TITLE OF THE INVENTION

Synthesis of Substituted Arylmethylureas, Analogues, and Crystalline Forms Thereof and

Methods of Using Same

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/121,021, filed December 3, 2020, and U.S. Provisional Application No. 63/040,211, filed June 17, 2020, all of which applications are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

Hepatitis B is one of the world's most prevalent diseases, being listed by the National Institute of Allergy and Infectious Diseases (NIAID) as a high priority area of interest. 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.

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 HB V 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. There is thus a pressing need for the introduction of novel, safe, and effective therapies for chronic hepatitis B.

Hepatitis B is caused by hepatitis B virus (HBV), a noncytopathic, liver tropic DNA virus belonging to Hepadnaviridae family. 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. Inhibition of pg RNA encapsidation may block HBV replication and provide a new therapeutic approach to HBV treatment. A capsid inhibitor acts by inhibiting the expression and/or function of a capsid protein either directly or indirectly: for example, it may inhibit capsid assembly, induce formation of non-capsid polymers, promote excess capsid assembly or misdirected capsid assembly, affect capsid stabilization, and/or inhibit RNA encapsidation. A capsid inhibitor may also act by inhibiting capsid function in one or more downstream events within the replication process, such as, but not limited to, viral DNA synthesis, transport of relaxed circular DNA (rcDNA) into the nucleus, covalently closed circular DNA (cccDNA) formation, virus maturation, budding and/or release.

Hepatitis D virus (HDV) is a small circular enveloped RNA virus that can propagate only in the presence of 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, 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.

Clinically, inhibition of pg RNA encapsidation, or more generally inhibition of nucleocapsid assembly, may offer certain therapeutic advantages for treatment of hepatitis B and/or hepatitis D. In one aspect, inhibition of pg RNA encapsidation may complement the current medications by providing an option for a subpopulation of patients that do not tolerate or benefit from the current medications. In another aspect, based on their distinct antiviral mechanism, inhibition of pg RNA encapsidation may be effective against HBV and/or HDV variants resistant to the currently available DNA polymerase inhibitors. In yet another aspect, combination therapy of the pg RNA encapsidation inhibitors with DNA polymerase inhibitors may synergistically suppress HBV and/or HDV replication and prevent drug resistance emergence, thus offering a more effective treatment for chronic hepatitis B and/or hepatis D infection.

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, as only about one- quarter of patients is serum HDV RNA undetectable 6 months post therapy.

There is thus a need in the art for the identification of novel compounds that can be used to treat and/or prevent HBV and/or HDV infection in a subject. In certain embodiments, the novel compounds inhibit HBV and/or HDV nucleocapsid assembly. In other embodiments, the novel compounds can be used in patients that are HBV and/or HBV-HDV infected, patients who are at risk of becoming HBV and/or HBV-HDV infected, and/or patients that are infected with drug -resistant HBV and/or HDV. The present invention addresses this need.

BRIEF SUMMARY OF THE INVENTION

The present disclosure includes, in one aspect, methods of preparing (i?)-3-(3-cyano- 4-fluorophenyl)- 1 -(1 -(6, 7-difluoro- 1 -oxo- 1 ,2-dihy droisoquinolin-4-yl)ethyl)- 1 -methylurea, also known as compound (X), or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer (such as, in a non-limiting example, an enantiomer or any mixtures thereof, such as, in a non-limiting example, mixtures in any proportions of enantiomers thereof), and/or tautomer, and any mixtures thereof:

In another aspect, the present disclosure provides developable forms of (X), or a solvate thereof, that are useful to treat, ameliorate, and/or prevent HBV (and/or HBV-HDV) infection and related conditions in a subject In other embodiments, the present disclosure provides polymorphs of (X), which can include any solvates thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of illustrative embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, certain illustrative embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIG. l is a non-limiting illustration of a polarized light microscopy (PLM) image of (R)-3 -(3 -cy ano-4-fluorophenyl)- 1 -( 1 -(6, 7-difluoro- 1 -oxo- 1 ,2-dihy droi soquinolin-4-yl)ethyl)- 1-methylurea (X) crystalline pattern 1.

FIGs. 2A-2K are non-limiting illustrations of hot-stage microscopy images of crystalline pattern 1 of (X) at several temperatures: (A) 25 °C; (B) 140 °C; (C) 150 °C; (D) 158 °C; (E) 170 °C; (F) 178 °C; (G) 185 °C; (H) 193 °C; (I) 217 °C; (J) 220 °C; and (K) 222 °C.

FIG. 3 is a non-limiting illustration of a XRPD spectrum of crystalline pattern 1 of (X).

FIG. 4 is a non-limiting illustration of an overlaid XRPD spectrum of (a) crystalline pattern 1 of (X) at 25 °C; (b) crystalline pattern 2 of (X) at 25 °C; (c) and crystalline pattern 1 of (X) at 160 °C.

FIG. 5 is a non-limiting illustration of a TG-FTIR analysis of crystalline pattern 1 of (X).

FIG. 6 is a non-limiting illustration of a DSC analysis of crystalline pattern 1 of (X).

FIG. 7 is a non-limiting illustration of a DVS analysis of crystalline pattern 1 of (X).

FIG. 8 is a non-limiting illustration of a DVS analysis of crystalline pattern 1 of (X).

FIG. 9 is a non-limiting illustration of an overlaid XRPD spectrum of crystalline pattern 1 of (X): (a) before DVS analysis; and (b) after DVS analysis (crystalline pattern 9).

FIG. 10 is a non-limiting illustration of a TG-FTIR analysis of crystalline pattern 1 of (X) after DVS analysis (crystalline pattern 9).

FIG. 11 is a non-limiting illustration of a polarized light microscopy (PLM) image of crystalline pattern 2 of (X).

FIG. 12 is a non-limiting illustration of a XRPD spectrum of crystalline pattern 2 of (X).

FIG. 13 is a non-limiting illustration of an overlaid XRPD spectrum of: (a) crystalline pattern 1 of (X); (b) a first sample of crystalline pattern 2 of (X), and (c) a second sample of crystalline pattern 2 of (X).

FIG. 14 is a non-limiting illustration of a TG-FTIR analysis of crystalline pattern 2 of (X).

FIG. 15 is a non-limiting illustration of a DSC analysis of crystalline pattern 2 of (X).

FIG. 16 is a non-limiting illustration of a DVS analysis of crystalline pattern 2 of (X).

FIG. 17 is a non-limiting illustration of a DVS analysis of crystalline pattern 2 of (X).

FIG. 18 is a non-limiting illustration of a XRPD spectrum of crystalline pattern 3 of (X).

FIG. 19 is a non-limiting illustration of a TG-FTIR analysis of crystalline pattern 3 of (X).

FIG. 20 is a non-limiting illustration of a XRPD spectrum of crystalline pattern 4 of

(X). FIG. 21 is a non-limiting illustration of a TG-FTIR analysis of crystalline pattern 4 of (X).

FIG. 22 is a non-limiting illustration of a XRPD spectrum of crystalline pattern 5 of (X).

FIG. 23 is a non-limiting illustration of a TG-FTIR analysis of crystalline pattern 5 of (X).

FIG. 24 is a non-limiting illustration of a XRPD spectrum of crystalline pattern 6 of (X).

FIG. 25 is a non-limiting illustration of a TG-FTIR analysis of crystalline pattern 6 of (X).

FIG. 26 is a non-limiting illustration of a XRPD spectrum of crystalline pattern 7 of (X).

FIG. 27 is a non-limiting illustration of a TG-FTIR analysis of crystalline pattern 7 of (X).

FIG. 28 is a non-limiting illustration of a XRPD spectrum of crystalline pattern 8 of (X).

FIG. 29 is a non-limiting illustration of an overlaid XRPD spectrum of (a) crystalline pattern 4 of (X) and (b) crystalline pattern 8 of (X).

FIG. 30 is a non-limiting illustration of a TG-FTIR analysis of crystalline pattern 8 of (X).

FIG. 31 is a non-limiting illustration of results of competitive slurry experiments as overlaid XRPD spectra of (a) crystalline pattern 2 of (X); (b) crystalline pattern 3 of (X); as well as the products of competitive slurry experiments comprising crystalline patterns 2 of (X) and 3 of (X) in acetone at: (c) rt; and (d) 50 °C.

FIG. 32 is a non-limiting illustration of an overlaid XRPD spectrum of (a) crystalline pattern 1 of (X); (b) crystalline pattern 9 after DVS of crystalline pattern 1 of (X); (c) crystalline pattern 2 of (X); and (d) crystalline pattern 4 of (X).

FIG. 33 is a non-limiting illustration of an overlaid XRPD spectrum of (a) crystalline pattern 1 of (X); (b) crystalline pattern 2 of (X); (c) crystalline pattern 5 of (X); and (d) crystalline pattern 7 of (X).

FIG. 34 is a non-limiting illustration of an overlaid XRPD spectrum of several independent samples of crystalline pattern 2 of (X) collected from different solvents and recrystallization techniques comprising (a) water slurry; (b) 2-methyl THF slurry; (c) 2- propanol slurry; and (d) «-heptane slurry. FIG. 35 is a non-limiting illustration of an overlaid XRPD spectrum comprising (a) crystalline pattern 3 obtained from a methanol slurry of crystalline pattern 1 of (X); (b) crystalline pattern 8 obtained from an ethanol slurry of crystalline pattern 1 of (X) and subsequently dried; (c) crystalline pattern 2 obtained from a toluene slurry; and (d) crystalline pattern 2 obtained from a water slurry.

FIG. 36 is a non-limiting illustration of an overlaid XRPD spectrum of (a) post-DVS treatment crystalline pattern 1 of (X); and (b) crystalline solid collected from a water slurry of crystalline pattern 1 of (X) which was heated at 40 °C for 6 days.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure relates, in certain aspects, to the discovery of scalable synthetic routes that allow for reproducible multi-gram synthesis of certain substituted urea-containing compounds. The disclosure relates, in other aspects, to developable forms of certain substituted urea compounds that are useful to treat, ameliorate, and/or prevent HBV (and/or HBV-HDV) infection and related conditions in a subject.

The present disclosure includes, in one aspect, methods of preparing (f?)-3-(3-cyano- 4-fluorophenyl)- 1 -(1 -(6,7-difluoro- 1 -oxo- 1 ,2-dihydroisoquinolin-4-yl)ethyl)- 1 -methylurea, also known as compound (X), or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer (such as, in a non-limiting example, an enantiomer or any mixtures thereof, such as, in a non-limiting example, mixtures in any proportions of enantiomers thereof), and/or tautomer, and any mixtures thereof:

In another aspect, the present disclosure provides developable forms of (X), or a solvate thereof. In other embodiments, the present disclosure provides polymorphs of (X) (which can include any solvates thereof).

Certain crystalline polymorph forms of (X), are described herein. Certain polymorph forms were identified during a crystallization screen from solvents such as acetone, tetrahydrofuran, ethanol, dichloromethane, dimethylformamide/water, DMF//-butyl methyl ether, water, tetrahydrofuran, acetone/water, 2-methyl tetrahydrofuran, 2-propanol, n- heptane, methanol, toluene, dimethylsulfoxide/methyl ethyl ketone/toluene, ethyl acetate, and tetrahydrofuran/water. Solvates comprising ethanol, ethyl acetate, methanol, a possible mixed solvate/hydrate or acetone solvate, and a possible non-stoichiometric hydrate (crystalline patterns 4, 5, 6, 7, and 9, respectively) and non-solvated forms (crystalline patterns 3 and 8) were identified in such screens. A dichloromethane solvate (Form 1) and a stable non-solvated form (Form 2) were identified prior to such screens.

The disclosures of PCT International Application No. PCT/US2019/065756 filed December 11, 2019 (published as WO 2020/123674 on June 18, 2020), U.S. Provisional Application No. 62/896,237 filed September 5, 2019, and U.S. Provisional Application No. 62/778,471 filed December 12, 2018 are incorporated herein in their entireties by reference.

Definitions

Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes are described as having, including, or comprising specific process steps, it is contemplated that compositions of the present teachings also consist essentially of, or consist of, the recited components, and that the processes of the present teachings also consist essentially of, or consist of, the recited processing steps.

In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components.

As used herein, unless defined otherwise, all technical and scientific terms generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, virology, biochemistry and pharmaceutical sciences are those well-known and commonly employed in the art. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B."

As used herein, the term "about" will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein, "about" when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

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 (Ci-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., Ci-Cio means one to ten carbon atoms) and includes straight, branched chain, or cyclic substituent groups. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, /c/V-butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl. A specific embodiment is (Ci-Ce)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-CºCH. The term "homopropargylic" refers to a group exemplified by -CH2CH2-CºCH.

As used herein, the term "aromatic" refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, /. e. , having (4n+2) delocalized p (pi) electrons, where 'h' 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-(Ci-C ₆ )alkyl" refers to a functional group wherein a one-to-six carbon alkylene chain is attached to an aryl group, e.g. , -CFhCFh-phenyl or -CFh- phenyl (or benzyl). Specific examples are aryl-CFh- and aryl-CE^CEE)-. The term "substituted aryl-(Ci-C ₆ )alkyl" refers to an aryl-(Ci-C ₆ )alkyl functional group in which the aryl group is substituted. A specific example is substituted aryl(CH2)-. Similarly, the term "heteroaryl-(Ci-C ₆ )alkyl" refers to a functional group wherein a one-to-three carbon alkylene chain is attached to a heteroaryl group, e.g., -CFhCFh-pyridyl. A specific example is heteroaryl-(CH2)-. The term "substituted heteroaryl-(Ci-C ₆ )alkyl" refers to a heteroaryl-(Ci- C ₆ )alkyl functional group in which the heteroaryl group is substituted. A specific example is substituted heteroaryl-(CH2)-.

For the purposes of the present invention the terms "compound," "analog," and "composition of matter" stand equally well for the prodrug agent described herein, including all enantiomeric forms, diastereoisomeric forms, salts, and the like, and the terms "compound," "analog," and "composition of matter" are used interchangeably throughout the present specification.

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-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, 2,3-dihydroxycyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctanyl, decalinyl, 2,5-dimethylcyclopentyl, 3,5- dichlorocyclohexyl, 4-hydroxy cyclohexyl, 3,3,5-trimethylcyclohex-l-yl, octahydropentalenyl, octahydro- l//-indenyl, 3a,4,5,6,7,7a-hexahydro-3//-inden-4-yl, decahydroazulenyl; bicyclo[6.2.0]decanyl, decahydronaphthalenyl, and dodecahydro-1 //- 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, l,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, an "effective amount," "therapeutically effective amount" or "pharmaceutically 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.

"Instructional material," as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression that can be used to communicate the usefulness of the composition and/or compound of the invention in a kit. The instructional material of the kit may, for example, be affixed to a container that contains the compound and/or composition of the invention or be shipped together with a container that contains the compound and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively. Delivery of the instructional material may be, for example, by physical delivery of the publication or other medium of expression communicating the usefulness of the kit, or may alternatively be achieved by electronic transmission, for example by means of a computer, such as by electronic mail, or download from a website. As used herein, the term "halide" refers to a halogen atom bearing a negative charge. The halide anions are fluoride (F ), chloride (CU), bromide (Br ), and iodide (G).

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 "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 quaternized. Up to two heteroatoms may be placed consecutively. Examples include - CH=CH-0-CH ₃ , -CH=CH-CH ₂ -0H, -CH ₂ -CH=N-0CH3, -CH=CH-N(CH ₃ )-CH ₃ , and -CH ₂ - CH=CH-CH ₂ -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 quaternized. 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: -OCEbCEbCEb, - CH ₂ CH ₂ CH ₂ OH, -CH ₂ CH ₂ NHCH ₃ , -CH ₂ SCH ₂ CH ₃ , and -CH ₂ CH ₂ S(=0)CH _3. Up to two heteroatoms may be consecutive, such as, for example, -CEbNEl-OCEb, or -CEbCEbSSCEb.

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 quaternized. 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-l,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, 2-, 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 "NMT" refers to Not More Than.

As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound useful within the invention 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 invention, 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 invention 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 invention, 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 corn 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, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; 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 invention, 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 invention.

Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention 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 bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, 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, a "patient" or "subject" may be 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.

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," or "substituted alkynyl" refers to alkyl, cycloalkyl, alkenyl, or alkynyl, 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, -ML·, -NH(CI-C ₆ alkyl), -N(CI-C ₆ alkyl)2, 1 -methyl -imidazol-2-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, - C(=0)0H, -C(=0)0(Ci-C ₆ )alkyl, trifluoromethyl, -CºN, -C(=0)ML·, -C(=0)NH(Ci- Ce)alkyl, -C(=0)N((Ci-C ₆ )alkyl)2, -SO2NH2, -S0 ₂ NH(Ci-C ₆ alkyl), -S0 ₂ N(Ci-C ₆ alkyl) ₂ , - C(=NH)ML·, and -NO2, in certain embodiments containing one or two substituents independently selected from halogen, -OH, alkoxy, - L·, trifluoromethyl, -N(CH3)2, and - C(=0)0H, in certain embodiments independently selected from halogen, alkoxy and -OH. Examples of substituted alkyls include, but are not limited to, 2,2-difluoropropyl, 2- carboxy cyclopentyl and 3-chloropropyl.

For aryl, aryl-(Ci-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 ., R ² and R ³ 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 "Ci- ₆ alkyl" is specifically intended to individually disclose Ci, C2, C3, C4, Cs, Ce, 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 Cs-Ce alkyl.

The term "treat," "treating" or "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.

The term “vol” as used herein to describe an amount of a solvent, wherein no specified volume is provided, means 1 L solvent per 100 g of limiting reagent. For example, in the context of the statement, “compound A (100 g) was dissolved in water (10 vol),” the amount of water used to dissolve compound A is 1 L.

The term “V” as used herein to describe amount of solvent, wherein no specified volume is provided, means 1 L solvent per 1 kg of limiting reagent. For example, in the context of the statement “compound A (100 g) was dissolved in water (10 V),” the amount of water used to dissolve compound A is 100 mL (0.1 L).

Certain abbreviations used herein follow: cccDNA, covalently closed circular DNA; CPME, cyclopentylmethane; DMF, dimethylformamide; DMSO, dimethylsulfoxide; DNA, deoxyribonucleic acid; DSC, differential scanning calorimetry; GC, gas chromatography; GPC, gel-permeation chromatography; HBsAg, HBV surface antigen; HBV, hepatitis B virus; HDV, hepatitis D virus; HPLC, high pressure liquid chromatography; MEK, methyl ethyl ketone; MTBE, methyl tert-butyl ether; NARTI or NRTI, nucleoside reverse- transcriptase inhibitor; NtARTI or NtRTI, nucleotide analog reverse-transcriptase inhibitor; NMR, Nuclear Magnetic Resonance; PLM, polarized light microscopy; RH, relative humidity; rcDNA, relaxed circular DNA; sAg, surface antigen; SFC, supercritical fluid chromatography; TGA, thermogravimetric analysis; THF, tetrahydrofuran; TLC, thin layer chromatography; XRPD, X-ray powder diffraction; XRDP X-ray diffraction pattern.

Ranges: throughout this disclosure, various aspects of the present invention 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 present invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges 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 subranges 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. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement "about X to Y" has the same meaning as "about X to about Y," unless indicated otherwise.

Likewise, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z," unless indicated otherwise. This applies regardless of the breadth of the range.

Compounds and Synthesis

The present disclosure further provides methods of preparing 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 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 ., 'H or ¹³ C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or by chromatography such as high-performance 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, etal. , 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 disclosure includes methods of preparing (f?)-3-(3-cyano-4-fluorophenyl)-l-(l- (6,7-difluoro-l-oxo-l,2-dihydroisoquinolin-4-yl)ethyl)-l-met hylurea, also known as compound (X), or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer (such as, in a non-limiting example, an enantiomer or any mixtures thereof, such as, in a non- limiting example, mixtures in any proportions of enantiomers thereof), and/or tautomer, and any mixtures thereof:

In certain embodiments, the compound of formula (X), or a salt, solvate, prodrug, isotopically labelled derivative, and/or tautomer thereof, can be prepared according to the non-limiting synthetic scheme outlined in Scheme 1.

Commercially available bromide (A) can be converted to the corresponding ketone (B) by Lewis-acid catalyzed coupling (using in a non-limiting example a copper (I) salt) with acetylacetone in the presence of base (such as, but not limited to, an alkali alkoxide).

Ketone (B) can undergo acid-catalyzed cyclization in dichloromethane (DCM) to yield isochromenone (C), which can be reacted with formamide in basic solution to yield isoquinolinone (D). Alternatively, under acidic conditions in the presence of methanol, ketone (B) can undergo both acid-catalyzed esterification to yield ester (C) and acid- catalyzed cyclization to yield isochromene (C), either of which can be independently reacted with triazine under basic conditions to yield isoquinolinone (D).

Isoquinolinone (D) can be converted to the corresponding 1-chloroisoquinoline (E) using a chlorinating agent, such as but not limited to phosphorous oxychloride, thionyl chloride, and so forth, in the presence of a tertiary base such as but not limited to triethylamine (TEA), Hiinig's base (diisopropylethylamine or DIPEA), pyridine, and so forth, in an inert solvent such as but not limited to acetonitrile, methylene chloride, and so forth.

1-Chloroisoquinoline (E) can be converted to the corresponding 1- methoxyisoquinoline (F) using for example an alkali methoxide in methanol solution.

1-Methoxyisoquinoline (F) can be converted to sulfmamide (H), for example in a two-step process, wherein (F) is reacted with (G) in the presence of a Lewis acid, such as but not limited to titanium (IV) alkoxide, to generate an N-alkylidene sulfmamide, which can be reduced to sulfmamide (H) using a reducing agent such as but not limited to a borohydride.

Sulfmamide (H) can be alkylated at the sulfmamide nitrogen using a methyl donor, such as but not limited to a methyl halide, triflate, sulfate, mesyalte, or tosylate, to generate chiral intermediate (I), which can be hydrolyzed to the corresponding isoquinolinone (J) using an acid, such as but not limited to hydrochloric acid, sulfuric acid, and so forth. Carbamate (K) can be prepared by reacting 3-cyano-4-fluoroaniline and phenyl chloroformate in the presence of a tertiary base such as but not limited to triethylamine (TEA), Hiinig's base (diisopropylethylamine or DIPEA), pyridine, and so forth.

Compound (J) and compound (K) can be coupled in the presence of a tertiary amine base such as but not limited to triethylamine (TEA), Hiinig's base (diisopropylethylamine or

DIPEA), pyridine, and so forth, to generate compound (X). In certain embodiments, (X) is isolated as free acid/base.

Scheme 1.

In certain embodiments, the at least one compound of the invention is a component of a pharmaceutical composition further including at least one pharmaceutically acceptable carrier.

The compounds contemplated in the invention may possess one or more stereocenters, and each stereocenter may exist independently in either the ( R ) or (S) 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. In certain embodiments, a mixture of one or more isomers is utilized as the therapeutic compound described herein. In other embodiments, compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis and/or separation of a mixture of enantiomers and/or diastereoisomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.

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 ² H, ³ H, ^U C, ¹³ C, ¹⁴ C, ³⁶ C1, ¹⁸ F, ¹²³ I, ¹²⁵ I, ¹³ N, ¹⁵ N, ¹⁵ 0, ¹⁷ 0, ¹⁸ 0, ³² P, and ³⁵ S. In certain embodiments, isotopically-labeled compounds are useful in drug and/or substrate tissue distribution studies. In other embodiments, substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements). In yet other embodiments, substitution with positron emitting isotopes, such as ^U C, ¹⁸ F, ¹⁵ 0 and ¹³ N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. 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.

The compounds described herein, and other related compounds having different substituents are synthesized using techniques and materials described herein and/or as described, for example, in Fieser & Fieser's Reagents for Organic Synthesis, Vol. 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Vol. 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Vol. 1-40 (John Wiley and Sons, 1991), Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989), March, Advanced Organic Chemistry 4 ^th Ed., (Wiley 1992); Carey & Sundberg, Advanced Organic Chemistry, 4 ^th Ed., Vols. A and B (Plenum 2000,2001), and Green & Wuts, Protective Groups in Organic Synthesis 3rd Ed., (Wiley 1999) (all of which are incorporated by reference for such disclosure). General methods for the preparation of compound as described herein are modified by the use of appropriate reagents and conditions, for the introduction of the various moieties found in the formula as provided herein.

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 profiles 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, /V,/V -dibenzyl ethylene- 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 invention are useful within the methods of the invention in combination with one or more additional agents useful for treating 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, 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 vaccine.

(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, ( l//,2//,3//,5//)-3-(6-amino-9//-9-purinyl)-2-fluoro-5- (hydroxymethyl)-4-methylenecyclopentan-l-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 (li?,2i?,3i?,5i?)-3-(6-amino-9F/-9-purinyl)-2-fluoro-5- (hydroxymethyl)-4-methylenecyclopentan-l-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, US 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 ((((!//, 3//, 4//, 5//)-3 -(6-ami no-9//-purin-9-yl)-4-fluoro-5-hydroxy-2- ethylenecyclopentyl) methoxyXphenoxy) phosphoryl)-(D or L)-alaninate and methyl ((((l//,2//,3//,4//)-3-fluoro-2- hydroxy-5-methylene-4-(6-oxo- 1 , 6-dihydro-9//-purin-9-yl)cyclopentyl)m ethoxy Xphenoxy) phosphoryl)-(D or L)-alaninate. Also included are the individual diastereomers thereof, which include, for example, methyl ((//)-((( l//,3//,4//,5//)-3-(6-amino-9//-purin-9-yl)-4-fluoro-5- hydroxy-2-methylenecyclopentyl)methoxy)(phenoxy)phosphoryl)- (D or L)-alaninate and methyl (fV)-(((l//,3//,4//,5//)-3-(6-amino-9//-purin-9-yl)-4-fluoro -5-hydroxy-2- methylenecyclopentyl) methoxy)(phenoxy)phosphoryl)-(D or L)-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, and WO2014089296, 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 Inf 1 Patent Application Publication No. WO 2013144129), AT-61 (see Int'l Patent Application Publication No. WO 1998033501; and King, etal. , 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, 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) Intntu n ostimulators

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-limiting 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 invention. 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 HB V. 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 invention are set forth in published international application PCT/CA2017/050447 (PCT Application Publication number WO/2017/177326, published on October 19, 2017) which is hereby incorporated by reference in its entirety.

(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.

Methods

The current disclosure, in one aspect, provides a method of treating or preventing hepatitis virus infection in a subject. In certain embodiments, the infection comprises hepatitis B virus (HBV) 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 invention. 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 infection. In yet other embodiments, the at least one additional agent comprises at least one selected from the group consisting of reverse transcriptase inhibitor; capsid inhibitor; cccDNA formation inhibitor; RNA destabilizer; oligomeric nucleotide targeted against the HBV genome; immunostimulator, such as checkpoint inhibitor (e.g, PD- L1 inhibitor); GalNAc-siRNA conjugate targeted against an HBV gene transcript; and therapeutic vaccine. 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 invention further provides a method of inhibiting expression and/or function of a viral capsid protein either directly or indirectly in a 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 invention. In 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 HBV infection. In yet other embodiments, the at least one additional agent comprises at least one selected from the group consisting of reverse transcriptase inhibitor; capsid inhibitor; cccDNA formation inhibitor; RNA destabilizer; oligomeric nucleotide targeted against the HBV genome; immunostimulator, such as checkpoint inhibitor (e.g, PD-L1 inhibitor); GalNAc-siRNA conjugate targeted against an HBV gene transcript; and therapeutic vaccine. 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 a mammal. In other embodiments, the mammal is a human.

Pharmaceutical Compositions and Formulations

The invention provides pharmaceutical compositions comprising at least one compound of the invention or a salt or solvate thereof, which are useful to practice methods of the invention. Such a pharmaceutical composition may consist of at least one compound of the invention 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 invention 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 invention 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 invention 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 invention 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 invention 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 invention 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 invention 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 invention 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 invention 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 invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of at least one compound of the invention 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., RECOMB UMIN®), 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 invention 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 invention 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 invention 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 invention 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 invention 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 (i.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 invention 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 invention 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 invention 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 invention 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 invention 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 invention 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 invention 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 invention 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 invention 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 invention for administration may be in the range of from about 1 pg to about 7,500 mg, about 20 pg to about 7,000 mg, about 40 pg to about 6,500 mg, about 80 pg to about 6,000 mg, about 100 pg to about 5,500 mg, about 200 pg to about 5,000 mg, about 400 pg to about 4,000 mg, about 800 pg 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 invention is from about 0.5 pg and about 5,000 mg. In some embodiments, a dose of a compound of the invention 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 invention is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the invention, 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, /. 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 invention 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 invention 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 invention 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, corn 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 invention 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 invention, and a further layer providing for the immediate release of one or more compounds useful within the methods of the invention. 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 invention 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 corneum layer of the epidermis. The stratum corneum is a highly resistant layer comprised of protein, cholesterol, sphingolipids, free fatty acids and various other lipids, and includes cornified and living cells. One of the factors that limit the penetration rate (flux) of a compound through the stratum corneum 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, or N-methyl-2-pyrrolidone.

One acceptable vehicle for topical delivery of some of the compositions of the invention may contain liposomes. The composition of the liposomes and their use are known in the art (z.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 corneum 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 corneum, and thus allows increased transport across the stratum corneum. 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 invention 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 micrometers, 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 invention 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 invention 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 invention 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 invention also include dosage forms as described in U.S. Patent Applications Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Additional dosage forms of this invention 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 invention 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 invention 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 invention, the compounds useful within the invention 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 invention 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.

The following examples further illustrate aspects of the present invention. However, they are in no way a limitation of the teachings or disclosure of the present invention as set forth herein.

EXAMPLES

The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.

Methods of Characterization X-ray Powder Diffraction (XRPD)

Powder X-ray diffraction was carried out with a Stoe Stadi P diffractometer equipped with a Mythen IK detector operating with Cu-Kai radiation. The measurements with this instrument were performed in transmission at a tube voltage of 40 kV and 40 mA tube power. A curved Ge monochromator allows testing with Cu-K _ai radiation. The following parameters were set: 0.02° 20 step size, 12 s step time, 1.5-50.5° 20 scanning range, and 1°20 detector step (detector mode in step scan). For a typical sample preparation, about 10 mg of sample was placed between two acetate foils and mounted into a Stoe transmission sample holder. The sample was rotated during the measurement. All sample preparation and measurement was done in an ambient air atmosphere. Thermogravimetric Infrared Spectroscopy (TG-FTIR)

Thermogravimetric measurements were carried out with a Netzsch Thermo- Microbalance TG 209 coupled to a Bruker FTIR Spectrometer Vector 22 (sample pans with a pinhole, N2 atmosphere, heating rate 10 K/min).

Microscopy

Light microscopy was performed on a Leitz Orthoplan polarized microscope part #130880, generally a 10 x 10 magnification was applied.

Nuclear Magnetic Resonance (' H-NMR)

Compounds of the present disclosure were characterized by ¹ H-NMR spectroscopy using at least one NMR spectrometer having an operating frequency selected from the group consisting of 300 MHz, 400 MHz, and 600 MHz; solvent peak used for referencing; chemical shifts reported on the TMS scale.

Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry was carried out with a TA Instruments Q2000 instrument (sample pan with a pinhole in the lid, heating rate 10 K/min). The melting point is understood as the peak maximum.

Dynamic Vapor Sorption (DVS)

DVS measurements were performed with an SPS11-lOOn "Sorptions Priisystem" from ProUmid (formerly "Projekt Messtechnik"), August-Nagel-Str. 23, 89079 Ulm (Germany). About 5-20 mg of sample were put into an aluminum sample pan. Humidity change rates of 5% per hour were used.

The sample was placed on an aluminum or platinum holder on top of a microbalance and allowed to equilibrate at 50% RH before starting the pre-defmed humidity programs:

2 h at 50% RH

50 to 95% RH (5%/h); 5 h at 95% RH 95 to 0% RH (5%/h); 5 h at 0% RH 0 to 95% RH (5%/h); 5 h at 95% RH 95 to 50% RH (5%/h); 2 h at 50% RH

The hygroscopicity was classified based on the mass gain at 85% RH relative to the initial mass as follows: deliquescent (sufficient water adsorbed to form a liquid), very hygroscopic (mass increase of >15%), hygroscopic (mass increase <15% but >2%), slightly hygroscopic (mass increase <2% but >0.2%), or non-hygroscopic (mass increase <0.2%).

Raman Spectroscopy

FT-Raman spectra were recorded on a Bruker MultiRAM FT-Raman system with a near infrared Nd:YAG laser operating at 1064 nm and a liquid nitrogen-cooled germanium detector. 64 scans with a resolution of 2 cm ¹ were accumulated in the range from 3500 to - 50 cm ¹ ; however, only data above 100 cm ¹ are evaluated due to filter cutoff effects.

Nominal laser powers are typically 100 or 300 mW.

Solubility

Approximate solubility was determined by incremental addition of solvent to about 10 mg of the compound. If the substance was not dissolved by addition of a total of at least 10 mL solvent, the solubility is indicated as <1 mg/ml. Due to the experimental error inherent in this method, the solubility values are intended to be regarded as rough estimates and were used solely for the design of crystallization experiments.

Example 1: Synthesis of (X)

Step 1 — Synthesis of 4,5-difluoro-2-(2-oxopropyl)benzoic acid (B)

To a suspension of 2-bromo-4,5-difluorobenzoic acid (compound A, 400 g, 1.0 eq), CuBr (24 g, 0.1 eq), and acetylacetone (338 g, 2.0 eq) in EtOH (4 L, 10 vol) was added a solution of 21% NaOEt in EtOH (1.75 L, 3.0 eq) dropwise. The mixture was refluxed at 80 °C for 14 h. A LCMS sample of reaction mixture showed one main peak with the desired mass. The mixture was cooled to room temperature (rt), filtered through CELITE®, and washed with a small amount of EtOH. The filtrate was concentrated in vacuo. The residue was diluted with dichloromethane (DCM, 15 vol) and washed with 2 N HC1 (5 vol, pH=2); the aqueous layer was back extracted with DCM (2 x 600 mL). The combined organic layer was dried over Na2S04, filtered, and concentrated in vacuo. The crude product was triturated with hexane (3 vol), filtered, and dried to afford 330 g (90.9% yield) of compound (B). 'H NMR (400 MHz, Chloroform- ) d 8.04 - 7.92 (m, 1H), 7.04 (t, J= 8.9 Hz, 1H), 4.09 (s, 2H),

2.27 (s, 3H).

Step 2 — Synthesis of 6,7-difluoro-3-methyl-lH-isochromen-l-one (C)

B C

The above 4,5-difluoro-2-(2-oxopropyl)benzoic acid (compound B, 330 g, 1.0 eq) was dissolved in DCM (3.3 L, 10 vol) and cone. H2SO4 (33 mL) was slowly added at rt. The reaction was stirred at rt for 14 h. A LCMS of the reaction mixture showed 1 main peak with desired product mass. The reaction was cooled to 5 °C and saturated NaHCCb (5 vol) was slowly added (exothermic gas release observed). The mixture was stirred at rt 30 min until off-gassing subsided. The phases settled and were separated. The organic layer was washed with brine. The organic layer was dried over Na2S04, filtered, and concentrated. The crude product was triturated with hexane (3 vol), filtered, and dried to afford 274 g (90.7% yield) of 6,7-difluoro-3-methyl-lH-isochromen-l-one (C) as a white solid. ¾ NMR (400 MHz, Chloroform - ) d 8.02 (dd, J= 10.0, 7.9 Hz, 1H), 7.12 (dd, J= 10.1, 7.1 Hz, 1H), 6.20 (s, 1H),

2.28 (s, 3H).

Step 2 ' — Synthesis of 6, 7-difluoro-3-methyl-lH-isochromen-l-one (C) and methyl 4,5- difluoro-2- (2-oxopropyl) benzoate (C)

Compound B (221 mg) was dissolved in MeOH (3 mL) and cone. H2SO4 (1 mL) was slowly added at rt. The reaction was heated at 60 °C overnight. LCMS of the reaction mixture showed two main peaks, one having the desired product mass. The reaction was cooled to rt and slowly added to a mixture of MeOH (10 mL) and saturated NaHC03 (10 mL) at 0 °C (exotherm and vigorous off-gassing observed). The mixture was stirred 30 min until off-gassing subsided. The mixture was concentrated in vacuo until mostly water remained. The suspension was dissolved in EtOAc and water. The phases were split and the aqueous was back extracted with EtOAc twice. The organic layers were combined, dried over Na2S04, filtered, and concentrated to afford a yellow semi-solid/oil. The crude product was loaded onto a 4 g silica gel column and eluted with 0-100% EtOAc in hexanes. Pure product fraction (second peak off ISCO) was concentrated to afford 63 mg (48% yield) methyl 4,5- difluoro-2-(2-oxopropyl)benzoate (C) as a clear, yellow oil. 'H NMR (400 MHz,

Chloroform - ) d 7.88 (dd, J= 11.0, 8.2 Hz, 1H), 6.99 (ddd, J= 10.6, 7.5, 0.4 Hz, 1H), 4.07 (s, 2H), 3.84 (s, 4H), 2.30 (s, 3H). The first peak fraction off the ISCO was concentrated to afford 38 mg (29% yield) of 6, 7-difluoro-3 -methyl -///-isochromen-1 -one (C) as a white solid. LCMS: m/z: C10H5F2O2: M+l calc.: 197.04 found: 197.05 along with 'H NMR appeared to afford 6,7-difluoro-3-methyl-lH-isochromen-l-one. ¾ NMR (400 MHz, Chloroform - ) d 8.03 (dd, J= 10.0, 7.9 Hz, 1H), 7.12 (dd, J= 10.1, 7.1 Hz, 1H), 6.20 (s, 1H), 2.29 (s, 3H).

Step 3 — Synthesis of 4-acetyl-6, 7-difluoroisoquinolin-l (2H)-one (D)

To a solution of 6,7-difluoro-3-methyl-lH-isochromen-l-one (compound C, 280 g,

1.0 eq) in 2-methyltetrahydrofuran (MeTHF, 2.8 L, 10 vol) was added K2CO3 (749 g, 3.8 eq) and the system stirred at rt. Formamide (193 g, 3.0 eq) was added dropwise over a period of 30 min, and an exothermic reaction was observed. The reaction mixture was heated at 55-60 °C for 14 h. A LCMS sample of reaction mixture showed one major peak with the desired product mass. The reaction mixture was cooled to 25 °C and filtered. The solid was slurried with cold water (10 vol) at 0 °C and then the pH was slowly adjusted over 1.5 h to pH 6 using 2 N HC1 (4.3 L). The light brown slurry was stirred for 1 h, filtered and washed with water, cold MeOH, and hexane, and dried to afford 200 g (63% yield) of 4-acetyl-6,7- difluoroisoquinolin-l(2H)-one (D) as an off-white solid. 'H NMR (400 MHz, DMSO-r/r,) d 12.19 (s, 1H), 8.92 - 8.81 (m, 1H), 8.24 (s, 1H), 8.12 - 8.02 (m, 1H), 2.48 (s, 3H). Note: Triazine can be used in place of formamide; alternative bases such as NaOMe may be used; and solvents such as MeOH may be used.

Step 3 ' — Synthesis of 4-acetyl-6, 7-difluoroisoquinolin-l (2H)-one (D)

To a solution of methyl 4,5-difluoro-2-(2-oxopropyl)benzoate (compound C, 63 mg, 0.28 mmol) and triazine (27 mg, 0.33 mmol) in MeOH (2 mL) was added a solution of 25% NaOMe in MeOH (298 mg, 1.38 mmol). The mixture was stirred at rt for 1 h.

LCMS sample of reaction mixture showed one major peak with the desired product mass. After 2 h, the reaction was quenched with 5 mL 10% ammonium chloride solution, and stirred for 15 min. The mixture was concentrated in vacuo to remove MeOH. The slurry was then dissolved with EtOAc and water. The phases were separated, and the aqueous was back extracted with EtOAC. The combined organic phase was dried over Na2S04, filtered, and concentrated to afford 58.6 mg (95% yield) of 4-acetyl-6,7-difluoroisoquinolin-l(2H)-one (D) as an off-white solid.

Step 3" — Synthesis of 4-acetyl-6, 7-difluoroisoquinolin-l (2H)-one (D)

To a solution of 6,7-difluoro-3-methyl-lH-isochromen-l-one (compound C, 38 mg, 0.19 mmol) and triazine (19 mg, 0.23 mmol) in MeOH (1 mL) was added a solution of 25% NaOMe in MeOH (209 mg, 0.97 mmol). The mixture was stirred at rt for 1 h.

LCMS sample of reaction mixture showed one major peak with the desired product mass. After 2 h at rt, the reaction was quenched with 5 mL 10% ammonium chloride solution, and stirred for 15 min. The mixture was concentrated in vacuo to remove MeOH. The slurry was then dissolved with EtOAc and water. The phases were separated and the aqueous was back extracted with EtOAc. The combined organic phase was dried over Na2S04, filtered, and concentrated to afford 32.3 mg (75% yield) of 4-acetyl-6,7-difluoroisoquinolin-l(2H)-one (D) as an off-white solid.

Step 4 — Synthesis of l-(l-chloro-6,7-difluoroisoquinolin-4-yl)ethan-l-one (E)

D E

To a suspension of 4-acetyl-6,7-difluoro-2H-isoquinolin-l-one (compound D, 650 g, 2.91 mol) in acetonitrile (ACN, 11 L, 17 V) was added triethylamine (TEA, 406 mL, 2.95 mol, 1.00 eq) at it. POCb (327 mL, 3.50 mol, 1.20 eq) was added dropwise slowly, at which time an exotherm was observed from 25 to 32 °C, and jacket cooling was applied. After the addition was completed, the reaction mixture was heated to internal batch temperature of 75 °C for 18 h. The reaction was sampled by HPLC, showing 0.92% starting material, 97.22% desired product, and 0.74% of a later-eluting peak (assumed to be an over-chlorination by product). The reaction was cooled to 40 °C, and very slowly quenched with 6.6 L water.

The reaction was stirred at 40 °C for 1 h. The reaction mixture was cooled to 20 °C and stirred for 1 h, then cooled again to 0 °C and aged for 1 h at 0 °C. The slurry was filtered.

The solid was washed with 2 L water followed by 1.5 L ACN chilled to 0 °C. The solid was dried under vacuum in an oven at 40-45 °C overnight to afford 660 g (94% yield) of compound (E) as a tan solid. ¾ NMR (400 MHz, Chloroform- ) d 8.95 (dd, J= 12.4, 8.0 Hz, 1H), 8.85 (s, 1H), 8.19 (dd, J= 10.5, 8.0 Hz, 1H), 2.77 (s, 3H). Note: Equivalents of POCb, addition of base, and temperature are important parameters. Larger amounts of POCb and higher temperatures resulted in high levels of impurities.

Step 5 — Synthesis of l-(6,7-difluoro-l-methoxyisoquinolin-4-yl)ethan-l-one (F)

E F To a slurry of l-(l-chloro-6,7-difluoro-4-isoquinolyl)ethenone (compound E, 965 g, 3.99 mol) in 14.5 L (15 V) MeOH and 14.5 L (15 V) THF was added a solution of 25% NaOMe in MeOH (1.29 L, 5.99 mol, 1.5 eq) slowly, maintaining internal batch temp at 0-3 °C. The solids did not completely dissolve, but yielded a yellow slurry. The reaction mixture was stirred at 0 °C. The reaction was sampled by HPLC after 1 h, at which time the desired product was observed as major peak, with 1.1% di- substituted impurity (dimethoxy impurity and SM co-eluted on the HPLC method initially used). After no more than 2 h, the reaction was slowly quenched with a solution of 10% citric acid in water (14 L). An exotherm was observed and the white slurry was stirred for 1 h at 0 °C. The slurry was filtered and solids were washed with water to provide a wet cake. The wet cake was dried under vacuum in an oven at 35-40 °C for 64-80 h to afford 881 g (93% yield) of compound (F) as an off-white fluffy solid. ¾NMR (400 MHz, Chloroform- ) d 9.01 (dd, J= 13.1, 8.2 Hz, 1H), 8.74 (s, 1H), 8.02 (dd, J= 10.6, 8.3 Hz, 1H), 4.19 (s, 3H), 2.70 (s, 3H).

Step 6 — Preparation of (R)-N-((R)-l-(6,7-difluoro-l-methoxyisoquinolin-4-yl)ethyl)- 2- methylpropane-2-sulfinamide (H)

A reactor was charged with (A)-2-methylpropane-2-sulfmamide (compound G, 495.1 g, 4085.0 mmol, 1.9 eq), l-(6,7-difluoro-l-methoxyisoquinolin-4-yl)ethan-l-one (compound F, 510.0 g, 2150.0 mmol, 1.0 eq), MeTHF (2.6 L, 5.0 V), and Ti(OEt) (1471.3 g, 6450.0 mmol, 3 eq). The mixture was heated to 82 °C and agitated. After 1.5 h the reaction mixture was concentrated to about 3 V. Fresh MeTHF (2 V) was charged and heating was continued. This process was repeated twice over the next 3-5 h until the reaction was deemed complete by HPLC, wherein no more than (NMT) 8% of residual level of (F) was observed by HPLC. The reaction mixture was adjusted to 50 °C, MeTHF (5.1 L, 10 V), and EtOH (495.3 g, 5.0 eq) were charged and then cooled to -18 °C. NaBH ₄ (81.3 g, 1.0 eq) was charged to the reactor in 10 portions over 1 h while maintaining a temperature between -18 °C and -15 °C. The mixture was agitated for about 4 h until the reaction was deemed complete, with NMT 1% of the imine intermediate observed by HPLC. In another reactor, a sodium citrate solution was prepared by charging the reactor with citric acid monohydrate (1672.8 g, 3.28 S), purified water (8.0 L, 15.72 V), and sodium hydroxide (255 g, 0.5 S). The sodium citrate solution was cooled to 10 °C and the reaction mixture transferred into the sodium citrate solution, with agitation, while maintaining a temperature between 10 °C and 16 °C. The mixture was then adjusted to 25 °C and agitated for about 18 h. Layers were separated, the organic layer was washed successively with purified water (2.5 L), a 8.0 wt% NaHCCh (2. 5 L) solution, and a 24.0 wt% brine solution (2.5 L). The organic layer was dried over Na2SC>4 and filtered. The filtrate was evaporated to about 2 V while maintaining an internal temperature of about 30 °C to 36 °C. The residue was solvent swapped with cyclopentyl methyl ether (CPME, 1.5 L, 3 V) twice. To the resulting residue was slowly charged n- heptane (3.0 L, 6 V) and the mixture heated to 50 °C to 60 °C for 2 h, cooled to 20 °C to 30 °C over 2 h, agitated at this temperature for 2 h, then filtered and washed with n-heptane (3 V). Solids were dried at 45 °C to obtain compound (H) (584.6 g, 79.4%) as light yellow solid. The diastereomer ratio (dr) of compound (H) obtained was 97:3. m/z 343.2 [M+H] ⁺ . ¾NMR (400 MHz, Chloroform-i/) d 8.11 - 7.99 (m, 2H), 7.91 (dd, J= 12.0, 7.6 Hz, 1H), 5.03 - 4.93 (m, 1H), 4.11 (s, 3H), 3.48 (s, 1H), 1.69 (d, J= 6.6 Hz, 3H), 1.23 (s, 9H). Alternately crude material of compound H may be used in the next step without further purification.

Step 7 — Preparation of (R)-N-((R)-l-(6,7-difluoro-l-methoxyisoquinolin-4-yl)ethyl)- N,2- dimethylpropane-2-sulfinamide (I)

A reactor was charged with compound (H) (480 g, 1401.9 mmol) and MeTHF (6.72 L, 14 V). The mixture was agitated to obtain a clear solution and then cooled to -6 °C. KOH powder (463.0 g, 7009.35 mmol, 5.0 eq) was charged and agitated for 30 min. Mel (458.0 g, 3224.3 mmol, 2.3 eq) was slowly charged over 1 h while maintaining a temperature below 0 °C. The reaction mixture was agitated at this temperature for 30 min and then warmed to 20 °C to 25 °C. The reaction mixture was agitated for 13 h until the reaction was deemed complete, wherein NMT 1% of compound (H) was observed by HPLC. The reaction mixture was cooled to 0 °C to 5 °C, and purified water (3.84 L, 8 V) was slowly charged over 2 h.

The mixture was warmed to 20 °C to 25 °C and agitated for 10 min. The aqueous layer was separated and back extracted with MeTHF twice (2.4 L, 5 V). The organic layers were pooled and washed sequentially with a 5.0 wt% brine solution (2.4 L, 5 V), then purified water (2.4 L, 5 V). The organic layer was dried over anhydrous Na2SC>4 and filtered. The filtrate was concentrated to about 3 V and solvent swapped twice with MTBE (3.84 L, 8 V) to about 2 V. MTBE was charged to the residue to adjust the volume to 3 V, then the mixture was heated to 45 °C. Next, n-heptane (2.88 L, 6V) was charged, and the mixture was heated at 45 °C for 1 h, then slowly cooled to 0 °C over 2 h. Solids were filtered and washed with 10% MTBE/n-heptane (1.44 L, 3 V). Solids were dried at 45 °C to obtain compound I (386.6 g, 77.5 %) as off-white solid. The diastereomeric ratio (dr) of compound (I) obtained was 99.5:0.5. m/z 357.2 [M+H] ⁺ . ¾NMR (400 MHz, Chloroform-d) d 8.08 - 7.97 (m, 2H), 7.72 (dd, J = 12.0, 7.6 Hz, 1H), 4.68 (q, J = 6.9 Hz, 1H), 4.11 (s, 3H), 2.66 (s, 3H), 1.75 (d, J = 6.9 Hz, 3H), 1.65 (s, 1H), 0.98 (s, 9H). Compound I can be alternately crystallized from isopropyl acetate (IP Ac), EtOAc, and/or acetonitrile.

Step 8 — Preparation of (R)-6,7-difluoro-4-(l-(methylamino)ethyl)isoquinolin-l(2H)-o ne

(J)

A reactor was charged with methanol (2.04 L, 6.0 V) and cooled to 0 °C. To the methanol was charged acetyl chloride (599.1 g, 448.9 mmol, 8.0 eq) while maintaining temperature below 20 °C. This mixture was agitated at 20 °C for 1 h. In another reactor, compound (I) (340.0 g, 56.11 mmol, 1.0 eq) and methanol (2.72 L, 8.0 V) were charged, agitated for 10 min and then cooled to 5 °C. Methanolic HC1 was slowly transferred to the solution of compound (I) while maintaining temperature below 30 °C. The reaction mixture was heated at 60 °C for 4 h until reaction was deemed complete, wherein NMT 1% of compound (I) was observed by HPLC. The reaction mixture was cooled to 35 °C and concentrated to 3 V and solvent swapped with MeTHF (3.4 L, 10 V) and reduced to 2.5 V.

To the resulting residue were charged MeTHF (3.4 L, 10 V) and purified water (3.4 L, 10 V), then the system was agitated for 30 min, and the organic layer was separated. The aqueous layer was washed with MTBE (1.7 L, 5 V) and adjusted to -pH 7.4 by slow addition of NaHCCb (205.6 g, 2.85 eq). The aqueous layer was agitated for 1 h, then 0.1 L of THF was charged, and agitation was continued at 20 °C for 14 h. The resulting solids were filtered, washed with purified water (610 mL, 2 V), and dried at 45 °C. Compound (J) (177.0 g, 86.5%) was obtained as beige solid m/z 239.1 [M+H] ⁺ . ¾ NMR (400 MHz, DMSO-d6) d 11.84 (s, 1H), 9.35 (s, 1H), 8.17 - 8.07 (m, 2H), 7.64 (d, J = 4.5 Hz, 1H), 4.75 (q, J = 6.8 Hz, 1H), 2.48 (s, 3H), 1.57 (d, J = 6.7 Hz, 3H).

Step 9 — Preparation of (R)-3-(3-cyano-4-fluorophenyl)-l-(l-(6,7-difluoro-l-oxo-l,2- dihydroisoquinolin-4-yl)ethyl)-l-methylurea (X)

Compounds (J), (K), and MeTHF were charged into a reactor. After adjusting to 15- 25 °C, triethylamine was charged. Then, the reaction mixture was heated to 50 °C (45-55 °C) and agitated for NLT 6 h. Once the reaction was complete, the temperature was adjusted to 15 °C (10-20 °C) and 1 N HC1 solution was slowly charged to the reaction mixture while maintaining an internal temperature of 10-20 °C. After phase separation, the organic layer was washed sequentially with additional 1 N HC1 solution, 1 N NaOH solution (x 2), purified water, 8 wt % aqueous NaHC03 solution, and purified water. Then, the organic layer was concentrated to 4.5-5.5 V while maintaining a jacket temperature of NMT 60 °C. After charging with MeTHF, the concentration was repeated to 8-10 V. The contents were filtered via a cartridge filter and concentrated to 4.5-5.5 V while maintaining a jacket temperature of NMT 60 °C. After additional concentration with MeTHF, the solvent swap with EtOAc was performed while maintaining an internal temperature of 30-55 °C. After adjusting to 4.5-6.0 V (target 5 V) by charging EtOAc, the slurry was heated to 50 °C (45-55 °C) and agitated at the same temperature for NLT 2 h. Then, the contents were slowly adjusted to 15 °C (10-20 °C) for NLT 3 h and agitated for NLT 3 h. The resulting slurry was filtered, and the wet cake was washed with EtOAc. After deliquoring, the filtered cake was dried at NMT 55 °C under vacuum to afford compound (X) in 75-90% yield as a white to pale tan solid. 'H NMR (400 MHz, DMSO- e) d 11.61 (d, J= 5.6 Hz, 1H), 8.63 (s, 1H), 8.06 (dd, J= 10.9, 8.5 Hz, 1H), 7.99 (dd, J= 5.8, 2.7 Hz, 1H), 7.85 (ddd, J= 9.3, 4.8, 2.8 Hz, 1H), 7.66 (dd, J= 12.7, 7.5 Hz, 1H), 7.42 (t, J= 9.1 Hz, 1H), 7.20 (d, J= 5.7 Hz, 1H), 5.75 (q, J= 6.8 Hz, 1H), 2.60 (s, 3H), 1.41 (d, J= 6.8 Hz, 3H).

Example 2: Preparation of phenyl (3-cyano-4-fluorophenyl)carbamate (K)

5-Amino-2-fluorobenzonitrile and MeTHF were charged into a reactor and the temperature was adjusted to 15-25 °C. Then, pyridine was charged at 15-25 °C. After cooling to a temperature of -10 °C to -5 °C, phenyl chloroformate was slowly charged to the reactor while maintaining an internal temperature of NMT 0 °C. After adjusting to 0 °C (-5 °C to 5 °C), the reaction mixture was agitated at 0 °C (-5 °C to 5 °C) for NLT 1 h until the reaction was complete. Purified water was slowly charged to the reaction mixture while maintaining an internal temperature of NMT 15 °C. After charging EtOAc, the contents were adjusted to 15-25 °C and agitated for NLT 10 minutes. After phase separation, the aqueous layer was back extracted with EtOAc. The combined organic layer was washed sequentially with a 1 N HC1 solution (x 2), then a 5 wt.% NaCl solution. The organic layer was concentrated to 4.5-5.5 V while maintaining an internal temperature of NMT 35 °C. After charging EtOAc, the contents were concentrated to 4.5-5.5 V once again. After filtration via a cartridge, the contents were concentrated to 1-2 V. After adjusting the volume of contents to approximately 2.5 V by charging EtOAc, the contents were adjusted to 40 °C (35-45 °C). Then, n-heptane was slowly charged to the contents while maintaining an internal temperature of 35-45 °C. After agitation at 35-45 °C for NLT 0.5 h, the contents were slowly adjusted to 15-20 °C over 1-2 h, and agitated at the same temperature for NLT 2 h. Then, the product slurry was filtered, and the wet cake was washed with 10 v/v% EtOAc in n-heptane. After deliquoring, the filtered cake was dried at NMT 40 °C under vacuum to afford compound (K) in 75-95% yield as an off-white to tan solid. ¾ NMR (400 MHz,

Chloroform - ) d 7.79 (dd, J = 5.5, 2.9 Hz, 1H), 7.65 (dt, J = 9.2, 3.6 Hz, 1H), 7.46 - 7.36 (m, 2H), 7.32 - 7.18 (m, 3H), 7.18 - 7.14 (m, 1H), 7.12 (s, 1H).

Example 3: Purification and Crystallization of (X) Form 1 To a suspension of (f?)-6,7-difluoro-4-(l-(methylamino)ethyl)isoquinolin-l(2F/) -one hydrochloride (0.91 g, 3.31 mmol, 1.0 eq.) in 27 mL of THF was added triethylamine (0.97 mL, 6.94 mmol, 2.1 eq) followed by phenyl N-(3-cyano-4-fluoro-phenyl)carbamate (0.85 g, 3.31 mmol, 1.0 eq). The reaction was stirred at rt for 5 min and then heated to 50 °C for 5 h. When the reaction was complete the mixture was concentrated in vacuo and the oil was diluted with EtOAc. The organic layer was washed with 0.2 N HC1 solution and the aqueous was back extracted with EtOAc. The combined organic layer was washed with NaHC03 solution, dried over Na2S04, filtered, and concentrated to an oil. The oil was diluted with 3 mL DCM at which point the product began to precipitate. The isolated solids were combined with 2 smaller batches prepared by the same method and dissolved in 10% MeOH in DCM. The solvent was removed in vacuo to afford 1.99 g (75.6% combined yield) compound (X) after drying at 50 °C in a vacuum oven for 24 h. LCMS: m/z found 401.10 [M+H] ⁺ . 'H NMR (400 MHz, DMSO-ifc): d 11.61 (d, J= 5.6 Hz, 1H), 8.64 (s, 1H), 8.06 (dd, J= 10.9, 8.5 Hz, 1H), 7.99 (dd, J= 5.8, 2.7 Hz, 1H), 7.85 (ddd, J= 9.3, 4.8, 2.8 Hz, 1H), 7.66 (dd, J= 12.7,

7.5 Hz, 1H), 7.42 (t, J= 9.1 Hz, 1H), 7.20 (d, J= 5.7 Hz, 1H), 5.75 (q, J= 6.8 Hz, 1H), 2.60 (s, 3H), 1.41 (d, J= 6.8 Hz, 3H).

Example 4: Characterization of (X) Crystalline Form 1

Initial characterization was performed on dichlorom ethane solvate crystalline Form 1, including polarized light microscopy (PLM), x-ray powder diffraction (XRPD), thermoanalytical characterization (TG-FTIR), dynamic vapor sorption (DVS), and solubility studies.

Polarized light microscopy analysis shows that the particles are crystalline, very small and partially agglomerated (FIG. 1). Hot-stage microscopy was performed by heating from 25 °C to 250 °C at 10 K/min. Optical images were collected at several temperatures (FIGs. 2A-2K). The observations made in this experiment suggest that the sample undergoes desolvation and loses crystallinity in the temperature range from about 140 °C to 158 °C. However, between 170 °C and 193 °C, the sample seems to recrystallize before melting between 217 °C to 222 °C. Thus, a phase transformation to a desolvated form may have occurred.

The XRPD analysis of Form 1 confirmed the crystalline nature of the starting material (FIG. 3). The peak intensities are low and the peaks are fairly broad, potentially indicating poor crystallinity, however these features may be attributable to the small particle sizes of the sample and not be inherent to the solvate itself. Table 1. XRPD Peak Table for Form 1

Form 1 was further subjected to Hot-stage XRPD, wherein the sample was heated from 25 °C to 185 °C at 10 K/min and XRPD patterns were collected at several temperatures (FIG. 4). While the temperature was varied between measurements, the temperature was kept constant for the duration of each XRPD measurement. XRPD patterns were collected at 160 °C, 165 °C, 170 °C, 180 °C, and 185 °C. At higher temperatures, the sample color changed from white to brown. Furthermore, overlaid XRPD patterns demonstrate that Form 1 had converted to Form 2 when the temperature reached 160 °C. At higher temperatures (>170 °C), the sample began to lose crystallinity and melt.

Thermogravimetry coupled with FT-IR spectroscopy was performed on Form 1. The TG-FTIR shows that the sample contains about 10% DCM, which corresponds well to the stoichiometric amount for a hemi-solvate (FIG. 5). The analysis further suggests that the sample decomposes at temperatures above about 200 °C. Informed of the high solvent content of Form 1 by TG-FTIR, DSC was carried out in a sample pan with a pin hole, and a drying step was introduced in order to obtain a solvent-free sample. DSC revealed a glass transition near 130 °C with a ACp of about 0.4 J/(g-K) which is typical for an amorphous pharmaceutical compound (FIG. 6). At higher temperatures, a small part of a crystalline fraction seems to melt and the exothermic signal might correspond to a recrystallization.

Dynamic vapor sorption (DVS) studies indicated that Form 1 lost almost all DCM during the first cycle (FIG. 7 and FIG. 8). It is unlikely that all DCM was replaced by water at the end of the test. The XRPD patterns before and after the DVS experiments are rather similar and a substantial degree of isomorphism is suggested. A conversion from a solvate to a hydrate occurred, and this new form was designated as Form 9. The most pronounced difference is the appearance of a strong reflection at 4.0° 2Q (FIG. 9). Additionally, the TG- FTIR of the sample after DVS (Form 9) confirms the replacement of the dichlorom ethane with water, and the water content was about 3.8% (FIG. 10). This result may indicate that the starting material is likely a channel solvate. The DCM was completely removed during the measurement.

Approximate solubility was determined for Form 1 at room temperature. These values were obtained by addition of small aliquots of solvent to approximately 10 mg of solid to achieve dissolution by shaking and/or sonicating for a short period of time. The maximum amount of solvent that was used for these determinations was 10 mL. It is noted that these values are only approximations and do not necessarily correspond to the thermodynamic solubility values.

Table 2. Solubility studies of Form 1 of (X)

Example 5: Purification and Crystallization of (X) Form 2

A reactor was charged with compound (J) (1.0 kg), compound (K) (1.0 kg, 0.93 eq.), and 2-MeTHF (25.2 kg). The reactor was further charged with triethylamine (0.47 kg, 1.1 eq.) at 15-25 °C, and an additional 0.43 kg of 2-MeTHF was used to rinse the line used. The reactor contents were heated to 50 °C (45-55 °C) for no less than 6 h. The reaction was monitored by HPLC and considered complete when no more than 2% (J) remained, and no more than 0.5% (K) remained.

The contents of the reactor were cooled to an internal temperature of 15 °C (10-20 °C) and the internal temperature was maintained as the reactor was slowly charged with a 1 N HC1 solution (5.1 kg). The contents of the reactor were stirred at 10-20 °C, then agitation was stopped, the phases were separated, and the bottom aqueous layer was discarded. The reactor was charged again with a 1 N HC1 solution (5.1 kg), while the temperature was maintained at 10-20 °C. The contents of the reactor were stirred at 10-20 °C, then agitation was stopped, the phases were separated, and the bottom aqueous layer was discarded. Next, the reactor was charged with a 1 N NaOH solution (5.2 kg), while the temperature was maintained at 10-25 °C. The contents of the reactor were agitated at 20-25 °C, then agitation was stopped, the phases were separated, and the bottom aqueous layer was discarded. The reactor was again charged with a 1 N NaOH solution (5.2 kg), while the temperature was maintained at 10-25 °C. The contents of the reactor were agitated at 20-25 °C, then agitation was stopped, the phases were separated, and the bottom aqueous layer was discarded. Next, the reactor was charged with purified water (5.0 kg). The reactor was agitated, then agitation was stopped, the phases were separated, and the bottom aqueous phase was discarded. Next, the reactor was charged with a NaHC03 solution (5.3 kg, 8 wt.% aq.), while the temperature was maintained at 20-25 °C. The contents of the reactor were agitated at 20-25 °C, then agitation was stopped, the phases were separated, and the bottom aqueous layer was discarded. Next, the reactor was charged with purified water (5 L), agitated, then agitation was stopped, the phases were separated, and the final aqueous layer was discarded.

The organic phase was concentrated under vacuum to 4.5-5.5 L while maintaining a jacket temperature of no more than 60 °C. Next, the organic phase was charged with 2- MeTHF (8.84 kg). The material was concentrated again under vacuum to 8-10 L while maintaining a jacket temperature of no more than 60 °C. The organic material was Polish filtered via a cartridge filter and the filtrate was transferred to a reactor. All lines and the filter were rinsed with 2-MeTHF (0.85 kg).

The organic phase was concentrated under vacuum to 4.5-5.5 L while maintaining a jacket temperature of no more than 60 °C, then 2-MeTHF (4.27 kg) was added. The organic phase was concentrated under vacuum to 4.5-5.5 L while maintaining a jacket temperature of no more than 60 °C, then 2-MeTHF (4.27 kg) was added. The temperature of the contents of the reactor was adjusted to 35-45 °C, then the contents were concentrated under vacuum to 3.5-4.5 L while maintaining an internal temperature of 30-55 °C.

Next, EtOAc (5 L) was added, and the temperature of the contents of the reactor was adjusted to 35-45 °C. The contents of the reactor were concentrated under vacuum to 3.5-4.5 L while maintaining an internal temperature of 30-55 °C, then EtOAc (5 L) was added. The temperature of the contents of the reactor was adjusted to 35-45 °C. The contents of the reactor were concentrated under vacuum to 3.5-4.5 L while maintaining an internal temperature of 30-55 °C. The volume of the contents of the reactor was adjusted to 4.5-6.0 L by charging with EtOAc, then the contents of the reactor were agitated for no less than 5 min. Next, the internal temperature of the reactor was adjusted to 45-55 °C and agitated for no less than 2 h. The contents of the reactor were slowly cooled to 10-20 °C for no less than 3 h and stirred for no less than 3 h. The resulting slurry was filtered and the wet cake was deliquored. The wet cake was washed with EtOAc (2 x 2 L), then the wet cake was dried at no more than 55 °C, under vacuum, for no less than 12 h until residual 2-MeTHF and EtOAc were each present in an amount no greater than 5000 ppm. Following this procedure, (R)-3-(3-cyano-4- fluorophenyl)- 1 -( 1 -(6,7-difluoro- 1 -oxo- 1 ,2-dihydroisoquinolin-4-yl)ethyl)- 1 -methylurea, compound (X), is typically obtained in 79-86% yield from compound (J) with no less than 99.0% purity as a white to off-white solid.

Example 6: Characterization of (X) Crystalline Form 2 Polarized light microscopy analysis demonstrated that the particles are crystalline

(FIG. 11). Compared with Form 1, the particles of Form 2 appear significantly larger and exhibit stronger birefringence. The crystalline nature of Form 2 is further demonstrated by XRPD analysis (FIG. 12). The high crystallinity of Form 2 is in contrast to the less degree of crystallinity observed in Form 1 (FIG. 13). Table 3. XRPD Peak Table for Form 2 TG-FTIR was performed for Form 2 and no mass loss was detected at temperatures up to about 200 °C, indicating that this is an anhydrous polymorph of (X) (FIG. 14).

Differential scanning calorimetry of Form 2 revealed a fairly sharp endotherm with a peak maximum at 219 °C, which is attributable to melting and associated with an enthalpy of fusion of about 84 J/g (FIG. 15).

The behavior of Form 2 in the presence of variable water vapor pressure was investigated using dynamic vapor sorption measurements. The results of the DVS measurement indicate that the change in water content is fairly small over the entire relative humidity range (FIG. 16 and FIG. 17). Less than 0.2% water was absorbed at 95% relative humidity, and therefore Form 2 is considered non-hygroscopic

Example 7: Polymorph Screen of (X)

Unless otherwise indicated, crystalline Form 1 of (X) was used as a starting material for a solvent-mediated polymorph screen, containing slurry experiments in a number of stressed conditions, with varying temperature, solvent and water activity. New crystalline patterns were observed and labeled forms III- VIII. The general conditions used to obtain new crystalline forms are provided in Table 4. At least one detailed description for the preparation of each crystalline form is provided herein. Table 4. Polymorph screen of free Form (1) with slurry experiments

^a Form 4 used as starting material; ^b Form 7 used as starting material.

Example 8: Crystallization and Characterization of (X) Crystalline Form 3 A sample of (X) Form 1 (54.2 mg) was dissolved in 4.6 mL THF at 50 °C. The resulting colorless solution was cooled to 20 °C for 10 h and stirred at 20 °C for about 9 h. Then the clear solution was cycled (1 cycle) between 20 °C and 5 °C, followed by cooling to 5 °C ( e.g 20 °C to 5 °C 10 h; 5 °C to 20 °C 10 h; 20 °C to 5 °C 10 h). After one day stirring at 5 °C the resulting dilute white suspension was concentrated under N2 for 2 h, then stirring was continued at 5 °C for 2 days. Then the suspension was filter centrifuged and a white solid product was obtained.

The solid collected was analyzed by XRPD and TG-FTIR. The low intensity XRPD reflections observed for Form 3 may indicate poor crystallinity (FIG. 18). The TG-FTIR showed no significant mass loss (FIG. 19). Table 5. XRPD Peak Table for Form 3

Example 9: Characterization of (X) Crystalline Form 4

A sample of (X) Form 1 (55.6 mg) was suspended in 0.6 mL ethanol and the resulting white suspension was stirred at rt for 6 days. The suspension was filter centrifuged and a white solid was obtained.

The solid collected was analyzed by XRPD and TG-FTIR. The XRPD pattern indicated that Form 4 is crystalline in nature (FIG. 20). The TG-FTIR revealed that Form 4 contained about 18% ethanol, but no pronounced step is observed, thus complicating the differentiation of surface absorbed and lattice bound ethanol. However, the fact that temperatures above 170 °C were needed to remove all solvent suggests that Form 4 is an ethanol solvate of (X) (FIG. 21).

Table 6. XRPD Peak Table for Form 4

Example 10: Characterization of (X) Crystalline Form 5

A sample of (X) Form 1 (48.5 mg) was suspended in 0.6 mL EtOAc, and the resulting white suspension was stirred at rt for 6 days. The suspension was filter centrifuged and a white solid was obtained.

The solid collected was analyzed by XRPD and TG-FTIR. The XRPD pattern indicated that Form 5 is crystalline in nature (FIG. 22). The TG-FTIR indicates that the sample contains about 9% EtOAc, which is close to the theoretical value of 9.9% for a hemi- solvate (FIG. 23). Table 7. XRPD Peak Table for Form 5

Example 11: Characterization of (X) Crystalline Form 6

A sample of (X) Form 1 (49.6 mg) was suspended in 0.3 mL methanol and the resulting white suspension was stirred at 40 °C for 6 days. Then the suspension was filter centrifuged and a white solid was obtained.

The solid collected was analyzed by XRPD and TG-FTIR. The XRPD pattern indicated that Form 6 is crystalline in nature (FIG. 24). The TG-FTIR indicates that the sample contained about 6.4% methanol, which released within the temperature range from rt to about 150 °C (FIG. 25). No pronounced step was observed, thus complicating the differentiation of surface absorbed and lattice bound methanol. However, the fact that a temperature >100 °C was required to remove all solvent indicated that Form 6 is a methanol solvate.

Table 8. XRPD Peak Table for Form 6

Example 12: Characterization of (X) Crystalline Form 7

A sample of (X) Form 1 (47.9 mg) was dissolved in 2.7 mL acetone/water (4/1) at 50 °C. The resulting colorless solution was cooled to 20 °C for 10 h and stirred at 20 °C for about 9 h. Then the clear solution was cycled (1 cycle) between 20 °C and 5 °C, followed by cooling to 5 °C ( e.g ., 20 °C to 5 °C 10 h; 5 °C to 20 °C 10 h; 20 °C to 5 °C 10 h). After one day stirring at 5 °C, the resulting dilute white suspension was concentrated under N2 for 2 h, then stirring was continued at 5 °C for 2 days. Then the suspension was filter centrifuged and a white solid product was obtained.

The solid collected was analyzed by XRPD and TG-FTIR. The XRPD pattern indicated that Form 7 is crystalline in nature (FIG. 26). The TG-FTIR indicates that the sample contained about 38.7% water and acetone, which released within the temperature range from rt to about 130 °C (FIG. 27). The fact that a temperature >130 °C is required to remove all solvent, as well as the presence of a second step at about 110 °C, indicated that Form 7 is an acetone solvate of (X), or a mixed solvate/hydrate. Table 9. XRPD Peak Table for Form 7

Example 13: Characterization of (X) Crystalline Form 8

The sample of (X) Form 4 was dried at rt under vacuum for 2 days, then the sample was dried at 40 °C under vacuum for one day. The solid was analyzed by XRPD and TG-FTIR. The XRPD pattern indicated that

Form 8 is crystalline in nature (FIG. 28). Additionally, an overlay comparing Form 8 and Form 4 (prior to drying) has been provided (FIG. 29). TG-FTIR analysis revealed a water content of about 2.7%, and an absence of ethanol (FIG. 30). The observed mass loss is close to the expected theoretical water content for a hemihydrate, which is 2.25%. However, further studies are necessary to clarify this form.

Table 10. XRPD Peak Table for Form 8

Example 14: Characterization of (X) Crystalline Form 9

A sample of (X) Form 1 was subjected to dynamic vapor sorption (DVS) experiments, which indicated that crystalline form 1 of (X) loses almost all DCM during the first cycle and the DCM is likely replaced by water at the end of the test, yielding crystalline form 9 of (X).

The solid recovered post-DVS was analyzed by XRPD and TG-FTIR. Although the XRPD patterns before and after DVS are similar, a substantial degree of isomorphism is suggested (FIG. 9). Crystalline form 9 may be the result of the conversion of a solvate to a hydrate. The most pronounced difference is the appearance of a strong reflection at 4.0° 2Q (FIG. 9). TG-FTIR of the sample after DVS confirmed the replacement of DCM with water, wherein the water content is approximately 3.8%, indicating that crystalline form 9 may be a channel solvate (FIG. 10).

Crystalline form 9 of (X) was also prepared by recrystallization from water. A sample of (X) Form 1 (51.8 mg) was suspended in 0.8 mL water. The resulting white suspension was stirred at 40 °C for 6 days, then the suspension was filter centrifuged to obtain a white solid.

Table 11. XRPD Peak Table for Form 9

Example 15: Competitive slurries of (X)

Competitive suspension equilibration experiments were used to determine the thermodynamically stable polymorph form under the tested conditions (Table 12). Two experiments were carried out with acetone as the solvent at rt and at 50 °C, independently. In each case, a sample of (X) Form 2 was suspended in acetone and seeded with Form 3 and the resulting suspensions were stirred for 7 days at the designated temperatures (i.e., rt or 50 °C).

Comparison of the XRPD patterns of the two starting materials and the two products from the experiments demonstrates that at room temperature and 50 °C, Form 2 is the thermodynamically stable form (FIG. 31). Table 12. Competitive slurry experiments

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 method of preparing (i?)-3-(3-cyano-4-fluorophenyl)-l-(l- (6,7-difluoro-l-oxo-l,2-dihydroisoquinolin-4-yl)ethyl)-l-met hylurea (X), or a salt or solvate thereof: the method comprising reacting phenyl (3-cyano-4-fluorophenyl)carbamate (K): and (//)-6,7-difluoro-4-(l -(methyl ami no)ethyl)isoquinolin- l (2H)-one (J): so as to generate a first reaction system comprising (X).

Embodiment 2 provides the method of Embodiment 1, wherein the reaction of the (K) and the (J) is performed in the presence of a base.

Embodiment 3 provides the method of Embodiment 2, wherein the base comprises triethylamine, diisopropylethylamine, and/or pyridine.

Embodiment 4 provides the method of any one of Embodiments 1-3, wherein the reaction of the (K) and the (J) is performed in a solvent comprising 2-methyltetrahydrofuran. Embodiment 5 provides the method of any one of Embodiments 1-4, wherein purification of the (X) comprises neutralizing at least a fraction of the base in the first reaction system and forming a first solution of the generated (X) in 2-methyltetrahydrofuran.

Embodiment 6 provides the method of Embodiment 5, wherein purification of the (X) further comprises exchanging at least a fraction of the 2-methyltetrahydrofuran in the first solution with ethyl acetate, thereby forming a second reaction system.

Embodiment 7 provides the method of Embodiment 6, wherein at least partial concentration of the second reaction system yields solid (X).

Embodiment 8 provides the method of any one of Embodiments 1-7, wherein (K) is prepared by reacting 5-amino-2-fluorobenzonitrile and phenyl chloroformate.

Embodiment 9 provides the method of Embodiment 8, wherein the 5-amino-2- fluorobenzonitrile and the phenyl chloroformate are reacted in the presence of a base.

Embodiment 10 provides the method of Embodiment 9, wherein the base comprises triethylamine, diisopropylethylamine, and/or pyridine.

Embodiment 11 provides the method of any one of Embodiments 1-10, wherein the (J) is prepared by reacting (//)-N-((//)- l -(6,7-difluoro-l -methoxyisoquinolin-4-yl)ethyl)-N,2- dimethylpropane-2-sulfmamide (I) with an acid:

Embodiment 12 provides the method of Embodiment 11, wherein the acid comprises an ethereal solution of HC1 and/or a solution of acetyl chloride in methanol.

Embodiment 13 provides the method of any one of Embodiments 11-12, wherein the (I) is prepared by methylating (//)-N-((//)- l -(6,7-difluoro- l -methoxyisoquinolin-4-yl)ethyl)- 2-methylpropane-2-sulfmamide (H):

Embodiment 14 provides the method of Embodiment 13, wherein the methylating agent comprises methyl iodide, methyl chloride, methyl triflate, and/or dimethyl sulfate. Embodiment 15 provides the method of any one of Embodiments 13-14, wherein the (H) is prepared by reacting (f?)-2-methylpropane-2-sulfmamide (G) and l-(6,7-difluoro-l-methoxyisoquinolin-4-yl)ethan-l-one (F) so as to form the corresponding N-alkylene sulfmamide, and treating the N-alkylene sulfmamide with a reducing agent to form (H).

Embodiment 16 provides the method of Embodiment 15, wherein (F) and the (G) are reacted with in the presence of a titanium (IV) alkoxide to form the N-alkylene sulfmamide.

Embodiment 17 provides the method of any one of Embodiments 15-16, wherein the reducing agent comprises a borohydride.

Embodiment 18 provides the method of any one of Embodiments 15-17, wherein the (F) is prepared by reacting l-(l-chloro-6,7-difluoro-4-isoquinolyl)ethenone (E) with an alkali methoxide.

Embodiment 19 provides the method of Embodiment 18, wherein the (E) is prepared by reacting 4-acetyl-6,7-difluoro-2H-isoquinolin-l-one (D) with a chlorinating agent.

Embodiment 20 provides the method of Embodiment 19, wherein the chlorinating agent comprises POCb. Embodiment 21 provides the method of Embodiment 20, wherein the POCb : (D) molar ratio is about 1.2 : 1.

Embodiment 22 provides the method of any one of Embodiments 19-21, wherein the (D) and the chlorinating agent are reacted in the presence of a base.

Embodiment 23 provides the method of Embodiment 22, wherein the base comprises triethylamine, diisopropylethylamine, and/or pyridine.

Embodiment 24 provides the method of any one of Embodiments 19-23, wherein the (D) and the chlorinating agent are contacted in a temperature not to exceed about 35 °C.

Embodiment 25 provides the method of Embodiment 24, wherein the mixture comprising the (D) and the chlorinating agent is kept at a temperature not to exceed about 80 °C.

Embodiment 26 provides the method of any one of Embodiments 19-25, wherein the (D) is prepared by reacting 6,7-difluoro-3-methyl-lH-isochromen-l-one (C) or methyl 4,5- difluoro-2-(2-oxopropyl)benzoate (C) with at least one agent selected from formamide and triazine.

Embodiment 27 provides the method of Embodiment 26, wherein the (C) or the (O) and the at least one agent are reacted in the presence of a base.

Embodiment 28 provides the method of Embodiment 27, wherein the base comprises potassium carbonate, sodium carbonate, sodium methoxide, and/or potassium methoxide.

Embodiment 29 provides the method of any one of Embodiments 27-28, wherein the (C) or the (C) and the at least one agent are reacted in the presence of a solvent.

Embodiment 30 provides the method of Embodiment 29, wherein the solvent comprises 2-methyltetrahydrofuran and/or methanol.

Embodiment 31 provides the method of any one of Embodiments 26-30, wherein the (C) or the (C) is prepared by reacting 4,5-difluoro-2-(2-oxopropyl)benzoic acid (B) with an acid.

Embodiment 32 provides the method of Embodiment 31, wherein the (B) and the acid are reacted in the presence of a solvent.

Embodiment 33 provides the method of Embodiment 32, wherein the solvent is dichloromethane (DCM) or methanol (MeOH).

Embodiment 34 provides the method of Embodiment 31, wherein the (B) is prepared by reacting 2-bromo-4,5-difluorobenzoic acid (A) with acetyl acetone.

Embodiment 35 provides the method of Embodiment 34, wherein the (A) and acetylacetone are reacted in presence of a Lewis acid.

Embodiment 36 provides the method of Embodiment 35, wherein the Lewis acid comprises a copper (I) salt.

Embodiment 37 provides the method of any one of Embodiments 34-36, wherein the (A) and acetylacetone are reacted in presence of a base.

Embodiment 38 provides the method of Embodiment 37, wherein the base comprises an alkali alkoxide.

Embodiment 39 provides a (i?)-3-(3-cyano-4-fluorophenyl)-l-(l-(6,7-difluoro-l-oxo- l,2-dihydroisoquinolin-4-yl)ethyl)-l-methylurea (X) crystalline solid, which is characterized by an X-ray diffraction pattern (XRDP) selected from the group consisting of:

(a) Crystalline Form 1, with a X-ray powder diffraction spectrum comprising 2Q values (in degrees) of about: 7.1, 8.1, 25.8, 26.1, and 26.5;

(b) Crystalline Form 2, with a X-ray powder diffraction spectrum comprising 2Q values (in degrees) of about: 11.29, 12.02, 17.51, 21.84, 22.09, and 22.72;

(c) Crystalline Form 3, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 10.78, 11.28, 17.65, 19.10, and 26.45;

(d) Crystalline Form 4, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 8.0, 10.8, 13.2, 25.2, 25.5, and 26.5;

(e) Crystalline Form 5, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 7.09, 8.06, 15.57, 15.86, 25.72, 26.07, 26.42, and 26.50;

(f) Crystalline Form 6, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 5.2, 7.8, 10.1, 11.3, 13.8, and 25.7;

(g) Crystalline Form 7, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 9.0, 12.9, 13.5, 20.4, and 26.0;

(h) Crystalline Form 8, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 5.2, 7.8, and 13.9; and

(i) Crystalline Form 9, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 4.0, 7.2, and 8.1; wherein the XRDP is measured with a Copper X-ray source.

Embodiment 40 provides the solid of Embodiment 39, which is characterized by an X-ray diffraction pattern (XRDP) selected from the group consisting of:

(a) Crystalline Form 1, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 10.8, 16.5, 16.9, 17.3, 17.7, 18.3, 19.5, 21.0, 21.4, 21.9, 22.9, 24.0,

24.3, 25.3, 27.9, 28.6, 30.5, and 32.6;

(b) Crystalline Form 2, with a X-ray powder diffraction spectrum further comprising 20 values (in degrees) of about: 8.26, 18.32, 19.58, 21.25, 25.19, 28.15, and 29.54;

(c) Crystalline Form 3, with a X-ray powder diffraction spectrum further comprising 20 values (in degrees) of about: 8.34, 12.49, 14.37, 18.65, 22.66, 23.20, and 24.03;

(d) Crystalline Form 4, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 5.1, 8.6, 9.7, 10.3, 12.5, 16.9, 18.4, 23.6, 25.9, and 30.1;

(e) Crystalline Form 5, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 9.58, 12.12, 13.17, 14.15, 26.77, and 27.67;

(f) Crystalline Form 6, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 7.4, 9.5, 10.9, 11.5, 12.7, 19.1, 22.5, 22.7, 26.0, 26.5, 26.7, and 27.9;

(g) Crystalline Form 7, with a X-ray powder diffraction spectrum comprising 20 values

(in degrees) of about: 4.0, 7.0, 7.6, 7.8, 9.6, 10.1, 10.7, 12.1, 14.7, 19.9, 20.7, 21.0, 22.9, 25.2,

26.4, 27.0, 27.8, and 28.8;

(h) Crystalline Form 8, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 7.4, 9.5, 10.1, 11.4, 11.6, 25.7, 26.9, and 28.1; and

(i) Crystalline Form 9, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 6.5, 9.6, 10.8, 11.1, 12.1, 13.2, 14.4, 15.4, 15.7, 19.1, 26.0, and 26.4.

Embodiment 41 provides the solid of any one of Embodiments 39-40, which is characterized by an X-ray diffraction pattern (XRDP) selected from the group consisting of: (a) Crystalline Form 1, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 6.5, 7.1, 8.1, 9.6, 10.8, 12.1, 13.0, 13.2, 14.3, 15.5, 16.5, 16.9, 17.3, 17.7, 18.3, 19.1, 19.5, 21.0, 21.4, 21.9, 22.9, 24.0, 24.3, 25.3, 25.8, 26.1, 26.5, 27.0, 27.9,

28.6, 30.5, and 32.6;

(b) Crystalline Form 2, with a X-ray powder diffraction spectrum further comprising 20 values (in degrees) of about: 12.2, 12.4, 14.27, 17.22, 18.00, 20.06, 24.72, 27.49, 31.74, 32.10, and 33.06;

(c) Crystalline Form 3, with a X-ray powder diffraction spectrum further comprising 20 values (in degrees) of about: 24.76, 25.27, 28.37, 30.52, and 32.67;

(d) Crystalline Form 4, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 11.3, 12.9, 14.3, 15.2, 16.0, 16.6, 17.4, 17.6, 18.7, 19.2, 20.2, 20.5,

21.1, 21.3, 21.6, 21.8, 22.5, 23.0, 24.0, 24.3, 26.9, 27.3, 27.8, 28.4, 28.8, 29.5, 30.5, 31.3,

31.6, 32.1, 32.4, 32.9, 33.5, 33.9, 34.5, 35.4, 35.7, 36.1, 36.7, 38.0, 38.2, 39.4, 39.9, and 40.7;

(e) Crystalline Form 5, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 4.03, 6.45, 17.34, 19.27, 19.80, 20.79, 21.62, 22.13, 22.51, 23.53, 24.40, 27.94, 28.55, 29.24, 29.54, 30.38, 30.73, and 32.71;

(f) Crystalline Form 6, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 8.4, 13.5, 14.3, 14.9, 15.1, 15.5, 16.3, 16.7, 17.1, 17.7, 18.3, 18.7, 19.7,

20.1, 20.7, 21.7, 21.9, 23.3, 24.0, 24.5, 25.0, 25.5, 27.3 28.5, 29.7, 29.9, 30.6, 31.0, 31.6, 32.0, 32.3, 32.5, 33.1, 33.7, 34.3, 34.6, 34.9, 36.0, 36.6, 37.1, 38.7, 40.1, 40.7, and 41.2;

(g) Crystalline Form 7, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 11.3, 11.6, 12.5, 13.8, 14.2, 15.1, 15.6, 16.1, 16.9, 17.7, 18.0, 18.4,

18.7, 19.1, 19.5, 21.5, 22.3, 22.6, 23.3, 24.1, 24.4, 27.2, 28.2, 28.4, 29.2, 30.0, 30.5, 30.7, 31.4, 31.8, 32.6, 32.9, 33.3, 33.7, 34.9, 35.3, 35.8, 36.6, 37.3, 37.5, 38.2, 38.9, 39.4, 40.1, 40.3, and 41.2;

(h) Crystalline Form 8, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 7.2, 8.3, 9.1, 10.9, 12.7, 15.2, 15.6, 16.7, 17.7, 18.3, 18.7, 19.1, 20.8,

21.8, 21.9, 22.6, 22.8, 23.2, 24.0, 25.0, 26.1, 26.5, 27.4, 30.0, 30.8, 31.8, 32.5, 33.0, 33.7,

34.9, 35.8, 36.1, 38.7, and 41.2; and

(i) Crystalline Form 9, with a X-ray powder diffraction spectrum comprising 20 values (in degrees) of about: 16.7, 17.6, 22.7, 23.2, 24.1, 24.4, 27.3, 28.2, 28.8, 30.8, and 32.7.

Embodiment 42 provides the solid of Embodiment 39(a), wherein the solid is obtained by crystallizing (X) in at least one solvent selected from the group consisting of methanol and dichloromethane.

Embodiment 43 provides the solid of Embodiment 39(a), comprising dichloromethane. Embodiment 44 provides the solid of Embodiment 39(b), wherein the solid is obtained by crystallizing (X) in at least one solvent selected from the group consisting of water, ethyl acetate, acetone/water mixture, 2-methyltetrahydrofuran, 2-propanol, «-heptane, toluene, acetone, water/tetrahydrofuran mixture, and dichloromethane.

Embodiment 45 provides the solid of Embodiment 44, wherein the water/tetrahydrofuran mixture has a ratio of water : tetrahydrofuran of about 1 : 1 (v/v).

Embodiment 46 provides the solid of Embodiment 44, wherein the acetone/water mixture has a ratio of acetone : water of about 4 : 1 (v/v) and the solid was vacuum dried at room temperature for about 1 day.

Embodiment 47 provides the solid of Embodiment 39(c), wherein the solid is obtained by crystallizing (X) in at least one solvent selected from the group consisting of acetone, dichloromethane, dimethylformamide//er/-butyl methyl ether mixture, and tetrahydrofuran.

Embodiment 48 provides the solid of Embodiment 47, wherein the di methyl form a i de//tv7-butyl methyl ether mixture has a ratio of dimethylformamide : tert- butyl methyl ether of about 1 : 10 (v/v).

Embodiment 49 provides the solid of Embodiment 39(d), wherein the solid is obtained by crystallizing (X) in ethanol.

Embodiment 50 provides the solid of Embodiment 39(d), comprising ethanol.

Embodiment 51 provides the solid of Embodiment 39(e), wherein the solid is obtained by crystallizing (X) in ethyl acetate.

Embodiment 52 provides the solid of Embodiment 39(e), comprising ethyl acetate.

Embodiment 53 provides the solid of Embodiment 39(f), wherein the solid is obtained by crystallizing (X) in methanol.

Embodiment 54 provides the solid of Embodiment 39(f), comprising methanol.

Embodiment 55 provides the solid of Embodiment 39(g), wherein the solid is obtained by crystallizing (X) in an acetone/water mixture.

Embodiment 56 provides the solid of Embodiment 39(g), wherein the acetone/water mixture has a ratio of acetone : water of about 4 : 1 (v/v).

Embodiment 57 provides the solid of Embodiment 39(h), wherein the solid is obtained by crystallizing (X) in ethanol and vacuum drying at room temperature for about 4 days.

Embodiment 58 provides the solid of Embodiment 39(i), wherein the solid is obtained by performing at least one cycle of a dynamic vapor sorption (DVS) experiment on crystalline form 1 of (X).

Embodiment 59 provides the solid of Embodiment 39(i), wherein the solid is obtained by crystallizing (X) in water.

Embodiment 60 provides the solid of any one of Embodiments 39-46, wherein at least one applies: the solid in (a) is characterized by a Differential Scanning Calorimetry (DSC) thermogram has having a glass transition at about 127.9 °C; the solid in (b) is characterized by a DSC thermogram having a single maximum value at about 219 °C.

Embodiment 61 provides a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and the solid of any one of Embodiments 39-60.

Embodiment 62 provides the pharmaceutical composition of Embodiment 61, which is in solid dosage form for oral administration.

Embodiment 63 provides the pharmaceutical composition of any one of Embodiments 61-62, which is part of a tablet, dragee, drop, suppository, capsule, caplet, and/or gelcap.

Embodiment 64 provides the pharmaceutical composition of any one of Embodiments 61-63, further comprising at least one additional agent useful for treating hepatitis virus infection.

Embodiment 65 provides the pharmaceutical composition of Embodiment 64, wherein the at least one additional agent comprises at least one selected from the group consisting of reverse transcriptase inhibitor; capsid inhibitor; cccDNA formation inhibitor; RNA destabilizer; oligomeric nucleotide targeted against the HBV genome; immunostimulator, such as checkpoint inhibitor ( e.g ., PD-L1 inhibitor); GalNAc-siRNA conjugate targeted against an HBV gene transcript; and therapeutic vaccine.

Embodiment 66 provides a method of treating or preventing hepatitis B virus (HBV) infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the solid of any of Embodiments 39-60 or the pharmaceutical composition of any one of Embodiments 61-65.

Embodiment 67 provides a method of inhibiting expression and/or function of a viral capsid protein directly or indirectly in a hepatitis B virus-infected subject, the method comprising administering to the subject a therapeutically effective amount of the solid of any one of Embodiments 39-60 or the pharmaceutical composition of any one of Embodiments 61-65.

Embodiment 68 provides the method of any one of Embodiments 65-67, wherein the subject is further administered at least one additional agent useful for treating HBV infection.

Embodiment 69 provides the method of Embodiment 68, wherein the solid or pharmaceutical composition, and the at least one additional agent, are coformulated.

Embodiment 70 provides the method of any of Embodiments 65-69, wherein the subject is further infected with hepatitis D virus (HDV).

Embodiment 71 provides the method of any of Embodiments 65-70, wherein the subject is a mammal.

Embodiment 72 provides the method of Embodiments 71, 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 the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.