeAb presence if monitoring HBeAg as the determinant for seroconversion, or defined as serum HBsAg absence if monitoring HBsAg as the determinant for seroconversion, as determined by currently available detection limits of commercial ELISA systems.
[00121 ] In certain embodiments there is provided a method for reducing an amount of HBV DNA and an amount of HBV antigen in a mammal infected with a hepatitis B virus by administering a therapeutically active amount of a first therapeutic agent that is a compound of Formula (I) as described, and administering a second therapeutic agent, wherein the first therapeutic agent, the second therapeutic agent are administered either together in a single formulation, or administered separately in different formulations, and wherein the administration of the first therapeutic agent and the second therapeutic agent is done concomitantly, or in series. In particular related embodiments, the method as described further comprises administering a therapeutically effective amount of an optional third therapeutic agent, wherein the optional third therapeutic agent is selected from an HBV agent, an HCV agent, a chemotherapeutic agent, an antibiotic, an analgesic, a non-steroidal anti-inflammatory (NSAID) agent, an antifungal agent, an antiparasitic agent, an anti-nausea agent, an anti-diarrheal agent, and an immunosuppressant agent. In more particular related embodiments, the optional third therapeutic agent is selected from interferon alpha-2b; interferon alpha-2a; and interferon alphacon-1 (pegylated and unpegylated); ribavirin; an HBV RNA replication inhibitor; an HBV antigen production inhibitor; an HBV therapeutic vaccine; an HBV prophylactic vaccine; lamivudine (3TC); entecavir (ETV); tenofovir diisoproxil fumarate (TDF); telbivudine (LdT); adefovir; or an HBV antibody therapy (monoclonal or polyclonal); an HCV agent selected from an HCV RNA replication inhibitor (e.g., ViroPharma's VP50406 series); an HCV therapeutic vaccine; an HCV protease inhibitor; an HCV helicase inhibitor; or an HCV antibody therapy (monoclonal or polyclonal).
[00122]Another embodiment provides a method for promoting seroconversion of a hepatitis B virus in a mammal infected with HBV, the method comprising administering a therapeutically effective amount of a compound of Formula (I) as described herein to a mammal infected with hepatitis B; the method comprising monitoring for the presence of HBeAg plus HBeAb in a serum sample of the mammal, or monitoring for the presence of HBsAg in a serum sample of the mammal, such that the absence of HBeAg plus the presence of HBeAb in the serum sample if monitoring HBeAg as the determinant for seroconversion, or the absence of HBsAg in the serum sample if monitoring HBsAg as the determinant for seroconversion, as determined by current detection limits of commercial ELISA systems, is indication of seroconversion in the mammal.
[00123] In particular embodiments described herein for treating a hepatitis B virus, hepatitis B virus infection, a hepatitis B virus-related condition or a hepatitis B/hepatitis D virus coinfection in a mammal in need of such treatment, the method comprises administering a first therapeutic agent that is a compound of Formula (I) as described herein or a pharmaceutically acceptable salt or prodrug thereof, and a second therapeutic agents, wherein the second therapeutic agents may be an anti-inflammatory agent; a chemotherapeutic agent or anti-infection agent; an HBV agent; an HCV agent; an antibiotic; an analgesic; a non-steroidal anti-inflammatory (NSAID) agent; an antifungal agent; an antiparasitic agent; an anti-nausea agent; an anti-diarrheal agent; an immunomodulatory; or an immunosuppressant agent.
[00124]Certain embodiments provide the use of a combination of a compound of Formula (I) as described herein, suitably a compound of Table 1 , or pharmaceutically acceptable salt or prodrug thereof and a second therapeutic agents or compositions as described herein in the manufacture of a medicament for treating, ameliorating, delaying or preventing an HBV-related disease, disorder or condition in an animal.
[00125]Certain embodiments provide the use of a combination of a first therapeutic agent that is a compound of Formula (I) as described herein or a
pharmaceutically acceptable salt or prodrug thereof, and a second therapeutic agent or composition as described herein in the manufacture of a medicament for treating, ameliorating, delaying or preventing liver disease in an animal.
[00126]For any of the embodiments described herein which provide a method of treating a hepatitis B virus infection or hepatitis B virus-related condition, a method for reducing an amount of HBV DNA and an amount of HBV antigen in a mammal infected with a hepatitis B virus, or a method for promoting seroconversion of a hepatitis B virus in a mammal infected with HBV, wherein the method comprises administration of a therapeutically effective amount of a compound of Formula (I) as described herein or a pharmaceutically acceptable salt or prodrug thereof, including a therapeutically effective amount of a compound from Table 1 or pharmaceutically acceptable salt thereof, whether alone or in combination, and whether present in a pharmaceutical formulation or simply present in a diluent, the administration may be oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal or intratracheal administration.
[00127] In pharmaceutical dosage forms, the administration of a compound of Formula (I) as described herein, including a compound of Table 1 , may be done alone, or in appropriate association, as well as in combination with other pharmaceutically active agents, such as other HBV agents known in the field. The following methods and excipients are merely exemplary and are in no way limiting. The STING compounds of Formula (I) as described herein and in Table 1 , or pharmaceutically acceptable salt thereof, can be administered in combination with a conventional pharmaceutical carrier, excipient or the like (e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and the like). If desired, the pharmaceutical composition can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like (e.g., sodium acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, and the like).
[00128]For oral preparations, the compounds described herein can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents. [00129]Furthermore, the compounds described herein can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water- soluble bases, and can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
[00130] In general, the compounds of Formula (I) as described herein, including the compounds of Table 1 , or a pharmaceutically acceptable salt or prodrug thereof, for use in combination with the therapeutic agents and HBV agents described herein, will be administered in a therapeutically effective amount by any of the accepted modes of administration for therapeutic agents that serve similar utilities.
[00131]Depending on the nature of the specific composition of the combination of a STING compound or pharmaceutically acceptable salt or prodrug thereof as described herein, and a second therapeutic agent, the composition may be administered to the host using any convenient means capable of resulting in the desired reduction of target viral transcripts, viral genome amount or load in the target cell. Thus, a compound as described herein or pharmaceutically acceptable salt or prodrug thereof, can be incorporated into a variety of formulations for therapeutic administration. More particularly, the compounds that as described herein or a pharmaceutically acceptable salt or prodrug thereof, and combinations thereof described herein, can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents. In certain embodiments, compounds described herein or a pharmaceutically acceptable salt of prodrug thereof, and combinations thereof described herein, may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. In certain embodiments, administration of a compound as described herein or a pharmaceutically acceptable salt or prodrug thereof, and combinations thereof described herein, may be achieved in various ways, including, but not limited to, oral, buccal, rectal, parenteral, intraperitoneal, intravenous, intradermal, transdermal or intratracheal administration, and also administration may be via microneedle patch delivery or topical formulation.
[00132] For oral preparations, the compound as described herein or a
pharmaceutically acceptable salt or prodrug thereof described herein can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
[00133]The compounds of Formula (I) as described herein, including compounds of Table 1 , or a pharmaceutically acceptable salt or prodrug thereof as described herein, can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. Liquid pharmaceutically administrable compositions of STING compounds as described herein can, for example, be prepared by dissolving, dispersing, etc. at least compound as described herein and optional pharmaceutical adjuvants in a carrier (e.g., water, saline, aqueous dextrose, glycerol, glycols, ethanol or the like) to form a solution or suspension. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, as emulsions, or in solid forms suitable for dissolution or suspension in liquid prior to injection. The percentage of compound contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject. However, percentages of active ingredient of 0.01 % to 10% in solution are employable, and will be higher if the composition is a solid which will be subsequently diluted to the above percentages. In certain embodiments, the composition will comprise from about 0.2 to 2% of the active agent in solution.
[00134]The actual amount of the therapeutic compound(s) - i.e. the therapeutic ingredient(s), will depend upon numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compounds used, the route and form of administration, and other factors. The pharmaceutical composition can be administered more than once a day, such as once or twice a day. In particular embodiments, the HBV pharmaceutical formulation will be administered once or twice per week over the course of 24, 36, or 48 weeks or longer, as needed, to effectively treat the HBV infection, including to reduce viral load, to reduce viral antigens, produce seroconversion, bolster the subjects immune response and/or decrease HBV DNA levels and normalize alanine transferase (ALT) levels.
[00135]Unit dosage forms for oral or rectal administration of a compound as described herein or a pharmaceutically acceptable salt thereof, or combinations thereof described herein, such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration of a compound as described herein or a pharmaceutically acceptable salt thereof, or combinations thereof described herein, may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
[00136]The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
[00137]The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
[00138]Those of skill in the art will readily appreciate that dose levels can vary as a function of the specific therapeutic agent, the nature of the delivery vehicle, and the like. Preferred dosages for a given therapeutic agent, including for a specific compound as described herein including in combination with a second therapeutic agent, are readily determinable by those of skill in the art by a variety of means.
[00139]Examples of delivery methods for administration of the therapeutic agents described herein, as well as details of pharmaceutical formulations, solvates, hydrates, salts and prodrugs such as esters are well-known to one of skill in the art and are described in the literature.
[00140]Ultimately, the choice of formulation and dosage depends on various factors such as the mode of drug administration and bioavailability of the drug substance.
[00141]The methods of the present invention will work in any mammalian cell, where representative mammal cells of interest include, but are not limited to cells of: ungulates or hooved animals, e.g., cattle, goats, pigs, sheep, etc.; rodents, e.g., hamsters, mice, rats, etc.; lagomorphs, e.g., rabbits; primates, e.g., monkeys, baboons, humans, etc.; and the like.
[00142]Pharmaceutical formulations of compounds described herein for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal, may comprises at least one STING compound of Formula (I) as described herein or pharmaceutically acceptable salt or prodrug thereof, including a compound from Table 1 or a pharmaceutically acceptable salt thereof, and a second therapeutic agent as described herein, as an active ingredient. It should be understood that the pharmaceutical composition may include a diluent, and optionally may comprises a pharmaceutical carrier; and that the pharmaceutical composition optionally may comprise further compounds, such as chemotherapeutic compounds, anti-inflammatory compounds, antiviral compounds, analgesics, NSAIDs, narcotics, antibiotics, antifungal compounds, antiparasitic compounds, and/or immuno-modulating compounds.
[00143] The compounds described herein for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal can be used "as is" or in the form of a variety of pharmaceutically acceptable salts or prodrugs. As used herein, the term "pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the herein-identified compounds and exhibit minimal undesired toxicological effects. Non-limiting examples of such salts can be formed with organic amino acid and base addition salts formed with metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and the like, or with a cation formed from ammonia, N,N-dibenzylethylene-diamine, D-glucosamine, tetraethylammonium, or ethylenediamine.
[00144] In a particular embodiment of the invention, the compound that as described herein may be in the form of a pro-drug. The prodrug may include a cleavable moiety connected to the compound, by a cleavable bond. The cleavable moiety may be a moiety that directs the compound as described herein to the liver. Examples of such liver-targeting cleavable moieties include carbohydrate moieties, including mannose or galactose moieties, or N-acetylmannosamine or more particularly N-acetylgalactosamine (GalNAc) moieties. In certain embodiments there may be a carbohydrate cluster or GalNac cluster as the cleavable moiety that is connected to a compound of Formula (I) as described herein, suitably a compound of Table 1 by a cleavable bond. A GalNAc cluster may include 2 GalNAc moieties, 3 GalNac moieties or 4 GalNac moieties.
[00145]0ther moieties and techniques that can be used for carrier-mediated delivery to target compounds that as described herein to the liver include the use of cholesterol, polyethyleneglycol (PEG), dextran, lipid nanoparticles, masked endosomal compounds that incorporate both targeting iigands and endosomal escape moieties, for exampie by conjugating poiy(vinyiether) to GaiNAc and PEG and then iinking the
GaiNAc-PEG to the compound to be delivered through a cleavable bond, such as a disulfide bond. A discussion of techniques for targeting siRNAs to the liver, which one of skiii in the art would understand is applicable for targeting compounds as described herein lo the liver is found in Nature Reviews Genetics 16, 543-553 (2015) (A Wittrup and J Ueberma), the entire contents of which are incorporated by reference herein.
Delivery of STING agonists of Formula (I) as described herein, suitably a compound of Table 1 , to tissue may also be enhanced by carrier-mediated delivery including, but not limited to, cationic liposomes, cyclodextrins, porphyrin derivatives, branched chain dendrimers, polyethylenimine polymers, nanoparticles and microspheres (Dass, C R. J Pharm Pharmacol 2002; 54(1 ):3-27), incorporated by reference herein. Other discussions of targeting drug delivery to the liver can be found in“Efficient Hepatic Delivery of Drugs: Novel Strategies and Their Significance’ BioMed Research international vol. 2013 (2013), Article ID 328184, 20 pages, by N. ishra et a!., the entire contents of which are incorporated by reference herein.
[00146]Pharmaceutical compositions of the compounds described herein for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal include, but are not limited to, solutions, emulsions, and liposome-containing
formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self- emulsifying semisolids. Pharmaceutical formulations of compounds described herein for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. The compounds described herein for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels and suppositories.
[00147]The compounds described herein for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers. The compounds described herein for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal may also be conjugated to active drug substances, for example, aspirin, ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic, and an anticancer drug substance. In addition, the compounds described herein for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal, may be administered as a combination therapy together with, or separately in conjunction with, drug substances useful for treating cancer, inflammation, pain, bacterial, fungal and/or parasitic infections, alcoholism, substance abuse, diabetes, as well as with drug substances useful for treating other viral infections such as HIV, HCV and the like.
[00148]There are two general types of hepatitis B viral infections: acute, and chronic. Subjects who have experienced a HBV infection may also recover and become non-symptomatic carriers. Acute hepatitis B results when a person exposed to the hepatitis B virus begins to develop the signs and symptoms of viral hepatitis. This period of time, called the incubation period, is an average of 90 days, but could be as short as 45 days or as long as 6 months. For most people this infection will cause mild to moderate discomfort but will go away by itself because of the body's immune response succeeds in fighting the virus. However, some people, particularly those with
compromised immune systems, such as persons suffering from AIDS, undergoing chemotherapy, taking immunosuppressant drugs, or taking steroids, have very serious problems as a result of the acute HBV infection, and go on to develop more severe conditions such as fulminant liver failure.
[00149]Chronic hepatitis B occurs when a person initially suffers from an acute infection but is then unable to fight off the infection. Whether the disease becomes chronic or completely resolves depends mostly on the age of the infected person. About 90% of infants infected at birth will progress to chronic disease. However, as a person ages, the risk of chronic infection decreases such that between 20%-50% of children and less than 10% of older children or adults will progress from acute to chronic infection. Chronic HBV infections are the primary treatment goal for embodiments of the present invention, although compounds of the present invention are also capable of treating HBV- related conditions, such as inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, and more.
[00150]There is a 95% chance that a child will develop immune tolerance and the condition will progress to chronic hepatitis; whereas if the patient contracts HBV as an adult, there is only a 5% chance that the condition will become chronic.
[00151] There are four phases of chronic HBV infection. The first phase is the immune tolerant phase, where minimal fibrosis and inflammation of the liver is exhibited, coupled with high HBV DNA and normal alanine aminotransferase (ALT) levels. The second phase is an immune clearance phase, where active inflammation of the liver is observable upon histology, which may also include fluctuating levels of ALT activity and fluctuating levels of HBV DNA. A third inactive carrier state phase may follow, in which infected subjects may exhibit mild hepatitis and minimal fibrosis upon histology. A fourth is also reported, termed reactivation of HBV. This last phase is characterized by active inflammation of the liver, as observed by biopsy, despite hepatitis B e antigen (HBeAg) negativity and anti-HBeAg positivity.
[00152]Chronic HBV disease is of several types. The first, known as HBeAg positive, or“wild type” HBV infection, is characterized as anti-HBeAg negative and exhibiting quantities of HBV DNA > 20,000 lU/mL (> 105 copies/mL). A second, known as HBeAg negative, or“mutant core” HBV infection, is characterized as anti-HBeAg positive and exhibiting HBV DNA >2,000 lU/mL (> 104 copies/mL). These two types of chronic HBV infection have historically evaded therapeutic approaches.
[00153]Patients suffering from acute HBV infection produce a vigorous, polyclonal, and multispecific Cytotoxic T Lymphocyte (CTL) response to viral antigens, whereas chronically infected patients have a weak or undetectable CTL response.
Hepatitis occurs when a weak HBV-specific immune response is activated that is sufficiently strong to destroy HBV-infected hepatocytes. Little is known regarding the mechanism responsible for T cell hyporesponsiveness or tolerance, but it is thought that it may involve the following mechanisms: negative selection (neonates); immunological ignorance; peripheral anergy or lack of co-stimulatory molecules, e.g. regulatory T cells (Tregs); exhaustion (up-regulation of PD-1). A common factor for all these suggested mechanisms is high levels of antigen persisting in the patient.
[00154]The HBV antigen HBeAg is a secreted, non-particulate form of HBV core protein. HBV antigens HBeAg and HBeAg share primary amino acid sequences, so show cross-reactivity at the T cell level. HBeAg is not required for viral assembly or replication, although studies suggest they may be required for establishment of chronic infection.
[00155]Neonatal infection with HBeAg-negative mutant often results in fulminant acute rather than chronic HBV infection (Terezawa et al (1991) Pediatr. Res. 29:5), whereas infection of young woodchucks with WHeAg-negative mutant results in a much lower rate of chronic WHV infection (Cote et al (2000) Hepatology 31 :190). HBeAg may possibly function as a toleragen by inactivating core specific T cells through deletion or clonal anergy (Milich et al (1998) J. Immunol. 160:8102). There is a positive correlation between reduction of HBV viral load and antigens, and a decrease of expression, by T cells, of the inhibitory receptor programmed death-1 (PD-1 ; also known as PDCD1), a negative regulator of activated T cells, upon antiviral therapy and HBeAg seroconversion (Evans et al (2008) Hepatology 48:759). [00156]HBV surface antigen, or HBsAg, is the envelope protein of infectious HBV viral particles but is also secreted as a non-infectious particle with serum levels 1000-fold higher than HBV viral particles. The serum levels of HBsAg in an infected person or animal can be as high as 1000 pg/mL (Kann and Gehrlich (1998) Topley & Wilson’s Microbiology and Microbial Infections, 9th ed. 745). In acute HBV infections, the half-life of HBsAg in the serum, or serum t½, is 8.3 days (Chulanov et al (2003) J. Med. Virol. 69: 313). Internalization of HBsAg by myeloid dendritic cells inhibits up-regulation of costimulatory molecules (i.e. B7) and inhibits T cell stimulatory capacity (den Brouw et al (2008) Immunology 126:280), and dendritic cells from chronically infected patients also show deficits in expression of co-stimulatory molecules, secretion of IL-12, and stimulation of T cells in the presence of HBsAg (Zheng et al (2004) J. Viral Hepatitis 1 1 :217).
[00157]HBsAg specific CD8 cells from CHB patients show altered tetramer binding. These CD8 cells are not anergic but may have TCR topology that confers partial tolerance or ignorance (Reignat et al (2002) J. Exp. Med. 195:1089). Moreover, reduction in serum HBsAg > 1 log at week 24 has a high predictive value (92%) for sustained virological response (SVR - defined as nondetectable HBV DNA by PCR at 1 year after treatment) during Peg-IFNa2a therapy (Moucari et al (2009) Hepatology 49:1 151 ).
[00158]Due to overlapping transmission routes, many people have been exposed to both hepatitis B virus (HBV) and hepatitis C virus (HCV), and a smaller proportion are chronically infected with both viruses, especially in regions such as Asia where HBV is endemic. Estimates suggest that up to 10% of people with HCV may also have HBV, while perhaps 20% of people with HBV are coinfected with HCV. However, treatment of hepatitis B or hepatitis B in HBV-HCV coinfected individuals has not been well studied.
[00159]Treatment is complicated by the fact that HCV and HBV appear to inhibit each other's replication (though not all studied have observed this interaction). Therefore, treatment that fully suppresses HBV could potentially allow HCV to re-emerge, or vice versa.
[00160]Therefore, use of compounds of Formula (I) as described herein, suitably a compound of Table 1 , or a pharmaceutically acceptable salt or prodrug thereof, in combination with a second therapeutic agent that may be a therapeutic agent as described herein, including an HVC agent as described herein, for preventing, ameliorating or treating liver disease, or symptoms thereof may advantageously be used for treating humans infected with both HBV and HCV. Exemplary treatment options for hepatitis C (HCV) include interferons, e.g., interferon alpha-2b, interferon alpha-2a, and interferon alphacon-1. Less frequent interferon dosing can be achieved using pegylated interferon (interferon attached to a polyethylene glycol moiety which significantly improves its pharmacokinetic profile). Combination therapy with interferon alpha-2b (pegylated and unpegylated) and ribavirin has also been shown to be efficacious for some patient populations. Other agents currently being developed include HCV RNA replication inhibitors (e.g., ViroPharma's VP50406 series), HCV antisense agents, HCV therapeutic vaccines, HCV protease inhibitors, HCV helicase inhibitors and HCV antibody therapy (monoclonal or polyclonal).
[00161 ] In another embodiment, a STING compound of Formula (I) as described herein, suitably a compound of Table 1 , or a pharmaceutically acceptable salt or prodrug thereof as described herein for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal may be administered to a human subject infected with HBV, in combination with a second therapeutic agent, wherein the second therapeutic agent may be administered in the same drug formulation as the STING compound, or may be administered in a separate formulation. The second therapeutic agent may also be administered simultaneously with an optional third therapeutic agent, or may be administered separately, so that a dose of each of the STING compound of Formula (I) as described herein, suitably a compound of Table 1 , or a pharmaceutically acceptable salt thereof, the second therapeutic agent, and the optional third therapeutic agent overlap, in time, within the subject’s body. In related embodiments, the second and optional third therapeutic agent may be therapeutic agent as described herein, including an HBV agent selected from a compound described in WO2019/069293, thymosin a1 , interferon b-1 a, interferon b-1 b, interferon g-1 b, interferon g1 , interferon g2, interferon g3, interferon a- 2a, interferon a-2b, pegylated interferon p-2a, pegylated interferon p-2b, pegylated interferon g1 , pegylated interferon g2, GS-9620 (4-amino-2-butoxy-8-[[3-(pyrrolidin-1- ylmethyl)phenyl]methyl]-5,7-dihydropteridin-6-one), dehydroepiandrosterone, androstenediol or androstenetrial; interferon alphacon-1 (pegylated and unpegylated), ribavirin; an HBV RNA replication inhibitor; an HBV therapeutic vaccine; an HBV prophylactic vaccine; lamivudine (3TC); entecavir (ETV); tenofovir diisoproxil fumarate (TDF); telbivudine (LdT); adefovir dipivoxil; and an HBV antibody therapy (monoclonal or polyclonal).
[00162]Use of pharmaceutical compositions of the present invention
encompassing compounds as described herein or a pharmaceutically acceptable salt or prodrug thereof in combination with a second therapeutic agent as described herein, wherein the optional second therapeutic agent may be an HBV agent described herein, in use with an optional third therapeutic agent that may be an HBV agent described herein, and in combination with an optional fourth therapeutic agent that may be an HBV agent as described herein, will thus offer patients suffering from chronic HBV infections an improved therapeutic treatment that may result in complete seroconversion, ultimately leading to a complete cure for chronic HBV infections. Use of compounds as described herein or a pharmaceutically acceptable salt or prodrug thereof and a second therapeutic agent, and optionally with a third therapeutic agent, and optionally with a fourth therapeutic agent, is an approach that is also expected to reduce HBV-related liver conditions, such as cirrhosis, hepatocellular carcinoma (HCC), and fibrosis and inflammation, resulting in the need for fewer liver transplants and helping to prevent deaths from liver disease.
[00163] In addition, beyond an improved reduction in viral load, use of the compounds as described herein will provide other benefits. HBeAg is believed to have an immunosuppressive role, and HBsAg is thought to contribute to T cell exhaustion.
Therefore, treatment with a compound as described herein or a pharmaceutically acceptable salt or prodrug thereof in combination with a second therapeutic agent, in combination with an optional third therapeutic agent, in combination with an optional fourth therapeutic agent, will bolster a patient’s immune response by reducing HBeAg and HBsAg levels, allowing the immune system to better control infection. The second, third and fourth therapeutic agent may be an HBV agent, selected from a compound described in WO2019/069293, thymosin a1 , interferon b-1 a, interferon b-1 b, interferon g-1 b, interferon g1 , interferon g2, interferon g3, interferon a-2a, interferon a-2b, pegylated interferon p-2a, pegylated interferon p-2b, pegylated interferon g1 , pegylated interferon g2, GS-9620 (4-amino-2-butoxy-8-[[3-(pyrrolidin-1 -ylmethyl)phenyl]methyl]-5,7- dihydropteridin-6-one), dehydroepiandrosterone, androstenediol or androstenetrial;
interferon alphacon-1 (pegylated and unpegylated), ribavirin; an HBV RNA replication inhibitor; an HBV therapeutic vaccine; an HBV prophylactic vaccine; lamivudine (3TC); entecavir (ETV); tenofovir diisoproxil fumarate (TDF); telbivudine (LdT); adefovir dipivoxil; and an HBV antibody therapy (monoclonal or polyclonal). It is also expected that reduction of HBV antigen presentation in the liver will help to minimize the extent of HBV infection flare-ups, a result of the immune system restoration, all of which will increase the chances of a complete cure of chronic HBV infection.
[00164] An additional list of therapeutic agents that may be useful when used in combination with a compound described herein or a pharmaceutically acceptable salt or prodrug thereof, are indicated in Table 3.
[00165]An embodiment of the invention provides a method for treating hepatitis B virus infection and reducing hepatitis B viral genome in a target cell of a mammal is provided, comprising administering a therapeutically effective amount of a STING compound as described herein or a pharmaceutically acceptable salt or prodrug thereof, administering a therapeutically effective amount of a second therapeutic agent, and administering a therapeutically effective amount of an optional third therapeutic agent and optional fourth therapeutic agent which may each be an HBV agent, thereby treating the hepatitis B virus infection. Also provided are pharmaceutical compositions for use in practicing the subject methods. Embodiments of the invention comprising use of the compounds as described herein or a pharmaceutically acceptable salt or prodrug thereof and a second, therapeutic agent, an optional third therapeutic agent and an optional fourth therapeutic agent as described herein finds use in a variety of applications, including the treatment of subjects suffering from a viral mediated disease condition, e.g., an HBV mediated disease condition. Such conditions include, but are not limited to, fibrosis and inflammation of the liver, cirrhosis of the liver, and hepatocellular cancer (liver cancer).
[00166] In an embodiment there is provided, use of a first therapeutic agent that is a STING compound of Formula (I) as described herein, suitably a compound of Table 1 , or a pharmaceutically acceptable salt thereof, in combination with a second therapeutic agent that is an HBV agent , in combination with an optional third agent that is an HBV agent, in combination with an optional fourth therapeutic agent that is an HBV agent for use in combination for preventing, ameliorating or treating liver disease, or symptoms thereof in an animal.
[00167] In another embodiment, there is provided a method of treating an HBV infection in a mammal, including a human, wherein treating comprises administering a therapeutically effective dose of a first therapeutic agent that is a compound as described herein or a pharmaceutically acceptable salt or prodrug thereof, administering a therapeutically effective amount of a second therapeutic agent that is an HBV agent, and administering a therapeutically effective amount of an optional third therapeutic agent that is an HBV agent, and administering a therapeutically effective amount of an optional fourth therapeutic agent that is an HBV agent so as to treat the HBV infection and promote seroconversion in the mammal, including a human. The effective dose or each therapeutic agent may range from 0.01 mg/kg body weight, to approximately 100 mg/kg body weight, administered once or twice weekly, for 24, 36 or 48 weeks, or longer, as determined by a physician. Efficacy of treatment may be determined using quantification of viral load or other evidence of infection, such as through measurement of HBeAg, HBsAg, HBV DNA levels, ALT activity levels, serum HBV levels, and the like, thereby allowing adjustment of treatment dose, treatment frequency, and treatment length. [00168] EXAMPLES
[00169] GENERAL SYNTHETIC METHODS
[00170] The compounds of this invention may be prepared using the synthetic procedures illustrated in the reaction schemes below, which can be readily adapted to prepare other compounds of the invention by drawing on the knowledge of a skilled organic chemist. The syntheses provided in these schemes are applicable for producing compounds of the invention having a variety of R groups employing appropriate precursors, which are suitably protected if needed, to achieve compatibility with the reactions outline herein. Subsequent deprotection, where needed, affords compounds of the nature generally disclosed. Intermediately may also be present as salts.
[00171] The naming program used for chemical compounds was ChemDraw® Professional 16.0.
[00172] GENERAL SYNTHETIC SCHEME
Scheme 1
Figure imgf000049_0001
[00173]The compounds exemplified herein can be prepared by the general sequence outlined in Scheme 1. T reatment of amine A with 1 -fluoro-2-nitrobenzene B under basic conditions affords intermediate C. Notably, inorganic bases (K2C03), organic bases (DIPEA), aprotic solvents (DMF) and protic solvents (MeOH, iPrOH) have all proven effective for this transformation. Subjection of intermediate C to acidic conditions (HCI, TEA) unmasks the pendant amine moiety (Note: in the syntheses of a number of exemplars, the protection of an amine functionality as the corresponding fe/ -butyl carbamate (Boc) prior to the formation of D was unnecessary). With amine D in hand, treatment with 1 -flu oro-2-nitro benzene E under basic conditions generates intermediate F. The nitro functionalities of F can be reduced to the corresponding anilines by palladium-catalyzed hydrogenation (10% Pd/C, H2), sodium hydrosulfite (sodium dithionite) in the presence of ammonium hydroxide, or zinc under acidic conditions (acetic acid). Treatment of bis-aniline G with 1 -ethyl-3-methyl-1 /-/-pyrazole-5-carbonyl isothiocyanate (i) followed by EDC/DIPEA assembles bis-benzimidazole H. In the ultimate step, methylation under standard conditions (Mel, Cs2C03) provides targeted compounds of generic structure J.
[00174] INTERMEDIATES
[00175] Intermediate i. 1-Ethyl-3-methyl-1 H-pyrazole-5-carbonyl
isothiocyanate
Figure imgf000050_0001
[00176] 1 -Ethyl-3-methyl-1 H-pyrazole-5-carbonyl chloride
Figure imgf000050_0002
[00177]To a suspension of 1 -ethyl-3-methyl-1 /-/-pyrazole-5-carboxylic acid (14.7 g, 95 mmol) in dichloromethane (DCM) (300 ml_) under an atmosphere of N2 was added A/,A/-dimethylformamide (0.185 ml_, 2.38 mmol), followed by oxalyl chloride (9.18 ml_, 105 mmol) dropwise. The mixture was stirred at RT for 2 h. Following this duration, the mixture was concentrated to dryness and the crude title compound (17.18 g) was carried forward without any further purification.
[00178]1 -Ethyl-3-methyl-1 H-pyrazole-5-carbonyl isothiocyanate (i)
Figure imgf000050_0003
[00179]To a solution of 1 -ethyl-3-methyl-1 /-/-pyrazole-5-carbonyl chloride (17.2 g, 96 mmol) in acetone (300 ml_) under an atmosphere of N2 at 0 °C was added potassium thiocyanate (11.2 g, 115 mmol) in equal portions. The mixture was stirred at RT for 4 h. Following this duration, the mixture was concentrated to dryness and the crude product was taken up in hexanes (100 ml_) and filtered through Celite. The solid was washed with hexanes (2x) and filtered. The combined filtrate was concentrated under vacuum to afford the title compound as an orange oil (16.1 g, 82 mmol, 86% yield). 1H NMR (400 MHz, CHLOROFORM-d) d ppm 1.38 - 1.47 (m, 3 H) 2.29 - 2.35 (m, 3 H) 4.52 (q, J= 7.25 Hz, 2 H) 6.75 (s, 1 H).
[00180] COMPOUND EXAMPLES
[00181]Example 1. N,LG-((2E,2Έ)-((E)^iiί-2-bhb-1 ,4-diyl)bis(3-methyl-1 ,3- dihydro-2H-benzo[d]imidazole-1 -yl-2-ylidene))bis(1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamide)
Figure imgf000051_0001
[00182]ferf-Butyl (£)-(4-((2-nitrophenyl)amino)but-2-en-1 -yl)carbamate
Figure imgf000051_0002
[00183]To a solution of fe/ -butyl (E)-(4-aminobut-2-en-1-yl)carbamate (1 g, 5.4 mmol) in isopropanol (10.7 ml) was added 1-fluoro-2-nitro benzene (0.57 ml, 5.4 mmol) and DIPEA (2.0 ml, 1 1.3 mmol). The reaction vessel was capped and heated to 70 °C. After stirring for ~1 hour, additional fe/ -butyl (E)-(4-aminobut-2-en-1-yl)carbamate (500 mg, 2.68 mmol) and DIPEA (0.938 ml, 5.37 mmol) were added and the reaction temperature was increased to 80 °C. After 3 h, anhydrous sodium bicarbonate (1 g, 11.9 mmol) was added and the mixture was stirred at 80 °C. After 5 h, the mixture cooled to room temperature, giving a precipitate. The solids were filtered off and the filtrate was concentrated via a nitrogen blowdown unit to give the title compound as an orange solid. The material was carried forward without purification. LC-MS m/z 330.1 (M+Na)+, 1 .14 min (ret. time); Ή NMR (400 MHz, CDCI3) d ppm 8.22 (dd, J=8.62, 1 .52 Hz, 1 H) 8.18 (br. s„ 1 H) 7.42 - 7.51 (m, 1 H) 6.85 (d, J=8.62 Hz, 1 H) 6.71 (ddd, J=8.49, 7.10, 1 .14 Hz, 1 H) 5.70 - 5.88 (m, 3 H) 4.64 (br. s„ 1 H) 3.95 - 4.05 (m, 2 H) 3.69 - 3.87 (m, 3 H) 1 .37 - 1 .60 (m, 9 H).
[00184](E)-N7-(2-Nitrophenyl)but-2-ene-1 ,4-diamine
Figure imgf000052_0001
[00185]fe/?-Butyl (E)-(4-((2-nitrophenyl)amino)but-2-en-1 -yl)carbamate (430 mg,
1 .4 mmol) was dissolved in dichloromethane (DCM) (7.0 ml_). To this clear orange solution was added HCI (1 .8 ml_, 7.0 mmol) as a 4 M solution in dioxane. The mixture was allowed to stir at room temperature. After 18 h, the volatiles were removed via a nitrogen blowdown unit, affording a yellow solid. This yellow solid was dried under vacuum to give the title compound (400 mg, 1 .4 mmol, 97% yield). LC-MS m/z 208.1 (M+H)+, 0.53 min (ret. time); Ή NMR (400 MHz, DMSO -d6) d ppm 8.28 (br. s., 2 H) 8.08 (dd, J=8.62, 1 .52 Hz, 2 H) 7.53 (ddd, J=8.68, 6.91 , 1 .65 Hz, 1 H) 6.98 (dd, J=8.74, 0.89 Hz, 1 H) 6.71 (ddd, J=8.43, 7.03, 1 .27 Hz, 1 H) 5.82 - 6.03 (m, 1 H) 5.63 - 5.82 (m, 1 H) 4.07 (d, J=3.80 Hz, 1 H) 3.34 - 3.54 (m, 2 H).
[00186](E)-Ni,AM-bis(2-Nitrophenyl)but-2-ene-1 ,4-diamine
Figure imgf000052_0002
[00187](E)-A/f-(2-nitrophenyl)but-2-ene-1 ,4-diamine, 2Hydrochloride (395 mg, 1 .410 mmol) was dissolved in isopropanol (4.7 mL) and anhydrous sodium bicarbonate (415 mg, 4.9 mmol) was subsequently added. To this heterogenous yellow solution was added 1 -fluoro-2-nitrobenzene (0.15 mL, 1 .4 mmol). The reaction vessel was capped and the mixture was heated to 80 °C. After 4 h, additional 1 -fluoro-2-nitrobenzene (37 pL, 0.352 mmol) was added and the mixture was allowed to stir at 80 °C for approximately 14 h. Following this duration, the reaction mixture cooled to room temperature to give a precipitate, which was then filtered. The yellow solid was rinsed with isopropanol followed by water and dried in a vacuum oven for 4 hours at 50 °C to afford the title compound as a yellow/orange solid (517 mg, 1 .50 mmol, 106% yield). LC-MS m/z 329.1 (M+H)+, 1 .27 min (ret. time); Ή NMR (400 MHz, DMSO -d6) d ppm 8.28 (t, J=5.58 Hz, 2 H) 8.05 (dd, J=8.62, 1 .52 Hz, 2 H) 7.47 (td, J= 7.73, 1 .27 Hz, 2 H) 6.86 - 7.06 (m, 2 H) 6.68 (ddd, J=8.43, 7.03, 1 .01 Hz, 2 H) 5.77 (t, J=2.41 Hz, 2 H) 3.90 - 4.16 (m, 4 H). (benzene-1 ,2-diamine)
Figure imgf000053_0001
[00189](E)-A/1 ,A/4-bis(2-nitrophenyl)but-2-ene-1 ,4-diamine (517 mg, 1 .6 mmol) was dissolved in methanol (5 ml_) and acetic acid (1 ml_). To this yellow heterogenous solution was added zinc (721 mg, 1 1 .02 mmol) in equal portions. The heterogenous mixture was stirred at room temperature. After 15 minutes stirring at room temperature, the reaction was filtered through Celite and the filtrate was concentrated. The crude material was purified by silica gel chromatography (2-10% MeOH:DCM with NH3 as a modifier). The product-containing fractions were combined and concentrated to give the title compound as an off-white solid (92 mg, 0.336 mmol, 21 % yield). LC-MS mlz 269.3 (M+H)+, 0.47 min (ret. time); Ή NMR (400 MHz, DMSO -d6) d ppm 6.66 - 6.80 (m, 4 H) 6.52 - 6.66 (m, 4 H) 5.91 (dt, J=2.66, 1 .46 Hz, 2 H) 3.69 - 3.89 (m, 4 H).
[00190](£)-N,N'-(But-2-ene-1 ,4-diylbis(1 H-benzo[d]imidazole-1 ,2-diyl))bis(1 - ethyl-3-methyl-1 H-pyrazole-5-carboxamide)
Figure imgf000053_0002
[00191](E)-A/f,A/r-(but-2-ene-1 ,4-diyl)bis(benzene-1 ,2-diamine) (92 mg, 0.343 mmol) was dissolved in A/,A/-dimethylformamide (DMF) (3.4 ml_). The resulting light orange homogenous solution was cooled to 0 °C. After stirring for 5 minutes at 0 °C, 1 - ethyl-3-methyl-1 H-pyrazole-5-carbonyl isothiocyanate (0.50 ml_, 0.50 mmol) was added as a 1 M solution in 1 ,4-dioxane. The mixture was stirred at 0 °C. After 15 minutes, EDC (164 mg, 0.857 mmol) was added followed by DIPEA (0.30 ml_, 1 .71 mmol). The reaction mixture was heated to 45 °C. After 30 minutes, the reaction was cooled to room temperature. A 4:1 water/saturated aqueous NH4CI solution (20 ml_) was added and a light brown solid precipitated. The solid was filtered off and rinsed with water (2 x 10 ml_). The solid was dried in a vacuum oven (50 °C) for 4 hours to obtain the title compound as a light brown solid (130 mg, 0.216 mmol, 63% yield). LC-MS mlz 591 .3 (M+H)+, 1 .04 min (ret. time); 1 H NMR (400 MHz, DMSO -d6) d ppm 12.68 (br. s., 2 H) 7.50 (d, J= 7.35 Hz, 2 H) 7.39 (d, J= 7.86 Hz, 2 H) 6.93 - 7.30 (m, 4 H) 6.55 (s, 2 H) 5.90 (br. s., 2 H) 4.83 (br. s., 4 H) 4.54 (q, J=7.10 Hz, 4 H) 2.13 (s, 6 H) 1 .28 (t, J=7.10 Hz, 6 H).
[00192]N,Ar-((2E,2'£)-((£)-But-2-ene-1 ,4-diyl)bis(3-methyl-1 ,3-dihydro-2H- benzo[d]imidazole-1 -yl-2-ylidene))bis(1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide)
Figure imgf000054_0001
[00193](E)-/V,/V'-(but-2-ene-1 ,4-diylbis(1 /-/-benzo[d]imidazole-1 ,2-diyl))bis(1 -ethyl- 3-methyl-1 /-/-pyrazole-5-carboxamide) (1 10 mg, 0.186 mmol) was dissolved in N,N- dimethylformamide (DMF) (2 mL). To the resulting brown solution was added cesium carbonate (203 mg, 0.623 mmol) followed by methyl iodide (29.1 mI_, 0.466 mmol). The reaction vessel was capped and the mixture was stirred at room temperature. After stirring for 16 hours at room temperature, additional cesium carbonate (203 mg, 0.623 mmol) and methyl iodide (29.1 mI_, 0.466 mmol) were added and the mixture was heated to 45 °C. After 1 h, the mixture was diluted with a 3:1 chloroform/EtOH solution (15 mL) and water (15 mL). The layers were separated and the aqueous layer was back-extracted twice more with the 3:1 chloroform/EtOH solution (10 mL / each). The combined organic phases were dried over anhydrous MgS04, filtered and concentrated. The crude material was purified by silica gel chromatography (35-100% (3:1 EtOAc/EtOH)/Heptanes as the eluent; compound came out at ~90% (3:1 EtOAc/EtOH)/Heptanes). The product- containing fractions were combined and concentrated yielding a light brown solid, which was then triturated to provide the title compound as an off-white solid (42.2 mg, 0.067 mmol, 35.9 % yield). LC-MS m/z 619.4 (M+H)+, 0.77 min (ret. time); Ή NMR (400 MHz, DMSO-cfe) d ppm 7.56 (d, J= 7.86 Hz, 2 H) 7.42 (d, J=8.1 1 Hz, 2 H) 7.26 - 7.36 (m, 2 H) 7.16 - 7.26 (m, 2 H) 6.43 (s, 2 H) 5.82 (br. s„ 2 H) 4.74 (br. s., 4 H) 4.45 (q, J=7.10 Hz, 4 H) 3.53 (s, 6 H) 2.13 (s, 6 H) 1 .22 (t, J=7.10 Hz, 6 H).
[00194]Example 2. Methyl (£)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2, 3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3-methyl-2,3-dihydro- 1H-benzo[d]imidazole-5-carboxylate
Figure imgf000055_0001
[00195]2,2,2-Trifluoro-JV-(2-nitrophenyl)acetamide
Figure imgf000055_0002
[00196]To a stirred solution of 2-nitroaniline (50 g, 362 mmol) in dichloromethane (DCM) (700 mL) was added DIPEA (190 ml_, 1086 mmol) followed by TFAA (1 12 ml_, 796 mmol) dropwise at 0°C. The resulting reaction mixture was stirred for 3 h at 28 °C. After 3 h, water (500 mL) was added to the reaction mixture. The aqueous layer was extracted with DCM (3 x 500 mL). The combined organic layers were washed with cold water (2 x 300 mL) followed by brine (500 mL) and dried over anhydrous sodium sulfate. After filtration, the organic layer was concentrated under reduced pressure to afford crude material. Water (600 mL) was added to the crude material and the mixture was stirred at RT for 1 h. The obtained yellow solid was filtered and dried under vacuum to afford the title compound as a yellow solid (70 g, 294 mmol, 81 % yield). LC-MS mlz 233 (M-H)+, 2.05 min (ret. time).
[00197] (E)-N-(4-bromobut-2-en-1 -yl)-2,2,2-trifluoro-N-(2- nitrophenyl)acetamide
Figure imgf000055_0003
[00198]To a stirred solution of 2,2,2-trifluoro-A/-(2-nitrophenyl)acetamide (70 g, 299 mmol) in acetonitrile (1400 ml_) was added potassium carbonate (45.5 g, 329 mmol) followed by (E)-1 ,4-dibromobut-2-ene (160 g, 747 mmol) at 0°C. The reaction mixture was stirred for 3 h at 28 °C. After 3 h, the reaction mixture was filtered and washed with DCM (1000 ml). The filtrate was concentrated under reduced pressure to afford a brown oil (250 g). This was absorbed onto silica gel (500 g) and purified by silica gel chromatography (10-15% EtOAc in petroleum ether) to give the title compound as a yellow semi-solid (75 g, 202 mmol, 68% yield). LC-MS mlz 287 (M-Br)+, 2.38 min (ret. time).
[00199] Methyl 3-nitro-4-(2,2,2-trifluoroacetamido)benzoate
Figure imgf000056_0001
[00200]To a stirred solution of methyl 4-amino-3-nitrobenzoate (20 g, 102 mmol) in dichloromethane (DCM) (300 ml_) was added DIPEA (53.4 ml_, 306 mmol) followed by TFAA (31.7 ml_, 224 mmol) dropwise at 0°C. The resulting reaction mixture was stirred for 3 h at 28 °C. After 3 h, water (500 ml_) was added to the reaction mixture and it was extracted with DCM (3 x 200 ml_). The combined organic layers were washed with cold water (2 x 200 ml_) followed by brine (200 ml_) and dried over sodium sulfate. After filtration, the organic layer was concentrated under reduced pressure to afford the crude title compound as a brown liquid (30 g). This was then absorbed onto silica gel (60 g) and purified by silica gel chromatography (10-15% EtOAc in petroleum ether) to provide the title compound as a yellow solid (18 g, 59 mmol, 58% yield). LC-MS mlz 290.9 (M+H)+, 2.22 min (ret. time).
[00201]Methyl (£)-3-nitro-4-((4-(2,2,2-trifluoro-N-(2- nitrophenyl)acetamido)but-2-en-1-yl)amino)benzoate
Figure imgf000056_0002
[00202]To a stirred solution of methyl 3-nitro-4-(2,2,2-trifluoroacetamido)benzoate (15 g, 51 .3 mmol) in acetonitrile (250 ml_) was added potassium carbonate (15.6 g, 113 mmol) followed by (E)-A/-(4 bromobut-2-en-1-yl)-2,2,2-trifluoro-A/-(2- nitrophenyl)acetamide (22.6 g, 61 .6 mmol) at RT. The reaction was stirred for 16 h at 70 °C. Following this duration, the reaction mixture was filtered and washed with DCM (300 ml). The filtrate was concentrated under reduced pressure to afford the crude title compound as a brown oil (40 g). This was adsorbed onto silica gel (80 g) and purified by silica gel chromatography (20-25% EtOAc in petroleum ether) to afford the title compound as a brown liquid (20 g, 34.9 mmol, 68% yield). LC-MS mlz 482.9 (M+H)+, 2.62 min (ret. time).
[00203]Methyl (£)-3-nitro-4-((4-((2-nitrophenyl)amino)but-2-en-1 - yl)amino)benzoate
Figure imgf000057_0001
[00204]To a stirred solution of methyl (E)-3-nitro-4-((4-(2,2,2-trifluoro-A/-(2- nitrophenyl)acetamido)but-2-en-1 -yl)amino)benzoate (20 g, 41 .5 mmol) in methanol (150 mL) and dichloromethane (DCM) (300 mL) was added potassium carbonate (14.3 g, 104 mmol) at 28 °C. The reaction was stirred at 28 °C for 16 h. After 16 h, the reaction mixture was filtered and washed with DCM (300 ml). The filtrate was concentrated under reduced pressure to afford a residue. Water (200 mL) was added to the residue and the mixture was stirred at RT for 1 h. The obtained yellow solid was filtered and dried under vacuum to afford the title compound as a yellow solid (13 g, 32.9 mmol, 79 % yield). LC-MS mlz 387 (M+H)+, 2.54 min (ret. time).
[00205] Methyl (£)-3-amino-4-((4-((2-aminophenyl)amino)but-2-en-1 - yl)amino)benzoate
Figure imgf000057_0002
[00206] To a stirred solution of methyl (E)-3-nitro-4-((4-((2-nitrophenyl)amino)but-2- en-1 -yl)amino)benzoate (13 g, 33.6 mmol) in methanol (300 mL) was added ammonium hydroxide (1 14 mL, 673 mmol) dropwise at 0°C. A solution of sodium dithionite (58.6 g, 336 mmol) in water (150 mL) was subsequently added dropwise at 0°C. The reaction was stirred at 28 °C for 32 h. After 32 h, the reaction mixture was filtered through Celite and washed with ethyl acetate (500 ml). The filtrate was evaporated under reduced pressure to afford a residue. Water was added to the residue and the resulting mixture was stirred for 1 h at RT. The obtained solid was filtered and dried under vacuum to afford the title compound as a brown solid (7.5 g, 22.4 mmol, 66.7 % yield). LC-MS mlz 327 (M+H)+,
1 .28 min (ret. time).
[00207]Methyl (E)-2-(1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-1 -(4-(2-(1 - ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)- 1 H-benzo[d]imidazole-5-carboxylate
Figure imgf000058_0001
[00208]To a stirred solution of methyl (E)-3-amino-4-((4-((2- aminophenyl)amino)but-2-en-1 -yl)amino)benzoate (4 g, 12.25 mmol) in N,N- dimethylformamide (DMF) (80 mL) was added 1 -ethyl-3-methyl-1 /-/-pyrazole-5 carbonyl isothiocyanate (7.2 g, 36.8 mmol) at 28 °C and stirred for 5 h. After 5 h, EDC (14.1 g, 73.5 mmol) followed by triethylamine (27.3 mL, 196 mmol) was added to the reaction mixture at 0 °C and the resulting reaction mixture was stirred at 28 °C for 16 h. After 16 h, water (500 ml) was added to the reaction mixture and stirred at RT for 1 h. The obtained solid was filtered, washed with diethyl ether (200 ml) and dried under vaccum to afford the title compound as an off-white solid (4.5 g, 6.7 mmol, 55% yield). LC-MS mlz 648.9 (M+H)+, 2.43 min (ret. time).
[00209]Methyl (£)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-1 -((£)- 4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H- benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazole-5- carboxylate
Figure imgf000058_0002
[00210]To a stirred solution of methyl (E)-2-((1 -ethyl-3-methyl-1 /-/-pyrazole-5- carbonyl)imino)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3- dihydro-1 Hbenzo[d]imidazol-1 -yl)but-2-en-1 -y l)-2 , 3-d i hyd ro- 1 H-benzo[d]imidazole-5- carboxylate (3.5 g, 5.4 mmol) in A/,A/-dimethylformamide (DMF) (50 ml_) was added cesium carbonate (7.0 g, 21 .6 mmol) followed by methyl iodide (1 .3 ml_, 21 .6 mmol) at 0°C. The reaction was stirred in a sealed tube at 28 °C for 3 h. After 3 h, water (500 ml) was added to the reaction mixture and stirred at RT for 1 h. The obtained solid was filtered, washed with diethyl ether (200 ml) and dried under vacuum to afford the title compound as an off-white solid (2.2 g, 3.0 mmol, 56% yield). LC-MS mlz 677.2 (M+H)+, 1 .95 min (ret. time); Ή NMR (400 MHz, DMSO -cfe) d ppm 8.07 (d, J = 1 .2 Hz, 1 H), 7.82
(dd, J = 1 .60, 8.4 Hz, 1 H), 7.55 (d, J = 8.0 Hz, 1 H), 7.50 (d, J = 8.4 Hz, 1 H), 7.42 (d, J = 8.0 Hz, 1 H), 7.29-7.29 (m, 1 H), 7.19-7.20 (m, 1 H), 6.44 (s, 1 H), 6.40 (d, J = 0.4 Hz, 1 H), 5.81 (q, J = 4.4 Hz, 2H), 4.75 (t, J = 4.0 Hz, 4H), 4.42-4.42 (m, 4H), 3.90 (s, 3H), 3.57 (s, 3H), 3.53 (s, 3H), 2.13 (s, 3H), 2.12 (s, 3H), 1 .23 (t, J = 2.8 Hz, 3H), 1 .21 (t, J = 4.8 Hz, 3H).
[00211]Example 3. 1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en- 1 -yl)-4-methoxy-1 -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide
Figure imgf000059_0001
[00212] 2,2,2-Trifluoro-N-(2-methoxy-6-nitrophenyl)acetamide
Figure imgf000059_0002
[00213]To a stirred solution of 2-methoxy-6-nitroaniline (1 .5 g, 8.9 mmol) in dichloromethane (DCM) (18 mL) was added DIPEA (4.7 ml_, 26.8 mmol) followed by TFAA (2.77 mL, 19.63 mmol) dropwise at 0°C. The resulting reaction mixture was stirred for 3 h at 28 °C. After this duration, the volatiles were removed under reduced pressure to give a brown, viscous material. Water (15 mL) was added and the resulting mixture was stirred at RT for 1 h. The obtained pale yellow precipitate was filtered and dried under vacuum to afford the title compound as a pale yellow solid (2 g, 7.46 mmol, 84 % yield). LC-MS m/z 263 (M+H)+, 1 .84 min (ret. time).
[00214](£)-2,2,2-Trifluoro-N-(2-methoxy-6-nitrophenyl)-N-(4-(2,2,2-trifluoro-N- (2-nitrophenyl)acetamido)but-2-en-1 -yl)acetamide
Figure imgf000060_0001
[00215]To a solution of 2,2,2-trifluoro-A/-(2-methoxy-6-nitrophenyl)acetamide (1 .0 g, 3.8 mmol) in acetonitrile (15 mL) was added (E)-A/-(4-bromobut-2-en-1 -yl)-2,2,2- trifluoro-A/-(2-nitrophenyl)acetamide ( vide supra, 1 .7 g, 4.5 mmol) and potassium carbonate (1 .2 g, 8.3 mmol) under an atmosphere of nitrogen at room temperature. The resulting reaction mixture was stirred at 70 °C for 16 h. Following this duration, the reaction mixture was filtered through Celite and the filtrate was concentrated under reduced pressure. The obtained residue was dissolved in ethyl acetate (20 mL) and washed with water (20 mL). The aqueous layer was extracted with EtOAc (2 x 20 mL) and the combined organics were washed with brine (20 mL) and dried over anhydrous sodium sulfate. After filtration, the organic layer was concentrated under reduced pressure to obtain a brown liquid. This was subsequently absorbed onto silica gel (5 g) and purified by silica gel chromatography (5-20% EtOAc in petroleum ether) to afford the title compound as a pale orange liquid (700 mg, 1 .2 mmol, 31 % yield). LC-MS m/z 551 (M+H)+, 2.60 min (ret. time).
[00216](E)-N7-(2-Methoxy-6-nitrophenyl)-N4-(2-nitrophenyl)but-2-ene-1 ,4- diamine
Figure imgf000060_0002
[00217]To a suspension of (E)-2,2,2-trifluoro-A/-(2-methoxy-6-nitrophenyl)-A/-(4-
(2,2,2-trifluoro-A/-(2-nitrophenyl)acetamido)but-2-en-1 -yl)acetamide (1 .1 g, 2.0 mmol) in a
1 :2 mixture of methanol:DCM (30 mL) under an atmosphere of nitrogen at room temperature was added potassium carbonate (0.691 g, 5.0 mmol). The reaction mixture was stirred at 27 °C for 16 h. After this duration, the reaction mixture was filtered through
Celite. The filtrate was evaporated under reduced pressure and the resulting residue was dissolved in EtOAc (100 mL) and washed with water (50 mL) and brine (50 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated to afford a brown residue. This was subsequently adsorbed onto silica gel (1 .2 g) and purified by silica gel chromatography (15% EtOAc in petroleum ether) to afford the title compound as a brown gum (0.45 g, 1 .22 mmol, 61 % yield). LC-MS mlz 359 (M+H)+, 2.60 min (ret. time).
[00218] (E)-Ni-(4-((2-Aminophenyl)amino)but-2-en-1 -yl)-6-methoxybenzene- 1 , 2-diamine
Figure imgf000061_0001
[00219]To a suspension of (E)-A/f-(2-methoxy-6-nitrophenyl)-A/4-(2- nitrophenyl)but-2-ene-1 ,4-diamine (0.4 g, 1 .1 mmol) in methanol (16 mL) stirred under an atmosphere of nitrogen at room temperature was added ammonium hydroxide (0.78 g, 22.3 mmol) and a solution of sodium dithionite (1 .9 g, 1 1 .2 mmol) in water (8 mL). The reaction mixture was stirred at room temperature for 8 h. After this duration, the reaction mixture was filtered through Celite and washed with MeOH (2 x 10 mL). The filtrate was evaporated and the resulting material was dissolved in water (30 mL), followed by extraction with EtOAc (2 x 50 mL). The combined organics were washed with brine (30 mL) and dried over sodium sulfate. After filtration, the organic layer was concentrated under reduced pressure to afford a brown residue. This was subsequently adsorbed onto silica gel and purified by silica gel chromatography (80 % EtOAc in petroleum ether) to afford the title compound as a brown gum (300 mg, 0.95 mmol, 85% yield). LC-MS mlz 299 (M+H)+, 0.96 min (ret. time).
[00220](E)-1 -Ethyl-N-(1 -(4-(2-(1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamido)- 1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-7-methoxy-1 H-benzo[d]imidazol-2-yl)-3- methyl-1 H-pyrazole-5-carboxamide
Figure imgf000061_0002
[00221] A solution of (E)-A/f-(4-((2-aminophenyl)amino)but-2-en-1 -yl)-6- methoxybenzene-1 ,2-diamine (0.25 g, 0.84 mmol) and 1 -ethyl-3-methyl-1 /-/-pyrazole-5- carbonyl isothiocyanate (0.82 g, 4.2 mmol) in A/,A/-dimethylformamide (DMF) (12 mL) was stirred under nitrogen at room temperature for 4 h. After this duration, triethylamine (1 .4 g, 13.4 mmol) and 3-(((ethylimino)methylene)amino)-/V,/V-dimethylpropan-1 -amine- hydrochloride (1 .1 g, 5.9 mmol) were added at 0°C and the resulting reaction mixture was stirred at room temperature for 16 h. After this duration, water (30 ml) was added and the resulting reaction mixture was stirred at room temperature for 1 h. The mixture was filtered and the collected solid was washed with pentane (30 ml) and dried under vacuum to afford the title compound as a white solid (300 mg, 0.42 mmol, 58% yield). LC-MS m/z 621 (M+H)+, 2.40 min (ret. time).
[00222] 1 -Ethyl-N-((£)-3-((£)-4-((£)-2-((1 -ethyl-3-methy 1-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4- methoxy-1 -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide
Figure imgf000062_0001
[00223]To a stirred suspension of 1 -ethyl-A/-((£)-1 -((£)-4-((£)-2-((1 -ethyl-3- methyl-1 /-/-pyrazole-5-carbonyl)imino)-2,3-dihydro-1 /-/-benzo[d]imidazol-1 -yl)but-2-en-1 - yl)-7-methoxy-1 ,3-dihydro-2/-/-benzo[d]imidazol-2-ylidene)-3-methyl-1 /-/-pyrazole-5- carboxamide (0.3 g, 0.48 mmol), and cesium carbonate (0.787 g, 2.42 mmol) in N,N- dimethylformamide (DMF) (12 mL) was added iodomethane (0.343 g, 2.42 mmol) at 0 °C. The resulting reaction mixture was stirred at room temperature for 18 h. Following this duration, the reaction was warmed to 45 °C and stirred for 1 h. Following this duration, the reaction mixture was filtered through Celite and washed with EtOAc (2 x 20 mL). The filtrate was concentrated under reduced pressure. This was dissolved in 20 mL of 3:1 DCME:EtOH and washed with water (10 mL), brine (20 mL) and dried over anhydrous sodium sulfate. After filtration, the organic layer was concentrated under reduced pressure to afford the crude compound. This was subsequently purified by reverse-phase HPLC (Sunfire C18 (19*150)mm 5 micron column, eluent 0.1 % TFA in watenacetonitrile, 10 mL/min flow rate) to afford the title compound with TFA contaminant. The material was then stirred in aqueous ammonia (1 ml_), separated and filtered, and the collected solid was washed with water and pentane and then dried under vacuum to give the title compound as an off-white solid (25 mg, 0.038 mmol, 8% yield). LC-MS mlz 649 (M+H)+, 2.29 min (ret. time); 1H NMR (400 MHz, DMSO -cfe) d ppm 7.56 (d, J =7.6 Hz, 1 H), 7.39 (d, J = 8.0 Hz, 1 H), 7.31 (dd, J = 8.0, 7.6 Hz, 1 H), 7.27-7.15 (m, 3H), 6.89 (d, J = 8.0 Hz, 1 H), 6.41 (s, 3H), 6.40 (s, 1 H), 5.89-5.83 (m, 1 H), 5.67-5.62 (m, 1 H), 4.83 (q, J = 5.6 Hz, 2H),
4.73 (q, J = 5.6 Hz, 2H), 4.48-4.42 (m, 4H), 3.71 (s, 1 H), 3.53 (s, 3H), 3.49(s, 3H).
[00224]Example 4. 1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-7-(3-hydroxypropoxy)-3-methyl-2,3-dihydro-1 H- benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-methoxy-1 -methyl-1 ,3-dihydro-2H- benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide
Figure imgf000063_0001
[00225]tert-Butyl (E)-(4-((2-methoxy-6-nitrophenyl)amino)but-2-en-1 - yl)carbamate
Figure imgf000063_0002
[00226] To a mixture of te/ -butyl (E)-(4-aminobut-2-en-1 -yl)carbamate (1 .2 g, 6.4 mmol; Friedman Ohana, R., et al., ACS Chem. Biol. 2016, 2608-2617) and DIPEA (1 .5 ml_, 8.8 mmol) in isopropanol (25 ml_) was added 2-fluoro-1 -methoxy-3-nitrobenzene (1 g, 5.8 mmol). The mixture was stirred at 60 °C for 66 h, then concentrated. The residue was dissolved in ethyl acetate and washed with water and brine. The organic layers were dried (anhydrous sodium sulfate), filtered and concentrated. The residue was purified by silica gel chromatography (RediSep Gold 40g 10-30% ethyl acetate/heptanes) to provide the title compound as an orange solid. LC-MS m/z 369,1 (M+Na)+, 1 .09 min (ret. time).
[00227]fert-Butyl (E)-(4-((2-amino-6-methoxyphenyl)amino)but-2-en-1 - yl)carbamate
Figure imgf000064_0001
[00228]To a solution of fe/ -butyl (E)-(4-((2-methoxy-6-nitrophenyl)amino)but-2- en-1 -yl)carbamate (450 mg, 1 .3 mmol) in methanol (10 ml_) was added sodium hydrosulfite (1 .4 g, 6.7 mmol) in water (4 ml_). The mixture was stirred at room
temperature for 1 h, then concentrated. The residue was partitioned between water and dichloromethane. The layers were separated and the aqueous layer was extracted with dichloromethane. The combined organic layers were washed with brine, dried (anhydrous sodium sulfate), filtered, and concentrated to provide the title compound as a yellow oil. LC-MS m/z 308.3 (M+H)+, 0.58 min (ret. time).
[00229]fert-Butyl (£)-( 4-(2-(1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-7- methoxy-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)carbamate
Figure imgf000064_0002
[00230]To a solution of fe/ -butyl (E)-(4-((2-amino-6-methoxyphenyl)amino)but-2- en-1 -yl)carbamate (6.1 g, 19.8 mmol) in A/,A/-dimethylformamide (DMF) (60 mL) cooled in an ice bath was added 1 -ethyl-3-methyl-1 /-/-pyrazole-5-carbonyl isothiocyanate (21 .8 mL, 21 .8 mmol) dropwise. After stirring for 60 min, TEA (5.5 mL, 39.7 mmol) and EDC (4.57 g,
23.81 mmol) were added and the ice-bath removed. The mixture was stirred at room temperature for 18 h, then diluted with water. The mixture was extracted with dichloromethane. The combined organic layer was washed with water and brine, dried (anhydrous sodium sulfate), filtered, and concentrated to provide the crude product as a yellow solid (1 1 .9 g, 25.4 mmol, 128%). LC-MS m/z 469.5 (M+H)+, 1 .02 min (ret. time).
[00231 ]fert-Butyl ((£)-4-((£)-2-((1-ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-7-methoxy-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2- en-1-yl)carbamate
Figure imgf000065_0001
[00232]To a solution of fe/ -butyl ((E)-4-((E)-2-((1 -ethyl-3-methyl-1 /-/-pyrazole-5- carbonyl)imino)-7-methoxy-2,3-dihydro-1 /-/-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)carbamate (3 g, 5.3 mmol) in A/,A/-dimethylformamide (DMF) (20 mL) cooled in an ice bath was added cesium carbonate (2.60 g, 7.97 mmol) and methyl iodide (0.4 mL, 6.4 mmol) sequentially. The ice-bath was removed and the mixture was allowed to stir at room temperature for 3 days. Additional cesium carbonate (1 .7 g, 5.3 mmol) and methyl iodide (0.4 mL, 6.4 mmol) were added and stirring was continued for 4 h. After dilution with water and dichloromethane, the layers were separated and the aqueous layer was extracted with dichloromethane. The combined organic layers were washed with water and brine, dried (anhydrous sodium sulfate), filtered, and concentrated. The crude material was purified by silica gel chromatography [RediSep Gold 80g, 10-60% (3:1 ethyl acetate:ethanol)/dichloromethane] to provide the title compound as a white foamy solid (1 .6 g, 63% yield). LC-MS m/z 483.5 (M+H)+, 0.83 min (ret. time).
[00233]ierf-Butyl(3-(2-fluoro-3-nitrophenoxy)propoxy)dimethylsilane
Figure imgf000065_0002
[00234]To a mixture of 2-fluoro-3-nitrophenol (1 g, 6.4 mmol) and potassium carbonate (1 .9 g, 14.0 mmol) in A/,A/-dimethylformamide (DMF) (15 mL) was added tert- butyl(3-(2-fluoro-3-nitrophenoxy)-propoxy)dimethylsilane (2.0 g, 6.1 mmol). The mixture was stirred at 80 °C for 2 h, then diluted with water and extracted with diethyl ether (3x). The organic layer was washed sequentially with water, 10% aqueous lithium chloride, and water. The organic layer was dried (anhydrous sodium sulfate), filtered, and
concentrated. The residue was purified by silica gel chromatography (Redisep Rf Gold cartridge 40g, 0-30% ethyl acetate/heptanes) to provide the title compound as an amber- colored liquid (2.1 g, 6.1 mmol, 96% yield). 1H NMR (400 MHz, DMSO-cfe) d ppm 0.02 (s, 6 H) 0.84 - 0.88 (m, 9 H) 1 .85 - 2.04 (m, 2 H) 3.72 - 3.81 (m, 2 H) 4.13 - 4.30 (m, 2 H)
7.27 - 7.42 (m, 1 H) 7.54 - 7.71 (m, 2 H).
[00235]N-((£)-3-((£)-4-Aminobut-2-en-1 -yl)-4-methoxy-1 -methyl-1 ,3-dihydro- 2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1H-pyrazole-5-carboxamide, hydrochloride
Figure imgf000066_0001
[00236]To a suspension of te/ -butyl ((E)-4-((E)-2-((1-ethyl-3-methyl-1 /-/-pyrazole- 5-carbonyl)imino)-7-methoxy-3-methyl-2,3-dihydro-1 /-/-benzo[d]imidazol-1 -yl)but-2-en-1 - yl)carbamate (1.5 g, 3.0 mmol) in methanol (15 ml_) was added HCI (4M dioxane solution) (3.0 ml_, 12.0 mmol). The mixture was allowed to warm to room temperature and stirred for 16 h, then concentrated to provide the title compound, which was carried forward without further purification (1.3 g). LC-MS mlz 383.3 (M+H)+, 0.50 min (ret. time).
[00237]N-((£)-3-((£)-4-((2-(3-((fert-Butyldimethylsilyl)oxy)propoxy)-6- nitrophenyl)amino)but-2-en-1-yl)-4-methoxy-1 -methyl-1 ,3-dihydro-2H- benzo[d]imidazol-2-ylidene)-1-ethyl-3-methyl-1H-pyrazole-5-carboxamide
Figure imgf000066_0002
[00238]To a mixture of A/-((E)-3-((E)-4-aminobut-2-en-1-yl)-4-methoxy-1-methyl- 1 ,3-dihydro-2/-/-benzo[d]imidazol-2-ylidene)-1-ethyl-3-methyl-1 /-/-pyrazole-5-carboxamide, Hydrochloride (1 3g, 3 mmol), DIPEA (1 .6 ml_, 9.0 mmol), and isopropanol (25 ml_) was added fe/?-butyl(3-(2-fluoro-3-nitrophenoxy)propoxy)dimethylsilane (0.946 g, 2.87 mmol). The mixture was stirred at 60 °C for 3 days, then cooled to room temperature and concentrated. The crude was dissolved in dichloromethane and washed with water and brine. The organic layer was dried (anhydrous sodium sulfate), filtered, and concentrated to provide a mixture of silylated and desilylated products (2 g). To a solution of this product mixture (2 g) in A/,A/-dimethylformamide (DMF) (12 mL) was added imidazole (0.354 g, 5.19 mmol) and TBDMS-CI (0.783 g, 5.19 mmol). The mixture was stirred at room temperature for 2 h, then diluted with ethyl acetate and water. The organic layer was washed with water and brine, then dried (anhydrous sodium sulfate), filtered, and concentrated to provide the title compound, which was carried forward without further purification (2.5 g). LC-MS m/z 692.4 (M+H)+, 1.34 min (ret. time).
[00239]N-((£)-3-((£)-4-((2-Amino-6-(3-((fert- butyldimethylsilyl)oxy)propoxy)phenyl)amino)but-2-en-1-yl)-4-methoxy-1 -methyl- 1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamide
Figure imgf000067_0001
[00240]To a solution of A/-((£)-3-((£)-4-((2-(3-((terf- butyldimethylsilyl)oxy)propoxy)-6-nitrophenyl)amino)but-2-en-1-yl)-4-methoxy-1-methyl- 1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1-ethyl-3-methyl-1 /-/-pyrazole-5-carboxamide (2.4 g, 3.5 mmol) in methanol (20 ml_) cooled in an ice bath was added sodium hydrosulfite (2.8 g, 13.9 mmol) in water (8.4 ml_). The ice bath was removed and the mixture was stirred at room temperature for 30 min and then concentrated. The residue was partitioned between water and 3:1 chloroform/ethanol. The layers were separated and the aqueous layer was extracted with 3:1 chloroform/ethanol. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated to provide the title compound as an amber colored oil (2.3 g, 3.4 mmol, 98%). LC-MS m/z 662.4 (M+H)+, 1.03 min (ret. time).
[00241 ]N-((£)-7-(3-((fert-Butyldimethylsilyl)oxy)propoxy)-1-((£)-4-((£)-2-((1 - ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-methoxy-3-methyl-2,3-dihydro-1H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1- ethyl-3-methyl-1 H-pyrazole-5-carboxamide
Figure imgf000068_0001
[00242]To solution of A/-((E)-3-((E)-4-((2-amino-6-(3-((te/ - butyldimethylsilyl)oxy)propoxy)-phenyl)amino)but-2-en-1 -yl)-4-methoxy-1 -methyl-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide (2.2 g, 3.3 mmol) in A/,A/-dimethylformamide (DMF) (20 mL) cooled in an ice bath was added 1 -ethyl-3-methyl-1 /-/-pyrazole-5-carbonyl isothiocyanate (3.7 mL, 3.7 mmol) dropwise. After stirring for 90 min, TEA (0.927 mL, 6.65 mmol) and EDC (0.765 g, 3.99 mmol) were added and the ice-bath was removed. The mixture was stirred at room temperature for 18 h, then diluted with water. The mixture was extracted with dichloromethane. The combined organic layer was washed with water and brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude material was purified by silica gel chromatography [RediSep Gold 80g, 0-50% (3:1 ethyl acetate:ethanol)/heptane] to provide the title compound as a foamy solid (1 .5 g, 1 .8 mmol, 55% yield). LC-MS m/z 823.3 (M+H)+, 1 .28 min (ret. time).
[00243]N-((£)-3-((£)-4-((£)-7-(3-((tert-butyldimethylsilyl)oxy)propoxy)-2-((1 - ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H- benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-methoxy-1 -methyl-1 ,3-dihydro-2H- benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide
Figure imgf000068_0002
[00244]To a solution of A/-((E)-7-(3-((fe/?-butyldimethylsilyl)oxy)propoxy)-1 -((E)-4- ((E)-2-((1 -ethyl-3-methyl-1 /-/-pyrazole-5-carbonyl)imino)-7-methoxy-3-methyl-2, 3-dihydro- 1 /-/-benzo[d]imidazol-1-yl)but-2-en-1-yl)-1 ,3-dihydro-2/-/-benzo[d]imidazol-2-ylidene)-1- ethyl-3-methyl-1 /-/-pyrazole-5-carboxamide (1.5 g, 1 .8 mmol) in A/,A/-dimethylformamide (DMF) (12 mL) cooled in an ice bath was added cesium carbonate (0.891 g, 2.73 mmol) and methyl iodide (0.137 mL, 2.2 mmol) sequentially. The ice-bath was removed and the mixture was allowed to stir at room temperature for 64 h. After dilution with water, the precipitate was filtered and rinsed with water. The collected solid was dried in vacuo to provide the title compound, which was carried forward without further purification (1.4 g, 92% yield). LC-MS m/z 837.4 (M+H)+, 1 .17 min (ret. time).
[00245] 1 -Ethyl-N-((£)-3-((£)-4-((£)-2-((1 -ethyl-3-methy 1-1 H-pyrazole-5- carbonyl)imino)-7-(3-hydroxypropoxy)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 - yl)but-2-en-1 -yl)-4-methoxy-1 -methyl-1 , 3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3- methyl-1 H-pyrazole-5-carboxamide
Figure imgf000069_0001
[00246]To a solution of A/-((E)-3-((E)-4-((E)-7-(3-((te/ - butyldimethylsilyl)oxy)propoxy)-2-((1 -ethyl-3-methyl-1 /-/-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 /-/-benzo[d]imidazol-1-yl)but-2-en-1-yl)-4-methoxy-1 -methyl- 1 ,3- dihydro-2/-/-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 /-/-pyrazole-5-carboxamide (150 mg, 0.179 mmol) in methanol (2 ml_) was added HCI methanol solution (0.29 ml_, 0.36 mmol). The mixture was stirred at room temperature for 2 h, then HCI methanol solution (0.72 ml_, 0.90 mmol) was added and stirring was continued for 16 h. After concentration, the residue was purified by reversed phase HPLC/MS [Waters XSelect 5 pm C18, 150 X 30 mm, 15-55% CH3CN:H20, 0.1 % TFA as modifier]. The fractions containing the desired product were passed through a PL-HC03 MP SPE cartridge. The eluate was concentrated to provide the title compound (64 mg, 49% yield). LC-MS m/z 723.3 (M+H)+, 0.72 min (ret. time). 1H NMR (400 MHz, DMSO -d6) d ppm1.21 (td, J=7.15, 3.01 Hz, 6 H) 1 .69 (quin, J=6.21 Hz, 2 H) 2.12 (s, 3 H) 2.12 (s, 3 H) 3.43 (t, J=6.15 Hz, 2 H) 3.49 (s, 3 H) 3.50 (s, 3 H) 3.64 (s, 3 H) 3.96 (t, J=6.40 Hz, 2 H) 4.40 - 4.49 (m, 4 H) 4.77 - 4.87 (m, 4 H) 5.64 - 5.78 (m, 2 H) 6.39 (s, 1 H) 6.40 (s, 1 H) 6.85 (dd, J= 7.78, 2.76 Hz, 2 H) 7.13 - 7.18 (m, 2 H) 7.20 - 7.27 (m, 2 H).
[00247] Exam pie 5. (E)-1 -Ethyl-N-(3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en- 1 -yl)-4-(hydroxymethyl)-1 -methyl-1 H-benzo[d]imidazol-2(3H)-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide
Figure imgf000070_0001
[00248]ferf-Butyl (£)-(4-((2-nitrophenyl)amino)but-2-en-1 -yl)carbamate
Figure imgf000070_0002
[00249]A mixture of 1 -fluoro-2-nitrobenzene (0.749 ml_, 7.09 mmol), te/ -butyl (E)- (4-aminobut-2-en-1 -yl)carbamate (1 .6 g, 8.5 mmol) and DIPEA (2.476 ml_, 14.17 mmol) in isopropanol (20 ml_) was stirred for 3 days at room temperature. The mixture was stirred for additional 20 hours at 50 °C. The mixture was concentrated and the residue was redissolved in ethyl acetate and washed with water. The organic layer was dried over anhydrous MgS04. It was filtered and the filtrate was concentrated. The crude product was purified by silica gel chromatography eluting with a gradient of 0 to 40% ethyl acetate in heptane. The title compound (1 .9 g, 6.2 mmol, 87% yield) was obtained as an orange oil. LC-MS m/z 308.1 (M+H)+, 1 .10 min (ret. time).
[00250]2-Fluoro-1 -((methoxymethoxy)methyl)-3-nitrobenzene
Figure imgf000070_0003
[00251]To a solution of (2-fluoro-3-nitrophenyl)methanol (1 .0 g, 5.84 mmol) in dichloromethane (DCM) (20 ml_) was added DIPEA (2.041 ml_, 1 1.69 mmol), followed by MOM-CI (0.577 ml_, 7.60 mmol). The reaction mixture was stirred for 3 days at room temperature. The mixture was diluted with water and extracted with dichloromethane. The organic extract was dried over anhydrous MgS04. It was filtered and the filtrate was concentrated. The crude product was purified by silica gel chromatography eluting with a gradient of 0 to 30% ethyl acetate in heptane. The title compound (1.1 g, 5.1 mmol, 87% yield) was obtained as a light yellow oil. 1H NMR (400 MHz, CHLOROFORM-d) d ppm 3.44 (s, 3 H) 4.75 (s, 2 H) 4.78 (s, 2 H) 7.29 - 7.35 (m, 1 H) 7.75 - 7.83 (m, 1 H) 7.97 - 8.06 (m, 1 H).
[00252](E)-N7-(2-((Methoxymethoxy)methyl)-6-nitrophenyl)-AM-(2- nitrophenyl)but-2-ene-1 ,4-diamine
Figure imgf000071_0001
[00253]A mixture of 2-fluoro-1-((methoxymethoxy)methyl)-3-nitrobenzene (500 mg, 2.3 mmol), (E)-/V7-(2-nitrophenyl)but-2-ene-1 ,4-diamine, 2hydrochloride (716 mg, 2.6 mmol) and DIPEA (2.0 ml_, 11.6 mmol) in isopropanol (20 ml_) was stirred for 20 hours at 70 °C. After cooling to room temperature, the mixture was concentrated and the residue was diluted with water and extracted with ethyl acetate. The organic extract was washed with water and dried over anhydrous MgSC It was filtered and the filtrate was concentrated. The crude product was purified by silica gel chromatography eluting with a gradient of 0 to 40% ethyl acetate in heptane. The title compound (920 mg, 2.3 mmol, 98% yield) was obtained as a red oil. LC-MS mlz 403.3 (M+H)+, 1.25 min (ret. time).
[00254](E)-N7-(4-((2-Aminophenyl)amino)but-2-en-1-yl)-6- ((methoxymethoxy)methyl)benzene-1 ,2-diamine
Figure imgf000071_0002
[00255]To a suspension of (E)-/V7-(2-((methoxymethoxy)methyl)-6-nitrophenyl)- A/4-(2-nitrophenyl)but-2-ene-1 ,4-diamine (0.9 g, 2.2 mmol) and ammonium hydroxide (12.4 ml_, 89 mmol) in methanol (30 ml_) was added sodium hydrosulfite (3.89 g, 22.37 mmol) in water (10 ml_). The reaction mixture was stirred for 18 h at room temperature. Methanol was evaporated and the residue was extracted with ethyl acetate. The organic extract was washed with water and dried over anhydrous MgS04. It was filtered and the filtrate was concentrated. The crude product was purified by silica gel chromatography eluting with a gradient of 0 to 100% ethyl acetate in heptane. The title compound (495 mg, 1.4 mmol, 65% yield) was obtained as a colorless oil. LC-MS mlz 343.3 (M+H)+, 0.46 min (ret. time).
[00256] 1 -Ethyl-N-((£)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-7- ((methoxymethoxy)methyl)-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl- 1H-pyrazole-5-carboxamide
Figure imgf000072_0001
[00257]To a solution of (E)-A/f-(4-((2-aminophenyl)amino)but-2-en-1-yl)-6- ((methoxymethoxy)methyl)benzene-1 ,2-diamine (490 mg, 1.4 mmol) in N,N- dimethylformamide (DMF) (6.0 mL) was added 1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl isothiocyanate 1 .0 M in dioxane (3.15 mL, 3.15 mmol). The reaction mixture was stirred for 3 hours at room temperature. EDC (823 mg, 4.29 mmol) and TEA (1.197 mL, 8.59 mmol) were added and the mixture was stirred for 18 hours at room temperature. It was diluted with water (10 mL) and extracted with ethyl acetate (15 mL). The organic extract was washed with water and dried over anhydrous MgS04. It was filtered and the filtrate was concentrated. The resulting solid was triturated with ethyl acetate/hexanes (1 :1) and filtered to give the title compound (654 mg, 0.984 mmol, 69% yield) as a white solid. LC- MS m/z 665.4 (M+H)+, 1 .15 min (ret. time).
[00258] 1 -Ethyl-N-((£)-3-((£)-4-((£)-2-((1 -ethyl-3-methy 1-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4- ((methoxymethoxy)methyl)-1 -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3- methyl-1H-pyrazole-5-carboxamide
Figure imgf000073_0001
[00259]To a mixture of 1 -ethyl-A/-((£)-1 -((£)-4-((£)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-7- ((methoxymethoxy)methyl)-1 ,3-dihydro-2/-/-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide (650 mg, 0.978 mmol) and cesium carbonate (1274 mg, 3.91 mmol) in A/,A/-dimethylformamide (DMF) (4.0 mL) was added iodomethane (0.183 ml_, 2.93 mmol) at 0 °C and stirred for 30 min. The mixture was filtered and the filtrate concentrated and purified by reverse-phase HPLC [Waters XSelect 5 pm C18, 150 X 30 mm, 30-85% CH3CN:H20, 0.1 % TFA as modifier]. The title compound (452 mg, 0.65 mmol, 67% yield) was obtained as a white powder. LC-MS m/z 693.5 (M+H)+, 0.91 min (ret. time).
[00260](£)-1 -Ethyl-N-(3-((£)-4-((£)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4- (hydroxymethyl)-l -methyl-1 H-benzo[d]imidazol-2(3H)-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide
Figure imgf000073_0002
[00261]A mixture of 1 -ethyl-A/-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 /-/-pyrazole- 5-carbonyl)imino)-3-methyl-2,3-dihydro-1 /-/-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4- ((methoxymethoxy)methyl)-1 -methyl-1 ,3-dihydro-2/-/-benzo[d]imidazol-2-ylidene)-3- methyl-1 /-/-pyrazole-5-carboxamide (450 mg, 0.650 mmol) and HCI 4.0 M in dioxane (1 .6 mL, 6.5 mmol) in methanol (4 ml_) was stirred for 20 hours at 70 °C. After cooling to room temperature, the mixture was concentrated and the crude product was purified by reverse-phase HPLC [Waters XSelect 5 pm C18, 150 X 30 mm, 15-55% CH3CN:H20, 0.1 % formic acid as modifier] to give the title compound (420 mg, 0.647 mmol, 100% yield) as a white solid. LC-MS m/z 649.4 (M+H)+, 0.79 min (ret. time). Ή NMR (400 MHz, DMSO-cfe) d ppm 1 .16 - 1 .23 (m, 6 H) 2.13 (s, 6 H) 3.53 (d, J= 1 .47 Hz, 6 H) 4.43 (q,
J=7.01 Hz, 4 H) 4.59 (d, J=5.14 Hz, 2 H) 4.72 (d, J=5.14 Hz, 2 H) 4.98 (d, J=3.18 Hz, 2 H) 5.43 - 5.51 (m, 1 H) 5.53 (t, J= 5.26 Hz, 1 H) 5.88 - 5.96 (m, 1 H) 6.39 (s, 1 H) 6.40 (s,
1 H) 7.19 - 7.25 (m, 2 H) 7.26 - 7.35 (m, 2 H) 7.41 (d, J=8.07 Hz, 1 H) 7.54 (dd, J=1 1 .49, 8.07 Hz, 2 H).
[00262] Example 6. 1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en- 1 -yl)-1 -methyl-4-(morpholinomethyl)-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3- methyl-1 H-pyrazole-5-carboxamide
Figure imgf000074_0001
[00263]N-((£)-1 -((£)-4-((£)-7-(chloromethyl)-2-((1 -ethyl-3-methyl-1 H-pyrazole- 5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3- methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamide
Figure imgf000075_0001
[00264]To a suspension of 1 -ethyl-/V-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 /-/-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)- 4-(hydroxymethyl)-1 -methyl-1 ,3-dihydro-2/-/-benzo[d]imidazol-2-ylidene)-3-methyl-1 /-/- pyrazole-5-carboxamide (200 mg, 0.308 mmol) in 1 ,2-dichloroethane (DCE) (15 ml_) was added thionyl chloride (0.135 ml_, 1 .85 mmol). The reaction mixture was stirred for 20 hours at 90 °C. After cooling to room temperature, it was filtered to give the title compound (200 mg, 0.3 mmol, 97% yield) as a white solid. LC-MS mlz 667.4 (M+H)+, 0.98 min (ret. time).
[00265] 1-Ethyl-N-((£)-3-((£)-4-((£)-2-((1-ethyl-3-methy 1-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-1 - methyl-4-(morpholinomethyl)-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-
1H-pyrazole-5-carboxamide
Figure imgf000075_0002
[00266]A mixture of /V-((E)-1 -((E)-4-((E)-7-(chloromethyl)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 /-/-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)- 3-methyl-1 ,3-dihydro-2/-/-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 /-/-pyrazole-5- carboxamide (30 mg, 0.045 mmol), morpholine (0.020 ml_, 0.225 mmol) and DIPEA (0.024 ml_, 0.135 mmol) in ethanol (1 .5 ml_) was stirred for 3 hours at 70 °C. It was concentrated and the residue was purified by reverse-phase HPLC [Waters XSelect 5 pm C18, 150 X 30 mm, 15-55% CH3CN:H20, 0.1 % formic acid as modifier] to give the title compound (26 mg, 0.036 mmol, 81 % yield) as white solid. LC-MS m/z 718.5 (M+H)+, 0.73 min (ret. time). Ή NMR (400 MHz, DMSO -d6) d ppm 1 .21 (td, J=7.09, 3.18 Hz, 6 H) 2.13 (s, 6 H) 2.22 (br. s., 4 H) 3.45 (br. s., 6 H) 3.52 (s, 3 H) 3.54 (s, 3 H) 4.40 - 4.50 (m, 4 H)
4.73 (d, J=4.89 Hz, 2 H) 5.10 (br. s., 2 H) 5.35 - 5.45 (m, 1 H) 5.88 - 5.98 (m, 1 H) 6.40 (s, 1 H) 6.41 (s, 1 H) 7.1 1 (d, J= 7.58 Hz, 1 H) 7.20 - 7.28 (m, 2 H) 7.32 (t, J= 7.70 Hz, 1 H) 7.44 (d, J= 7.82 Hz, 1 H) 7.55 (t, J=7.34 Hz, 2 H).
[00267]Example compounds 7-9 in Table 1 were prepared analogously to the synthetic sequence described for Example 6:
[00268]Example 10. 1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en- 1 -yl)-4-(methoxymethyl)-1 -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3- methyl-1 H-pyrazole-5-carboxamide
Figure imgf000076_0001
[00269]A mixture of /V-((E)-1 -((E)-4-((E)-7-(chloromethyl)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 /-/-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)- 3-methyl-1 ,3-dihydro-2/-/-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamide (30 mg, 0.045 mmol) and sodium methoxide (25 mg, 0.463 mmol) in methanol (1 .5 mL) was stirred for 20 hours at 70°C. After cooling to room temperature, the mixture was concentrated and the crude product was purified by reverse-phase HPLC [Waters XSelect 5 pm C18, 150 X 30 mm, 30-85% CH3CN:H20, 0.1 % formic acid as modifier] to give the title compound (14 mg, 0.021 mmol, 47% yield) as a white solid. LC- MS m/z 663.5 (M+H)+, 0.92 min (ret. time). 1 H NMR (400 MHz, DMSO -cfe) d ppm 1 .21 (t, J= 7.09 Hz, 6 H) 2.13 (s, 6 H) 3.17 (s, 3 H) 3.53 (d, J=1 .96 Hz, 6 H) 4.44 (q, J= 7.09 Hz, 4 H) 4.49 (s, 2 H) 4.73 (d, J=5.14 Hz, 2 H) 4.87 (br. s., 2 H) 5.42 - 5.51 (m, 1 H) 5.85 - 5.94 (m, 1 H) 6.40 (s, 1 H) 6.41 (s, 1 H) 7.20 - 7.26 (m, 2 H) 7.27 - 7.35 (m, 2 H) 7.43 (d,
J= 8.07 Hz, 1 H) 7.57 (t, J=8.68 Hz, 2 H). [00270] Exam pie 11. (rac)-1-Ethyl-N-((£)-3-((£)-4-((£)-2-((1-ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1H-benzo[d]imidazol-1 -yl)but-2-en- 1 -yl)-4-(1 -hydroxyethyl)-1 -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3- methyl-1H-pyrazole-5-carboxamide
Figure imgf000077_0001
[00271 ]1 -Ethyl-N-((£)-3-((£)-4-((£)-2-((1-ethyl-3-methy 1-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4- formyl-1 -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole- 5-carboxamide
Figure imgf000077_0002
[00272]To a suspension of 1 -ethyl-A/-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 /-/-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)- 4-(hydroxymethyl)-1 -methyl-1 ,3-dihydro-2/-/-benzo[d]imidazol-2-ylidene)-3-methyl-1 /-/- pyrazole-5-carboxamide (80 mg, 0.123 mmol) in dichloromethane (DCM) (20 ml_) was added activated manganese dioxide (214 mg, 2.466 mmol). The mixture was stirred for 2.0 hours at 40 °C. It was subsequently filtered and the filtrate was concentrated to give the title compound (80 mg, 0.124 mmol, 100% yield) as a light yellow solid. LC-MS m/z 647.4 (M+H)+, 0.88 min (ret. time). [00273] 1 -Ethyl-N-((£)-3-((£)-4-((£)-2-((1 -ethyl-3-methy 1-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(1 - hydroxyethyl)-1 -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide
Figure imgf000078_0001
[00274]To a suspension of 1 -ethyl-/V-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 /-/-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)- 4-formyl-1 -methyl-1 ,3-dihydro-2/-/-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide (80 mg, 0.124 mmol) in tetrahydrofuran (THF) (2.0 mL) was added methylmagnesium bromide 3.4 M in 2-THF (0.109 mL, 0.371 mmol). The mixture was stirred for 2.0 hours at room temperature. To the mixture was added additional methylmagnesium bromide 3.4 M in 2-THF (0.109 mL, 0.371 mmol) and the reaction mixture was stirred for 30 min. The mixture was diluted with saturated NH4CI (aq) and extracted with ethyl acetate. The organic extract was concentrated and the crude product was purified by reverse-phase HPLC [Waters XSelect 5 pm C18, 150 X 30 mm, 15-55% CH3CN:H20, 0.1 % formic acid as modifier] to give the title compound as white solid. LC- MS m/z 663.4 (M+H)+, 0.83 min (ret. time). 1 H NMR (400 MHz, DMSO -cfe) d ppm 1 .21 (t, J= 7.09 Hz, 6 H) 1 .31 (d, J=6.36 Hz, 3 H) 2.12 (s, 3 H) 2.13 (s, 3 H) 3.52 (s, 3 H) 3.53 (s, 3 H) 4.44 (q, J= 7.09 Hz, 4 H) 4.73 (d, J=5.14 Hz, 2 H) 4.85 - 4.94 (m, 1 H) 5.02 - 5.1 1 (m, 2 H) 5.40 (d, J=5.38 Hz, 1 H) 5.42 - 5.51 (m, 1 H) 5.88 - 5.96 (m, 1 H) 6.40 (s, 2 H) 7.19 - 7.25 (m, 1 H) 7.28 - 7.35 (m, 2 H) 7.36 - 7.40 (m, 1 H) 7.42 (d, J= 7.82 Hz, 1 H) 7.50 (d, J=6.85 Hz, 1 H) 7.56 (d, J= 7.82 Hz, 1 H).
[00275]Example 12. 1 -Ethyl-N-((£)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en- 1 -yl)-4-(hydroxymethyl)-3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3- methyl-1 H-pyrazole-5-carboxamide
Figure imgf000079_0001
[00276] (£)-(2-Nitro-3-((4-((2-nitrophenyl)amino)but-2-en-1- yl)amino)phenyl)methanol
Figure imgf000079_0002
[00277]To a solution of (E)-A/f-(2-nitrophenyl)but-2-ene-1 ,4-diamine (630 mg,
3.04 mmol) in isopropanol (12 ml_) was added DIPEA (2.65 ml_, 15.20 mmol), followed by (3-fluoro-2-nitrophenyl)methanol (520 mg, 3.04 mmol). The reaction mixture was stirred for 2 days at 100 °C. After cooling to room temperature, the mixture was concentrated and the residue was diluted with water and extracted with ethyl acetate. The organic extract was dried over anhydrous MgS04. It was filtered and the filtrate was concentrated. The crude product was purified on by silica gel chromatography eluting with a gradient of 0 to 60% ethyl acetate in heptane to give the title compound (570 mg, 1.6 mmol, 52% yield) as an orange solid. LC-MS mlz 359.3 (M+H)+, 1.06 min (ret. time).
[00278](E)-(2-Amino-3-((4-((2-aminophenyl)amino)but-2-en-1 - yl)amino)phenyl)methanol
Figure imgf000079_0003
[00279]To a suspension of (E)-(2-nitro-3-((4-((2-nitrophenyl)amino)but-2-en-1 - yl)amino)phenyl)methanol (560 mg, 1.6 mmol) in methanol (30 ml_) was added ammonium hydroxide (28% in water) (8.69 ml_, 62.5 mmol), followed by a solution of sodium hydrosulfite (2721 mg, 15.6 mmol) in water (10 ml_). The reaction mixture was stirred for 2 hours at room temperature. It was filtered, washed with methanol and water. The filtrate was concentrated and extracted with ethyl acetate. The organic extract was washed with water and dried over anhydrous MgS04. It was filtered and the filtrate was concentrated to give the title compound (350 mg, 1 .2 mmol, 75% yield) as an off-white solid. LC-MS mlz 299.4 (M+H)+, 0.36 min (ret. time).
[00280](£)-1 -Ethyl-N-(1 -(4-(2-(1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamido)- 1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(hydroxymethyl)-1 H-benzo[d]imidazol-2- yl)-3-methyl-1 H-pyrazole-5-carboxamide
Figure imgf000080_0001
[00281]To a solution of (E)-(2-amino-3-((4-((2-aminophenyl)amino)but-2-en-1 - yl)amino)phenyl)methanol (350 mg, 1 .2 mmol) in A/,A/-dimethylformamide (DMF) (10 mL) was added 1 -ethyl-3-methyl-1 /-/-pyrazole-5-carbonyl isothiocyanate 1 .0 M in dioxane (2.3 mL, 2.3 mmol). The mixture was stirred for 3 hours, EDC (675 mg, 3.52 mmol) was added, followed by TEA (0.981 mL, 7.04 mmol). The reaction mixture was stirred for 3 days at room temperature. The mixture was added to vigorously-stirring water dropwise. The resulting precipitate was filtered, washed with water and dried to give the title compound (652 mg, 1 .05 mmol, 90% yield) as a white solid. LC-MS mlz 621 .4 (M+H)+, 1 .06 min (ret. time).
[00282] 1 -Ethyl-N-((£)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4- (hydroxymethyl)-3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide
Figure imgf000081_0001
[00283]To a solution of (E)-1 -ethyl-A/-(1 -(4-(2-(1 -ethyl-3-methyl-1 /-/-pyrazole-5- carboxamido)-1 /-/-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(hydroxymethyl)-1 H- benzo[d]imidazol-2-yl)-3-methyl-1 /-/-pyrazole-5-carboxamide (650 mg, 1 .5 mmol) and cesium carbonate (1365 mg, 4.19 mmol) in A/,A/-dimethylformamide (DMF) (12 mL) was added iodomethane (0.196 mL, 3.14 mmol) at 0 °C. The ice bath was removed and the reaction mixture was stirred for 1 hour. The mixture was added to water with stirring. The resulting precipitate was filtered and washed with water. The solid was purified by reverse-phase [Waters XSelect 5 pm C18, 150 X 30 mm, 15-55% CH3CN:H20, 0.1 % formic acid as modifier] to give the title compound (280 mg, 0.432 mmol, 41 % yield) as a white solid. LC-MS m/z 649.5 (M+H)+, 0.80 min (ret. time). Ή NMR (400 MHz, DMSO -cfe) d ppm 1 .23 (q, J= 7.09 Hz, 6 H) 2.14 (d, J= 1 .22 Hz, 6 H) 3.53 (s, 3 H) 3.77 (s, 3 H) 4.46 (quin, J= 7.09 Hz, 4 H) 4.74 (br. s., 4 H) 4.84 (d, J=5.14 Hz, 2 H) 5.54 (t, J=5.38 Hz, 1 H) 5.80 (s, 2 H) 6.43 (s, 1 H) 6.45 (s, 1 H) 7.13 - 7.27 (m, 3 H) 7.29 - 7.34 (m, 1 H) 7.39 (t, J=8.44 Hz, 2 H) 7.56 (d, J= 8.07 Hz, 1 H).
[00284] Exam pie 13-1. 1 -Ethyl-N-((£)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en- 1-yl)-3-methyl-4-(morpholinomethyl)-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3- methyl-1 H-pyrazole-5-carboxamide
Figure imgf000081_0002
N-((£)-4-(Chloromethyl)-1 -((£)-4-((£)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 - yl)-3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H- pyrazole-5-carboxamide
Figure imgf000082_0001
[00285]To a solution of 1 -ethyl-A/-((£)-1 -((£)-4-((£)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 /-/-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)- 4-(hydroxymethyl)-3- methyl-1 ,3-dihydro-2/-/-benzo[d]imidazol-2-ylidene)-3-methyl-1 /-/- pyrazole-5-carboxamide (155 mg, 0.239 mmol) in dichloromethane (DCM) (2.0 ml_) was added thionyl chloride (0.105 ml_, 1 .433 mmol). The reaction mixture was stirred for 2 hours at room temperature. The mixture was concentrated to give the title compound (160 mg, 0.24 mmol, 100% yield) as a white solid. LC-MS m/z 667.4 (M+H)+, 0.97 min (ret. time).
[00286] 1 -Ethyl-N-((£)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3- methyl-4-(morpholinomethyl)-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl- 1H-pyrazole-5-carboxamide
Figure imgf000082_0002
[00287]A mixture of A/-((E)-4-(chloromethyl)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 /-/-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)- 3-methyl-1 ,3-dihydro-2/-/-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamide (35 mg, 0.052 mmol), morpholine (0.023 mL, 0.262 mmol) and DIPEA (0.027 mL, 0.157 mmol) in ethanol (1 .5 mL) was stirred for 2 hours at 70 °C. It was concentrated and the residue was purified by reverse-phase HPLC [Waters XSelect 5 pm C18, 150 X 30 mm, 15-55% CH3CN:H20, 0.1 % formic acid as modifier] to give the title compound (28 mg, 0.039 mmol, 74% yield) as a white solid. LC-MS mlz 718.5 (M+H)+, 0.68 min (ret. time). Ή NMR (400 MHz, DMSO -cfe) d ppm 1 .22 (td, J=7.09, 3.67 Hz, 6 H) 2.13 (s, 6 H) 2.40 (br. s., 4 H) 3.53 (s, 3 H) 3.54 (br. s„ 4 H) 3.76 (s, 2 H) 3.86 (s, 3 H)
4.44 (qd, J=7.01 , 2.69 Hz, 4 H) 4.73 (br. s., 4 H) 5.80 - 5.86 (m, 2 H) 6.42 (s, 1 H) 6.44 (s, 1 H) 7.1 1 - 7.16 (m, 2 H) 7.18 - 7.23 (m, 1 H) 7.31 (t, J=7.83 Hz, 1 H) 7.37 - 7.42 (m, 2 H) 7.56 (d, J= 8.07 Hz, 1 H).
[00288]Example compounds 13-2, 14 and 15 in Table 1 were prepared analogously to the synthetic sequence described for Example 13-1 .
Example 16. (rac)-1 -Ethyl-N-((£)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1- yl)but-2-en-1 -yl)-4-(1 -hydroxyethyl)-3-methyl-1 ,3-dihydro-2H- benzo[d]imidazol-2-ylidene)-3-methyl-1H-pyrazole-5-carboxamide
Figure imgf000083_0001
1 -Ethyl-N-((£)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 - yl)-4-formyl-3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl- 1 H-pyrazole-5-carboxamide
Figure imgf000083_0002
[00289]To a solution of 1 -ethyl-A/-((E)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 /-/-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)- 4-(hydroxymethyl)-3- methyl-1 ,3-dihydro-2/-/-benzo[d]imidazol-2-ylidene)-3-methyl-1 /-/- pyrazole-5-carboxamide (60 mg, 0.092 mmol) in dichloromethane (DCM) (2 mL) was added activated manganese dioxide (161 mg, 1 .850 mmol). The mixture was stirred for 20 hours at room temperature. It was filtered and the filtrate was concentrated to give the title compound as a light yellow solid. LC-MS m/z 647.4 (M+H)+, 0.90 min (ret. time).
1 -Ethyl-N-((£)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(1-hydroxyethyl)-3- methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1H-pyrazole-5- carboxamide
Figure imgf000084_0001
[00290]To a suspension of 1 -ethyl-A/-((E)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 /-/-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)- 4-formyl-3-methyl-1 ,3-dihydro-2/-/-benzo[d]imidazol-2-ylidene)-3-methyl-1 /-/-pyrazole-5- carboxamide (50 mg, 0.077 mmol) in tetrahydrofuran (THF) (2.0 mL) was added methylmagnesium bromide 3.4 M in 2-THF (0.1 14 mL, 0.387 mmol). The mixture was stirred for 30 min at room temperature. The mixture was diluted with saturated NH4CI (aq) and extracted with ethyl acetate. The organic extract was concentrated and the crude product was purified by reverse-phase HPLC [Waters XSelect 5 pm C18, 150 X 30 mm, 15-55% CH3CN:H20, 0.1 % formic acid as modifier] to give the title compound (32 mg, 0.048 mmol, 63% yield) as a white solid. LC-MS m/z 663.5 (M+H)+, 0.84 min (ret. time).
1H NMR (400 MHz, DMSO -cfe) d ppm 1 .23 (td, J=7.09, 5.14 Hz, 6 H) 1 .50 (d, J=6.36 Hz, 3 H) 2.14 (d, J=0.98 Hz, 6 H) 3.53 (s, 3 H) 3.76 (s, 3 H) 4.46 (quin, J= 6.72 Hz, 4 H) 4.73 (br. s., 4 H) 5.36 - 5.44 (m, 1 H) 5.47 (d, J=4.89 Hz, 1 H) 5.80 (br. s., 2 H) 6.43 (s, 1 H) 6.45 (s, 1 H) 7.21 (q, J= 7.50 Hz, 2 H) 7.29 - 7.36 (m, 2 H) 7.40 (dd, J= 7.70, 3.79 Hz, 2 H) 7.56 (d, J= 7.82 Hz, 1 H).
Example 17. N,Ar-((2E,2'£)-Butane-1 ,4-diylbis(3-methyl-1 ,3-dihydro-2H- benzo[d]imidazole-1 -yl-2-ylidene))bis(1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide)
Figure imgf000085_0001
[00291 ]JV7,JV4-Bis(2-nitrophenyl)butane-1 ,4-diamine
Figure imgf000085_0002
[00292]A solution of 1 -fluoro-2-nitrobenzene (2.6 g, 18.2 mmol) and butane-1 ,4- diamine (800 mg, 9.1 mmol) in isopropanol (50 ml_) was stirred for 18 h at room temperature to form an orange suspension. The suspension was filtered and the filtrate was washed with isopropanol (3 x 50 mL), then dried under vacuum for 18 h to give the title compound ( 2.0 g, 6.1 mmol, 67% yield) which was carried forward without further purification. LC-MS m/z 353.1 (M+Na)+, 1 .29 min (ret. time). 1H NMR (400 MHz, DMSO- cfe) 5 ppm 8.16 (t, J=5.5 Hz, 2H), 8.06 (dd, J=8.7, 1 .6 Hz, 2H), 7.48-7.58 (m, 2H), 7.08 (d, J=8.0 Hz, 2H), 6.68 (ddd, J=8.4, 7.0, 1 .1 Hz, 2H), 3.43 (d, J=5.8 Hz, 4H), 1 .66-1 .80 (m, 4H).
[00293]N1 ,N1 '-(Butane-1 ,4-diyl)bis(benzene-1 ,2-diamine)
Figure imgf000085_0003
[00294]A suspension of A/f,A/4-bis-(2-nitrophenyl)butane-1 ,4-diamine (1 .9 g, 6.0 mmol) and zinc (0.4 g, 6.0 mmol) in methanol (60 mL) and acetic acid (6 mL) was stirred at 40 °C for 3 hours. After this duration, the reaction mixture was filtered and washed with DCM/MeOH (ca. 5:1 , 3 x 100 mL) to afford a solid residue and filtrate. The filtrate was concentrated, then dissolved in DCM (200 mL) and washed with saturated aqueous sodium bicarbonate solution (150 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated. The solid residue was then triturated sequentially with saturated aqueous sodium bicarbonate (50 mL) and DCM/MeOH (ca. 5:1 , 3 x 50 mL) and this procedure was repeated three times. The resulting biphasic mixture was separated and the aqueous was extracted with additional DCM (100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The two samples were combined to afford the title compound as a dark brown residue (1 .2 g, 4.4 mmol, 74% yield). LC-MS m/z 271 .2 (M+H)+, 0.46 min (ret. time). 1H NMR (400 MHz, DMSO-cfe) d ppm 6.47-6.57 (m, 4H), 6.32-6.46 (m, 4H), 4.47 (br. s„ 4H), 4.34 (t, J=4.9 Hz, 2H), 3.06 (d, J=5.0 Hz, 4H), 1 .61 -1 .81 (m, 4H).
[00295] W,W'-(Butane-1 ,4-diylbis(1 H-benzo[d]imidazole-1 ,2-diyl))bis(1 -ethyl-3- methyl-1 H-pyrazole-5-carboxamide)
Figure imgf000086_0001
[00296]To a solution of N1,N1 -(butane- 1 ,4-diyl)bis(benzene-1 ,2-diamine) (1 .2 g, 4.4 mmol) in A/,A/-dimethylformamide (DMF) (10 ml_) at room temperature was added 1 - ethyl-3-methyl-1 /-/-pyrazole-
[00297]5-carbonyl isothiocyanate (1 M in dioxane) (9.8 ml_, 9.8 mmol). The reaction was stirred at room temperature for 3 hours. Following this duration, EDC (2.6 g, 13.3 mmol) and Et3N (3.7 ml_, 26.6 mmol) were added and the reaction was stirred for 18 h at room temperature. The reaction mixture was then diluted with water (300 ml_) and stirred for 30 minutes. The resulting precipitate was filtered and washed with water and dried under vacuum to afford a brown residue. The residue was purified by silica gel chromatography (0-100% EtOAc in hexanes, 80 g column, elution at 100% EtOAc) to afford an off-white residue (1 .2 g, 2.0 mmol, 45% yield). LC-MS m/z 593.2 (M+H)+, 1 .03 min (ret. time). 1H NMR (400 MHz, DMSO -cfe) d ppm 12.68 (s, 2H), 7.39-7.58 (m, 4H), 7.08-7.30 (m, 4H), 6.59 (s, 2H), 4.57 (q, J=7.0 Hz, 4H), 4.27 (br. s., 4H), 2.1 1 (s, 6H),
1 .87 (br. s., 4H), 1 .30 (t, J=7.0 Hz, 6H).
[00298]N,N -((2£,2'£)-Butane-1 ,4-diylbis(3-methyl-1 ,3-dihydro-2H- benzo[d]imidazole-1 -yl-2-ylidene))bis(1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide)
Figure imgf000087_0001
[00299]A solution of A/,A/'-(butane-1 ,4-diylbis(1 /-/-benzo[d]imidazole-1 ,2-diyl))bis(1 - ethyl-3-methyl-1 /-/-pyrazole-5-carboxamide) (200 mg, 0.44 mmol), Mel (0.04 mL, 0.68 mmol), Cs2C03 (440 mg, 1 .4 mmol) in A/,A/-dimethylformamide (DMF) (2 mL) was stirred at room temperature for 18 h. Following this duration, the reaction was filtered and concentrated. The resulting residue was purified by reverse-phase HPLC (15-55% acetonitrile in water, 0.1 % formic acid) to afford the title compound as an off-white solid (122 mg, 0.20 mmol, 58% yield). LC-MS m/z 621 .3 (M+H)+, 0.75 min (ret. time). Ή NMR (400 MHz, DMSO-cfe) d ppm 7.47-7.58 (m, 4H), 7.21 -7.37 (m, 4H), 6.44 (s, 2H), 4.47 (q, J=7.1 Hz, 4H), 4.15 (br. s., 4H), 3.51 (s, 6H), 2.13 (s, 6H), 1 .75 (br. s., 4H), 1 .23 (t , J=7.0
Hz, 6H).
[00300]The compound of Example 18 is made generally according to the above scheme and the processes described in the Examples.
Example 19. 1 -Ethyl-N-((E)-3-(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-1 -methyl-4- (morpholinomethyl)-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide, Formic acid salt
Figure imgf000087_0002
tert-Butyl (4-((2-(hydroxymethyl)-6-nitrophenyl)amino)butyl)carbamate A mixture of commercially-available (2-fluoro-3-nitrophenyl)methanol (1 .05 g, 6.14 mmol), commercially-available tert-butyl (4-aminobutyl)carbamate (1.271 g, 6.75 mmol), and DIPEA (3.21 mL, 18.41 mmol) in isopropanol (10 mL) was stirred for 20 hours at 70 °C. The mixture was cooled room temperature and was concentrated. The residue was diluted with water and was extracted with ethyl acetate. The organic extract was washed with water, was dried over anhydrous MgS04, was filtered, and the filtrate was concentrated. Purification on the Combiflash eluting with a gradient of 0 to 40% ethyl acetate/ethanol (3:1) in heptane provided the title compound as a red oil (2.05 g, 6.04 mmol, 98% yield). LC-MS m/z 340.3 (M+H)+. 1H NMR (400 MHz, CHLOROFORM-d) d ppm 1.45 (s, 9 H) 1.54 - 1.60 (m, 2 H) 1 .62 - 1.71 (m, 2 H) 2.78 (br s, 1 H) 3.16 (q, J=6.6 Hz, 2 H) 3.32 (q, J=6.6 Hz, 2
H) 4.58 (br s, 1 H) 4.79 (d, J=5.4 Hz, 2 H) 6.87 (dd, J=8.5, 7.3 Hz, 1 H) 6.94 (br s, 1 H) 7.55 (dd, J= 7.3, 1.6 Hz, 1 H) 8.03 (dd, J=8.5, 1 .6 Hz, 1 H).
(2-((4-Aminobutyl)amino)-3-nitrophenyl)methanol, Dihydrochloride salt
Figure imgf000088_0001
To a solution of tert-butyl (4-((2-(hydroxymethyl)-6-nitrophenyl)amino)butyl)carbamate (2.04 g, 6.01 mmol) in methanol (15 ml_) was added 4.0 M HCI in dioxane (15.03 mL,
60.1 mmol). The reaction mixture was stirred for 2.0 hours at room temperature and was concentrated. The residue was triturated with dichloromethane and was filtered to provide the title compound as a white solid (1 .88 g, 6.02 mmol, 100% yield). LC-MS m/z 240.2 (M+H)+. Ή NMR (400 MHz, DMSO -cfe) d ppm 1 .53 - 1.62 (m, 4 H) 2.75 (br d, J=5.4 Hz, 2 H) 3.05 - 3.12 (m, 2 H) 3.17 (s, 1 H) 4.56 (s, 2 H) 6.83 (dd, J=8.3, 7.2 Hz, 1 H) 7.56 (dd, J= 7.2, 1.6 Hz, 1 H) 7.79 (dd, J=8.3, 1.6 Hz, 1 H) 8.01 (br s, 3 H).
(3-Nitro-2-((4-((2-nitrophenyl)amino)butyl)amino)phenyl)methanol
Figure imgf000088_0002
A mixture of (2-((4-aminobutyl)amino)-3-nitrophenyl)methanol, Dihydrochloride salt (651 mg, 2.086 mmol), commercially-available 1-fluoro-2-nitrobenzene (0.20 mL, 1 .897 mmol), and DIPEA (1 .656 mL, 9.48 mmol) in isopropanol (10 mL) was stirred for 20 hours at 80 °C. The mixture was cooled to room temperature and was concentrated. The residue was diluted with water, was extracted with ethyl acetate. The organic extract was washed with water, was dried over anhydrous MgS04, was filtered, and the filtrate was concentrated. Purification on the Combiflash eluting with a gradient of 0 to 50% ethyl acetate in heptane provided the title compound as an orange solid (485 mg, 1 .346 mmol, 71 % yield). LC-MS m/z 361 .3 (M+H)+. 1H NMR (400 MHz, CHLOROFORM-d) d ppm 1 .83 (m, 4 H) 2.27 (t, J=5.9 Hz, 1 H) 3.38 (q, J=6.4 Hz, 4 H) 4.80 (d, J= 597 Hz, 2 H) 6.68 (td, J= 7.7, 1 .2 Hz, 1 H) 6.78 - 6.93 (m, 2 H) 6.93 - 6.98 (m, 1 H) 7.46 (t, J=6.9 Hz, 1 H) 7.55 (d, J=5.9 Hz, 1 H) 8.03 (dd, J=8.3, 1 .4 Hz, 1 H) 8.06 (br s, 1 H) 8.19 (dd, J=8.8, 1 .5 Hz, 1 H).
(3-Amino-2-((4-((2-aminophenyl)amino)butyl)amino)phenyl)methanol
Figure imgf000089_0001
To a solution of (3-nitro-2-((4-((2-nitrophenyl)amino)butyl)amino)phenyl)methanol (0.48 g, 1 .332 mmol) in methanol (40 ml_) was added ammonium hydroxide (7.41 ml_, 53.3mmol), followed by sodium hydrosulfite (2.319 g, 13.32 mmol) in water (10 ml_). The resulting mixture was stirred for 1 hour at room temperature and was concentrated. The residue was diluted with water and was extracted with ethyl acetate. The organic extract was washed with water, was dried over anhydrous MgS04, was filtered, and the filtrate was concentrated to give the title compound as a clear colorless oil (0.345 g, 1 .148 mmol, 86% yield). LC-MS m/z 301 .3 (M+H)+. Ή NMR (400 MHz, CHLOROFORM-d) d ppm 1 .74 - 1 .86 (m, 4 H) 3.05 (t, J=6.6 Hz, 2 H) 3.19 (t, J=6.6 Hz, 3 H) 3.26 - 3.57 (m, 2 H) 3.88 (br s, 2 H) 4.67 (s, 2 H) 6.64 (d, J= 1 .5 Hz, 1 H) 6.66 (d, J= 1 .5 Hz, 1 H) 6.67 - 6.69 (m, 1 H) 6.69 - 6.72 (m, 2 H) 6.74 (d, J= 1 .5 Hz, 1 H) 6.76 (d, J= 1 .5 Hz, 1 H) 6.82 - 6.92 (m, 2 H).
1 -Ethyl-N-(1 -(4-(2-(1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-1 H- benzo[d]imidazol-1 -yl)butyl)-7-(hydroxymethyl)-1 H-benzo[d]imidazol-2-yl)-3-methyl- 1 H-pyrazole-5-carboxamide
Figure imgf000089_0002
To a solution of (3-amino-2-((4-((2-aminophenyl)amino)butyl)amino)phenyl)methanol (0.34 g, 1 .132 mmol) in N,N-dimethylformamide (DMF) (6.0 mL) was added 1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl isothiocyanate 1 .0 M in dioxane (2.377 mL, 2.377 mmol). The mixture was stirred for 18 hours at room temperature, EDC»HCI (0.651 g, 3.40 mmol) was added, followed by TEA (0.947 mL, 6.79 mmol). The reaction mixture was stirred for an additional 18 hours at room temperature and was diluted with water. The resulting precipitate was filtered, was washed with ethyl acetate, and was dried to give the title compound as a white solid (447 mg, 0.718 mmol, 63.4% yield). LC-MS m/z 623.4 (M+H)+. Ή NMR (400 MHz, DMSO-cfe) d ppm 1 .27 - 1 .33 (m, 6 H) 1 .90 (br s, 3 H) 2.09 (s, 3 H) 2.10 (s, 3 H) 2.52 - 2.55 (m, 1 H) 4.18 - 4.38 (m, 2 H) 4.45 - 4.51 (m, 2 H) 4.57 (dq, J= 1 1 , 7.1 Hz, 4 H) 4.75 (d, J=5.4
Hz, 2 H) 5.53 (t, J=5.4 Hz, 1 H) 6.56 (s, 1 H) 6.59 (s, 1 H) 7.13 - 7.30 (m, 4 H) 7.48 - 7.57 (m, 3 H) 12.70 (br s, 1 H) 12.77 (br d, J= 7.8 Hz, 1 H).
1 -Ethyl-N-((E)-3-(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl- 2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-4-(hydroxymethyl)-1 -methyl-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide
Figure imgf000090_0001
To a mixture of 1 -ethyl-N-(1 -(4-(2-(1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamido)-1 H- benzo[d]imidazol-1 -yl)butyl)-7-(hydroxymethyl)-1 H-benzo[d]imidazol-2-yl)-3-methyl- 1 Hpyrazole-5-carboxamide (400 mg, 0.642 mmol) and cesium carbonate (837 mg, 2.57 mmol) in N,N-dimethylformamide (DMF) (8.0 mL) was added iodomethane (0.120 ml_, 1 .927 mmol) and mixture was stirred for 30 min at the room temperature. The mixture was filtered, was diluted with water, and was extracted with ethyl acetate. The organic extract was washed with water, was dried over anhydrous MgS04, was filtered, and the filtrate was concentrated. Purification on the Combiflash eluting with a gradient of 0 to 40% methanol/dichloromethane (1 :4) in dichloromethane provided the title compound as a white solid (250 mg, 0.384 mmol, 59.8% yield). LC-MS m/z 651 .4 (M+H)+. Ή NMR (400 MHz, DMSO-cfe) d ppm 1 .23 (t, J= 7.1 Hz, 6 H) 1 .67 - 1 .85 (m, 4 H) 2.13 (d, J= 1 .2 Hz, 6 H) 3.50 (s, 3 H) 3.51 (s, 3 H) 4.16 (t, J=6.4 Hz, 2 H) 4.36 (t, J= 6.7 Hz, 2 H) 4.47(q, J=7A Hz, 4 H) 4.71 (d, J=5.1 Hz, 2 H) 5.53 (t, J=5.3 Hz, 1 H) 6.44 (s, 1 H) 6.45 (s, 1 H) 7.24 - 7.35 (m, 4 H) 7.48 - 7.56 (m, 3 H).
N-((E)-1 -(4-((E)-7-(chloromethyl)-2-((1-ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)- 3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-3-methyl-1 ,3-dihydro-2H- benzo[d]imidazol-2-ylidene)-1-ethyl-3-methyl-1 H-pyrazole-5-carboxamide
Figure imgf000091_0001
To a solution of 1 -ethyl-N-((E)-3-(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-4-(hydroxymethyl)- 1 -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide (35 mg, 0.054 mmol) in dichloromethane (DCM) (1 .0 ml_) was added thionyl chloride (0.020 ml_, 0.269 mmol). The reaction mixture was stirred for 1 .0 hour at room temperature and was concentrated to provide the title compound as a white solid (35 mg, 0.052 mmol, 97% yield). LC-MS m/z 669.3 (M+H)+.
1 -Ethyl-N-((E)-3-(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl- 2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-1 -methyl-4-(morpholinomethyl)-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide, Formic acid salt
Figure imgf000091_0002
To a solution of N,N-((E)-1 -(4-((E)-7-(chloromethyl)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-3-methyl-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide (35 mg, 0.052 mmol) in ethanol (1 .5 ml_) was added morpholine (0.023 ml_, 0.261 mmol), followed by DIPEA (0.027 ml_, 0.157 mmol). The mixture was stirred for 2.0 hours at 70 °C, was cooled to room temperature, and was concentrated. Purification on the MDAP eluting with a gradient of 15 to 55% acetonitrile in water containing 0.1 % formic acid provide the title compound as a white solid (31 mg, 0.043 mmol, 82% yield). LC-MS m/z 720.4 (M+H)+.
Ή NMR (400 MHz, DMSO -d6) d ppm 1 .23 (dt, J=1 1 , 7.1 Hz, 6 H) 1 .78 (br s, 4 H) 2.13 (s,
3 H) 2.14 (s, 3 H) 2.20 - 2.28 (m, 4 H) 3.43 (br s, 4 H) 3.49 (s, 3 H) 3.52 (s, 3 H) 3.57 (s, 2 H) 4.15 - 4.22 (m, 2 H) 4.36 - 4.44 (m, 2 H) 4.44 - 4.52 (m, 4 H) 6.44 (s, 1 H) 6.45 (s, 1 H) 7.15 (d, J= 7.1 Hz, 1 H) 7.22 - 7.35 (m, 3 H) 7.48 - 7.57 (m, 3 H).
[00301 ]The compounds of Examples 20 to 31 are made generally according to the above scheme and the processes described in the Examples.
[00302] Exam pie 32 N-((£)-1 -((£)-4-((£)-7-(1 ,2-Dihydroxyethyl)-2-((1 -ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 - yl)but-2-en-1 -yl)-3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3- methyl-1 H-pyrazole-5-carboxamide
Figure imgf000092_0001
2-Fluoro-1 -nitro-3-vinylbenzene
Figure imgf000092_0002
A mixture of commercially-available 3-bromo-2-fluoronitrobenzene (2.0 g, 9.09 mmol), tributyl(vinyl)stannane (3.99 mL, 13.64 mmol), and
tetrakis(triphenylphosphine)palladium(0) (0.525 g, 0.455 mmol) in toluene (12 mL) was stirred for 20 hours at 100 °C. The mixture was cooled to room temperature, was diluted with water, and was extracted with ether. The organic extract was washed with brine, was dried over anhydrous MgSC>4, was filtered, and the filtrate was concentrated. Purification on the Combiflash eluting with a gradient of 0 to 5% ethyl acetate in heptane provided the title compound as clear, light-yellow liquid (1 .15 g, 6.88 mmol, 76% yield). 1H NMR (400 MHz, CHLOROFORM-d) d ppm 5.58 (d, J= 1 1 Hz, 1 H) 5.94 (d, J=18 Hz, 1 H) 6.90 - 6.98 (m, 1 H) 7.25 - 7.31 (m, 2 H) 7.80 (ddd, J= 7.7, 6.2, 1 .7 Hz,1 H) 7.94 (ddd, J=8.3, 6.9, 1 .5 Hz, 1 H). 1 -(2-Fluoro-3-nitrophenyl)ethane-1 ,2-diol
Figure imgf000093_0001
To a solution of 2-fluoro-1 -nitro-3-vinylbenzene (1 .12 g, 6.70 mmol) in tetrahydrofuran (THF) (10 ml_) was added osmium(VIII) oxide (2.5 wt.% in t-butanol) (4.21 ml_, 0.335 mmol), followed by NMO (1 .178 g, 10.05 mmol). The mixture was stirred for 18 hours at room temperature, was diluted with saturated Na2S203(aq), and was extracted with ethyl acetate. The organic extract was washed with water, was dried over anhydrous MgS04, was filtered, and the filtrate was concentrated to provide the title compound as yellow oil (1 .3 g, 6.46 mmol, 96% yield). Ή NMR (400 MHz, CHLOROFORM-d) d ppm 2.15 (br s, 1 H) 2.93 (br s, 1 H) 3.66 (br dd, J =1 1 , 7.8 Hz, 1 H) 3.97 (br d, J=10 Hz, 1 H)
5.26 (dd, J= 7.3, 2.5 Hz, 1 H) 7.35 (td, J=8.0, 1 .2 Hz, 1 H) 7.92 (ddd, J= 7.8, 5.6, 2.0 Hz, 1 H) 8.01 (td, J= 7.7, 1 .1 Hz, 1 H).
4-(2-Fluoro-3-nitrophenyl)-2,2-dimethyl-1 ,3-dioxolane
Figure imgf000093_0002
A mixture of 1 -(2-fluoro-3-nitrophenyl)ethane-1 ,2-diol (1 .25 g, 6.21 mmol), 2,2- dimethoxypropane (2.292 ml_, 18.64 mmol), and p-toluenesulfonic acid monohydrate (0.1 18 g, 0.621 mmol) in A/,A/-dimethylformamide (DMF) (10 ml_) was stirred for 2 days at room temperature, was diluted with saturated NaHC03(aq), and was extracted with ethyl acetate. The organic extract was washed with water, was dried over anhydrous MgS04, was filtered, and the filtrate was concentrated. Purification on the Combiflash eluting with a gradient of 0 to 20% ethyl acetate in heptane provided the title compound as clear, light-yellow oil (1 .13 g, 4.68 mmol, 75% yield). 1H NMR (400 MHz, CHLOROFORM-d) d ppm 1 .54 (s, 3 H) 1 .57 (s, 3 H) 3.75 -3.79 (m, 1 H) 4.53 (ddd, J=8.3, 6.0, 1 .7 Hz, 1 H) 5.42 (t, J=6.9 Hz, 1 H) 7.34 (td, J=8.1 , 1 .0 Hz, 1 H) 7.87 - 7.94 (m, 1 H) 8.00 (t, J= 7.6 Hz, 1 H). (E)-N7-(2-(2,2-dimethyl-1 ,3-dioxolan-4-yl)-6-nitrophenyl)-AM-(2- nitrophenyl)but-2-ene-1 ,4-diamine
Figure imgf000094_0001
A mixture of (E)-A/7-(2-nitrophenyl)but-2-ene-1 ,4-diamine, dihydrochloride salt (607 mg, 2.166 mmol), 4-(2-fluoro-3-nitrophenyl)-2,2-dimethyl-1 ,3-dioxolane (475 mg, 1.969 mmol) and DIPEA (1.720 ml_, 9.85 mmol) in isopropanol (5.0 ml_) was stirred for 20 hours at 80 °C. The mixture was cooled to room temperature and was concentrated. The residue was diluted with water and was extracted with ethyl acetate. The organic extract was dried over anhydrous MgS04, was filtered, and the filtrate was concentrated.
Purification on the Combiflash eluting with a gradient of 0 to 30% ethyl acetate in heptane provided the title compound as an orange oil (730 mg, 1.704 mmol, 87% yield). LC-MS m/z 429.2 (M+H)+.
(E)-N7-(4-((2-aminophenyl)amino)but-2-en-1 -yl)-6-(2,2-dimethyl-1 ,3-dioxolan- 4-yl)benzene-1 ,2-diamine
Figure imgf000094_0002
To a solution of (E)-A/1-(2-(2,2-dimethyl-1 ,3-dioxolan-4-yl)-6-nitrophenyl)-A/4-(2- nitrophenyl)but-2-ene-1 ,4-diamine (0.73 g, 1.704 mmol) in methanol (45 ml_) was added ammonium hydroxide (9.48 ml_, 68.2 mmol), followed by sodium dithionite (2.97 g, 17.04 mmol) in water (15 ml_). The reaction mixture was stirred for 1.0 hour at room
temperature and was concentrated. The residue was diluted with brine and was extracted with ethyl acetate. The organic extract was dried over anhydrous MgS04, was filtered, and the filtrate was concentrated. Purification on the Combiflash eluting with a gradient of 0 to 40% ethyl acetate/ethanol (3:1) in heptane provided the title compound as colorless oil (380 mg, 1.031 mmol, 60.5% yield). LC-MS m/z 369.3 (M+H)+. N-((£)-7-(2,2-dimethyl-1 ,3-dioxolan-4-yl)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamide
Figure imgf000095_0001
To a solution of (£)-A/1 -(4-((2-aminophenyl)amino)but-2-en-1 -yl)-6-(2,2-dimethyl- 1 ,3-dioxolan-4-yl)benzene-1 ,2-diamine (380 mg, 1 .031 mmol) in A/,A/-dimethylformamide (DMF) (6.0 mL) was added 1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl isothiocyanate 1 .0 M in dioxane (2.269 mL, 2.269 mmol). The mixture was stirred for 3 hours at room temperature, EDOHCI (593 mg, 3.09 mmol) was added, followed by TEA (0.862 mL,
6.19 mmol). The reaction mixture was stirred for 3 days at room temperature, was diluted with water, and was extracted with ethyl acetate. The organic extract was washed with water, was over anhydrous MgSC>4, was filtered, and the filtrate was concentrated.
Purification on the Combiflash eluting with a gradient of 0 to 40% ethyl acetate/ethanol (3:1) in heptane provided the title compound as white solid (560 mg, 0.81 1 mmol, 79% yield). LC-MS m/z 691 .6 (M+H)+. Ή NMR (400 MHz, DMSO -cfe) d ppm 1 .23 - 1 .32 (m, 6 H) 1 .40 (s, 2 H) 2.13 (d, J=7.34 Hz, 4 H) 2.74 (s, 2 H) 2.90 (s, 2 H) 3.77 (dd, J=8.1 , 7.09 Hz, 2 H) 4.18 (dd, J=8.3, 6.4 Hz, 1 H) 4.44 - 4.61 (m, 4 H) 4.82 (br d, J=4.9 Hz, 3 H) 4.88 (br s, 1 H) 5.10 (br d, J=16 Hz, 1 H) 5.41 (t, J=6.4 Hz, 1 H) 5.50 - 5.60 (m, 1 H) 5.94 - 6.03 (m, 1 H) 6.53 (d, J=8.3 Hz, 1 H) 7.1 1 - 7.25 (m, 3 H) 7.28 - 7.32 (m, 1 H) 7.38 (d,
J= 7.8 Hz, 1 H) 7.45 - 7.59 (m, 2 H) 7.96 (s, 1 H) 12.67 (br s, 1 H) 12.83 (br s, 1 H).
N-((E)-1 -((£)-4-((£)-7-(2,2-dimethyl-1 ,3-dioxolan-4-yl)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1-yl)but-2-en- 1 -yl)-3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H- pyrazole-5-carboxamide
Figure imgf000096_0001
To a solution of A/-((E)-7-(2,2-dimethyl-1 ,3-dioxolan-4-yl>-1 -((E)-4-((£)-2-((1 -ethyl- 3-methyl-1 H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 - yl)-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamide (560 mg, 0.81 1 mmol) in A/,A/-dimethylformamide (DMF) (6.0 mL) was added cesium carbonate (1056 mg, 3.24 mmol), followed by iodomethane (0.152 mL, 2.432 mmol). The reaction mixture was stirred for 1 .0 hour at room temperature, was diluted with water, and was extracted with ethyl acetate. The organic extract was washed with water, was dried over anhydrous MgS04, was filtered, and the filtrate was concentrated. Purification on the Combiflash eluting with a gradient of 20 to 80% ethyl acetate/ethanol (3:1) in heptane provided the title compound as white solid (400 mg,
0.556 mmol, 68.6% yield). LC-MS m/z 719.4 (M+H)+. Ή NMR (400 MHz, DMSO -cfe) d ppm 1 .18 - 1 .24 (m, 6 H) 1 .26 (s, 3 H) 1 .40 (s, 3 H) 2.12 (s, 3 H) 2.14 (s, 3 H) 3.53 (s, 3 H) 3.53 (s, 3 H) 3.80 (dd, J=8.6, 6.6 Hz, 1 H) 4.20 (dd, J=8.3, 6.4 Hz, 1 H) 4.44 (qd, J= 7.0, 3.9 Hz, 4 H) 4.72 - 4.81 (m, 3 H) 4.97 (br s, 1 H) 5.40 (t, J=6.4 Hz, 1 H) 5.52 (br d, J=16 Hz, 1 H) 5.94 (br d, J=16 Hz, 1 H) 6.39 (s, 1 H) 6.41 (s, 1 H) 7.19 - 7.24 (m, 1 H) 7.29 - 7.38 (m, 2 H) 7.38 - 7.41 (m, 1 H) 7.43 (d, J= 7.8 Hz, 1 H) 7.56 (d, J= 7.3 Hz, 2 H).
N-((E)-1 -((E)-4-((E)-7-(1 ,2-Dihydroxyethyl)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3- methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamide
Figure imgf000097_0001
To a solution of A/-((£)-1 -((£)-4-((£)-7-(2,2-dimethyl-1 ,3-dioxolan-4-yl)-2-((1 -ethyl- 3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 - yl)but-2-en-1 -yl)-3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl- 1 H-pyrazole-5-carboxamide (380 mg, 0.529 mmol) in methanol (8.0 ml_) was added 4.0 M HCI in dioxane (2.0 mL, 8.00 mmol). The reaction mixture was stirred for 2.0 hours at room temperature, and the mixture was concentrated to provide the title compound as white solid (350 mg, 0.516 mmol, 98% yield). LC-MS m/z 679.5 (M+H)+.
[00303]Example 33. 1 -Ethyl-N-((£)-3-((£)-4-((£)-2-((1 -Ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en- 1 -yl)-4-(1 -hydroxy-2 -morpholinoethyl)-1 -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2- ylidene)-3-methyl-1 H-pyrazole-5-carboxamide
Figure imgf000097_0002
2-((E)-2-((1-Ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-((E)-4-((E)- 2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H- benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-1 -methyl-2, 3-dihydro-1 H- benzo[d]imidazol-4-yl)-2-hydroxyethyl methanesulfonate
Figure imgf000098_0001
To a suspension of N-((E)-1 -((E)-4-((E)-7-(1 ,2-dihydroxyethyl)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)- 3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamide (0.9 g, 1 .326 mmol) in Dichloromethane (DCM) (25 ml_) was added TEA (1 .109 ml_, 7.96 mmol) followed by mesyl-CI (0.207 ml_, 2.65 mmol). The reaction mixture was stirred for 10 min at room temperature. The reaction was monitored by LC-MS. To the mixture 0.2 ml_ of TEA was added and the reaction was stirred for 5 min. The reaction was monitored by LC-MS again. To the mixture was added additional 0.2 mL of TEA and 0.05 mL of mesyl-CI and it was stirred for 5 min. The reaction was determined complete. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic extract was washed with water and dried over anhydrous MgS04. It was filtered and the filtrate was concentrated to give the title compound (1 .0 g, 1 .189 mmol, 90% purity, 90% yield) as white solid used as the intermediate without further purification. LC-MS m/z 757.6 (M+H)+, 0.82 min (ret. time).
1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 - yl)-1 -methyl-4-(oxiran-2-yl)-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3- methyl-1 H-pyrazole-5-carboxamide
Figure imgf000098_0002
To a solution of 2-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-((E)-4-((E)-2-
((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro- 1 Hbenzo[d]imidazol-1 -yl)but-2-en-1 -yl)-1 -methyl-2, 3-dihydro- 1 H-benzo[d]imidazol-4-yl)-2- hydroxyethyl methanesulfonate (1 .0 g, 1 .321 mmol) in Methanol (25 mL) was added potassium carbonate (0.548 g, 3.96 mmol). The reaction mixture was stirred for 2 hours at room temperature. It was filtered through celite and the filtrate was concentrated. The residue was diluted with water and extracted with ethyl acetate. The organic extract was washed with water and dried over anhydrous MgS04 and the slurry was filtered. The filtrate was concentrated to give the title compound (0.9 g, 1 .308 mmol, 99% yield) used as the intermediate without further purification. LC-MS m/z 661 .7 (M+H)+, 0.88 min (ret. time). Ή NMR (400 MHz, CHLOROFORM-d) d ppm 1 .41 (td, J=7.09, 3.42 Hz, 6 H) 2.29 (s, 3 H) 2.30 (s, 3 H) 2.83 (dd, J=5.38, 2.45 Hz, 1 H) 3.01 - 3.12 (m, 2 H) 3.62 (s, 3 H)3.63
(s, 3 H) 4.60 - 4.67 (m, 4 H) 4.71 - 4.77 (m, 2 H) 5.04 (br dd, J=12.47, 3.18 Hz, 2 H) 5.68 (br d, J=15.65 Hz, 1 H) 5.90 (br d, J=15.65 Hz, 1 H) 6.57 (s, 1 H) 6.58 (s, 1 H) 7.15 - 7.20 (m, 3 H) 7.25 - 7.31 (m, 4 H).
[00304] 1 -Ethyl-N-((£)-3-((£)-4-((£)-2-((1 -Ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(1 - hydroxy-2 -morpholinoethyl)-1 -methyl-1 , 3-dihydro-2H-benzo[d]imidazol-2 -ylidene)- 3-methyl-1 H-pyrazole-5-carboxamide
Figure imgf000099_0001
To a solution of 1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-1 -methyl-4- (oxiran-2-yl)-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide (0.9 g, 1 .362 mmol) in Ethanol (6.0 mL) was added morpholine (1 .187 mL, 13.62 mmol). The reaction mixture was stirred for 20 hours at 75 °C. The mixture was concentrated and the residue was purified on the mass directed autopurification (MDAP) system (formic acid, extended method B, 8 injections) to give the title compound (612 mg, 0.771 mmol, 56.6% yield) as white solid. LC-MS m/z 748.5 (M+H)+, 0.59 min (ret. time). 1H NMR (400 MHz, DMSO-cfe) d ppm 1 .20 (t, J=6.85 Hz, 6 H) 2.12 (s, 3 H) 2.13 (s, 3 H) 2.19 - 2.26 (m, 2 H) 2.27 - 2.34 (m, 2 H) 2.47 - 2.55 (m, 1 H) 2.56 - 2.64 (m, 1 H) 3.39 (t, J=4.28 Hz, 4 H) 3.52 (s, 3 H) 3.54 (s, 3 H) 4.42 (quin, J= 7.09 Hz, 4 H) 4.68 - 4.78 (m, 2 H) 4.87 - 4.96 (m, 1 H) 5.07 (d, J=19.56 Hz, 2 H) 5.36 (d, J=4.16 Hz, 1 H) 5.48 - 5.58 (m,
1 H) 5.94 (d, J=15.65 Hz, 1 H) 6.38 (s, 1 H) 6.39 (s, 1 H) 7.19 - 7.24 (m, 1 H) 7.33 (t,
J= 7.83 Hz, 2 H) 7.44 (dd, J=12.47, 7.58 Hz, 2 H) 7.50 (d, J= 8.07 Hz, 1 H) 7.57 (d, J=8.07 Hz, 1 H).
[00305]The compounds of Examples 34 to 38 are made generally according to the above scheme and the processes described in the Examples.
[00306] BIOLOGICAL ASSAYS
[00307] Cell Culture
[00308] Human and mouse macrophage cell lines and Kupffer cells are maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). An immortalized human hepatocyte-derived cell line supporting high level of HBV replication in a tetracycline (tet)-inducible manner, is maintained as described in Ladner, et al AAC 1997. Cryopreserved primary human hepatocytes (PHH) from Lonza are used in these experiments. Vials of cryopreserved PHH are placed in a 37°C water bath for approximately 70 seconds, just until thawed. The cells are pooled, resuspended gently in differentiation medium (Williams medium containing differentiation supplement, GlutaMax-1™, and penicillin/streptomycin) and counted using a
hemacytometer. The cells are pelleted by centrifugation at 100 X g for 10 min and resuspended to a density of 5.5 X 105 cells/mL in differentiation medium. 100 mί of cell suspension are plated in each well of collagen-coated 96-well plates. The plates are incubated at 37°C and 5% C02 for 2-4 days prior to infection.
[00309] Reagents
[00310] STING compounds can be made as described herein and as disclosed in International Patent Application PCT/IB2017/051945 filed April 5, 2016, titled
"HETEROCYCLIC AMIDES USEFUL AS PROTEIN MODULATORS”, the entire contents of which are incorporated by reference herein.
[00311]Recombinant murine IFN-a, IL-1 , IL-6 and TNF-a. can be purchased from PBL InterferonSource. Antibody against carboxyl terminal 14 amino acid of HBV core protein can be prepared as described previously in Xu C, et al. Interferons accelerate decay of replication-competent nucleocapsids of hepatitis B virus. J Virol 2010; 84:9332- 40. Antibodies against b-actin and mouse IFNAR-1 were obtained from Sigma-Aldrich and Santa Cruz Biotechnology, respectively. Antibodies against Human and mouse STING, TBK1 , S172-phosphorylated TBK1 , IkBa p38, phosphorylated-p38, JNK, phosphorylated-JNK, ERK, phosphorylated-ERK, can be purchased from Cell Signaling Technology.
[00312] Exam pie 39 [00313]HEK WT agonist assay:
[00314] Activation of STING in cells may be determined using a luciferase reporter assay in human embryonic kidney cells (HEK293T) co-transfected with plasmids expressing STING and the enzyme firefly luciferase driven by the interferon stimulated response element promoter (pISRE-Luc) (Agilent Technologies). Full-length human STING (Gene ID 340061) and full-length human cyclic guanine adenine synthase (cGAS) (reference sequence NIVM 38441.2) can be cloned into mammalian cell expression vectors containing a cytomegalovirus promoter. Transfections can be prepared using a cell suspension with Fugene® 6 following the manufacturer’s instructions (3:1
Fugene®:DNA). Fifty microliters of the transfection suspension are dispensed into wells of a 384-well plate containing 250 nl_ of a compound of Formula (I) as described herein, suitably a compound of Table 1 , or a pharmaceutically acceptable salt thereof. The final well composition contains ~20,000 cells/well, 1 ng STING, 20 ng pISRE-Luc, and empty vector pcDNA3.1 (lnvitrogen) to bring the total DNA concentration to 125 ng. Control wells expected to generate maximal activation of STING were cotransfected with a cGAS expression plasmid. Plates are sealed and incubated for 24 hours at 37 °C. The expression of firefly luciferase is processed using Steady-Glo® luciferase assay system (Promega) and can be analyzed using a standard laboratory luminescence plate reader. Data is normalized to luminescence response in the presence of cGAS, is plotted as a function of compound concentration, and can be fit using a standard model of receptor activation to derive the pEC5o.
[00315] Exam pie 40
[00316] Analyses of HBVDNA, RNA, HBsAg, and HBeAg
[00317]HBsAg levels can be measured using the Abazyme HBV s Ag kit, catalog # EL10018, according to the manufacturer’s instructions. HBeAg levels can be measured using the BioChain HBV e Ag kit, catalog #K031006096 according to the manufacturer’s instructions. For analysis of HBV DNA in the supernatant, DNA is extracted from 50 pL of supernatant using the QiaAmp 96 DNA Blood Kit (Qiagen) according to the
manufacturer’s instructions. 5 pl_ of DNA is used for TaqMan analysis using primers and conditions as described [Thimme R et al (2003) J. Virol. 77, 68-76] A standard curve based on known quantities of a plasmid clone of HBV DNA can be used to calculate HBV genome equivalents secreted in the media. Inhibition of cell viability, HBV DNA, and secreted antigens are normalized using values from cells undergoing the transfection procedure in the absence of oligonucleotide. Data are analyzed by non-linear regression and fit to a log(inhibitor) vs response curve using GraphPad Prism5.
[00318]Rea/-T/me PCR and RT-PCR Assays For cytokine gene expression analysis, total RNA is extracted using TRIzol reagent or RNeasy kit. cDNAs are synthesized using Superscript III (Invitrogen). Quantitative realtime PCR analysis is performed using a LightCycler 480 II (Roche).
[00319] Isolation of Non Parenchymal Cells from the Mouse Liver
[00320] The non-parenchymal cells (NPCs) such as liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC), natural killer (NK) and NK-T cells, dendritic cells, CD4+ and CD8+ T cells and hepatic stellate cells (HSC) are isolated from the liver as descried in Mohar et.al., Methods in Molecular Biology, 2015. This protocol permits the collection of peripheral blood, intact liver tissue, and hepatocytes, in addition to NPCs. In situ perfusion via the portal vein leads to efficient liver digestion. NPCs are enriched from the resulting single-cell suspension by differential and gradient centrifugation. The NPCs can be analyzed or sorted into highly enriched populations using flow cytometry. The isolated cells are suitable for flow cytometry, protein, and mRNA analyses as well as primary culture. For flow cytometry based analysis, epitopes of CD8a, CD4, CD11 b, NK1.1 , Tie2, F4/80, GR1 can be used for major liver NPC and leukocytes.
[00321]Example 41
[00322 ]lntracellular cytokine staining (ICS) assay
[00323] ICS assays can be performed on both splenocytes and liver mononuclear cells. Cells are seeded in Ubottom 96-well plates. Plates with cells are incubated overnight at 37 °C either in complete medium alone as negative control or with the peptides of HLA-A2/DR1 restricted HBV epitopes at a concentration of 2 pg/ml. The list of HBV specific epitopes are described in Bertoletti et al., Gastroenterology 1997, Pajot et al., Microbes Infect 2006, Sette et al., J Immuno 1994, Rehermann et al., J Exp Med 1995, Nayersina et al., J Immunol 1993, Loirat et al., J Immunol 2000, and Mizukoshi et al., J Immunol 2004. Brefeldin A at 2pg/ml_ (Sigma, B6542) is added after one hour of incubation. After the overnight culture, cells are washed with PBS FAGS and incubated with 5 pL of PBS FAGS containing rat anti-mouse CD16/CD32 antibody and a viability marker LD fixable yellow, Thermofisher, L34959 for 10 min in the dark at 4'C. Then, cells are stained for 20 min in the dark at 4'C with 25 pL of PBS FAGS containing Mab. The mix is composed of monoclonal antibodies against CD3 (hamster Mab anti-mouse CD3- PerCP, BD Biosciences, 553067), CD8 (rat Mab anti-mouse CD8-APC-H7, BD
Biosciences, 560182), CD4 (rat Mab anti-mouse CD4- PE-Cy7, BD Biosciences,
552775), and NK cells (Rat Mab anti mouse NK P46 BV421 , Biolegend, 137612). Cells are fixed after several washes and permeabilized for 20 min in the dark at room temperature with Cytofix/Cytoperm, washed with Perm/Wash solution (BD Biosciences, 554714) at 4 'C. 20 Intracellular cytokine staining with antibodies against IFNy (rat Mab anti-mouse IFNy-APC, clone XMG1 .2, BD Biosciences, 554413) and tumor necrosis factor alpha (TNFa) (rat Mab antimouse TNFa— FITC, clone MP6-XT22; 1/250 (BD Biosciences 554418) is performed for 30 min in the dark at 4 'C. Before analysis by flow cytometry using the MACSQuant Analyzer, cells are washed with Perm/Wash and resuspended in PBS FAGS containing 1 % Formaldehyde. Live CD3+CD8+CD4- and cells CD3+CD8-CD4+ are gated and presented on dot-plot. Two regions can be defined to gate for positive cells for each cytokine. Numbers of events found in these gates can be divided by total number of events in parental population to yield percentages of responding T cells. For each mouse, the percentage is obtained in medium alone is considered as background and subtracted from the percentage obtained with peptide stimulations. Threshold of positivity is defined according to experiment background i.e. the mean percentage of stained cells is obtained for each group in medium alone condition plus two standard deviations. Only percentage of cytokine representing at least 5 events is considered as positive.
[00324] Exam pie 42
[00325] Antiviral Efficacy of STING agonist in AA V-HB V Mouse Model
[00326] Male C57BL/6 mice (4-6 weeks old), specific pathogen free, are purchased from SLAG (Shanghai Laboratory Animal Center of Chinese Academy of Sciences) and housed in an animal care facility in individually ventilated cages.
Guidelines are followed for the care and of animals as indicated by WuXi IACUC
(Institutional Animal Care and Use Committee, WUXI IACUC protocol number
R20131126-Mouse). Mice are allowed to acclimate to the new environment for 3 days and are grouped according to the experimental design. Recombinant AAV-HBV is diluted in PBS, 200 pL per injection. This recombinant virus carriesl .3 copies of the HBV genome (genotype D, serotype ayw). On day 0, all mice is injected through tail vein with 200 pL AAV-HBV. On days 6, 13 and 20 after AAV injection, all mice in is
submandibularly bled (0.1 ml blood/mouse) for serum collection. On day 22 post injection, mice with stable viremia are ready for treatment.
[00327] Animals are tested for the efficacy of STING agonist in vivo at 30 ug as bolus intravenously for one week every 3-4 days. A group of mice are then sacrificed 24 h after treatment for detection of intrahepatic upregulation of cytokines (IFN-I, II, IL6, TNF, IL12, CXCL10) and interferon stimulated genes (OAS1 , IFH1) by measuring mRNA expression by real-time RT-PCR assay. Weight of individual mouse is monitored before and 24 h after the treatment. Liver tissues are collected from mice and fixed in 3.7% formaldehyde. Each liver is divided into six parts that are individually embedded in paraffin and sectioned. Liver sections are stained with hematoxylineosin or immunostained with a rabbit anti-HBcAg primary antibody (Dako, France). HBcAg- positive hepatocytes are analyzed by using ImageJ software (version 1 .43; W. S.
Rasband, U.S. NIH, Bethesda, MD). HBsAg and HBeAg in mouse sera are assayed with commercial enzyme-linked immunosorbent assay (ELISA) kits. The HBsAg concentration is calculated in ng/ml by reference to a standard curve established with known concentrations of HBsAg. Serum HBeAg levels are determined in 10-fold-diluted sera. Antibodies are quantified by ELISA as previously described in Mancini et.al., J. Med. Virol 1993. Alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) activities in sera of AAV8-HBV- and PBS injected mice are measured.
[00328] Exam pie 43
[00329 Establishment of Cell Culture System to Evaluate PRR Agonist-Induced Antiviral Response Against HBV
[00330] Unlike RIG-l-like receptors that are ubiquitously expressed in many types of somatic cells, expression of other PRRs, such as TLRs, cGAS STING, is usually restricted to macrophages, dendritic cells and a few other cell types. Due to the lack of expression or expression in a low amount of PRRs, such as STING and TLRs, treatment of hepatocytes usually does not induce a robust cytokine response. For instance, direct treatment of hepatocytes with TLR agonists induces a negligible cytokine response. However, liver resident dendritic cells, macrophages (Kupffer cells) and other hepatic nonparenchymal cells (NPCs) express high levels of TLRs and thus respond to TLR agonists and produce inflammatory cytokines.
[00331] To screen small molecular PRR agonists for treatment of chronic hepatitis B, a cell-based assay mimicking the intrahepatic environment can be used. HBV infected primary human hepatocytes (PHHs) are treated with testing compounds and the conditioned media of treated macrophages are then applied to HBV infected PHHs to test the compound-induced antiviral cytokine response in macrophages.
[00332] Exam pie 44
[00333] STING agonists Induce Antiviral Response in nonparenchymal cells in the liver to Suppress HBV Replication in Hepatocytes
[00334] Using the assay system described above, STING compounds of Formula (I), suitably a compound of Table 1 , or a pharmaceutically acceptable salt thereof, are tested for indirect, but not direct, antiviral activity against HBV. Briefly, HBV infected PHHs can be treated for 2 days with the varying concentrations of STING compound (Direct Treatment), and 50% of the conditioned media can be harvested from isolated human liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC), or NK cells or isolated human macrophages (to be treated with each of the STING compounds for 12 h) (Indirect Treatment). HBV infected PHH, RAW264.7 or macrophage cells can be mock-treated or treated with 0.5 mM to 125 mM of STING compound or 1 m9/hiI of LPS for 30 and 60 min. Expression and activation of STING is determined by Western blot assay. A shift in gel mobility will be seen as a result of STING phosphorylation b-actin can serve as a loading control. Intracellular HBV RNA is determined by qRT-PCR. HBV core protein is determined by Western blot assay using antibody against carboxyl terminal 14 amino acid of HBV core protein (b actin serves as loading control). HBV nucleocapsids are examined by a particle gel assay to detect both the intact nucleocapsids and capsid-associated HBV DNA. Encapsidated HBV (core DNA) replication intermediates (RC, DSL and SS) are determined by Southern blot assay.
[00335] Any antiviral response induced by STING compounds in nonparenchymal cell types such as LSEC, NK, KC is expected to post-transcriptionally reduce the amounts of HBV capsid protein and the assembled capsids. Consequentially, the amounts of HBV DNA replication intermediates are also expected to be decreased.
[00336] Exam pie 45
[00337] STI NG Compounds Are Expected to Induce a Distinct Profile of Cytokine Response
[00338] To determine the antiviral mechanism of the conditioned media that can be harvested from STING compound-treated LSEC, NK, KC cells, the signaling pathway activation and cytokine profiles induced by STING compounds in treated cells can be analyzed.
[00339] Cells are treated with STING compound in dose response for the varying times. Total cellular proteins can be fractioned by SDS-PAGE and transferred onto PVDF membranes. Total and phosphorylated STING, TBK1 , p38, JNK and Erk as well as IKBa can be detected by Western blot assays with their specific antibodies b-actin serves as a loading control.
[00340] STING compounds are expected to induce STING phosphorylation, which should be detectable in cells treated with STING compounds for more than 30 min.
STING compounds are expected to induce phosphorylation of TBK1 , a kinase essential for IRF3 phosphorylation and induction of IFN-b. in the STING pathways. Also, STING compounds are expected to induce degradation of IKBa.
[00341] The cytokine profiles induced by STING compounds can be determined by qRT-PCR assays. Cells are treated with STING compounds in dose response for varying times. The amounts of mRNA specifying the specific cytokines and chemokines can then be quantified by real-time RT-PCR assays. Data (mean.+-.standard deviation, N=3) can be expressed as fold induction of gene expression relative to untreated controls.
[00342] qRT-PCR assays is expected to show whether STING compounds induce an IFN response, and/or an inflammatory cytokine response.
[00343]_Example 46
[00344]Testing for Type I IFNs as Mediators of STING Compound-Induced Antiviral Response Against HBV.
[00345] To determine the role of type I IFNs and other cytokines in STING compound-induced antiviral response, one can investigate whether blockade of type I IFN response with a monoclonal antibody that specifically recognizes type I IFN receptor might attenuate any antiviral activity that may be induced by STING compounds in macrophages.
[00346] HBV-infected PHHs are incubated either with or without 10 pg/ml of a monoclonal antibody against type I interferon receptor IFNAR1 (Ab INFAR) at 37°C. for 1 h, followed by treatment for 2 days with varying concentrations of IFN-a. or 50% of conditioned media that can be harvested from macrophages (treated with the same varying concentrations of STING compounds for 12 h). Cytoplasmic HBV DNA can be quantified by a real-time PCR assay and data (mean. +-. standard deviation, N=4) can be represented as percentage of mock-treated controls. HBV-infected PHHs can be treated with the varying concentrations of IL-1 , IL-6 or TNF- a. for 4 days. Cytoplasmic HBV core DNA can then be analyzed by qPCR or Southern blot hybridization.
[00347] Blockade of type I IFN receptor in HBV-infected PHHs is expected to significantly reduced an antiviral response by IFN-a. Treatment of HBV-infected PHHs with the type I IFN receptor antibody may also attenuate an antiviral response by conditioned media from STING compound-treated macrophages, which could then indicate that type I IFNs may be mediators of STING compound-induced antiviral response against HBV.
[00348] To determine the role of other cytokines in STING compound-induced antiviral response, the antiviral effects of IL-1 , IL-6 and TNF-a can be tested to see which inhibit HBV DNA replication and which may thus play a role in any STING compound- induced antiviral response observed against HBV.
[00349] Exam pie 47
[00350] In vivo PK/PD of STING agonist in mice
[00351] To determine the target engagement upon STING agonist treatment in vivo, pharmacokinetics and pharmacodynamic relationship upon administration of STING agonist intravenously or orally is examined in C57BL/6 mice. Varying doses of STING agonist is administered along with vehicle only arm in 3 mice per group. At time points of 0, 1 h, 3h, 4h, 6h, and 24h, the mice are euthanized and blood, sera, liver, spleen, and draining lymph node are collected for measuring drug concentration in the liver and sera as well as responses of type I and II IFNs, and various cytokines including IL6, TNFa, IL12, IL7, CXCL10 in collected tissues.
[00352] To further understand which cell types are responsible for induction of cytokines in the liver, the nonparenchymal cells (NPC) is collected for qRT-PCR and flow cytometry as described earlier. Using cellular markers specific to each cell type, the cell types inducing type I IFNs can be identified.
[00353] The activation of STING is expected to induce strong upregulation of type I responses at early time points upon administration of the compound and to decline over time by 24 hours. The response is more prone to NPCs rather than hepatocytes, which is parenchymal cells in the liver.
[00354] Exam pie 48
[00355] STING Compound Inhibition of HBV Replication in Mice
[00356] To further validate the antiviral effects of STING compounds in vivo, C57BL/6 mice can be infected with AAV8-HBV to establish HBV replication in mice. After a month after infection, mice can be either treated with 3 doses of STING compound at 30ug or 100 ug or treated with just vehicle, intravenously for a week every 3 days.
Twenty-four hours after the last dose, livers and sera are collected and analyzed for HBsAg, HBV DNA, and cytokine responses. Four mice may be included in each of the control group and the STING compound treatment group. The number of HBV DNA copies/ml from each animal after subtraction of the copies from input plasmid can be plotted. Total RNA are extracted from livers and mRNA levels of cytokines can be analyzed by real-time RT-PCR assay. Plots are prepared to represent mRNA levels from each animal. All the data are presented in boxplots to indicate medians, interquartiles as well as ranges (min, max), and can be statistically analyzed by t-test.
[00357] Compared to the vehicle-treated control group, treatment of STING compound in mice is expected to reduce systemic and intrahepatic HBV DNA by 1- to 2- log after the course of treatment. In agreement with the anticipated antiviral mechanism, expression of representative IFN-stimulated genes (ISGs) and cytokines are expected to be induced in the livers of STING compound treated animals.
[00358] Example 49
[00359]/n vivo effect of STING Compound in a combination with an HBV inhibitor in Mice [00360]To understand the effect of STING agonist as immune modulator to break immune tolerance against HBV, it is examined along with an HBV antiviral treatment using AAV8-HBV mouse model. C57BL/6 mice are infected with AAV8-HBV for 30 days. Mice with persistent HBV infection are grouped and treated with a compound of Formula (I) as described herein, suitably a compound of Table 1 , or a pharmaceutically acceptable salt thereof, at 10mg/kg B.I.D daily or vehicle for 8 weeks. At 8 weeks post treatment, the treatment is combined with either STING agonist or vehicle for additional week at 30 ug intravenously over 3 days. Mice are maintained for 4 more weeks without further treatment. Sera are collected at scheduled times and a subset of mice in the group is euthanized at scheduled times and collected for tissues. All of the mice are euthanized at the end of the study and processed for HBV end points such as HBsAg, HBeAg, HBV DNA, intrahepatic HBV mRNA and cellular responses such as cytokine responses are measured by RT-PCR, ELISA, or qPCR. NPC are isolated and analyzed for the activation of STING pathway. The levels of HBV specific CD4 and CD8 T cells are determined using known HBV epitopes by tetramer staining. Anti-HBs are determined from collected sera.
[00361] Treatment of an HBV antiviral inhibitor known to suppress the level of HBsAg is expected to reduce the viral barrier so that the host immune response can be rejuvenated upon treatment of an immune modulator. After 8 week-long treatment of an antiviral followed by co-treatment with STING agonist, sustained response of
seroclearance and seroconversion is an indication of breaking immunotolerance against HBV. It can be confirmed by the increase of the frequency of IFNy producing CD4+ and CD8+ T cells in the liver and spleen that are specific to HBV epitopes.
[00362] Exam pie 50
[00363JFRET assay: The binding potency of molecules to the C-terminal Domain (CTD) of human STING was determined using a competition binding assay as described in WO2017/175156. In this assay, STING recombinant protein with a C-terminal biotinylated Avi-tag was employed. When bound to STING, an Alexa488-labeled orthosteric site probe accepted the 490 nm emission from Tb-Streptavidin-Avi-STING and an increase in fluorescence was measured at 520 nm. Molecules that compete for the probe binding site resulted in a low 520nm signal. The assay was run in Greiner black 384-well plates (Catalog # 784076) containing 125nL compounds in neat DMSO. A solution of 500pM STING, 500pM Streptavidin-Lumi4-Tb, and 100nM Alexa488 probe in phosphate buffered saline containing 0.02% (w/v) pluronic F127 and 0.02% (w/v) bovine serum albumin was added to the plate using a Combi liquid handler
(ThermoFisher). Plates were centrifuged for 1 min at 500rpm, incubated for 15min at room temperature, and then fluorescence emission at 520 nm following 337nm laser excitation on a PheraSTAR plate reader (BMG Labtech) was measured.
[00364]The fluorescence raw counts at 490nm and 520nm were used to calculate the HTRF ratio (fluorescence at 520nm/490nm) and the ratio was then expressed as % inhibition using the formula, %/
Figure imgf000109_0001
where U is the unknown value, C1 is the average low control response of the mixture without truncated STING (corresponding to amino acidsl 49-379 of the human STING enzyme) recombinant protein, and C2 is the average of maximum response. Curve fitting was performed using the equation Y = A + where A is the minimum response, B is the maximum response, C is the log™ *
Figure imgf000109_0002
XC5o, D is the slope factor, and x is the log™ compound concentration [M] in ABASE XE.
[00365] Exam pie 51
[00366]PBMC agonist assay: Activation of STING by compounds was determined by measuring levels of IFNp secreted from human peripheral blood mononuclear cells (PBMC, homozygous WT-STING) treated with different doses of compounds. Frozen PBMC cells were thawed, resuspended in media (RPMI-1640 with L-glutamine, 1 .5 g/L NaHC03, 4.5 g/L glucose, 10 mM Hepes, and 1 mM NaPyruvate + 10% FBS + 1 % Glutamax Supplement) to a final concentration of 3X105 cells/mL and dispensed into a 384-well tissue culture plate (Griener 781073) at a density of 15,000 cells per well containing 250 nl_ of compound diluted in DMSO. The level of IFNp protein secreted into the growth media was measured after four hours of incubation at 37 °C using a human IFNp electrochemiluminescence kit (Meso Scale Diagnostics) following the manufacturer’s instructions. For dose-response, the results of each test well were expressed as % activation, using the following normalization equation: N = 100 - 100*(CMPD-CTRL2)/(CTRL1 -CTRL2) where CMPD is the unknown value, CTRL1 is the average of the 100% activation control wells (using 750nM or a known full agonist), and C2 is the average of the 0% activation control wells (DMSO). Curve fitting was performed using the following equation: y = A+((B-A)/(1 +(10Ax/10AC)AD)) where A is the minimum response, B is the maximum response, C is the log™EC5o, and D is the Hill slope. The results for each compound were recorded as pECso values (-C in the above equation).
The results for each of example compounds 1 -17 were recorded (pEC5o) in Table 2, along with values for PBMC against %max, STING FRET IC50, and STING FRET %max.
[00367]The embodiments of the invention described above are intended to be merely exemplary; numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention as defined in any appended claims. Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the methods and that such changes and modifications can be made without departing from the spirit of the disclosed methods. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the methods.
[00368] The disclosures of each patent, patent application, and publication cited or described in this document are hereby incorporated herein by reference, in its entirety.
Table 1
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Table 2: Biological data
PBMC agonist assay: EC5o < 1 nM = +++++; 1 nM < EC5o < 10 nM = ++++; 10 nM < EC5o
< 100 nM = +++; 100 nM < EC5o < 500 nM = ++; EC5o ³ 500 nM
STING FRET assay: IC50 < 1 nM = +++++; 1 nM < IC50 < 10 nM = ++++; 10 nM < IC50 < 100 nM = +++; 100 nM < IC50 < 500 nM = ++; IC50 ³ 500 nM
Figure imgf000121_0001
The compound of Example 32 was tested in the above assays and found to have an activity (EC5o) of 5 nM in the above PBMC agonist assay, and an activity (IC5o) of 1 nM in the above STING FRET assay. Table 3:
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001

Claims

WHAT IS CLAIMED:
A compound according to Formula (I):
when
Figure imgf000125_0001
wherein said C2-C6alkyl or said C2-C6alkenyl is each independently optionally substituted by 1 -2 substituents selected from -Rc, -OH, and -ORc;
R1 and R2 are independently absent or Ci-C3alkyl;
W1, X1, Y1, and Z1 are each independently -CR3, -CR3', -CR3", -CR3"' or N, with the proviso that no more than two of W1, X1, Y1 and Z1 may be N;
W2, X2, Y2, and Z2 are each independently -CR4, -CR4', -CR4", -CR4"' or N, with the proviso that no more than two of W2, X2, Y2 and Z2 may be N;
R3, R3', R3", R3"', R4, R4', R4", R4"', R9, R10, R11, and R12 are each independently selected from H, Ci_4 alkoxyl, Ci_4 alkyl, -COO-Ci_4alkyl, -C4.7heterocycloalkyl, -Ci-4alkylC4. 7heterocycloalkyl, and -OCi-4alkylC4-7heterocycloalkyl, wherein the -C4-7heterocycloalkyl, the
-C4.7heterocycloalkyl of the -Ci-4alkylC4-7heterocycloalkyl, or the C4.7heterocycloalkyl of the
-OCi-4alkylC4.7heterocycloalkyl comprises one or more heteroatoms selected from O, and N,
wherein the Ci_4 alkyl, Ci_4 alkoxyl, the Ci_4 alkyl of the -Ci-4alkylC4.
7heterocycloalkyl or the Ci_4 alkyl of the -OCi-4alkylC4.7heterocycloalkyl is optionally substituted by 1 -4 substituents independently selected from -Rc, -OH and -ORc, and wherein the C4.7heterocycloalkyl, the C4.7heterocycloalkyl of the -Ci-4alkylC4. 7heterocycloalkyl, or the C4.7heterocycloalkyl of the -OCi-4alkylC4.7heterocycloalkyl is substituted by 1 -4 substituents R’, R”, R’”, and R””, wherein R’, R”, R’”, and R”” are each independently selected from -Rc, -OH and -ORc, or any two of R’, R”, R’”, and R”” may combine with the ring atoms to which they are attached to form a 3-7 membered ring;
R5 and R6 are independently Ci-C4alkyl;
R7 and R8 are each independently H or Ci-C4alkyl;
each Rc is independently Ci-C4alkyl optionally substituted by a substituent selected from -OH or Ci-4 alkoxyl;
or a salt thereof.
2. A compound of Formula (I) according to claim 1 , wherein R1 and R2 are -CH2- and R5 and R6 are -CH3, or a salt thereof.
3. A compound of Formula (I) according to any of claims 1 or 2, wherein W1 , X1 , Y1 , and Z1 are each independently -CR3, -CR3', -CR3" or -CR3"' and W2, X2, Y2, and Z2 are each independently -CR4, -CR4', -CR4" or -CR4"', or a salt thereof.
4. A compound of Formula (I) according to any of claims 1 to 3, wherein W1 is CR3 and W2 is CR4, wherein R3 and R4 are each independently selected from H, -C4.
yheterocycloalkyl, -Ci-4alkylC4-7heterocycloalkyl, -OCi-4alkylC4-7heterocycloalkyl, or a salt thereof.
5. A compound of Formula (I) according to any of claims 1 to 3, wherein W1 is CR3 and W2 is CR4, wherein R3 and R4 are each independently selected from H, Ci-4 alkoxyl or Ci_4 alkyl, wherein the Ci_4 alkyl or Ci_4 alkoxyl is optionally substituted by 1 -4 substituents independently selected from -Rc, -OH and -ORc, or a salt thereof.
6. A compound of Formula (I) according to any of claims 1 to 5, wherein A is
C2-C6alkylene, or a salt thereof.
7. A compound of Formula (I) according to any of claims 1 to 5, wherein A is
C2alkenylene, or a salt thereof.
8. A compound of Formula (I) according to any of claims 1 to 5, wherein R1 and R2 are each
-CH2- and A is C2-alkenylene, or a salt thereof.
9. A compound of Formula (I) according to any of claims 1 to 8, wherein R6 and R7 are each independently H or C2alkyl, or a salt thereof.
10. A compound, selected from the group consisting of:
N,N'-((2E,2'E)-((E)-but-2-ene-1 ,4-diyl)bis(3-methyl-1 , 3-dihydro-2H-benzo[d]imid azole-1 -yl- 2-ylidene))bis(1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide);
Methyl (E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-1 -((E)-4-((E)-2-((1 -ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but- 2-en-1 -yl)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazole-5-carboxylate;
1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-
2.3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-methoxy-1 -methyl- 1 ,3-dihydro-2H- benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-7-(3- hydroxypropoxy)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4- methoxy-1 -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
(E)-1 -Ethyl-N-(3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-
2.3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(hydroxymethyl)-1 -methyl-1 H- benzo[d]imidazol-2(3H)-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-
2.3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-1 -methyl-4-(morpholinomethyl)-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-
2.3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-1 -methyl-4-(piperidin-1 -ylmethyl)-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
N-((E)-1 -((E)-4-((E)-7-(azepan-1 -ylmethyl)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3-methyl-
1 .3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide; N-((E)-1 -((E)-4-((E)-7-((3-oxa-6-azabicyclo[3.1 .1 ]heptan-6-yl)methyl)-2-((1 -ethyl-3-methyl- 1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 - yl)-3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamide;
1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-
2.3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(methoxymethyl)-1 -methyl-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide; 1 -Ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-
2.3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(1 -hydroxyethyl)-1 -methyl-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-
2.3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(hydroxymethyl)-3- methyl- 1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-
2.3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3-methyl-4-(morpholinomethyl)-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-
2.3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3-methyl-4-(piperidin-1 -ylmethyl)-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
N-((E)-4-((3-oxa-6-azabicyclo[3.1 .1 ]heptan-6-yl)methyl)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl- 1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 - yl)-3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamide;
1 -Ethyl-N-((E)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-
2.3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(1 -hydroxyethyl)-3-methyl-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
N,N'-((2E,2'E)-Butane-1 ,4-diylbis(3-methyl-1 ,3-dihydro-2H-benzo[d]imidazole-1 -yl-2- ylidene))bis(1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide);
1 -ethyl-N-((E)-3-(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3- dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-4-(hydroxymethyl)-1 -methyl-1 ,3-dihydro-2H- benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3- dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-1 -methyl-4-(morpholinomethyl)-1 ,3-dihydro-2H- benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3- dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-1 -methyl-4-(piperidin-1 -ylmethyl)-1 , 3-d i hydro-2 H- benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
N-((E)-1 -(4-((E)-7-((3-oxa-6-azabicyclo[3.1 .1 ]heptan-6-yl)methyl)-2-((1 -ethyl-3-methyl-1 H- pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-3- methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5- carboxamide; 1 -ethyl-N-((E)-1 -(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3- dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-4-(hydroxymethyl)-3-methyl-1 ,3-dihydro-2H- benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-1 -(4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3- dihydro-1 H-benzo[d]imidazol-1 -yl)butyl)-3-methyl-4-(morpholinomethyl)-1 ,3-dihydro-2H- benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((2R,5R)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2, 3-dihydro- 1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-4-(hydroxymethyl)-1 -methyl-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((2R,5R)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-1 -methyl-4-(morpholinomethyl)-
1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((2S,5S)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-4-(hydroxymethyl)-1 -methyl-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((2S,5S)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-1 -methyl-4-(morpholinomethyl)-
1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((2S,5R)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-4-(hydroxymethyl)-1 -methyl-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((2S,5R)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-1 -methyl-4-(morpholinomethyl)-
1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((2R,5S)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-4-(hydroxymethyl)-1 -methyl-1 ,3- dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((2R,5S)-5-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)hexan-2-yl)-1 -methyl-4-(morpholinomethyl)-
1 .3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
N-((E)-1 -((E)-4-((E)-7-(1 ,2-dihydroxyethyl)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3-methyl-
1 .3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide; 1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-
2.3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(1 -hydroxy-2-morpholinoethyl)-1 - methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide; 1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl- 7-(morpholinomethyl)-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4- (hydroxymethyl)-l -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-1 -methyl-
1 .2-dihydro-3H-imidazo[4,5-b]pyridin-3-yl)but-2-en-1 -yl)-1 -methyl-4-(morpholinomethyl)-
1 .3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-1 -methyl-
1 .2-dihydro-3H-imidazo[4,5-c]pyridin-3-yl)but-2-en-1 -yl)-1 -methyl-4-(morpholinomethyl)-
1 .3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-
2.3-dihydro-1 H-imidazo[4,5-c]pyridin-1 -yl)but-2-en-1 -yl)-1 -methyl-4-(morpholinomethyl)-
1 .3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((E)-4-((Z)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-
2.3-dihydro-1 H-imidazo[4,5-b]pyridin-1 -yl)but-2-en-1 -yl)-1 -methyl-4-(morpholinomethyl)-
1 .3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl- 4-(morpholinomethyl)-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-1 -methyl-4- (morpholinomethyl)-l ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5- carboxamide;
or a salt thereof.
1 1 . A compound, selected from the group consisting of:
(E)-1 -Ethyl-N-(3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(hydroxymethyl)-1 -methyl- 1 H-benzo[d]imidazol-2(3H)-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
1 -Ethyl-N-((E)-1 -((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(hydroxymethyl)-3-methyl-
1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H-pyrazole-5-carboxamide;
N-((E)-4-((3-oxa-6-azabicyclo[3.1 .1 ]heptan-6-yl)methyl)-1 -((E)-4-((E)-2-((1 -ethyl-3- methyl-1 H-pyrazole-5-carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-
2-en-1 -yl)-3-methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H- pyrazole-5-carboxamide;
N-((E)-1 -((E)-4-((E)-7-(1 ,2-dihydroxyethyl)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5- carbonyl)imino)-3-methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-3-methyl- 1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-1 -ethyl-3-methyl-1 H-pyrazole-5-carboxamide; and
1 -ethyl-N-((E)-3-((E)-4-((E)-2-((1 -ethyl-3-methyl-1 H-pyrazole-5-carbonyl)imino)-3- methyl-2,3-dihydro-1 H-benzo[d]imidazol-1 -yl)but-2-en-1 -yl)-4-(1 -hydroxy-2- morpholinoethyl)-1 -methyl-1 ,3-dihydro-2H-benzo[d]imidazol-2-ylidene)-3-methyl-1 H- pyrazole-5-carboxamide;
or a pharmaceutically acceptable salt thereof.
12. The compound of any of claims 1 -9 wherein the compound is a compound of Table 1 .
13. The compound or a salt thereof according to any of claims 1 -10 or 12, wherein the salt is a pharmaceutically acceptable salt.
14. A method of inhibiting a hepatitis B virus in an animal comprising:
administering to the animal a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, according to claim 1 1 or 13.
15. A method of treating a Hepatitis B infection in a human infected with or at risk of being infected with a hepatitis B virus, the method comprising:
administering to the human a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, according to claim 1 1 or 13.
16. A method for reducing an amount of hepatitis B DNA and an amount of hepatitis B antigen in a mammal infected with a hepatitis B virus, the method comprising:
administering a therapeutically effective amount of a compound according to claim 1 1 or 13, or a pharmaceutically acceptable salt thereof, wherein the reduction of hepatitis B DNA and the reduction of Hepatitis B antigen is calculated by comparing to the amount of hepatitis B DNA and the amount of HBV antigen in the mammal before said
administration of the compound.
17. The method of claim 16, wherein the amount of hepatitis B DNA and an amount of hepatitis B antigen is reduced at least 90% compared to the amount before said administration of the compound.
18. A method of promoting seroconversion of a Hepatitis B virus in a mammal, the method comprising: administering a compound according to claim 1 1 or 13, or a pharmaceutically acceptable salt thereof;
monitoring for the presence of HBeAg plus HBeAb in a serum sample of the mammal; or
monitoring for the presence of HBsAg in a serum sample of the mammal, wherein the absence of HBeAg plus the presence of HBeAb in the serum sample, if monitoring HBeAg as the determinant for seroconversion, or
wherein the absence of HBsAg plus serum HBsAb presence in the serum sample, if monitoring HBsAg as the determinant for seroconversion, is evidence of seroconversion in the mammal, as determined by current detection limits of commercial ELISA systems.
19. Use of a compound of according to claim 11 or 13, in the manufacture of a medicament for use in therapy.
20. Use of a compound according to claim 11 or 13, in the manufacture of a
medicament for use in the treatment of HBV.
21 . A compound according to claim 11 or 13, for use in therapy.
22. A compound of according to claim 11 or 13, for use in the treatment of hepatitis B virus.
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021206158A1 (en) 2020-04-10 2021-10-14 小野薬品工業株式会社 Method of cancer therapy
US11155567B2 (en) 2019-08-02 2021-10-26 Mersana Therapeutics, Inc. Sting agonist compounds and methods of use
CN114163420A (en) * 2021-10-26 2022-03-11 中山大学附属第一医院 Endoplasmic reticulum Golgi matrix targeting small molecule, conjugate and application thereof
WO2023025256A1 (en) * 2021-08-26 2023-03-02 成都先导药物开发股份有限公司 Sting agonist suitable for use as effector molecule of antibody drug conjugate
WO2024182414A1 (en) * 2023-02-27 2024-09-06 Biontech Us Inc. Sting agonists containing hydrazide, hydrazine, and hydroxamic acid functional groups
WO2024186626A1 (en) * 2023-03-03 2024-09-12 Bolt Biotherapeutics, Inc. Aza-bicyclic sting agonist immunoconjugates, and uses thereof
US12156870B2 (en) 2020-04-02 2024-12-03 Mersana Therapeutics, Inc. Antibody drug conjugates comprising sting agonists

Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005087238A2 (en) 2004-03-15 2005-09-22 Karaolis David K R Method for stimulating the immune, inflammatory or neuroprotective response
WO2007054279A2 (en) 2005-11-08 2007-05-18 Helmholtz-Zentrum für Infektionsforschung GmbH Cyclic-dinucleotides and its conjugates as adjuvants and their uses in pharmaceutical compositions
WO2013166000A1 (en) 2012-04-30 2013-11-07 Barber Glen N Modulating immune responses
WO2013185052A1 (en) 2012-06-08 2013-12-12 Aduro Biotech Compostions and methods for cancer immunotherapy
WO2014093936A1 (en) 2012-12-13 2014-06-19 Aduro Biotech, Inc. Compositions comprising cyclic purine dinucleotides having defined stereochemistries and methods for their preparation and use
US20140341976A1 (en) 2013-05-18 2014-11-20 Aduro Biotech, Inc. Compositions and methods for inhibiting "stimulator of interferon gene" -dependent signalling
WO2014189805A1 (en) 2013-05-18 2014-11-27 Auro Biotech, Inc. Compositions and methods for activating "stimulator of interferon gene"-dependent signalling
WO2015077354A1 (en) 2013-11-19 2015-05-28 The University Of Chicago Use of sting agonist as cancer treatment
WO2017106740A1 (en) 2015-12-16 2017-06-22 Aduro Biotech, Inc. Methods for identifying inhibitors of "stimulator of interferon gene"-dependent interferon production
WO2017175156A1 (en) 2016-04-07 2017-10-12 Glaxosmithkline Intellectual Property Development Limited Heterocyclic amides useful as protein modulators
WO2017175147A1 (en) 2016-04-07 2017-10-12 Glaxosmithkline Intellectual Property Development Limited Heterocyclic amides useful as protein modulators
WO2019069293A1 (en) 2017-10-05 2019-04-11 Glaxosmithkline Intellectual Property Development Limited Chemical compounds
WO2019069270A1 (en) * 2017-10-05 2019-04-11 Glaxosmithkline Intellectual Property Development Limited Modulators of stimulator of interferon genes (sting)
WO2019134705A1 (en) * 2018-01-08 2019-07-11 成都先导药物开发股份有限公司 Immunomodulator

Patent Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005087238A2 (en) 2004-03-15 2005-09-22 Karaolis David K R Method for stimulating the immune, inflammatory or neuroprotective response
WO2007054279A2 (en) 2005-11-08 2007-05-18 Helmholtz-Zentrum für Infektionsforschung GmbH Cyclic-dinucleotides and its conjugates as adjuvants and their uses in pharmaceutical compositions
WO2013166000A1 (en) 2012-04-30 2013-11-07 Barber Glen N Modulating immune responses
WO2013185052A1 (en) 2012-06-08 2013-12-12 Aduro Biotech Compostions and methods for cancer immunotherapy
WO2014093936A1 (en) 2012-12-13 2014-06-19 Aduro Biotech, Inc. Compositions comprising cyclic purine dinucleotides having defined stereochemistries and methods for their preparation and use
US20140341976A1 (en) 2013-05-18 2014-11-20 Aduro Biotech, Inc. Compositions and methods for inhibiting "stimulator of interferon gene" -dependent signalling
WO2014189805A1 (en) 2013-05-18 2014-11-27 Auro Biotech, Inc. Compositions and methods for activating "stimulator of interferon gene"-dependent signalling
WO2015077354A1 (en) 2013-11-19 2015-05-28 The University Of Chicago Use of sting agonist as cancer treatment
WO2017106740A1 (en) 2015-12-16 2017-06-22 Aduro Biotech, Inc. Methods for identifying inhibitors of "stimulator of interferon gene"-dependent interferon production
WO2017175156A1 (en) 2016-04-07 2017-10-12 Glaxosmithkline Intellectual Property Development Limited Heterocyclic amides useful as protein modulators
WO2017175147A1 (en) 2016-04-07 2017-10-12 Glaxosmithkline Intellectual Property Development Limited Heterocyclic amides useful as protein modulators
WO2019069293A1 (en) 2017-10-05 2019-04-11 Glaxosmithkline Intellectual Property Development Limited Chemical compounds
WO2019069270A1 (en) * 2017-10-05 2019-04-11 Glaxosmithkline Intellectual Property Development Limited Modulators of stimulator of interferon genes (sting)
WO2019134705A1 (en) * 2018-01-08 2019-07-11 成都先导药物开发股份有限公司 Immunomodulator

Non-Patent Citations (53)

* Cited by examiner, † Cited by third party
Title
AGUIRRE ET AL., PLOS PATHOG, vol. 8, no. 10, 2012, pages e1002934
BARBER ET AL., NAT. REV. IMMUNOL., vol. 15, no. 2, 2015, pages 87 - 103
BERGE ET AL., J. PHARM. SCI., vol. 66, 1977, pages 1 - 19
BERTOLETTI ET AL., GASTROENTEROLOGY, 1997
BURDETTE D LVANCE R E, NATURE IMMUNOLOGY, vol. 14, 2013, pages 19 - 26
CAI X ET AL., MOLECULAR CELL, vol. 54, 2014, pages 289 - 296
CHEN ET AL., PROTEIN CELL, vol. 5, no. 5, 2014, pages 369 - 81
CHULANOV ET AL., J. MED. VIROL., vol. 69, 2003, pages 313
COTE ET AL., HEPATOLOGY, vol. 31, 2000, pages 190
CROW YJ ET AL., NAT. GENET., vol. 38, no. 8, 2006, pages 38917 - 920
DASS, C R., J PHARM PHARMACOL, vol. 54, no. 1, 2002, pages 3 - 27
DEN BROUW ET AL., IMMUNOLOGY, vol. 126, 2008, pages 280
DING ET AL., J HEPATOL, vol. 59, no. 1, 2013, pages 52 - 8
EVANS ET AL., HEPATOLOGY, vol. 48, 2008, pages 759
FRIEDMAN OHANA, R. ET AL., ACS CHEM. BIOL., 2016, pages 2608 - 2617
GAO ET AL., SCIENCE, vol. 341, no. 6148, 2013, pages 903 - 71
GAO P ET AL., CELL, vol. 153, 2013, pages 1094 - 1107
HERZNER ET AL., NAT IMMUNOL, vol. 16, no. 10, 2015, pages 1025 - 33
HOLM ET AL., NAT COMM., vol. 7, 2016, pages 10680
HUBER J.P. ET AL., J IMMUNOL, vol. 185, no. 5, 2010, pages 2551 - 817
ISAACSLINDEMANN, J. VIRUS INTERFERENCE. PROC. R. SOC. LON. SER. B. BIOL. SCI., vol. 147, 1957, pages 258 - 267
ISHIKAWA ET AL., NATURE, vol. 461, no. 7265, 2009, pages 788 - 92
ISHIKAWA HBARBER G N, NATURE, vol. 455, 2008, pages 674 - 678
JIN ET AL., J IMMUNOL., vol. 187, no. 5, 2011, pages 2595 - 601
KANNGEHRLICH: "Topley & Wilson's Microbiology and Microbial Infections", 1998, pages: 745
LIBANOVA R. ET AL., MICROBIAL BIOTECHNOLOGY, vol. 5, 2012, pages 168 - 176
LIU ET AL., J VIROL, vol. 90, no. 20, 2016, pages 9406 - 19
LOIRAT ET AL., J IMMUNOL, 2000
MA ET AL., PNAS, vol. 112, no. 31, 2015, pages E4306 - E4315
MADAMANIA, CELL HOST & MICROBE, vol. 19, no. 2, 2016, pages 150 - 158
MANCINI, J. MED. VIROL, 1993
MILICH ET AL., J. IMMUNOL., vol. 160, 1998, pages 8102
MIZUKOSHI ET AL., J IMMUNOL, 2004
MOHAR, METHODS IN MOLECULAR BIOLOGY, 2015
MOUCARI ET AL., HEPATOLOGY, vol. 49, 2009, pages 1151
N. MISHRA: "Efficient Hepatic Delivery of Drugs: Novel Strategies and Their Significance", BIOMED RESEARCH INTERNATIONAL, vol. 2013, 2013, pages 20
NATURE REVIEWS GENETICS, vol. 16, 2015, pages 543 - 553
NAYERSINA ET AL., J IMMUNOL, 1993
NITTA ET AL., HEPATOLOGY, vol. 57, no. 1, 2013, pages 46 - 58
PAJOT ET AL., MICROBES INFECT, 2006
REHERMANN ET AL., J EXP MED, 1995
REIGNAT ET AL., J. EXP. MED., vol. 195, 2002, pages 1089
SETTE ET AL., J IMMUNO, 1994
SHARMA ET AL., IMMUNITY, vol. 35, no. 2, 2011, pages 194 - 207
STETSON DB ET AL., CELL, vol. 134, 2008, pages 587 - 598
STOREK ET AL., J IMMUNOL., vol. 194, no. 7, 2015, pages 3236 - 45
SUN ET AL., PLOS ONE, vol. 7, no. 2, 2012, pages e30802
TAKEUCHI O. ET AL., CELL, vol. 140, 2010, pages 805 - 820
TEREZAWA ET AL., PEDIATR. RES., vol. 29, 1991, pages 5
THIMME R ET AL., J. VIROL., vol. 77, 2003, pages 68 - 76
WASSERMANN ET AL., CELL HOST MICROBE, vol. 17, no. 6, 2015, pages 811 - 810
XU C ET AL.: "Interferons accelerate decay of replication-competent nucleocapsids of hepatitis B virus", J VIROL, vol. 84, 2010, pages 9332 - 40, XP055367954, DOI: 10.1128/JVI.00918-10
ZHENG ET AL., J. VIRAL HEPATITIS, vol. 11, 2004, pages 217

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11155567B2 (en) 2019-08-02 2021-10-26 Mersana Therapeutics, Inc. Sting agonist compounds and methods of use
US11939343B2 (en) 2019-08-02 2024-03-26 Mersana Therapeutics, Inc. Sting agonist compounds and methods of use
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