BACKGROUND

Bile acids (BAs) and their derivatives modulate famesoid X receptor (FXR) and regulate FXR-mediated diseases and conditions. Natural bile acids such as chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), and the taurine and glycine conjugates thereof are known FXR ligands. A semi-synthetic bile acid analogue, 3a,7a-dihydroxy-6a-ethyl-5P-cholan-24-oic acid (6-ethyl-chenodeoxycholic acid (6-ECDCA) or obeticholic acid (OCA)), disclosed in WO 2002/075298, is a highly potent FXR modulator, which is currently marketed as OCALIVA ^® for the treatment of primary biliary cholangitis (PBC). Another semi-synthetic bile acid analog, 3a,7a,11b- trihydroxy-6a-ethyl-5P-cholan-24-oic acid (Compound 100) while being a potent FXR agonist, also showed specificity against G protein-coupled receptor TGR5 (GP-BAR1, M- BAR, GPBAR, or GPR131): 100

Identification of potent and selective bile acid-based FXR agonists is fundamental not only to further explore the physiological roles and pathological implications of bile acid signaling, but also to advance novel therapeutic opportunities associated with the selective modulation of FXR by bile acid analogs. More efficacious and selective bile acid-based FXR agonists may demonstrate added therapeutic value by avoiding potential side effects associated with TGR5 activation ( e.g ., itching, gallbladder filling, and cholesterol gallstone formation).

Although methods of synthesizing Compound 100 and its analogs are described in WO 2014/184271 and WO 2017/062763, there remains a need for more efficient methods of preparing selective FXR modulators, such as Compound 100 and its analogs, including processes with a reduced number of steps and increased yields, and providing high purity intermediates and final products. The present application addresses this need.

SUMMARY

The present application provides methods of preparing bile acid derivatives.

In one aspect, the present application relates to a method of preparing a compound of formula I: or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, wherein R ¹ , R ² , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , m, n, and p are as described herein.

In another aspect, the present application relates to a method of preparing a compound of formula II: or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, wherein R ¹ , R ² , R ⁴ , R ⁵ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , m, n, and p are as described herein.

In another aspect, the present application relates to a method of preparing a compound of formula III: or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, wherein R ² , R ⁴ , R ⁵ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , m, n, and p are as described herein.

In one aspect, the present application relates to Compound A, Compound B, Compound B’, Compound C, Compound C’, Compound D, Compound El, Compound EG, Compound El”, EE”, Compound E2a, Compound E2b, Compound E3a, Compound E3b, or Compound F.

In one aspect, the present application relates to Compound A-l, Compound B-l, Compound B-E, Compound C, Compound C’, Compound D, Compound El, Compound EE, Compound El”, Compound EE”, Compound E2a, Compound E2b, Compound E3a, Compound E3b, or Compound F.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and are not intended to be limiting. In the case of conflict, the present specification, including definitions, will control. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. The references cited herein are not admitted to be prior art to the claimed invention.

Other features and advantages of the application will be apparent from the following detailed description. DETAILED DESCRIPTION

Definitions

Certain terms used in the specification and claims are collected here.

As used herein, the phrase “a compound of the disclosure” or “a compound of the application” refers to a compound of any one of formulae 1, 1-9, 1-9a, II, III, Ilia, or Illb, or any other compound explicitly disclosed herein.

The term “C1-C6 alkyl” or “Aik” or “alkyl”, as used herein, refers to a straight- chain or branched hydrocarbon moiety having 1, 2, 3, 4, 5, or 6 carbon atoms. Examples of Ci-Ce alkyl moieties include, but are not limited to, methyl, ethyl, «-propyl, isopropyl, cyclopropyl, «-butyl, isobutyl, sec-butyl, /-butyl, «-pentyl, isopentyl, and «-hexyl. ’’C1-C4 alkyl” refers to a straight-chain or branched hydrocarbon moiety having 1, 2, 3, or 4 carbon atoms.

The term “alkenyl” refers to a straight-chain or branched hydrocarbon moiety containing at least one carbon-carbon double bond. Both the trans and cis isomers of the carbon-carbon double bond are encompassed under the term “alkenyl”. Examples of alkenyl moieties include, but are not limited to, vinyl, allyl, 1-butenyl, 2-butenyl, 3- butenyl, and 2-hexenyl.

As used herein, “alkynyl” refers to a straight-chain or branched hydrocarbon moiety containing at least one carbon-carbon triple bond. Examples of alkynyl moieties include, but are not limited to, ethynyl, 2-propynyl, 5-but-l-en-3-ynyl, and 3-hexynyl.

The term “alkoxy” refers to a straight-chain or branched saturated hydrocarbon covalently attached to an oxygen atom. Examples of alkoxy moieties include, but are not limited to, methoxy, ethoxy, isopropyloxy, «-propoxy, «-butoxy, /-butoxy, and pentoxy.

As used herein, the term “halogen” or “Hal” refers to fluorine, bromine, chlorine, and iodine.

As used herein, “carbocycle”, “carbocyclic”, or “carbocyclic ring” is intended to include any stable monocyclic or bicyclic ring having the specified number of carbons, any of which may be saturated, unsaturated, or aromatic. Carbocyclic ring includes cycloalkyl and aryl. For example, a C3-C8 carbocyclic ring is intended to include a monocyclic or bicyclic ring having 3,4, 5, 6, 7, or 8 carbon atoms. Examples of carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptenyl, cycloheptyl, cycloheptenyl, adamantyl, cyclooctyl, cyclooctenyl, and phenyl.

As used herein, “heterocycle”, “heterocyclic”, or “heterocyclic group” includes any ring structure (saturated, unsaturated, or aromatic) which contains at least one ring heteroatom ( e.g ., N, O or S). Heterocycle includes heterocycloalkyl and heteroaryl. Examples of heterocycles include, but are not limited to, morpholine, pyrrolidine, tetrahydrothiophene, piperidine, piperazine, oxetane, pyran, tetrahydropyran, azetidine, and tetrahydrofuran. Examples of heterocyclic groups include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, lH-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, pyridinyl, pyridyl, and pyrimidinyl.

As used herein, the term "cycloalkyl" refers to a saturated or unsaturated nonaromatic hydrocarbon mono- or multi-ring (e.g., fused, bridged, or spiro rings) system having 3 to 10 carbon atoms (e.g, C3-C6). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, and cycloheptenyl.

As used herein, any recited moiety which includes, but is not limited to, alkyl, alkenyl, alkynyl, alkoxy, carbocyclic ring, heterocyclic ring, cycloalkyl, etc. can be optionally substituted. The term "optionally substituted" refers to the indicated moiety which may or may not be substituted, and when substituted is mono-, di-, or tri- substituted, such as with 1, 2, or 3 substituents. In some instances, the substituent is halogen or OH.

As used herein, the term “protecting group” refers to an appropriate moiety for masking, for example, a hydroxyl functionality, which is stable/non-reactive under the reaction condition (e.g, non-reactive with an agent used in the reaction). One skilled in the art will recognize the particular moieties employed for protecting certain functional groups, e.g, hydroxyl group, instead of another functionality, e.g, carboxylic acid. The protecting group reagents include, but are not limited to, acylating agents (e.g, acetic anhydride, benzoyl chloride, pivaloyl chloride, etc.), silylating agents ( e.g ., TMS-C1, TES- Cl, TBDMS-C1, etc.), ether forming reagents (MOM-C1, MEM-C1, dihydropyran, ethyl vinyl ether, haloalkanes such as iodomethane, bromomethane, iodoethane, bromoethane, etc.), chloroformates (methyl chloroformate, ethyl chloroformate, isobutyl chloroformate, benzyl chloroformate, etc.), in the presence of an appropriate base (e.g., carbonate salts, bicarbonate salts, pyridine, triethylamine, diisopropyl ethylamine, N-methylmorpholine, etc.). Alternatively, an ester-based solvent (e.g, methyl acetate, ethyl acetate, isopropyl acetate, ethyl formate, methyl trifluoroacetate, methyl propionate, etc.) can be used in conjunction with an acid (e.g, methanesulfonic acid, p-toluenesulfonic acid, cone sulfuric acid, etc.) to selectively acylate the disclosed compounds, e.g, at C-3 position.

As used herein, the term "leaving group" or “LG” refers to a labile functionality that has a propensity to dissociate from carbon (e.g, Cl, Br, I, sulfonated alcohols such as methane sulfonates, p-toluenesulfonates, trifluoromethane sulfonates, trifluoroacetates, sulforylated alcohols, phosphorylated alcohols, etc.). The leaving groups can be either replaced with another functional group or eliminated, e.g, to produce an unsaturated compound.

The phrase “pharmaceutically acceptable carrier” is art-recognized, and includes, for example, pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition from one organ, or portion of the body, to another organ, or portion of the body. Each carrier is “acceptable” in the sense of being compatible with the other ingredients of a subject composition and not injurious to the patient. In certain embodiments, a pharmaceutically acceptable carrier is non-pyrogenic. Some examples of materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer’s solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

As used herein, the term "pharmaceutically acceptable excipient" refers to an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use.

A “pharmaceutical composition” is a formulation containing a compound of the present application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial. The quantity of active ingredient ( e.g ., a formulation of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it may be necessary to make routine variations to the dosage depending, for example, on the age and condition of the patient. The dosage will also depend on the route of administration.

A variety of routes are contemplated, including oral, ocular, ophthalmic, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, and the like. Dosage forms for the topical or transdermal administration of a compound of this application include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. In another embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.

As used herein, the term “amino acid conjugates” refers to conjugates of a compound of the application with any suitable amino acid. Taurine (-NH^CThjiSCbH), glycine (-NHCH2CO2H), and sarcosine (-N^CHajCtfcCC H) are examples of amino acid conjugates. Suitable amino acid conjugates of the compounds have the added advantage of enhanced integrity in bile or intestinal fluids. Suitable amino acids include, but are not limited to taurine, glycine, and sarcosine. The amino acid conjugates of the compounds of the application can be prepared according to methods known in the art. For example, a free or protected bile acid or bile acid derivative can be coupled to an amino acid (protected or unprotected), e.g ., glycine, sarcosine, or taurine amino acid, using standard peptide coupling conditions (e.g, in the presence of a base (e.g, tri ethyl amine, diisopropyl ethylamine (DIPEA), etc.) and specific coupling reagents, for example, N-Ethoxycarbonyl- 2-ethoxy-l,2-dihydroquinoline (EEDQ), 4-(4,6-Dimethoxy-l,3,5-triazin-2-yl)-4- methylmorpholinium chloride (DMTMM), etc.).

As defined herein, the term “metabolite” refers to glucuroni dated and sulfated derivatives of the compounds described herein, wherein one or more glucuronic acid or sulfate moieties are linked to the compound of the application. Glucuronic acid moieties may be linked to the compounds through glycosidic bonds with the hydroxyl groups of the compounds (e.g, 3-hydroxyl, 7-hydroxyl, 11-hydroxyl, and/or the hydroxyl of the R ⁷ group). Sulfated derivatives of the compounds may be formed through sulfation of the hydroxyl groups (e.g, 3-hydroxyl, 7-hydroxyl, 11-hydroxyl, and/or the hydroxyl of the R ⁷ group). Examples of metabolites include, but are not limited to, 3-O-glucuronide, 7-0- glucuronide, 11-O-glucuronide, 3-0-7-0-diglucuronide, 3-0-11-O-triglucuronide, 7-0-11- O-triglucuronide, and 3-0-7-0-11-O-triglucuronide, of the compounds described herein, and 3-sulfate, 7-sulfate, 11-sulfate, 3, 7-bi sulfate, 3, 11-bi sulfate, 7, 11-bi sulfate, and 3,7,11- trisulfate, of the compounds described herein. Many drug molecules have been conjugated to glucuronic acid in order to obtain the required derivatives as tools for improving insights on their absorption, metabolism and bioavailability. Isolation of the metabolites is often laborious and analytical standards are necessary as reference compounds for quantification of metabolite levels in clinical samples and for further pharmacological evaluation. The study of metabolites of drugs can contribute to the toxicity, research, and safety assessment of the drug molecules. Some glucuronides have similar or even greater biological activity compared to their corresponding parent drug molecules. For example, well-known active glucuronide is morphine 6-0-glucuronide, which has even more analgesic action than morphine. Methods of chemical and enzymatic synthesis of glucuronides are well-known in the art. The Koenigs-Knorr reaction is one of the most widely applied methods for the synthesis of alkyl and aryl O-glucuronide compounds. In this reaction, the aglycone (starting alcohol or phenol) is coupled with, for example, methyl acetobromo-a-D- glucuronate in the presence of, for example, silver salts. If the substrate molecule (aglycone) has multiple glucuronidation sites, chemical synthesis can yield a mixture of mono- and polyglucuronides unless the unwanted glucuronidation sites are protected. The reaction gives glucuronides in variable yields depending on the catalyst, solvent, aglycone, and the ratio of the starting materials used. Other methods have been used for the synthesis of glucuronides including flow methods (Mostarda, et al. Org. Biomol. Chem. 12 (2014) 9592-9600); the main differences between the reactions are in the glycosyl donor (Stachulski, e/a/., J. Med. Chem. 49 (2006) 6931-6945; Kaspersen, e/a/., Xenobiotica 17 (1987) 1451-1471 (methods of chemical synthesis of sulfate and glucuronide conjugates.); Stachulski, e/a/., Nat. Prod. Rep. 15 (1998) 173-186).

The term “prodrug” as used herein, refers to a bile acid derivative or compound that, after administration, is metabolized (z.e., converted within the body) into a pharmacologically active drug. Inactive prodrugs are pharmacologically inactive medications that are metabolized into an active form within the body. Instead of administering a drug directly, a corresponding prodrug might be used instead to improve how a medicine is absorbed, distributed, metabolized, and excreted (ADME). Prodrugs are often designed to improve bioavailability when a drug itself is poorly absorbed from the gastrointestinal tract. A prodrug may be used to improve how selectively the drug interacts with cells or processes that are not its intended target. This can reduce adverse or unintended effects of a drug, especially important in treatments having severe unintended and undesirable side effects.

The term “treating”, as used herein, refers to relieving, lessening, reducing, eliminating, modulating, or ameliorating, z.e., causing regression of the disease state or condition.

The term “preventing”, as used herein, refers to completely or almost completely stop a disease state or condition, from occurring in a patient or subject, especially when the patient or subject is predisposed to such or at risk of contracting a disease state or condition. Preventing can also include inhibiting, z.e., arresting the development, of a disease state or condition, and relieving or ameliorating, z.e., causing regression of the disease state or condition, for example when the disease state or condition may already be present.

The phrase “reducing the risk of’, as used herein, refers to lowering the likelihood or probability of a central nervous system disease, inflammatory disease and/or metabolic disease from occurring in a patient, especially when the subject is predisposed to such occurrence.

“Combination therapy” (or “co-therapy”) refers to the administration of a compound of the application and at least a second agent as part of a specific treatment regimen intended to provide the beneficial effect from the co-action of these therapeutic agents (i.e., the compound of the application and at least a second agent). The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents. Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected). “Combination therapy” may, but generally is not, intended to encompass the administration of two or more of these therapeutic agents as part of separate monotherapy regimens that incidentally and arbitrarily result in the combinations of the present application. “Combination therapy” is intended to embrace administration of these therapeutic agents in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single capsule having a fixed ratio of each therapeutic agent or in multiple, single capsules for each of the therapeutic agents. Sequential or substantially simultaneous administration of each therapeutic agent can be affected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination selected may be administered by intravenous injection while the other therapeutic agents of the combination may be administered orally. Alternatively, for example, all therapeutic agents may be administered orally or all therapeutic agents may be administered by intravenous injection. The sequence in which the therapeutic agents are administered is not narrowly critical.

“Combination therapy” also embraces the administration of the therapeutic agents as described above in further combination with other biologically active ingredients and non-drug therapies ( e.g surgery or mechanical treatments). Where the combination therapy further comprises a non-drug treatment, the non-drug treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and non-drug treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non-drug treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks. As used herein, “combination therapy” means that a compound of the application can be administered in conjunction with another therapeutic agent. “In conjunction with” refers to administration of one treatment modality in addition to another treatment modality, such as administration of a compound of the application as described herein in addition to administration of another therapeutic agent to the same subject. As such, “in conjunction with” refers to administration of one treatment modality before, during, or after delivery of a second treatment modality to the subject.

An “effective amount” of a compound of the application, or a combination of compounds is an amount (quantity or concentration) of compound or compounds. In one embodiment, when a therapeutically effective amount of a compound is administered to a subject in need of treatment symptoms arising from the disease are ameliorated immediately or after administration of the compound one or more times. The amount of the compound to be administered to a subject will depend on the particular disorder, the mode of administration, coadministered compounds, if any, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, body weight, and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.

The term “prophylactically effective amount” means an amount (quantity or concentration) of a compound of the present application, or a combination of compounds, that is administered to prevent or reduce the risk of a disease - in other words, an amount needed to provide a preventative or prophylactic effect. The amount of the present compound to be administered to a subject will depend on the particular disorder, the mode of administration, coadministered compounds, if any, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, body weight, and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.

A “subject” includes mammals, e.g ., humans, companion animals ( e.g. , dogs, cats, birds, and the like), farm animals (e.g, cows, sheep, pigs, horses, and the like), and laboratory animals (e.g, rats, mice, guinea pigs, and the like). Typically, the subject is human.

As used herein, farnesoid X receptor or FXR refers to all mammalian forms of such receptor including, for example, alternative splice isoforms and naturally occurring isoforms (see, e.g, Huber etal., Gene 290:35-43 (2002)). Representative FXR species include, without limitation rat FXR (GenBank Accession No. NM 021745), mouse FXR (GenBank Accession No. NM 009108), and human FXR (GenBank Accession No. NM 005123).

The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in some embodiments ±5%, in some embodiments ±1%, and in some embodiments ±0.1% from the specified value, as such variations are appropriate to practice the disclosed methods or to make and used the disclosed compounds and in the claimed methods.

Methods of the application

Previous methods of preparing compounds of formula I were described, e.g. in U.S Patent Nos. 10,815,267 and 11,034,717. The methods of the present application employ fewer steps and do not use cryogenic conditions.

In one aspect, the present application relates to a method of preparing a compound of formula I: or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, wherein: each is independently a single bond or a double bond;

R ¹ is OH, alkoxy, or oxo;

R ² and R ³ are each independently H, OH, OSO3H, 00(0)0¾, OPO3H2, halogen, or alkyl optionally substituted with one or more halogen or OH; or

R ² and R ³ taken together with the carbon atom to which they are attached form a carbonyl;

R ⁴ is H, halogen, alkyl optionally substituted with one or more halogen or OH, alkenyl, or alkynyl;

R ⁵ and R ⁶ are each independently H, OH, OSO ₃ H, 0C(0)CH ₃ , OPO ₃ H ₂ , halogen, or alkyl optionally substituted with one or more halogen or OH; or

R ⁵ and R ⁶ taken together with the carbon atom to which they are attached form a carbonyl;

R ⁷ is OH, OSO ₃ H, SO ₃ H, OSO ₂ NH ₂ , SO ₂ NH ₂ , OPO ₃ H ₂ , PO ₃ H ₂ , CO ₂ H, C(0)NH0H, NH(CH ₂ ) ₂ S0 ₃ H, NHCH ₂ CO ₂ H, tetrazolyl, oxadiazolyl, thiadiazolyl, 5-oxo-

1.2.4-oxadiazolyl, 5-oxo-l,2,4-thiadiazolyl, oxazolidine-dionyl, thiazolidine-dionyl, 3- hydroxyisoxazolyl, 3-hydroxyisothiazolyl, pyrimidine, 3,5-difluoro-4-hydroxyphenyl, or

2.4-difluoro-3-hydroxyphenyl;

R ⁸ , R ⁹ , and R ¹⁰ are each independently H, OH, halogen, or alkyl optionally substituted with one or more halogen or OH; or

R ¹⁰ is H, OH, halogen, or alkyl optionally substituted with one or more halogen or OH; and R ⁸ and R ⁹ , taken together with the carbon atoms to which they are attached, form a 3- to 6-membered carbocyclic or heterocyclic ring comprising 1 or 2 heteroatoms selected from N, O, and S; or R ⁸ is H, OH, halogen, or alkyl optionally substituted with one or more halogen or OH; and R ⁹ and R ¹⁰ , taken together with the carbon atoms to which they are attached, form a 3- to 6-membered carbocyclic or heterocyclic ring comprising 1 or 2 heteroatoms selected from N, O, and S; m is 0, 1, or 2; n is 0 or 1; and p is 0 or 1; the method comprising at least one of the following steps:

Step 1-1. Converting Compound A-l to Compound B-l:

Compound A-1 Compound B-1 wherein indicates a bond that is either a single or a double bond, Step 2-1. Converting Compound B-1 to Compound C:

Compound B-1 wherein each PG independently indicates a protecting group, Step 3. Converting Compound C to Compound D:

Compound C Compound D

Step 4-1. Converting Compound D to Compound El :

Step 5-1. Converting Compound El to Compound F: and

Step 6. Converting Compound F to Compound 100:

In one aspect, the present application relates to a method of preparing a compound of formula I: or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, wherein R ¹ , R ² , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , m, n, and p are each as defined above, the method comprising at least one of the following steps: Step 1-2. Converting Compound A to Compound B:

Compound A Compound B wherein indicates a bond that is either a single or a double bond, Step 2-2. Protecting Compound B to form Compound C:

Compound B Compound C wherein each PG independently indicates a protecting group,

Step 3. Converting Compound C to Compound D:

Compound C Compound D

Step 4-1. Converting Compound D to Compound El :

Step 6. Converting Compound F to Compound 100:

Compound F Compound 100

In one aspect, the present application relates to a method of preparing a compound of formula I: or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, wherein R ¹ , R ² , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , m, n, and p are each as defined above, the method comprising at least one of the following steps: Step 1-2. Converting Compound A to Compound B:

Compound A Compound B wherein ' indicates a bond that is either a single or a double bond, Step 2-2. Protecting Compound B to form Compound C:

Compound B Compound C wherein each PG independently indicates a protecting group, Step 3. Converting Compound C to Compound D:

Compound C Compound D

Step 4-2a. Converting Compound D to Compound E2a:

Compound D Compound E2a

?

Step 4-2b. Converting Compound E2a to Compound E2b:

Step 5-2. Hydrogenating Compound E2b to form Compound F:

Step 6. converting Compound F to Compound 100:

Compound F Compound 100

In one aspect, the present application relates to a method of preparing a compound of formula I: or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, wherein R ¹ , R ² , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , m, n, and p are each as defined above, the method comprising at least one of the following steps: Step 1-2. Converting Compound A to Compound B:

Compound A Compound B wherein ' indicates a bond that is either a single or a double bond, Step 2-2. Protecting Compound B to form Compound C:

Compound B Compound C wherein each PG independently indicates a protecting group, Step 3. Converting Compound C to Compound D:

Compound C Compound D

Step 4-3 a. Converting Compound D to Compound E3a:

Step 4-3b. Converting Compound E3a to Compound E3b:

Step 5-3. Hydrogenating Compound E3b to form Compound F:

Compound E3b Compound F , and

Step 6. Converting Compound F to Compound 100:

Compound F Compound 100

In one embodiment, Step 1-1 comprises converting Compound A-l to Compound B-l by mixing Compound A with a monooxygenase. In one embodiment, the monooxygenase is a cytochrome P450 enzyme (“CYP450”). In one embodiment, the CYP450 is an animal CYP450, i.e., a CYP450 obtained from an animal. In another embodiment, the CYP450 is a plant CYP450, i.e., a CYP450 obtained from a plant.

In one embodiment, Step 1-1 comprises converting Compound A-l to Compound B-l under microbial conditions. In one embodiment, Step 1-1 comprises converting Compound A-l to Compound B-l by mixing Compound A with a natural or genetically modified mircroorganism (e.g. a bacterium, a fungus, an algae, a prokaryotic cell, a eukaryotic cell, an insect caell, or a mammalian cell (e.g. a human cell)) which expresses a cytochrome P450 monooxygenase (e.g. CYP7A, more prefereably CYP11B1) or other enzyme capable of catalyzing a stereoselective oxidation reaction. In one embodiment, the stereoselective oxidative catalysis by a cytochrome P450 monooxygenase or other enzyme is conducted by a microorganism. In one embodiment, the microorganism is selected from the group consisting of Absidia, Aspergillus , Cephalosporium , Cunningamella , Curvularia , Diplodia , Dothideales , Fusarium , Gibberella , Helminthosporium , Hypocreales , Mucor, Mucorales, Rhizopus , Saccharomyces . In one embodiment, the microorganism is selected from Cephalosporium aphidicola , Cladosporium herbarum , Colletotrichum lini , Fusarium culmorum , A. moniliforme , A. oxysporum, Mucor piriformis , M plumbeus , Rhizopus stolonifer , Botryodiplodia theobromae IFO 6469, Diplodia gossypina ATCC 28570, DSM 62-678, DSM 62-679, Botryosphaeria ribis ATCC 22802, Botryosphaeria berengeriana ATCC 12557, and Botryosphaeria rhodina CBS 374.54, CBS 287.47 and CBS 306.58. In one embodiment, the microorganism is selected from the Pleosporaceae family (e.g., Curvularia lunata VKPM F-981, Alternaria alternata , or Bipolaris sorokiniana ( =Helminthosporium )), the Hypocreaceae family (e.g, Fusarium sp.), and the Mucoraceae family (e.g, Rhizopus nigricans), Arthrobacter sp. (e.g. , Arthrobacter polychromogene, Arthrobacter niigatensis, Arthrobacter defluvii), Rhodococcus sp. (e.g, Rhodococcus pyridinivorans, Rhodococcus erythropolis, Rhodococcus opacus, Rhodococcus ruber, Rhodococcus globerulus, Rhodococcus wratislaviensis), Pseudomonas sp. (e.g, Pseudomonas syringiae, Pseudomonas fluorescens), Lactobacillus sp. (e.g, Lactobacillus mesenter, Lactobacillus sake, Lactobacillus farciminis, Lactobacillus kefiri), Burkholderia sp. (e.g, Burkholderia pyrrocinia, Burkholderia xenovorans, Burkholderia multivorans), Xanthobacter sp. (e.g. , Xanthobacter autotrophicus, Xanthobacter tagetidis ), Furasium sp. (e.g, Fusarium oxysporum ), Chlorophyceae (e.g., Dunaliella minuta, Coccomyxa elongata, Trebouxia decolorans, Chlorella ellipsoidea, Chlorella saccharophila, Chlorella pringsheimii, Trebouxia sp., Dunaliella primolectd), Prasinophyceae (e.g, Tetraselmis tetrathele, Tetraselmis chui, Tetraselmis sueica, Pyramimonas gelidicold), Cyanobacteria (e.g, Anacystis nidulans, Fremyella diplosiphon, Cvanidium caldarium, Microcystis aeruginosa, Anabaena cylindrica, Spirulina platensis, Spirulina sp., Calothrix sp., Nostoc commune), Chrysophyceae (e.g. , Ochromonas danica, Ochromonas malhamensis, Ochromonas sociabilis), Xanthophyceae (e.g., Botrydium granulatum, Monodus subterraneus, Tribonema aequale), Euglenophyceae (e.g., Euglena gracilis, Astasia longd), Bangiophyceae (e.g, Goniotrichum elegans, Porphyridium cruentum, Porphyridium aeurigeum), Cryptophyceae (e.g., Cryptomonas sp., Nematochrysopsis roscoffensis), Raphidophyceae (Fibrocapsa japonica ), Chrysochromulina polylepis,Prymnesium patellifera, Ochrosphaera neapolitana, Ochrosphaera verrucosa, Pavlova lutheri, Pavlova lutheri, Emiliania huxleyi, Isochrysis galbanajsochrysis galbana, Isochrysis sp ., Isochrysis sp.,ChrysoliIa lamellosa,Chrysotila lamellosa,

Chrysotila stipitata,Hymenomomas carterae,Coccolithus pelagicus, Nitzschia longissima, Melosira granulats, Thalassionema nitzschoides, Nitzschia frustulum, Chaetoceros simplex, Skeletonema costatum, Thalassiosira fluviatilis, Fragilaria sp., Asterionella glacialis, Biddulphia sinensis, Ciclotella nana, Vavicula pelliculosa, Nitzschia closterium, Phaeodactylum tricornutum, Phaeodactylum tricornutum, Stauroneis amphioxys, Nitzschia ovalis, Biddulphia aurita, Chaetoceros sp., Thalassiosira pseudonana, Thalassiosira pseudonana, Amphora exigua, Amphora sp., Nitzschia alba, Rhizoselenium spp., Gonyaulax spp., Peridinium foliaceum, Peridinium foliaceum, Gonyaulax diegensis, Pyrocystis lunula, Gonyaulax polygramma, Gymnodinium wilczeki, Glenodinium hallii, Noctiluca milaris, Gymnodinium simplex, and Prorocentrum cor datum. In one embodiment, the microorganism is Curvularia lunata VKPM F-981. In one embodiment, the microorganism is Curvularia lunata VKPM F-981 as disclosed in Andryushina, V.A., Yaderets, V.V., Stytsenko, T.S. etal. Effect of the steroid molecule structure on the direction of its hydroxylation by the fungus Curvularia lunata. Appl Biochem Microbiol 49, 386-394 (2013) In one embodiment, Step 2-1 further comprises Steps 2-la and 2-lb:

Step 2- la. Olefmation of compound B-l and installation of a protected carboxy group to form Compound B-G

Compound B-1 Compound B-1 ' _anc j

Step 2-lb. Protecting the Cl 1 hydroxy group of Compound B-G to produce Compound C

In one embodiment, the olefmation of Step 2- la is a Wittig reaction. In one embodiment, the olefmation of Step 2- la is a Horner Wadsworth Emmons reaction.

In one embodiment, Step 2-lb comprises reacting the hydroxy group with an agent to form a protecting group selected from acetyl, benzoyl, b-methoxyethoxymethylether, dimethoxytrityl, methyl ether, methoxymethyl ether, methoxyethyl ether, ethoxyethyl ether, ethoxymethyl ether, allyl ether, /-butyl ether, methoxytrityl, / -methoxybenzyl ether, methylthiomethyl ether, tetrahydropyranyl ether, tetrahydrofuranyl ether, trityl, silyl ether ( e.g ., trimethyl silyl (TMS), /-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), and triisopropyl silyl (TIPS) ethers), methyl ester, pivaloyl, and benzoic acid ester. In one embodiment, the agent is an acylating agent (e.g., acetic anhydride, benzoyl chloride, pivaloyl chloride, etc.), a silylating agent (e.g, TMS-C1, TES-C1, TBDMS-C1, etc.), an ether forming reagent (e.g, MOM-C1, MEM-Cl, dihydropyran, ethyl vinyl ether, haloalkanes such as iodomethane, bromomethane, iodoethane, bromoethane, etc.), or a chloroformate (methyl chloroformate, ethyl chloroformate, isobutyl chloroformate, benzyl chloroformate, etc.). In one embodiment, the agent is selected from MOM-C1, MEM-Cl, dihydropyran, ethyl vinyl ether, /-butyldiphenylsilyl, methoxymethyl acetal, benzylhailde (e.g, benzyl fluoride, benzyl chloride, benzylbromide, or benzyliodide), benzoylhailde (e.g, benzoylfluoride, benzoyl chloride, benzoylbromide, or benzoyliodide), and haloalkane ( e.g ., iodomethane, bromom ethane, iodoethane, or bromoethane).

In one embodiment, the agent in Step 2- lb is a silylating agent. In one embodiment, the agent is TMS-C1. In one embodiment, the agent is TES-C1. In one embodiment, the agent is TBDMS-C1. In one embodiment, the agent is TIPS-Cl. In one embodiment, the agent is TOM-C1. In one embodiment, the agent is TMS-OTf. In one embodiment, the agent is TES-OTf. In one embodiment, the agent is TBDMS-OTf. In one embodiment, the agent is TIPS-OTf. In one embodiment, the agent is TOM-OTf. In one embodiment, the hydroxy protecting group is a silyl ether. In one embodiment, the silyl ether is TMS. In one embodiment, the silyl ether is TBDMS. In one embodiment, the silyl ether is TES. In one embodiment, the silyl ether is TIPS. In one embodiment, the silyl ether is TOM.

In one embodiment, the reaction of Step 2-lb is carried out for greater than 12 hours. In one embodiment, the reaction is carried out for between 12 and 24 hours. In one embodiment, the reaction is carried out between 16 and 20 hours. In one embodiment, the reaction is carried out for about 18 hours.

In one embodiment, Step 2-2 comprises protecting the hydroxy group and/or carboxyl group in Compound B with a protecting group. In some embodiments, Step 2-2 comprises protecting the hydroxy group in Compound B with a protecting group. In some embodiments, Step 2-2 comprises protecting the carboxyl group in Compound B with a protecting group. In some embodiments, Step 2-2 comprises protecting the hydroxy group in Compound B with a protecting group and protecting the carboxyl group in Compound B with a protecting group. In some embodiments, Step 2-2 comprises protecting the carboxyl group in Compound B with a protecting group, followed by protecting the hydroxy group in Compound B with a protecting group. Protecting groups for carrying out Step 2-2 are well known in the art.

In one embodiment, Step 2-2 further comprises Steps 2-2a and 2-2b:

Step 2 -2a. Protecting the carboxy group of Compound B to produce Compound B’

Compound B Compound B' _anc j

Step 2 -2b. Protecting the Cl 1 hydroxy group of Compound B’ to produce Compound C

Compound B' Compound C

In one embodiment, Step 2-2a comprises reacting the carboxyl group with an agent to form a protecting group selected from methyl ester, benzyl ester, /-butyl ester, silyl ester, orthoester, and oxazoline. In one embodiment, the agent is an acylating agent ( e.g ., acetic anhydride, benzoyl chloride, pivaloyl chloride, etc.), a silylating agent (e.g., TMS-C1, TES- Cl, TBDMS-C1, etc.), or a chloroformate (methyl chloroformate, ethyl chloroformate, isobutyl chloroformate, benzyl chloroformate, etc.). In one embodiment, Step 2-2a comprises dissolving Compound B in an alcoholic solvent and adding a catalytic amount of acid. In one embodiment, the alcoholic solvent is selected from methanol, ethanol, and isopropanol. In one embodiment, the alcoholic solvent is methanol. In one embodiment, the acid is selected from H2SO4, HC1, methanesulfonic acid, and toluenesulfonic acid. In one embodiment, Step 2-2a is conducted at a temperature between ambient and reflux. In one embodiment, Step 2-2a is conducted at ambient temperature. In one embodiment, Step 2-2a is conducted at reflux.

In one embodiment, Step 2-2b comprises reacting the hydroxy group with an agent to form a protecting group selected from acetyl, benzoyl, b-methoxyethoxymethylether, dimethoxytrityl, methyl ether, methoxymethyl ether, methoxyethyl ether, ethoxyethyl ether, ethoxymethyl ether, allyl ether, /-butyl ether, methoxytrityl, /;-methoxybenzyl ether, methylthiomethyl ether, tetrahydropyranyl ether, tetrahydrofuranyl ether, trityl, silyl ether (e.g., trimethyl silyl (TMS), /-butyl di methyl si lyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), and triisopropyl silyl (TIPS) ethers), methyl ester, pivaloyl, and benzoic acid ester. In one embodiment, the agent is an acylating agent (e.g, acetic anhydride, benzoyl chloride, pivaloyl chloride, etc.), a silylating agent (e.g, TMS-C1, TES-C1, TBDMS-C1, etc.), an ether forming reagent (e.g, MOM-C1, MEM-C1, dihydropyran, ethyl vinyl ether, haloalkanes such as iodomethane, bromomethane, iodoethane, bromoethane, etc.), or a chloroformate (methyl chloroformate, ethyl chloroformate, isobutyl chloroformate, benzyl chloroformate, etc.). In one embodiment, the agent is selected from MOM-C1, MEM-C1, dihydropyran, ethyl vinyl ether, /-butyl di phenyl si lyl , methoxymethyl acetal, benzylhailde (e.g, benzyl fluoride, benzyl chloride, benzylbromide, or benzyliodide), benzoylhailde (e.g, benzoylfluoride, benzoyl chloride, benzoylbromide, or benzoyliodide), and haloalkane (e.g, iodomethane, bromomethane, iodoethane, or bromoethane).

In one embodiment, the agent in Step 2-2b is a silylating agent. In one embodiment, the agent is TMS-C1. In one embodiment, the agent is TES-C1. In one embodiment, the agent is TBDMS-C1. In one embodiment, the agent is TIPS-Cl. In one embodiment, the agent is TOM-C1. In one embodiment, the agent is TMS-OTf. In one embodiment, the agent is TES-OTf. In one embodiment, the agent is TBDMS-OTf. In one embodiment, the agent is TIPS-OTf. In one embodiment, the agent is TOM-OTf. In one embodiment, the hydroxy protecting group is a silyl ether. In one embodiment, the silyl ether is TMS. In one embodiment, the silyl ether is TBDMS. In one embodiment, the silyl ether is TES. In one embodiment, the silyl ether is TIPS. In one embodiment, the silyl ether is TOM.

In one embodiment, the reaction of Step 2-2b is carried out for greater than 12 hours. In one embodiment, the reaction is carried out for between 12 and 24 hours. In one embodiment, the reaction is carried out between 16 and 20 hours. In one embodiment, the reaction is carried out for about 18 hours.

In one embodiment, the protecting reaction is carried out in the presence of an appropriate solvent. In one embodiment, the protection reaction is carried out in the presence of a basic solvent (e.g. pyridine, pyridine derivatives, trialkylamines, N- methylmorpholine, etc.). In one embodiment, the protecting reaction is carried out in the presence of an appropriate base (e.g, carbonate salts, bicarbonate salts, pyridine, triethylamine, diisopropyl ethylamine, N-methylmorpholine, etc.). In one embodiment, the protecting reaction is carried out in the presence of an ester-based solvent ( e.g ., methyl acetate, ethyl acetate, isopropyl acetate, ethyl formate, methyl trifluoroacetate, methyl propionate, etc.). In one embodiment, the ester-based solvent is used in conjunction with an acid (e.g., methanesulfonic acid, p-toluenesulfonic acid, cone sulfuric acid, etc.).

Other conditions for carrying out the protection of the hydroxy and carboxyl groups in Compound B can be determined and optimized according to techniques and methods well known in the art.

In one embodiment, Step 3 further comprises Steps 3a and 3b: Step 3a. Oxidizing Compound C to form Compound C’

Compound C Compound D

In one embodiment, Step 3a comprises reacting Compound C with an oxidant to form compound D. In one embodiment, the oxidant is a 1-4 benzoquinone derivative. In one embodiment, the oxidant is chloranil. In one embodiment, the oxidant is 2,3-dichloro- 5,6-dicyano-l,4-benzoquinone (DDQ). In one embodiment, Step 3a comprises treatment with N-bromosuccinimide or dibromo-dimethylhydantoin, followed by treatment with a base.

In one embodiment, the Step 3a is carried out in the presence of an appropriate solvent. In one embodiment, the solvent is an organic solvent. In one embodiment, the solvent is a polar organic solvent. In one embodiment, the solvent is a polar protic organic solvent. In one embodiment, the solvent is an alcohol. In one embodiment, the solvent is methanol. In one embodiment, the solvent is ethanol. In one embodiment, the solvent is isopropanol. In one embodiment, the solvent is butanol. In one embodiment, the solvent is isobutanol, tert- butanol, or .svc-butanol.

In one embodiment, Step 3a is carried out at a temperature above room temperature. In one embodiment, the temperature is between 40 °C and 100 °C. In one embodiment, the temperature is between 40 °C and 80 °C. In one embodiment, the temperature is between 40 °C and 60 °C.

In one embodiment, Step 3a is carried out for between 1 and 8 hours. In one embodiment, Step 3a is carried out for between 2 and 7 hours. In one embodiment, Step 3a is carried out for between 3 and 6 hours. In one embodiment, Step 3a is carried out for between 4 and 6 hours. In one embodiment, Step 3a is carried out for between 4 and 5 hours.

In one embodiment, Step 3b comprises reacting Compound C’ with an oxidant. In one embodiment, the oxidant is an peroxide-based oxidant. In one embodiment, the oxidant is a peroxide. In one embodiment, the oxidant is a peroxy acid. In one embodiment, the oxidant is a peroxycarboxylic acid. In one embodiment, the oxidant is peroxybenzoic acid. In one embodiment, the oxidant is peracetic acid. In one embodiment, the oxidant is trifluoroperacetic acid. In one embodiment, the oxidant is dimethyldioxirane. In one embodiment, the oxidant is methyl(trifluoromethyl)dioxirane. In one embodiment, the oxidant is weto-chloroperoxybenzoic acid (mCPBA).

In one embodiment, Step 3b is carried out in the presence of a radical scavenger. In one embodiment, the radical scavenger is a phenolic antioxidant. In one embodiment, the radical scavenger is butylated hydroxytoluene (BHT, also known as 2,6-di-/er/-butyl-4- methylphenol) In one embodiment, the radical scavenger is butylated hydroxyanisole (BHA, which comprises a mixture of 2-/er/-butyl-4-hydroxyanisole and 3-/er/-butyl-4- hydroxyanisole). In one embodiment, the radical scavenger is tertiary butylhydroquinone (TBHQ).

In one embodiment, Step 3b is carried out in the presence of an appropriate solvent. In one embodiment, the solvent is an organic solvent. In one embodiment, the solvent is an aqueous solvent. In one embodiment, the solvent is a mixture of organic and aqueous solvents. In one embodiment, the organic solvent is a polar solvent. In one embodiment, the organic solvent is a polar aprotic solvent. In one embodiment, the organic solvent is an ester-based solvent. In one embodiment, the organic solvent is isopropyl acetate. In one embodiment, the organic solvent is ethyl acetate. In one embodiment, the aqueous solvent is water. In one embodiment, the solvent is a mixture of water and ethyl acetate.

In one embodiment, Step 3b is carried out at a temperature above room temperature. In one embodiment, the temperature is between 40 °C and 100 °C. In one embodiment, the temperature is between 70 °C and 100 °C. In one embodiment, the temperature is between 70 °C and 80 °C.

In one embodiment, Step 3b is carried out for between 1 and 5 hours. In one embodiment, Step 3b is carried out for between 1 and 4 hours. In one embodiment, Step 3b is carried out for between 2 and 4 hours. In one embodiment, Step 3b is carried out for between 2 and 3 hours.

In one embodiment, Step 4-1 comprises alkylating Compound D with an organometallic reagent to form Compound El.

In one embodiment, the organometallic reagent is an alkykllithium reagent.

In one embodiment, the organometallic reagent is a Gilman reagent formed by reaction of an alkyl lithium compound of the following formula:

R ⁴ -Li, wherein R ⁴ is as defined in formula I, and a copper (I) salt, including a copper (I) halide such as copper (I) iodide.

In one embodiment, Step 4-1 is carried out in an organic solvent. In one embodiment, the reaction is carried out in a polar organic solvent. In one embodiment, the reaction is carried out in a polar aprotic solvent. In one embodiment, the reaction is carried out in an ether solvent. In one embodiment, the reaction is carried out in THF. In one embodiment, the reaction is carried out in cyclopentyl methyl ether. In one embodiment, the reaction is carried out in 2-methyl THF. In one embodiment, the reaction is carried out in tBME. In one embodiment, the reaction is carried out in diethyl ether. In one embodiment, the reaction is carried out in a mixture of two or more of the foregoing solvents

In one embodiment, the organometallic reagent is a Grignard reagent: R ⁴ MgX, where R ⁴ is as defined in formula I, and X is halogen. In one embodiment, the Grignard reagent is ethylmagnesium bromide.

In one embodiment, Step 4-1 is carried out in the presence of a zinc (II) salt, such as zinc chloride, and a catalytic amount of a copper (I) or copper(II) salt or complex, such as copper (I) chloride, copper (II) chloride, or a copper(I) or copper (II) acetyl acetonate (acac) complex.

In one embodiment, Step 4-1 may be carried out in an organic solvent, for example, an ether such as THF, cyclopentyl methyl ether, 2-methyl THF, methyl tert-butyl ether (tBME), or diethyl ether or a mixture thereof. In one embodiment, the reaction is carried out in a polar organic solvent. In one embodiment, the reaction is carried out in a polar aprotic solvent. In one embodiment, the reaction is carried out in an ether solvent. In one embodiment, the reaction is carried out in THF. In one embodiment, the reaction is carried out in cyclopentyl methyl ether. In one embodiment, the reaction is carried out in 2-methyl THF. In one embodiment, the reaction is carried out in tBME. In one embodiment, the reaction is carried out in diethyl ether. In one embodiment, the reaction is carried out in a mixture of two or more of the foregoing solvents.

In one embodiment, Step 4-1 is carried out at a temperature below room temperature. In one embodiment, the temperature is between -78 °C and 0 °C. In one embodiment, the temperature is between -40 °C and 0 °C. In one embodiment, the temperature is between -20 °C and 0 °C. In one embodiment, the temperature is between - 20°C and -10 °C. In one embodiment, the temperature is about -20 °C.

In one embodiment, Step 4-1 is carried out between 30 minutes and 2 hours. In one embodiment, Step 4-1 is carried out between 30 and 90 minutes. In one embodiment, Step 4-1 is carried out between 60 and 90 minutes.

In one embodiment, Step 4-2a comprises reacting Compound D with an acetylide to form Compound E2a. In one embodiment, the acetylide is a lithium acetylide, a copper acetylide, a calcium carbide, or a silver acetylide. In one embodiment, the acetylide is a lithium acetylide. In one embodiment, the lithium acetylide is a lithium alkylsilyl acetylide. In one embodiment, the lithium alkylsilyl acetylide is lithium trimethyl silyl acetylide. In one embodiment, the reaction between Compound D and acetylide is carried out in the presence of a Lewis acid. In one embodiment, the Lewis acid is an aluminum based Lewis acid, a boron based Lewis acid, an iron based Lewis acid, a tin based Lewis acid, or a zinc based Lewis acid. In one embodiment, the Lewis acid is AIBn, AICL, aluminum isopropoxide, BCL, boron trichloride methyl sulfide, BF3, boron trifluoride acetic acid, boron trifluoride dibutyl etherate, boron trifluoride acetonitrile, boron trifluoride diethyl etherate, boron trifluoride methyl etherate, boron trifluoride methyl sulfide, FeBn, FeCL, ZnCh, or SnCL. In one embodiment, the Lewis acid is ZnCh or BF3.

In one embodiment, Step 4-2b comprises treating Compound E2a with a isomerization catalyst to form Compound E2b. In one embodiment, the isomerization catalyst is Fe(CO)s, Pd(OAc)2, a ruthenium salt, a Lewis acid, or a protic acid.

In one embodiment, Step 4-3a comprises reacting Compound D with a vinyl Grignard reagent to form Compound E3a. In one embodiment, the vinyl Grignard reagent is ethenylmagnesium bromide.

In one embodiment, the reaction between Compound D and vinyl Grignard reagent is carried out in the presence of a Lewis acid. In one embodiment, the Lewis acid is an aluminum based Lewis acid, a boron based Lewis acid, an iron based Lewis acid, a tin based Lewis acid, or a zinc based Lewis acid. In one embodiment, the Lewis acid is AIBn, AlCb, aluminum isopropoxide, BCb, boron trichloride methyl sulfide, BF3, boron trifluoride acetic acid, boron trifluoride dibutyl etherate, boron trifluoride acetonitrile, boron trifluoride diethyl etherate, boron trifluoride methyl etherate, boron trifluoride methyl sulfide, FeBn, FeCL, ZnCh, or SnCL. In one embodiment, the Lewis acid is ZnCh or BF3.

In one embodiment, Step 4-3b comprises treating Compound E3a with a isomerization catalyst to form Compound E3b. In one embodiment, the isomerization catalyst is Fe(CO)s, Pd(OAc)2, a ruthenium salt, a Lewis acid, or a protic acid.

In one embodiment, Step 5-1 comprises Step 5- la.

Step 5-la. Reducing Compound El to form Compound EL:

Compound E1 Compound E1 '

In one embodiment, Step 5- la comprises reacting Compound El with a hydrogenating agent to form Compound EG. In one embodiment, the hydrogenating agent is hydrogen gas, an alcohol ( e.g ., methanol, ethanol, or isopropanol), or an acid (e.g, formic acid). In one embodiment, the hydrogenating agent is hydrogen gas.

In one embodiment, Step 5- la is carried out in the presence of a catalyst. Catalysts for hydrogenation is well known in the art. In one embodiment, the catalyst is a nickel-, platinum-, palladium-, rhodium-, or ruthenium-based catalyst. In one embodiment, the catalyst is Pd/C, Pd/CaCCE, Pd/AhCE, Pd/Pt, or Raney nickel.

In one embodiment, Step 5- la is carried out in the presence of an organic solvent, for example, an alcoholic solvent such as methanol, ethanol or isopropanol; ethyl acetate; pyridine; acetic acid; cyclopentyl methyl ether (CPME); or N,N-dimethylformamide (DMF). In some embodiments, the solvent is a polar organic solvent. In some embodiments the organic solvent is a polar protic solvent. In some embodiments, the organic solvent is a polar aprotic solvent. In some embodiments, the organic solvent is an alcohol. In some embodiments, the organic solvent is methanol. In some embodiments, the organic solvent is ethanol. In some embodiments, the organic solvent is isopropanol. In some embodiments, the organic solvent is ethyl acetate. In some embodiments, the organic solvent is pyridine. In some embodiments, the organic solvent is acetic acid. In some embodiments, the organic solvent is CPME. In some embodiments, the organic solvent is DMF. In some embodiments, the organic solvent is DMSO. In some embodiments, the organic solvent is THF. In some embodiments, the organic solvent is dichloromethane. In some embodiments, the organic solvent is acetonitrile. The organic solvent may optionally be mixed with a co-solvent such as acetone or water and/or a base such as triethylamine. In some embodiments, Step 5- la is carried out at ambient pressure. In some embodiments, Step 5- la is carried out at elevated pressure.

In some embodiments, Step 5- la is carried out at a temperature below room temperature. In some embodiments, the temperature is between -20 and 20 °C. In some embodiments, the temperature is between -10 and 10 °C. In some embodiments, the temperature is between 0 and 10 °C. In some embodiments, the temperature is between 0 and 5 °C.

In some embodiments, Step 5-la is carried out for between 1-10 hours. In some embodiments, Step 5- la is carried out for between 1-5 hours. In some embodiments, Step 5- la is carried out for between 2-5 hours. In some embodiments, Step 5- la is carried out for between 3-5 hours.

In one embodiment, Step 5-1 further comprises Step 5-lb Step 5-lb. Oxidizing the 7-hydroxyl in Compound EG to form Compound El”:

Step 5-lb may be carried out with any suitable methods known in the art. In one embodiment, Step 5- lb is carried out in the presence of an oxidant and a co-oxidant. In one embodiment, the oxidant is an iodine-based oxidant. In one embodiment, the co-oxidant is a ruthenium salt. In one embodiment, Step 5-lb is carried out in the presence of RuCh and H5IO6. In one embodiment, the RuCl·, is present in a sub-stoichiometric quantity. In one embodiment, the oxidization reagent is a Dess-Martin periodinane ( 1,1,1 -triacetoxy- 1,1- dihydro- 1,2-benziodoxol) oxidation. In one embodiment, the Dess-Martin periodinane oxidation is carried out in a chlorinated solvent such as chloroform or dichloromethane. In one embodiment, the oxidization reaction comprises reaction with hypochlorite, for example, sodium hypochlorite, under acidic conditions, for example, provided by acetic acid. In one embodiment, the oxidization reaction is carried out in an aqueous solvent. In one embodiment, the oxidization reaction is a Jones reaction using sodium dichromate or chromic trioxide in dilute sulfuric acid.

In some embodiments, Step 5-lb is carried out at a temperature below room temperature. In some embodiments, the temperature is between -20 and 20 °C. In some embodiments, the temperature is between -10 and 10 °C. In some embodiments, the temperature is between 0 and 10 °C. In some embodiments, the temperature is between 0 and 5 °C.

In one embodiment, Step 5-1 further comprises Step 5-lc.

Step 5-lc. Deprotection of C-l 1 Alcohol of Compound El” to form Compound EE”:

In one embodiment, Step 5-lc involves deprotection of the Cl 1 alcohol by treating Compound El” with a deprotecting agent. In one embodiment, the deprotecting agent is a fluoride-based deprotecting agent. In one embodiment, the deprotecting agent is NFER

In one embodiment, Step 5-lc is carried out at a temperature above room temperature. In one embodiment, the temperature is between 40 °C and 100 °C. In one embodiment, the temperature is between 30 °C and 60 °C. In one embodiment, the temperature is between 35 °C and 45 °C. In one embodiment, the temperature about 40 °C.

In some embodiments, Step 5-lc is carried out for about 1-8 hours. In some embodiments, Step 5-lc is carried out for about 2-7 hours. In some embodiments, Step 5- lc is carried out for about 3-6 hours. In some embodiments, Step 5-lc is carried out for about 4-6 hours.

In one embodiment, Step 5-1 further comprises Step 5-ld.

Step 5-ld. Isomerization of C6 alkyl of EG” to form Compound F:

In one embodiment, Step 5-ld comprises treating Compound EG” with a base. In one embodiment, the base is an inorganic salt. In one embodiment, the base is NaOH. In one embodiment, the base is used as an aqueous solution. In one embodiment, the base used is a 10% aqueous solution of NaOH. In one embodiment, the base is used above room temperature. In one embodiment, the base is used in the presence of a polar protic solvent. In one embodiment, the base is used in the presence of an alcoholic solvent. In one embodiment, the alcoholic solvent is methanol. In one embodiment, the alcoholic solvent is ethanol. In one embodiment, the alcoholic solvent is isopropanol.

In one embodiment, Step 5-ld is carried out at a temperature above room temperature. In one embodiment, the temperature is between 30 °C and 100 °C. In one embodiment, the temperature is between 30 °C and 60 °C. In one embodiment, the temperature is between 45 °C and 55 °C. In one embodiment, the temperature about 50 °C.

In one embodiment, Step 5-ld comprises treating Compound EG” with a isomerization catalyst. In one embodiment, the isomerization catalyst is Fe(CO)s, Pd(OAc)2, a ruthenium salt, a Lewis acid, or a protic acid. In one embodiment, the isomerization comprises treating Compound El” with a base.

In one embodiment, Step 5-2 comprises reacting Compound E2b with a hydrogenating agent to form Compound F. In one embodiment, the hydrogenating agent is hydrogen gas, an alcohol ( e.g ., methanol, ethanol, or isopropanol), or an acid (e.g, formic acid). In one embodiment, the hydrogenating agent is hydrogen gas.

In one embodiment, the hydrogenation of Compound E2b is carried out in the presence of a catalyst. Catalysts for hydrogenation is well known in the art. In one embodiment, the catalyst is a nickel-, platinum-, palladium-, rhodium-, or ruthenium-based catalyst. In one embodiment, the catalyst is Pd/C, Pd/CaCCE, Pd/AhCE, Pd/Pt, or Raney nickel. In one embodiment, the hydrogenation of Compound E2b is carried out in the presence of an organic solvent, which may be an alcoholic solvent such as methanol, ethanol or isopropanol; ethyl acetate; pyridine; acetic acid; cyclopentyl methyl ether (CPME); or N,N-dimethylformamide (DMF). The organic solvent may optionally be mixed with a co-solvent such as acetone or water and/or a base such as triethylamine.

In one embodiment, Step 5-3 comprises reacting Compound E3b with a hydrogenating agent to form Compound F. In one embodiment, the hydrogenating agent is hydrogen gas, an alcohol ( e.g ., methanol, ethanol, or isopropanol), or an acid (e.g, formic acid). In one embodiment, the hydrogenating agent is hydrogen gas.

In one embodiment, the hydrogenation of Compound E3b is carried out in the presence of a catalyst. Catalysts for hydrogenation is well known in the art. In one embodiment, the catalyst is a nickel-, platinum-, palladium-, rhodium-, or ruthenium-based catalyst. In one embodiment, the catalyst is Pd/C, Pd/CaCCE, Pd/AhCE, Pd/Pt, or Raney nickel.

In one embodiment, the hydrogenation of Compound E3b is carried out in the presence of an organic solvent, which may be an alcoholic solvent such as methanol, ethanol or isopropanol; ethyl acetate; pyridine; acetic acid; cyclopentyl methyl ether (CPME); or N,N-dimethylformamide (DMF). The organic solvent may optionally be mixed with a co-solvent such as acetone or water and/or a base such as triethylamine.

In one embodiment, Step 6 comprises reacting Compound F with a reducing agent to reduce the 7-keto to 7-OH and the 3-keto to 3-OH. In one embodiment, Step 6 reduction of Compound F comprises reducing the 7-keto to 7-a-OH. In one embodiment, Step 6 reduction of Compound F comprises reducing the 3-keto to 3-a-OH. In one embodiment, Step 6 reduction of Compound F comprises reducing the 7-keto to 7-a-OH and the 3-keto to 3-a-OH.

Reducing agents suitable for carrying out Step 6 are well known in the art. In one embodiment, the reducing agent is a hydride. In one embodiment, the reducing agent is lithium aluminum hydride (LiAlH4). In one embodiment, the reducing agent is sodium borohydride (NaBHi). In one embodiment, the reducing agent is hydrogen gas (without or with a suitable catalyst). In one embodiment, the reducing agent is sodium amalgam (Na(Hg)). In one embodiment, the reducing agent is sodium-lead alloy (Na + Pb). In one embodiment, the reducing agent is zinc amalgam (Zn(Hg)). In one embodiment, the reducing agent is diborane. In one embodiment, the reducing agent is compounds containing the Fe2+ ion, such as iron(II) sulfate, compounds containing the Sn2+ ion, such as tin(II) chloride. In one embodiment, the reducing agent is sulfite. In one embodiment, the reducing agent is a dithionate ( e.g ., Na ₂ S ₂ 0 ₆ ). In one embodiment, the reducing agent is a thiosulfate (e.g., Na ₂ S ₂ C> ₃ ). In one embodiment, the reducing agent is an iodide. In one embodiment, the reducing agent is hydrazine, diisobutylaluminium hydride. In one embodiment, the reducing agent is oxalic acid. In one embodiment, the reducing agent is formic acid. In one embodiment, the reducing agent is ascorbic acid. In one embodiment, the reducing agent is phosphites. In one embodiment, the reducing agent is hypophosphites. In one embodiment, the reducing agent is phosphorous acid. In one embodiment, the reducing agent is carbon monoxide. In one embodiment, the reducing agent is carbon (C). In one embodiment, the reducing agent is or tris-2- carboxyethylphosphine hydrochloride (TCEP). In one embodiment, the reducing agent is NaBH _4.

In one embodiment, Step 6 is carried out under a basic condition. In one embodiment, Step 6 is carried out in the presence of a strong base. In one embodiment, the strong base is sodium. In one embodiment, the strong base is potassium hydroxide.

Any suitable solvent may be used to carry out Step 6. In one embodiment, the solvent comprises tetrahydrofuran, water, or a mixture thereof. In one embodiment, the solvent is an aqueous solvent. In one embodiment, the solvent is water.

In one embodiment, Step 6 is carried out at a temperature above room temperature. In one embodiment, the temperature is between 50 °C and 100 °C. In one embodiment, the temperature is between 80 °C and 10 °C. In one embodiment, the temperature is between 90 °C and 100 °C. In one embodiment, the temperature is between 90 °C and 95 °C.

In some embodiments, Step 6 is carried out for about 1-8 hours. In some embodiments, Step 6 is carried out for about 1-7 hours. In some embodiments, Step 6 is carried out for about 2-6 hours. In some embodiments, Step 6 is carried out for about 3-6 hours.

In some embodiments, compounds of the application, or pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof are isotopically labeled (or radiolabeled). Examples of isotopes that can be incorporated into compounds of the application, or pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof include isotopes of hydrogen, carbon, nitrogen, fluorine, such as ² H, ¾, ^U C, ¹³ C , ¹⁴ C, and ¹⁸ F. In some embodiments, compounds of the application are deuterated, i.e., incorporate ² H, tritiated, i.e., incorporate ¾, and radiolabeled with carbon-14, i.e., ¹⁴ C. Isotopically labeled compounds of the application, or pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof can generally be prepared by carrying out the procedures herein, by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

In certain embodiments, the present application relates to a method of preparing a compound of formula 1-9 or I-9a: or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, wherein R ² -R ¹⁰ are as described herein.

In some embodiments, the compound of formula 1, 1-9, or I-9a, wherein R ⁷ is OSCriH, SO ₃ H, OSO ₂ NH ₂ , SO ₂ NH ₂ , OPO ₃ H ₂ , PO ₃ H ₂ , CO ₂ H, C(0)NH0H, tetrazolyl, oxadiazolyl, thiadiazolyl, 5-oxo-l,2,4-oxadiazolyl, 5-oxo-l,2,4-thiadiazolyl, oxazolidinedionyl, thiazolidinedionyl, 3-hydroxyisoxazolyl, 3-hydroxyisothiazolyl, pyrimidine, 3,5-difluoro-4-hydroxyphenyl or 2,4-difluoro-3-hydroxyphenyl, all of which can be optionally further substituted, can be prepared using synthetic procedures described in WO 2017/062763, US20160130297, US20160145295, US20160145296, US20160185815, US20160229886, US20160289262, and WO 2018/081285 or using other procedure known in the art. The presently disclosed method provides an efficient synthesis of intermediates that can be further elaborated to various side chain analogs, including, but not limited to compounds with the following side chains: where Z is any same or different appropriate substituent, Y is a heteroatom (e.g., O, N, or S), and he is any appropriate heterocycle (e.g., aromatic or non-aromatic 4-6-memberd ring), which, for example, can include, but is not limited to the following groups In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-1.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-1 and Step 3, and optionally Step 2-1.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-1, Step 3, Step 4-1, and optionally Step 2-1.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-1, Step 3, Step 4-1, Step 5-1, and optionally Step 2-1.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-1, Step 3, Step 4-1, Step 5-1, Step 6, and optionally Step 2-1.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-1, Step 3, Step 4-2a, Step 4-2b, and optionally Step 2-1.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-1, Step 3, Step 4-2a, Step 4-2b, Step 5-2, and optionally Step 2-1.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-1, Step 3, Step 4-2a, Step 4-2b, Step 5-2, Step 6, and optionally Step 2-1. In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-1, Step 3, Step 4-3a, Step 4-3b, and optionally Step 2-1.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-1, Step 3, Step 4-3a, Step 4-3b, Step 5-3, and optionally Step 2-1.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-1, Step 3, Step 4-3a, Step 4-3b, Step 5-3, Step 6, and optionally Step 2-1.

It is understood that when Step 2-1 is omitted, the protecting groups (PG) shown in Compound C, Compound D, Compound El, Compound EG, Compound El”, Compound E2a, Compound E2b, Compound E3a, Compound E3b, and Compound F, would be replaced with H.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-2.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 3.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 4-1, Step 4- 2a and Step 4-2b, or Step 4-3 a and Step 4-3b.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 5-1, Step 5- 2, or Step 5-3. In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 6.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-2 and Step 3, and optionally Step 2-2.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-2, Step 3, Step 4-1, and optionally Step 2-2.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-2, Step 3, Step 4-1, Step 5-1, and optionally Step 2-2.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-2, Step 3, Step 4-1, Step 5-1, Step 6, and optionally Step 2-2.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-2, Step 3, Step 4-2a, Step 4-2b, and optionally Step 2-2.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-2, Step 3, Step 4-2a, Step 4-2b, Step 5-2, and optionally Step 2-2.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-2, Step 3, Step 4-2a, Step 4-2b, Step 5-2, Step 6, and optionally Step 2-2. In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-2, Step 3, Step 4-3a, Step 4-3b, and optionally Step 2-2.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-2, Step 3, Step 4-3a, Step 4-3b, Step 5-3, and optionally Step 2-2.

In certain embodiments, the present application relates to a method of preparing a compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, comprising Step 1-2, Step 3, Step 4-3a, Step 4-3b, Step 5-3, Step 6, and optionally Step 2-2.

It is understood that when Step 2-2 is omitted, the protecting groups (PG) shown in Compound C, Compound D, Compound El, Compound EG, Compound El”, Compound E2a, Compound E2b, Compound E3a, Compound E3b, and Compound F, would be replaced with H.

The synthetic processes of the present application can tolerate a wide variety of functional groups, therefore various substituted starting materials can be used. The processes generally provide the desired final compound at or near the end of the overall process, although it may be desirable in certain instances to further convert the compound to a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof.

In one aspect, the present application relates to Compound A, Compound B, Compound B’, Compound C, Compound C’, Compound D, Compound El, Compound EE, Compound El”, EE”, Compound E2a, Compound E2b, Compound E3a, Compound E3b, or Compound F.

In one aspect, the present application relates to Compound A-l, Compound B-l, Compound B-E, Compound C, Compound C’, Compound D, Compound El, Compound EE, Compound El”, EE”, Compound E2a, Compound E2b, Compound E3a, Compound E3b, or Compound F.

In one embodiment, the present application relates to Compound B. In one embodiment, the present application relates to Compound B\

In one embodiment, the present application relates to Compound B-l.

In one embodiment, the present application relates to Compound B-G

In one embodiment, the present application relates to Compound C.

In one embodiment, the present application relates to Compound C’.

In one embodiment, the present application relates to Compound D.

In one embodiment, the present application relates to Compound El, Compound EG, Compound El”, or Compound EE”.

In one embodiment, the present application relates to Compound E2a or Compound

E2b.

In one embodiment, the present application relates to Compound E3a or Compound

E3b.

In one embodiment, the present application relates to Compound F.

In some embodiments, the compound of formula 1-9 or I-9a is further transformed into the compound of formula I, wherein R ⁷ is OSO3H, SO3H, OSO2NH2, SO2NH2, OPO3H2, PO3H2, C(0)NH0H, tetrazolyl, oxadiazolyl, thiadiazolyl, 5-oxo-l,2,4- oxadiazolyl, 5-oxo-l,2,4-thiadiazolyl, oxazolidinedionyl, thiazolidinedionyl, 3- hydroxyisoxazolyl, 3-hydroxyisothiazolyl, or 2,4-difluoro-3-hydroxyphenyl, and R ¹ is alkoxy or oxo using known synthetic procedures. In some embodiments, the compound of formula 1-9 or I-9a is further transformed into the compound of formula I, wherein R ⁷ is OSO3H, SO3H, OSO2NH2, SO2NH2, OPO3H2, PO3H2, CO2H, C(0)NH0H, tetrazolyl, oxadiazolyl, thiadiazolyl, 5-oxo-l,2,4-oxadiazolyl, 5-oxo-l,2,4-thiadiazolyl, oxazolidinedionyl, thiazolidinedionyl, 3-hydroxyisoxazolyl, 3-hydroxyisothiazolyl, pyrimidine, 3,5-difluoro-4-hydroxyphenyl or 2,4-difluoro-3-hydroxyphenyl, all of which can be optionally further substituted, and R ¹ is alkoxy or oxo using synthetic procedures described in WO 2017/062763, US20160130297, US20160145295, US20160145296, US20160185815, US20160229886, US20160289262, and WO2018/081285 or using other procedure known in the art.

For example, compounds wherein R ⁷ is tetrazolyl, oxadiazolyl, thiadiazolyl, 5-oxo- 1,2,4-oxadiazolyl, 5-oxo-l,2,4-thiadiazolyl, oxazolidinedionyl, thiazolidinedionyl, 3- hydroxyisoxa-zolyl, 3-hydroxyisothiazolyl, or 2,4-difluoro-3-hydroxyphenyl can be prepared from the corresponding carboxylic acid via a coupling with the required R ⁷ - containing boronic acids:

In some embodiments, R ¹¹ protecting group is selected from C(0)Ci-C ₄ alkyl, Ci- C ₆ alkoxycarbonyl, optionally substituted aryloxycarbonyl, benzoyl, benzyl, pivaloyl, tetrahydropyranyl ether, tetrahydrofuranyl, 2-methoxyethoxymethyl ether, methoxymethyl ether, ethoxyethyl ether, p-methoxybenzyl ether, methylthiomethyl ether, triphenylmethyl, dimethoxytrityl, methoxytrityl, and silyl ether. In one embodiment, the silyl ether is selected from trimethyl silyl ether, triethylsilyl ether, triisopropyl silyl ether, tert- butyldimethylsilyl ether, and /c77-buty 1 di pheny 1 si 1 y 1 ether. In one embodiment, the R ¹¹ protecting group is benzoyl or acetyl. In one embodiment, the R ¹¹ protecting group is C(0)Ci-C ₄ alkyl. In one embodiment, the R ¹¹ protecting group is acetyl. In some embodiments R ¹¹ is H.

In certain embodiments, the process of the present application provides a compound of formula II: or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof.

In certain embodiments, the process of present application provides a compound of formula III: in, or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof.

In some embodiments, the present application relates to a method of making a compound of formula Ilia: Ilia, or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof.

In some embodiments, the present application relates to a method of making a compound of formula Illb: mb, or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof.

In certain embodiments, of the present application relates to a method of preparing compound of formula I according to the following biocatalytic process:

A wide variety of microbial organisms are capable of catabolizing xenobiotics. Organisms which are capable of biooxidation can be drawn from both mesophiles or extremeophiles, including but not limited to either wild type or genetically modified prokaryotes and eukaryotes. In some cases, the organisms are classified in the same genus but differ in type strain based on isolation source or growth conditions. Examples of microbial organisms include but are not limited to the following: bacteria, yeast, fungi, algea, and molds. Fermentation can take place during any phase of the microbial lifecycle including the lag phase, exponential phase, or stationary phase, using either aerobic and anaerobic conditions. Table 1 shows several references that identified microorganisms capable of biooxidation of sterioid derivatives.

Table 1: Biooxidation of steroid derivatives

The suitable organisms include, but are not limited to Streptomyces diastatochromogenes, Streptomyces griseus, Streptomyces sp, Streptomyces rimosus, Streptomyces albidoflavus, Streptomyces avermitilis, Streptomyces fradiae, Streptomyces griseolus, Streptomyces platensis, Streptomyces violascens, Streptomyces ochraceiscleroticus, Methylobacterium extorquens, Methylophaga thalassica, Rhizopus stolonifer, Absidia coerulea , Beauveria has si ana, Cunninghamella elegans, Rhizopus oryzae, Gliocladium roseum, Verticillium lecanii, Fusarium oxysporum, Curvularia lunata, Mortierella isabellina, Cunninghamella blakesleeana, Mortierella ramanniana, Mucor rouxii, Rhodococcus sp. , Streptomyces griseus, Gluconobacter oxydans, Sporobolomyces salmonicolor, Saccharyomyces cerevisiae, Candida parapsilosis, Rhodococcus erythropolis, Rhodotorula glutinis, Kluyveromyces lactis, Debrayomyces hansenii , Pichia angusta, Kluyveromyces polysporus, Pichia guiliermondii , Saccharyomyces cerevisie S288c, Lactobacillus brevis, Ruegeria pomeroyi (Silicibacter pomeroyi), Leucanostoc mesenteroides, Burkholderia thailandensis, Bradyrhizobium sp., Rhodopseudomonas palustris, Nakamurella multipartite, Sphingomonas wittichii, Rhodopiruellula baltica, Rhodococcus opacus, Helicostylum piriforme, Agrobacterium sp.,Streptomyces lincolnensis, Bacillus megaterium, Pseudomonas sp., Penicillium chrysogenum, Nonomuraea recticatena, Verticillium theobromae, Cunninghamella echinulate, Syncephalastrum racemosum, Absidia pseudocylindrospora, Petromyces alliaceus, Aspergillus ochraceus, Aspergillus oryzae, Mucor plumbeus, Cyathus striatus, Absidia corymbifera, Gliocladium viride, Geotrichum candidum, Kluyveromyces marxianus, Cladophialophora psammophila, Cladophialophora immunda, Pseudeurotium zonatum, Cunninghamella echinulate, Cladosporium sphaerospermum, Streptomyces sp., Azoarcus toluvorans, Pseudomonas chlororaphis, Phanerochaete chrysosporium, Pseudomonas putida mt-2, Cupriavidus necator, Cupriavidus basilensis, Novosphingobium subterraneum, Novosphingobium aromaticivorans, Rhodococcus rhodochrous, Novosphingobium stygium, Burkholderia sp., Pseudoxanthomonas spadix, Mycobacterium gilvum, Delftia acidovorans, Paracoccus denitrificans, Mycobacterium neoaurum, Streptomyces rimosus, Streptomyces ambofaciens, Pleurotis sapidus, Emericella nidulans, Fusarium solani, Comamonas tester oni, Fusarium graminearum, Fusarium longipes, Fusarium cerealis, Fusarium sporotrichiodes, Fusarium equiseti, Fusarium cerealis, Fusarium incarnatum, Nonomuraea dietziae, Methyloccus capsulatus, Rhizopus stolonifera, Mucor flavus, Streptomyces lilacinus, Xanthobacter sp., Rhizopus microspores, Sporidiobolus johnsonii, Bradyrhizobium japonicum, Ogataea methanolica, Bacillus benzeovorans, Cupriavidus necator, Mycobacterium parafortuitum, Actinoplanes sp., Paracoccus pantotrophus, Streptomyces rochii, Mycoplasma lipofaciens, Aspergillus niger 402, Agrocybe aegerita, Agrocybe aegerita, Caldariomyces fumago, Mycobacterium sp., Rhodococcus sp., Rhodococcus rhodochrous, Rhizopus oryzae, Phanerochaete chrysosporium, Fusarium ciliatum, Escherichia coli, Mucor griseocyanus, Rhodopirellula baltica.

In one of the embodiments, the microorganisms are grown in about 50 ml conical centrifuge tubes containing about 10 ml of the required growth medium. In some embodiments, the growth media can be Nutrient Broth ( e.g. , 15 g Peptone, 3 g Yeast extract, 6 g NaCl, 1 g Glucose); Gym Streptomyces media (e.g, 4 g Glucose, 4 g Yeast extract, 10 g Malt extract); Malt extract peptone ( e.g ., 30 g Malt extract, 3 g Peptone); or Potato dextrose media (e.g., 30 g Potato extract, 10 g Glucose). Following inoculation of media from microbial glycerol stocks, the cultures are incubated for 7 days at about 28°C with shaking. After that, about 1 ml of each microbial culture was transferred into the wells of deep 96 well plates. The plates are incubated at about 28 °C with agitation for 48 hours before addition of OCA dissolved in DMSO (to the final concentration of about 2 mg/ml). The plates are incubated for a further 36 hours before addition of about 1 ml of 100% acetonitrile, after which the plates are incubated at room temperature before centrifugation at 9,000 xg for 15 mins. About 200 pL of supernatant are transferred to a clean 96 well plate before analysis by UHPLC (ultra-high performance liquid chromatography). In one of the embodiments, the biotransformation process can utilize the method(s) reported in Ishida, et ah, Chem. Pharm. Bull. 46 (1998), 12-16 for natural product 3a,7a,1 la-trihydroxy-5 -cholan-24-oic acid (1 la-OH CDCA).

In one of the embodiments, the present application provides compounds of formula

1. 1-9, II, III, Ilia, or Illb, wherein R ⁴ is in the a-position. In one embodiment, the present application provides compounds of formula 1, 1-9, 1-9a, II, III, Ilia, or Illb, wherein R ⁴ is C ₁ -C ₄ alkyl. In one of the embodiments, the present application provides compounds of formula 1, 1-9, 1-9a, II, III, Ilia, or Illb, wherein R ⁴ is methyl, ethyl, or propyl. In one embodiment, R ⁴ is ethyl. In another embodiment, R ⁴ is alpha-ethyl. In one of the embodiments, the present application provides compounds of formula 1, 1-9, 1-9a, II, III, Ilia, or Illb, wherein R ⁴ is H or halogen. In one of the embodiments, the present application provides compounds of formula 1, 1-9, 1-9a, II, III, Ilia, or Illb, wherein R ⁴ is C1-C6 alkyl optionally substituted with one or more halogen or OH. In one of the embodiments, the present application provides compounds of formula 1, 1-9, 1-9a, II, III, Ilia, or Illb, wherein R ⁴ is C2-C6 alkenyl or C2-C6 alkynyl.

In one of the embodiments, the present application provides compounds of formula

1. 1-9, 1-9a, II, III, Ilia, or Illb, wherein R ⁷ is OH, OSO ₃ H, SO ₃ H, OSO ₂ NH ₂ , SO ₂ NH ₂ , OPO ₃ H ₂ , PO ₃ H ₂ , CO ₂ H, or C(0)NHOH.

In one of the embodiments, the present application provides compounds of formula 1, 1-9, 1-9a, II, III, Ilia, or Illb, wherein R ⁷ is OH, OSO ₃ H, OSO ₂ NH ₂ , OPO ₃ H ₂ , or C0 ₂ H. In one of the embodiments, the present application provides compounds of formula

1, 1-9, 1-9a, II, III, Ilia, or Illb, wherein R ⁷ is OH.

In one of the embodiments, the present application provides compounds of formula

1, 1-9, 1-9a, II, III, Ilia, or Illb, wherein R ⁷ is C0 ₂ H.

In one of the embodiments, the present application provides compounds of formula

1, 1-9, 1-9a, II, III, Ilia, or Illb, wherein R ⁷ is 0S0 ₃ H.

In one of the embodiments, the present application provides compounds of formula

1, 1-9, 1-9a, II, III, Ilia, or Illb, wherein R ⁷ is SO ₃ H.

In one of the embodiments, the present application provides compounds of formula

1, 1-9, 1-9a, II, III, Ilia, or Illb, wherein R ⁷ is OSO2NH2 or SO2NH2.

In one of the embodiments, the present application provides compounds of formula

1, 1-9, 1-9a, II, III, Ilia, or Illb, wherein R ⁷ is OPO ₃ H ₂ , PO ₃ H ₂ , or C(0)NH0H.

In one of the embodiments, the present application provides compounds of formula

1, 1-9, 1-9a, II, III, Ilia, or Illb, wherein R ⁷ is tetrazolyl, oxadiazolyl, thiadiazolyl, 5-oxo-

1.2.4-oxadiazolyl, 5-oxo-l,2,4-thiadiazolyl, oxazolidine-dionyl, thiazolidine-dionyl, 3- hydroxyisoxazolyl, 3-hydroxyisothiazolyl, or 2,4-difluoro-3-hydroxyphenyl.

In one of the embodiments, the present application provides compounds of formula 1, 1-9, 1-9a, II, III, Ilia, or Illb, wherein R ⁷ is OH, OSO ₃ H, OSO ₂ NH ₂ , OPO ₃ H ₂ , C0 ₂ H, tetrazolyl, oxadiazolyl, thiadiazolyl, 5-oxo-l,2,4-oxadiazolyl, 5-oxo-l,2,4-thiadiazolyl, oxazolidine-dionyl, thiazolidine-dionyl, 3-hydroxyisoxazolyl, 3-hydroxyisothiazolyl, or

2.4-difluoro-3 -hy droxyphenyl .

In one of the embodiments, the present application provides compounds of formula

1, 1-9, 1-9a, II, III, Ilia, or Illb, wherein R ⁵ is OSO ₃ H, 0C(0)CH , or OPO ₃ H ₂ .

In one of the embodiments, the present application provides compounds of formula

1, 1-9, 1-9a, II, III, Ilia, or Illb, wherein R ⁵ and R ⁶ taken together with the carbon atom to which they are attached form a carbonyl.

In one of the embodiments, the present application provides compounds of formula I, la, lb, 1-9, II, or III, wherein m is 0.

In one of the embodiments, the present application provides compounds of formula I, la, lb, 1-9, II, or III, wherein m is 1. In one of the embodiments, the present application provides compounds of formula I, la, lb, 1-9, II, or III, wherein m is 2.

In one of the embodiments, the present application provides compounds of formula I, la, lb, 1-9, II, or III, wherein n is 1.

In one of the embodiments, the present application provides compounds of I, la, lb, 1-9, II, or III, wherein p is 0.

In one of the embodiments, the present application provides compounds of formula I, la, lb, 1-9, or II, , wherein R ¹ is in the b-position (beta-position).

In one embodiment, the compound prepared by the methods of the present application is Compound 100:

In one aspect, the method of the present application produces a substantially pure compound of formula I, or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof.

The term “purity” as used herein refers to the amount of compound of formula I based on analytic methods commonly used in the art ( e.g ., HPLC). In some embodiments, the compound of formula I has a purity of greater than about 90%. In one embodiment, the compound of formula I has a purity of greater than about 95%. In one embodiment, the compound of formula I has a purity of greater than about 98%. For example, the purity of the synthesized compound of Formula I is about 96.0%, about 97.0%, about 98.0%, about 99.0%, or about 100%. For example, the purity of the synthesized compound of formula I is 98.5%, 99.0%, or 99.5%. In one embodiment, the purity is determined by HPLC.

The present application provides methods for the synthesis of highly pure compounds of formula I which are safe and which can produce compounds of formula I on a large scale. In one embodiment, the method of the present application produces compounds of formula I in high yield (>98%) and with limited number of impurities. The compounds of the application have asymmetric centers and can be isolated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials. Many geometric isomers of olefins, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present application. Cis and trans geometric isomers of the compounds of the application and can be isolated as a mixture of isomers or as separate isomeric forms. All chiral, diastereomeric, racemic, and geometric isomeric forms of a structure are intended, unless specific stereochemistry or isomeric form is specifically indicated. All processes used to prepare compounds of the present application and intermediates made therein are considered to be part of the present application. All tautomers of shown or described compounds are also considered to be part of the present application. Furthermore, the application also includes metabolites of the compounds described herein.

The application also comprehends isotopically-labeled compounds of the application, or pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, which are identical to those recited in formulae of the application and following, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature. Examples of isotopes that can be incorporated into compounds of the application, or pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof include isotopes of hydrogen, carbon, nitrogen, fluorine, such as ² H, ¾, ^U C, ¹³ C, ¹⁴ C, and ¹⁸ F.

Deuterated, i.e., ² H, tritiated, /. e. , ¾, and carbon-14, /. e. , ¹⁴ C, isotopes may be used for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., ² H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be used in some circumstances. Isotopically labeled compounds of the application, or pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof can generally be prepared by carrying out the procedures disclosed in the Schemes and/or in the Examples, by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent. However, one skilled in the art will recognize that not all isotopes can be included by substitution of the non-isotopically labeled reagent. In one embodiment, compounds of the application, or pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof are not isotopically labeled. In one embodiment, deuterated compounds of the application are useful for bioanalytical assays. In another embodiment, compounds of the application, or pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof are radiolabeled.

Pharmaceutical Compositions

A "pharmaceutical composition" is a formulation containing one or more compounds of the application in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. It can be advantageous to formulate compositions 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 subject to be treated; each unit containing a predetermined quantity of active reagent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms are dictated by and directly dependent on the unique characteristics of the active reagent and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active agent for the treatment of individuals.

Possible formulations include those suitable for oral, sublingual, buccal, parenteral (e.g, subcutaneous, intramuscular, or intravenous), rectal, topical including transdermal, intranasal and inhalation administration. Most suitable means of administration for a particular patient will depend on the nature and severity of the disease being treated or the nature of the therapy being used and on the nature of the active compound, but where possible, oral administration may be used for the prevention and treatment of FXR mediated diseases and conditions. Formulations suitable for oral administration may be provided as discrete units, such as tablets, capsules, cachets, lozenges, each containing a predetermined amount of the active compound; as powders or granules; as solutions or suspensions in aqueous or non-aqueous liquids; or as oil-in- water or water-in-oil emulsions. Formulations suitable for sublingual or buccal administration include lozenges comprising the active compound and, typically a flavored base, such as sugar and acacia or tragacanth and pastilles comprising the active compound in an inert base, such as gelatin and glycerin or sucrose acacia.

Formulations suitable for parenteral administration typically comprise sterile aqueous solutions containing a predetermined concentration of the active compound; the solution may be isotonic with the blood of the intended recipient. Additional formulations suitable for parenteral administration include formulations containing physiologically suitable co-solvents and/or complexing agents such as surfactants and cyclodextrins. Oil- in-water emulsions are also suitable formulations for parenteral formulations. Although such solutions may be administered intravenously, they may also be administered by subcutaneous or intramuscular injection.

Formulations suitable for rectal administration may be provided as unit-dose suppositories comprising the active ingredient in one or more solid carriers forming the suppository base, for example, cocoa butter.

Formulations suitable for topical or intranasal application include ointments, creams, lotions, pastes, gels, sprays, aerosols, and oils. Suitable carriers for such formulations include petroleum jelly, lanolin, polyethylene glycols, alcohols, and combinations thereof.

Formulations of the application may be prepared by any suitable method, typically by uniformly and intimately admixing the active compound with liquids or finely divided solid carriers or both, in the required proportions and then, if necessary, shaping the resulting mixture into the desired shape.

For example, a tablet may be prepared by compressing an intimate mixture comprising a powder or granules of the active ingredient and one or more optional ingredients, such as a binder, lubricant, inert diluent, or surface-active dispersing agent, or by molding an intimate mixture of powdered active ingredient and inert liquid diluent. Suitable formulations for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers, or insufflators. For pulmonary administration via the mouth, the particle size of the powder or droplets is typically in the range of 0.5-10 pm, or may be about 1-5 pm, to ensure delivery into the bronchial tree. For nasal administration, a particle size in the range of 10-500 pm may be used to ensure retention in the nasal cavity.

Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of the active ingredient in a liquefied propellant.

During use, these devices discharge the formulation through a valve adapted to deliver a metered volume, typically from 10 to 150 pm, to produce a fine particle spray containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and mixtures thereof. The formulation may additionally contain one or more co-solvents, for example, ethanol surfactants, such as oleic acid or sorbitan trioleate, anti-oxidants, and suitable flavoring agents.

Nebulizers are commercially available devices that transform solutions or suspensions of the active ingredient into a therapeutic aerosol mist either by means of acceleration of a compressed gas typically air or oxygen, through a narrow venturi orifice, or by means of ultrasonic agitation. Suitable formulations for use in nebulizers consist of the active ingredient in a liquid carrier and comprise up to 40% w/w of the formulation, preferably less than 20% w/w. The carrier is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride. Optional additives include preservatives if the formulation is not prepared sterile, for example, methyl hydroxy-benzoate, anti-oxidants, flavoring agents, volatile oils, buffering agents, and surfactants.

Suitable formulations for administration by insufflation include finely comminuted powders which may be delivered by means of an insufflator or taken into the nasal cavity in the manner of a snuff. In the insufflator, the powder is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually- operated pump. The powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant. The active ingredient typically comprises from 0.1 to 100 % w/w of the formulation.

In a further embodiment, the present application provides a pharmaceutical composition comprising, as active ingredient, a compound of the application together, and/or in admixture, with at least one pharmaceutical carrier or diluent. These pharmaceutical compositions may be used in the prevention or treatment of the foregoing diseases or conditions.

The carrier is pharmaceutically acceptable and must be compatible with, i.e., not have a deleterious effect upon, the other ingredients in the composition. The carrier may be a solid or liquid and is preferably formulated as a unit dose formulation, for example, a tablet which may contain from 0.05 to 95% by weight of the active ingredient. If desired, other physiologically active ingredients may also be incorporated in the pharmaceutical compositions of the application.

In addition to the ingredients specifically mentioned above, the formulations of the present application may include other agents known to those skilled in the art of pharmacy, having regard for the type of formulation in issue. For example, formulations suitable for oral administration may include flavoring agents and formulations suitable for intranasal administration may include perfumes.

In one of the embodiments, the present application provides a pharmaceutical composition comprising the compounds of formula I and a pharmaceutically acceptable carrier or excipient.

Methods of Treatment

The compounds of the application ( e.g ., compounds of formula I, la, lb, 1-9, II, III, Ilia, Illb, and 100) are useful for therapy in subjects such as mammals, including humans. In particular, the compounds of the application are useful in a method of treating or preventing a disease or condition in a subject comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the disease or condition is FXR-mediated (e.g., FXR plays a role in the initiation or progress of the disease or condition). In one embodiment, the disease or condition is mediated by decreased FXR activity. In one embodiment, the disease or condition is selected from cardiovascular disease, chronic liver disease, lipid disorder, gastrointestinal disease, renal disease, metabolic disease, cancer, and neurological disease.

In one embodiment, the application relates to a method of treating or preventing cardiovascular disease in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the application relates to a method of treating cardiovascular disease. In one embodiment, cardiovascular disease selected from atherosclerosis, arteriosclerosis, dyslipidemia, hypercholesteremia, hyperlipidemia, hyperlipoproteinemia, and hypertriglyceridemia.

The term "hyperlipidemia" refers to the presence of an abnormally elevated level of lipids in the blood. Hyperlipidemia can appear in at least three forms: (1) hypercholesterolemia, i.e., an elevated cholesterol level; (2) hypertriglyceridemia, /. e. , an elevated triglyceride level; and (3) combined hyperlipidemia, i.e., a combination of hypercholesterolemia and hypertriglyceridemia.

The term "dyslipidemia" refers to abnormal levels of lipoproteins in blood plasma including both depressed and/or elevated levels of lipoproteins ( e.g ., elevated levels of LDL, VLDL and depressed levels of HDL).

In one embodiment, the application relates to a method selected from reducing cholesterol levels or modulating cholesterol metabolism, catabolism, absorption of dietary cholesterol, and reverse cholesterol transport in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof.

In another embodiment, the application relates to a method of treating or preventing a disease affecting cholesterol, triglyceride, or bile acid levels in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the application relates to a method of lowering triglycerides in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof.

In one embodiment, the application relates to a method of treating or preventing a disease state associated with an elevated cholesterol level in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the application relates to a method of treating a disease state associated with an elevated cholesterol level in a subject. In one embodiment, the application relates to a method of preventing a disease state associated with an elevated cholesterol level in a subject. In one embodiment, the disease state is selected from coronary artery disease, angina pectoris, carotid artery disease, strokes, cerebral arteriosclerosis, and xanthoma.

In one embodiment, the application relates to a method of treating or preventing a lipid disorder in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the application relates to a method of treating a lipid disorder. In one embodiment, the application relates to a method of preventing a lipid disorder.

Lipid disorders are the term for abnormalities of cholesterol and triglycerides.

Lipid abnormalities are associated with an increased risk for vascular disease, and especially heart attacks and strokes. Abnormalities in lipid disorders are a combination of genetic predisposition as well as the nature of dietary intake. Many lipid disorders are associated with being overweight. Lipid disorders may also be associated with other diseases including diabetes, the metabolic syndrome (sometimes called the insulin resistance syndrome), underactive thyroid or the result of certain medications (such as those used for anti -rejection regimens in people who have had transplants).

In one embodiment, the application relates to a method of treating or preventing one or more symptoms of disease affecting lipid metabolism (z.e., lipodystrophy) in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the application relates to a method of treating one or more symptoms of a disease affecting lipid metabolism. In one embodiment, the application relates to a method of preventing one or more symptoms of a disease affecting lipid metabolism.

In one embodiment, the application relates to a method of decreasing lipid accumulation in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof.

In one embodiment, the application relates to a method of treating or preventing liver disease in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the application relates to a method of treating chronic liver disease. In one embodiment, the application relates to a method of preventing chronic liver disease. In one embodiment, the FXR mediated liver disease is selected from a cholestatic liver disease such as primary biliary cirrhosis (PBC) also known as primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), chronic liver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), hepatitis C infection, alcoholic liver disease, liver damage due to progressive fibrosis, and liver fibrosis. Other examples of FXR mediated diseases also include portal hypertension, bile acid diarrhea, hyperlipidemia, high LDL-cholesterol, high HDL cholesterol, high triglycerides, and cardiovascular disease. Other liver diseases include cerebrotendinous xanthomatosis (CTX), drug induced cholestasis, intrahepatic cholestasis of pregnancy, parenteral nutrition associated cholestasis (PNAC), bacterial overgrowth or sepsis associated cholestasis, autoimmune hepatitis, chronic viral hepatitis, liver transplant associated graft versus host disease, living donor transplant liver regeneration, congenital hepatic fibrosis, choledocholithiasis, granulomatous liver disease, intra- or extrahepatic malignancy, Sjogren's syndrome, Sarcoidosis, Wilson's disease, Gaucher's disease, hemochromatosis, and alpha 1 -antitrypsin deficiency. In one embodiment, the application relates to a method of treating or preventing one or more symptoms of cholestasis, including complications of cholestasis in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the application relates to a method of treating one or more symptoms of cholestasis. In one embodiment, the application relates to preventing one or more symptoms of cholestasis.

Cholestasis is typically caused by factors within the liver (intrahepatic) or outside the liver (extrahepatic) and leads to the accumulation of bile salts, bile pigment bilirubin, and lipids in the blood stream instead of being eliminated normally. Intrahepatic cholestasis is characterized by widespread blockage of small ducts or by disorders, such as hepatitis, that impair the body's ability to eliminate bile. Intrahepatic cholestasis may also be caused by alcoholic liver disease, primary biliary cirrhosis, cancer that has spread (metastasized) from another part of the body, primary sclerosing cholangitis, gallstones, biliary colic, and acute cholecystitis. It can also occur as a complication of surgery, serious injury, cystic fibrosis, infection, or intravenous feeding or be drug induced. Cholestasis may also occur as a complication of pregnancy and often develops during the second and third trimesters. Extrahepatic cholestasis is most often caused by choledocholithiasis (Bile Duct Stones), benign biliary strictures (non-cancerous narrowing of the common duct), cholangiocarcinoma (ductal carcinoma), and pancreatic carcinoma. Extrahepatic cholestasis can occur as a side effect of many medications.

A compound of the application may be used for treating or preventing one or more symptoms of intrahepatic or extrahepatic cholestasis, including without limitation, biliary atresia, obstetric cholestasis, neonatal cholestasis, drug induced cholestasis, cholestasis arising from Hepatitis C infection, chronic cholestatic liver disease such as primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC).

In one embodiment, the application relates to a method of enhancing liver regeneration in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the method is enhancing liver regeneration for liver transplantation. In one embodiment, the application relates to a method of treating or preventing fibrosis in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the application relates to a method of treating fibrosis. In one embodiment, the application relates to a method of preventing fibrosis.

Accordingly, as used herein, the term fibrosis refers to all recognized fibrotic disorders, including fibrosis due to pathological conditions or diseases, fibrosis due to physical trauma ("traumatic fibrosis"), fibrosis due to radiation damage, and fibrosis due to exposure to chemotherapeutics. As used herein, the term "organ fibrosis" includes but is not limited to liver fibrosis, fibrosis of the kidneys, fibrosis of lung, and fibrosis of the intestine. "Traumatic fibrosis" includes but is not limited to fibrosis secondary to surgery (surgical scarring), accidental physical trauma, bums, and hypertrophic scarring.

As used herein, "liver fibrosis" includes liver fibrosis due to any cause, including but not limited to virally-induced liver fibrosis such as that due to hepatitis B or C vims; exposure to alcohol (alcoholic liver disease), certain pharmaceutical compounds including but not limited to methotrexate, some chemotherapeutic agents, and chronic ingestion of arsenicals or vitamin A in megadoses, oxidative stress, cancer radiation therapy or certain industrial chemicals including but not limited to carbon tetrachloride and dimethylnitrosamine; and diseases such as primary biliary cirrhosis, primary sclerosing cholangitis, fatty liver, obesity, non-alcoholic steatohepatitis, cystic fibrosis, hemochromatosis, auto-immune hepatitis, and steatohepatitis. Current therapy in liver fibrosis is primarily directed at removing the causal agent, e.g ., removing excess iron (e.g, in the case of hemochromatosis), decreasing viral load (e.g, in the case of chronic viral hepatitis), or eliminating or decreasing exposure to toxins (e.g, in the case of alcoholic liver disease). Anti-inflammatory drugs such as corticosteroids and colchicine are also known for use in treating inflammation that can lead to liver fibrosis. As is known in the art, liver fibrosis may be clinically classified into five stages of severity (SO, SI, S2, S3, and S4), usually based on histological examination of a biopsy specimen. SO indicates no fibrosis, whereas S4 indicates cirrhosis. While various criteria for staging the severity of liver fibrosis exist, in general early stages of fibrosis are identified by discrete, localized areas of scarring in one portal (zone) of the liver, whereas later stages of fibrosis are identified by bridging fibrosis (scarring that crosses zones of the liver).

In one embodiment, the application relates to a method of treating or preventing organ fibrosis in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the fibrosis is liver fibrosis.

In one embodiment, the application relates to a method of treating or preventing gastrointestinal disease in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the application relates to a method of treating gastrointestinal disease. In one embodiment, the application relates to a method of preventing gastrointestinal disease. In one embodiment, the gastrointestinal disease is selected from inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), bacterial overgrowth, malabsorption, post-radiation colitis, and microscopic colitis. In one embodiment, the inflammatory bowel disease is selected from Crohn's disease and ulcerative colitis.

In one embodiment, the application relates to a method of treating or preventing renal disease in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the application relates to a method of treating renal disease. In one embodiment, the application relates to a method of preventing renal disease. In one embodiment, the renal disease is selected from diabetic nephropathy, focal segmental glomerulosclerosis (FSGS), hypertensive nephrosclerosis, chronic glomerulonephritis, chronic transplant glomerulopathy, chronic interstitial nephritis, and polycystic kidney disease.

In one embodiment, the application relates to a method of treating or preventing metabolic disease in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the application relates to a method of treating renal disease. In one embodiment, the application relates to a method of preventing renal disease. In one embodiment, the metabolic disease is selected from insulin resistance, hyperglycemia, diabetes mellitus, diabesity, and obesity. In one embodiment, the diabetes mellitus is type I diabetes. In one embodiment, the diabetes mellitus is type II diabetes.

Diabetes mellitus, commonly called diabetes, refers to a disease or condition that is generally characterized by metabolic defects in production and utilization of glucose which result in the failure to maintain appropriate blood sugar levels in the body.

In the case of type II diabetes, the disease is characterized by insulin resistance, in which insulin loses its ability to exert its biological effects across a broad range of concentrations. This resistance to insulin responsiveness results in insufficient insulin activation of glucose uptake, oxidation and storage in muscle and inadequate insulin repression of lipolysis in adipose tissue and of glucose production and secretion in liver. The resulting condition is elevated blood glucose, which is called "hyperglycemia". Uncontrolled hyperglycemia is associated with increased and premature mortality due to an increased risk for microvascular and macrovascular diseases, including retinopathy (the impairment or loss of vision due to blood vessel damage in the eyes); neuropathy (nerve damage and foot problems due to blood vessel damage to the nervous system); and nephropathy (kidney disease due to blood vessel damage in the kidneys), hypertension, cerebrovascular disease, and coronary heart disease. Therefore, control of glucose homeostasis is a critically important approach for the treatment of diabetes.

Insulin resistance has been hypothesized to unify the clustering of hypertension, glucose intolerance, hyperinsulinemia, increased levels of triglyceride and decreased HDL cholesterol, and central and overall obesity. The association of insulin resistance with glucose intolerance, an increase in plasma triglyceride and a decrease in high-density lipoprotein cholesterol concentrations, hypertension, hyperuricemia, smaller denser low- density lipoprotein particles, and higher circulating levels of plasminogen activator inhibitor- 1, has been referred to as "Syndrome X". Accordingly, methods of treating or preventing any disorders related to insulin resistance including the cluster of disease states, conditions or disorders that make up "Syndrome X" are provided. In one embodiment, the application relates to a method of treating or preventing metabolic syndrome in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the application relates to a method of treating metabolic syndrome. In another embodiment, the application relates to a method of preventing metabolic syndrome.

In one embodiment, the application relates to a method of treating or preventing cancer in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the application relates to a method of treating cancer. In one embodiment, the application relates to a method of preventing cancer. In one embodiment, the cancer is selected from hepatocellular carcinoma, colorectal cancer, gastric cancer, renal cancer, prostate cancer, adrenal cancer, pancreatic cancer, breast cancer, bladder cancer, salivary gland cancer, ovarian cancer, uterine body cancer, and lung cancer. In one embodiment, the cancer is hepatocellular carcinoma. In one embodiment, the cancer is colorectal cancer. In one embodiment, the cancer is gastric cancer. In one embodiment, the cancer is renal cancer. In one embodiment, the cancer is prostate cancer. In one embodiment, the cancer is adrenal cancer. In one embodiment, the cancer is pancreatic cancer. In one embodiment, the cancer is breast cancer. In one embodiment, the cancer is bladder cancer. In one embodiment, the cancer is salivary gland cancer. In one embodiment, the cancer is ovarian cancer. In one embodiment, the cancer is uterine body cancer. In one embodiment, the cancer is lung cancer.

In another embodiment, at least one of an agent selected from Sorafenib, Sunitinib, Erlotinib, or Imatinib is co-administered with the compound of the application to treat cancer. In one embodiment, at least one of an agent selected from Abarelix, Aldeleukin, Allopurinol, Altretamine, Amifostine, Anastozole, Bevacizumab, Capecitabine, Carboplatin, Cisplatin, Docetaxel, Doxorubicin, Erlotinib, Exemestane, 5-Fluorouracil, Fulvestrant, Gemcitabine, Goserelin Acetate, Irinotecan, Lapatinib Ditosylate, Letozole, Leucovorin, Levamisole, Oxaliplatin, Paclitaxel, Panitumumab, Pemetrexed Disodium, Profimer Sodium, Tamoxifen, Topotecan, and Trastuzumab is co-administered with the compound of the application to treat cancer. Appropriate treatment for cancers depends on the type of cell from which the tumor derived, the stage and severity of the malignancy, and the genetic abnormality that contributes to the tumor.

Cancer staging systems describe the extent of cancer progression. In general, the staging systems describe how far the tumor has spread and puts patients with similar prognosis and treatment in the same staging group. In general, there are poorer prognoses for tumors that have become invasive or metastasized.

In one type of staging system, cases are grouped into four stages, denoted by Roman numerals I to IV. In stage I, cancers are often localized and are usually curable. Stage II and IIIA cancers are usually more advanced and may have invaded the surrounding tissues and spread to lymph nodes. Stage IV cancers include metastatic cancers that have spread to sites outside of lymph nodes.

Another staging system is TNM staging which stands for the categories: Tumor, Nodes, and Metastases. In this system, malignancies are described according to the severity of the individual categories. For example, T classifies the extent of a primary tumor from 0 to 4 with 0 representing a malignancy that does not have invasive activity and 4 representing a malignancy that has invaded other organs by extension from the original site. N classifies the extent of lymph node involvement with 0 representing a malignancy with no lymph node involvement and 4 representing a malignancy with extensive lymph node involvement. M classifies the extent of metastasis from 0 to 1 with 0 representing a malignancy with no metastases and 1 representing a malignancy with metastases.

These staging systems or variations of these staging systems or other suitable staging systems may be used to describe a tumor such as hepatocellular carcinoma. Few options only are available for the treatment of hepatocellular cancer depending on the stage and features of the cancer. Treatments include surgery, treatment with Sorafenib, and targeted therapies. In general, surgery is the first line of treatment for early stage localized hepatocellular cancer. Additional systemic treatments may be used to treat invasive and metastatic tumors.

In one embodiment, the application relates to a method of treating or preventing gallstones in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the application relates to a method of treating gallstones. In one embodiment, the application relates to a method of preventing gallstones.

A gallstone is a crystalline concretion formed within the gallbladder by accretion of bile components. These calculi are formed in the gallbladder but may distally pass into other parts of the biliary tract such as the cystic duct, common bile duct, pancreatic duct, or the ampulla of Vater. Rarely, in cases of severe inflammation, gallstones may erode through the gallbladder into adherent bowel potentially causing an obstruction termed gallstone ileus. Presence of gallstones in the gallbladder may lead to acute cholecystitis, an inflammatory condition characterized by retention of bile in the gallbladder and often secondary infection by intestinal microorganisms, predominantly Escherichia coli , and Bacteroides species.

Presence of gallstones in other parts of the biliary tract can cause obstruction of the bile ducts, which can lead to serious conditions such as ascending cholangitis or pancreatitis.

In one embodiment, the application relates to a method of treating or preventing cholesterol gallstone disease in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the application relates to a method of treating cholesterol gallstone disease. In one embodiment, the application relates to a method of preventing cholesterol gallstone disease.

In one embodiment, the application relates to a method of treating or preventing neurological disease in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the application relates to a method of treating neurological disease. In one embodiment, the application relates to a method of preventing neurological disease. In one embodiment, the neurological disease is stroke. In one embodiment, the application relates to a method as described herein and further wherein, the compound is administered by a route selected from oral, parenteral, intramuscular, intranasal, sublingual, intratracheal, inhalation, ocular, vaginal, rectal, and intracerebroventricular. In one embodiment, the route is oral.

In one embodiment, the compound utilized in one or more of the methods described herein is an FXR agonist. In one embodiment, the compound is a selective FXR agonist. In another embodiment, the compound does not activate TGR5. In one embodiment, the compound does not activate other nuclear receptors involved in metabolic pathways ( e.g ., as measured by an AlphaScreen assay). In one embodiment, such other nuclear receptors involved in metabolic pathways are selected from LXRp, PXR, CAR, PPARa, PPAR5, PPARy, RAR, RARa, VDR, TR, PR, RXR, GR, and ER. In one embodiment, the compound induces apoptosis.

In one embodiment, the application relates to a method of regulating the expression level of one or more genes involved in bile acid homeostasis.

In one embodiment, the application relates to a method of down regulating the expression level of one or more genes selected from CYP7al and SREBP-IC in a cell by administering to the cell a compound of the application. In one embodiment, the application relates to a method of up regulating the expression level of one or more genes selected from OSTa, OSTp, BSEP, SHP, UGT2B4, MRP2, FGF-19, PPARy, PLTP, APOCII, and PEPCK in a cell by administering to the cell a compound of the application.

The application also relates to the manufacture of a medicament for treating or preventing a disease or condition (e.g., a disease or condition mediated by FXR), wherein the medicament comprises a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the application relates to the manufacture of a medicament for treating or preventing any one of the diseases or conditions described herein above, wherein the medicament comprises a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof.

The application also relates to a composition for use in a method for treating or preventing a disease or condition (e.g, a disease or condition mediated by FXR), wherein the composition comprises a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof. In one embodiment, the application relates to a composition for use in a method for treating or preventing any one of the diseases or conditions described herein above, wherein the composition comprises a compound of the application or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof.

The methods of the application comprise the step of administering an effective amount of a compound of the application. As used herein, the term an "effective amount" refers to an amount of a compound of the application which is sufficient to achieve the stated effect. Accordingly, an effective amount of a compound of the application used in a method for the prevention or treatment of FXR mediated diseases or conditions will be an amount sufficient to prevent or treat the FXR mediated disease or condition.

Similarly, an effective amount of a compound of the application for use in a method for the prevention or treatment of a cholestatic liver disease or increasing bile flow will be an amount sufficient to increase bile flow to the intestine. The amount of the compound of the application which is required to achieve the desired biological effect will depend on a number of factors such as the use for which it is intended, the means of administration, and the recipient, and will be ultimately at the discretion of the attendant physician or veterinarian. In general, a typical daily dose for the treatment of a FXR mediated disease and condition, for instance, may be expected to lie in the range of from about 0.01 mg/kg to about 100 mg/kg. This dose may be administered as a single unit dose or as several separate unit doses or as a continuous infusion. Similar dosages would be applicable for the treatment of other diseases, conditions and therapies including the prevention and treatment of cholestatic liver diseases.

In one of the embodiments, the present application proves a method of treating or preventing a disease or condition in a subject in need thereof comprising administering an effective amount of the compound of formula I or a pharmaceutically acceptable salt, solvate, amino acid conjugate, sulfate, glucuronide conjugate, or prodrug thereof, and wherein the disease or condition is mediated by FXR.

In one of the embodiments, the present application proves a method of treating or preventing a disease or condition in a subject in need thereof comprising administering an effective amount of the compound of formula I, wherein the disease is selected from cardiovascular disease, chronic liver disease, lipid disorder, gastrointestinal disease, renal disease, metabolic disease, cancer, and neurological disease.

In one of the embodiments, the present application proves a method of treating or preventing a disease or condition in a subject in need thereof comprising administering an effective amount of the compound of formula I, wherein the disease is cardiovascular disease selected from atherosclerosis, arteriosclerosis, dyslipidemia, hypercholesteremia, hyperlipidemia, hyperlipoproteinemia, and hypertriglyceridemia.

In one of the embodiments, the present application proves a method of treating or preventing a disease or condition in a subject in need thereof comprising administering an effective amount of the compound of formula I, wherein the disease is liver disease selected from a cholestatic liver disease such as primary biliary cirrhosis (PBC) also known as primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), chronic liver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), hepatitis C infection, alcoholic liver disease, liver damage due to progressive fibrosis, and liver fibrosis. Other examples of FXR mediated diseases also include portal hypertension, bile acid diarrhea, hyperlipidemia, high LDL-cholesterol, high HDL cholesterol, high triglycerides, and cardiovascular disease. Other liver diseases include cerebrotendinous xanthomatosis (CTX), drug induced cholestasis, intrahepatic cholestasis of pregnancy, parenteral nutrition associated cholestasis (PNAC), bacterial overgrowth or sepsis associated cholestasis, autoimmune hepatitis, chronic viral hepatitis, liver transplant associated graft versus host disease, living donor transplant liver regeneration, congenital hepatic fibrosis, choledocholithiasis, granulomatous liver disease, intra- or extrahepatic malignancy, Sjogren's syndrome, Sarcoidosis, Wilson's disease, Gaucher' s disease, hemochromatosis, and alpha 1 -antitrypsin deficiency.

In one of the embodiments, the present application proves a method of treating or preventing a disease or condition in a subject in need thereof comprising administering an effective amount of the compound of formula I, wherein the disease is gastrointestinal disease selected from inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), bacterial overgrowth, malabsorption, post-radiation colitis, and microscopic colitis.

In one of the embodiments, the present application proves a method of treating or preventing a disease or condition in a subject in need thereof comprising administering an effective amount of the compound of formula I, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

In one of the embodiments, the present application proves a method of treating or preventing a disease or condition in a subject in need thereof comprising administering an effective amount of the compound of formula I, wherein the disease is renal disease selected from diabetic nephropathy, focal segmental glomerulosclerosis (FSGS), hypertensive nephrosclerosis, chronic glomerulonephritis, chronic transplant glomerulopathy, chronic interstitial nephritis, and polycystic kidney disease.

In one of the embodiments, the present application proves a method of treating or preventing a disease or condition in a subject in need thereof comprising administering an effective amount of the compound of formula I, wherein the disease is metabolic disease selected from insulin resistance, hyperglycemia, diabetes mellitus, diabesity, and obesity.

In one of the embodiments, the present application proves a method of treating or preventing a disease or condition in a subject in need thereof comprising administering an effective amount of the compound of formula I, wherein the disease is cancer selected from hepatocellular carcinoma, colorectal cancer, gastric cancer, renal cancer, prostate cancer, adrenal cancer, pancreatic cancer, breast cancer, bladder cancer, salivary gland cancer, ovarian cancer, uterine body cancer, and lung cancer.

EXAMPLES

The following examples are intended to illustrate certain embodiments of the present application, but do not exemplify the full scope of the application. A summary of one synthetic route to Compound 100 is shown below in Procedure A:

Procedure A:

Abbreviations

Example 1 Compound 20 may be prepared by, e.g. bioconversion from a plant sterol.

200 mL of sterile Curvularia lunata culture medium containing 0.1% K ₂ HPO ₄ , 0.05% MgSCri, 0.05% FeSCL, 5% malt extract, 0.05% KC1, 0.01% sucrose, and 0.2% NaNCh are added to a 500 mL flask. A solution of 20 (100 mg) in acetone is added and shaken at 25-35 °C. The mixture is extracted with dichloromethane and the extracts are dried over sodium sulfate and concentrated. The residue is purified by silica gel chromatography (5-20% MTBE in heptane) and product-rich fractions are pooled and concentrated to give 21

Compound 21 may be converted to compound 22 by olefmation using techniques known in the art, such as those disclosed in US 2,624,748 and Uekawa et al. (2004) Short- step Synthesis of Chenodiol from Stigmasterol. Bioscience, biotechnology, and biochemistry, 68, 1332-1337.

To a solution of 22 (1.00 g, 2.5 mmol) in pyridine (6 mL) at 5-10 °C was added a solution of triethylsilyl trifluoromethanesulfonate (1.32 g, 5 mmol, 2.5 equiv.) in pyridine (2 mL) portionwise. The mixture was gradually warmed to 15-20 °C and stirred 18 h. The reaction was quenched with aqueous NTLCl (4 mL) and stirring continued for 4-6 h. The mixture was diluted with MTBE and washed with water. The organic layer was dried over Na ₂ SC> ₄ and concentrated under vacuum to afford 13 as a solid (1.16 g, 2.25 mmol, 90% yield). Example 2

To a solution of 13 (1.1 g, 2.14 mmol) in tert-butanol (5 mL) was added chloranil (605 mg, 2.46 mmol, 1.15 equiv.) and the reaction was heated between 40-60 °C for 5-6 h. The mixture was cooled to approximately 10 °C and the solids were filtered and washed with DCM. The filtrate was concentrated under vacuum and the residue was chromatographed on silica gel (5-25% heptane:ethyl acetate) to afford 24c as a solid (735 mg, 1.43 mmol, 67% yield).

Example 3

Chemical Formula: C ₃₁ H4 ₈ 0 ₅ Si

Chemical Formula: C ₃₁ H4 ₈ 0 ₄ Si Molecular Weight: 528.81 Molecular Weight: 512.81

A mixture of 24c (730 mg, 1.42 mmol), BHT (9.4 mg, 0.04 mmol, 3 mol%), ethyl acetate (6 mL) and water (2 mL) was heated to 70-80 °C. To the stirred mixture was slowly added a solution of mCPBA (431 mg, 2.50 mmol, 1.7 equiv.) in ethyl acetate (4 mL). The reaction mixture was stirred at 65-70 °C for 2-3 h, then cooled to 15-20 °C and washed with dilute NaOH (aq), then 5% aqueous NaiSO . The organic layer was dried over Na ₂ SC> ₄ , then concentrated to a solid. The solids were triturated with ethyl acetate at 50-60 °C, then cooled, filtered and dried under vacuum to afford 25c (570 mg, 1.08 mmol, 76% yield).

Example 4 A solution of EtMgBr in THF (1M, 1.85 mL, 1.85 mmol, 1.9 equiv.) was added portionwise to a pre-cooled (-20 °C) solution of ZnCh (0.5M, 1.85 mL, 0.93 mmol, 0.96 equiv.) in THF (1.85 mL). Copper iodide (9.2 mg, 0.048 mmol, 0.05 equiv.) was added in one portion, followed by portionwise addition of 25c (513 mg, 0.97 mmol) in THF (1.85 mL). The reaction was stirred at -20 °C for 60-90 min. Additional Cul (9.2 mg, 0.048 mmol, 0.05 equiv.) and EtMgBr in THF (1M, 0.98 mL, 0.49 mmol, 0.5 equiv.) is added, if needed, to drive the reaction to completion. The reaction is quenched with aqueous NH ₄ CI (1 mL) and the solids were filtered. The filtrate was extracted with MTBE and washed with aqueous NH ₄ CI (3 x 1 mL). The organic layer was dried over NaiSCL, filtered and concentrated under vacuum to afford a 26c as a solid (492 mg, 0.88 mmol, 91% yield). Example 5

A suspension of 26c (486 mg, 0.87 mmol), 10% Pd/C (50% wet, 92 mg, 5 mol%) in DMF (15 mL) was cooled to 0-5 °C. The suspension was vigorously stirred under a hydrogen atmosphere (ambient pressure) for 3-5 h. the mixture was diluted with MTBE and filtered. The filtrate was washed with water and the organic layer was dried with Na ₂ SC> ₄ , filtered, and concentrated under vacuum to afford 27c as a solid (467 mg, 0.83 mmol).

Example 6

To a stirred suspension of H5IO6 (274 mg, 1.2 mmol, 1.5 equiv.) in water (8 mL) containing RuCh (5 mg, 0.024 mmol, 0.03 equiv.) at 0-5 °C was added a solution of 27c (450 mg, 0.80 mmol) in THF (4 mL) and acetone (4 mL) in portions. The reaction mixture was stirred for 2-5 h. The reaction was filtered and the filtrate was diluted with MTBE and washed with water until a negative starch-iodine test was achieved.

Removal of the TES-protecting group: The organic layer was concentrated under vacuum to an oil which was taken up into THF (4 mL). To the THF solution was added a solution of MRF (59 mg, 1.6 mmol, 2 equiv.) in water (0.1 mL), and the mixture was stirred at 40 °C for 4-6 h. The mixture was diluted with MTBE and washed with water.

The organic layer was concentrated under vacuum and diluted with methanol diluted with methanol and concentrated to an oil.

Hydrolysis of the ester and isomerization of the C-6 ethyl moiety: The oil from the above step was taken up into methanol (5 mL) and heated to approximately 50 °C for 8-16 h in the presence of 10% aqueous NaOH (0.96 g, 2.4 mmol, 3 equiv.). The mixture was diluted with water and concentrated to a residue. The residue was diluted with water and acidified with 2N HC1. The resulting suspension was filtered, washed with water and dried under vacuum to afford 28c (294 mg, 0.68 mmol, 85% yield).

Example 7

Compound 100

Chemical Formula: C ₆ H ₄₀ O Molecular Weight: 432.60

To a solution of NaOH (104 mg, 2.6 mmol, 4 equiv.) in water (6 mL) was added 28c (280 mg, 0.65 mmol). The mixture was stirred at 15-20 °C until a solution was obtained. To the solution was added NaB¾ (49 mg, 1.95 mmol, 2 equiv.) in portions, and the mixture was stirred at 15-20 °C. The mixture was then heated to 90-95 °C and additional NaB¾ (25 mg, 0.65 mmol, 1 equiv.) was added. The mixture was stirred for 3- 6 h and cooled to ambient temperature, quenched with HC1 and extracted with MTBE, and the MTBE layer was washed with water and the aqueous layers were discarded. The product was isolated from the MTBE layer by warming to 50 °C, then adding heptane (20- 30% v/v). The mixture was cooled to 15-20 °C and filtered and dried to produce Compound 100 as a solid (227 mg, 0.52 mmol, 80% yield). Optionally, Compound 100 can be isolated from water by stirring the MTBE layer was stirred with INNaOH (aq) (2-2.5 equiv.) for 30 min, then separating and collecting the aqueous layer. The MTBE layer is washed with water and then discarded, while and the aqueous washings are combined with the basic aqueous layer. The combined aqueous layers are concentrated under vacuum to remove residual MTBE and the residue is diluted with water and acidified to pH 2-3 with IN HC1 (aq). The suspension is heated to 40 °C for 30-60 min, then cooled. The suspension is filtered, washed with water and dried under vacuum to afford Compound 100.