WO2022214937A1 - Substituted tetracyclic carboxylic acids, analogues thereof, and methods using same - Google Patents

Substituted tetracyclic carboxylic acids, analogues thereof, and methods using same Download PDF

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Publication number
WO2022214937A1
WO2022214937A1 PCT/IB2022/053112 IB2022053112W WO2022214937A1 WO 2022214937 A1 WO2022214937 A1 WO 2022214937A1 IB 2022053112 W IB2022053112 W IB 2022053112W WO 2022214937 A1 WO2022214937 A1 WO 2022214937A1
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Prior art keywords
butyl
tert
imidazo
tetrahydropyrido
oxo
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PCT/IB2022/053112
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French (fr)
Inventor
Shuai Chen
Andrew G. Cole
Bruce D. Dorsey
Min Gao
Dimitar B. Gotchev
Troy Harasym
Michael J. Sofia
Sunny Ho-Kwong TANG
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Arbutus Biopharma Corporation
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Publication of WO2022214937A1 publication Critical patent/WO2022214937A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
    • C07D471/14—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses

Definitions

  • Hepatitis B is one of the world's most prevalent diseases. Although most individuals resolve the infection following acute symptoms, approximately 30% of cases become chronic. 350-400 million people worldwide are estimated to have chronic hepatitis B, leading to 0.5-1 million deaths per year, due largely to the development of hepatocellular carcinoma, cirrhosis, and/or other complications. Hepatitis B is caused by hepatitis B virus (HBV), a noncytopathic, liver tropic DNA virus belonging to Hepadnaviridae family.
  • HBV hepatitis B virus
  • a limited number of drugs are currently approved for the management of chronic hepatitis B, including two formulations of alpha-interferon (standard and pegylated) and five nucleoside/nucleotide analogues (lamivudine, adefovir, entecavir, telbivudine, and tenofovir) that inhibit HBV DNA polymerase.
  • the first-line treatment choices are entecavir, tenofovir, or peg-interferon alfa-2a.
  • peg-interferon alfa-2a achieves desirable serological milestones in only one third of treated patients and is frequently associated with severe side effects.
  • Entecavir and tenofovir require long-term or possibly lifetime administration to continuously suppress HBV replication, and may eventually fail due to emergence of drug-resistant viruses.
  • HBV is an enveloped virus with an unusual mode of replication, centering on the establishment of a covalently closed circular DNA (cccDNA) copy of its genome in the host cell nucleus.
  • Pregenomic (pg) RNA is the template for reverse transcriptional replication of HBV DNA. The encapsidation of pg RNA, together with viral DNA polymerase, into a nucleocapsid is essential for the subsequent viral DNA synthesis.
  • HBV envelope is a major factor in the disease process.
  • serum levels of HBV surface antigen can be as high as 400 ⁇ g/ml, driven by the propensity for infected cells to secrete non-infectious subviral particles at levels far in excess of infectious (Dane) particles.
  • HBsAg comprises the principal antigenic determinant in HBV infection and is composed of the small, middle and large surface antigens (S, M and L, respectively). These proteins are produced from a single open reading frame as three separate N-glycosylated polypeptides through utilization of alternative transcriptional start sites (for L and M/S mRNAs) and initiation codons (for L, M and S).
  • HBsAg Although the viral polymerase and HBsAg perform distinct functions, both are essential proteins for the virus to complete its life cycle and be infectious. HBV lacking HBsAg is completely defective and cannot infect or cause infection. HBsAg protects the virus nucleocapsid, begins the infectious cycle, and mediates morphogenesis and secretion of newly forming virus from the infected cell.
  • HBV people chronically infected with HBV are usually characterized by readily detectable levels of circulating antibody specific to the viral capsid (HBc), with little, if any detectable levels of antibody to HBsAg.
  • chronic carriers produce antibodies to HBsAg, but these antibodies are complexed with the circulating HBsAg, which can be present in mg/mL amounts in a chronic carrier's circulation. Reducing the amount of circulating levels of HBsAg might allow any present anti-HBsA to manage the infection. Further, even if nucleocapsids free of HBsAg were to be expressed or secreted into circulation (perhaps as a result of cell death), the high levels of anti-HBc would quickly complex with them and result in their clearance.
  • Hepatitis D virus is a small circular enveloped RNA virus that can propagate only in the presence of the hepatitis B virus (HBV).
  • HBV hepatitis B virus
  • HDV requires the HBV surface antigen protein to propagate itself. Infection with both HBV and HDV results in more severe complications compared to infection with HBV alone. These complications include a greater likelihood of experiencing liver failure in acute infections and a rapid progression to liver cirrhosis, with an increased chance of developing liver cancer in chronic infections.
  • hepatitis D In combination with hepatitis B virus, hepatitis D has the highest mortality rate of all the hepatitis infections.
  • the routes of transmission of HDV are similar to those for HBV. Infection is largely restricted to persons at high risk of HBV infection, particularly injecting drug users and persons receiving clotting factor concentrates.
  • the compounds can be used in patients that are HBV infected, patients who are at risk of becoming HBV infected, and/or patients that are infected with drug-resistant HBV.
  • the HBV-infected subject is further HDV-infected. The present invention addresses this need.
  • the disclosure provides a compound selected from: wherein R 1 , R 2 , R 3a , R 3b , R 4a , R 4b , R 6I , R 6II , R 6III , and R 6IV , and any other applicable variables, are defined elsewhere herein.
  • the disclosure further provides a pharmaceutical composition comprising at least one compound of the disclosure.
  • the disclosure further provides a method of treating, ameliorating, and/or preventing hepatitis B virus infection in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of at least one compound and/or at least one pharmaceutical composition of the disclosure.
  • the disclosure further provides a method of reducing or minimizing levels of at least one selected from the group consisting of hepatitis B virus surface antigen (HBsAg), hepatitis B e-antigen (HBeAg), hepatitis B core protein, and pregenomic (pg) RNA, in a HBV-infected subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of at least one compound and/or at least one pharmaceutical composition of the disclosure.
  • HBsAg hepatitis B virus surface antigen
  • HeAg hepatitis B e-antigen
  • pg pregenomic
  • FIG. 1 illustrates a non-limiting ORTEP drawing of Compound 1 in the asymmetric unit showing labeling scheme.
  • FIG. 2 illustrates non-limiting ORTEP representations of the four molecules of Compound 1 in the asymmetric unit.
  • FIG. 3 illustrates a non-limiting ORTEP drawing of Compound 19 showing labeling scheme.
  • FIG. 4 illustrates non-limiting ORTEP representations of the four molecules of Compound 19 in the asymmetric unit.
  • the disclosure relates, in certain aspects, to the discovery of certain substituted tetracyclic compounds that are useful to treat, ameliorate, and/or prevent HBV and/or HBV- HDV infection and related conditions in a subject.
  • the compounds inhibit and/or reduce HBsAg secretion in a HBV-infected subject.
  • the compounds reduce or minimize levels of HBsAg in a HBV-infected subject.
  • the compounds reduce or minimize levels of HBeAg in a HBV-infected subject.
  • the compounds reduce or minimize levels of hepatitis B core protein in a HBV-infected subject.
  • the compounds reduce or minimize levels of pg RNA in a HBV-infected subject.
  • the compounds have a developable metabolic stability.
  • the compounds have a developable liver-to-plasma distribution ratio.
  • the HBV-infected subject is further HDV-infected.
  • cccDNA covalently closed circular DNA
  • HBc hepatitis B capsid
  • HBV hepatitis B virus
  • HBeAg hepatitis B e-antigen
  • HBsAg hepatitis B virus surface antigen
  • MLM mouse liver microsome
  • pg RNA pregenomic RNA.
  • the articles “a” and “an” refer to one or to more than one (i.e. , to at least one) of the grammatical object of the article.
  • an element means one element or more than one element.
  • alkenyl employed alone or in combination with other terms, means, unless otherwise stated, a stable monounsaturated or diunsaturated straight chain or branched chain hydrocarbon group having the stated number of carbon atoms. Examples include vinyl, propenyl (or allyl), crotyl, isopentenyl, butadienyl, 1,3-pentadienyl, 1,4-pentadienyl, and the higher homologs and isomers.
  • alkoxy employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined elsewhere herein, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (or isopropoxy), and the higher homologs and isomers.
  • oxygen atom such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (or isopropoxy), and the higher homologs and isomers.
  • a specific example is (C 1 -C 3 )alkoxy, such as, but not limited to, ethoxy and methoxy.
  • alkyl by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e.. C 1 -C 10 means one to ten carbon atoms) and includes straight, branched chain, or cyclic substituent groups. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert- butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl. A specific embodiment is (C 1 -C 6 )alkyl. such as, but not limited to, ethyl, methyl, isopropyl, isobutyl, «-pentyl, «-hexyl, and cyclopropylmethyl.
  • alkynyl employed alone or in combination with other terms means, unless otherwise stated, a stable straight chain or branched chain hydrocarbon group with a triple carbon-carbon bond, having the stated number of carbon atoms. Non-limiting examples include ethynyl and propynyl, and the higher homologs and isomers.
  • the term “propargylic” refers to a group exemplified by -CH 2 -C ⁇ CH .
  • homopropargylic refers to a group exemplified by -CH 2 CH 2 -C ⁇ CH .
  • aromatic refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, i. e. , having (4n+2) delocalized ⁇ (pi) electrons, where 'n' is an integer.
  • aryl employed alone or in combination with other terms means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two or three rings) wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples include phenyl, anthracyl, and naphthyl.
  • Aryl groups also include, for example, phenyl or naphthyl rings fused with one or more saturated or partially saturated carbon rings (e.g ., bicyclo [4.2.0] octa- 1,3,5-trienyl, or indanyl), which can be substituted at one or more carbon atoms of the aromatic and/or saturated or partially saturated rings.
  • phenyl or naphthyl rings fused with one or more saturated or partially saturated carbon rings (e.g ., bicyclo [4.2.0] octa- 1,3,5-trienyl, or indanyl), which can be substituted at one or more carbon atoms of the aromatic and/or saturated or partially saturated rings.
  • aryl-(C 1 -C 6 )alkyl refers to a functional group wherein a one to six carbon alkanediyl chain is attached to an aryl group, e.g., -CH 2 CH 2 -phenyl or - CH 2 -phenyl (or benzyl). Specific examples are aryl-CH 2 - and aryl-CH(CH 3 )-.
  • substituted aryl-(C 1 -C 6 )alkyl refers to an aryl-(C 1 -C 6 )alkyl functional group in which the aryl group is substituted.
  • heteroaryl-(C 1 -C 6 )alkyl refers to a functional group wherein a one to three carbon alkanediyl chain is attached to a heteroaryl group, e.g., -CH 2 CH 2 -pyridyl.
  • heteroaryl-(CH 2 )- refers to a heteroaryl- (C 1 -C 6 )alkyl functional group in which the heteroaryl group is substituted.
  • a specific example is [substituted heteroaryl]-( CH 2 )-.
  • co-administered and “co-administration” as relating to a subject refer to administering to the subject a compound and/or composition of the disclosure along with a compound and/or composition that may also treat or prevent a disease or disorder contemplated herein.
  • the co-administered compounds and/or compositions are administered separately, or in any kind of combination as part of a single therapeutic approach.
  • the co-administered compound and/or composition may be formulated in any kind of combinations as mixtures of solids and liquids under a variety of solid, gel, and liquid formulations, and as a solution.
  • cycloalkyl by itself or as part of another substituent refers to, unless otherwise stated, a cyclic chain hydrocarbon having the number of carbon atoms designated (i.e., C 3 -C 6 refers to a cyclic group comprising a ring group consisting of three to six carbon atoms) and includes straight, branched chain, or cyclic substituent groups.
  • Examples of (C 3 -C 6 )cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl rings can be optionally substituted.
  • Non-limiting examples of cycloalkyl groups include: cyclopropyl, 2-methyl -cyclopropyl, cyclopropenyl, cyclobutyl,
  • decahydroazulenyl bicyclo[6.2.0]decanyl, decahydronaphthalenyl, and dodecahydro-1H- fluorenyl.
  • cycloalkyl also includes bicyclic hydrocarbon rings, non-limiting examples of which include bicyclo-[2.1.1]hexanyl, bicyclo[2.2.1]heptanyl, bicyclo[3.1.1]heptanyl, 1,3 -dimethyl [2.2.1] heptan-2-yl, bicyclo[2.2.2]octanyl, and bicyclo [3.3.3 ]undecanyl .
  • a "disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject's health continues to deteriorate.
  • a disorder in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health.
  • halide refers to a halogen atom bearing a negative charge.
  • the halide anions are fluoride (F-), chloride (Cl-), bromide (Br-), and iodide (I-).
  • halo or halogen alone or as part of another substituent refers to, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
  • Hepatitis B virus refers to a virus species of the genus Orthohepadnavirus, which is a part of the Hepadnaviridae family of viruses, and that is capable of causing liver inflammation in humans.
  • Hepatitis D virus refers to a virus species of the genus Deltaviridae, which is capable of causing liver inflammation in humans.
  • the HDV particle comprises an envelope, which is provided by HBV and surrounds the RNA genome and the HDV antigen.
  • the HDV genome is a single, negative stranded, circular RNA molecule nearly 1.7 kb in length.
  • the genome contains several sense and antisense open reading frames (ORFs), only one of which is functional and conserved.
  • the RNA genome is replicated through an RNA intermediate, the antigenome.
  • the genomic RNA and its complement, the antigenome can function as ribozymes to carry out self-cleavage and self- ligation reactions.
  • a third RNA present in the infected cell also complementary to the genome, but 800 bp long and polyadenylated, is the mRNA for the synthesis of the delta antigen (HDAg).
  • heteroalkenyl by itself or in combination with another term refers to, unless otherwise stated, a stable straight or branched chain monounsaturated or diunsaturated hydrocarbon group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quatemized. Up to two heteroatoms may be placed consecutively.
  • heteroalkyl by itself or in combination with another term refers to, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quatemized.
  • the heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group.
  • Up to two heteroatoms may be consecutive, such as, for example, -CH 2 NH-OCH 3 , or -CH 2 CH 2 SSCH 3 .
  • heteroaryl or “heteroaromatic” refers to a heterocycle having aromatic character.
  • a polycyclic heteroaryl may include one or more rings that are partially saturated. Examples include tetrahydroquinoline and 2,3-dihydrobenzofuryl.
  • heterocycle or “heterocyclyl” or “heterocyclic” by itself or as part of another substituent refers to, unless otherwise stated, an unsubstituted or substituted, stable, mono- or multi-cyclic heterocyclic ring system that comprises carbon atoms and at least one heteroatom selected from the group consisting of N, O, and S, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quatemized.
  • the heterocyclic system may be attached, unless otherwise stated, at any heteroatom or carbon atom that affords a stable structure.
  • a heterocycle may be aromatic or non-aromatic in nature. In certain embodiments, the heterocycle is a heteroaryl.
  • non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazoline, pyrazolidine, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3- dioxane, homopiperazine, homopiperidine, 1,3-dioxepane, 4,7-dihydro- 1, 3 -dioxepin and hexamethyleneoxide
  • heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl (such as, but not limited to, 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.
  • polycyclic heterocycles include indolyl (such as, but not limited to, 3-, 4- , 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (such as, but not limited to, 1- and 5 -isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (such as, but not limited to, 2- and 5 -quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (such as, but not limited to, 3-, 4-, 5-, 6- and 7 -benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzo
  • heterocyclyl and heteroaryl moieties are intended to be representative and not limiting.
  • composition refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition facilitates administration of the compound to a subject.
  • the term "pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound useful within the disclosure, and is relatively non-toxic, i.e., the material may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • the term "pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the subject such that it may perform its intended function.
  • a pharmaceutically acceptable material, composition or carrier such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the subject such that it may perform its intended function.
  • Such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the disclosure, and not injurious to the subject.
  • materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic s
  • pharmaceutically acceptable carrier also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the disclosure, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.
  • the "pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the disclosure.
  • Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the disclosure are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.
  • pharmaceutically acceptable salt refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and/or bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates (including hydrates), and clathrates thereof.
  • a “pharmaceutically effective amount,” “therapeutically effective amount,” or “effective amount” of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered.
  • prevent means avoiding or delaying the onset of symptoms associated with a disease or condition in a subject that has not developed such symptoms at the time the administering of an agent or compound commences.
  • Disease, condition and disorder are used interchangeably herein.
  • RNA Destabilizer refers to a molecule, or a salt or solvate thereof, that reduces the total amount of HBV RNA in mammalian cell culture or in a live human subject.
  • an RNA Destabilizer reduces the amount of the RNA transcript(s) encoding one or more of the following HBV proteins: surface antigen, core protein, RNA polymerase, and e antigen.
  • the terms “subject” and “individual” and “patient” can be used interchangeably, and may refer to a human or non-human mammal or a bird.
  • Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals.
  • the subject is human.
  • substituted refers to that an atom or group of atoms has replaced hydrogen as the substituent attached to another group.
  • substituted refers to any level of substitution, namely mono-, di-, tri-, tetra-, or penta-substitution, where such substitution is permitted.
  • the substituents are independently selected, and substitution may be at any chemically accessible position. In certain embodiments, the substituents vary in number between one and four. In other embodiments, the substituents vary in number between one and three. In yet another embodiments, the substituents vary in number between one and two.
  • the substituents are independently selected from the group consisting of C 1 -C 6 alkyl, -OH, C 1 -C 6 alkoxy, halogen, amino, acetamido, and nitro.
  • the carbon chain may be branched, straight or cyclic.
  • the ring when two substituents are taken together to form a ring having a specified number of ring atoms (e.g. , two groups taken together with the nitrogen to which they are attached to form a ring having from 3 to 7 ring members), the ring can have carbon atoms and optionally one or more (e.g., 1 to 3) additional heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • the ring can be saturated or partially saturated, and can be optionally substituted.
  • substituents of compounds are disclosed in groups or in ranges. It is specifically intended that the description include each and every individual subcombination of the members of such groups and ranges.
  • C 1-6 alkyl is specifically intended to individually disclose C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 1 -C 6 , C 1 -C 5 , C 1 -C 4 , C 1 -C 3 , C 1 -C 2 , C 2 -C 6 , C 2 -C 5 , C 2 -C 4 , C 2 -C 3 , C 3 -C 6 , C 3 -C 5 , C 3 -C 4 , C 4 -C 6 , C 4 -C 5 , and C 5 -C 6 alkyl.
  • treat means reducing the frequency or severity with which symptoms of a disease or condition are experienced by a subject by virtue of administering an agent or compound to the subject.
  • ranges throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual and partial numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • the disclosure includes certain compound recited herein, as well as any salt, solvate, geometric isomer (such as, in a non-limiting example, any geometric isomer and any mixtures thereof, such as, in a non-limiting example, mixtures in any proportions of any geometric isomers thereof), stereoisomer (such as, in a non-limiting example, any enantiomer or diastereoisomer, and any mixtures thereof, such as, in a non-limiting example, mixtures in any proportions of any enantiomers and/or diastereoisomers thereof), tautomer (such as, in a non-limiting example, any tautomer and any mixtures thereof, such as, in a non-limiting example, mixtures in any proportions of any tautomers thereof), and any mixtures thereof.
  • geometric isomer such as, in a non-limiting example, any geometric isomer and any mixtures thereof, such as, in a non-limiting example, mixtures in any proportions of any geometric iso
  • the disclosure includes one of the following compounds, or a salt, solvate, geometric isomer, stereoisomer, tautomer and any mixtures thereof:
  • R 2 is selected from the group consisting of H, optionally substituted C 1 -C 6 alkyl (e.g., optionally substituted benzyl, or C 1 -C 6 alkyl optionally substituted with 1-3 independently selected halogen groups), and optionally substituted C 3 -C 8 cycloalkyl;
  • R 3a , R 3b , R 4a , and R 4b are each independently selected from the group consisting of H, alkyl-substituted oxetanyl, optionally substituted C 1 -C 6 alkyl (e.g., optionally substituted with 1-3 groups independently selected from the group consisting of F, Cl, Br, I, OH, and OMe), and optionally substituted C 3 -C 8 cycloalkyl (e.g., optionally substituted with 1-3 groups independently selected from the group consisting of F, Cl, Br, I, OH, and OMe); or one pair selected from the group consisting of R 3a / R 3b , R 4a / R 4b , and R 3a / R 4a combine to form a divalent group selected from the group consisting of C 1 -C 6 alkanediyl, -(CH 2 )nO(CH 2 )n-, -(CH 2 ) n NR 7 (CH 2
  • R 6I is -CX 3 , -CHX 2 , -OCX 3 , or -OCHX 2 , wherein each occurrence of X is independently F, Cl, Br, or I;
  • R 6II and R 6III are selected such that: one of them is -CX 3 , -CHX 2 , -OCX 3 , -OCHX 2 , F, Cl, Br, I, C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkyl, and optionally substituted C 3 -C 8 cycloalkyl, wherein each occurrence of X is independently F, Cl, Br, or I; and the other is H, -CX 3 , -CHX 2 , -OCX 3 , -OCHX 2 , F, Cl, Br, I, C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkyl, and optionally substituted C 3 -C 8 cycloalkyl, wherein each occurrence of X is independently F, Cl, Br, or I; R 6IV is H; and each occurrence of R 7 is independently selected from the group consisting of H and C 1 -C 6 alkyl (e
  • the compound is selected from the group consisting of: or a salt, solvate, geometric isomer, stereoisomer, tautomer and any mixtures thereof.
  • R 6I is -CX 3 . In certain embodiments, R 6I is -CHX 2 . In certain embodiments, R 6I is -OCX 3 . In certain embodiments, R 6I is -OCHX 2 . In certain embodiments, in R 6I at least one X is F. In certain embodiments, in R 6I at least one X is Cl. In certain embodiments, in R 6I at least one X is Br. In certain embodiments, in R 6I at least one X is I. In certain embodiments, in R 6I at least two X's are F. In certain embodiments, in R 6I at least two X's are Cl. In certain embodiments, in R 6I at least two X's are Br.
  • each X is F. In certain embodiments, in R 6I each X is Cl. In certain embodiments, in R 6I each X is Br. In certain embodiments, in R 6I each X is I.
  • R 6II is H. In certain embodiments, R 6II is -CX 3 . In certain embodiments, R 6II is -CHX 2 . In certain embodiments, R 6II is -OCX 3 . In certain embodiments, R 6II is -OCHX 2 . In certain embodiments, R 6II is F. In certain embodiments, R 6II is Cl. In certain embodiments, R 6II is Br. In certain embodiments, R 6II is I. In certain embodiments, R 6II is C 1 -C 6 alkoxy. In certain embodiments, R 6II is optionally substituted C 1 - C 6 alkyl.
  • R 6II is optionally substituted C 3 -C 8 cycloalkyl.
  • in R 6II at least one X is F.
  • in R 6II at least one X is Cl.
  • in R 6II at least one X is Br.
  • in R 6II at least one X is I.
  • in R 6II at least two X's are F.
  • in R 6II at least two X's are Cl.
  • in R 6II at least two X's are Br.
  • in R 6II at least two X's are I.
  • each X is F. In certain embodiments, in R 6II each X is Cl. In certain embodiments, in R 6II each X is Br. In certain embodiments, in R 6II each X is I. In certain embodiments, R 6III is H. In certain embodiments, R 6III is -CX 3 . In certain embodiments, R 6III is -CHX 2 . In certain embodiments, R 6III is -OCX 3 . In certain embodiments, R 6III is -OCHX 2 . In certain embodiments, R 6III is F. In certain embodiments, R 6III is Cl. In certain embodiments, R 6III is Br. In certain embodiments, R 6III is I.
  • R 6III is C 1 -C 6 alkoxy. In certain embodiments, R 6III is optionally substituted C 1 -C 6 alkyl. In certain embodiments, R 6III is optionally substituted C 3 -C 8 cycloalkyl. In certain embodiments, in R 6III at least one X is F. In certain embodiments, in R 6III at least one X is Cl. In certain embodiments, in R 6III at least one X is Br. In certain embodiments, in R 6III at least one X is I. In certain embodiments, in R 6III at least two X's are F. In certain embodiments, in R 6III at least two X's are Cl.
  • At least two X's are Br. In certain embodiments, in R 6III at least two X's are I. In certain embodiments, in R 6III each X is F. In certain embodiments, in R 6III each X is Cl. In certain embodiments, in R 6III each X is Br. In certain embodiments, in R 6III each X is I.
  • R 2 is H. In certain embodiments, R 2 is methyl. In certain embodiments, R 2 is ethyl. In certain embodiments, R 2 is n-propyl. In certain embodiments, R 2 is isopropyl. In certain embodiments, R 2 is cyclopropyl. In certain embodiments, R 2 is cyclobutyl. In certain embodiments, R 2 is cyclopentyl. In certain embodiments, R 2 is cyclohexyl.
  • At least one of R 3a or R 3b is independently optionally substituted C 1 -C 6 alkyl or optionally substituted C 3 -C 8 cycloalkyl. In certain embodiments, at least one of R 3a or R 3b is independently selected from the group consisting of n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl. In certain embodiments, at least one of R 3a or R 3b is n-propyl. In certain embodiments, at least one of R 3a or R 3b is isopropyl. In certain embodiments, at least one of R 3a or R 3b is n-butyl. In certain embodiments, at least one of R 3a or R 3b is isobutyl. In certain embodiments, at least one of R 3a or R 3b is sec-butyl.
  • At least one of R 3a or R 3b is t-butyl.
  • R 3a is H. In certain embodiments, R 3a is not H. In certain embodiments, R 3a is alkyl-substituted oxetanyl. In certain embodiments, R 3a is optionally substituted C 1 -C 6 alkyl. In certain embodiments, R 3a is optionally substituted C 3 -C 8 cycloalkyl. In certain embodiments, R 3b is H. In certain embodiments, R 3b is not H. In certain embodiments, R 3b is alkyl-substituted oxetanyl. In certain embodiments, R 3b is optionally substituted C 1 -C 6 alkyl.
  • R 3b is optionally substituted C 3 - C 8 cycloalkyl.
  • R 4a is H. In certain embodiments, R 4a is not H. In certain embodiments, R 4a is alkyl-substituted oxetanyl. In certain embodiments, R 4a is optionally substituted C 1 -C 6 alkyl. In certain embodiments, R 4a is optionally substituted C 3 - C 8 cycloalkyl.
  • R 4b is H. In certain embodiments, R 4b is not H. In certain embodiments, R 4b is alkyl-substituted oxetanyl. In certain embodiments, R 4b is optionally substituted C 1 -C 6 alkyl. In certain embodiments, R 4b is optionally substituted C 3 - C 8 cycloalkyl.
  • R 3a is H and R 3b is H. In certain embodiments, R 3a is H and R 3b is isopropyl. In certain embodiments, R 3a is H and R 3b is tert-butyl. In certain embodiments, R 3a is methyl and R 3b is isopropyl. In certain embodiments, R 3a is methyl and R 3b is tert-butyl. In certain embodiments, R 3a is methyl and R 3b is methyl. In certain embodiments, R 3a is methyl and R 3b is ethyl. In certain embodiments, R 3a is ethyl and R 3b is ethyl.
  • R 4a is H and R 4b is H. In certain embodiments, R 4a is H and R 4b is isopropyl. In certain embodiments, R 4a is H and R 4b is tert-butyl. In certain embodiments, R 4a is methyl and R 4b is isopropyl. In certain embodiments, R 4a is methyl and R 4b is tert-butyl. In certain embodiments, R 4a is methyl and R 4b is methyl. In certain embodiments, R 4a is methyl and R 4b is ethyl. In certain embodiments, R 4a is ethyl and R 4b is ethyl.
  • one pair selected from the group consisting of R 3a / R 3b , R 4a / R 4b , and R 3a / R 4a combine to form C 1 -C 6 alkanediyl.
  • one pair selected from the group consisting of R 3a / R 3b , R 4a / R 4b , and R 3a / R 4a combine to form - (CH 2 )nO(CH 2 )n-, which is optionally substituted with at least one C 1 -C 6 alkyl or halogen, wherein each occurrence of n is independently selected from the group consisting of 1 and 2.
  • one pair selected from the group consisting of R 3a / R 3b , R 4a / R 4b , and R 3a / R 4a combine to form -(CH 2 )nNR 9 (CH 2 )n-, which is optionally substituted with at least one C 1 -C 6 alkyl or halogen, wherein each occurrence of n is independently selected from the group consisting of 1 and 2.
  • R 3a and R 3b are independently selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, hydroxymethyl, 2-hydroxy-ethyl, 2-methoxy-ethyl, methoxymethyl, 2 -methyl- 1-methoxy- prop-2-yl, 2-methyl- 1 -hydroxy-prop-2 -yl, and trifluoroethyl.
  • R 4a and R 4b are independently selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, hydroxymethyl, 2-hydroxy-ethyl, 2-methoxy- ethyl, methoxymethyl, and 2 -methyl- l-methoxy-prop-2-yl.
  • R 4a is selected from the group consisting of H, methyl, ethyl, 2-hydroxy-ethyl, and 2-methoxy- ethyl.
  • R 3a and R 3b combine to form 1,1-methanediyl (i.e., an exocyclic double bond). In certain embodiments, R 3a and R 3b combine to form 1,2- ethanediyl. In certain embodiments, R 3a and R 3b combine to form 1,3-propanediyl. In certain embodiments, R 3a and R 3b combine to form 1,4-butanediyl. In certain embodiments, R 3a and R 3b combine to form 1,5-pentanediyl. In certain embodiments, R 3a and R 3b combine to form 1,6-hexanediyl.
  • R 3a and R 4a combine to form 1,2-ethanediyl. In certain embodiments, R 3a and R 4a combine to form 1,2-propanediyl. In certain embodiments, R 3a and R 4a combine to form 1,3-propanediyl. In certain embodiments, R 3a and R 4a combine to form (1 -methyl or 2-methyl)- 1,4-butanediyl. In certain embodiments, R 3a and R 4a combine to form (1,1- dimethyl / 1,2- dimethyl / 1,3- dimethyl / or 2,2-dimethyl)- 1,3-propanediyl. In certain embodiments, R 3a and R 4a combine to form 1,5-pentanediyl. In certain embodiments, R 3a and R 4a combine to form 1,6-hexanediyl.
  • each occurrence of alkyl, alkenyl, cycloalkyl, or acyl is independently optionally substituted with at least one substituent selected from the group consisting of C 1 -C 6 alkyl, halogen, -OR", phenyl (thus yielding, in non-limiting examples, optionally substituted phenyl-(C 1 -C 3 alkyl), such as, but not limited to, benzyl or substituted benzyl), and -N(R")(R"), wherein each occurrence of R" is independently H, C 1 -C 6 alkyl or C 3 -C 8 cycloalkyl.
  • each occurrence of aryl or heteroaryl is independently optionally substituted with at least one substituent selected from the group consisting of C 1 - C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 haloalkoxy, halogen, -CN, -OR", -N(R")(R"), and C 1 -C 6 alkoxycarbonyl, wherein each occurrence of R" is independently H, C 1 -C 6 alkyl or C 3 -C 8 cycloalkyl.
  • the C 1 -C 6 alkyl is optionally substituted with 1-3 groups independently selected from the group consisting of F, Cl, Br, I, OH, and OMe.
  • the C 3 -C 8 cycloalkyl is optionally substituted with 1-3 groups independently selected from the group consisting of F, Cl, Br, I, OH, and OMe.
  • the compounds of the disclosure or a salt, solvate, stereoisomer (such as, in a non-limiting example, an enantiomer or diastereoisomer thereof), any mixture of one or more stereoisomers (such as, in a non-limiting example, mixtures in any proportion of enantiomers thereof, and/or mixtures in any proportion of diastereoisomers thereof), tautomer, and/or any mixture of tautomers thereof, are recited in Table 1.
  • stereoisomer such as, in a non-limiting example, an enantiomer or diastereoisomer thereof
  • any mixture of one or more stereoisomers such as, in a non-limiting example, mixtures in any proportion of enantiomers thereof, and/or mixtures in any proportion of diastereoisomers thereof
  • tautomer and/or any mixture of tautomers thereof
  • the compound is selected form the group consisting of:
  • the compounds of the disclosure may possess one or more stereocenters, and each stereocenter may exist independently in either the (R) or (5) configuration.
  • compounds described herein are present in optically active or racemic forms.
  • the compounds described herein encompass racemic, optically active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase.
  • a compound illustrated herein by the racemic formula further represents either of the two enantiomers or mixtures thereof, or in the case where two or more chiral center are present, all diastereomers or mixtures thereof.
  • the compounds of the disclosure exist as tautomers. All tautomers are included within the scope of the compounds recited herein.
  • Compounds described herein also include isotopically labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes suitable for inclusion in the compounds described herein include and are not limited to 2 H, 3 H, n C, 13 C, 14 C, 36 C1, 18 F, 123 I, 125 I, 13 N, 15 N, 15 O, 17 O, 18 O, 32 P, and 35 S. In certain embodiments, substitution with heavier isotopes such as deuterium affords greater chemical stability.
  • Isotopically labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically labeled reagent in place of the non-labeled reagent otherwise employed.
  • the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.
  • salts embraces addition salts of free acids or bases that are useful within the methods of the disclosure.
  • pharmaceutically acceptable salt refers to salts that possess toxicity profdes within a range that affords utility in pharmaceutical applications.
  • the salts are pharmaceutically acceptable salts.
  • Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present disclosure, such as for example utility in process of synthesis, purification or formulation of compounds useful within the methods of the disclosure.
  • Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid.
  • inorganic acids include sulfate, hydrogen sulfate, hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate).
  • organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4- hydroxybenzoic, phenylacetic, mandelic, embonic (or pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, sulfanilic, 2-hydroxyethanesulfonic, trifluoromethanesulfonic, p-toluenesulfonic, cyclohexylaminosulfonic, stearic, alginic, b- hydroxybutyric
  • Suitable pharmaceutically acceptable base addition salts of compounds of the disclosure include, for example, ammonium salts and metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts.
  • Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N'-dibenzylethylene- diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (or N- methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.
  • the compounds of the disclosure are useful within the methods of the disclosure in combination with one or more additional agents useful for treating, ameliorating, and/or preventing HBV and/or HDV infections.
  • additional agents may comprise compounds or compositions identified herein, or compounds (e.g., commercially available compounds) known to treat, prevent, and/or reduce the symptoms of HBV and/or HDV infections.
  • Non-limiting examples of one or more additional agents useful for treating HBV and/or HDV infections include: (a) reverse transcriptase inhibitors; (b) capsid inhibitors; (c) cccDNA formation inhibitors; (d) RNA destabilizers; (e) oligomeric nucleotides targeted against the HBV genome; (f) immunostimulators, such as checkpoint inhibitors (e.g, PD-L1 inhibitors); (g) GalNAc-siRNA conjugates targeted against an HBV gene transcript; and (h) therapeutic vaccines.
  • the reverse transcriptase inhibitor is a reverse -transcriptase inhibitor (NARTI or NRTI). In other embodiments, the reverse transcriptase inhibitor is a nucleotide analog reverse-transcriptase inhibitor (NtARTI or NtRTI).
  • Reported reverse transcriptase inhibitors include, but are not limited to, entecavir, clevudine, telbivudine, lamivudine, adefovir, and tenofovir, tenofovir disoproxil, tenofovir alafenamide, adefovir dipovoxil, ( 1R,2R,3R,5R)-3-(6-ammo-9H-9-piirinyl)-2-fluoro-5- (hydroxymethyl)-4-methylenecyclopentan-1-ol (described in U.S. Patent No.
  • Reported reverse transcriptase inhibitors further include, but are not limited to, entecavir, lamivudine, and ( 1R,2R,3R,5R)-3-(6-ammo-9H-9-piirinyl)-2-fluoro-5- (hydroxymethyl)-4-methylenecyclopentan- 1 -ol.
  • Reported reverse transcriptase inhibitors further include, but are not limited to, a covalently bound phosphoramidate or phosphonamidate moiety of the above-mentioned reverse transcriptase inhibitors, or as described in for example U.S. Patent No. 8,816,074,
  • Reported reverse transcriptase inhibitors further include, but are not limited to, nucleotide analogs that comprise a phosphoramidate moiety, such as, for example, methyl (((( 1R,3R,4R,5R)-3-(6-amino-9H-purin-9-yl)-4-fluoro-5-hydroxy-2-mcthylcnccyclopcntyl) methoxyXphenoxy) phosphoryl)-(D or U)-alaninate and methyl (((( 1 R.2R.3R.4R)-3-fluoro-2- hydroxy-5-methylene-4-(6-oxo-1,6-dihydro-9H-purin-9-yl)cyclopentyl)methoxy)(phenoxy) phosphoryl)-(D or U)-alaninate.
  • nucleotide analogs that comprise a phosphoramidate moiety, such as, for example, methyl (((( 1R,3R,4
  • the individual diastereomers thereof include, for example, methyl ((R)-((( 1R.3R.4R.5R)-3-(6-ammo-9H-purin-9-yl)-4-fluoro-5- hydroxy-2-methylenecyclopentyl)methoxy)(phenoxy)phosphoryl)-(D or U)-alaninate and methyl ((S)-((( 1R.3R.4R.5R)-3-(6-ammo-9H-purin-9-yl)-4-fluoro-5-hydroxy-2- methylenecyclopentyl) methoxyXphenoxy )phosphoryl)-(D or U)-alaninate.
  • Reported reverse transcriptase inhibitors further include, but are not limited to, compounds comprising a phosphonamidate moiety, such as, for example, tenofovir alafenamide, as well as those described in U.S. Patent Application Publication No. US 2008/0286230 Al, incorporated herein in its entirety by reference.
  • Methods for preparing stereoselective phosphoramidate or phosphonamidate containing actives are described in, for example, U.S. Patent No. 8,816,074, as well as U.S. Patent Application Publications No. US 2011/0245484 Al and US 2008/0286230 Al, all of which incorporated herein in their entireties by reference.
  • capsid inhibitor includes compounds that are capable of inhibiting the expression and/or function of a capsid protein either directly or indirectly.
  • a capsid inhibitor may include, but is not limited to, any compound that inhibits capsid assembly, induces formation of non-capsid polymers, promotes excess capsid assembly or misdirected capsid assembly, affects capsid stabilization, and/or inhibits encapsidation of RNA (pgRNA).
  • Capsid inhibitors also include any compound that inhibits capsid function in a downstream event(s) within the replication process (e.g., viral DNA synthesis, transport of relaxed circular DNA (rcDNA) into the nucleus, covalently closed circular DNA (cccDNA) formation, virus maturation, budding and/or release, and the like).
  • the inhibitor detectably inhibits the expression level or biological activity of the capsid protein as measured, e.g., using an assay described herein.
  • the inhibitor inhibits the level of rcDNA and downstream products of viral life cycle by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or at least 90%.
  • Reported capsid inhibitors include, but are not limited to, compounds described in International Patent Applications Publication Nos WO 2013006394, WO 2014106019, WO 2014089296, WO 2018052967, WO 201817285, WO 2020023710 and WO 2020123674, all of which incorporated herein in their entireties by reference.
  • Reported capsid inhibitors also include, but are not limited to, the following compounds and pharmaceutically acceptable salts and/or solvates thereof: Bay-41-4109 (see Int'l Patent Application Publication No. WO 2013144129), AT-61 (see Int'l Patent Application Publication No. WO 1998033501; and King, et al., 1998, Antimicrob. Agents Chemother. 42(12):3179—3186), DVR-01 and DVR-23 (see Int'l Patent Application Publication No. WO 2013006394; and Campagna, et al., 2013, J. Virol. 87(12):6931, all of which incorporated herein in their entireties by reference.
  • capsid inhibitors include, but are not limited to, those generally and specifically described in U.S. Patent Application Publication Nos. US 2015/0225355, US 2015/0132258, US 2016/0083383, US 2016/0052921 and Int'l Patent Application Publication Nos. WO 2013096744, WO 2014165128, WO 2014033170, WO 2014033167, WO 2014033176, WO 2014131847, WO 2014161888, WO 2014184350, WO 2014184365, WO 2015059212, WO 2015011281, WO 2015118057, WO 2015109130, WO 2015073774,
  • WO 2015180631 WO 2015138895, WO 2016089990, WO 2017015451, WO 2016183266, WO 2017011552, WO 2017048950, WO 2017048954, WO 2017048962, WO 2017064156 and are incorporated herein in their entirety by reference.
  • cccDNA Covalently closed circular DNA
  • cccDNA formation inhibitor includes compounds that are capable of inhibiting the formation and/or stability of cccDNA either directly or indirectly.
  • a cccDNA formation inhibitor may include, but is not limited to, any compound that inhibits capsid disassembly, rcDNA entry into the nucleus, and/or the conversion of rcDNA into cccDNA.
  • the inhibitor detectably inhibits the formation and/or stability of the cccDNA as measured, e.g., using an assay described herein.
  • the inhibitor inhibits the formation and/or stability of cccDNA by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or at least 90%.
  • Reported cccDNA formation inhibitors include, but are not limited to, compounds described in Int'l Patent Application Publication No. WO 2013130703, and are incorporated herein in their entirety by reference.
  • cccDNA formation inhibitors include, but are not limited to, those generally and specifically described in U.S. Patent Application Publication No. US 2015/0038515 Al, and are incorporated herein in their entirety by reference.
  • RNA destabilizer refers to a molecule, or a salt or solvate thereof, that reduces the total amount of HBV RNA in mammalian cell culture or in a live human subject.
  • an RNA destabilizer reduces the amount of the RNA transcript(s) encoding one or more of the following HBV proteins: surface antigen, core protein, RNA polymerase, and e antigen.
  • the RNA destabilizer reduces the total amount of HBV RNA in mammalian cell culture or in a live human subject by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or at least 90%.
  • RNA destabilizers include compounds described in U.S. Patent No. 8,921,381, as well as compounds described in U.S. Patent Application Publication Nos. US 2015/0087659 and US 2013/0303552, all of which are incorporated herein in their entireties by reference.
  • RNA destabilizers include, but are not limited to, those generally and specifically described in Int'l Patent Application Publication Nos. WO 2015113990, WO 2015173164, US 2016/0122344, WO 2016107832, WO 2016023877, WO 2016128335, WO 2016177655, WO 2016071215, WO 2017013046, WO 2017016921, WO 2017016960, WO 2017017042, WO 2017017043, WO 2017102648, WO 2017108630, WO 2017114812, WO 2017140821, WO 2018085619, WO 2019177937, WO 2019222238, WO 2020150366, and WO 2021025976, and are incorporated herein in their entirety by reference. (e) Oligomeric Nucleotides Targeted against the HBV Genome
  • Reported oligomeric nucleotides targeted against the HBV genome include, but are not limited to, Arrowhead-ARC-520 (see U.S. Patent No. 8,809,293; and Wooddell et al., 2013, Molecular Therapy 21(5):973-985, all of which incorporated herein in their entireties by reference).
  • the oligomeric nucleotides can be designed to target one or more genes and/or transcripts of the HBV genome.
  • Oligomeric nucleotide targeted to the HBV genome also include, but are not limited to, isolated, double stranded, siRNA molecules, that each include a sense strand and an antisense strand that is hybridized to the sense strand.
  • the siRNA target one or more genes and/or transcripts of the HBV genome.
  • checkpoint inhibitor includes any compound that is capable of inhibiting immune checkpoint molecules that are regulators of the immune system (e.g., stimulate or inhibit immune system activity).
  • some checkpoint inhibitors block inhibitory checkpoint molecules, thereby stimulating immune system function, such as stimulation of T cell activity against cancer cells.
  • a non-limting example of a checkpoint inhibitor is a PD-L1 inhibitor.
  • the term "PD-L1 inhibitor” includes any compound that is capable of inhibiting the expression and/or function of the protein Programmed Death-Ligand 1 (PD-L1) either directly or indirectly.
  • PD-L1 also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a type 1 transmembrane protein that plays a major role in suppressing the adaptive arm of immune system during pregnancy, tissue allograft transplants, autoimmune disease, and hepatitis.
  • PD-L1 binds to its receptor, the inhibitory checkpoint molecule PD-1 (which is found on activated T cells, B cells, and myeloid cells) so as to modulate activation or inhibition of the adaptive arm of immune system.
  • the PD-L1 inhibitor inhibits the expression and/or function of PD-L1 by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or at least 90%.
  • Reported PD-L1 Inhibitors include, but are not limited to, compounds recited in one of the following patent application publications: US 2018/0057455; US 2018/0057486; WO 2017/106634; WO 2018/026971; WO 2018/045142; WO 2018/118848; WO 2018/119221; WO 2018/119236; WO 2018/119266; WO 2018/119286; WO 2018/121560; WO 2019/076343; WO 2019/087214; and are incorporated herein in their entirety by reference.
  • GalNAc is the abbreviation for N-acetylgalactosamine
  • siRNA is the abbreviation for small interfering RNA.
  • An siRNA that targets an HBV gene transcript is covalently bonded to GalNAc in a GalNAc-siRNA conjugate useful in the practice of the present disclosure. While not wishing to be bound by theory, it is believed that GalNAc binds to asialoglycoprotein receptors on hepatocytes thereby facilitating the targeting of the siRNA to the hepatocytes that are infected with HBV. The siRNA enter the infected hepatocytes and stimulate destruction of HBV gene transcripts by the phenomenon of RNA interference.
  • GalNAc-siRNA conjugates useful in the practice of this aspect of the present disclosure are set forth in published PCT International Applications No.
  • PCT/CA2017/050447 PCT Application Publication No. WO/2017/177326, published October 19, 2017
  • PCT/US2018/026918 PCT Application Publication No. WO2018/191278, published October 18, 2018. all of which are hereby incorporated by reference in their entireties.
  • administration of a therapeutic vaccine is useful in the practice of the present disclosure for the treatment of a viral disease in a subject.
  • the viral disease is a hepatitis virus.
  • the hepatitis virus is at least one selected from the group consisting of hepatitis B virus (HBV) and hepatitis D virus (HDV).
  • the subject is a human.
  • a synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-Emax equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114: 313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22:27-55).
  • Each equation referred to elsewhere herein may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination.
  • the corresponding graphs associated with the equations referred to elsewhere herein are the concentration-effect curve, isobologram curve and combination index curve, respectively.
  • the present disclosure further provides methods of preparing the compounds of the present disclosure.
  • Compounds of the present teachings can be prepared in accordance with the procedures outlined herein, from commercially available starting materials, compounds known in the literature, or readily prepared intermediates, by employing standard synthetic methods and procedures known to those skilled in the art. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be readily obtained from the relevant scientific literature or from standard textbooks in the field. It should be contemplated that the disclosure includes each and every one of the synthetic schemes described and/or depicted herein.
  • reaction temperatures i.e.. reaction temperatures, times, mole ratios of reactants, solvents, pressures, and so forth
  • Optimum reaction conditions can vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
  • Those skilled in the art of organic synthesis will recognize that the nature and order of the synthetic steps presented can be varied for the purpose of optimizing the formation of the compounds described herein.
  • product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or by chromatography such as high pressure liquid chromatography (HPLC), gas chromatography (GC), gel-permeation chromatography (GPC), or thin layer chromatography (TLC).
  • spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or by chromatography such as high pressure liquid chromatography (HPLC), gas chromatography (GC), gel-permeation chromatography (GPC), or thin layer chromatography (TLC).
  • HPLC high pressure liquid chromatography
  • GC gas chromatography
  • GPC gel-permeation chromatography
  • TLC thin layer chromatography
  • Preparation of the compounds can involve protection and deprotection of various chemical groups.
  • the need for protection and deprotection and the selection of appropriate protecting groups can be readily determined by one skilled in the art.
  • the chemistry of protecting groups can be found, for example, in Greene, et al. , Protective Groups in Organic Synthesis, 2d. Ed. (Wiley & Sons, 1991), the entire disclosure of which is incorporated by reference herein for all purposes.
  • Suitable solvents typically are substantially nonreactive with the reactants, intermediates, and/or products at the temperatures at which the reactions are carried out, i. e. , temperatures that can range from the solvent's freezing temperature to the solvent's boiling temperature.
  • a given reaction can be carried out in one solvent or a mixture of more than one solvent.
  • suitable solvents for a particular reaction step can be selected.
  • a compound of formula (I) can be prepared, for example, according to the synthetic methods outlined in Scheme 1.
  • a compound of formula (I) can be prepared, for example, according to the synthetic methods outlined in Scheme 2.
  • the disclosure provides a method of treating, ameliorating, and/or preventing hepatitis virus infection in a subject.
  • the infection comprises hepatitis B virus (HBV) infection.
  • the infection comprises hepatitis D virus (HDV) infection.
  • the method comprises administering to the subject in need thereof a therapeutically effective amount of at least one compound of the disclosure.
  • the compound of the disclosure is the only antiviral agent administered to the subject.
  • the at least one compound is administered to the subject in a pharmaceutically acceptable composition.
  • the subject is further administered at least one additional agent useful for treating the hepatitis virus infection.
  • the at least one additional agent comprises at least one selected from the group consisting of reverse transcriptase inhibitors, capsid inhibitors, cccDNA formation inhibitors, RNA destabilizers, oligomeric nucleotides targeted against the HBV genome, immunostimulators, GalNAc-siRNA conjugates targeted against an HBV gene transcript, and therapeutic vaccines.
  • the subject is co-administered the at least one compound and the at least one additional agent.
  • the at least one compound and the at least one additional agent are coformulated.
  • the disclosure further provides a method of inhibiting and/or reducing HBV surface antigen (HBsAg) secretion either directly or indirectly in a subject.
  • the disclosure further provides a method of reducing or minimizing levels of HBsAg in a HBV-infected subject.
  • the disclosure further provides a method of reducing or minimizing levels of HBeAg in a HBV-infected subject.
  • the disclosure further provides a method of reducing or minimizing levels of hepatitis B core protein in a HBV-infected subject.
  • the disclosure further provides a method of reducing or minimizing levels of pg RNA in a HBV-infected subject.
  • the method comprises administering to the subject in need thereof a therapeutically effective amount of at least one compound of the disclosure.
  • the at least one compound is administered to the subject in a pharmaceutically acceptable composition.
  • the compound of the disclosure is the only antiviral agent administered to the subject.
  • the subject is further administered at least one additional agent useful for treating the hepatitis infection.
  • the at least one additional agent comprises at least one selected from the group consisting of reverse transcriptase inhibitors, capsid inhibitors, cccDNA formation inhibitors, RNA destabilizers, oligomeric nucleotides targeted against the HBV genome, immunostimulators, GalNAc-siRNA conjugates targeted against an HBV gene transcript, and therapeutic vaccines.
  • the subject is co-administered the at least one compound and the at least one additional agent.
  • the at least one compound and the at least one additional agent are coformulated.
  • the subject is infected with HBV. In other embodiments, the subject is infected with HDV. In yet other embodiments, the subject is infected with HBV and HDV.
  • the subject is a mammal. In other embodiments, the mammal is a human.
  • compositions comprising at least one compound of the disclosure or a salt or solvate thereof, which are useful to practice methods of the disclosure.
  • a pharmaceutical composition may consist of at least one compound of the disclosure or a salt or solvate thereof, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one compound of the disclosure or a salt or solvate thereof, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these.
  • At least one compound of the disclosure may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
  • the pharmaceutical compositions useful for practicing the method of the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In other embodiments, the pharmaceutical compositions useful for practicing the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 1,000 mg/kg/day.
  • compositions of the disclosure will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • compositions that are useful in the methods of the disclosure may be suitably developed for nasal, inhalational, oral, rectal, vaginal, pleural, peritoneal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, epidural, intrathecal, intravenous or another route of administration.
  • a composition useful within the methods of the disclosure may be directly administered to the brain, the brainstem, or any other part of the central nervous system of a mammal or bird.
  • Other contemplated formulations include projected nanoparticles, microspheres, liposomal preparations, coated particles, polymer conjugates, resealed erythrocytes containing the active ingredient, and immunologically- based formulations.
  • compositions of the disclosure are part of a pharmaceutical matrix, which allows for manipulation of insoluble materials and improvement of the bioavailability thereof, development of controlled or sustained release products, and generation of homogeneous compositions.
  • a pharmaceutical matrix may be prepared using hot melt extrusion, solid solutions, solid dispersions, size reduction technologies, molecular complexes (e.g., cyclodextrins, and others), microparticulate, and particle and formulation coating processes. Amorphous or crystalline phases may be used in such processes.
  • the route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.
  • the formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology and pharmaceutics. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single-dose or multi-dose unit.
  • a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one- third of such a dosage.
  • the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
  • compositions suitable for ethical administration to humans are principally directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the disclosure is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.
  • compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers.
  • the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of at least one compound of the disclosure and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, recombinant human albumin (e.g., RECOMBUMIN®), solubilized gelatins (e.g., GELOFUSINE®), and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), recombinant human albumin, solubilized gelatins, suitable mixtures thereof, and vegetable oils.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, are included in the composition.
  • Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.
  • Formulations may be employed in admixtures with conventional excipients, i. e. , pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, inhalational, intravenous, subcutaneous, transdermal enteral, or any other suitable mode of administration, known to the art.
  • the pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or fragrance-conferring substances and the like.
  • additional ingredients include, but are not limited to, one or more ingredients that may be used as a pharmaceutical carrier.
  • the composition of the disclosure may comprise a preservative from about 0.005% to 2.0% by total weight of the composition.
  • the preservative is used to prevent spoilage in the case of exposure to contaminants in the environment.
  • Examples of preservatives useful in accordance with the disclosure include but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and combinations thereof.
  • One such preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.
  • the composition may include an antioxidant and a chelating agent which inhibit the degradation of the compound.
  • Antioxidants for some compounds are BHT, BHA, alpha- tocopherol and ascorbic acid in the exemplary range of about 0.01% to 0.3%, or BHT in the range of 0.03% to 0.1% by weight by total weight of the composition.
  • the chelating agent may be present in an amount of from 0.01% to 0.5% by weight by total weight of the composition.
  • Exemplary chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01% to 0.20%, or in the range of 0.02% to 0.10% by weight by total weight of the composition.
  • the chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are exemplary antioxidant and chelating agent, respectively, for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.
  • Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle.
  • Aqueous vehicles include, for example, water, and isotonic saline.
  • Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
  • Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents.
  • Oily suspensions may further comprise a thickening agent.
  • suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl cellulose.
  • Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively).
  • naturally-occurring phosphatides such as lecithin
  • condensation products of an alkylene oxide with a fatty acid with a long chain aliphatic alcohol
  • with a partial ester derived from a fatty acid and a hexitol or with a partial ester derived from a fatty acid and a hexito
  • emulsifying agents include, but are not limited to, lecithin, acacia, and ionic or non-ionic surfactants.
  • Known preservatives include, but are not limited to, methyl, ethyl, or «-propyl para-hydroxybenzoates, ascorbic acid, and sorbic acid.
  • Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.
  • Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent.
  • an "oily" liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water.
  • Liquid solutions of the pharmaceutical composition of the disclosure may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent.
  • Aqueous solvents include, for example, water, and isotonic saline.
  • Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
  • Powdered and granular formulations of a pharmaceutical preparation of the disclosure may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, ionic and non-ionic surfactants, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.
  • a pharmaceutical composition of the disclosure may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion.
  • the oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these.
  • compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally- occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate.
  • emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.
  • Methods for impregnating or coating a material with a chemical composition include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (/. e. , such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.
  • Methods for mixing components include physical milling, the use of pellets in solid and suspension formulations and mixing in a transdermal patch, as known to those skilled in the art.
  • the regimen of administration may affect what constitutes an effective amount.
  • the therapeutic formulations may be administered to the patient either prior to or after the onset of a disease or disorder. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • compositions of the present disclosure may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder contemplated herein.
  • An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • an effective dose range for a therapeutic compound of the disclosure is from about 0.01 mg/kg to 100 mg/kg of body weight/per day.
  • One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
  • the compound may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less.
  • the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days.
  • a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
  • the frequency of the dose is readily apparent to the skilled artisan and depends upon a number of factors, such as, but not limited to, type and severity of the disease being treated, and type and age of the animal.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • a medical doctor e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • physician or veterinarian could start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle.
  • the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disease or disorder in a patient.
  • compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more.
  • the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two days, every three days to once a week, and once every two weeks.
  • the frequency of administration of the various combination compositions of the disclosure will vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors.
  • the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient will be determined by the attending physician taking all other factors about the patient into account.
  • Compounds of the disclosure for administration may be in the range of from about 1 pig to about 7,500 mg, about 20 pig to about 7,000 mg, about 40 pig to about 6,500 mg, about 80 pig to about 6,000 mg, about 100 pig to about 5,500 mg, about 200 pig to about 5,000 mg, about 400 pig to about 4,000 mg, about 800 pig to about 3,000 mg, about 1 mg to about 2,500 mg, about 2 mg to about 2,000 mg, about 5 mg to about 1,000 mg, about 10 mg to about 750 mg, about 20 mg to about 600 mg, about 30 mg to about 500 mg, about 40 mg to about 400 mg, about 50 mg to about 300 mg, about 60 mg to about 250 mg, about 70 mg to about 200 mg, about 80 mg to about 150 mg, and any and all whole or partial increments there-in- between.
  • the dose of a compound of the disclosure is from about 0.5 pig and about 5,000 mg. In some embodiments, a dose of a compound of the disclosure used in compositions described herein is less than about 5,000 mg, or less than about 4,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg.
  • a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
  • the present disclosure is directed to a packaged pharmaceutical composition
  • a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.
  • the term “container” includes any receptacle for holding the pharmaceutical composition or for managing stability or water uptake.
  • the container is the packaging that contains the pharmaceutical composition, such as liquid (solution and suspension), semisolid, lyophilized solid, solution and powder or lyophilized formulation present in dual chambers.
  • the container is not the packaging that contains the pharmaceutical composition, i.e.. the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition.
  • packaging techniques are well known in the art.
  • the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product.
  • the instructions may contain information pertaining to the compound's ability to perform its intended function, e.g., treating, preventing, or reducing a disease or disorder in a patient.
  • Routes of administration of any of the compositions of the disclosure include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, epidural, intrapleural, intraperitoneal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
  • inhalational e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, epidural, intrapleural, intraperitone
  • compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, emulsions, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.
  • compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic, generally recognized as safe (GRAS) pharmaceutically excipients which are suitable for the manufacture of tablets.
  • GRAS inert, non-toxic, generally recognized as safe
  • excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate.
  • Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient.
  • a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets.
  • tablets may be coated using methods described in U.S. Patents Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotically controlled release tablets.
  • Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide for pharmaceutically elegant and palatable preparation.
  • Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin.
  • the capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.
  • Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.
  • an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.
  • Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin from animal-derived collagen or from a hypromellose, a modified form of cellulose, and manufactured using optional mixtures of gelatin, water and plasticizers such as sorbitol or glycerol.
  • a physiologically degradable composition such as gelatin from animal-derived collagen or from a hypromellose, a modified form of cellulose, and manufactured using optional mixtures of gelatin, water and plasticizers such as sorbitol or glycerol.
  • Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.
  • the compounds of the disclosure may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents; fillers; lubricants; disintegrates; or wetting agents.
  • the tablets may be coated using suitable methods and coating materials such as OPADRY ® film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY® OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY® White, 32K18400). It is understood that similar type of film coating or polymeric products from other companies may be used.
  • a tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients.
  • Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent.
  • Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture.
  • compositions used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents.
  • Known dispersing agents include, but are not limited to, potato starch and sodium starch glycolate.
  • Known surface-active agents include, but are not limited to, sodium lauryl sulphate.
  • Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate.
  • Known granulating and disintegrating agents include, but are not limited to, com starch and alginic acid.
  • binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose.
  • Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.
  • Granulating techniques are well known in the pharmaceutical art for modifying starting powders or other particulate materials of an active ingredient.
  • the powders are typically mixed with a binder material into larger permanent free-flowing agglomerates or granules referred to as a "granulation.”
  • solvent-using "wet" granulation processes are generally characterized in that the powders are combined with a binder material and moistened with water or an organic solvent under conditions resulting in the formation of a wet granulated mass from which the solvent must then be evaporated.
  • Melt granulation generally consists in the use of materials that are solid or semi-solid at room temperature (i.e.. having a relatively low softening or melting point range) to promote granulation of powdered or other materials, essentially in the absence of added water or other liquid solvents.
  • the low melting solids when heated to a temperature in the melting point range, liquefy to act as a binder or granulating medium.
  • the liquefied solid spreads itself over the surface of powdered materials with which it is contacted, and on cooling, forms a solid granulated mass in which the initial materials are bound together.
  • the resulting melt granulation may then be provided to a tablet press or be encapsulated for preparing the oral dosage form.
  • Melt granulation improves the dissolution rate and bioavailability of an active (i.e., drug) by forming a solid dispersion or solid solution.
  • U.S. Patent No. 5,169,645 discloses directly compressible wax-containing granules having improved flow properties.
  • the granules are obtained when waxes are admixed in the melt with certain flow improving additives, followed by cooling and granulation of the admixture.
  • certain flow improving additives such as sodium bicarbonate
  • the present disclosure also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds useful within the methods of the disclosure, and a further layer providing for the immediate release of one or more compounds useful within the methods of the disclosure.
  • a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.
  • Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions.
  • the liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non- aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl para-hydroxy benzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats
  • emulsifying agent e.g., lecithin or acacia
  • non- aqueous vehicles e.g., almond oil, oily esters or ethyl alcohol
  • preservatives e.g., methyl or propyl para-hydroxy benzoates or sorb
  • parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue.
  • Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
  • parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrastemal injection, and kidney dialytic infusion techniques.
  • Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline.
  • a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline.
  • Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration.
  • Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multidose containers containing a preservative. Injectable formulations may also be prepared, packaged, or sold in devices such as patient-controlled analgesia (PCA) devices.
  • PCA patient-controlled analgesia
  • Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (i.e.. powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • Such sterile injectable formulations may be prepared using a non toxic parenterally acceptable diluent or solvent, such as water or 1,3-butanediol, for example.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • stratum comeum layer of the epidermis An obstacle for topical administration of pharmaceuticals is the stratum comeum layer of the epidermis.
  • the stratum comeum is a highly resistant layer comprised of protein, cholesterol, sphingolipids, free fatty acids and various other lipids, and includes comified and living cells.
  • One of the factors that limit the penetration rate (flux) of a compound through the stratum comeum is the amount of the active substance that can be loaded or applied onto the skin surface. The greater the amount of active substance which is applied per unit of area of the skin, the greater the concentration gradient between the skin surface and the lower layers of the skin, and in turn the greater the diffusion force of the active substance through the skin. Therefore, a formulation containing a greater concentration of the active substance is more likely to result in penetration of the active substance through the skin, and more of it, and at a more consistent rate, than a formulation having a lesser concentration, all other things being equal.
  • Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions.
  • Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent.
  • Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
  • Enhancers of permeation may be used. These materials increase the rate of penetration of drugs across the skin. Typical enhancers in the art include ethanol, glycerol monolaurate, PGML (polyethylene glycol monolaurate), dimethylsulfoxide, and the like. Other enhancers include oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids, dimethylsulfoxide, polar lipids, orN-methyl-2-pyrrolidone.
  • compositions of the disclosure may contain liposomes.
  • the composition of the liposomes and their use are known in the art (i.e., U.S. Patent No. 6,323,219).
  • the topically active pharmaceutical composition may be optionally combined with other ingredients such as adjuvants, anti-oxidants, chelating agents, surfactants, foaming agents, wetting agents, emulsifying agents, viscosifiers, buffering agents, preservatives, and the like.
  • a permeation or penetration enhancer is included in the composition and is effective in improving the percutaneous penetration of the active ingredient into and through the stratum comeum with respect to a composition lacking the permeation enhancer.
  • compositions may further comprise a hydrotropic agent, which functions to increase disorder in the structure of the stratum comeum, and thus allows increased transport across the stratum comeum.
  • hydrotropic agents such as isopropyl alcohol, propylene glycol, or sodium xylene sulfonate, are known to those of skill in the art.
  • the topically active pharmaceutical composition should be applied in an amount effective to affect desired changes.
  • amount effective shall mean an amount sufficient to cover the region of skin surface where a change is desired.
  • An active compound should be present in the amount of from about 0.0001% to about 15% by weight volume of the composition. For example, it should be present in an amount from about 0.0005% to about 5% of the composition; for example, it should be present in an amount of from about 0.001% to about 1% of the composition.
  • Such compounds may be synthetically-or naturally derived.
  • a pharmaceutical composition of the disclosure may be prepared, packaged, or sold in a formulation suitable for buccal administration.
  • Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) of the active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein.
  • formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient.
  • Such powdered, aerosolized, or aerosolized formulations when dispersed, may have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.
  • the examples of formulations described herein are not exhaustive and it is understood that the disclosure includes additional modifications of these and other formulations not described herein, but which are known to those of skill in the art.
  • a pharmaceutical composition of the disclosure may be prepared, packaged, or sold in a formulation suitable for rectal administration.
  • a composition may be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic irrigation.
  • Suppository formulations may be made by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e.. about 20°C) and which is liquid at the rectal temperature of the subject (i.e.. about 37°C in a healthy human).
  • Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides.
  • Suppository formulations may further comprise various additional ingredients including, but not limited to, antioxidants, and preservatives.
  • Retention enema preparations or solutions for rectal or colonic irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier.
  • enema preparations may be administered using, and may be packaged within, a delivery device adapted to the rectal anatomy of the subject.
  • Enema preparations may further comprise various additional ingredients including, but not limited to, antioxidants, and preservatives.
  • Additional dosage forms of this disclosure include dosage forms as described in U.S. Patents Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790. Additional dosage forms of this disclosure also include dosage forms as described in U.S. Patent Applications Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos.
  • compositions and/or formulations of the present disclosure may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
  • sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period.
  • the period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
  • the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds.
  • the compounds for use the method of the disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
  • the compounds useful within the disclosure are administered to a subject, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
  • delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that may, although not necessarily, include a delay of from about 10 minutes up to about 12 hours.
  • pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
  • immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.
  • short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
  • rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
  • reaction conditions including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, are within the scope of the present application.
  • range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • mice Female CD-I mice (30 ⁇ - 3 g) were ordered from Hilltop Lab Animals Inc., then acclimated for at least 3 days before the commencement of the pharmacokinetic studies. Compounds of interest were dissolved into a cosolvent formulation amenable to oral dosing. The formulation consisted of 60% polyethylene glycol 400 (Spectrum Chemical, NF grade), 10% propylene glycol (Spectrum Chemical, NF grade), 5% ethanol (Spectrum Chemical,
  • the terminal collections consisted of ⁇ 500 ⁇ L of blood obtained by cardiac puncture and transferred to microtubes containing anti-coagulant K2EDTA. The blood was then centrifuged (16,000 x g), and the plasma isolated. In addition, livers were also harvested. All samples were stored at -80 °C prior to analysis. Plasma was used as collected. Liver samples were weighed and homogenized in four volumes (w/v) of phosphate buffered saline, pH 7.4 (PBS). Plasma and liver homogenate samples were precipitated with 30 and 7.5 volumes of acetonitrile, respectively.
  • PBS phosphate buffered saline
  • calibration curve samples were prepared by spiking known concentrations (0.24 - 4,000ng/mL) of compounds into blank CD-I matrices of plasma and liver homogenate.
  • the samples were centrifuged (1,200 x g) and the supernatants were collected. The supernatants were injected then into an LC-MS/MS system for analysis.
  • the LC-MS/MS detection is performed using a SCIEX 5500 Qtrap instrument.
  • Each compound was analyzed by reverse phase HPLC using an ACE Ultracore SuperC182.5 pm, 2.1 mm x 50 mm column.
  • Mobile phases consisted of Solvent A: water with 0.1% formic acid, and Solvent B: acetonitrile with 0.1% formic acid.
  • a calibration curve for each compound was constructed by fitting the known analyte concentration versus the analyte response using the least-squares linear regression analysis with a weighting scheme of 1/x 2 .
  • Compound concentrations in plasma and liver homogenate samples were quantified by interpolation on the calibration curve of the relevant matrix. Interpolated plasma concentrations values were directly reported. Liver homogenate concentration values were multiplied by five to account for the additional four volumes of PBS used during homogenization, in order to extrapolate the original concentration in liver tissue only.
  • the formulation consisted of 60% polyethylene glycol 400 (Spectrum Chemical, NF grade), 10% propylene glycol (Spectrum Chemical, NF grade), 5% ethanol (Spectrum Chemical, USP grade, 200 proof) and 25% phosphate buffered saline, pH 7.4 (Thermoscientific).
  • the formulation consisted of 60% polyethylene glycol 400 (Spectrum Chemical, NF grade), 10% propylene glycol (Spectrum Chemical, NF grade), and 30% 100 mM bicarbonate buffer, pH 9 (Thermoscientific).
  • compounds were either prepared at 1 mg/mL or 2 mg/mL. Animals were dosed orally based on body weight at a volume of 5 mL/kg. Per study design, doses administered were 5 mg/kg and/or 10 mg/kg.
  • the treated animals were terminated at 12 or 24 hours post dose.
  • the terminal collections consisted of ⁇ 0.5-1.0 mL of blood obtained by cardiac puncture and transferred to microtubes containing anti-coagulant K2EDTA. The blood was then centrifuged (16,000 x g) at 4 °C for 5 min, and the plasma was isolated. In addition, livers were also harvested. All samples were stored at -80 °C prior to analysis. Plasma was used as collected. Liver samples were weighed and homogenized in four volumes (w/v) of phosphate buffered saline, pH 7.4 (PBS). Plasma and liver homogenate samples were precipitated with 26.7 and 10.0 volumes of acetonitrile containing internal standard, respectively.
  • PBS phosphate buffered saline
  • a calibration curve for each compound was constructed by fitting the known analyte concentration versus the analyte response using the least-squares linear regression analysis with a weighting scheme of 1/x 2 .
  • Compound concentrations in plasma and liver homogenate samples were quantified by interpolation on the calibration curve of the relevant matrix. Interpolated plasma concentrations values were directly reported. Liver homogenate concentration values were multiplied by five to account for the additional four volumes of PBS used during homogenization, in order to extrapolate the original concentration in liver tissue only.
  • the in vitro metabolic stability of compounds in a liver microsome system was used to assess their susceptibility to hepatic degradation. Stability was assessed in mouse liver microsomes or rat liver microsomes (respectively, MLM or RLM; BioIVT) at 0.3 ⁇ M for each compound containing 0.5 mg/mL of protein, at 37 °C in the presence of the co-factor, NADPH, which initiates the reaction. Depletion of the test compounds was monitored over a 60-minute incubation period, with time point collections at 0, 5, 10, 20, 30, and 60 min. At each collection, the reaction was terminated by the addition of acetonitrile containing internal standard.
  • the reaction mixture was slowly warmed to rt and stirred for 16 h.
  • the mixture was quenched with saturated aqueous NaHCO 3 solution (20 mL) and fdtered.
  • the fdtrate was extracted with CH 2 CI 2 (3 x 40 mL).
  • the reaction mixture was slowly warmed to rt and stirred for 4h.
  • the mixture was quenched with saturated aqueous NaHCO 3 solution (4 mL), the phases were separated, and the aqueous layer was extracted further with CH 2 CI 2 (3 x 10 mL).
  • the combined organic phase was dried over Na 2 SO 4 , fdtered and concentrated under reduced pressure.
  • the compounds were prepared in a similar manner as 5-(tert-butyl)-11- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid from an appropriately substituted methyl 5-(tert-butyl)-1-(2,4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate, followed by ester hydrolysis and debenzylation.
  • the compounds were prepared in a similar manner as 5-(tert-butyl)-11- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid from an appropriately substituted methyl 5-(tert-butyl)-1-(2,4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate, followed by ester hydrolysis and debenzylation.
  • the compounds were prepared in a similar manner as 5-(tert-butyl)- 11- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid from an appropriately substituted methyl 5-tert -butyl)-1-(2,4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate, followed by ester hydrolysis and debenzylation.
  • the compounds were prepared in a similar manner as 5-(tert-butyl)-11- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid from an appropriately substituted methyl 5-(tert-butyl)-1-(2,4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate, followed by ester hydrolysis and debenzylation.
  • the compounds were prepared in a similar manner as 5-(tert-butyl)-11- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid from an appropriately substituted methyl 5-(tert-butyl)-1-(2,4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate, followed by ester hydrolysis and debenzylation.
  • the compounds were prepared in a similar manner as 5-(tert-butyl)-11- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid from an appropriately substituted methyl 5 -(tert-butyl)- 1 -(2.4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate, followed by ester hydrolysis and debenzylation.
  • Example 17 5-(tert-Butyl)-9-chloro-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic (single enantiomer I)
  • Example 18 5-(tert-Butyl)-9-chloro-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic (single enantiomer II)
  • the compounds were prepared in a similar manner as 5-(tert-butyl)- 11- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid from an appropriately substituted methyl 5-(tert-butyl)-1-(2,4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate, followed by ester hydrolysis and debenzylation.
  • the compounds were prepared in a similar manner as 5-(tert-butyl)-11- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid from an appropriately substituted methyl 5-(tert-butyl)-1-(2,4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate, followed by ester hydrolysis and debenzylation.
  • the compounds were prepared in a similar manner as 5-(tert-butyl)- 11- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid from an appropriately substituted methyl 5-(tert-butyl)-1-(2,4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate, followed by ester hydrolysis and debenzylation.
  • HBsAg Inhibition of HBsAg was determined in HepG2.2.15 cells.
  • Cells were maintained in culture medium containing 10% fetal calf serum, G414, Glutamine, penicillin/streptomycin.
  • Cells were seeded in 96-well collagen-coated plate at a density of 30,000 cells/well. Serially diluted compounds were added to cells next day at the final DMSO concentration of 0.5%. Cells were incubated with compounds for 2-3 days, after which medium was removed. Fresh medium containing compounds was added to cells for additional 3-4 days.
  • HBsAg immunoassay microplate- based chemiluminescence immunoassay kits, CLIA, Autobio Diagnosics Co., Zhengzhou, China, Catalog # CL0310-2
  • CLIA microplate- based chemiluminescence immunoassay kits
  • EC 50 value effective concentrations that achieved 50% inhibitory effect
  • the compounds of the present disclosure are useful for the treatment, amelioration, and/or prevention of Hepatitis B (and/or Hepatitis D) in mammals.
  • the virus infects hepatocyte cells in the liver.
  • a compound of the present disclosure should preferentially accumulate in the liver upon administration to the subject.
  • preferential liver accumulation of the compound maximizes inhibition of HBV/HDV proliferation and/or inhibition of cell infection by HBV/HDV. In certain embodiments, preferential liver accumulation of the compound minimizes and/or eliminates accumulation in tissues and/or organs other than the liver. In certain embodiments, preferential liver accumulation of the compound minimizes and/or eliminates exposure of the compound to tissues and/or organs other than the liver. In certain embodiments, preferential liver accumulation of the compound minimizes and/or eliminates at least one the following: off-target effects, undesirable exposure, undesirable pharmacokinetics, and/or undesirable metabolism.
  • a clinically developable compound should be metabolically stable upon administration to the subject, such that the compound is not metabolized to metabolites that are quickly eliminated from the liver and/or excreted from the subject, metabolites that have undesirable biological activities, and/or metabolites that are devoid of desirable biological activities.
  • liver accumulation potential of a compound can be ascertained through liver-to- plasma distribution studies, using methods that are known in the art and/or described elsewhere herein.
  • a high liver-to-plasma ratio is desirable, as it indicates that the compound tends to accumulate in the liver over other tissues or organs.
  • an important developability parameter is the ratio between the compound concentration in the liver (C liver ) and the compound concentration that would be required to inhibit the virus infection by 90% (EC 90 ).
  • C liver the compound concentration in the liver
  • EC 90 the compound concentration that would be required to inhibit the virus infection by 90%
  • AUCiast Area Under the Curve (AUC) time curve for the compound's plasma concentration up to the last measurable concentration (AUCiast).
  • AUCiast value is desirable, as it indicates that the compound does not accumulate in the blood/plasma.
  • a low plasma AUCiast value is consistent with the compound's preferential accumulation in the liver, especially when coupled with a high liver-to-plasma ratio and/or a high C liver /EC 90 ratio.
  • the susceptibility of a compound to undergo hepatic degradation can be ascertained through in vitro intrinsic clearance studies, using methods that are known in the art and/or described elsewhere herein.
  • MLM mouse liver microsome
  • RLM rat liver microsome
  • a low MLM/RLM intrinsic clearance rate is desirable, as it indicates that the compound is not degraded and/or modified by the MLMs/RLMs and thus has good metabolic stability.
  • PK data for oral administration to the mouse were collected for selected compounds of the disclosure (all of which are substituted 5,6-dihydropyrido[2',1':2,3]imidazo[4,5-h]quinolin-2(1H)-ones, as shown below), as well as comparative compounds with the same tetracyclic structure:
  • mice Exemplary data for such determinations in mice are provided in Table 2.
  • the antiviral activity, liver-to-plasma distribution, and metabolic stability of the tested compounds varied widely with their particular substitution on the terminal dihydropyridyl ring.
  • biological activity can be ascertained by their EC 50 value for HBsAg inhibition.
  • Their liver accumulation behavior can be ascertained by their liver-to- plasma ratio and their plasma AUCiast values, as well as by the ratio of measured liver concentration and the EC 90 value for HBsAg inhibition.
  • Compounds A-C are provided herein as comparative compounds.
  • Compound A with a methoxy substituent at position 11, showed good HBsAg inhibition, but a moderate liver-to-plasma distribution ratio and high MLM intrinsic clearance, leading to a poor C liver /EC 90 ratio.
  • Introduction of an additional methoxy group at position 10 slightly increased the HBsAg inhibition and reduced MLM intrinsic clearance of the compound, but decreased its liver-to-plasma distribution ratio to the point where the C liver ,/EC 90 ratio decreased by an order of magnitude relative to Compound A.
  • PK data were obtained for oral administration of selected compounds of the disclosure to the rat (Table 3).
  • Compound B showed low liver-to-plasma distribution ratio in the rat, as measured by the C liver ,/ EC 90 ratio, and in fact the liver-to- plasma ratio for that compound at the 24 hour time point was below the detection level.
  • Compound C showed improved liver-to-plasma distribution ratio and reduced plasma AUCiast in the rat (as compared to Compound B), but had undesirable increase in RUM intrinsic clearance.
  • Compounds 1 and 19 showed good HBsAg inhibition, low RUM clearance, and promising plasma AUCiast values and C liver , /EC 90 ratios in the rat.
  • the compounds of the disclosure combine potent antiviral activity against HBV/HDV, preferential accumulation in the liver, and good hepatic stability (as evidenced by low intrinsic clearance values), and are thus good candidates for development as clinically validated Hepatitis B (and/or Hepatitis D) treatments.
  • Rotation frames were integrated using CrysAlisPro [CrysAlisPro 1.171.41.104a: Rigaku Oxford Diffraction, Rigaku Corporation, Oxford, UK. (2020)], producing a listing of unaveraged F 2 and ⁇ (F 2 ) values.
  • the intensity data were corrected for Uorentz and polarization effects and for absorption using SCALE3 ABSPACK [SCALE3 ABSPACK v1.0.7: an Oxford Diffraction program; Oxford Diffraction Ltd: Abingdon, UK, 2005] (minimum and maximum transmission 0.7195, 1.0000).
  • SCALE3 ABSPACK v1.0.7 an Oxford Diffraction program; Oxford Diffraction Ltd: Abingdon, UK, 2005] (minimum and maximum transmission 0.7195, 1.0000).
  • the structure was solved by direct methods - ShelXT [SHELXT v2014/4: Sheldrick, G.M., Acta Cryst, A, 71, 3-8 (2015)].
  • the asymmetric unit consists of four crystallographically-independent molecules (see FIG. 2).
  • the maximum D/s in the final cycle of least squares was 0.010 and the two most prominent peaks in the final difference Fourier were +0.39 and -0.28 e/ ⁇ 3 .
  • the Flack parameter [Flack, H.D., Acta Cryst. A39, 876-881 (1983)] refined to a similar value of -0.02(5).
  • FIGs. 1-2 are ORTEP representations of the molecules with 30% probability thermal ellipsoids displayed.
  • Rotation frames were integrated using CrysAlisPro [CrysAlisPro 1.171.41.104a: Rigaku Oxford Diffraction, Rigaku Corporation, Oxford, UK. (2020)], producing a listing of unaveraged F 2 and ⁇ (F 2 ) values.
  • SCALE3 ABSPACK vl.0.7 an Oxford Diffraction program; Oxford Diffraction Ltd: Abingdon, UK, 2005] (minimum and maximum transmission 0.7168, 1.0000).
  • the structure was solved by direct methods - ShelXT [SHELXT v2014/4: Sheldrick, G.M., Acta Cryst, A, 71, 3-8 (2015)].
  • the asymmetric unit consists of 4 molecules of the title compound plus 4 molecules of water and two molecules of disordered iso-propyl alcohol. Refinement was by full-matrix least squares based on F 2 using SHELXL-2018 [SHELXL-2018/3: Sheldrick,
  • the maximum D/s in the final cycle of least squares was 0.001 and the two most prominent peaks in the final difference Fourier were ⁇ 0.77 and -0.39 e/ ⁇ 3 .
  • the Flack parameter [Flack,
  • FIG. 3 is an ORTEP representation of the molecule with 50% probability thermal ellipsoids displayed.
  • R 2 is selected from the group consisting of H, optionally substituted C 1 -C 6 alkyl, and optionally substituted C 3 -C 8 cycloalkyl;
  • R 3a , R 3b , R 4a , and R 4b are each independently selected from the group consisting of H, alkyl- substituted oxetanyl, optionally substituted C 1 -C 6 alkyl, and optionally substituted C 3 -C 8 cycloalkyl; or one pair selected from the group consisting of R 3a / R 3b , R 4a / R 4b , and R 3a / R 4a combine to form a divalent group selected from the group consisting of C 1 -C 6 alkanediyl,
  • each occurrence of n is independently 1 or 2 and wherein each divalent group is optionally substituted with at least one C 1 -C 6 alkyl or halogen; each occurrence of R 5 is independently selected from the group consisting of H, optionally substituted C 1 -C 6 alkyl, and optionally substituted C 3 -C 8 cycloalkyl;
  • R 6I is -CX 3 , -CHX 2 , -OCX 3 , or -OCHX 2 , wherein each occurrence of X is independently F, Cl,
  • R 6II and R 6III are selected such that: one of them is -CX 3 , -CHX 2 , -OCX 3 , -OCHX 2 , F, Cl, Br, I, C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkyl, and optionally substituted C 3 -C 8 cycloalkyl, wherein each occurrence of X is independently F, Cl, Br, or I; and the other is H, -CX 3 , -CHX 2 , -OCX 3 , -OCHX 2 , F, Cl, Br, I, C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkyl, and optionally substituted C 3 -C 8 cycloalkyl, wherein each occurrence of X is independently F, Cl, Br, or I;
  • R 6IV is H; and each occurrence of R 7 is independently selected from the group consisting of H and C 1 -C 6 alkyl; or a salt, solvate, geometric isomer, stereoisomer, tautomer and any mixtures thereof.
  • Embodiment 2 provides the compound of Embodiment 1, wherein at least one of R 3a or R 3b is independently selected from the group consisting of optionally substituted C 1 -C 6 alkyl and optionally substituted C 3 -C 8 cycloalkyl.
  • Embodiment 3 provides the compound of any one of Embodiments 1-2, wherein each occurrence of alkyl or cycloalkyl is independently optionally substituted with at least one substituent selected from the group consisting of C 1 -C 6 alkyl, halogen, -OR”, phenyl, and - N(R”)(R”), wherein each occurrence of R” is independently H, C 1 -C 6 alkyl, or C 3 -C 8 cycloalkyl.
  • Embodiment 4 provides the compound of any one of Embodiments 1-3, wherein at least one applies: R 3a is H and R 3b is isopropyl; R 3a is H and R 3b is tert-butyl; R 3a is methyl and R 3b is isopropyl; R 3a is methyl and R 3b is tert-butyl; R 3a is methyl and R 3b is methyl; R 3a is methyl and R 3b is ethyl; and R 3a is ethyl and R 3b is ethyl.
  • Embodiment 5 provides the compound of any one of Embodiments 1-4, wherein R 2 is H, methyl, or cyclopropyl.
  • Embodiment 6 provides the compound of any one of Embodiments 1-5, wherein R 3a and R 3b are not H.
  • Embodiment 7 provides the compound of any one of Embodiments 1-6, wherein one of R 6II and R 6III is H.
  • Embodiment 8 provides the compound of any one of Embodiments 1-7, which is selected from the group consisting of:
  • Embodiment 9 provides a pharmaceutical composition comprising at least one compound of any one of Embodiments 1-8 and a pharmaceutically acceptable carrier.
  • Embodiment 10 provides the pharmaceutical composition of Embodiment 9, further comprising at least one additional agent useful for treating, ameliorating, and/or preventing hepatitis B virus infection.
  • Embodiment 11 provides the pharmaceutical composition of Embodiment 10, wherein the at least one additional agent comprises at least one selected from the group consisting of reverse transcriptase inhibitors, capsid inhibitors, cccDNA formation inhibitors, RNA destabilizers, oligomeric nucleotides targeted against the HBV genome, immunostimulators, GalNAc-siRNA conjugates targeted against an HBV gene transcript, and therapeutic vaccines.
  • the at least one additional agent comprises at least one selected from the group consisting of reverse transcriptase inhibitors, capsid inhibitors, cccDNA formation inhibitors, RNA destabilizers, oligomeric nucleotides targeted against the HBV genome, immunostimulators, GalNAc-siRNA conjugates targeted against an HBV gene transcript, and therapeutic vaccines.
  • Embodiment 12 provides the pharmaceutical composition of Embodiment 11, wherein the oligomeric nucleotide comprises one or more siRNAs.
  • Embodiment 13 provides the pharmaceutical composition of any one of Embodiments 10-12, wherein the at least one additional agent further treats, ameliorates, and/or prevents hepatitis D virus infection.
  • Embodiment 14 provides a method of treating, ameliorating, and/or preventing hepatitis B virus infection in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of at least one compound of any one of Embodiments 1-8 and/or at least one pharmaceutical composition of any one of Embodiments 9-13.
  • Embodiment 15 provides the method of Embodiment 14, wherein the subject is further infected with hepatitis D virus (HDV).
  • HDV hepatitis D virus
  • Embodiment 16 provides a method of reducing or minimizing levels of at least one selected from the group consisting of hepatitis B virus surface antigen (HBsAg), hepatitis B e- antigen (HBeAg), hepatitis B core protein, and pregenomic (pg) RNA, in a HBV-infected subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of at least one compound any one of Embodiments 1-8 and/or at least one pharmaceutical composition of any one of Embodiments 9-13.
  • HsAg hepatitis B virus surface antigen
  • HeAg hepatitis B e- antigen
  • pg pregenomic RNA
  • Embodiment 17 provides the method of any one of Embodiments 14-16, wherein the at least one compound is administered to the subject in a pharmaceutically acceptable composition.
  • Embodiment 18 provides the method of any one of Embodiments 14-17, wherein the subject is further administered at least one additional agent useful for treating, ameliorating, and/or preventing the HBV infection.
  • Embodiment 19 provides the method of Embodiment 18, wherein the at least one additional agent comprises at least one selected from the group consisting of reverse transcriptase inhibitors, capsid inhibitors, cccDNA formation inhibitors, RNA destabilizers, oligomeric nucleotides targeted against the HBV genome, immunostimulators, GalNAc-siRNA conjugates targeted against an HBV gene transcript, and therapeutic vaccines.
  • the at least one additional agent comprises at least one selected from the group consisting of reverse transcriptase inhibitors, capsid inhibitors, cccDNA formation inhibitors, RNA destabilizers, oligomeric nucleotides targeted against the HBV genome, immunostimulators, GalNAc-siRNA conjugates targeted against an HBV gene transcript, and therapeutic vaccines.
  • Embodiment 20 provides the method of Embodiment 19, wherein the oligomeric nucleotide comprises one or more siRNAs.
  • Embodiment 21 provides the method of any one of Embodiments 18-20, wherein the subject is co-administered the at least one compound and the at least one additional agent.
  • Embodiment 22 provides the method of any one of Embodiments 18-21, wherein the at least one compound and the at least one additional agent are coformulated.
  • Embodiment 23 provides the method of any one of Embodiments 14-22, wherein the subject is further infected with HDV.
  • Embodiment 24 provides the method of any one of Embodiments 14-23, wherein the subject is a mammal.
  • Embodiment 25 provides the method of Embodiment 24, wherein the mammal is a human.

Abstract

The present disclosure provides substituted tetracyclic carboxylic acids, analogues thereof, and compositions comprising the same, which can be used to treat and/or prevent hepatitis B virus (HBV) infection and/or hepatitis D virus (HDV) in a patient. Compounds provided are of formulae:

Description

Substituted Tetracyclic Carboxylic Acids, Analogues Thereof, and Methods Using Same
CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63/170,920, filed April 5, 2021, which application is being incorporated by reference in its entirety herein.
BACKGROUND
Hepatitis B is one of the world's most prevalent diseases. Although most individuals resolve the infection following acute symptoms, approximately 30% of cases become chronic. 350-400 million people worldwide are estimated to have chronic hepatitis B, leading to 0.5-1 million deaths per year, due largely to the development of hepatocellular carcinoma, cirrhosis, and/or other complications. Hepatitis B is caused by hepatitis B virus (HBV), a noncytopathic, liver tropic DNA virus belonging to Hepadnaviridae family.
A limited number of drugs are currently approved for the management of chronic hepatitis B, including two formulations of alpha-interferon (standard and pegylated) and five nucleoside/nucleotide analogues (lamivudine, adefovir, entecavir, telbivudine, and tenofovir) that inhibit HBV DNA polymerase. At present, the first-line treatment choices are entecavir, tenofovir, or peg-interferon alfa-2a. However, peg-interferon alfa-2a achieves desirable serological milestones in only one third of treated patients and is frequently associated with severe side effects. Entecavir and tenofovir require long-term or possibly lifetime administration to continuously suppress HBV replication, and may eventually fail due to emergence of drug-resistant viruses.
HBV is an enveloped virus with an unusual mode of replication, centering on the establishment of a covalently closed circular DNA (cccDNA) copy of its genome in the host cell nucleus. Pregenomic (pg) RNA is the template for reverse transcriptional replication of HBV DNA. The encapsidation of pg RNA, together with viral DNA polymerase, into a nucleocapsid is essential for the subsequent viral DNA synthesis.
Aside from being a critical structural component of the virion, the HBV envelope is a major factor in the disease process. In chronically infected individuals, serum levels of HBV surface antigen (HBsAg) can be as high as 400 μg/ml, driven by the propensity for infected cells to secrete non-infectious subviral particles at levels far in excess of infectious (Dane) particles. HBsAg comprises the principal antigenic determinant in HBV infection and is composed of the small, middle and large surface antigens (S, M and L, respectively). These proteins are produced from a single open reading frame as three separate N-glycosylated polypeptides through utilization of alternative transcriptional start sites (for L and M/S mRNAs) and initiation codons (for L, M and S).
Although the viral polymerase and HBsAg perform distinct functions, both are essential proteins for the virus to complete its life cycle and be infectious. HBV lacking HBsAg is completely defective and cannot infect or cause infection. HBsAg protects the virus nucleocapsid, begins the infectious cycle, and mediates morphogenesis and secretion of newly forming virus from the infected cell.
People chronically infected with HBV are usually characterized by readily detectable levels of circulating antibody specific to the viral capsid (HBc), with little, if any detectable levels of antibody to HBsAg. There is evidence that chronic carriers produce antibodies to HBsAg, but these antibodies are complexed with the circulating HBsAg, which can be present in mg/mL amounts in a chronic carrier's circulation. Reducing the amount of circulating levels of HBsAg might allow any present anti-HBsA to manage the infection. Further, even if nucleocapsids free of HBsAg were to be expressed or secreted into circulation (perhaps as a result of cell death), the high levels of anti-HBc would quickly complex with them and result in their clearance.
Studies have shown that the presence of subviral particles in a culture of infected hepatocytes may have a transactivating function on viral genomic replication, and the circulating surface antigen suppresses virus-specific immune response. Furthermore, the scarcity of virus-specific cytotoxic T lymphocytes (CTLs), that is a hallmark of chronic HBV infection, may be due to repression of MHC I presentation by intracellular expression of L and M in infected hepatocytes. Existing FDA-approved therapies do not significantly affect HBsAg serum levels.
Hepatitis D virus (HDV) is a small circular enveloped RNA virus that can propagate only in the presence of the hepatitis B virus (HBV). In particular, HDV requires the HBV surface antigen protein to propagate itself. Infection with both HBV and HDV results in more severe complications compared to infection with HBV alone. These complications include a greater likelihood of experiencing liver failure in acute infections and a rapid progression to liver cirrhosis, with an increased chance of developing liver cancer in chronic infections. In combination with hepatitis B virus, hepatitis D has the highest mortality rate of all the hepatitis infections. The routes of transmission of HDV are similar to those for HBV. Infection is largely restricted to persons at high risk of HBV infection, particularly injecting drug users and persons receiving clotting factor concentrates.
Currently, there is no effective antiviral therapy available for the treatment of acute or chronic type D hepatitis. Interferon-alfa, given weekly for 12 to 18 months, is the only licensed treatment for hepatitis D. Response to this therapy is limited-in only about one- quarter of patients is serum HDV RNA undetectable 6 months post therapy.
There is thus a need in the art for novel compounds and/or compositions that can be used to treat, ameliorate, and/or prevent HBV infection in a subject. In certain embodiments, the compounds can be used in patients that are HBV infected, patients who are at risk of becoming HBV infected, and/or patients that are infected with drug-resistant HBV. In other embodiments, the HBV-infected subject is further HDV-infected. The present invention addresses this need.
BRIEF SUMMARY
The disclosure provides a compound selected from:
Figure imgf000005_0001
wherein R1, R2, R3a, R3b, R4a, R4b, R6I, R6II, R6III, and R6IV, and any other applicable variables, are defined elsewhere herein. The disclosure further provides a pharmaceutical composition comprising at least one compound of the disclosure. The disclosure further provides a method of treating, ameliorating, and/or preventing hepatitis B virus infection in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of at least one compound and/or at least one pharmaceutical composition of the disclosure. The disclosure further provides a method of reducing or minimizing levels of at least one selected from the group consisting of hepatitis B virus surface antigen (HBsAg), hepatitis B e-antigen (HBeAg), hepatitis B core protein, and pregenomic (pg) RNA, in a HBV-infected subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of at least one compound and/or at least one pharmaceutical composition of the disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
The following detailed description of exemplary embodiments will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating, non-limiting embodiments are shown in the drawings. It should be understood, however, that the instant specification is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
FIG. 1 illustrates a non-limiting ORTEP drawing of Compound 1 in the asymmetric unit showing labeling scheme.
FIG. 2 illustrates non-limiting ORTEP representations of the four molecules of Compound 1 in the asymmetric unit.
FIG. 3 illustrates a non-limiting ORTEP drawing of Compound 19 showing labeling scheme.
FIG. 4 illustrates non-limiting ORTEP representations of the four molecules of Compound 19 in the asymmetric unit.
DETAILED DESCRIPTION
The disclosure relates, in certain aspects, to the discovery of certain substituted tetracyclic compounds that are useful to treat, ameliorate, and/or prevent HBV and/or HBV- HDV infection and related conditions in a subject. In certain embodiments, the compounds inhibit and/or reduce HBsAg secretion in a HBV-infected subject. In other embodiments, the compounds reduce or minimize levels of HBsAg in a HBV-infected subject. In yet other embodiments, the compounds reduce or minimize levels of HBeAg in a HBV-infected subject. In yet other embodiments, the compounds reduce or minimize levels of hepatitis B core protein in a HBV-infected subject. In yet other embodiments, the compounds reduce or minimize levels of pg RNA in a HBV-infected subject. In yet other embodiments, the compounds have a developable metabolic stability. In yet other embodiments, the compounds have a developable liver-to-plasma distribution ratio. In yet other embodiments, the HBV-infected subject is further HDV-infected.
Definitions
As used herein, each of the following terms has the meaning associated with it in this section.
Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in animal pharmacology, pharmaceutical science, separation science, and organic chemistry are those well-known and commonly employed in the art. It should be understood that the order of steps or order for performing certain actions is immaterial, so long as the present teachings remain operable. Moreover, two or more steps or actions can be conducted simultaneously or not.
The following non-limiting abbreviations are used herein: cccDNA, covalently closed circular DNA; HBc, hepatitis B capsid; HBV, hepatitis B virus; HBeAg, hepatitis B e-antigen; HBsAg, hepatitis B virus surface antigen; MLM, mouse liver microsome; pg RNA, pregenomic RNA.
As used herein, the articles "a" and "an" refer to one or to more than one (i.e. , to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.
As used herein, the term "alkenyl," employed alone or in combination with other terms, means, unless otherwise stated, a stable monounsaturated or diunsaturated straight chain or branched chain hydrocarbon group having the stated number of carbon atoms. Examples include vinyl, propenyl (or allyl), crotyl, isopentenyl, butadienyl, 1,3-pentadienyl, 1,4-pentadienyl, and the higher homologs and isomers. A functional group representing an alkene is exemplified by -CH2-CH=CH2.
As used herein, the term "alkoxy" employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined elsewhere herein, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (or isopropoxy), and the higher homologs and isomers. A specific example is (C1-C3)alkoxy, such as, but not limited to, ethoxy and methoxy.
As used herein, the term "alkyl" by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e.. C1-C10 means one to ten carbon atoms) and includes straight, branched chain, or cyclic substituent groups. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert- butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl. A specific embodiment is (C1-C6)alkyl. such as, but not limited to, ethyl, methyl, isopropyl, isobutyl, «-pentyl, «-hexyl, and cyclopropylmethyl.
As used herein, the term "alkynyl" employed alone or in combination with other terms means, unless otherwise stated, a stable straight chain or branched chain hydrocarbon group with a triple carbon-carbon bond, having the stated number of carbon atoms. Non-limiting examples include ethynyl and propynyl, and the higher homologs and isomers. The term "propargylic" refers to a group exemplified by -CH2-C≡CH . The term "homopropargylic" refers to a group exemplified by -CH2CH2-C≡CH .
As used herein, the term "aromatic" refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, i. e. , having (4n+2) delocalized π (pi) electrons, where 'n' is an integer.
As used herein, the term "aryl" employed alone or in combination with other terms means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two or three rings) wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples include phenyl, anthracyl, and naphthyl. Aryl groups also include, for example, phenyl or naphthyl rings fused with one or more saturated or partially saturated carbon rings ( e.g ., bicyclo [4.2.0] octa- 1,3,5-trienyl, or indanyl), which can be substituted at one or more carbon atoms of the aromatic and/or saturated or partially saturated rings.
As used herein, the term "aryl-(C1-C6)alkyl" refers to a functional group wherein a one to six carbon alkanediyl chain is attached to an aryl group, e.g., -CH2CH2-phenyl or - CH2-phenyl (or benzyl). Specific examples are aryl-CH2- and aryl-CH(CH3)-. The term "substituted aryl-(C1-C6)alkyl" refers to an aryl-(C1-C6)alkyl functional group in which the aryl group is substituted. A specific example is [substituted aryl]-(CH2)-. Similarly, the term "heteroaryl-(C1-C6)alkyl" refers to a functional group wherein a one to three carbon alkanediyl chain is attached to a heteroaryl group, e.g., -CH2CH2-pyridyl. A specific example is heteroaryl-(CH2)-. The term "substituted heteroaryl-(C1-C6)alkyl" refers to a heteroaryl- (C1-C6)alkyl functional group in which the heteroaryl group is substituted. A specific example is [substituted heteroaryl]-( CH2)-.
In one aspect, the terms "co-administered" and "co-administration" as relating to a subject refer to administering to the subject a compound and/or composition of the disclosure along with a compound and/or composition that may also treat or prevent a disease or disorder contemplated herein. In certain embodiments, the co-administered compounds and/or compositions are administered separately, or in any kind of combination as part of a single therapeutic approach. The co-administered compound and/or composition may be formulated in any kind of combinations as mixtures of solids and liquids under a variety of solid, gel, and liquid formulations, and as a solution.
As used herein, the term "cycloalkyl" by itself or as part of another substituent refers to, unless otherwise stated, a cyclic chain hydrocarbon having the number of carbon atoms designated (i.e., C3-C6 refers to a cyclic group comprising a ring group consisting of three to six carbon atoms) and includes straight, branched chain, or cyclic substituent groups. Examples of (C3-C6)cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl rings can be optionally substituted. Non-limiting examples of cycloalkyl groups include: cyclopropyl, 2-methyl -cyclopropyl, cyclopropenyl, cyclobutyl,
2,3 -dihydroxy cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctanyl, decalinyl, 2,5-dimethylcyclopentyl, 3,5- dichlorocyclohexyl, 4-hydroxy cyclohexyl, 3 ,3 ,5 -trimethylcyclohex- 1 -yl, octahydropentalenyl, octahydro-1H-indenyl, 3a.4.5.6.7.7a-hexahydro-3H-inden-4-yl. decahydroazulenyl; bicyclo[6.2.0]decanyl, decahydronaphthalenyl, and dodecahydro-1H- fluorenyl. The term "cycloalkyl" also includes bicyclic hydrocarbon rings, non-limiting examples of which include bicyclo-[2.1.1]hexanyl, bicyclo[2.2.1]heptanyl, bicyclo[3.1.1]heptanyl, 1,3 -dimethyl [2.2.1] heptan-2-yl, bicyclo[2.2.2]octanyl, and bicyclo [3.3.3 ]undecanyl .
As used herein, a "disease" is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject's health continues to deteriorate.
As used herein, a "disorder" in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health.
As used herein, the term "halide" refers to a halogen atom bearing a negative charge. The halide anions are fluoride (F-), chloride (Cl-), bromide (Br-), and iodide (I-).
As used herein, the term "halo" or "halogen" alone or as part of another substituent refers to, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
As used herein, the term "Hepatitis B virus" (or HBV) refers to a virus species of the genus Orthohepadnavirus, which is a part of the Hepadnaviridae family of viruses, and that is capable of causing liver inflammation in humans.
As used herein, the term "Hepatitis D virus" (or HDV) refers to a virus species of the genus Deltaviridae, which is capable of causing liver inflammation in humans. The HDV particle comprises an envelope, which is provided by HBV and surrounds the RNA genome and the HDV antigen. The HDV genome is a single, negative stranded, circular RNA molecule nearly 1.7 kb in length. The genome contains several sense and antisense open reading frames (ORFs), only one of which is functional and conserved. The RNA genome is replicated through an RNA intermediate, the antigenome. The genomic RNA and its complement, the antigenome, can function as ribozymes to carry out self-cleavage and self- ligation reactions. A third RNA present in the infected cell, also complementary to the genome, but 800 bp long and polyadenylated, is the mRNA for the synthesis of the delta antigen (HDAg).
As used herein, the term "heteroalkenyl" by itself or in combination with another term refers to, unless otherwise stated, a stable straight or branched chain monounsaturated or diunsaturated hydrocarbon group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quatemized. Up to two heteroatoms may be placed consecutively. Examples include - CH=CH-O-CH3, -CH=CH-CH2-OH, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, and -CH2- CH=CH-CH2-SH.
As used herein, the term "heteroalkyl" by itself or in combination with another term refers to, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quatemized. The heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples include: -OCH2CH2CH3, - CH2CH2CH2OH, -CH2CH2NHCH3, -CH2SCH2CH3, and -CH2CH2S(=O)CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2NH-OCH3, or -CH2CH2SSCH3.
As used herein, the term "heteroaryl" or "heteroaromatic" refers to a heterocycle having aromatic character. A polycyclic heteroaryl may include one or more rings that are partially saturated. Examples include tetrahydroquinoline and 2,3-dihydrobenzofuryl. As used herein, the term "heterocycle" or "heterocyclyl" or "heterocyclic" by itself or as part of another substituent refers to, unless otherwise stated, an unsubstituted or substituted, stable, mono- or multi-cyclic heterocyclic ring system that comprises carbon atoms and at least one heteroatom selected from the group consisting of N, O, and S, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quatemized. The heterocyclic system may be attached, unless otherwise stated, at any heteroatom or carbon atom that affords a stable structure. A heterocycle may be aromatic or non-aromatic in nature. In certain embodiments, the heterocycle is a heteroaryl.
Examples of non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazoline, pyrazolidine, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3- dioxane, homopiperazine, homopiperidine, 1,3-dioxepane, 4,7-dihydro- 1, 3 -dioxepin and hexamethyleneoxide .
Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl (such as, but not limited to, 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.
Examples of polycyclic heterocycles include indolyl (such as, but not limited to, 3-, 4- , 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (such as, but not limited to, 1- and 5 -isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (such as, but not limited to, 2- and 5 -quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (such as, but not limited to, 3-, 4-, 5-, 6- and 7 -benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (such as, but not limited to, 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (such as, but not limited to, 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl, benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.
The aforementioned listing of heterocyclyl and heteroaryl moieties is intended to be representative and not limiting.
As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a subject.
As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound useful within the disclosure, and is relatively non-toxic, i.e., the material may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the subject such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the disclosure, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the disclosure, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions. The "pharmaceutically acceptable carrier" may further include a pharmaceutically acceptable salt of the compound useful within the disclosure. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the disclosure are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.
As used herein, the language "pharmaceutically acceptable salt" refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and/or bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates (including hydrates), and clathrates thereof.
As used herein, a "pharmaceutically effective amount," "therapeutically effective amount," or "effective amount" of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered.
The term "prevent," "preventing," or "prevention" as used herein means avoiding or delaying the onset of symptoms associated with a disease or condition in a subject that has not developed such symptoms at the time the administering of an agent or compound commences. Disease, condition and disorder are used interchangeably herein.
As used herein, the term "RNA Destabilizer" refers to a molecule, or a salt or solvate thereof, that reduces the total amount of HBV RNA in mammalian cell culture or in a live human subject. In a non-limiting example, an RNA Destabilizer reduces the amount of the RNA transcript(s) encoding one or more of the following HBV proteins: surface antigen, core protein, RNA polymerase, and e antigen.
By the term "specifically bind" or "specifically binds" as used herein is meant that a first molecule preferentially binds to a second molecule (e.g. , a particular receptor or enzyme), but does not necessarily bind only to that second molecule.
As used herein, the terms "subject" and "individual" and "patient" can be used interchangeably, and may refer to a human or non-human mammal or a bird. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. In certain embodiments, the subject is human.
As used herein, the term "substituted" refers to that an atom or group of atoms has replaced hydrogen as the substituent attached to another group.
As used herein, the term "substituted alkyl," "substituted cycloalkyl," "substituted alkenyl," "substituted alkynyl," or "substituted acyl" refers to alkyl, cycloalkyl, alkenyl, alkynyl, or acyl, as defined elsewhere herein, substituted by one, two or three substituents independently selected from the group consisting of halogen, -OH, alkoxy, tetrahydro-2-H- pyranyl, -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, l-methyl-imidazol-2-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, -C(=O)OH, -C(=O)O(C1-C6)alkyl, trifluoromethyl, -C≡N, - C(=O)NH2, -C(=O)NH(C1-C6)alkyl, -C(=O)N((C1-C6)alkyl)2, -SO2NH2, -SO2NH(C1-C6 alkyl), -SO2N(C1-C6 alkyl)2, -C(=NH)NH2, and -NO2, in certain embodiments containing one or two substituents independently selected from halogen, -OH, alkoxy, -NH2, trifluoromethyl, -N(CH3)2, and -C(=O)OH, in certain embodiments independently selected from halogen, alkoxy, and -OH. Examples of substituted alkyls include, but are not limited to, 2,2- difluoropropyl, 2-carboxycyclopentyl and 3-chloropropyl.
For aryl, aryl-(C1-C3)alkyl and heterocyclyl groups, the term "substituted" as applied to the rings of these groups refers to any level of substitution, namely mono-, di-, tri-, tetra-, or penta-substitution, where such substitution is permitted. The substituents are independently selected, and substitution may be at any chemically accessible position. In certain embodiments, the substituents vary in number between one and four. In other embodiments, the substituents vary in number between one and three. In yet another embodiments, the substituents vary in number between one and two. In yet other embodiments, the substituents are independently selected from the group consisting of C1-C6 alkyl, -OH, C1-C6 alkoxy, halogen, amino, acetamido, and nitro. As used herein, where a substituent is an alkyl or alkoxy group, the carbon chain may be branched, straight or cyclic.
Unless otherwise noted, when two substituents are taken together to form a ring having a specified number of ring atoms (e.g. , two groups taken together with the nitrogen to which they are attached to form a ring having from 3 to 7 ring members), the ring can have carbon atoms and optionally one or more (e.g., 1 to 3) additional heteroatoms independently selected from nitrogen, oxygen, or sulfur. The ring can be saturated or partially saturated, and can be optionally substituted.
Whenever a term or either of their prefix roots appear in a name of a substituent the name is to be interpreted as including those limitations provided herein. For example, whenever the term "alkyl" or "aryl" or either of their prefix roots appear in a name of a substituent (e.g., arylalkyl, alkylamino) the name is to be interpreted as including those limitations given elsewhere herein for "alkyl" and "aryl" respectively.
In certain embodiments, substituents of compounds are disclosed in groups or in ranges. It is specifically intended that the description include each and every individual subcombination of the members of such groups and ranges. For example, the term "C1-6 alkyl" is specifically intended to individually disclose C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6 alkyl.
The terms "treat," "treating" and "treatment," as used herein, means reducing the frequency or severity with which symptoms of a disease or condition are experienced by a subject by virtue of administering an agent or compound to the subject.
Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual and partial numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
Compounds
The disclosure includes certain compound recited herein, as well as any salt, solvate, geometric isomer (such as, in a non-limiting example, any geometric isomer and any mixtures thereof, such as, in a non-limiting example, mixtures in any proportions of any geometric isomers thereof), stereoisomer (such as, in a non-limiting example, any enantiomer or diastereoisomer, and any mixtures thereof, such as, in a non-limiting example, mixtures in any proportions of any enantiomers and/or diastereoisomers thereof), tautomer (such as, in a non-limiting example, any tautomer and any mixtures thereof, such as, in a non-limiting example, mixtures in any proportions of any tautomers thereof), and any mixtures thereof.
The disclosure includes one of the following compounds, or a salt, solvate, geometric isomer, stereoisomer, tautomer and any mixtures thereof:
Figure imgf000015_0001
Figure imgf000016_0001
wherein:
R1 is -C(=O)OR5;
R2 is selected from the group consisting of H, optionally substituted C1-C6 alkyl (e.g., optionally substituted benzyl, or C1-C6 alkyl optionally substituted with 1-3 independently selected halogen groups), and optionally substituted C3-C8 cycloalkyl;
R3a, R3b, R4a, and R4b are each independently selected from the group consisting of H, alkyl-substituted oxetanyl, optionally substituted C1-C6 alkyl (e.g., optionally substituted with 1-3 groups independently selected from the group consisting of F, Cl, Br, I, OH, and OMe), and optionally substituted C3-C8 cycloalkyl (e.g., optionally substituted with 1-3 groups independently selected from the group consisting of F, Cl, Br, I, OH, and OMe); or one pair selected from the group consisting of R3a / R3b, R4a / R4b, and R3a / R4a combine to form a divalent group selected from the group consisting of C1-C6 alkanediyl, -(CH2)nO(CH2)n-, -(CH2)nNR7(CH2)n-, -(CH2)nS(CH2)n-, - (CH2)nS(=O)(CH2)n-, and -(CH2)nS(=O)2(CH2)n-, wherein each occurrence of n is independently 1 or 2 and wherein each divalent group is optionally substituted with at least one C1-C6 alkyl or halogen; each occurrence of R5 is independently selected from the group consisting of H, optionally substituted C1-C6 alkyl, and optionally substituted C3-C8 cycloalkyl;
R6I is -CX3, -CHX2, -OCX3, or -OCHX2, wherein each occurrence of X is independently F, Cl, Br, or I;
R6II and R6III are selected such that: one of them is -CX3, -CHX2, -OCX3, -OCHX2, F, Cl, Br, I, C1-C6 alkoxy, optionally substituted C1-C6 alkyl, and optionally substituted C3-C8 cycloalkyl, wherein each occurrence of X is independently F, Cl, Br, or I; and the other is H, -CX3, -CHX2, -OCX3, -OCHX2, F, Cl, Br, I, C1-C6 alkoxy, optionally substituted C1-C6 alkyl, and optionally substituted C3-C8 cycloalkyl, wherein each occurrence of X is independently F, Cl, Br, or I; R6IV is H; and each occurrence of R7 is independently selected from the group consisting of H and C1-C6 alkyl (e.g., methyl or ethyl).
In certain embodiments, the compound is selected from the group consisting of:
Figure imgf000017_0001
or a salt, solvate, geometric isomer, stereoisomer, tautomer and any mixtures thereof.
In certain embodiments, R6I is -CX3. In certain embodiments, R6I is -CHX2. In certain embodiments, R6I is -OCX3. In certain embodiments, R6I is -OCHX2. In certain embodiments, in R6I at least one X is F. In certain embodiments, in R6I at least one X is Cl. In certain embodiments, in R6I at least one X is Br. In certain embodiments, in R6I at least one X is I. In certain embodiments, in R6I at least two X's are F. In certain embodiments, in R6I at least two X's are Cl. In certain embodiments, in R6I at least two X's are Br. In certain embodiments, in R6I at least two X's are I. In certain embodiments, in R6I each X is F. In certain embodiments, in R6I each X is Cl. In certain embodiments, in R6I each X is Br. In certain embodiments, in R6I each X is I.
In certain embodiments, R6II is H. In certain embodiments, R6II is -CX3. In certain embodiments, R6II is -CHX2. In certain embodiments, R6II is -OCX3. In certain embodiments, R6II is -OCHX2. In certain embodiments, R6II is F. In certain embodiments, R6II is Cl. In certain embodiments, R6II is Br. In certain embodiments, R6II is I. In certain embodiments, R6II is C1-C6 alkoxy. In certain embodiments, R6II is optionally substituted C1- C6 alkyl. In certain embodiments, R6II is optionally substituted C3-C8 cycloalkyl. In certain embodiments, in R6II at least one X is F. In certain embodiments, in R6II at least one X is Cl. In certain embodiments, in R6II at least one X is Br. In certain embodiments, in R6II at least one X is I. In certain embodiments, in R6II at least two X's are F. In certain embodiments, in R6II at least two X's are Cl. In certain embodiments, in R6II at least two X's are Br. In certain embodiments, in R6II at least two X's are I. In certain embodiments, in R6II each X is F. In certain embodiments, in R6II each X is Cl. In certain embodiments, in R6II each X is Br. In certain embodiments, in R6II each X is I. In certain embodiments, R6III is H. In certain embodiments, R6III is -CX3. In certain embodiments, R6III is -CHX2. In certain embodiments, R6III is -OCX3. In certain embodiments, R6III is -OCHX2. In certain embodiments, R6III is F. In certain embodiments, R6III is Cl. In certain embodiments, R6III is Br. In certain embodiments, R6III is I. In certain embodiments, R6III is C1-C6 alkoxy. In certain embodiments, R6III is optionally substituted C1-C6 alkyl. In certain embodiments, R6III is optionally substituted C3-C8 cycloalkyl. In certain embodiments, in R6III at least one X is F. In certain embodiments, in R6III at least one X is Cl. In certain embodiments, in R6III at least one X is Br. In certain embodiments, in R6III at least one X is I. In certain embodiments, in R6III at least two X's are F. In certain embodiments, in R6III at least two X's are Cl. In certain embodiments, in R6III at least two X's are Br. In certain embodiments, in R6III at least two X's are I. In certain embodiments, in R6III each X is F. In certain embodiments, in R6III each X is Cl. In certain embodiments, in R6III each X is Br. In certain embodiments, in R6III each X is I.
In certain embodiments, R1 is -C(=O)OH. In certain embodiments, R1 is - C(=O)O(C1-C6 alkyl). In certain embodiments, R1 is selected from the group consisting of - C(=O)OH, -C(=O)OMe, -C(=O)OEt, -C(=O)O-nPr, -C(=O)O-iPr, -C(=O)O-cyclopentyl, and -C(=O)O-cyclohexyl .
In certain embodiments, R2 is H. In certain embodiments, R2 is methyl. In certain embodiments, R2 is ethyl. In certain embodiments, R2 is n-propyl. In certain embodiments, R2 is isopropyl. In certain embodiments, R2 is cyclopropyl. In certain embodiments, R2 is cyclobutyl. In certain embodiments, R2 is cyclopentyl. In certain embodiments, R2 is cyclohexyl.
In certain embodiments, at least one of R3a or R3b is independently optionally substituted C1-C6 alkyl or optionally substituted C3-C8 cycloalkyl. In certain embodiments, at least one of R3a or R3b is independently selected from the group consisting of n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl. In certain embodiments, at least one of R3a or R3b is n-propyl. In certain embodiments, at least one of R3a or R3b is isopropyl. In certain embodiments, at least one of R3a or R3b is n-butyl. In certain embodiments, at least one of R3a or R3b is isobutyl. In certain embodiments, at least one of R3a or R3b is sec-butyl.
In certain embodiments, at least one of R3a or R3b is t-butyl.
In certain embodiments, R3a is H. In certain embodiments, R3a is not H. In certain embodiments, R3a is alkyl-substituted oxetanyl. In certain embodiments, R3a is optionally substituted C1-C6 alkyl. In certain embodiments, R3a is optionally substituted C3-C8 cycloalkyl. In certain embodiments, R3b is H. In certain embodiments, R3b is not H. In certain embodiments, R3b is alkyl-substituted oxetanyl. In certain embodiments, R3b is optionally substituted C1-C6 alkyl. In certain embodiments, R3b is optionally substituted C3- C8 cycloalkyl. In certain embodiments, R4a is H. In certain embodiments, R4a is not H. In certain embodiments, R4a is alkyl-substituted oxetanyl. In certain embodiments, R4a is optionally substituted C1-C6 alkyl. In certain embodiments, R4a is optionally substituted C3- C8 cycloalkyl. In certain embodiments, R4b is H. In certain embodiments, R4b is not H. In certain embodiments, R4b is alkyl-substituted oxetanyl. In certain embodiments, R4b is optionally substituted C1-C6 alkyl. In certain embodiments, R4b is optionally substituted C3- C8 cycloalkyl.
In certain embodiments, R3a is H and R3b is H. In certain embodiments, R3a is H and R3b is isopropyl. In certain embodiments, R3a is H and R3b is tert-butyl. In certain embodiments, R3a is methyl and R3b is isopropyl. In certain embodiments, R3a is methyl and R3b is tert-butyl. In certain embodiments, R3a is methyl and R3b is methyl. In certain embodiments, R3a is methyl and R3b is ethyl. In certain embodiments, R3a is ethyl and R3b is ethyl.
In certain embodiments, R4a is H and R4b is H. In certain embodiments, R4a is H and R4b is isopropyl. In certain embodiments, R4a is H and R4b is tert-butyl. In certain embodiments, R4a is methyl and R4b is isopropyl. In certain embodiments, R4a is methyl and R4b is tert-butyl. In certain embodiments, R4a is methyl and R4b is methyl. In certain embodiments, R4a is methyl and R4b is ethyl. In certain embodiments, R4a is ethyl and R4b is ethyl.
In certain embodiments, one pair selected from the group consisting of R3a / R3b, R4a / R4b, and R3a / R4a combine to form C1-C6 alkanediyl. In certain embodiments, one pair selected from the group consisting of R3a / R3b, R4a / R4b, and R3a / R4a combine to form - (CH2)nO(CH2)n-, which is optionally substituted with at least one C1-C6 alkyl or halogen, wherein each occurrence of n is independently selected from the group consisting of 1 and 2. In certain embodiments, one pair selected from the group consisting of R3a / R3b, R4a / R4b, and R3a / R4a combine to form -(CH2)nNR9(CH2)n-, which is optionally substituted with at least one C1-C6 alkyl or halogen, wherein each occurrence of n is independently selected from the group consisting of 1 and 2.
In certain embodiments, R3a and R3b are independently selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, hydroxymethyl, 2-hydroxy-ethyl, 2-methoxy-ethyl, methoxymethyl, 2 -methyl- 1-methoxy- prop-2-yl, 2-methyl- 1 -hydroxy-prop-2 -yl, and trifluoroethyl. In certain embodiments, R4a and R4b are independently selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, hydroxymethyl, 2-hydroxy-ethyl, 2-methoxy- ethyl, methoxymethyl, and 2 -methyl- l-methoxy-prop-2-yl. In certain embodiments, R4a is selected from the group consisting of H, methyl, ethyl, 2-hydroxy-ethyl, and 2-methoxy- ethyl. In certain embodiments, R3a and R3b combine to form 1,1-methanediyl (i.e., an exocyclic double bond). In certain embodiments, R3a and R3b combine to form 1,2- ethanediyl. In certain embodiments, R3a and R3b combine to form 1,3-propanediyl. In certain embodiments, R3a and R3b combine to form 1,4-butanediyl. In certain embodiments, R3a and R3b combine to form 1,5-pentanediyl. In certain embodiments, R3a and R3b combine to form 1,6-hexanediyl. In certain embodiments, R3a and R4a combine to form 1,2-ethanediyl. In certain embodiments, R3a and R4a combine to form 1,2-propanediyl. In certain embodiments, R3a and R4a combine to form 1,3-propanediyl. In certain embodiments, R3a and R4a combine to form (1 -methyl or 2-methyl)- 1,4-butanediyl. In certain embodiments, R3a and R4a combine to form (1,1- dimethyl / 1,2- dimethyl / 1,3- dimethyl / or 2,2-dimethyl)- 1,3-propanediyl. In certain embodiments, R3a and R4a combine to form 1,5-pentanediyl. In certain embodiments, R3a and R4a combine to form 1,6-hexanediyl.
In certain embodiments, each occurrence of alkyl, alkenyl, cycloalkyl, or acyl is independently optionally substituted with at least one substituent selected from the group consisting of C1-C6 alkyl, halogen, -OR", phenyl (thus yielding, in non-limiting examples, optionally substituted phenyl-(C1-C3 alkyl), such as, but not limited to, benzyl or substituted benzyl), and -N(R")(R"), wherein each occurrence of R" is independently H, C1-C6 alkyl or C3-C8 cycloalkyl.
In certain embodiments, each occurrence of aryl or heteroaryl is independently optionally substituted with at least one substituent selected from the group consisting of C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, halogen, -CN, -OR", -N(R")(R"), -NO2, - S(=O)2N(R")(R"), acyl, and C1-C6 alkoxycarbonyl, wherein each occurrence of R" is independently H, C1-C6 alkyl or C3-C8 cycloalkyl.
In certain embodiments, each occurrence of aryl or heteroaryl is independently optionally substituted with at least one substituent selected from the group consisting of C1- C6 alkyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, halogen, -CN, -OR", -N(R")(R"), and C1-C6 alkoxycarbonyl, wherein each occurrence of R" is independently H, C1-C6 alkyl or C3-C8 cycloalkyl.
In certain embodiments, the C1-C6 alkyl is optionally substituted with 1-3 groups independently selected from the group consisting of F, Cl, Br, I, OH, and OMe. In certain embodiments, the C3-C8 cycloalkyl is optionally substituted with 1-3 groups independently selected from the group consisting of F, Cl, Br, I, OH, and OMe.
In certain embodiments, the compounds of the disclosure, or a salt, solvate, stereoisomer (such as, in a non-limiting example, an enantiomer or diastereoisomer thereof), any mixture of one or more stereoisomers (such as, in a non-limiting example, mixtures in any proportion of enantiomers thereof, and/or mixtures in any proportion of diastereoisomers thereof), tautomer, and/or any mixture of tautomers thereof, are recited in Table 1.
In certain embodiments, the compound is selected form the group consisting of:
(R)-5-(tert-butyl)- 11 -(difluoromethoxy)-9-methoxy-2-oxo- 1 ,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5 -(tert-butyl)- 11 -(difluoromethoxy)-9-methoxy-2-oxo- 1 ,2,5 ,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5-(tert-butyl)- 11 -(difluoromethoxy)-9-fluoro-2-oxo- 1 ,2,5,6- tetrahydropyrido [2',1' : 2,3] imidazo [4,5 -h] quinoline-3 -carboxylic acid;
(S)-5-(tert-butyl)-11-(difluoromethoxy)-9-fluoro-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5 -(tert-butyl)-2-oxo- 11 -(trifluoromethyl)- 1 ,2,5 ,6-tetrahydropyrido [2',1' : 2,3]imidazo [4,5 - h] quinoline -3 -carboxylic acid;
(S)-5-(tert-butyl)-2-oxo-11-(trifluoromethyl)-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid;
(R)-5-(tert-butyl)-11-(difluoromethyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid;
(S)-5-(tert-butyl)-11-(difluoromethyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid;
(R)-5 -(tert-butyl)-9-(difluoromethoxy)- 11 -methoxy-2-oxo- 1 ,2,5 ,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5 -(tert-butyl)-9-(difluoromethoxy)- 11 -methoxy-2-oxo-1,2,5 ,6- tetrahydropyrido [2', 1 ' :2, 3] imidazo [4,5 -h]quinoline-3 -carboxylic acid;
(R)-5-(tert-butyl)-9, 11-bis(difluoromethoxy)-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5-(tert-butyl)-9, 11-bis(difluoromethoxy)-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5 -(tert-butyl)- 11 -(difluoromethoxy)- 10-methoxy-2-oxo- 1 ,2,5 ,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid; (S)-5-(tert-butyl)- 11 -(difluoromethoxy)- 10-methoxy-2-oxo- 1 ,2,5,6- tetrahydropyrido[2', 1 ' : 2,3]imidazo [4,5 -h] quinoline-3 -carboxylic acid;
(R)-5-(tert-butyl)-9-chloro-2-oxo- 11 -(trifluoromethyl)- 1 ,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5-(tert-butyl)-9-chloro-2-oxo- 11 -(trifluoromethyl)- 1 ,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5-(tert-butyl)-9-cyclopropyl-2-oxo- 11 -(trifluoromethyl)- 1 ,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5 -(tert-butyl)-9-cyclopropyl-2-oxo- 11 -(trifluoromethyl)- 1 ,2,5 ,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5-(tert-butyl)-9-methoxy-2-oxo- 11 -(trifluoromethyl)- 1,2, 5, 6- tetrahydropyrido [2',1' : 2,3] imidazo [4,5-h]quinoline-3 -carboxylic acid;
(S)-5 -(tert-butyl)-9-methoxy-2-oxo- 11 -(trifluoromethyl)- 1 ,2,5 ,6- tetrahydropyrido [2',1' : 2,3] imidazo [4,5-h]quinoline-3 -carboxylic acid;
(R)-5-(tert-butyl)-9-chloro-1-methyl-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5 -(tert-butyl)-9-chloro- 1 -methyl-2-oxo- 11 -(trifluoromethyl)- 1 ,2,5 ,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid; or a salt, solvate, geometric isomer, stereoisomer, tautomer and any mixtures thereof.
The compounds of the disclosure may possess one or more stereocenters, and each stereocenter may exist independently in either the (R) or (5) configuration. In certain embodiments, compounds described herein are present in optically active or racemic forms. The compounds described herein encompass racemic, optically active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. A compound illustrated herein by the racemic formula further represents either of the two enantiomers or mixtures thereof, or in the case where two or more chiral center are present, all diastereomers or mixtures thereof.
In certain embodiments, the compounds of the disclosure exist as tautomers. All tautomers are included within the scope of the compounds recited herein.
Compounds described herein also include isotopically labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include and are not limited to 2H, 3H, nC, 13C, 14C, 36C1, 18F, 123I, 125I, 13N, 15N, 15O, 17O, 18O, 32P, and 35S. In certain embodiments, substitution with heavier isotopes such as deuterium affords greater chemical stability. Isotopically labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically labeled reagent in place of the non-labeled reagent otherwise employed.
In certain embodiments, the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.
In all of the embodiments provided herein, examples of suitable optional substituents are not intended to limit the scope of the claimed disclosure. The compounds of the disclosure may contain any of the substituents, or combinations of substituents, provided herein.
Salts
The compounds described herein may form salts with acids or bases, and such salts are included in the present disclosure. The term "salts" embraces addition salts of free acids or bases that are useful within the methods of the disclosure. The term "pharmaceutically acceptable salt" refers to salts that possess toxicity profdes within a range that affords utility in pharmaceutical applications. In certain embodiments, the salts are pharmaceutically acceptable salts. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present disclosure, such as for example utility in process of synthesis, purification or formulation of compounds useful within the methods of the disclosure.
Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include sulfate, hydrogen sulfate, hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate). Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4- hydroxybenzoic, phenylacetic, mandelic, embonic (or pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, sulfanilic, 2-hydroxyethanesulfonic, trifluoromethanesulfonic, p-toluenesulfonic, cyclohexylaminosulfonic, stearic, alginic, b- hydroxybutyric, salicylic, galactaric, galacturonic acid, glycerophosphonic acids and saccharin (e.g., saccharinate, saccharate). Salts may be comprised of a fraction of one, one or more than one molar equivalent of acid or base with respect to any compound of the disclosure.
Suitable pharmaceutically acceptable base addition salts of compounds of the disclosure include, for example, ammonium salts and metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N'-dibenzylethylene- diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (or N- methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.
Combination Therapies
In one aspect, the compounds of the disclosure are useful within the methods of the disclosure in combination with one or more additional agents useful for treating, ameliorating, and/or preventing HBV and/or HDV infections. These additional agents may comprise compounds or compositions identified herein, or compounds (e.g., commercially available compounds) known to treat, prevent, and/or reduce the symptoms of HBV and/or HDV infections.
Non-limiting examples of one or more additional agents useful for treating HBV and/or HDV infections include: (a) reverse transcriptase inhibitors; (b) capsid inhibitors; (c) cccDNA formation inhibitors; (d) RNA destabilizers; (e) oligomeric nucleotides targeted against the HBV genome; (f) immunostimulators, such as checkpoint inhibitors (e.g, PD-L1 inhibitors); (g) GalNAc-siRNA conjugates targeted against an HBV gene transcript; and (h) therapeutic vaccines.
(a) Reverse Transcriptase Inhibitors
In certain embodiments, the reverse transcriptase inhibitor is a reverse -transcriptase inhibitor (NARTI or NRTI). In other embodiments, the reverse transcriptase inhibitor is a nucleotide analog reverse-transcriptase inhibitor (NtARTI or NtRTI).
Reported reverse transcriptase inhibitors include, but are not limited to, entecavir, clevudine, telbivudine, lamivudine, adefovir, and tenofovir, tenofovir disoproxil, tenofovir alafenamide, adefovir dipovoxil, ( 1R,2R,3R,5R)-3-(6-ammo-9H-9-piirinyl)-2-fluoro-5- (hydroxymethyl)-4-methylenecyclopentan-1-ol (described in U.S. Patent No. 8,816,074, incorporated herein in its entirety by reference), emtricitabine, abacavir, elvucitabine, ganciclovir, lobucavir, famciclovir, penciclovir, and amdoxovir.
Reported reverse transcriptase inhibitors further include, but are not limited to, entecavir, lamivudine, and ( 1R,2R,3R,5R)-3-(6-ammo-9H-9-piirinyl)-2-fluoro-5- (hydroxymethyl)-4-methylenecyclopentan- 1 -ol.
Reported reverse transcriptase inhibitors further include, but are not limited to, a covalently bound phosphoramidate or phosphonamidate moiety of the above-mentioned reverse transcriptase inhibitors, or as described in for example U.S. Patent No. 8,816,074,
U.S. Patent Application Publications No. US 2011/0245484 Al, and US 2008/0286230A1, all of which incorporated herein in their entireties by reference.
Reported reverse transcriptase inhibitors further include, but are not limited to, nucleotide analogs that comprise a phosphoramidate moiety, such as, for example, methyl (((( 1R,3R,4R,5R)-3-(6-amino-9H-purin-9-yl)-4-fluoro-5-hydroxy-2-mcthylcnccyclopcntyl) methoxyXphenoxy) phosphoryl)-(D or U)-alaninate and methyl (((( 1 R.2R.3R.4R)-3-fluoro-2- hydroxy-5-methylene-4-(6-oxo-1,6-dihydro-9H-purin-9-yl)cyclopentyl)methoxy)(phenoxy) phosphoryl)-(D or U)-alaninate. Also included are the individual diastereomers thereof, which include, for example, methyl ((R)-((( 1R.3R.4R.5R)-3-(6-ammo-9H-purin-9-yl)-4-fluoro-5- hydroxy-2-methylenecyclopentyl)methoxy)(phenoxy)phosphoryl)-(D or U)-alaninate and methyl ((S)-((( 1R.3R.4R.5R)-3-(6-ammo-9H-purin-9-yl)-4-fluoro-5-hydroxy-2- methylenecyclopentyl) methoxyXphenoxy )phosphoryl)-(D or U)-alaninate.
Reported reverse transcriptase inhibitors further include, but are not limited to, compounds comprising a phosphonamidate moiety, such as, for example, tenofovir alafenamide, as well as those described in U.S. Patent Application Publication No. US 2008/0286230 Al, incorporated herein in its entirety by reference. Methods for preparing stereoselective phosphoramidate or phosphonamidate containing actives are described in, for example, U.S. Patent No. 8,816,074, as well as U.S. Patent Application Publications No. US 2011/0245484 Al and US 2008/0286230 Al, all of which incorporated herein in their entireties by reference.
(b) Capsid Inhibitors
As described herein, the term "capsid inhibitor" includes compounds that are capable of inhibiting the expression and/or function of a capsid protein either directly or indirectly. For example, a capsid inhibitor may include, but is not limited to, any compound that inhibits capsid assembly, induces formation of non-capsid polymers, promotes excess capsid assembly or misdirected capsid assembly, affects capsid stabilization, and/or inhibits encapsidation of RNA (pgRNA). Capsid inhibitors also include any compound that inhibits capsid function in a downstream event(s) within the replication process (e.g., viral DNA synthesis, transport of relaxed circular DNA (rcDNA) into the nucleus, covalently closed circular DNA (cccDNA) formation, virus maturation, budding and/or release, and the like). For example, in certain embodiments, the inhibitor detectably inhibits the expression level or biological activity of the capsid protein as measured, e.g., using an assay described herein. In certain embodiments, the inhibitor inhibits the level of rcDNA and downstream products of viral life cycle by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or at least 90%.
Reported capsid inhibitors include, but are not limited to, compounds described in International Patent Applications Publication Nos WO 2013006394, WO 2014106019, WO 2014089296, WO 2018052967, WO 201817285, WO 2020023710 and WO 2020123674, all of which incorporated herein in their entireties by reference.
Reported capsid inhibitors also include, but are not limited to, the following compounds and pharmaceutically acceptable salts and/or solvates thereof: Bay-41-4109 (see Int'l Patent Application Publication No. WO 2013144129), AT-61 (see Int'l Patent Application Publication No. WO 1998033501; and King, et al., 1998, Antimicrob. Agents Chemother. 42(12):3179—3186), DVR-01 and DVR-23 (see Int'l Patent Application Publication No. WO 2013006394; and Campagna, et al., 2013, J. Virol. 87(12):6931, all of which incorporated herein in their entireties by reference.
In addition, reported capsid inhibitors include, but are not limited to, those generally and specifically described in U.S. Patent Application Publication Nos. US 2015/0225355, US 2015/0132258, US 2016/0083383, US 2016/0052921 and Int'l Patent Application Publication Nos. WO 2013096744, WO 2014165128, WO 2014033170, WO 2014033167, WO 2014033176, WO 2014131847, WO 2014161888, WO 2014184350, WO 2014184365, WO 2015059212, WO 2015011281, WO 2015118057, WO 2015109130, WO 2015073774,
WO 2015180631, WO 2015138895, WO 2016089990, WO 2017015451, WO 2016183266, WO 2017011552, WO 2017048950, WO 2017048954, WO 2017048962, WO 2017064156 and are incorporated herein in their entirety by reference.
(c) cccDNA Formation Inhibitors
Covalently closed circular DNA (cccDNA) is generated in the cell nucleus from viral rcDNA and serves as the transcription template for viral mRNAs. As described herein, the term "cccDNA formation inhibitor" includes compounds that are capable of inhibiting the formation and/or stability of cccDNA either directly or indirectly. For example, a cccDNA formation inhibitor may include, but is not limited to, any compound that inhibits capsid disassembly, rcDNA entry into the nucleus, and/or the conversion of rcDNA into cccDNA. For example, in certain embodiments, the inhibitor detectably inhibits the formation and/or stability of the cccDNA as measured, e.g., using an assay described herein. In certain embodiments, the inhibitor inhibits the formation and/or stability of cccDNA by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or at least 90%.
Reported cccDNA formation inhibitors include, but are not limited to, compounds described in Int'l Patent Application Publication No. WO 2013130703, and are incorporated herein in their entirety by reference.
In addition, reported cccDNA formation inhibitors include, but are not limited to, those generally and specifically described in U.S. Patent Application Publication No. US 2015/0038515 Al, and are incorporated herein in their entirety by reference.
(d) RNA Destabilizer
As used herein, the term "RNA destabilizer" refers to a molecule, or a salt or solvate thereof, that reduces the total amount of HBV RNA in mammalian cell culture or in a live human subject. In a non-limiting example, an RNA destabilizer reduces the amount of the RNA transcript(s) encoding one or more of the following HBV proteins: surface antigen, core protein, RNA polymerase, and e antigen. In certain embodiments, the RNA destabilizer reduces the total amount of HBV RNA in mammalian cell culture or in a live human subject by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or at least 90%.
Reported RNA destabilizers include compounds described in U.S. Patent No. 8,921,381, as well as compounds described in U.S. Patent Application Publication Nos. US 2015/0087659 and US 2013/0303552, all of which are incorporated herein in their entireties by reference.
In addition, reported RNA destabilizers include, but are not limited to, those generally and specifically described in Int'l Patent Application Publication Nos. WO 2015113990, WO 2015173164, US 2016/0122344, WO 2016107832, WO 2016023877, WO 2016128335, WO 2016177655, WO 2016071215, WO 2017013046, WO 2017016921, WO 2017016960, WO 2017017042, WO 2017017043, WO 2017102648, WO 2017108630, WO 2017114812, WO 2017140821, WO 2018085619, WO 2019177937, WO 2019222238, WO 2020150366, and WO 2021025976, and are incorporated herein in their entirety by reference. (e) Oligomeric Nucleotides Targeted Against the HBV Genome
Reported oligomeric nucleotides targeted against the HBV genome include, but are not limited to, Arrowhead-ARC-520 (see U.S. Patent No. 8,809,293; and Wooddell et al., 2013, Molecular Therapy 21(5):973-985, all of which incorporated herein in their entireties by reference).
In certain embodiments, the oligomeric nucleotides can be designed to target one or more genes and/or transcripts of the HBV genome. Oligomeric nucleotide targeted to the HBV genome also include, but are not limited to, isolated, double stranded, siRNA molecules, that each include a sense strand and an antisense strand that is hybridized to the sense strand. In certain embodiments, the siRNA target one or more genes and/or transcripts of the HBV genome.
(f) I mm un ostim u I a tors
Checkpoint Inhibitors
As described herein, the term "checkpoint inhibitor" includes any compound that is capable of inhibiting immune checkpoint molecules that are regulators of the immune system (e.g., stimulate or inhibit immune system activity). For example, some checkpoint inhibitors block inhibitory checkpoint molecules, thereby stimulating immune system function, such as stimulation of T cell activity against cancer cells. A non-limting example of a checkpoint inhibitor is a PD-L1 inhibitor.
As described herein, the term "PD-L1 inhibitor" includes any compound that is capable of inhibiting the expression and/or function of the protein Programmed Death-Ligand 1 (PD-L1) either directly or indirectly. PD-L1, also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a type 1 transmembrane protein that plays a major role in suppressing the adaptive arm of immune system during pregnancy, tissue allograft transplants, autoimmune disease, and hepatitis. PD-L1 binds to its receptor, the inhibitory checkpoint molecule PD-1 (which is found on activated T cells, B cells, and myeloid cells) so as to modulate activation or inhibition of the adaptive arm of immune system. In certain embodiments, the PD-L1 inhibitor inhibits the expression and/or function of PD-L1 by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or at least 90%.
Reported PD-L1 Inhibitors include, but are not limited to, compounds recited in one of the following patent application publications: US 2018/0057455; US 2018/0057486; WO 2017/106634; WO 2018/026971; WO 2018/045142; WO 2018/118848; WO 2018/119221; WO 2018/119236; WO 2018/119266; WO 2018/119286; WO 2018/121560; WO 2019/076343; WO 2019/087214; and are incorporated herein in their entirety by reference. (g) GalNAc-siRNA Conjugates Targeted Against an HBV Gene Transcript
"GalNAc" is the abbreviation for N-acetylgalactosamine, and "siRNA" is the abbreviation for small interfering RNA. An siRNA that targets an HBV gene transcript is covalently bonded to GalNAc in a GalNAc-siRNA conjugate useful in the practice of the present disclosure. While not wishing to be bound by theory, it is believed that GalNAc binds to asialoglycoprotein receptors on hepatocytes thereby facilitating the targeting of the siRNA to the hepatocytes that are infected with HBV. The siRNA enter the infected hepatocytes and stimulate destruction of HBV gene transcripts by the phenomenon of RNA interference.
Examples of GalNAc-siRNA conjugates useful in the practice of this aspect of the present disclosure are set forth in published PCT International Applications No.
PCT/CA2017/050447 (PCT Application Publication No. WO/2017/177326, published October 19, 2017) and No. PCT/US2018/026918 (PCT Application Publication No. WO2018/191278, published October 18, 2018). all of which are hereby incorporated by reference in their entireties.
(h) Therapeutic Vaccines
In certain embodiments, administration of a therapeutic vaccine is useful in the practice of the present disclosure for the treatment of a viral disease in a subject. In certain embodiments, the viral disease is a hepatitis virus. In certain embodiments, the hepatitis virus is at least one selected from the group consisting of hepatitis B virus (HBV) and hepatitis D virus (HDV). In certain embodiments, the subject is a human.
A synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-Emax equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114: 313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22:27-55). Each equation referred to elsewhere herein may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to elsewhere herein are the concentration-effect curve, isobologram curve and combination index curve, respectively.
Synthesis
The present disclosure further provides methods of preparing the compounds of the present disclosure. Compounds of the present teachings can be prepared in accordance with the procedures outlined herein, from commercially available starting materials, compounds known in the literature, or readily prepared intermediates, by employing standard synthetic methods and procedures known to those skilled in the art. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be readily obtained from the relevant scientific literature or from standard textbooks in the field. It should be contemplated that the disclosure includes each and every one of the synthetic schemes described and/or depicted herein.
It is appreciated that where typical or preferred process conditions (i.e.. reaction temperatures, times, mole ratios of reactants, solvents, pressures, and so forth) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions can vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures. Those skilled in the art of organic synthesis will recognize that the nature and order of the synthetic steps presented can be varied for the purpose of optimizing the formation of the compounds described herein.
The processes described herein can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or by chromatography such as high pressure liquid chromatography (HPLC), gas chromatography (GC), gel-permeation chromatography (GPC), or thin layer chromatography (TLC).
Preparation of the compounds can involve protection and deprotection of various chemical groups. The need for protection and deprotection and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Greene, et al. , Protective Groups in Organic Synthesis, 2d. Ed. (Wiley & Sons, 1991), the entire disclosure of which is incorporated by reference herein for all purposes.
The reactions or the processes described herein can be carried out in suitable solvents that can be readily selected by one skilled in the art of organic synthesis. Suitable solvents typically are substantially nonreactive with the reactants, intermediates, and/or products at the temperatures at which the reactions are carried out, i. e. , temperatures that can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected.
The following Schemes illustrate non-limiting synthetic routes that allow for the preparation of certain compounds of the disclosuren. It should be noted that substituents in these Schemes, such as but not limited to Ra-Re, are non-limiting in nature and correspond to substituents defined elsewhere herein, as would be contemplated by one skilled in the art.
In certain embodiments, a compound of formula (I), can be prepared, for example, according to the synthetic methods outlined in Scheme 1.
Figure imgf000031_0001
In certain embodiments, a compound of formula (I), can be prepared, for example, according to the synthetic methods outlined in Scheme 2.
Figure imgf000032_0001
Methods The disclosure provides a method of treating, ameliorating, and/or preventing hepatitis virus infection in a subject. In certain embodiments, the infection comprises hepatitis B virus (HBV) infection. In yet other embodiments, the infection comprises hepatitis D virus (HDV) infection. In other embodiments, the method comprises administering to the subject in need thereof a therapeutically effective amount of at least one compound of the disclosure. In yet other embodiments, the compound of the disclosure is the only antiviral agent administered to the subject. In yet other embodiments, the at least one compound is administered to the subject in a pharmaceutically acceptable composition. In yet other embodiments, the subject is further administered at least one additional agent useful for treating the hepatitis virus infection. In yet other embodiments, the at least one additional agent comprises at least one selected from the group consisting of reverse transcriptase inhibitors, capsid inhibitors, cccDNA formation inhibitors, RNA destabilizers, oligomeric nucleotides targeted against the HBV genome, immunostimulators, GalNAc-siRNA conjugates targeted against an HBV gene transcript, and therapeutic vaccines. In yet other embodiments, the subject is co-administered the at least one compound and the at least one additional agent. In yet other embodiments, the at least one compound and the at least one additional agent are coformulated.
The disclosure further provides a method of inhibiting and/or reducing HBV surface antigen (HBsAg) secretion either directly or indirectly in a subject. The disclosure further provides a method of reducing or minimizing levels of HBsAg in a HBV-infected subject.
The disclosure further provides a method of reducing or minimizing levels of HBeAg in a HBV-infected subject. The disclosure further provides a method of reducing or minimizing levels of hepatitis B core protein in a HBV-infected subject. The disclosure further provides a method of reducing or minimizing levels of pg RNA in a HBV-infected subject.
In certain embodiments, the method comprises administering to the subject in need thereof a therapeutically effective amount of at least one compound of the disclosure. In other embodiments, the at least one compound is administered to the subject in a pharmaceutically acceptable composition. In yet other embodiments, the compound of the disclosure is the only antiviral agent administered to the subject. In yet other embodiments, the subject is further administered at least one additional agent useful for treating the hepatitis infection. In yet other embodiments, the at least one additional agent comprises at least one selected from the group consisting of reverse transcriptase inhibitors, capsid inhibitors, cccDNA formation inhibitors, RNA destabilizers, oligomeric nucleotides targeted against the HBV genome, immunostimulators, GalNAc-siRNA conjugates targeted against an HBV gene transcript, and therapeutic vaccines. In yet other embodiments, the subject is co-administered the at least one compound and the at least one additional agent. In yet other embodiments, the at least one compound and the at least one additional agent are coformulated.
In certain embodiments, the subject is infected with HBV. In other embodiments, the subject is infected with HDV. In yet other embodiments, the subject is infected with HBV and HDV.
In certain embodiments, the subject is a mammal. In other embodiments, the mammal is a human.
Pharmaceutical Compositions and Formulations
The disclosure provides pharmaceutical compositions comprising at least one compound of the disclosure or a salt or solvate thereof, which are useful to practice methods of the disclosure. Such a pharmaceutical composition may consist of at least one compound of the disclosure or a salt or solvate thereof, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one compound of the disclosure or a salt or solvate thereof, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. At least one compound of the disclosure may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
In certain embodiments, the pharmaceutical compositions useful for practicing the method of the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In other embodiments, the pharmaceutical compositions useful for practicing the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 1,000 mg/kg/day.
The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the disclosure will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.
Pharmaceutical compositions that are useful in the methods of the disclosure may be suitably developed for nasal, inhalational, oral, rectal, vaginal, pleural, peritoneal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, epidural, intrathecal, intravenous or another route of administration. A composition useful within the methods of the disclosure may be directly administered to the brain, the brainstem, or any other part of the central nervous system of a mammal or bird. Other contemplated formulations include projected nanoparticles, microspheres, liposomal preparations, coated particles, polymer conjugates, resealed erythrocytes containing the active ingredient, and immunologically- based formulations.
In certain embodiments, the compositions of the disclosure are part of a pharmaceutical matrix, which allows for manipulation of insoluble materials and improvement of the bioavailability thereof, development of controlled or sustained release products, and generation of homogeneous compositions. By way of example, a pharmaceutical matrix may be prepared using hot melt extrusion, solid solutions, solid dispersions, size reduction technologies, molecular complexes (e.g., cyclodextrins, and others), microparticulate, and particle and formulation coating processes. Amorphous or crystalline phases may be used in such processes.
The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like. The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology and pharmaceutics. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single-dose or multi-dose unit.
As used herein, a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one- third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the disclosure is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.
In certain embodiments, the compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of at least one compound of the disclosure and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers, which are useful, include, but are not limited to, glycerol, water, saline, ethanol, recombinant human albumin (e.g., RECOMBUMIN®), solubilized gelatins (e.g., GELOFUSINE®), and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey). The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), recombinant human albumin, solubilized gelatins, suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, are included in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.
Formulations may be employed in admixtures with conventional excipients, i. e. , pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, inhalational, intravenous, subcutaneous, transdermal enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or fragrance-conferring substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic, anxiolytics or hypnotic agents. As used herein, "additional ingredients" include, but are not limited to, one or more ingredients that may be used as a pharmaceutical carrier.
The composition of the disclosure may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the disclosure include but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and combinations thereof. One such preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.
The composition may include an antioxidant and a chelating agent which inhibit the degradation of the compound. Antioxidants for some compounds are BHT, BHA, alpha- tocopherol and ascorbic acid in the exemplary range of about 0.01% to 0.3%, or BHT in the range of 0.03% to 0.1% by weight by total weight of the composition. The chelating agent may be present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Exemplary chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01% to 0.20%, or in the range of 0.02% to 0.10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are exemplary antioxidant and chelating agent, respectively, for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.
Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl cellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, acacia, and ionic or non-ionic surfactants. Known preservatives include, but are not limited to, methyl, ethyl, or «-propyl para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.
Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. As used herein, an "oily" liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water. Liquid solutions of the pharmaceutical composition of the disclosure may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
Powdered and granular formulations of a pharmaceutical preparation of the disclosure may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, ionic and non-ionic surfactants, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.
A pharmaceutical composition of the disclosure may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally- occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.
Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (/. e. , such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying. Methods for mixing components include physical milling, the use of pellets in solid and suspension formulations and mixing in a transdermal patch, as known to those skilled in the art.
Administration/Dosing
The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the patient either prior to or after the onset of a disease or disorder. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
Administration of the compositions of the present disclosure to a patient, such as a mammal, such as a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder contemplated herein. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the disclosure is from about 0.01 mg/kg to 100 mg/kg of body weight/per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
The compound may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose is readily apparent to the skilled artisan and depends upon a number of factors, such as, but not limited to, type and severity of the disease being treated, and type and age of the animal.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disease or disorder in a patient.
In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two days, every three days to once a week, and once every two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure will vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient will be determined by the attending physician taking all other factors about the patient into account.
Compounds of the disclosure for administration may be in the range of from about 1 pig to about 7,500 mg, about 20 pig to about 7,000 mg, about 40 pig to about 6,500 mg, about 80 pig to about 6,000 mg, about 100 pig to about 5,500 mg, about 200 pig to about 5,000 mg, about 400 pig to about 4,000 mg, about 800 pig to about 3,000 mg, about 1 mg to about 2,500 mg, about 2 mg to about 2,000 mg, about 5 mg to about 1,000 mg, about 10 mg to about 750 mg, about 20 mg to about 600 mg, about 30 mg to about 500 mg, about 40 mg to about 400 mg, about 50 mg to about 300 mg, about 60 mg to about 250 mg, about 70 mg to about 200 mg, about 80 mg to about 150 mg, and any and all whole or partial increments there-in- between.
In some embodiments, the dose of a compound of the disclosure is from about 0.5 pig and about 5,000 mg. In some embodiments, a dose of a compound of the disclosure used in compositions described herein is less than about 5,000 mg, or less than about 4,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.
The term "container" includes any receptacle for holding the pharmaceutical composition or for managing stability or water uptake. For example, in certain embodiments, the container is the packaging that contains the pharmaceutical composition, such as liquid (solution and suspension), semisolid, lyophilized solid, solution and powder or lyophilized formulation present in dual chambers. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i.e.. the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound's ability to perform its intended function, e.g., treating, preventing, or reducing a disease or disorder in a patient.
Administration
Routes of administration of any of the compositions of the disclosure include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, epidural, intrapleural, intraperitoneal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, emulsions, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.
Oral Administration
For oral application, particularly suitable are tablets, dragees, liquids, drops, capsules, caplets and gelcaps. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, a paste, a gel, toothpaste, a mouthwash, a coating, an oral rinse, or an emulsion. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic, generally recognized as safe (GRAS) pharmaceutically excipients which are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate.
Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods described in U.S. Patents Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotically controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide for pharmaceutically elegant and palatable preparation. Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. The capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.
Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.
Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin from animal-derived collagen or from a hypromellose, a modified form of cellulose, and manufactured using optional mixtures of gelatin, water and plasticizers such as sorbitol or glycerol. Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.
For oral administration, the compounds of the disclosure may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents; fillers; lubricants; disintegrates; or wetting agents. If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY ® film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY® OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY® White, 32K18400). It is understood that similar type of film coating or polymeric products from other companies may be used.
A tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycolate. Known surface-active agents include, but are not limited to, sodium lauryl sulphate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, com starch and alginic acid. Known binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.
Granulating techniques are well known in the pharmaceutical art for modifying starting powders or other particulate materials of an active ingredient. The powders are typically mixed with a binder material into larger permanent free-flowing agglomerates or granules referred to as a "granulation." For example, solvent-using "wet" granulation processes are generally characterized in that the powders are combined with a binder material and moistened with water or an organic solvent under conditions resulting in the formation of a wet granulated mass from which the solvent must then be evaporated.
Melt granulation generally consists in the use of materials that are solid or semi-solid at room temperature (i.e.. having a relatively low softening or melting point range) to promote granulation of powdered or other materials, essentially in the absence of added water or other liquid solvents. The low melting solids, when heated to a temperature in the melting point range, liquefy to act as a binder or granulating medium. The liquefied solid spreads itself over the surface of powdered materials with which it is contacted, and on cooling, forms a solid granulated mass in which the initial materials are bound together. The resulting melt granulation may then be provided to a tablet press or be encapsulated for preparing the oral dosage form. Melt granulation improves the dissolution rate and bioavailability of an active (i.e., drug) by forming a solid dispersion or solid solution.
U.S. Patent No. 5,169,645 discloses directly compressible wax-containing granules having improved flow properties. The granules are obtained when waxes are admixed in the melt with certain flow improving additives, followed by cooling and granulation of the admixture. In certain embodiments, only the wax itself melts in the melt combination of the wax(es) and additives(s), and in other cases both the wax(es) and the additives(s) will melt.
The present disclosure also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds useful within the methods of the disclosure, and a further layer providing for the immediate release of one or more compounds useful within the methods of the disclosure. Using a wax/pH-sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.
Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non- aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl para-hydroxy benzoates or sorbic acid). Liquid formulations of a pharmaceutical composition of the disclosure which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use.
Parenteral Administration
As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrastemal injection, and kidney dialytic infusion techniques.
Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multidose containers containing a preservative. Injectable formulations may also be prepared, packaged, or sold in devices such as patient-controlled analgesia (PCA) devices. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e.. powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non toxic parenterally acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form in a recombinant human albumin, a fluidized gelatin, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
Topical Administration
An obstacle for topical administration of pharmaceuticals is the stratum comeum layer of the epidermis. The stratum comeum is a highly resistant layer comprised of protein, cholesterol, sphingolipids, free fatty acids and various other lipids, and includes comified and living cells. One of the factors that limit the penetration rate (flux) of a compound through the stratum comeum is the amount of the active substance that can be loaded or applied onto the skin surface. The greater the amount of active substance which is applied per unit of area of the skin, the greater the concentration gradient between the skin surface and the lower layers of the skin, and in turn the greater the diffusion force of the active substance through the skin. Therefore, a formulation containing a greater concentration of the active substance is more likely to result in penetration of the active substance through the skin, and more of it, and at a more consistent rate, than a formulation having a lesser concentration, all other things being equal.
Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
Enhancers of permeation may be used. These materials increase the rate of penetration of drugs across the skin. Typical enhancers in the art include ethanol, glycerol monolaurate, PGML (polyethylene glycol monolaurate), dimethylsulfoxide, and the like. Other enhancers include oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids, dimethylsulfoxide, polar lipids, orN-methyl-2-pyrrolidone.
One acceptable vehicle for topical delivery of some of the compositions of the disclosure may contain liposomes. The composition of the liposomes and their use are known in the art (i.e., U.S. Patent No. 6,323,219).
In alternative embodiments, the topically active pharmaceutical composition may be optionally combined with other ingredients such as adjuvants, anti-oxidants, chelating agents, surfactants, foaming agents, wetting agents, emulsifying agents, viscosifiers, buffering agents, preservatives, and the like. In other embodiments, a permeation or penetration enhancer is included in the composition and is effective in improving the percutaneous penetration of the active ingredient into and through the stratum comeum with respect to a composition lacking the permeation enhancer. Various permeation enhancers, including oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids, dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone, are known to those of skill in the art. In another aspect, the composition may further comprise a hydrotropic agent, which functions to increase disorder in the structure of the stratum comeum, and thus allows increased transport across the stratum comeum. Various hydrotropic agents such as isopropyl alcohol, propylene glycol, or sodium xylene sulfonate, are known to those of skill in the art.
The topically active pharmaceutical composition should be applied in an amount effective to affect desired changes. As used herein "amount effective" shall mean an amount sufficient to cover the region of skin surface where a change is desired. An active compound should be present in the amount of from about 0.0001% to about 15% by weight volume of the composition. For example, it should be present in an amount from about 0.0005% to about 5% of the composition; for example, it should be present in an amount of from about 0.001% to about 1% of the composition. Such compounds may be synthetically-or naturally derived.
Buccal Administration A pharmaceutical composition of the disclosure may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) of the active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient. Such powdered, aerosolized, or aerosolized formulations, when dispersed, may have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein. The examples of formulations described herein are not exhaustive and it is understood that the disclosure includes additional modifications of these and other formulations not described herein, but which are known to those of skill in the art.
Rectal Administration
A pharmaceutical composition of the disclosure may be prepared, packaged, or sold in a formulation suitable for rectal administration. Such a composition may be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic irrigation.
Suppository formulations may be made by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e.. about 20°C) and which is liquid at the rectal temperature of the subject (i.e.. about 37°C in a healthy human). Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides. Suppository formulations may further comprise various additional ingredients including, but not limited to, antioxidants, and preservatives.
Retention enema preparations or solutions for rectal or colonic irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, enema preparations may be administered using, and may be packaged within, a delivery device adapted to the rectal anatomy of the subject. Enema preparations may further comprise various additional ingredients including, but not limited to, antioxidants, and preservatives.
Additional Administration Forms
Additional dosage forms of this disclosure include dosage forms as described in U.S. Patents Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790. Additional dosage forms of this disclosure also include dosage forms as described in U.S. Patent Applications Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos. WO 03/35041, WO 03/35040, WO 03/35029, WO 03/35177, WO 03/35039, WO 02/96404, WO 02/32416, WO 01/97783, WO 01/56544, WO 01/32217, WO 98/55107, WO 98/11879, WO 97/47285, WO 93/18755, and WO 90/11757.
Controlled Release Formulations and Drug Delivery Systems
In certain embodiments, the compositions and/or formulations of the present disclosure may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method of the disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
In certain embodiments of the disclosure, the compounds useful within the disclosure are administered to a subject, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that may, although not necessarily, include a delay of from about 10 minutes up to about 12 hours.
The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.
As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this disclosure and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, with art- recognized alternatives and using no more than routine experimentation, are within the scope of the present application.
It is to be understood that, wherever values and ranges are provided herein, the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, all values and ranges encompassed by these values and ranges are meant to be encompassed within the scope of the present disclosure. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application. The description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range and, when appropriate, partial integers of the numerical values within ranges. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
The following examples further illustrate aspects of the present disclosure. However, they are in no way a limitation of the teachings or disclosure of the present disclosure as set forth herein.
EXAMPLES The disclosure is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the disclosure is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.
Materials & Methods
The following procedures can be utilized in preparing and/or testing exemplary compounds of the disclosure.
Mouse In Vivo PK Assay
Female CD-I mice (30 ±- 3 g) were ordered from Hilltop Lab Animals Inc., then acclimated for at least 3 days before the commencement of the pharmacokinetic studies. Compounds of interest were dissolved into a cosolvent formulation amenable to oral dosing. The formulation consisted of 60% polyethylene glycol 400 (Spectrum Chemical, NF grade), 10% propylene glycol (Spectrum Chemical, NF grade), 5% ethanol (Spectrum Chemical,
USP grade, 200 proof) and 25% phosphate buffered saline, pH 7.4 (Thermoscientific). Compounds were either prepared at 0.02 mg/mL or 1 mg/mL. Animals were dosed orally based on body weight at a volume of 10 mL/kg. Per study design, doses administered were 0.2 mg/kg and/or 10 mg/kg. The treated animals were terminated at 12 or 24 hours post dose.
The terminal collections consisted of ~500 μL of blood obtained by cardiac puncture and transferred to microtubes containing anti-coagulant K2EDTA. The blood was then centrifuged (16,000 x g), and the plasma isolated. In addition, livers were also harvested. All samples were stored at -80 °C prior to analysis. Plasma was used as collected. Liver samples were weighed and homogenized in four volumes (w/v) of phosphate buffered saline, pH 7.4 (PBS). Plasma and liver homogenate samples were precipitated with 30 and 7.5 volumes of acetonitrile, respectively. In parallel, calibration curve samples were prepared by spiking known concentrations (0.24 - 4,000ng/mL) of compounds into blank CD-I matrices of plasma and liver homogenate. The samples were centrifuged (1,200 x g) and the supernatants were collected. The supernatants were injected then into an LC-MS/MS system for analysis. The LC-MS/MS detection is performed using a SCIEX 5500 Qtrap instrument. Each compound was analyzed by reverse phase HPLC using an ACE Ultracore SuperC182.5 pm, 2.1 mm x 50 mm column. Mobile phases consisted of Solvent A: water with 0.1% formic acid, and Solvent B: acetonitrile with 0.1% formic acid. A calibration curve for each compound was constructed by fitting the known analyte concentration versus the analyte response using the least-squares linear regression analysis with a weighting scheme of 1/x2. Compound concentrations in plasma and liver homogenate samples were quantified by interpolation on the calibration curve of the relevant matrix. Interpolated plasma concentrations values were directly reported. Liver homogenate concentration values were multiplied by five to account for the additional four volumes of PBS used during homogenization, in order to extrapolate the original concentration in liver tissue only.
Rat In Vivo PK Assay
Female Sprague Dawley rats (225 ± 25 g) were ordered from Hilltop Lab Animals Inc., then acclimated for at least 3 days before the commencement of the pharmacokinetic studies. Compounds of interest were dissolved into a cosolvent formulation amenable to oral dosing. For Compound B, the formulation was 0.5% methylcellulose (Fisher Scientific)/ 0.1% Tween80 (Thermo Scientific) in water. For Compound C and Compound 1, the formulation consisted of 60% polyethylene glycol 400 (Spectrum Chemical, NF grade), 10% propylene glycol (Spectrum Chemical, NF grade), 5% ethanol (Spectrum Chemical, USP grade, 200 proof) and 25% phosphate buffered saline, pH 7.4 (Thermoscientific). For Compound 19, the formulation consisted of 60% polyethylene glycol 400 (Spectrum Chemical, NF grade), 10% propylene glycol (Spectrum Chemical, NF grade), and 30% 100 mM bicarbonate buffer, pH 9 (Thermoscientific). For oral dose, compounds were either prepared at 1 mg/mL or 2 mg/mL. Animals were dosed orally based on body weight at a volume of 5 mL/kg. Per study design, doses administered were 5 mg/kg and/or 10 mg/kg.
The treated animals were terminated at 12 or 24 hours post dose.
The terminal collections consisted of ~0.5-1.0 mL of blood obtained by cardiac puncture and transferred to microtubes containing anti-coagulant K2EDTA. The blood was then centrifuged (16,000 x g) at 4 °C for 5 min, and the plasma was isolated. In addition, livers were also harvested. All samples were stored at -80 °C prior to analysis. Plasma was used as collected. Liver samples were weighed and homogenized in four volumes (w/v) of phosphate buffered saline, pH 7.4 (PBS). Plasma and liver homogenate samples were precipitated with 26.7 and 10.0 volumes of acetonitrile containing internal standard, respectively. In parallel, calibration curve samples were prepared by spiking known concentrations (0.49 - 8,000 ng/mL) of compounds into blank SD rat plasma and liver homogenate. The samples were centrifuged (1,200 x g) and the supernatants were collected. The supernatants were injected into an LC-MS/MS system for analysis. The LC-MS/MS detection is performed using a SCIEX 5500 Qtrap instrument. Each compound was analyzed by reverse phase HPLC using an ACE Ultracore SuperC18 2.5 pm, 2.1 mm x 50 mm column. Mobile phases consisted of Solvent A: water with 0.1% formic acid, and Solvent B: acetonitrile with 0.1% formic acid. A calibration curve for each compound was constructed by fitting the known analyte concentration versus the analyte response using the least-squares linear regression analysis with a weighting scheme of 1/x2. Compound concentrations in plasma and liver homogenate samples were quantified by interpolation on the calibration curve of the relevant matrix. Interpolated plasma concentrations values were directly reported. Liver homogenate concentration values were multiplied by five to account for the additional four volumes of PBS used during homogenization, in order to extrapolate the original concentration in liver tissue only.
Microsome Stability Assay
The in vitro metabolic stability of compounds in a liver microsome system was used to assess their susceptibility to hepatic degradation. Stability was assessed in mouse liver microsomes or rat liver microsomes (respectively, MLM or RLM; BioIVT) at 0.3 μM for each compound containing 0.5 mg/mL of protein, at 37 °C in the presence of the co-factor, NADPH, which initiates the reaction. Depletion of the test compounds was monitored over a 60-minute incubation period, with time point collections at 0, 5, 10, 20, 30, and 60 min. At each collection, the reaction was terminated by the addition of acetonitrile containing internal standard. Following centrifugation, the supernatant was analyzed by LC-MS/MS using a SCIEX 5500 Qtrap instrument. Each compound was analyzed by reverse phase HPLC using an ACE Ultracore SuperC182.5 pm, 2.1 mm x 50 mm column. Mobile phases consisted of Solvent A: water with 0.1% formic acid, and Solvent B: acetonitrile with 0.1% formic acid.
Compounds were quantified as a ratio of the analyte response relative to the response of the internal standard (A/IS ratio). Using Excel, the logarithmic value of the A/IS ratio was plotted against time. The elimination rate constant (K)(1/min) is defined as the slope of the logarithmic trend and was determined using the built-in Excel “SLOPE” function. The intrinsic clearance (Clint)(μL/min/mg protein) was calculated as -K/(protein concentration of incubation), or -K/(0.5 mg/mL) per above.
Example 1: (S)-5-(tert-butyl)-11-(difluoromethoxy)-9-methoxy-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer
I)
Example 2: (R)-5-tert -butyl)-11-(difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer
P)
Figure imgf000054_0001
(a) Synthesis of 3,5-dimethoxy-2-nitropyridine
Figure imgf000054_0002
To a solution of 3,5-dimethoxypyridine (10 g, 71.9 mmol) in concentrated H2SO4 (100 mL) was added fuming HNO3 (3 ml, 71.9 mmol) at 0 °C. The reaction mixture was stirred at rt for 1 h. The reaction mixture was quenched by pouring into ice cold water (100 mL). The resulting precipitate was filtered, washed with water (100 mL), followed by diethyl ether (2 x 50 mL) and dried to afford of 3,5-dimethoxy-2-nitropyridine as an off white solid (12 g, 90% yield, m/z: 185 [M+H]+ observed). 1H NMR (400 MHz, CDCl3): δ 7.83 (d, 1H), 7.43 (d, 1H), 3.97 (s, 6H).
(b) Synthesis of 5-methoxy-2-nitropyridin-3-ol
Figure imgf000054_0003
To a solution 3,5-dimethoxy-2-nitropyridine (10 g, 54.3 mmol) in methanol (150 mL) was added NaOH (IN, 163 ml) at 0 °C and the reaction mixture was stirred at 80 °C for 6 h. The mixture was cooled to rt and acidified with HC1 solution (IN, 100 mL). The aqueous layer was extracted with ethyl acetate (2 x 200 mL). The combined organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude residue was purified by normal phase SiO2 chromatography (0-100% EtO Ac/hexanes) to afford 5- methoxy-2-nitropyridin-3-ol as an off white solid (4 g, 43% yield, m/z: 171 [M+H]+ observed).
(c) Synthesis of 3-(difluoromethoxy)-5-methoxy-2-nitropyridine
Figure imgf000055_0001
To a solution of 5-methoxy-2-nitropyridin-3-ol (10 g, 58.8 mmol) and sodium chlorodifluoroacetate (35.9 g, 235 mmol) in DMSO (160 mL) and water (60 mL) was added K2CO3 (24.4 g, 176 mmol) at rt. The reaction mixture was stirred at 100 °C for 16 h. The mixture was cooled to rt, poured into ice water (200 mL) and extracted with EtOAc (2 x 300 mL). The combined organic layer was washed with saturated aqueous brine solution (100 mL), dried over anhydrous Na2SO4 and evaporated under reduced pressure. The crude mixture was basified with aqueous NaOH (IN) to pH 10 and extracted with EtOAc (2 x 100 mL). The combined organic phase was dried over anhydrous Na2S04 and concentrated under reduced pressure to afford 3-(difluoromethoxy)-5-methoxy-2-nitropyridine as a yellow liquid (5 g, 39% yield, m/z: 221 [M+H]+ observed). 1H NMR 400 MHz, CDCl3): δ 8.08 (d, 1H), 7.27 (d, 1H), 6.86-6.50 (m, 1H), 4.00 (s, 3H).
(d) Synthesis of 3-(difluoromethoxy)-5-methoxypyridin-2-amine
Figure imgf000055_0002
To a solution of 3-(difluoromethoxy)-5-methoxy-2-nitropyridine (4 g, 18.2 mmol) in MeOH (40 mL) was added palladium on carbon (10 wt%, 4 g, 1.82 mmol). The suspension was degassed using vacuum and purged with hydrogen gas (repeated cycle 2 times). The reaction was stirred at rt under hydrogen atmosphere for 16 h. The reaction mixture was fdtered through CELITE® and concentrated under reduced pressure. The crude residue was dissolved in CH2CI2, treated with activated charcoal (2 g), fdtered through CELITE® and concentrated under vacuum to afford 3-(difluoromethoxy)-5-methoxypyridin-2 -amine as a brown solid (2.5 g, 72% yield, m/z: 191 [M+H]+ observed).
(e) Synthesis of 4-tert-hutylcyclohexane-1,2-dione
Figure imgf000055_0003
To a mixture of 4-tert-butylcyclohexanone (38.5 g, 250 mmol) in 1,4-dioxane : water (1: 1, 200 mL) was added selenium dioxide (30.5 g, 275 mmol). The reaction mixture was stirred at rt for 30 minutes under nitrogen atmosphere, then warmed to 50 °C for 16 h. The mixture was cooled to rt and fdtered through CELITE®. The filtrtate was extracted with EtOAc (3 x 50 mL). The combined organic layer was dried over Na2SO4, fdtered through CELITE® and concentrated under reduced pressure. The residue was purified by normal phase SiO2 chromatography (0-40% EtO Ac/hexanes) to afford 4-tert-biitylcyclohexane- 1.2- dione as a yellow oil (18.9 g, 45% yield, m/z: 169 [M+H]+ observed). (f) Synthesis of 8-(tert-butyl)-4-(difluoromethoxy)-2-methoxy-8,9- dihydrobenzo[4,5]imidazo[1,2-a]pyridin-6(7H)-one
Figure imgf000056_0001
A mixture of 4-(tert-butyl (cyclohexane- 1 2-dionc (13.3 g, 79 mmol) and 3- (difluoromethoxy)-5-methoxypyridin-2-amine (6 g, 32 mmol) in acetic acid (60 mL) was heated to 115 °C under oxygen atmosphere for 12 h. The reaction mixture was cooled to rt and concentrated under reduced pressure. The residue was diluted with EtOAc (200 mL), washed with saturated aqueous NaHC03 solution (100 mL), followed by saturated aqueous brine solution (50 mL). The organic phase was dried over Na2S04, filtered, and evaporated under reduced pressure. The crude residue was triturated with diethyl ether (10 mL) to obtain 8-(tert-butyl)-4-(difluoromethoxy)-2-methoxy-8.9-dihydrobenzo|4.5 |imidazo| 1.2-a|pyridin- 6(7H)-one as a brown solid (2.5 g, 23% yield, m/z: 339 [M+H]+ observed.
(g) Synthesis of 8-(tert-butyl)-4-(difluoromethoxy)-N-(2,4-dimethoxybenzyl)-2- methoxy-8,9-dihydrobenzo[4,5]imidazo[1,2-a]pyridin-6(7H)-imine
Figure imgf000057_0001
To a solution of 8-tert -butyl )-4-(difluoromcthoxy )-2-mcthoxy-8.9- dihydrobenzo[4,5]imidazo[1,2-a]pyridin-6(7H)-one (1 g, 2.96 mmol) in CH2CI2 (15 ml) were added 2,4-dimethoxybenzylamine (0.66 mL, 4.4 mmol) and triethylamine (1.7 mL, 11.8 mmol). The reaction mixture was cooled to 0 °C and TiCl4 (1M solution in CH2CI2, 3.3 mL, 3.3 mmol) was added slowly over 10 min. The reaction mixture was slowly warmed to rt and stirred for 16 h. The mixture was quenched with saturated aqueous NaHCO3 solution (20 mL) and fdtered. The fdtrate was extracted with CH2CI2 (3 x 40 mL). The combined organic layer phase was dried over Na2SO4 and concentrated under reduced pressure to afford 8 -(tert- butyl)-4-(difluoromethoxy)-N-(2.4-dimethoxybenzyl)-2-methoxy-8.9- dihydrobenzo[4,5]imidazo[1,2-a]pyridin-6(7H)-imine as a brown solid which was used in the next step without further purification (1.1 g, 76% yield, m/z: 488 [M+H]+ observed).
(h) Synthesis of (S)-methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4- dimethoxybenzyl)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h]quinoline-3-carboxylate and (R)-methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4- dimethoxybenzyl)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h ]quin olin e-3-carboxylate
Figure imgf000057_0002
To a solution of 8-(tert-butyl )-4-(difluo romethoxy)-N-(2.4-di methoxybenzyl )-2- methoxy-8,9-dihydrobenzo[4,5]imidazo[1,2-a]pyridin-6(7H)-imine (1.1 g, 2.3 mmol) in diglyme (7 mL) was added dimethyl 2-(methoxymethylene)malonate (0.59 g, 3.4 mmol). The reaction mixture was heated to 180 °C for 30 min. The reaction was cooled to rt and allowed to stand for 16h. The precipitated solid was collected by filtration and washed with petroleum ether (2 x 10 mL). The crude residue was purified by normal phase SiO2 chromatography (0- 80% EtOAc/ petroleum ether) to afford methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4- dimethoxybenzyl)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylate as a yellow solid (200 mg, 15% yield, m/z: 598 [M+H]+ observed).
400 mg of the mixture of enantiomers was separated by SFC (supercritical fluid chromatography) on CHIRALCEL-OX-H column using 50% CELCN: MeOH to give (S)- methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-9-methoxy-2-oxo- 1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer I) as a yellow solid (faster eluting enantiomer, 120 mg, 30% yield, m/z: 598 [M+H]+ observed) and (R)-methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-9- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer II) as a yellow solid (slower eluting enantiomer, 120 mg, 30% yield, m/z: 598 [M+H]+ observed).
(i) Synthesis of (S)-5-(tert-Butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-9- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I)
Figure imgf000058_0001
To a solution of (S)-mcthyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4- dimethoxybenzyl)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h]quinoline-3-carboxylate (single enantiomer I) (100 mg, 0.17 mmol) in THF H2O (1: 1, 10 mL) was added lithium hydroxide monohydrate (28 mg, 0.67 mmol) at rt. The reaction mixture was heated to 40 °C for 2 h. The mixture was cooled to rt and 10% aqueous citric acid solution (50 mL) was added. The mixture was extracted with EtOAc (2 x 50 mL). The combined organic phase was dried over Na2SO4 and concentrated under reduced pressure to afford (S) -5-tert -butyl)- 11 -(difluoromethoxy)- 1 -(2,4-dimethoxybenzyl)-4-hydroxy-9- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) as a yellow solid (82 mg, 82% yield, m/z: 584 [M+H]+ observed).
(j) Synthesis of (S)-5-(tert-Butyl)-11-(difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) (Example 1)
Figure imgf000059_0001
To a solution of (S-)5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-9- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) (80 mg, 0.14 mmol) in CH2CI2 (1 mL) was added trifluoroacetic acid (0.1 mL) at 0 °C. The reaction mixture was slowly warmed to rt and stirred for 4 h. The mixture was quenched with saturated aqueous NaHCO3 solution (4 mL), the phases were separated, and the aqueous layer was extracted further with CH2CI2 (3 x 10 mL). The combined organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was triturated with diethyl ether to obtain (S)-5-(tert-butyl)- 1 1- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid (single enantiomer I) as a yellow solid (12 mg, 20% yield, m/z: 434 [M+H]+ observed). 1H NMR (400 MHz, DMSO-d6): δ 14.97 (s, 1H), 13.57 (s, 1H), 8.27- 8.05 (m, 3H), 6.97 (s, 1H) 3.88 (s, 3H), 3.66 (d, 1H), 3.18 (q, 1H), 3.06 (d, 1H), 0.74 (s, 9H).
(k) Synthesis of (R)-5-(tert-Butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-9- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II)
Figure imgf000060_0002
To a solution of (R)-methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4- dimethoxybenzyl)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylate (single enantiomer II) (100 mg, 0.17 mmol) in THF H2O (1:1, 10 mL) was added lithium hydroxide monohydrate (28 mg, 0.67 mmol) at rt. The reaction mixture was heated to 40 °C for 2 h. The mixture was cooled to rt and 10% aqueous citric acid solution (50 mL) was added. The mixture was extracted with EtOAc (2 x 50 mL). The combined organic phase was dried over Na2SO4 and concentrated under reduced pressure to afford (R)-5-(tert-butyl)- 11 -(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-4-hydroxy-9- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) as a yellow solid (80 mg, 81% yield, m/z: 584 [M+H]+ observed).
(1) (R)-5-(tert-Butyl)-11-(difluoromethoxy)-9-methoxy-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) (Example 2)
Figure imgf000060_0001
To a solution of (R)-5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-9- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) (80 mg, 0.14 mmol) in CH2CI2 (1 mL) was added trifluoroacetic acid (0.1 mL) at 0 °C. The reaction mixture was slowly warmed to rt and stirred for 4h. The mixture was quenched with saturated aqueous NaHCO3 solution (4 mL), the phases were separated, and the aqueous layer was extracted further with CH2CI2 (3 x 10 mL). The combined organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was triturated with diethyl ether to obtain (A)-5-(tert-butyl)-11- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid (single enantiomer II) as a yellow solid (12 mg, 20% yield, m/z: 434 [M+H]+ observed). 1H NMR (400 MHz, D DMSO-d6): δ 14.97 (s, 1H), 13.57 (s, 1H), 8.27-8.05 (m, 3H), 6.97 (s, 1H) 3.88 (s, 3H), 3.66 (d, 1H), 3.18 (q, 1H), 3.06 (d, 1H), 0.74 (s, 9H).
Example 3: 5-(tert-Butyl)-11-(difluoromethoxy)-9-fluoro-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer
I)
Example 4: 5-(tert-Butyl)-11-(difluoromethoxy)-9-fluoro-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer
P)
Figure imgf000061_0001
(a) Synthesis of 8-(tert-butyl)-2-fluoro-4-hydroxy-8,9-dihydrobenzo[4,5]imidazo[1,2- a]pyridin-6(7H)-one
Figure imgf000061_0002
A mixture of 4-tert-butylcyclohcxanc- 1 ,2-dionc (3.3 g, 19.5 mmol) and 2-amino-5- fluoropyridin-3-ol (1 g, 7.81 mol) in acetic acid (10 mL) was heated to 115 °C under oxygen atmosphere for 12 h. The reaction mixture was cooled to rt and concentrated under reduced pressure. The residue was diluted with EtOAc (200 mL), washed with saturated aqueous NaHCO3 solution (100 mL), followed by saturated aqueous brine solution (100 mL). The organic phase was dried over Na2SO4, fdtered, and evaporated under reduced pressure. The crude residue was triturated with diethyl ether : pentane (1: 1, 50 mL) and the resulting solid purified by normal phase SiO2 chromatography (0-8% MeOH/ CH2CI2) to afford 8 -(tert- butyl)-2-fluoro-4-hydroxy-8,9-dihydrobenzo[4,5 ]imidazo[1,2-a]pyridin-6(7H)-one as a brown solid (320 mg, 15% yield, m/z: 277 [M+H]+ observed). (b) Synthesis of 8-(tert-Butyl)-4-(difluoromethoxy)-2-fluoro-8,9- dihydrobenzo[4,5]imidazo[1,2-a]pyridin-6(7H)-one
Figure imgf000062_0001
To a stirred solution of 8-(tert-butyl)-2-fluoro-4-hydroxy-8,9-dihydrobenzo[4,5 ]imidazo[1,2-a]pyridin-6(7H)-one (3.4 g, 12.3 mmol) in DMF (35 mL) was added sodium chlorodifluoroacetate (9.4 g, 72.4 mmol), followed by K2CO3 (8.51 g, 61.6 mmol) at room temperature. The reaction mixture was stirred at 100 °C for 1 h. The reaction mixture cooled to rt, poured into ice water (200 mL) and extracted with EtOAc (2 x 300 mL). The organic layer was washed with saturated aqueous brine solution (100 mL), dried over Na2SO4 and evaporated under reduced pressure. The crude material was purified by normal phase S1O2 chromatography (0-50% EtOAc/ petroleum ether) to afford 8-(tert-butyl)-4- (difluoromethoxy)-2-fluoro-8,9-dihydrobenzo[4,5]imidazo[1,2-a]pyridin-6(7El)-one as a pale brown solid (1.28 g, 32% yield, m/z: 327 [M+H]+ observed).
(c) Synthesis of 8-(tert-Butyl)-4-(difluoromethoxy)-N-(2,4-dimethoxybenzyl)-2-fluoro- 8,9-dihydrobenzo[4,5]imidazo[1,2-a]pyridin-6(7H)-imine
Figure imgf000062_0002
To a solution of 8-(tert-butyl)-4-(difluoromethoxy)-2-fluoro-8,9- dihydrobenzo[4,5]imidazo[1,2-a]pyridin-6(7H)-one (1 g, 3.1 mmol) in CH2CI2 (10 ml) were added 2,4-dimethoxybenzylamine (0.7 mL, 4.6 mmol) and triethylamine (1.7 mL, 12.2 mmol). The reaction mixture was cooled to 0 °C and TiCl4 (1M solution in CH2CI2, 6.1 mL, 6.1 mmol) was added slowly over 10 min. The reaction mixture was slowly warmed to rt and stirred for 16 h. The mixture was quenched with saturated aqueous NaHCO3 solution (20 mL) and filtered. The filtrate was extracted with CH2CI2 (3 x 40 mL). The combined organic layer phase was dried over Na2SO4 and concentrated under reduced pressure to afford 8 -(tert- butyl)-4-(difluoromethoxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-8,9- dihydrobenzo[4,5]imidazo[1,2-a]pyridin-6(7H)-imine as a brown solid which was used in the next step without further purification (1.1 g, 76% yield, m/z: 476 [M+H]+ observed).
(d) Synthesis of methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-9- fluoro-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer I and single enantiomer II)
Figure imgf000063_0001
To a solution of 8-(tert-butyl)-4-(difluoromethoxy)-N-(2,4-dimethoxybenzyl)-2- fluoro-8,9-dihydrobenzo[4,5]imidazo[1,2-a]pyridin-6(7H)-imine (1.1 g, 2.3 mmol) in diglyme (10 mL) was added dimethyl 2-(methoxymethylene)malonate (1 g, 5.8 mmol). The reaction mixture was heated to 180 °C for 30 min. The reaction was cooled to rt and allowed to stand for 16 h. The precipitated solid was collected by filtration and washed with petroleum ether (2 x 10 mL). The crude residue was purified by normal phase SiO2 chromatography (0-80% EtOAc/ petroleum ether) to afford methyl 5-(tert-butyl)-11- (difluoromethoxy)-1-(2,4-dimethoxybenzyl)-9-fluoro-2-oxo- 1 ,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate as a yellow solid (150 mg, 11% yield, m/z: 586 [M+H]+ observed).
150 mg of methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-9- fluoro-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate was separated by SFC (supercritical fluid chromatography) on CHIRALCEL-OD-H column using 30% MeOHto give methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-9-fluoro-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer I) as a yellow solid (faster eluting enantiomer, 30 mg, 20% yield, m/z: 586 [M+H]+ observed) and methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)- 9-fluoro-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer II) as a yellow solid (slower eluting enantiomer, 30 mg, 20% yield, m/z: 586 [M+H]+ observed).
(e) Synthesis of 5-(tert-Butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-9-fluoro- 2-oxo- 1,2, 5, 6-tetrahydropyrido [2',1 ':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I)
Figure imgf000064_0001
To a solution of methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4- dimethoxybenzyl)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylate (single enantiomer I) (30 mg, 0.05 mmol) in THF H2O (4: 1, 2 mL) was added lithium hydroxide monohydrate (11 mg, 0.26 mmol) at rt. The reaction was heated to 40 °C for 2 h. The mixture was cooled to rt and 10% aqueous citric acid solution (30 mL) was added. The mixture was extracted with EtOAc (2 x 30 mL). The combined organic phase was dried over Na2SO4 and concentrated under reduced pressure to afford 5-(to7-butyl)- 11- (difluoromethoxy)-1-(2,4-dimethoxybenzyl)-9-fluoro-2-oxo- 1,2, 5, 6-tetrahydropyrido [2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) as a yellow solid (25 mg, 0.043 mmol, 85% yield, m/z: 572 [M+H]+ observed).
(f) Synthesis of 5-(tert-Butyl)-11-(difluoromethoxy)-9-fluoro-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) (Example 3)
Figure imgf000064_0002
To a solution of 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-9- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) (25 mg, 0.044 mmol) in CH2CI2 (1 mL) was added TFA (0.1 mL) at 0 °C. The reaction mixture was slowly warmed to rt and stirred for 4h. The mixture was quenched with saturated aqueous NaHCO3 solution (4 mL), the phases were separated, and the aqueous layer was extracted further with CH2CI2 (3 x 10 mL). The combined organic phase was dried over Na2SO4, fdtered and concentrated under reduced pressure. The residue was triturated with diethyl ether to afford 5-(tert-butyl)-11-(difluoromethoxy)-9-fluoro-2- oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) as a yellow solid (7 mg, 38% yield, m/z: 422 [M+H]+ observed). 1H NMR (400 MHz, DMSO-d6): δ 15.06 (s, 1H), 13.63 (s, 1H), 8.84 (s, 1H), 8.27-8.09 (m, 2H), 7.37 (d,
1H) 3.59 (d, 1H), 3.21-3.14 (m, 1H), 3.06 (d, 1H), 0.72 (s, 9 H).
(g) Synthesis of 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-9-fluoro- 2-oxo-1,2,5,6-tetrahydropyrido [2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II)
Figure imgf000065_0001
To a solution of methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4- dimethoxybenzyl)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylate (single enantiomer II) (30 mg, 0.05 mmol) in THF:H2O (4: 1, 2 mL) was added lithium hydroxide monohydrate (11 mg, 0.26 mmol) at rt. The reaction was heated to 40 °C for 2 h. The mixture was cooled to rt and 10% aqueous citric acid solution (30 mL) was added. The mixture was extracted with EtOAc (2 x 30 mL). The combined organic phase was dried over Na2SO4 and concentrated under reduced pressure to afford 5- (tert-butyl)- 11 -(difluoromethoxy)- 1 -(2,4-dimethoxybenzyl)-9-fluoro-2-oxo- 1 ,2,5,6- tetrahydropyrido [2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) as a yellow solid (25 mg, 0.043 mmol, 85% yield, m/z: 572 [M+H]+ observed). (h) Synthesis of 5-(tert-butyl)-11-(difluoromethoxy)-9-fluoro-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II): (Example 4)
Figure imgf000066_0001
To a solution of 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-9- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) (25 mg, 0.044 mmol) in CH2CI2 (1 mL) was added TFA (0.1 mL) at 0 °C. The reaction mixture was slowly warmed to rt and stirred for 4h. The mixture was quenched with saturated aqueous NaHCO3 solution (4 mL), the phases were separated, and the aqueous layer was extracted further with CH2CI2 (3 x 10 mL). The combined organic phase was dried over Na2SO4, fdtered and concentrated under reduced pressure. The residue was triturated with diethyl ether to afford 5-(tert-butyl)-11-(difluoromethoxy)-9-fluoro-2- oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) as a yellow solid (8 mg, 43% yield, m/z: 422 [M+H]+ observed). 1H NMR (400 MHz, DMSO-d6): δ 15.06 (s, 1H), 13.63 (s, 1H), 8.84 (s, 1H), 8.27-8.09 (m, 2H), 7.37 (d, 1H) 3.59 (d, 1H), 3.21-3.14 (m, 1H), 3.06 (d, 1H), 0.72 (s, 9H).
Example 5: 5-(tert-Butyl)-2-oxo-11-(trifluoromethyl)-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer
I)
Example 6: 5-(tert-Butyl)-2-oxo-11-(trifluoromethyl)-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer
P)
Figure imgf000066_0002
The compounds were prepared in a similar manner as 5-(tert-butyl)-11- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid from an appropriately substituted methyl 5-(tert-butyl)-1-(2,4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate, followed by ester hydrolysis and debenzylation.
(a) Synthesis of methyl 5-(tert-butyl)-1-(2,4-dimethoxybenzyl)-2-oxo-11- (trifluoromethyl)-1,2,5, 6-tetrahydropyrido[2 1 ':2,3]imidazo[4, 5-h ]quinoline-3-carboxylate (single enantiomer I and single enantiomer II)
Figure imgf000067_0001
200 mg of methyl 5 -(tert-butyl)- 1 -(2.4-dimcthoxybcnzyl)-2-oxo-11-(trifluoromcthyl)-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (prepared in a similar manner as methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-9- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate from 4-tert-biitylcyclohcxanc- 1 ,2-dionc and 3-(trifluoromethyl)pyridin-2-amine) was separated by SFC on CHIRALCEL-OD-H column using 40% MeOH to give methyl 5- (tert-butyl)-1-(2,4-dimethoxybenzyl)-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer I) as a yellow solid (faster eluting enantiomer, 80 mg, 40% yield, m/z: 570 [M+H]+ observed) and methyl 5-(tert-butyl)-1-(2,4-dimethoxybenzyl)-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer II) as a yellow solid (slower eluting enantiomer, 80 mg, 40% yield, m/z: 570 [M+H]+ observed).
(b) Synthesis of 5-(tert-Butyl)-2-oxo-11-(trifluoromethyl)-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) (Example 5)
Figure imgf000068_0001
To a solution of methyl 5-(tert-butyl)-1-(2,4-dimethoxybenzyl)-2-oxo-11- (trifluoromethyl)- 1 ,2,5 ,6-tetrahydropyrido [2',1' : 2,3]imidazo [4,5 -h] quinoline-3 -carboxylate (single enantiomer I) (80 mg, 0.14 mmol) in THF H2O (1: 1, 10 mL) was added lithium hydroxide monohydrate (59 mg, 1.41 mmol) at rt. The reaction mixture was heated to 40 °C for 2h. The mixture was cooled to rt and 10% aqueous citric acid solution (50 mL) was added. The mixture was extracted with EtOAc (2 x 50 mL). The combined organic phase was dried over Na2SO4 and concentrated under reduced pressure to afford 5-(to7-butyl)- 1 -(2.4- dimethoxybenzyl)-2-oxo-11-(trifluoromethyl)-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid (single enantiomer I) as a yellow solid (75 mg, 96% yield, m/z: 556 [M+H]+ observed).
To a solution of 5-(tert-butyl)-1-(2,4-dimethoxybenzyl)-2-oxo-11-(trifluoromethyl)-1, 2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) (75 mg, 0.135 mmol) in CH2CI2 (1 mL) was added trifluoroacetic acid (0.1 mL) at 0 °C. The reaction mixture was slowly warmed to rt and stirred for 4h. The mixture was quenched with saturated aqueous NaHCO3 solution (4 mL), the phases were separated, and the aqueous layer was extracted further with CH2CI2 (3 x 10 mL). The combined organic phase was dried over Na2SO4, fdtered and concentrated under reduced pressure. The residue was triturated with diethyl ether to afford 5-(to7-butyl)-2-oxo-11-(trifluoromethyl)-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) as a yellow solid (25 mg, 44% yield, m/z: 406 [M+H]+ observed). 1H NMR (400 MHz, DMSO- d6): S 14.90 (s, 1H), 13.50 (s, 1H), 8.90 (d, 1H), 8.28 (s, 1H), 7.80 (d, 1H), 7.19 (t, 1H), 3.65 (d, 1H), 3.25 (t, 1H), 3.14 (d, 1H), 0.72 (s, 9H).
(c) Synthesis of 5-(tert-Butyl)-2-oxo-11-(trifluoromethyl)-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II): (Example 6)
Figure imgf000069_0001
To a solution of methyl 5-(tert-butyl)-1-(2,4-dimethoxybenzyl)-2-oxo-11- (trifluoromethyl)- 1 ,2,5 ,6-tetrahydropyrido [2',1' : 2,3]imidazo [4,5 -h] quinoline-3 -carboxylate (single enantiomer II) (80 mg, 0.14 mmol) in THF H2O (1: 1, 10 mL) was added lithium hydroxide monohydrate (59 mg, 1.41 mmol) at rt. The reaction mixture was heated to 40 °C for 2 h. The mixture was cooled to rt and 10% aqueous citric acid solution (50 mL) was added. The mixture was extracted with EtOAc (2 x 50 mL). The combined organic phase was dried over Na2SO4 and concentrated under reduced pressure to afford 5-(tert-butyl)- 1 -(2.4- dimethoxybenzyl)-2-oxo-11-(trifluoromethyl)-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid (single enantiomer II) as a yellow solid (75 mg, 96% yield, m/z: 556 [M+H]+ observed).
To a solution of 5-(tert-butyl)-1-(2,4-dimethoxybenzyl)-2-oxo-11-(trifluoromethyl)-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) (75 mg, 0.135 mmol) in CH2CI2 (1 mL) was added trifluoroacetic acid (0.1 mL) at 0 °C. The reaction mixture was slowly warmed to rt and stirred for 4 h. The mixture was quenched with saturated aqueous NaHCO3 solution (4 mL), the phases were separated, and the aqueous layer was extracted further with CH2CI2 (3 x 10 mL). The combined organic phase was dried over Na2SO4, fdtered and concentrated under reduced pressure. The residue was triturated with diethyl ether to afford 5-(tert-butyl)-2-oxo-11-(trifluoromethyl)-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) as a yellow solid (22 mg, 40% yield, m/z: 406 [M+H]+ observed). 1H NMR (400 MHz, DMSO-d6): δ 14.90 (s, 1H), 13.50 (s, 1H), 8.90 (d, 1H), 8.28 (s, 1H), 7.80 (d, 1H), 7.19 (t, 1H), 3.65 (d, 1H), 3.25 (t, 1H), 3.14 (d, 1H), 0.72 (s, 9H).
Example 7: 5-(tert-Butyl)-11-(difluoromethyl)-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer
I)
Example 8: 5-(tert-Butyl)-11-(difluoromethyl)-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer
P)
Figure imgf000070_0001
The compounds were prepared in a similar manner as 5-(tert-butyl)-11- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid from an appropriately substituted methyl 5-(tert-butyl)-1-(2,4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate, followed by ester hydrolysis and debenzylation.
(a) Synthesis of methyl 5-(tert-butyl)-11-(difluoromethyl)-1-(2,4-dimethoxybenzyl)-2- oxo-1,2,5,6-tetrahydropyrido[2 ',1 ':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer I and single enantiomer II)
Figure imgf000070_0002
200 mg of methyl 5-(tert-butyl)-11-(difluoromethyl)-1-(2,4-dimethoxybenzyl)-2-oxo- 1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (prepared in a similar manner as methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-9- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate from 4-tert-biitylcyclohexane- 1 ,2-dionc and 3-(difluoromethyl)pyridin-2-amine) was separated by SFC on CHIRALCEL-OD-H column using 40% (MeOH) to give methyl 5-(tert- butyl)- 11 -(difluoromethyl)- 1 -(2,4-dimethoxybenzyl)-2-oxo- 1,2, 5, 6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer I) as a yellow solid (faster eluting enantiomer, 80 mg, 40% yield, m/z: 552 [M+H]+ observed) and methyl 5 -(tert- butyl)- 11 -(difluoromethyl)- 1 -(2,4-dimethoxybenzyl)-2-oxo- 1 ,2,5 ,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer II) as a yellow solid (slower eluting enantiomer, 80 mg, 40% yield, m/z: 552 [M+H]+ observed). (b) Synthesis of 5-(tert-butyl)-11-(difluoromethyl)-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) (Example 7)
Figure imgf000071_0001
To a solution of methyl 5-(tert-butyl)-11-(difluoromethyl)-1-(2,4-dimethoxybenzyl)- 2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer I) (80 mg, 0.15 mmol) in THF H2O (1: 1, 10 mL) was added lithium hydroxide monohydrate (30 mg, 0.73 mmol) at rt. The reaction mixture was heated to 40 °C for 2 h. The mixture was cooled to rt and 10% aqueous citric acid solution (50 mL) was added. The mixture was extracted with EtOAc (2 x 50 mL). The combined organic phase was dried over Na2SO4 and concentrated under reduced pressure to afford 5-(to7-butyl)-11-(difluoromethyl)- 1-(2, 4-dimethoxybenzyl)-2-oxo- 1,2,5, 6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylic acid (single enantiomer I) as a yellow solid (60 mg, 75% yield, m/z: 538 [M+H]+ observed).
To a solution of 5-(tert-butyl)-11-(difluoromethyl)-1-(2,4-dimethoxybenzyl)-2-oxo- 1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) (60 mg, 0.11 mmol) in CH2CI2 (1 mL) was added trifluoroacetic acid (0.1 mL) at 0 °C. The reaction mixture was slowly warmed to rt and stirred for 4h. The mixture was quenched with saturated aqueous NaHCO3 solution (4 mL), the phases were separated, and the aqueous layer was extracted further with CH2CI2 (3 x 10 mL). The combined organic phase was dried over Na2SO4, fdtered and concentrated under reduced pressure. The residue was triturated with diethyl ether to afford 5-(tert-butyl)-11-(difluoromethyl)-2 -oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) as a yellow solid (35 mg, 82% yield, m/z: 388 [M+H]+ observed). 1H NMR (400 MHz, DMSO- d6): S 15.18 (s, 1H), 13.57 (s, 1H), 8.77 (d, 1H), 8.22 (s, 1H), 7.61 (d, 2H), 7.16 (t, 1H), 3.62 (d, 1H), 3.23 (t, 1H), 3.06 (d, 1H), 0.72 (s, 9H).
(c) Synthesis of 5-(tert-Butyl)-11-(difluoromethyl)-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) (Example 8)
Figure imgf000072_0001
To a solution of methyl 5-(tert-butyl)-11-(difluoromethyl)-1-(2,4-dimethoxybenzyl)- 2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer II) (80 mg, 0.15 mmol) in THF : H2O (1: 1, 10 mL) was added lithium hydroxide monohydrate (30 mg, 0.73 mmol) at rt. The reaction mixture was heated to 40 °C for 2 h. The mixture was cooled to rt and 10% aqueous citric acid solution (50 mL) was added. The mixture was extracted with EtOAc (2 x 50 mL). The combined organic phase was dried over Na2SO4 and concentrated under reduced pressure to afford 5-(tert-butyl)-11-(difluoromethyl)- 1-(2, 4-dimethoxybenzyl)-2-oxo- 1,2,5, 6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylic acid (single enantiomer II) as a yellow solid (60 mg, 75% yield, m/z: 538 [M+H]+ observed).
To a solution of 5-(tert-butyl)-11-(difluoromethyl)-1-(2,4-dimethoxybenzyl)-2-oxo- 1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) (60 mg, 0.11 mmol) in CH2CI2 (1 mL) was added trifluoroacetic acid (0.1 mL) at 0 °C. The reaction mixture was slowly warmed to rt and stirred for 4h. The mixture was quenched with saturated aqueous NaHCO3 solution (4 mL), the phases were separated, and the aqueous layer was extracted further with CH2CI2 (3 x 10 mL). The combined organic phase was dried over Na2SO4, fdtered and concentrated under reduced pressure. The residue was triturated with diethyl ether to afford 5-(tert-butyl)-11-(difluoromethyl)-2 -oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) as a yellow solid (35 mg, 82% yield, m/z: 388 [M+H]+ observed). 1H NMR (400 MHz, DMSO-d6): δ 15.18 (s, 1H), 13.57 (s, 1H), 8.77 (d, 1H), 8.22 (s, 1H), 7.61 (d, 2H), 7.16 (t, 1H), 3.62 (d, 1H), 3.23 (t, 1H), 3.06 (d, 1H), 0.72 (s, 9H).
Comparative Example 9: 5-(tert-Butyl)-9,11-dimethoxy-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer
I)
Comparative Example 10: 5-(tert-Butyl)-9,11-dimethoxy-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer
P)
Figure imgf000073_0001
The compounds were prepared in a similar manner as 5-(tert-butyl)- 11- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid from an appropriately substituted methyl 5-tert -butyl)-1-(2,4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate, followed by ester hydrolysis and debenzylation.
(a) Synthesis of methyl 5-(tert-butyl)-1-(2,4-dimethoxybenzyl)-9,11-dimethoxy-2-oxo- l,2,5,6-tetrahydropyrido[2 ',1 ':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer I and single enantiomer II)
Figure imgf000073_0002
280 mg of methyl 5 -(tert-butyl)- 1 -(2.4-dimcthoxybcnzyl)-9,11 -dimethoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (prepared in a similar manner as methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-9- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate from 4-tert-butylcyclohcxanc- 1 ,2-dionc and 3,5-dimethoxypyridin-2-amine) was separated by SFC on Chiralpak IA column using 50% MeOH:CH3CN (1:1) to give methyl 5 -(tert- butyl)- 1 -(2,4-dimethoxybenzyl)-9, 11 -dimethoxy-2-oxo- 1 ,2,5 ,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer I) as a yellow solid (faster eluting enantiomer, 100 mg, 36% yield, m/z: 562 [M+H]+ observed) and methyl 5 -(tert-butyl)- 1 -(2.4-dimethoxybcnzyl)-9,11 -dimcthoxy-2-oxo- 1.2.5.6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer II) as a yellow solid (slower eluting enantiomer, 110 mg, 39% yield, m/z: 562 [M+H]+ observed). (b) Synthesis of 5-(tert-Butyl)-9, ll-dimethoxy-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) (Example 9)
Figure imgf000074_0001
To a solution of methyl 5-(tert-butyl)- 1 -(2.4-dimcthoxybcnzyl)-9,11 -dimethoxy-2- oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer I) (100 mg, 0.18 mmol) in THF H2O (1:1, 10 mL) was added lithium hydroxide monohydrate (30 mg, 0.72 mmol) at rt. The reaction mixture was heated to 40 °C for 2 h. The mixture was cooled to rt and 10% aqueous citric acid solution (50 mL) was added. The mixture was extracted with EtOAc (2 x 50 mL). The combined organic phase was dried over Na2SO4 and concentrated under reduced pressure to afford 5-(tert-butyl)- 1 -(2.4- dimethoxybenzyl)-9,11-dimethoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid (single enantiomer I) as a yellow solid (50 mg, 51% yield, m/z: 548 [M+H]+ observed).
To a solution of 5-(tert-butyl)-1-(2,4-dimethoxybenzyl)-9,11-dimethoxy-2-oxo- 1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) (50 mg, 0.09 mmol) in CH2CI2 (1 mL) was added trifluoroacetic acid (0.1 mL) at 0 °C. The reaction mixture was slowly warmed to rt and stirred for 4h. The mixture was quenched with saturated aqueous NaHCO3 solution (4 mL), the phases were separated, and the aqueous layer was extracted further with CH2CI2 (3 x 10 mL). The combined organic phase was dried over Na2SO4, fdtered and concentrated under reduced pressure. The residue was triturated with diethyl ether to afford 5-(tert-butyl)-9,11-dimethoxy-2 -oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) as a yellow solid (31 mg, 88% yield, m/z: 398 [M+H]+ observed). 1H NMR (400 MHz, DMSO- d6, TFA): δ 8.28 (s, 1H), 8.03 (d, 1H), 6.91 (s, 1H), 4.02 (s, 3H), 3.92 (s, 3H), 3.71 (d, 1H), 3.23-3.11 (m, 2H), 0.77 (s, 9H).
(c) Synthesis of 5-(tert-butyl)-9,11-dimethoxy-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) (Example 10)
Figure imgf000075_0001
To a solution of methyl 5-(tert-butyl)- 1 -(2.4-dimcthoxybcnzyl)-9,11 -dimethoxy-2- oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer II) (100 mg, 0.18 mmol) in THF H2O (1:1, 10 mL) was added lithium hydroxide monohydrate (30 mg, 0.72 mmol) at rt. The reaction mixture was heated to 40 °C for 2 h. The mixture was cooled to rt and 10% aqueous citric acid solution (50 mL) was added. The mixture was extracted with EtOAc (2 x 50 mL). The combined organic phase was dried over Na2SO4 and concentrated under reduced pressure to afford 5-(tert-butyl)- 1 -(2.4- dimethoxybenzyl)-9,ll-dimethoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid (single enantiomer II) as a yellow solid (50 mg, 50% yield, m/z: 548 [M+H]+ observed).
To a solution of 5-(tert-butyl)-1-(2,4-dimethoxybenzyl)-9,11-dimethoxy-2-oxo- 1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) (50 mg, 0.09 mmol) in CH2CI2 (1 mL) was added trifluoroacetic acid (0.1 mL) at 0 °C. The reaction mixture was slowly warmed to rt and stirred for 4h. The mixture was quenched with saturated aqueous NaHCO3 solution (4 mL), the phases were separated, and the aqueous layer was extracted further with CH2CI2 (3 x 10 mL). The combined organic phase was dried over Na2SO4, fdtered and concentrated under reduced pressure. The residue was triturated with diethyl ether to afford 5-(tert-butyl)-9,11-dimethoxy-2 -oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) as a yellow solid (31 mg, 88% yield, m/z: 398 [M+H]+ observed). 1H NMR (400 MHz, DMSO-d6, TFA): δ 8.28 (s, 1H), 8.03 (d, 1H), 6.91 (s, 1H), 4.02 (s, 3H), 3.92 (s, 3H), 3.71 (d, 1H), 3.23-3.11 (m, 2H), 0.77 (s, 9H).
Example 11: 5-(tert-Butyl)-9-(difluoromethoxy)-11-methoxy-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer
I) Example 12: 5-(tert-Butyl)-9-(difluoromethoxy)-11-methoxy-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer
P)
Figure imgf000076_0001
The compounds were prepared in a similar manner as 5-(tert-butyl)-11- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid from an appropriately substituted methyl 5-(tert-butyl)-1-(2,4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate, followed by ester hydrolysis and debenzylation.
(a) Synthesis of methyl 5-(tert-butyl)-9-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-11- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer I and single enantiomer II)
Figure imgf000076_0002
400 mg of methyl 5-(tert-butyl)-9-(difluoromethoxy)- 1 -(2.4-dimethoxybenzyl)-11- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (prepared in a similar manner as methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4- dimethoxybenzyl)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h]quinoline-3-carboxylate from 4-tert-biitylcyclohexane- 1 2-dionc and 5-(difluoromethoxy)- 3-methoxypyridin-2 -amine) was separated by SFC on DCPAK-P4VP column using 25% methanol to give methyl 5-(tert-butyl)-9-(difluoromethoxy)- 1 -(2.4-dimethoxybenzyl)- 11- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer I) as a yellow solid (faster eluting enantiomer, 130 mg, 33% yield, m/z: 598 [M+H]+ observed) and methyl 5-(tert-butyl)-9-(difluoromethoxy)-1-(2,4- dimethoxybenzyl)-11-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylate (single enantiomer II) as a yellow solid (slower eluting enantiomer, 170 mg, 43% yield, m/z: 598 [M+H]+ observed).
(b) Synthesis of 5-(tert-butyl)-9-(difluoromethoxy)-11-methoxy-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) (Example 11)
Figure imgf000077_0001
To a solution of methyl 5-(tert-butyl)-9-(difluoromcthoxy)- 1 -(2.4-dimethoxybenzyl)- 11-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer I) (130 mg, 0.23 mmol) in THF H2O (1:1, 10 mL) was added lithium hydroxide monohydrate (42 mg, 1 mmol) at rt. The reaction mixture was heated to 40 °C for 2 h. The mixture was cooled to rt and 10% aqueous citric acid solution (50 mL) was added. The mixture was extracted with EtOAc (2 x 50 mL). The combined organic phase was dried over Na2SO4 and concentrated under reduced pressure to afford 5 -( toy-butyl )-9- (difluoromethoxy)- 1 -(2,4-dimethoxybenzyl)- 11 -methoxy-2-oxo- 1 ,2,5 ,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) as a yellow solid (110 mg, 87% yield, m/z: 584 [M+H]+ observed).
To a solution of 5-(tert-butyl)-9-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-11- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) (110 mg, 0.19 mmol) in CH2CI2 (1 mL) was added trifluoroacetic acid (0.1 mL) at 0 °C. The reaction mixture was slowly warmed to rt and stirred for 4h. The mixture was quenched with saturated aqueous NaHCO3 solution (4 mL), the phases were separated, and the aqueous layer was extracted further with CH2CI2 (3 x 10 mL). The combined organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was triturated with diethyl ether to afford 5-(tert-butyl)-9- (difluoromethoxy)- 11 -methoxy-2-oxo- 1 ,2,5 ,6-tetrahydropyrido [2',1' : 2,3] imidazo [4,5 - h] quinoline -3 -carboxylic acid (single enantiomer I) as a yellow solid (35 mg, 43% yield, m/z: 434 [M+H]+ observed). TlNMR ^OO MHz, DMSO-d6): δ 15.22 (s, 1H), 13.63 (s, 1H), 8.34 (s, 1H), 8.19 (s, 1H), 7.46-7.09 (t, 1H), 6.77 (s, 1H), 3.99 (s, 3H), 3.58 (d, 1H), 3.19-3.00 (m, 2H), 0.71 (s, 9H).
5-(tert-Butyl)-9-( difluorometh oxy)-11 -methoxy-2-oxo-1,2, 5, 6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) (Example 12)
Figure imgf000078_0001
To a solution of methyl 5-(tert-butyl)-9-(difluoromethoxy)- 1 -(2.4-dimethoxybenzyl)- ll-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer II) (170 mg, 0.28 mmol) in THF H2O (1:1, 10 mL) was added lithium hydroxide monohydrate (42 mg, 1 mmol) at rt. The reaction mixture was heated to 40 °C for 2 h. The mixture was cooled to rt and 10% aqueous citric acid solution (50 mL) was added. The mixture was extracted with EtOAc (2 x 50 mL). The combined organic phase was dried over Na2SO4 and concentrated under reduced pressure to afford 5 -( toy-butyl )-9- (difluoromethoxy)- 1 -(2,4-dimethoxybenzyl)- 11 -methoxy-2-oxo- 1 ,2,5 ,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) as a yellow solid (140 mg, 84%, m/z: 584 [M+H]+ observed).
To a solution of 5-(tert-butyl)-9-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-11- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) (140 mg, 0.24 mmol) in CH2CI2 (1 mL) was added trifluoroacetic acid (0.1 mL) at 0 °C. The reaction mixture was slowly warmed to rt and stirred for 4h. The mixture was quenched with saturated aqueous NaHCO3 solution (4 mL), the phases were separated, and the aqueous layer was extracted further with CH2CI2 (3 x 10 mL). The combined organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was triturated with diethyl ether to afford 5-(toy-butyl)-9- (difluoromethoxy)- 11 -methoxy-2-oxo- 1 ,2,5 ,6-tetrahydropyrido [2',1' : 2,3] imidazo [4,5 - h] quinoline -3 -carboxylic acid (single enantiomer II) as a yellow solid (37 mg, 36% yield, m/z: 434 [M+H]+ observed). TlNMR (400 MHz, DMSO-d6): δ 15.22 (s, 1H), 13.63 (s, 1H), 8.34 (s, 1H), 8.19 (s, 1H), 7.46-7.09 (t, 1H), 6.77 (s, 1H), 3.99 (s, 3H), 3.58 (d, 1H), 3.19-3.00 (m, 2H), 0.71 (s, 9H). Example 13: 5-(tert-Butyl)-9,11-bis(difluoromethoxy)-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer
I)
Example 14: 5-(tert-Butyl)-9,11-bis(difluoromethoxy)-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II)
Figure imgf000079_0002
The compounds were prepared in a similar manner as 5-(tert-butyl)-11- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid from an appropriately substituted methyl 5-(tert-butyl)-1-(2,4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate, followed by ester hydrolysis and debenzylation.
(a) Synthesis of methyl 5-(tert-butyl)-9,11-bis(difluoromethoxy)-1-(2,4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate (single enantiomer I and single enantiomer II)
Figure imgf000079_0001
350 mg of methyl 5-(tert-butyl)-9,11 -bis(difluoromethoxy)-1-(2.4-dimethoxybenzyl)- 2-oco- 1 ,2,5 ,6-tetrahydropyrido [2',1' : 2,3] imidazo [4,5 -h] quinoline -3 -carboxylate (prepared in a similar manner as methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-9- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate from 4-to7-butylcyclohexane- 1 ,2-dionc and 3,5-dimethoxypyridin-2-amine) was separated by SFC on CHIRALPAK-IC column using 40% MeOH :CH3CN (1: 1) to give methyl 5 -(tert- butyl)-9, 11 -bis(difluoromethoxy)- 1 -(2,4-dimethoxybenzyl)-2-oxo- 1 ,2,5 ,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer I) as a yellow solid (faster eluting enantiomer, 160 mg, 46% yield, m/z: 634 [M+H]+ observed) and methyl 5-(to7-butyl)-9, 11 -bis(difluoromethoxy)- 1 -(2,4-dimethoxybenzyl)-2-oxo- 1 ,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer II) as a yellow solid (slower eluting enantiomer, 160 mg, 46% yield, m/z: 634 [M+H]+ observed).
(b) Synthesis of 5-(tert-butyl)-9,11-bis(difluoromethoxy)-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) (Example 13)
Figure imgf000080_0001
To a solution of methyl 5-(to7-butyl)-9,11 -bis(difliioromcthoxy)- 1 -(2.4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate (single enantiomer I) (160 mg, 0.25 mmol) in THF: H2O (1: 1, 10 mL) was added lithium hydroxide monohydrate (50 mg, 1.2 mmol) at rt. The reaction mixture was heated to 40 °C for 2 h. The mixture was cooled to rt and 10% aqueous citric acid solution (50 mL) was added. The mixture was extracted with EtOAc (2 x 50 mL). The combined organic phase was dried over Na2SO4 and concentrated under reduced pressure to afford 5-(tert-butyl)-9.11- bis(difluoromethoxy)- 1 -(2,4-dimethoxybenzyl)-2-oxo- 1 ,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) as a yellow solid (100 mg, 64% yield, m/z: 620 [M+H]+ observed).
To a solution of 5-(tert-butyl)-9,11-bis(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-2- oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) (100 mg, 0.16 mmol) in CH2CI2 (1 mL) was added trifluoroacetic acid (0.1 mL) at 0 °C. The reaction mixture was slowly warmed to rt and stirred for 4 h. The mixture was quenched with saturated aqueous NaHCO3 solution (4 mL), the phases were separated, and the aqueous layer was extracted further with CH2CI2 (3 x 10 mL). The combined organic phase was dried over Na2SO4, fdtered and concentrated under reduced pressure. The residue was triturated with diethyl ether to afford 5-(tert-butyl)-9,ll-bis(difluoromethoxy)-2-oxo-1, 2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) as a yellow solid (60 mg, 79% yield, m/z: 470 [M+H]+ observed). 1H NMR (400 MHz, DMSO-d6): δ 13.64 (s, 1H), 12.82 (s, 1H), 8.66 (d, 1H), 8.36-7.77 (m, 2H), 7.46- 7.09 (m, 2H), 3.59 (d, 1H), 3.18-3.11 (m, 1H), 2.98 (d, 1H), 0.71 (s, 9H).
(c) Synthesis of 5-(tert-butyl)-9,11-bis(difluoromethoxy)-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) (Example 14)
Figure imgf000081_0001
To a solution of methyl 5-(tert-butyl)-9,11 -bis(difliioromethoxy)- 1 -(2.4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate (single enantiomer II) (160 mg, 0.25 mmol) in THF:H2O (1: 1, 10 mL) was added lithium hydroxide monohydrate (50 mg, 1.2 mmol) at rt. The reaction mixture was heated to 40 °C for 2 h. The mixture was cooled to rt and 10% aqueous citric acid solution (50 mL) was added. The mixture was extracted with EtOAc (2 x 50 mL). The combined organic phase was dried over Na2SO4 and concentrated under reduced pressure to afford 5-(tert-butyl)-9,11- bis(difluoromethoxy)- 1 -(2,4-dimethoxybenzyl)-2-oxo- 1 ,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) as a yellow solid (100 mg, 64% yield, m/z: 620 [M+H]+ observed.
To a solution of 5-(tert-butyl)-9,11-bis(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-2- oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) (100 mg, 0.16 mmol) in CH2CI2 (1 mL) was added trifluoroacetic acid (0.1 mL) at 0 °C. The reaction mixture was slowly warmed to rt and stirred for 4h. The mixture was quenched with saturated aqueous NaHCO3 solution (4 mL), the layers were separated, and the aqueous layer was extracted further with CH2CI2 (3 x 10 mL). The combined organic phase was dried over Na2SO4, fdtered and concentrated under reduced pressure. The residue was triturated with diethyl ether to afford 5-(tert-butyl)-9,ll-bis(difluoromethoxy)-2-oxo-1, 2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) as a yellow solid (55 mg, 72% yield, m/z: 470 [M+H]+ observed). 1H NMR (400 MHz, DMSO-d6): δ 13.64 (s, 1H), 12.82 (s, 1H), 8.66 (d, 1H), 8.36-7.77 (m, 2H), 7.46- 7.09 (m, 2H), 3.59 (d, 1H), 3.18-3.11 (m, 1H), 2.98 (d, 1H), 0.71 (s, 9H).
Example 15: 5-(tert-Butyl)-11-(difluoromethoxy)-10-methoxy-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer
I)
Example 16: 5-(tert-Butyl)-11-(difluoromethoxy)-10-methoxy-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer
P)
Figure imgf000082_0001
The compounds were prepared in a similar manner as 5-(tert-butyl)-11- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid from an appropriately substituted methyl 5 -(tert-butyl)- 1 -(2.4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate, followed by ester hydrolysis and debenzylation.
(a) Synthesis of methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)~
10-meth oxy-2-oxo-1,2, 5, 6-tetrahydropyrido[2 1 ':2,3 ]imidazo[ 4, 5-h Jquinolin e-3- carboxylate (single enantiomer I and single enantiomer II)
Figure imgf000083_0001
130 mg of methyl 5 -(tert-butyl)-11-(difluoromcthoxy)- 1 -(2.4-dimcthoxybcnzyl)- 10- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (prepared in a similar manner as methyl 5-tert -butyl)-11-(difluoromethoxy)-1-(2,4- dimethoxybenzyl)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h]quinoline-3-carboxylate from 4-tert-butylcyclohexane- 1 ,2-dione and 2-amino-4- methoxypyridin-3-ol) was separated by SFC on CHIRALCEL-OD-H column using 40% MeOH to give methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-10- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer I) as a yellow solid (faster eluting enantiomer, 40 mg, 31% yield, m/z: 598 [M+H]+ observed) and methyl 5-tert -butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)- 10-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate (single enantiomer II) as a yellow solid (slower eluting enantiomer, 40 mg, 31% yield, m/z: 598 [M+H]+ observed).
(b) Synthesis of 5-(tert-butyl)-11-(difluoromethoxy)-10-methoxy-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I): (Example 15)
Figure imgf000083_0002
To a solution of methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4- dimethoxybenzyl)-10-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylate (single enantiomer I) (40 mg, 0.067 mmol) in THF H2O (1:1, 10 mL) was added lithium hydroxide monohydrate (14 mg, 0.33 mmol) at rt. The reaction mixture was heated to 40 °C for 2 h. The mixture was cooled to rt and 10% aqueous citric acid solution (50 mL) was added. The mixture was extracted with EtOAc (2 x 50 mL). The combined organic phase was dried over Na2SO4 and concentrated under reduced pressure to afford 5 -(tert- butyl)- 11 -(difluoromethoxy)- 1 -(2,4-dimethoxybenzyl)- 10-methoxy-2-oxo-1, 2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) as a yellow solid (33 mg, 84% yield, m/z: 584 [M+H]+ observed).
To a solution of 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-10- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) (33 mg, 0.056 mmol) in CH2CI2 (1 mL) was added trifluoroacetic acid (0.1 mL) at 0 °C. The reaction mixture was slowly warmed to rt and stirred for 4 h. The mixture was quenched with saturated aqueous NaHCO3 solution (4 mL), the layers were separated, and the aqueous layer was extracted further with CH2CI2 (3 x 10 mL). The combined organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was triturated with diethyl ether to afford 5-(tert-butyl)- 11- (difluoromethoxy)- 10-methoxy-2-oxo- 1 ,2,5 ,6-tetrahydropyrido [2',1' : 2,3] imidazo [4,5 - h] quinoline -3 -carboxylic acid (single enantiomer I) as a yellow solid (18 mg, 73% yield, m/z: 434 [M+H]+ observed). TlNMR (400 MHz, DMSO-d6): δ 14.89 (bs, 1H), 13.45 (bs, 1H), 8.55 (d, 1H), 8.25-7.86 (m, 2H), 7.24 (d, 1H), 3.97 (s, 3H), 3.59 (d, 1H), 3.22-3.15 (m, 1H), 3.05 (d, 1H), 0.74 (s, 9H).
(c) Synthesis of 5-(tert-butyl)-11-(difluoromethoxy)-10-methoxy-2-oxo-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) (Example 16)
Figure imgf000084_0001
To a solution of methyl 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4- dimethoxybenzyl)-10-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylate (single enantiomer II) (40 mg, 0.067 mmol) in THF:H2O (1: 1, 10 mL) was added lithium hydroxide monohydrate (14 mg, 0.33 mmol) at rt. The reaction mixture was heated to 40 °C for 2 h. The mixture was cooled to rt and 10% aqueous citric acid solution (50 mL) was added. The mixture was extracted with EtOAc (2 x 50 mL). The combined organic phase was dried over Na2S04 and concentrated under reduced pressure to afford 5-(tert-butyl)- 11 -(difluoromethoxy)- 1 -(2,4-dimethoxybenzyl)- 10-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) as a yellow solid (33 mg, 84% yield, m/z: 584 [M+H]+ observed).
To a solution of 5-(tert-butyl)-11-(difluoromethoxy)-1-(2,4-dimethoxybenzyl)-10- methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II) (33 mg, 0.056 mmol) in CH2CI2 (1 mL) was added trifluoroacetic acid (0.1 mL) at 0 °C. The reaction mixture was slowly warmed to rt and stirred for 4 h. The mixture was quenched with saturated aqueous NaHCO3 solution (4 mL), the layers were separated, and the aqueous layer was extracted further with CH2CI2 (3 x 10 mL). The combined organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was triturated with diethyl ether to afford 5-(tert-butyl)- 11- (difluoromethoxy)- 10-methoxy-2-oxo- 1 ,2,5 ,6-tetrahydropyrido [2',1' : 2,3] imidazo [4,5 - h] quinoline -3 -carboxylic acid (single enantiomer II) as a yellow solid (16 mg, 65% yield, m/z: 434 [M+H]+ observed). 1H NMR (400 MHz, DMSO-d6): δ 14.89 (bs, 1H), 13.45 (bs, 1H), 8.55 (d, 1H), 8.25-7.86 (m, 2H), 7.24 (d, 1H), 3.97 (s, 3H), 3.59 (d, 1H), 3.22-3.15 (m, 1H), 3.05 (d, 1H), 0.74 (s, 9H).
Example 17: 5-(tert-Butyl)-9-chloro-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic (single enantiomer I) Example 18: 5-(tert-Butyl)-9-chloro-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic (single enantiomer II)
Figure imgf000085_0001
The compounds were prepared in a similar manner as 5-(tert-butyl)- 11- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid from an appropriately substituted methyl 5-(tert-butyl)-1-(2,4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate, followed by ester hydrolysis and debenzylation.
60 mg of 5-(tert-butyl)-9-chloro-2 -oxo-11-(trifluoromethyl)-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid was separated by SFC on Chiralpak AS-3 column using iPrOH (5-40%, 0.05% iPrNH2 modifier) to give 5 -(tert- butyl)-9-chloro-2-oxo-11-(trifluoromethyl)-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h]quinoline-3-carboxylic acid (single enantiomer I) as a yellow solid (faster eluting enantiomer, 15 mg, 25% yield) and 5-(tert-butyl)-9-chloro-2-oxo-11-(trifluoromethyl)-1, 2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinobne-3-carboxybc acid (single enantiomer II) as a yellow solid (slower eluting enantiomer, 16 mg, 25% yield).
Single enantiomer I (Example 17): m/z: 440 [M+H]+ observed. 1H NMR (400 MHz, CDCl3): δ 8.43 (s, 1H), 8.17 (d, 1H), 7.60 (d, 1H), 3.41- 3.33 (m, 1H), 3.29 (dd, 1H), 3.00 (dd, 1H), 0.85 (s, 9H).
Single enantiomer II (Example 18): m/z: 440 [M+H]+ observed. 1H NMR (400 MHz, CDCl3): δ 8.43 (s, 1H), 8.17 (d, 1H), 7.60 (d, 1H), 3.41- 3.33 (m, 1H), 3.29 (dd, 1H), 3.00 (dd, 1H), 0.85 (s, 9H).
Example 19: (S)-5-(tert-Butyl)-9-cyclopropyl-2-oxo-11-(trifluoromethyl)-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer
I)
Example 20: (R)-5-tert -Butyl)-9-cyclopropyl-2-oxo-11-(trifluoromethyl)-1 ,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer
P)
Figure imgf000086_0001
The compounds were prepared in a similar manner as 5-(tert-butyl)-11- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid from an appropriately substituted methyl 5-(tert-butyl)-1-(2,4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate, followed by ester hydrolysis and debenzylation.
60 mg of 5-(tert-butyl)-9-cyclopropyl-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid was separated by SFC on Chiralpak IG-3 column using EtOH (5-50%, 0.1% iPrNH2 modifier) to give (S)-5-(tert- butyl)-9-cyclopropyl-2-oxo- 11 -(trifluoromethyl)- 1 ,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) as a yellow solid (faster eluting enantiomer, 18 mg, 30% yield) and (R)-5-(tert-butyl)-9- cyclopropyl-2-oxo- 11 -(trifluoromethyl)- 1 ,2,5 ,6-tetrahydropyrido [2',1' :2,3] imidazo [4,5 - h] quinoline -3 -carboxylic acid (single enantiomer II) as a yellow solid (slower eluting enantiomer, 20 mg, 33% yield).
Single enantiomer I (Example 19): m/z: 446 [M+H]+ observed. 1H NMR (400 MHz, CDCl3): δ 8.40 (s, 1H), 7.86 (s, 1H), 7.34 (s, 1H), 3.46 - 3.14 (m, 2H), 2.96 (d, 1H), 2.01 (ddd, 1H), 1.18 - 1.08 (m, 2H), 0.85 (s, 9H), 0.83 - 0.77 (m, 2H).
Figure imgf000087_0001
Single enantiomer II (Example 20): m/z: 446 [M+H]+ observed. 1H NMR (400 MHz, CDCl3): δ 8.40 (s, 1H), 7.86 (s, 1H), 7.34 (s, 1H), 3.46 - 3.14 (m, 2H), 2.96 (d, 1H), 2.01 (ddd, 1H), 1.18 - 1.08 (m, 2H), 0.85 (s, 9H), 0.83 - 0.77 (m, 2H).
Figure imgf000087_0002
Example 21: 5-(tert-Butyl)-9-methoxy-2-oxo-11-(trifluoromethyl)-1, 2,5,6- tetrahydropyrido [2',1 ' :2,3]imidazo [4,5-h] quinoline-3-carboxylic acid
Figure imgf000087_0003
The compounds were prepared in a similar manner as 5-(tert-butyl)- 11- (difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline -3 -carboxylic acid from an appropriately substituted methyl 5-(tert-butyl)-1-(2,4- dimethoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3- carboxylate, followed by ester hydrolysis and debenzylation. The title compound was obtained as a racemate: m/z: 436 [M+H]+ observed. 1H NMR (400 MHz, CDCl3) δ 10.46 (s, 1H), 8.41 (s, 1H), 7.50 (s, 1H), 7.42 (s, 1H), 3.95 (s, 3H), 3.42 - 3.31 (m, 1H), 3.25 (dd, 1H), 2.97 (d, 1H), 0.85 (s, 9H).
Example 22: 5-(tert-Butyl)-9-chloro-1-methyl-2-oxo-11-(trifluoromethyl)-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer
I)
Example 23: 5-(tert-Butyl)-9-chloro-1-methyl-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer
P)
Figure imgf000088_0001
(a) Synthesis of 8-(tert-butyl)-2-chloro-4-(trifluoromethyl)-8,9- dihydrobenzo[4,5]imidazo[1,2-a]pyridin-6(7H)-one
Figure imgf000088_0002
A mixture of 5-chloro-3-(trifluoromethyl)pyridin-2-amine (12.7 g, 64.8 mmol) and 4- tert-biitylcyclohcxanc- 1 ,2-dionc (21.7 g, 129 mmol) in acetic acid (25 mL) was heated to 120 °C under oxygen atmosphere for 72 h. The reaction was cooled to rt, fdtered through CELITE® and concentrated under vacuum. The resulting residue was kept at rt for 16h. The resulting off-white crystals were collected by fdtration and washed with EtO Ac/hexanes (1:4, 20 mL) to afford pure 8-tert-butyl-2-chloro-4-(trifluoromethyl)-8,9-dihydro-7H-pyrido[1,2- a]benzimidazol-6-one (3.1 g, batch 1). The fdtrate was concentrated under vacuum and dissolved in EtO Ac/hexanes (1:4, 50 mL). The solution was kept at 4 °C for 48 h. The resulting off-white crystals were collected by filtration and washed with EtO Ac/hexanes (1:4, 20 mL) to afford pure 8-tert-biityl-2-chloro-4-(trifluoromethyl)-8.9-dihydro-7H-pyrido[ 1.2- a]benzimidazol-6-one (3.1 g, batch 2). The two batches were combined to afford 8-tert-butyl- 2-chloro-4-(trifluoromethyl)-8,9-dihydro-7H-pyrido[1,2-a]benzimidazol-6-one (5.4 g, 24% yield, m/z: 345 [M+H]+ observed).
(b) Synthesis of 8-(tert-butyl)-2-chloro-N-methyl-4-(trifluoromethyl)-8,9- dihydrobenzo[4,5]imidazo[1,2-a]pyridin-6(7H)-imine
Figure imgf000089_0001
A mixture of 8-tert-butyl-2-chloro-4-(trifluoromethyl)-8,9-dihydro-7H-pyrido[1,2- a]benzimidazol-6-one (2.0 g, 5.8 mmol), methylamine (2M solution in THF, 11.6 mL, 23.2 mmol) and NaiSO3 (2 g) in 1,4-dioxane (10 mL) was heated at 95 °C in a sealed flask for 16 h. The reaction mixture was cooled to rt, filtered through CELITE® and concentrated under vacuum to afford crude 8-(tert-butyl)-2-chloro-N-methyl-4-(trifluoromethyl)-8,9- dihydrobenzo[4,5]imidazo[1,2-a]pyridin-6(7H)-imine as a brown oil, which was used in the next step without purification (2 g, 96% yield, m/z: 358 [M+H]+ observed).
(c) Synthesis of methyl 5-(tert-buty/)-9-ch loro- 1 -methy/-2-oxo- II -(trifluoromethy/)-
1,2, 5, 6-tetrah dropyrido[2 ', 1 ':2,3 ]imidazo[ 4, 5-h Jquinolin e-3-carboxylate
Figure imgf000089_0002
A mixture of 8-(tert-butyl)-2-chloro-N-methyl-4-(trifluoromethyl)-8,9- dihydrobenzo[4,5]imidazo[1,2-a]pyridin-6(7H)-imine (2 g, 5.6 mmol) and dimethyl 2- (methoxymethylene)propanedioate (2.9 g, 16.8 mmol) in diglyme (10 mL) was heated to 165 °C for 16 h. The reaction mixture was cooled to rt and concentrated under reduced pressure. The residue was purified by normal phase SiO2 chromatography (0-80% EtO Ac/hexanes) to afford methyl 5-(tert-butyl)-9-chloro- 1 -methyl-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylate as yellow solid (320 mg, 12% yield, m/z: 468 [M+H]+ observed).
(d) Synthesis of 5-(tert-Butyl)-9-chloro-1-methyl-2-oxo-11-(trifluoromethyl)-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I, Example 22) and 5-(tert-Butyl)-9-chloro-1-methyl-2-oxo-11-(trifluoromethyl)-1, 2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer II, Example 23)
Figure imgf000090_0001
A mixture of methyl 5-(tert-butyl)-9-chloro-1-methyl-2-oxo-11-(trifluoromethyl)-1, 2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinobne-3-carboxylate (180 mg, 0.38 mmol) and lithium hydroxide monohydrate (24 mg, 0.57 mmol) in 4-d1,ioxane/H2O (3: 1, 4 mL) was stirred at rt for overnight. The mixture was cooled to rt and IN HC1 was added to the solution to adjust the pH to 5. The reaction mixture was extracted with CH2CI2 (3 x 5 mL). The combined organic phase was concentrated under reduced pressure. The crude residue was purified by normal phase SiO2 chromatography (0-10%, MeOH/CH2CI2) to afford 5-(tert-butyl)-9-chloro- 1 -methyl -2 -oxo- 11 -(trifluoromethyl)- 1 ,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid as a yellow solid (120 mg, 69% yield, m/z: 454 [M+H]+ observed. 1H NMR (400 MHz, CDCl3): δ 8.37 (s, 1H), 8.18 (d, 1H), 7.58 (dd, 1H), 4.42 (s, 3H), 3.34 - 3.26 (m, 2H), 2.92 (q, 1H), 0.74 (d, 9H).
120 mg of 5-(tert-butyl)-9-chloro- 1 -methyl-2-oxo- 11 -(trifluoromethyl)- 1,2, 5, 6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid was separated by SFC on (S,S)-WHELK-O1 column using iPrOH (5-50%, 0.05% /PrNH2 modifier) to give 5 -(tert- butyl)-9-chloro- 1 -methyl-2-oxo- 11 -(trifluoromethyl)- 1 ,2,5 ,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid (single enantiomer I) as a yellow solid (faster eluting enantiomer, 40 mg, 33% yield) and 5-(to7-butyl)-9-chloro- 1 - methyl-2 -oxo-11-(trifluoromethyl)-1, 2,5, 6-tetrahydropyrido[2',1':2,3]imidazo[4, 5- h] quinoline -3 -carboxylic acid (single enantiomer II) as a yellow solid (slower eluting enantiomer, 38 mg, 32% yield). Single enantiomer I (Example 22): m/z: 454 [M+H]+ observed. 1H NMR (400 MHz, CDCl3): δ 8.37 (s, 1H), 8.18 (d, 1H), 7.58 (dd, 1H), 4.42 (s, 3H), 3.34 - 3.26 (m, 2H), 2.92 (q, 1H), 0.74 (d, 9H).
Single enantiomer II (Example 23): m/z: 454 [M+H]+ observed. 1H NMR (400 MHz, CDCl3): δ 8.37 (s, 1H), 8.18 (d, 1H), 7.58 (dd, 1H), 4.42 (s, 3H), 3.34 - 3.26 (m, 2H), 2.92 (q, 1H), 0.74 (d, 9H).
Example 24: Biological Activity
HBsAg Assay
Inhibition of HBsAg was determined in HepG2.2.15 cells. Cells were maintained in culture medium containing 10% fetal calf serum, G414, Glutamine, penicillin/streptomycin. Cells were seeded in 96-well collagen-coated plate at a density of 30,000 cells/well. Serially diluted compounds were added to cells next day at the final DMSO concentration of 0.5%. Cells were incubated with compounds for 2-3 days, after which medium was removed. Fresh medium containing compounds was added to cells for additional 3-4 days. At day 6 after exposure of compounds, supernatant was collected, the HBsAg immunoassay (microplate- based chemiluminescence immunoassay kits, CLIA, Autobio Diagnosics Co., Zhengzhou, China, Catalog # CL0310-2) was used to determine the level of HBsAg according to manufactory instruction. Dose-response curves were generated and the EC50 value (effective concentrations that achieved 50% inhibitory effect) were determined using XLfit software.
In addition, cells were seeded at a density of 5,000 cells/well for determination of cell viability in the presence and absence of compounds by using CellTiter-Glo reagent (Promega). Table 1 shows EC50 values obtained by the HBsAg assay for selected compounds.
Table 1.
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Example 25: Liver-to-Plasma Distribution and Metabolic Stability
The compounds of the present disclosure are useful for the treatment, amelioration, and/or prevention of Hepatitis B (and/or Hepatitis D) in mammals. As a key element of HBV's life cycle, the virus infects hepatocyte cells in the liver.
Thus, as an important criterion to determine whether a compound of the present disclosure can be developed as a clinically validated Hepatitis B (and/or Hepatitis D) treatment, such compound should preferentially accumulate in the liver upon administration to the subject.
In certain embodiments, preferential liver accumulation of the compound maximizes inhibition of HBV/HDV proliferation and/or inhibition of cell infection by HBV/HDV. In certain embodiments, preferential liver accumulation of the compound minimizes and/or eliminates accumulation in tissues and/or organs other than the liver. In certain embodiments, preferential liver accumulation of the compound minimizes and/or eliminates exposure of the compound to tissues and/or organs other than the liver. In certain embodiments, preferential liver accumulation of the compound minimizes and/or eliminates at least one the following: off-target effects, undesirable exposure, undesirable pharmacokinetics, and/or undesirable metabolism. Further, a clinically developable compound should be metabolically stable upon administration to the subject, such that the compound is not metabolized to metabolites that are quickly eliminated from the liver and/or excreted from the subject, metabolites that have undesirable biological activities, and/or metabolites that are devoid of desirable biological activities.
The liver accumulation potential of a compound can be ascertained through liver-to- plasma distribution studies, using methods that are known in the art and/or described elsewhere herein. For a compound of the present disclosure, a high liver-to-plasma ratio is desirable, as it indicates that the compound tends to accumulate in the liver over other tissues or organs.
Further, for an antiviral compound that exerts its biological activity by inhibiting a viral infection in the liver, an important developability parameter is the ratio between the compound concentration in the liver (Cliver) and the compound concentration that would be required to inhibit the virus infection by 90% (EC90). A high Cliver/EC90 ratio is desirable, as it indicates that the compound concentrates in the liver at levels above those required for effective inhibition of the viral infection.
Further, for an antiviral compound that exerts its biological activity by inhibiting a viral infection in the liver, another important developability parameter is the Area Under the Curve (AUC) time curve for the compound's plasma concentration up to the last measurable concentration (AUCiast). A low plasma AUCiast value is desirable, as it indicates that the compound does not accumulate in the blood/plasma. A low plasma AUCiast value is consistent with the compound's preferential accumulation in the liver, especially when coupled with a high liver-to-plasma ratio and/or a high Cliver/EC90 ratio.
The susceptibility of a compound to undergo hepatic degradation (which is an important component of the compound's overall metabolic stability) can be ascertained through in vitro intrinsic clearance studies, using methods that are known in the art and/or described elsewhere herein. As described elsewhere herein, one such study is the mouse liver microsome (MLM) intrinsic clearance assay or the rat liver microsome (RLM) intrinsic clearance assay, where the compound of interest is incubated with MLMs/RLMs in vitro and the compound concentration is measured as a function of time. In this assay, a low MLM/RLM intrinsic clearance rate is desirable, as it indicates that the compound is not degraded and/or modified by the MLMs/RLMs and thus has good metabolic stability.
Using the protocols described herein, pharmacokinetic (PK) data for oral administration to the mouse were collected for selected compounds of the disclosure (all of which are substituted 5,6-dihydropyrido[2',1':2,3]imidazo[4,5-h]quinolin-2(1H)-ones, as shown below), as well as comparative compounds with the same tetracyclic structure:
Figure imgf000097_0001
Exemplary data for such determinations in mice are provided in Table 2. The antiviral activity, liver-to-plasma distribution, and metabolic stability of the tested compounds varied widely with their particular substitution on the terminal dihydropyridyl ring. For these compounds, biological activity can be ascertained by their EC50 value for HBsAg inhibition. Their liver accumulation behavior can be ascertained by their liver-to- plasma ratio and their plasma AUCiast values, as well as by the ratio of measured liver concentration and the EC90 value for HBsAg inhibition. Compounds A-C are provided herein as comparative compounds.
Compound A, with a methoxy substituent at position 11, showed good HBsAg inhibition, but a moderate liver-to-plasma distribution ratio and high MLM intrinsic clearance, leading to a poor Cliver/EC90 ratio. Introduction of an additional methoxy group at position 10 (Compound B) slightly increased the HBsAg inhibition and reduced MLM intrinsic clearance of the compound, but decreased its liver-to-plasma distribution ratio to the point where the Cliver,/EC90 ratio decreased by an order of magnitude relative to Compound A. On the other hand, replacement of the methoxy group at position 11 with a difluoromethoxy group (Compound C) improved the compound's HBsAg inhibition and liver-to-plasma distribution ratio and reduced the plasma AUCiast (as compared to Compound B), but led to undesirable increase in MLM intrinsic clearance. This observed lower metabolic stability for Comparative Compound C is consistent with the fact that the Cliver,/EC90 ratio was 108 at 12 hours but that ratio dropped sharply by two orders of magnitude at 24 hours after administration, presumably because Compound C was quickly degraded in the liver over time. Consistently, the liver-to-plasma distribution ratio for Compound C was below detection levels by 24 hours after administration.
Unexpectedly, in one non-limiting aspect, it was discovered that compounds with attractive antiviral activity, liver-to-plasma distribution ratio, plasma AUCiast, and metabolic stability were obtained when small halogenated groups were introduced at position 11 and another specific substituent was added to position 9 and/or 10 of the terminal ring. All these compounds showed good HBsAg inhibition and promising plasma AUCiast values and Cliver,/ EC90) ratios. Further, administration of certain compounds of the disclosure at lower doses (0.2 mg/kg vs. 10 mg/kg) confirmed that the compounds concentrate primarily in the liver, as evidenced by higher liver-to-plasma ratios and much decreased plasma AUCiast values (see for example Compounds 1, 4, and 19) at the lower dose.
Similar pharmacokinetic (PK) data were obtained for oral administration of selected compounds of the disclosure to the rat (Table 3). Compound B showed low liver-to-plasma distribution ratio in the rat, as measured by the Cliver,/ EC90 ratio, and in fact the liver-to- plasma ratio for that compound at the 24 hour time point was below the detection level. Compound C showed improved liver-to-plasma distribution ratio and reduced plasma AUCiast in the rat (as compared to Compound B), but had undesirable increase in RUM intrinsic clearance. In contrast, Compounds 1 and 19 showed good HBsAg inhibition, low RUM clearance, and promising plasma AUCiast values and Cliver, /EC90 ratios in the rat.
Note that the pharmacokinetic effects observed in these studies (including the changes in liver-to-plasma distribution ratios and Cliver,/EC90 ratios) cannot be rationalized as a simple hydrophobic effect, as Compound 1 has a polar methoxy group at position 9 of the terminal ring and yet has one of the highest measured liver-to-plasma distribution ratios (compare with Compound C, which has a hydrogen at position 9, and Compound 4, which has a fluorine atom at position 9).
Taken together, these results highlight the unexpected and highly structurally- dependent effect identified by the present studies: the compounds of the disclosure combine potent antiviral activity against HBV/HDV, preferential accumulation in the liver, and good hepatic stability (as evidenced by low intrinsic clearance values), and are thus good candidates for development as clinically validated Hepatitis B (and/or Hepatitis D) treatments.
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
ho
Figure imgf000103_0001
Example 26: X-ray Structure Determination of Compound 1
Figure imgf000104_0001
Compound 1, C21H21F2N3O5, crystallizes in the triclinic space group P1 with a=12.5559(2)Å, b=13.4032(2)Å, c=13.5722(2)Å, α=101.3510(10)°, β=90.1130(10)°, γ=104.6510(10)°, V=2163.34(6)Å3, Z=4, and dcalc=1.331 g/cm3. X-ray intensity data were collected on a Rigaku XtaLAB Synergy-S diffractometer [CrysAlisPro 1.171.41.104a: Rigaku Oxford Diffraction, Rigaku Corporation, Oxford, UK. (2020)] equipped with an HPC area detector (HyPix-6000HE) and employing confocal multilayer optic-monochromated Cu-Ka radiation (λ= 1.54184 Å) at a temperature of 100K. Preliminary indexing was performed from a series of sixty 0.5° rotation frames with exposures of 0.25 sec. for θ = ±47.20° and 1 sec. for θ = 107.75°. A total of 10576 frames (129 runs) were collected employing w scans with a crystal to detector distance of 34.0 mm, rotation widths of 0.5° and exposures of 0.15 sec. for θ = ±47.20°, ±50.0°, ±54.0°, ±58.0°, ±62.0°, ±66.0°, ±74.0° and 0.5 sec. for θ = -86.25°, -74.0°, -78.0°, - 70.0°, ±107.75°, and ±90.0°, and ±107.75°.
Rotation frames were integrated using CrysAlisPro [CrysAlisPro 1.171.41.104a: Rigaku Oxford Diffraction, Rigaku Corporation, Oxford, UK. (2020)], producing a listing of unaveraged F2 and σ(F2) values. A total of 78482 reflections were measured over the ranges 6.652 ≤ 2θ ≤ 136.462°, -15 ≤ h ≤ 15, -16 ≤ k ≤ 16, -16 ≤ 1 ≤ 16 yielding 15296 unique reflections (Rint = 0.0509). The intensity data were corrected for Uorentz and polarization effects and for absorption using SCALE3 ABSPACK [SCALE3 ABSPACK v1.0.7: an Oxford Diffraction program; Oxford Diffraction Ltd: Abingdon, UK, 2005] (minimum and maximum transmission 0.7195, 1.0000). The structure was solved by direct methods - ShelXT [SHELXT v2014/4: Sheldrick, G.M., Acta Cryst, A, 71, 3-8 (2015)]. The asymmetric unit consists of four crystallographically-independent molecules (see FIG. 2). There was a region of disordered solvent for which a reliable disorder model could not be devised; the X-ray data were corrected for the presence of disordered solvent using SQUEEZE [PLATON (vl 10917): Spek, A.L., Acta Cryst, D65, 148-155 (2009)]. Refinement was by full-matrix least squares based on F2 using SHELXL-2018 [SHELXL-2018/3: Sheldrick, G.M., Acta Cryst, A, 71, 3-8 (2015)]. All reflections were used during refinement. The weighting scheme used was w=1/[σ2(Fo 2 )+ (0.0862P)2 + 0.4138P] where P = (Fo 2 + 2Fc 2)/3. Non-hydrogen atoms were refined anisotropically and hydrogen atoms were refined using a riding model. Refinement converged to R1 =0.0434 and wR2=0.1202 for 14697 observed reflections for which F > 4σ(F) and R1=0.0453 and wR2=0.1237 and GOF =1.031 for all 15296 unique, non- zero reflections and 1134 variables. The maximum D/s in the final cycle of least squares was 0.010 and the two most prominent peaks in the final difference Fourier were +0.39 and -0.28 e/Å3. The Hooft absolute structure parameter y was calculated using PLATON [Spek, A.L. J. Appl. Cryst, 41, 96-103 (2008)] The resulting value was y = 0.00(4) indicating that the absolute structure has been assigned correctly. The Flack parameter [Flack, H.D., Acta Cryst. A39, 876-881 (1983)] refined to a similar value of -0.02(5). If these parameters are equal to 0 (within 3 standard deviations) then the absolute structure has been assigned correctly; if they are 1, the opposite enantiomer has been modeled. Table 4 lists cell information, data collection parameters, and refinement data. Final positional and equivalent isotropic thermal parameters are given in Tables 5-6. Tables 7-8 list bond distances and bond angles. FIGs. 1-2 are ORTEP representations of the molecules with 30% probability thermal ellipsoids displayed.
Table 4. Summary of Structure Determination of Compound 1.
Figure imgf000105_0001
Figure imgf000106_0002
Table 5. Refined Positional Parameters for Compound 1.
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000114_0001
Figure imgf000115_0002
Table 9. Hydrogen Bonds for Compound 1
Figure imgf000115_0003
11 +X,1 +Y,1 +Z; 2-1+X,-1+Y,-1+Z; 3+X ,+Y,1+Z; 4+X ,+Υ,-1 +Z
Example 27: X-ray Structure Determination of Compound 19
Figure imgf000115_0001
Compound 19, C49H55F6N6O9, crystallizes in the triclinic space group PI with a=9.07533(5)Å, b=15.40352(9)Å, c= 17.04204(9) A, α=92.7942(5)°, β=90.5419(4)°, γ=97.6106(5)°, V=2358.24(2)Å3, Z=2, and dcalc=1.389 g/cm3. X-ray intensity data were collected on a Rigaku XtaLAB Synergy-S diffractometer [CrysAlisPro 1.171.41.104a: Rigaku Oxford Diffraction, Rigaku Corporation, Oxford, UK. (2020)] equipped with an HPC area detector (HyPix-6000HE) and employing confocal multilayer optic-monochromated Cu-Ka radiation (l= 1.54184 A) at a temperature of 100K. Preliminary indexing was performed from a series of sixty 0.5° rotation frames with exposures of 0.25 sec. for θ = ±47.29° and 1 sec. for θ = 107.75°. A total of 13712 frames (132 runs) were collected employing w scans with a crystal to detector distance of 34.0 mm, rotation widths of 0.5° and exposures of 0.05 sec. for θ = ±10.00°, ±47.04°, ±50.00°, ±54.00°, ±58.00°, ±62.00°, ±66.00°, ±70.00°, ±74.0° and 0.25 sec. for θ = - 70.00°, -74.00°, -78.00°, -82.00°, -86.25 and 107.75°.
Rotation frames were integrated using CrysAlisPro [CrysAlisPro 1.171.41.104a: Rigaku Oxford Diffraction, Rigaku Corporation, Oxford, UK. (2020)], producing a listing of unaveraged F2 and σ(F2) values. A total of 114601 reflections were measured over the ranges 5.192 ≤ 2θ ≤ 149°, -11 ≤ h ≤ l 1, -19 ≤ k ≤ 19, -21 ≤ 1 ≤ 21 yielding 18408 unique reflections (Rint = 0.0495). The intensity data were corrected for Uorentz and polarization effects and for absorption using SCALE3 ABSPACK [SCALE3 ABSPACK vl.0.7: an Oxford Diffraction program; Oxford Diffraction Ltd: Abingdon, UK, 2005] (minimum and maximum transmission 0.7168, 1.0000). The structure was solved by direct methods - ShelXT [SHELXT v2014/4: Sheldrick, G.M., Acta Cryst, A, 71, 3-8 (2015)]. The asymmetric unit consists of 4 molecules of the title compound plus 4 molecules of water and two molecules of disordered iso-propyl alcohol. Refinement was by full-matrix least squares based on F2 using SHELXL-2018 [SHELXL-2018/3: Sheldrick,
G.M., Acta Cryst., A, 71, 3-8 (2015)]. All reflections were used during refinement. The weighting scheme used was w=1/[σ2(Fo 2 )+ (0.0706P)2 ± 0.9046P] where P = (Fo 2 + 2Fc 2)/3. Non-hydrogen atoms were refined anisotropically and hydrogen atoms were refined using a riding model. Refinement converged to R1 =0.0407 and wR2=0.1094 for 17947 observed reflections for which F > 4 σ(F) and R1=0.0423 and wR2=0.1134 and GOF =1.016 for all 18408 unique, non-zero reflections and 1345 variables. The maximum D/s in the final cycle of least squares was 0.001 and the two most prominent peaks in the final difference Fourier were ±0.77 and -0.39 e/Å3. The Hooft absolute structure parameter y [Hooft, R.W.W., Straver, L.H., Spek, A.L. (2008) J. Appl.Cryst, 41, 96-103] was calculated using PLATON [Hooft, R.W.W., Straver, L.H., Spek, A.L. (2008) J. Appl.Cryst, 41, 96-103], The resulting value was y = 0.03(2) indicating that the absolute structure has been assigned correctly. The Flack parameter [Flack,
H.D., Acta Cryst. A39, 876-881 (1983)] refined to a similar value of 0.02(3). If these parameters are equal to 0 (within 3 standard deviations) then the absolute structure has been assigned correctly; if they are 1, the opposite enantiomer has been modeled.
Table 10 lists cell information, data collection parameters, and refinement data. Final positional and equivalent isotropic thermal parameters are given in Tables 11-12. Tables 13-14 list bond distances and bond angles. FIG. 3 is an ORTEP representation of the molecule with 50% probability thermal ellipsoids displayed.
Table 10. Summary of Structure Determination of Compound 19.
Figure imgf000117_0001
Table 11. Refined Positional Parameters for Compound 19.
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Table 13. Bond Distances in Compound 19, A.
Figure imgf000125_0002
Figure imgf000126_0002
Table 14. Bond Angles in Compound 19, °.
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Enumerated Embodiments
The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance:
Embodiment 1 provides a compound selected from:
Figure imgf000129_0001
R1 is -C(=O)OR5;
R2 is selected from the group consisting of H, optionally substituted C1-C6 alkyl, and optionally substituted C3-C8 cycloalkyl;
R3a, R3b, R4a, and R4b are each independently selected from the group consisting of H, alkyl- substituted oxetanyl, optionally substituted C1-C6 alkyl, and optionally substituted C3-C8 cycloalkyl; or one pair selected from the group consisting of R3a / R3b, R4a / R4b, and R3a / R4a combine to form a divalent group selected from the group consisting of C1-C6 alkanediyl,
-(CH2)nO(CH2)n-, -(CH2)nNR7(CH2)n-, -(CH2)nS(CH2)n-, -(CH2)nS(=O)(CH2)n-, and -
(CH2)nS(=O)2(CH2)n-, wherein each occurrence of n is independently 1 or 2 and wherein each divalent group is optionally substituted with at least one C1-C6 alkyl or halogen; each occurrence of R5 is independently selected from the group consisting of H, optionally substituted C1-C6 alkyl, and optionally substituted C3-C8 cycloalkyl;
R6I is -CX3, -CHX2, -OCX3, or -OCHX2, wherein each occurrence of X is independently F, Cl,
Br, or I; R6II and R6III are selected such that: one of them is -CX3, -CHX2, -OCX3, -OCHX2, F, Cl, Br, I, C1-C6 alkoxy, optionally substituted C1-C6 alkyl, and optionally substituted C3-C8 cycloalkyl, wherein each occurrence of X is independently F, Cl, Br, or I; and the other is H, -CX3, -CHX2, -OCX3, -OCHX2, F, Cl, Br, I, C1-C6 alkoxy, optionally substituted C1-C6 alkyl, and optionally substituted C3-C8 cycloalkyl, wherein each occurrence of X is independently F, Cl, Br, or I;
R6IV is H; and each occurrence of R7 is independently selected from the group consisting of H and C1-C6 alkyl; or a salt, solvate, geometric isomer, stereoisomer, tautomer and any mixtures thereof.
Embodiment 2 provides the compound of Embodiment 1, wherein at least one of R3a or R3b is independently selected from the group consisting of optionally substituted C1-C6 alkyl and optionally substituted C3-C8 cycloalkyl.
Embodiment 3 provides the compound of any one of Embodiments 1-2, wherein each occurrence of alkyl or cycloalkyl is independently optionally substituted with at least one substituent selected from the group consisting of C1-C6 alkyl, halogen, -OR”, phenyl, and - N(R”)(R”), wherein each occurrence of R” is independently H, C1-C6 alkyl, or C3-C8 cycloalkyl.
Embodiment 4 provides the compound of any one of Embodiments 1-3, wherein at least one applies: R3a is H and R3b is isopropyl; R3a is H and R3b is tert-butyl; R3a is methyl and R3b is isopropyl; R3a is methyl and R3b is tert-butyl; R3a is methyl and R3b is methyl; R3a is methyl and R3b is ethyl; and R3a is ethyl and R3b is ethyl.
Embodiment 5 provides the compound of any one of Embodiments 1-4, wherein R2 is H, methyl, or cyclopropyl.
Embodiment 6 provides the compound of any one of Embodiments 1-5, wherein R3a and R3b are not H.
Embodiment 7 provides the compound of any one of Embodiments 1-6, wherein one of R6II and R6III is H.
Embodiment 8 provides the compound of any one of Embodiments 1-7, which is selected from the group consisting of:
(R)-5-(tert-butyl)-11-(difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid; (S)-5-(tert-butyl)-11-(difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5-(tert-butyl)-11-(difluoromethoxy)-9-fluoro-2-oxo- 1 ,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5-(tert-butyl)-11-(difluoromethoxy)-9-fluoro-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5-(tert-butyl)-2-oxo-11-(trifluoromethyl)-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline-3 -carboxylic acid;
(S)-5-(tert-butyl)-2-oxo-11-(trifluoromethyl)-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline-3 -carboxylic acid;
(R)-5-(tert-butyl)-11-(difluoromethyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline-3 -carboxylic acid;
(S)-5-(tert-butyl)-11-(difluoromethyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline-3 -carboxylic acid;
(R)-5-(tert-butyl)-9-(difluoromethoxy)-11-methoxy-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5-(tert-butyl)-9-(difluoromethoxy)-11-methoxy-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5-(tert-butyl)-9,11 -bis(difluoromethoxy)-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5-(tert-butyl)-9,11 -bis(difluoromethoxy)-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5-(tert-butyl)-11-(difluoromethoxy)-10-methoxy-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5-(tert-butyl)-11-(difluoromethoxy)-10-methoxy-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5-(tert-butyl)-9-chloro-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5-(tert-butyl)-9-chloro-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5-(tert-butyl)-9-cyclopropyl-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid; (S)-5-(tert-butyl)-9-cyclopropyl-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5-(tert-butyl)-9-methoxy-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5-(tert-butyl)-9-methoxy-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5-(tert-butyl)-9-chloro-1-methyl-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5-(tert-butyl)-9-chloro- 1 -methyl-2-oxo- 11 -(trifluoromethyl)- 1 ,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid; or a salt, solvate, geometric isomer, stereoisomer, tautomer and any mixtures thereof.
Embodiment 9 provides a pharmaceutical composition comprising at least one compound of any one of Embodiments 1-8 and a pharmaceutically acceptable carrier.
Embodiment 10 provides the pharmaceutical composition of Embodiment 9, further comprising at least one additional agent useful for treating, ameliorating, and/or preventing hepatitis B virus infection.
Embodiment 11 provides the pharmaceutical composition of Embodiment 10, wherein the at least one additional agent comprises at least one selected from the group consisting of reverse transcriptase inhibitors, capsid inhibitors, cccDNA formation inhibitors, RNA destabilizers, oligomeric nucleotides targeted against the HBV genome, immunostimulators, GalNAc-siRNA conjugates targeted against an HBV gene transcript, and therapeutic vaccines.
Embodiment 12 provides the pharmaceutical composition of Embodiment 11, wherein the oligomeric nucleotide comprises one or more siRNAs.
Embodiment 13 provides the pharmaceutical composition of any one of Embodiments 10-12, wherein the at least one additional agent further treats, ameliorates, and/or prevents hepatitis D virus infection.
Embodiment 14 provides a method of treating, ameliorating, and/or preventing hepatitis B virus infection in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of at least one compound of any one of Embodiments 1-8 and/or at least one pharmaceutical composition of any one of Embodiments 9-13. Embodiment 15 provides the method of Embodiment 14, wherein the subject is further infected with hepatitis D virus (HDV).
Embodiment 16 provides a method of reducing or minimizing levels of at least one selected from the group consisting of hepatitis B virus surface antigen (HBsAg), hepatitis B e- antigen (HBeAg), hepatitis B core protein, and pregenomic (pg) RNA, in a HBV-infected subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of at least one compound any one of Embodiments 1-8 and/or at least one pharmaceutical composition of any one of Embodiments 9-13.
Embodiment 17 provides the method of any one of Embodiments 14-16, wherein the at least one compound is administered to the subject in a pharmaceutically acceptable composition.
Embodiment 18 provides the method of any one of Embodiments 14-17, wherein the subject is further administered at least one additional agent useful for treating, ameliorating, and/or preventing the HBV infection.
Embodiment 19 provides the method of Embodiment 18, wherein the at least one additional agent comprises at least one selected from the group consisting of reverse transcriptase inhibitors, capsid inhibitors, cccDNA formation inhibitors, RNA destabilizers, oligomeric nucleotides targeted against the HBV genome, immunostimulators, GalNAc-siRNA conjugates targeted against an HBV gene transcript, and therapeutic vaccines.
Embodiment 20 provides the method of Embodiment 19, wherein the oligomeric nucleotide comprises one or more siRNAs.
Embodiment 21 provides the method of any one of Embodiments 18-20, wherein the subject is co-administered the at least one compound and the at least one additional agent.
Embodiment 22 provides the method of any one of Embodiments 18-21, wherein the at least one compound and the at least one additional agent are coformulated.
Embodiment 23 provides the method of any one of Embodiments 14-22, wherein the subject is further infected with HDV.
Embodiment 24 provides the method of any one of Embodiments 14-23, wherein the subject is a mammal.
Embodiment 25 provides the method of Embodiment 24, wherein the mammal is a human.
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this disclosure has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this disclosure may be devised by others skilled in the art without departing from the true spirit and scope of the disclosure. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:
1. A compound selected from:
Figure imgf000135_0001
wherein:
R1 is -C(=O)OR5;
R2 is selected from the group consisting of H, optionally substituted C1-C6 alkyl, and optionally substituted C3-C8 cycloalkyl;
R3a, R3b, R4a, and R4b are each independently selected from the group consisting of H, alkyl- substituted oxetanyl, optionally substituted C1-C6 alkyl, and optionally substituted C3-C8 cycloalkyl; or one pair selected from the group consisting of R3a / R3b, R4a / R4b, and R3a / R4a combine to form a divalent group selected from the group consisting of C1-C6 alkanediyl, -(CH2)nO(CH2)n-, -(CH2)nNR7(CH2)n-, -(CH2)nS(CH2)n-, -(CH2)nS(=O)(CH2)n-, and - (CH2)nS(=O)2(CH2)n-, wherein each occurrence of n is independently 1 or 2 and wherein each divalent group is optionally substituted with at least one C1-C6 alkyl or halogen; each occurrence of R5 is independently selected from the group consisting of H, optionally substituted C1-C6 alkyl, and optionally substituted C3-C8 cycloalkyl; R6I is -CX3, -CHX2, -OCX3, or -OCHX2, wherein each occurrence of X is independently F, Cl, Br, or I; R6II and R6III are selected such that: one of them is -CX3, -CHX2, -OCX3, -OCHX2, F, Cl, Br, I, C1-C6 alkoxy, optionally substituted C1-C6 alkyl, and optionally substituted C3-C8 cycloalkyl, wherein each occurrence of X is independently F, Cl, Br, or I; and the other is H, -CX3, -CHX2, -OCX3, -OCHX2, F, Cl, Br, I, C1-C6 alkoxy, optionally substituted C1-C6 alkyl, and optionally substituted C3-C8 cycloalkyl, wherein each occurrence of X is independently F, Cl, Br, or I;
R6IV is H; and each occurrence of R7 is independently selected from the group consisting of H and C1-C6 alkyl; or a salt, solvate, geometric isomer, stereoisomer, tautomer and any mixtures thereof.
2. The compound of claim 1, wherein at least one of R3a or R3b is independently selected from the group consisting of optionally substituted C1-C6 alkyl and optionally substituted C3-C8 cycloalkyl.
3. The compound of claim 1, wherein each occurrence of alkyl or cycloalkyl is independently optionally substituted with at least one substituent selected from the group consisting of C1-C6 alkyl, halogen, -OR”, phenyl, and -N(R”)(R”), wherein each occurrence of R” is independently H, C1-C6 alkyl, or C3-C8 cycloalkyl.
4. The compound of claim 1, wherein at least one applies: R3a is H and R3b is isopropyl; R3a is H and R3b is tert-butyl; R3a is methyl and R3b is isopropyl; R3a is methyl and R3b is tert-butyl; R3a is methyl and R3b is methyl; R3a is methyl and R3b is ethyl; and R3a is ethyl and R3b is ethyl.
5. The compound of claim 1, wherein R2 is H, methyl, or cyclopropyl.
6 The compound of claim 1 , wherein R3a and R3b are not H.
7. The compound of claim 1 , wherein one of R6II and R6III is H.
8. The compound of claim 1, which is selected from the group consisting of:
(R)-5-(tert-butyl)-11-(difluoromethoxy)-9-methoxy-2-oxo-1 ,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5-(tert-butyl)-11-(difluoromethoxy)-9-methoxy-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5-(tert-butyl)-11-(difluoromethoxy)-9-fluoro-2-oxo- 1 ,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5-(tert-butyl)-11-(difluoromethoxy)-9-fluoro-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5-(tert-butyl)-2-oxo-11-(trifluoromethyl)-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline-3 -carboxylic acid;
(S)-5-(tert-butyl)-2-oxo-11-(trifluoromethyl)-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h]quinobne-3 -carboxylic acid;
(R)-5-(tert-butyl)-11-(difluoromethyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline-3 -carboxylic acid;
(S)-5-(tert-butyl)-11-(difluoromethyl)-2-oxo-1,2,5,6-tetrahydropyrido[2',1':2,3]imidazo[4,5- h] quinoline-3 -carboxylic acid;
(R)-5-(tert-butyl)-9-(difluoromethoxy)-11-methoxy-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5-(tert-butyl)-9-(difluoromethoxy)-11-methoxy-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5-(tert-butyl)-9,11 -bis(difluoromethoxy)-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5-(tert-butyl)-9,11 -bis(difluoromethoxy)-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5-(tert-butyl)-11-(difluoromethoxy)-10-methoxy-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5-(tert-butyl)-11-(difluoromethoxy)-10-methoxy-2-oxo-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid; (R)-5-(tert-butyl)-9-chloro-2-oxo-11-(trifluoromethyl)-1 ,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5-(tert-butyl)-9-chloro-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5-(tert-butyl)-9-cyclopropyl-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5-(tert-butyl)-9-cyclopropyl-2-oxo- 11 -(trifluoromethyl)-1 ,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5-(tert-butyl)-9-methoxy-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5-(tert-butyl)-9-methoxy-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(R)-5-(tert-butyl)-9-chloro-1-methyl-2-oxo-11-(trifluoromethyl)-1,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid;
(S)-5-(tert-butyl)-9-chloro- 1 -methyl-2-oxo- 11 -(trifluoromethyl)- 1 ,2,5,6- tetrahydropyrido[2',1':2,3]imidazo[4,5-h]quinoline-3-carboxylic acid; or a salt, solvate, geometric isomer, stereoisomer, tautomer and any mixtures thereof.
9. A pharmaceutical composition comprising at least one compound of claim 1 and a pharmaceutically acceptable carrier.
10. The pharmaceutical composition of claim 9, further comprising at least one additional agent useful for treating, ameliorating, and/or preventing hepatitis B virus infection.
11. The pharmaceutical composition of claim 10, wherein the at least one additional agent comprises at least one selected from the group consisting of reverse transcriptase inhibitors, capsid inhibitors, cccDNA formation inhibitors, RNA destabilizers, oligomeric nucleotides targeted against the HBV genome, immunostimulators, GalNAc-siRNA conjugates targeted against an HBV gene transcript, and therapeutic vaccines.
12. The pharmaceutical composition of claim 11, wherein the oligomeric nucleotide comprises one or more siRNAs.
13. The pharmaceutical composition of claim 10, wherein the at least one additional agent further treats, ameliorates, and/or prevents hepatitis D virus infection.
14. A method of treating, ameliorating, and/or preventing hepatitis B virus infection in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of at least one compound of claim 1 and/or at least one pharmaceutical composition of claim 9.
15. The method of claim 14, wherein the subject is further infected with hepatitis D virus (HDV).
16. A method of reducing or minimizing levels of at least one selected from the group consisting of hepatitis B virus surface antigen (HBsAg), hepatitis B e-antigen (HBeAg), hepatitis B core protein, and pregenomic (pg) RNA, in a HBV-infected subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of at least one compound of claim 1 and/or at least one pharmaceutical composition of claim 9.
17. The method of claim 14 or 16, wherein the at least one compound is administered to the subject in a pharmaceutically acceptable composition.
18. The method of claim 14 or 16, wherein the subject is further administered at least one additional agent useful for treating, ameliorating, and/or preventing the HBV infection.
19. The method of claim 18, wherein the at least one additional agent comprises at least one selected from the group consisting of reverse transcriptase inhibitors, capsid inhibitors, cccDNA formation inhibitors, RNA destabilizes, oligomeric nucleotides targeted against the HBV genome, immunostimulators, GalNAc-siRNA conjugates targeted against an HBV gene transcript, and therapeutic vaccines.
20. The method of claim 19, wherein the oligomeric nucleotide comprises one or more siRNAs.
21. The method of claim 18, wherein the subject is co-administered the at least one compound and the at least one additional agent.
22. The method of claim 18, wherein the at least one compound and the at least one additional agent are coformulated.
23. The method of claim 14 or 16, wherein the subject is further infected with HDV.
24. The method of claim 14 or 16, wherein the subject is a mammal.
25. The method of claim 24, wherein the mammal is a human.
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